2 SUMMARY

This paper addresses four sets of questions pertaining to the efficacy of rodenticide baits registered for

use to control commensal rodents (Norway rats, roof rats, and house mice) in the U.S. These questions

are related to possible impacts of EPA’s 2008 Risk Mitigation Decision (RMD) on rodenticide bait

products registered for use to control commensal rodents, with emphasis on the types of products that

are to be available or excluded from consumer type retail outlets.

This paper notes that all of the 10 rodenticides covered by the RMD were evaluated and found to meet

the U.S. criteria for effectiveness that existed at the time the agents initially were registered and that

the individual bait formulations registered for control of commensal rodents have been tested and

found to meet the efficacy requirements for effectiveness of such products. Although the various active

ingredients and bait formulations registered to control commensal rodents in the U.S. may not be

equally effective in all use situations (e.g., control of house mice with first

‐ generation anticoagulants),

effective commensal rodenticide bait products are available on the “consumer” and professional

markets following the June 4, 2011, deadline for compliance with the RMD.

Resistance to first generation anticoagulant rodenticides has been detected in populations of

commensal rodents in many nations, including the U.S.; but surveillance for resistance has not occurred

here in a systematic fashion for ~30 years. Resistance to first

‐ and some second

‐ generation

anticoagulants has been researched extensively in several European countries and found to be

genetically mediated, although simple genetic models do not explain all observations. The current

incidence of resistance to anticoagulant rodenticides in the U.S. is unknown. Therefore, it also is not

known to what extent resistance might render first

‐ generation anticoagulant baits ineffective in the U.S.

Although the RMD provides for removing second

‐ generation anticoagulants from “consumer” markets,

that policy is not expected to lead to widespread selection for resistance to first

‐ generation

anticoagulants in local populations of Norway rats, roof rats, and/or house mice because consumers

have options besides first

‐ generation anticoagulants for controlling commensal rodents and because it is

not expected that consumers, baiting locally and likely not in concert, would exert sustained selective

pressure. Consumers’ use of traps and rodenticides that are not anticoagulants instead of or in

alternation with first

‐ generation anticoagulants would limit the degree to which selection for resistance

occurred.

Differences in bait form (e.g., pelleted baits vs. bait blocks) may affect the attractiveness efficacy of

baits to commensal rodents in some situations. However, product

‐ specific efficacy data submitted to

and reviewed by EPA over its 41 years of existence indicate that bait block formulations sufficiently

palatable and lethal to laboratory rats and mice to meet or exceed the Agency’s efficacy criteria have

been produced for all of the active ingredients directly affected by the RMD except for Zinc Phosphide,

for which there are no bait

‐ block products registered. Bait blocks are held to the same efficacy criteria

as pelleted baits when the blocks are registered for use at sites (e.g., in and around buildings) similar to

those where pelleted baits are registered for use. Although initial acceptance of bait blocks might be

delayed for some individual rodents, bait block formulations are not inherently less effective against

commensal rodents than are small

‐ particle baits, including pelleted and meal formulations.3 Rodenticide baits are not rendered ineffective by being applied in tamper

‐ resistant bait stations rather

than in the open, which is prohibited by labeling in situations where open placements would be

accessible to children, pets, domestic animals, or nontargeted wildlife. In circumstances in which

commensal rodents do not enter bait stations and feed from them shortly after deployment, baiting in

tamper

‐ resistant bait stations may be somewhat less efficient in controlling commensal rodent

populations than open placements might be. However, open placements would be less well protected

from nontarget species and environmental factors that might shorten the effective life of the bait.

This paper also discusses whether potential deterrents (e.g., bittering agents) added to rodenticide baits

to reduce the number of incidents of exposure of young children to those baits would also adversely

affect the performance of the rodenticides against targeted species. Although there are published data

suggesting that certain baits containing the bittering agent Denatonium Benzoate performed similarly to

baits lacking that substance, there are some accounts that suggest otherwise. The paper does not

address whether baits containing Denatonium Benzoate at concentrations that commensal rodents will

accept are protective of children. As Denatonium Benzoate must be tasted to be perceived, its presence

in baits would not prevent initial exposures.4 CONTENTS

I. Introduction 5

A. Targeted Species 5

B. Major Provisions of the Risk Mitigation Decision (RMD) for Ten Rodenticide Compounds 5

II. Efficacy of the Rodenticide Active Ingredients Affected by the Risk Mitigation Decision 7

A. EPA’s Efficacy Data Requirements 8

1. New rodenticide active ingredients for controlling commensal rodents 9

2. New bait registrations for active ingredients registered for controlling commensal rodents 9

3. Laboratory screening studies vs. field studies 10

4. Special efficacy studies required by EPA as warranted by proposed claims 11

B. Speed of Kill 11

C. Palatability 12

D. Efficacy of Registered Commensal Rodenticide Baits, as Available to Consumers and 14

Other Users

III. Possible Effects of Resistance to First

‐ generation Anticoagulants on Rodent Control in 27

the U.S.

A. History of Anticoagulant Resistance 30

B. Mechanisms and Genetics of Anticoagulant Resistance 32

IV. Effects of Bait Form on the Effectiveness of Commensal Rodenticides 35

A. Efficacy Data Comparing Bait Blocks with Other Bait Forms 36

B. Selection of Bait Form for Controlling Commensal Rodent Populations 38

V. Possible Effects on Rodent Control of Limiting Applications to Bait Stations 40

A. Protective Rodenticide Bait Stations and U.S. Policies Pertaining to Them 40

B. Neophobia and Latency to Enter Rodenticide Bait Stations 43

C. Bait Stations vs. Placepacks and “non

‐ bait station baiting” 46

VI. Effects of Bittering Agents on the Effectiveness of Commensal Rodenticide Baits 47

A. Rationale for Adding Bittering Agents to Rodenticide Baits 48

B. Data on the Performance of Baits Containing Denatonium Benzoate 48

VII. References 52

APPENDIX 1 625 I. Introduction

The policies set forth in the Reregistration Eligibility Decisions (REDs) for the Rodenticide Cluster and

Zinc Phosphide and the Risk Mitigation Decision (RMD, EPA, 2008) for 10 rodenticide compounds are

intended to reduce the incidence of misuse of rodenticides. Such misuse has led to preventable

exposure incidents involving children, other humans, domesticated animals, and nontargeted wildlife.

This paper examines issues pertaining to the effectiveness of the active ingredients directly affected by

those REDs and the RMD as agents for controlling commensal rodents, and to the potential impact of

the RMD on commensal rodent control in the U.S. 1

A. Targeted Species

The three commensal rodent species that occur in the continental U.S., and are claimed to be controlled

by the products affected by the RMD, are the Norway (brown or sewer) rat ( Rattus norvegicus ), the roof

(black or ship) rat ( Rattus rattus ), and the house mouse ( Mus musculus ). These species are of Asian

origin and were brought to the Western Hemisphere, probably inadvertently, via the activities of

humans. The Polynesian (Pacific, kiore) rat ( Rattus exulans ) occurs in forests, fields, and commensally in

Hawaii and in various U.S. island territories in the Pacific Ocean. Its wide distribution on islands

throughout the Pacific may have resulted from deliberate introductions by Polynesian explorers and

colonizers (Stolzenburg, 2011).

Commensal rodent species, their biology, and methods and strategies for controlling them have been

described in articles and manuals published worldwide over the last several decades (e.g., Barnett,

1946; Bjornson and Wright, 1956; Brooks, 1973; Frantz and Davis, 1991; Timm, 1994a, b). The basic

tenets of commensal rodent control (e.g., identification, sanitation, exclusion wherever possible) have

changed little over that period of time, although traps, trapping, and other techniques have evolved,

and new rodenticides and bait station products have become available. Many of the rodenticides

mentioned in earlier works no longer are (or never were) registered in the U.S.

B. Major Provisions of the Risk Mitigation Decision (RMD) for Ten Rodenticide Compounds

The RMD established a policy which, after June 4, 2011, limits the types of bait products available at

“consumer” type retail outlets (e.g., grocery stores, drug stores, “big box” stores, hardware stores,

home

‐ improvement stores, club stores, convenience stores) to packages containing at least one ready

‐

to

‐ use bait station and no more than one pound of rodenticide bait. The baits sold in and with ready

‐ to

‐

use bait stations are to be in the form of bait blocks, pastes, or “other solid baits that achieve the safety

1 Commensal means “a companion at meals”. The biological term “commensalism” refers to a relationship

between two species from which on species benefits “and the other is neither benefited nor harmed” (Webster’s

New World Dictionary of American English

, Third College Edition, New York, NY: Webster’s New World

Dictionaries, Simon & Schuster, Inc., p. 280). The situation with commensal rodents is not fully consistent with this

definition. Absent control efforts, the rodents benefit from the relationship; but humans are harmed by it through

damage to property as well as potential adverse health effects. In the U.S., the term “commensal rodents” is

applied to several species of Old World rats and mice. As defined by Frantz and Davis (1991), “Commensal rats and

mice are those that live with or near human beings and depend upon them for at least part of their food and

shelter.”6 goal” (of “being reasonably expected to remain in the bait station”

– EPA, 2008, p. 11). Pelleted, whole

‐

grain, meal, and liquid bait formulations are not to be made available in consumer outlets. The RMD

limits the compounds that may be used in baits sold in consumer outlets for controlling commensal

rodents to the acute and semi

‐ acute rodenticides Bromethalin, Cholecalciferol, and Zinc Phosphide, and

the “first

‐ generation” anticoagulant rodenticides (Dubock and Kaukeinen, 1978) Chlorophacinone,

Diphacinone, and Warfarin. To be eliminated from the selection of commensal rodenticides available at

consumer outlets are the “second

‐ generation” anticoagulant rodenticides (Marsh, et al

, 1980)

Brodifacoum, Bromadiolone, Difenacoum, and Difethialone. These compounds were synthesized for the

purpose of creating anticoagulant rodenticides that would be effective against rodents resistant to first

‐

generation anticoagulants (Hadler and Shadbolt, 1975 Rowe and Bradfield, 1975; Dubock and

Kaukeinen, 1978).

The RMD also states that commensal rodenticide baits of any registered form and particle size and

containing any of the ten rodenticide compounds mentioned in the previous paragraph may be sold “in

farm stores or through direct sale to professional pest control operators, livestock producers, and other

professional users” provided that certain requirements for minimum package size are met. Baits

containing second

‐ generation anticoagulants that are limited by labeling to “use only inside of and

within 50 feet of agricultural buildings and not for use in and around homes” (EPA, 2008, p. 13) are to be

packaged and sold in retail containers that provide at least 8 pounds. The RMD further states that

rodenticide baits of any registered form and particle size that are “intended for use by professional

applicators” (EPA, 2008, p.13) and are labeled for controlling commensal rodents in or within 50 feet of

human residences, commercial and public buildings, and other structures may be sold at stores that

cater to professional pest control operators and others who use commensal rodenticides occupationally,

provided that packages of baits made with second

‐ generation anticoagulants hold at least 16 pounds of

bait, total. Packages providing at least 4 pounds, total, of any registered form of bait containing

Bromethalin, Chlorophacinone, Cholecalciferol, Diphacinone, Warfarin, or Zinc Phosphide may be sold

for structural uses (in or within 50 feet of buildings) on the agricultural and professional pest control

markets.

The RMD indicates that the bait stations to be included in retail packages of commensal rodenticides

sold at consumer type outlets are to be rated according to a four

‐ tiered system based upon the extent

to which the products have been shown to provide protection to children, to dogs, and against weather.

The scheme set forth in the RMD is summarized briefly below.

Tier 1

– Tamper

‐ resistant and weather

‐ resistant

– indoor and outdoor use (according to label)

Tier 2

– Tamper

‐ resistant (but not weather

‐ resistant)

– indoor use only

Tier 3

– Tamper

‐ resistant for young children (only)

– indoor use only

Tier 4

– Untested or tested without meeting any criteria for tamper resistance (beyond the self

‐

certification prescribed in the RMD)

– indoor use only7 This paper addresses the following efficacy

‐ related questions regarding the impacts of the RMD on

rodent control, with emphasis on the types of products that are to be available or excluded from

consumer type retail outlets:

Are the 10 rodenticides covered by the RMD equally or sufficiently effective in controlling commensal

rodents?

Does anticoagulant resistance render first

‐ generation anticoagulant baits ineffective in the U.S.? Would

the RMD’s limiting of the selection of rodenticide compounds in consumer outlets lead to greater

selection for resistance to first

‐ generation anticoagulants in local populations of Norway rats, roof rats,

and/or house mice?

Does bait form affect the efficacy of baits? Are bait block formulations inherently less effective against

commensal rodents than are small

‐ particle baits, including pelleted or meal formulations?

Are baits ineffective or significantly less efficient in controlling commensal rodents when the

formulations are applied in bait stations rather than in the open?

Would potential deterrents (e.g., bittering agents) added to baits to reduce the number of incidents of

exposure of young children to rodenticide baits also adversely affect the performance of the

rodenticides against targeted species?

II. Efficacy of the Rodenticide Active Ingredients Affected by the Risk Mitigation Decision

Are the 10 rodenticide compounds covered by the RMD equally or sufficiently effective in controlling

commensal rodents?

The 10 rodenticide active ingredients covered by the RMD are distinct chemical compounds. The seven

anticoagulants (Brodifacoum, Bromadiolone, Chlorophacinone, Difethialone, Diphacinone, Sodium Salt

of Diphacinone, Warfarin and Sodium Salt of Warfarin) share a mode of action but are distinct from one

another in molecular structure and in toxic effects. Although Brodifacoum is generally more toxic to a

wide array of target and nontarget species than other anticoagulants (Timm, 1994c), sensitivity to

anticoagulants currently registered in the U.S. varies considerably across species. Swine are relatively

sensitive to Warfarin (Timm, 1994c), whereas the mongoose Herpestes auropunctatus is extremely

sensitive to Diphacinone (Keith and Hirata, 1988). This brief discussion and much of the rest of this

paper serve to establish that the 10 rodenticide compounds covered by the RMD are not equal in all of

their effects.

The second part of the question concerns whether the 10 compounds are equally effective as

commensal rodenticides. All of the rodenticide compounds covered by the RMD met the Federal

requirements for efficacy data that were in place when the chemicals were registered. All, or nearly all,

commensal rodenticide bait products have met the product

‐ specific efficacy data requirements that are

currently in place for the type of product and the commensal rodenticide species for which control is

claimed on the products’ labels. Cholecalciferol, Difethialone, and Difenacoum were initially registered

after the cut

‐ off date (November 1, 1984) for reregistration but were registered according to the data8 requirements that EPA found to be applicable to them. The other seven rodenticide active ingredients

addressed by the RMD have been through the reregistration process, and all but Warfarin are currently

undergoing its final stage, product reregistration. 2

Thus, EPA has declared all of these active ingredients to be sufficiently effective to warrant registration

and/or continued registration. EPA further has found the commensal rodenticide bait products that

have been reregistered to be sufficiently effective to warrant continued registration. The bait

‐ block

formulations used in the ready

‐ to

‐ use bait station products that have been registered for sale on the

consumer market, in response to the RMD, have been subjected to laboratory choice

‐ feeding efficacy

studies (see below) which EPA has found to be acceptable. As discussed below, the rodenticides

registered for controlling commensal rodents in the U.S. are not identical in their effects; and problems

with controlling some species in some situations may be encountered for some of the compounds.

However, there are effective rodenticide bait products registered for use on the “consumer” and the

professional markets following the June 4, 2011, deadline for compliance with the provisions of the

RMD.

Information on the effectiveness of compounds and products used to control commensal rodents comes

to EPA primarily in the form of reports of research trials conducted to support claims of effectiveness

that have been proposed on labels included in applications for registering new products or for amending

existing registrations. Additional information regarding the efficacy of rodenticides is developed in the

contexts of basic and applied research conducted in the U.S. and in other countries, including but not

limited to studies conducted to meet registration requirements of other nations and to explore specific

issues with specific chemicals or chemical classes that have arisen over time. Among others, such issues

include relative effectiveness and effectiveness against rodents that are resistant to other commensal

rodenticide compounds. EPA does not routinely receive reports of studies not required by the Agency,

but some EPA personnel become aware of this research through their own reading and inquiries. At

times, entities seeking to inform or petition EPA in some fashion will include published papers and other

reports related to the efficacy of commensal rodenticides among the documents filed in support of their

petitions.

The remainder of this section addresses EPA’s efficacy data requirements for commensal rodenticides

and issues that affect the performance of bait formulations to susceptible rodents. Resistance to

registered active ingredients is addressed in section III.

A. EPA’s Efficacy Data Requirements

The information that EPA sees prior to making registration decisions regarding rodenticides and other

pesticides varies according to the nature of the chemical involved, its current registration status, the

sites where it is to be labeled for use, and the species that it is to be claimed to control. Commensal

2 EPA issued a Registration Standard for Warfarin in 1981 and a Reregistration Eligibility Decision (RED) for

Warfarin in 1991. Thus, Warfarin products have gone through two rounds of product reregistration. The RMD

required additional modifications for Warfarin registrations. EPA also issued a Registration Standard for Zinc

Phosphide.9 rodenticides are used in structural situations to control Norway rats, roof rats, and/or house mice.

Wherever they occur in association with humans, commensal rodents can be of public health

importance. Consequently, EPA requires submission of efficacy data to support claims for controlling

commensal rodents. This section summarizes EPA’s data requirements for commensal rodenticide baits,

in the process indicating the types of data that EPA reviews and the decisions that EPA makes when

claims of effectiveness are accepted for such products.

1. New rodenticide active ingredients for controlling commensal rodents

Since the 1972 amendments to the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) were

enacted, EPA has required a series of studies for demonstrating the effectiveness of new rodenticide

active ingredients that are to be applied in bait and/or tracking powder form. The studies required

include (1) acute oral toxicity testing of the active ingredient with wild

‐ type animals of the targeted

species; (2) laboratory efficacy screening of one or more bait or tracking

‐ powder formulations with wild

‐

type animals of the targeted species; and (3) indoor and outdoor field efficacy trials involving the

targeted species in actual use situations in different regions of the U.S. 3 If these studies are performed

appropriately and the results meet or exceed the relevant performance criteria, claims for control of the

species tested are accepted for the active ingredient and for the specific bait (formulation and form)

that was tested.

A rodenticide bait must be eaten by targeted rodents in amounts sufficient to kill enough of them to

meet or exceed the relevant performance criterion (or criteria) for the type of study that is being

conducted. EPA’s laboratory screening trials typically use a performance criterion of 90% mortality. In

field efficacy trials, the criterion typically is at least 70% reduction in activity, as demonstrated by two

independent census methods. Compounds, including first

‐ generation anticoagulants, which need

sustained feeding by rodents for several days to achieve maximum effectiveness also are required to

meet criteria for palatability (bait acceptance) which vary somewhat according to the nature of the test

being run. See II.C. below.

The commensal rodenticide compounds that were run through the full battery of EPA’s efficacy data

requirements include Pyriminil (aka Vacor, DLP

‐ 787, subsequently canceled), Brodifacoum,

Bromadiolone, Bromethalin, Cholecalciferol, and Difethialone.

2. New bait registrations for active ingredients registered for controlling commensal rodents

Once an active ingredient is registered for controlling a commensal rodent species using one bait

formulation, additional bait formulations may be registered on the basis of laboratory efficacy screening

trials using wild

‐ type animals of the same species. Protocols specific to various types of laboratory

screening tests are available from EPA’s Office of Pesticide Programs (Appendix I).

Applicants also may elect to run additional laboratory screening tests according to the relevant EPA

protocols using the originally accepted bait as the test material and laboratory strains of commensal

3 General guidance for the design of such studies appears on pages 307

‐ 310 of Subdivision G of EPA’s Pesticide

Assessment Guidelines, Office of Pesticides and Toxic Substances, Washington, DC, 1982.10 rodents as test subjects. The Wistar laboratory strain is preferred by EPA for the Norway rat. Swiss

‐

Webster is the preferred strain for the house mouse. If the results obtained with wild

‐ types and their

respective laboratory strains are similar with the same bait, trials with laboratory strains may be

substituted for trials with wild

‐ types in later laboratory screening trials. Once equivalency in laboratory

choice

‐ feeding trials has been established between wild

‐ type Norway rats and wild

‐ type roof rats, and

between wild

‐ type Norway rats and Wistar rats, additional bait formulations may be claimed to control

both Norway rats and roof rats on the basis of successful results in appropriately executed laboratory

efficacy trials with Wistar rats. Once equivalency in results of laboratory choice

‐ feeding trials has been

established between wild

‐ type and Swiss Webster strain house mice, additional bait formulations may

be registered for controlling house mice on the basis of acceptable performance in laboratory choice

‐

feeding trials using Swiss Webster mice.

The inferences drawn from comparable performance of a bait against wild

‐ type and laboratory

‐ strain

rodents in laboratory choice

‐ feeding trials do not extend beyond those described in the preceding

paragraph. Behavioral differences between laboratory strains and wild

‐ types have been observed in

other contexts, including neophobic responses by Norway rats to foods, bait stations, and other objects

(e.g., Barnett, 1946; Beck, et al 1988). Avoidance of new objects also has been observed with roof rats

(Cowan and Barnett, 1975).

For the rodenticide active ingredients registered after passage of the 1972 amendments to the Federal

Insecticide, Fungicide and Rodenticide Act (FIFRA), the baits run through the battery of laboratory and

field testing with wild

‐ types typically were pelleted formulations. For most of those active ingredients,

and for the first

‐ generation anticoagulants, the efficacy of bait block products has been supported by

the results of laboratory screening trials with laboratory strains, typically Wistar rats and Swiss

‐ Webster

mice, conducted subsequently to the laboratory and field efficacy trials that supported U.S. registration

of the active ingredient as a commensal rodenticide.

3. Laboratory Screening Studies vs. Field Studies

The suitability of an active ingredient for use as a toxic rodenticide applied in bait form is established

through the required series of trials (oral toxicity, choice feeding, and field testing) mentioned in II.A.1.,

plus any additional reported trials that may have been run to address related issues, including but not

limited to: mode of action, lethality in no

‐ choice feeding exposures, effectiveness against individuals

resistant to one or more other rodenticides, and relative palatability of candidate bait formulations.

Once an active ingredient is determined to be suitable for registration in commensal rodenticide baits,

laboratory screening tests typically are all that is required to support the registration of individual bait

formulations. Such tests assess the willingness of targeted rodents to ingest the new formulations,

which is essential to the effectiveness of rodenticide baits. These data are regarded as being specific to

the bait that was tested (bait formulation and bait form).

In accepting new bait formulations for registered rodenticide active ingredients, EPA makes the

assumption that the results of acceptable laboratory efficacy tests (with wild

‐ type or laboratory

‐ strain

subjects) are sufficient to support an inference that the new bait formulation is likely to perform as well11 as did the one used in the original field trials, other things being relatively equal. 4 EPA has not required

additional field trials when the difference in formulation types is between meal and pelleted baits,

pelleted and paraffinized block baits, or meal and block baits.

Field testing of new bait formulations of registered active ingredients only would be required if there

were something about the new formulation that caused EPA to question its suitability for use as a bait

under field conditions. As laboratory efficacy trials are surrogates for field trials, applicants and

registrants may elect to conduct field trials on new bait formulations in lieu of or in addition to

laboratory efficacy trials. Field trials necessarily involve wild

‐ types and assess the product under

conditions of actual use. Such conditions vary considerably from site to site and may include

uncontrollable confounding variables that render assessment of product performance difficult. While

the conditions of testing are artificial and wild

‐ type animals might not be used in them, laboratory trials

have the advantage of being able to be run under controlled conditions that should be replicable from

one testing facility to another.

4. Special Efficacy Studies Required by EPA as Warranted by Proposed Claims

Efficacy trials with wild

‐ types of commensal rodent species have been required to support claims of

effectiveness against rodents that are resistant to Warfarin and, by inference or demonstration, to other

first

‐ generation anticoagulants. Historically, such trials have involved subjects in which individuals’

resistance to Warfarin was confirmed through survival of no

‐ choice exposures, consistent with

procedures developed through the World Health Organization (WHO), to diets containing Warfarin

(WHO, 1970). Subsequently, it became possible to identify resistant individuals through the detection of

resistance

‐ conferring alleles (e.g., Kohn and Pelz, 1999).

When claims of special or unusual effectiveness are proposed, EPA typically gives applicants the

alternative of conducting efficacy trials designed specifically to assess the proposed claims or deleting

them. Procedures for screening baits for effectiveness when used in wet or damp areas have been

incorporated to Protocols 1.213 and 1.214, which typically are used to test bait

‐ block formulations.

B. Speed of kill

All anticoagulants kill indirectly through the effects of blood

‐ thinning and a suppressed clotting process.

The normal clotting process must be delayed for several days. Depending upon the specific

anticoagulant and the amount ingested, one to several days of bait consumption may be needed to

cause the death of a commensal rodent, which will occur three days to two weeks or more after bait

consumption begins. In summarizing laboratory trials with Brodifacoum

– the “single

‐ feeding”

anticoagulant that they were researching

‐‐ Dubock and Kaukeinen (1978, p. 128) observed that “Time

to death was similar to that expected from first generation anticoagulants.” Subsequent experience has

borne out this conclusion

– anticoagulant rodenticides all kill susceptible animals at essentially the same

4 If the new bait formulation is markedly different than any currently used with the active ingredient, especially if

the new bait seems likely to be prone to rapid fouling or degradation, EPA also might require limited field testing

to support the new formulation.12 pace, bait palatability and other factors being equal (e.g., Kaukeinen and Rampaud, 1986; Pitt, et al

,

2011; Witmer, 2007a,b). Documents prepared to provide guidance to rodenticide users typically make

the same point (e.g., Anonymous, 2002; Timm, 1994c).

Bromethalin, Cholecalciferol, and Zinc Phosphide typically kill more rapidly than do anticoagulant

rodenticides. Zinc Phosphide usually kills on the day of or on the next day after a lethal dosage has been

consumed. Ingestion of a toxic dose of Bromethalin induces anorexia, which is followed by death within

one to several days or by recovery. Cholecalciferol (Vitamin D 3 ) acts through excessive mobilization of

calcium, resulting in calcification of soft tissues, with death often occurring within two to several days,

but taking much longer for some subjects (e.g., Witmer, 2007a,b).

Under conditions of actual use, initial reluctance to accept a new food and social factors within rodent

populations may serve to extend the time from initial baiting to achieving the maximum effect of

treatment with anticoagulants to approximately a month and necessitate maintaining the active baiting

program for one to several weeks (e.g., Rennison and Dubock, 1978).

C. Palatability

In order to perform its intended function, a rodenticide bait must be ingested in sufficient quantity by

targeted rodents to ensure that all or nearly all of them die. When a rodenticide induces toxicosis prior

to the ingestion of a lethal dosage, the animal that ingests the bait containing that rodenticide may

associate the illness with the bait and refuse to ingest the bait again. Called “bait shyness” (e.g.,

Barnett, 1946; Hayes and Gaines, 1950), this effect may generalize to other baits containing the same

rodenticide or to other baits with the same feed components, depending upon whether the flavor of the

rodenticide or the rest of the bait was more salient to the animal.

Baits containing slow

‐ acting active ingredients and/or active ingredients that are not “stomach poisons”

are less likely to induce bait shyness. However, baits that are not very palatable might not be taken in

sufficient amounts or over sufficient numbers of days to control an individual rodent or a rodent

population. Baits containing active ingredients that require feeding over several consecutive days to

achieve maximum effectiveness are required to meet bait acceptance criteria when tested against a

standard challenge diet preparation.

Bait acceptance criteria are included in EPA’s laboratory efficacy protocols (e.g., Protocols 1.203, 1.204,

1.213, and 1.214

– Appendix I) for assessing the performance of anticoagulant baits if the duration of

exposure to the choice between the bait and the OPP standard rat and mouse challenge diet exceeds 24

hours. Acceptance is the percent of total feed consumption during the dietary choice phase of the test

that is comprised by the toxic bait. The standard rat and mouse challenge diet is comprised of 65%

whole yellow ground corn, 25% ground rolled oat groats, 5% corn oil, and 5% confectioner’s sugar.

Challenge diet is a preparation of moderate palatability to Norway rats and house mice, but was far

more palatable to them than was the laboratory rodent diet that was evaluated at the time when EPA’s

protocols for screening rodenticide baits under laboratory conditions were being developed (Palmateer,

1982). A standard challenge diet was established to promote consistency of results across laboratories

and fair comparisons among baits with respect to relative palatability.13 As noted above, the acceptance criterion was established because consumption of toxic bait over a

period of several days often is needed for anticoagulants to kill many of the animals exposed to them

(e.g., Dykstra, 1951; Saunders, et al 1955; Link, 1959). The need for multiple feedings for maximum

effectiveness is commonly seen among the first of the anticoagulant compounds that were developed as

rodenticides (Brooks, 1973), including all anticoagulant rodenticides registered in the U.S. prior to 1978.

Such compounds (e.g., Warfarin, Fumarin, Pindone [Pival], Valone [PMP], Diphacinone, and

Chlorophacinone) later became referred to collectively as “first generation anticoagulants” (Dubock and

Kaukeinen, 1978).

The acceptance criterion for meal and pelleted anticoagulant baits was set at 33% in Protocols 1.203 and

1.204, meaning that the toxic bait had to comprise at least one

‐ third of all measured feed consumption

in laboratory choice tests in which OPP rat and mouse diet was the only alternative feed. Put another

way, this criterion means that the toxic bait may not be rejected in favor of the challenge diet at a ratio

exceeding 2:1.

For paraffinized bait blocks containing anticoagulant rodenticides, the acceptance figure originally was

set at 25% (no greater than a 3:1 rejection); and the mortality criterion was set at 80%. Both of these

adjustments were adopted because bait blocks were formulated with paraffin, plus grain components

and other materials, for use in sewers and other wet and damp areas. It was felt that, while bait use in

sewers was necessary, a degree of compromise in effectiveness criteria likely would be needed for such

products due to the large amount of wax included in bait

‐ block formulations. Over time, however, EPA

learned that bait blocks tended to perform about as well in laboratory screening trials as did meal or

pelleted baits (e.g., Palmateer, 1974, discussed further below).

When registrants began proposing to register bait block products for use in the same types of situations

as those for which meal and pelleted baits were registered, however, EPA held such bait block products

to the same performance criteria (33% acceptance, 90% mortality) that were used for meal and pelleted

baits. The 25% acceptance and 80% mortality criteria were retained for baits that were claimed to be

effective when used in wet or damp areas, with baits having to be subjected to a standardized

weathering procedure (15 consecutive days at 90

‐ 100% relative humidity and ~100°F) before they were

used in bioassays to support such claims. These criteria are indicated in the current versions of

Protocols 1.213 and 1.214.

EPA does not require baits that contain acute rodenticide compounds to meet an acceptance figure

provided that the period of exposure to the choice between bait and challenge diet does not exceed 2

days (~48 hours, Protocols 1.209 and 1.210). However, baits with poor initial acceptance are not likely

to reach the 90% mortality figure for test groups in trials with short bait

‐ exposure periods.

Protocols 1.209 and 1.210 also provided for the screening of baits for which claims are sought to the

effect that a single night’s feeding on the bait is sufficient to kill the targeted species. Such claims often

are sought for baits containing second

‐ generation anticoagulants (see relevant discussion below). To

meet such claims, Protocols 1.209 and 1.210 are modified to shorten the bait

‐ exposure period to 24

hours, while the post

‐ exposure monitoring period usually is extended to allow for the protracted times14 to death typically observed with anticoagulant rodenticides. Although Protocols 1.209 and 1.210 lack an

acceptance criterion, baits with poor initial acceptance may be weeded out by trials with the exposure

to the choice between bait and challenge diet limited to one day.

Formulators use feed materials (usually one or more grains) and sweeteners to enhance the

attractiveness and sustained palatability of baits to rodents. The quality and shelf lives of these

ingredients can affect the attractiveness of the bait (e.g., Whisson, 1996). Other bait components

include the rodenticide compound itself and, perhaps, a dye, a binding agent, one or more

preservatives, a bittering agent, and/or an insecticide intended to limit the extent to which the bait

might be fouled and consumed by insects. If any of these ingredients

‐‐ typically present at low

concentrations

‐‐ affects the palatability of the bait formulation, such effects are likely to be negative

(e.g., Palmateer, 1979).

D. Efficacy of Registered Commensal Rodenticide Baits, as Available to Consumers and Other Users

In their review of worldwide research and operational use of Brodifacoum, Kaukeinen and Rampaud

(1986) observed that, during development as rodenticides, anticoagulant compounds initially were

screened for efficacy against Norway rats and that, perhaps as a consequence of that strategy, there is

“Generally poor efficacy with first

‐ generation anticoagulants against house mice” (p. 18). They add that

“brodifacoum retains good efficacy against M

. musculus

, particularly at 50

‐ ppm concentration in baits.”

Such accounts beg the question of whether bait products that meet the applicable criteria when

screened in laboratory tests and/or when in fresh condition retain such effectiveness over time and/or

under conditions of actual use.

On an acute (one

‐ dosage) LD 50 basis, house mice have been reported to be relatively insensitive to

Diphacinone (141

‐ 340 mg/kgbw) and to Warfarin (374 mg/kgbw, data cited by Timm, 1994c). 5

However, house mice are far more sensitive to these same multi

‐ dose anticoagulants if exposures to

them occur over several consecutive days. The 5

‐ day LD 50 values for laboratory strain house mice

reported for these same compounds are 1.41 mg/kg (0.42 mg/kgbw/day for males, 2.83 mg/kgbw/day

for females) for Diphacinone and 2.20 mg/kg (0.87 mg/kgbw/day for males and 8.00 mg/kgbw/day for

females) for Warfarin (Jackson and Ashton, 1992). A much lower sensitivity for females might lower the

effectiveness of marginally palatable baits against house mice. It would require 0.64 g of bait

consumption daily for 5 days to provide an 8

‐ mg/kgbw/day dosage of a 0.025% Warfarin bait to a 20

‐ g

female house mouse and 1.13 g of bait/day of a 0.005% Diphacinone bait to dose a 20

‐ g female house

mouse at 2.83 mg/kg.

Rowe and Redfern (1964) reported that up to four weeks of no

‐ choice feeding on a 0.025% Warfarin

diet were needed to kill some wild

‐ type house mice collected in England, with 4

‐ day feeding regimens

taking less than 50% of subjects and fixed durations ranging from 6 to 21 days killing more than 50% of

subjects but typically less than 100%. Using similar procedures but with feeding periods set at 3, 7, 14,

5 Reported results in acute oral LD 50 trials with house mice vary from study to study and strain to strain. On the

more sensitive end of the range of results with Diphacinone, Kusano (1974) reported LD 50 results of 30.0 and 28.3

mg/kgbw, respectively, for “dd

‐ strain” male and female house mice.15 and 18 days, Rowe and Redfern (1968) essentially replicated their earlier findings with 0.025% Warfarin,

and got similar results with Warfarin at 0.05%, with Diphacinone at 0.0125% (2.5 times the

concentration used in baits registered in the U.S.) and 0.025%, and with Chlorophacinone at 0.005% and

0.025%. In these trials, 100% kill of test group subjects (n

= 9 or 10) was achieved for the 18

‐ day feeding

duration for all groups but the one fed 0.0125% Diphacinone, in which 8 of 10 subjects were killed.

Replenishments of palatable baits would afford female house mice opportunities to ingest such dosages

in highly infested areas, but some advantage in controlling house mice with second

‐ generation could

result from there being less of a need to replenish baits as faithfully as with the first

‐ generation

anticoagulants (Dubock and Kaukeinen, 1978). The so

‐ called “single

‐ feeding” effect of second

‐

generation anticoagulants requires ample consumption of bait on the first day, but the extended

retention of those agents in the bodies of exposed animals may suppress the clotting process long

enough that further consumption is not needed to cause death (Jackson and Ashton, 1992).

Dubock and Kaukeinen (1978) state that Brodifacoum can act as “a potent multiple dose rodenticide” if

it is present in baits at relatively low concentrations (e.g., 0.001% or 0.002%). Presumably, such also

would be the case if only very smaller amounts of higher

‐ concentration baits are consumed daily.

Dubock and Kaukeinen (1978) cited 5

‐ day oral LD 50 s of 0.05

‐ 0.06 mg/kgbw/day for Norway rat vs. 0.26

mg/kgbw for acute oral LD 50 and 0.035 mg/kgbw/day for (albino) house mouse vs. 0.40 mg/kgbw for

acute oral LD 50

. 6 They also present data showing efficacy (5 of 5 rats killed) against “Homozygous

(Welsh) Resistant Norway Rats” in 10 day no

‐ choice feeding trials at formulation strengths of 0.0005%,

0.001%, and 0.002% Brodifacoum, whereas Warfarin at 0.025% and 0.020% killed none of 5,

Diphacinone at 0.025% killed none of 5, and Chlorophacinone at 0.025% killed 1 of 5. In (up to) 21

‐ day

no

‐ choice feeding trials, a 0.002% Brodifacoum diet killed all 12 resistant house mice to which it was

offered. Similar trials reportedly resulted in deaths of 14 of 15 resistant mice fed a 0.005% Difenacoum

diet, 6 of 13 resistant mice fed a 0.025% Chlorophacinone diet, and none of 9 resistant mice fed a

0.0125% Diphacinone diet.

Dubock and Kaukeinen (1978) rated “the 50 ppm concentration” of Brodifacoum as having “the optimal

potential for single

‐ feeding action with all pest rodents thus far investigated, while minimizing

environmental issues.” This conclusion is consistent with the “single

‐ feed” effect being the driving force

in selecting the 0.005% concentration for use in Brodifacoum (e.g., “TALON”) baits claimed to control

commensal rodents. That level was not needed for efficacy against resistant rodents, which was the

reason why second

‐ generation anticoagulants were developed. Dubock and Kaukeinen (1978, p. 134)

add that

The single feeding capability of the TALON pellet leaves more room for operator error,

manifest as underbaiting, which is so often the reason for failed treatments.

6 The selection of 0.005% as the concentration to be used in Brodifacoum baits was linked to supporting claims of

effectiveness after a “single

‐ feeding” rather than claims of effectiveness against rodents resistant to Warfarin and

other first

‐ generation anticoagulants. Concentrations which required multiple feedings on Brodifacoum baits for

full effectiveness also would be effective against resistant animals.16 Under no

‐ choice feeding conditions in trials conducted nearly two decades before those reported by

Dubock and Kaukeinen (1978), one day of exposure to a 0.025% Warfarin diet was sufficient to kill 9 of

12 wild

‐ type Norway rats; and one day of no

‐ choice exposure to a 0.005% Diphacinone diet killed 10 of

12 Norway rats (Bentley and Larthe, 1959).

For nearly the first 12 years of its existence, EPA maintained an operating facility for evaluating

rodenticide efficacy. The facility was located on the grounds of the U.S. Agricultural Research Center in

Beltsville, MD, and was operated by the U.S. Department of Agriculture (USDA) for nearly a decade prior

to the creation of EPA. Developmental research on methods for evaluating rodenticides and other

categories of vertebrate pesticides was conducted at the facility, which EPA inherited in 1970 when

primary authority for regulating pesticides in the U.S. was transferred to EPA from the USDA. Beginning

several years before its transfer to EPA, the facility established a program of market surveillance of

registered vertebrate pesticide products, with emphasis on the active

‐ ingredient composition and the

efficacy of baits registered for controlling commensal rodenticides. Through that program, USDA and,

later, EPA learned that many registered rodenticide bait products collected via inspections did not meet

the applicable criteria of relevant laboratory efficacy protocols (e.g., Palmateer, 1974).

During its history, the Beltsville facility conducted more than 5,000 studies, most of which involved

efficacy

‐ screening of registered rodenticide baits. Following the closing of the facility on June 30, 1982,

EPA contracted with the Denver Wildlife Research Center (DWRC), U.S. Department of the Interior, to

conduct efficacy screening of rodenticide bait products on a much smaller scale than had been done at

Beltsville. The contract with the DWRC was not renewed after the end of fiscal year 1985. Since then,

EPA has had no direct means for assessing the performance of market samples of registered rodenticide

baits.

Most of the trials conducted at Beltsville and Denver involved laboratory strains of Norway rats and

house mice. Although both facilities had limited capacity in this regard, neither of them had enough

animals to allow for routine screening of registered baits with wild

‐ type subjects. Beltsville maintained

outdoor colonies of Norway rats and roof rats and also housed wild

‐ type house mice. Some limited

testing with wild

‐ type Norway rats was conducted at Denver under EPA’s contract. Thus, when those

facilities were screening baits, most of the feedback obtained came from trials conducted with

laboratory strains.

Some data on the performance of rodenticide baits registered in the U.S. on wild

‐ type Rattus spp. rats

and house mice have become available recently. These data come from laboratory trials that were

conducted according to procedures different from those indicated in the protocols used at Beltsville and

Denver for evaluating bait and used in studies conducted by or for registrants to support claims for

controlling commensal rodents with bait products. However, the methods used in the recently reported

studies were adequate for assessing the relative performance of the products evaluated. The results

reported suggest that some registered products have limitations in controlling some of the species

against which they are claimed to be effective. Most of the baits tested were of pelleted formulations.17 Pitt, et al (2011) report on the effectiveness of certain rodenticide bait products that were registered in

the U.S. at the time of the research (~2008) on wild

‐ type roof rats, Polynesian rats ( Rattus exulans ), and

house mice caught on Hawaii. 7 Some of the products tested under laboratory conditions were not

registered in Hawaii for use to control all of the species against which they were tested. Using

procedures similar to those employed by Pitt, et al (2011), Witmer (2007a, b) ran laboratory efficacy

studies with wild

‐ type Norway rats and house mice collected in the Fort Collins, CO, area, plus offspring

of those rodents. Presented and discussed, below, the results reported by Witmer (2007a) and Pitt, et

al (2011) indicate effectiveness problems vs. wild

‐ type house mice for certain baits containing first

‐

generation anticoagulants as the active ingredient. Those results are consistent with the assessment by

Kaukeinen and Rampaud (1986) that is quoted above.

Witmer (2007b) acclimatized individually caged Norway rats for > 2 weeks, during which time they were

offered water, “rat chow”, chew sticks, and a piece of burlap. The test phase for solid

‐ bait products

involved exposure to the candidate bait plus “rat chow” for 3 consecutive days. After that, rats were

maintained on “rat chow” and observed for 10 additional days before being sacrificed. (Consequently,

rats that might have died >13 days after the onset of bait exposure were counted as survivors.) The one

liquid bait that Witmer (2007b) screened was presented, no

‐ choice, for 3 days as a substitute for water.

Witmer set 80% kill of test

‐ group subjects as the minimum level of performance for a bait to be

considered “efficacious”.

Witmer’s (2007b) results for diet consumption and mortality are summarized in the Table 1. Bait

acceptance

– the proportion comprised by the toxic bait of the total calculated consumption (bait plus

alternative diet)

– was moderate to very high for the anticoagulant baits tested. Such results are typical

when the alternative feed is a commercial laboratory rodent diet (e.g., Hayes and Gaines, 1950). All but

one Norway rat exposed to anticoagulant baits died as an apparent result, with all of the baits tested

meeting or exceeding Witmer’s criterion of 80% mortality in the test group. The survivor in the trial with

the Warfarin bait block product reportedly ate nearly 12.7 g. of the toxic bait over 3 days and accepted it

at 17.2% of total consumption. Ranges in times to death were similar among anticoagulant products,

with no apparent differences between the first

‐ generation and second

‐ generation compounds in speed

‐

to

‐ kill.

Composite acceptance of the Zinc Phosphide baits was low, although Witmer’s criterion of 80%

mortality was attained with the oat formulation. The survivor of the trial with the oat formulation

contributed 37.9 g. of rat chow consumption to the group’s total, presumably having learned to avoid

the toxic bait after a measured consumption of 0.5 g. Such learning may have occurred for all 5 rats

exposed to the pelleted Zinc Phosphide bait. None of them accepted it at greater than 8.1%, and all 5

survived. Acquired aversion (“bait shyness”) is commonly observed with Zinc Phosphide baits (e.g.,

Greaves, 1966). 8

7 Since 2008, registrations for many commensal rodenticide bait products have been amended to comply with

provisions of the RMD or have been canceled.

8 Prebaiting with a placebo for the toxic bait can overcome bait shyness to a degree by making rodents less hesitant

to consume the rodenticide once it is offered. Prebaiting is not required or suggested on labels for Zinc Phosphide18 Table 1. Bait acceptance (vs. a laboratory diet), over a 3

‐ day bait

‐ exposure period, and mortality results

reported by Witmer (2007b) for Norway rats ( Rattus norvegicus ), and descendents thereof, collected in

Fort Collins, CO, area.

Active

Ingredient Bait

Form Grams Chow

Consumed Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death*

First

‐ generation Anticoagulants

Diphacinone Pellets 118.0 230.4 66.1% 100% 5

‐ 8

Diphacinone, Na+ salt Liquid 296.5 n/a n/a 100% 4

‐ 6

Chlorophacinone Pellets 130.5 205.3 61.3% 100% 5

‐ 6

Warfarin Pellets 227.2 97.38 30.0% 80% 5

‐ 6

Second

‐ generation Anticoagulants

Brodifacoum CI† Pellets 94.3 224.9 70.5% 100% 5

‐ 9

Brodifacoum HA Pellets 35.8 209.9 85.4% 100% 5

‐ 6

Difethialone Pellets 31.6 190.07 85.7% 100% 5

‐ 7

Bromadiolone Pellets 42.9 206.8 82.8% 100% 4

‐ 6

Acute Toxicants

Zinc Phosphide Oats 46.0 7.5 14.0% 80% 1

Zinc Phosphide Pellets 199.3 9.44 4.5% 0%

‐

Bromethalin Pellets 25.0 45.51 64.5% 80% 1

‐ 4

Cholecalciferol Pellets 41.3 61.31 59.8% 100% 2

‐ 3

Control (10 rats, chow only)

None n/a 572.0 n/a n/a 0%

‐

*Animals euthanized at end of study are not included.

†This formulation was 0.0025% Brodifacoum rather than the 0.005% concentration typically used in

Brodifacoum baits registered in the U.S.

The survivor in the trial with the Bromethalin bait accepted it at 34%, while the 4 victims accepted it at

59.6% to 100%. As Bromethalin causes affected rodents to cease feeding for a time, acceptance scores

of victims reflect how much of each diet was take prior to the onset of that symptom. Animals that

recover from Bromethalin toxicosis do not necessarily avoid the same bait subsequently. The survivor in

Witmer’s (2007b) Bromethalin group might have succumbed (through recovery and subsequent

ingestion of the bait) if the bait

‐ exposure period had been longer than 3 days.

Under the conditions of testing, the Cholecalciferol bait used in these trials was accepted well and killed

all 5 rats to which it was offered.

products registered to control commensal rodents in residential areas but is required or recommended (depending

upon the targeted species) on labels for Zinc Phosphide products that are registered for above

‐ ground applications

to control “field” rodents (e.g., prairie dogs, ground squirrels, kangaroo rats).19 Witmer’s (2007a) trials with house mice initially were conducted using procedures similar to those that

he employed for Norway rats, but additional trials involving 7 days of exposure to bait were added to

the research project as a consequence of poor performance (<80% mortality) of many baits

‐‐ including

all baits containing first

‐ generation anticoagulants

‐‐ used in the initial trials with 3

‐ day bait

‐ exposure

periods.

Witmer’s (2007a) results regarding bait acceptance and mortality in the 3

‐ day trials are summarized in

Table 2. The 3 dry baits and the liquid bait that were made with first

‐ generation anticoagulants killed

zero or one (0

‐ 20%) of the test group mice. The dry baits all were accepted well versus the laboratory

‐

diet alternative, suggesting that they might yield higher levels of mortality if the bait

‐ exposure period

were lengthened.

Table 2. Bait acceptance (vs. a laboratory diet), over a 3

‐ day bait

‐ exposure period, and mortality results

reported by Witmer (2007b) for House mice ( Mus musculus ), and descendents thereof, collected in Fort

Collins, CO, area.

Active

Ingredient Bait

Form Grams Chow

Consumed Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death*

First

‐ generation Anticoagulants

Diphacinone Pellets 13.7 55.7 80.3% 0%

‐

Diphacinone, Na+ salt Liquid 41.3 n/a n/a 20% 5

Chlorophacinone Pellets 5.3 49.5 90.3% 20% 8

Warfarin Pellets 24.2 41.5 63.2% 0%

‐

Second

‐ generation Anticoagulants

Brodifacoum CI† Pellets 12.0 43.1 78.2% 80% 4

‐ 13

Brodifacoum HA Pellets 5.3 43.8 89.2% 100% 8

‐ 12

Difethialone Pellets 4.2 46.2 91.7% 80% 6

‐ 11

Bromadiolone Pellets 5.4 49.2 90.1% 40% 6

‐ 7

Acute Toxicants

Zinc Phosphide Oats 5.8 1.3 18.3% 100% 1

Zinc Phosphide Pellets 38.1 9.8 20.5% 40% 2

Bromethalin Pellets 6.4 11.6 64.4% 80% 1

‐ 3

Cholecalciferol Pellets 21.5 14.1 39.6% 20% 11

Control (5 mice, chow only)

None n/a 41.3 n/a n/a 0%

‐

*Animals euthanized at end of study are not included.

†This formulation was 0.0025% Brodifacoum rather than the 0.005% concentration typically used in

Brodifacoum baits registered in the U.S.

Three of the 4 baits containing second

‐ generation anticoagulants reached or surpassed Witmer’s 80%

mortality criterion, and all were accepted well. The Bromadiolone bait killed only 2 of 5 mice, however.20 The Zinc Phosphide oat bait killed all 5 house mice on the first day of the bait

‐ exposure period. As with

meadow voles (Eadie, 1950), it might be that a single particle of grain treated at 2% Zinc Phosphide

contains a fatal dosage for a house mouse, thereby limiting the effect of bait shyness on control of this

species. Despite more total calculated bait consumption than with the oat formulation, however, the

pelleted Zinc Phosphide killed only 2 of 5 (40%).

The Bromethalin bait was accepted well while mice were still feeding and killed 4 of 5 of them. The

Cholecalciferol bait was accepted fairly well but killed only one mouse.

In his 7

‐ day trials, Witmer (2007a) tested new groups of 5 house mice each with 6 of the 7 baits that did

not kill at least 4 (80%) of the mice used in the trials with the 3

‐ day bait

‐ exposure period. The Zinc

Phosphide pelleted bait was not used in the 7

‐ day trials. The bait acceptance and mortality results of

the 7

‐ day trials are shown in Table 3. Of those products, only the Bromadiolone bait reached the 80%

mortality level in the 7

‐ day trials. Despite very high acceptance figures, none of the dry bait products

containing first

‐ generation anticoagulants killed more than 2 of 5 mice after 7 days of bait exposure.

The only bait containing a second

‐ generation anticoagulant that was relegated to use in a 7

‐ day trial was

accepted well against the laboratory diet and killed 80% (4) of 5 mice.

Table 3. Bait acceptance (vs. a laboratory diet), over a 7

‐ day bait

‐ exposure period, and mortality results

reported by Witmer (2007a) for house mice ( Mus musculus ), and descendents thereof, collected in Fort

Collins, CO, area.

Active

Ingredient Bait

Form Grams Chow

Consumed‡ Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death*

First

‐ generation Anticoagulants

Diphacinone Pellets 12.0 81.4 87.2% 40% 5

‐ 8

Diphacinone, Na+ salt Liquid 129.5 n/a n/a 60% 6

‐ 8

Chlorophacinone Pellets 2.7 105.1 97.5% 40% 9

Warfarin Pellets 20.7 122.4 85.5% 20% 6

Second

‐ generation Anticoagulant

Bromadiolone Pellets 5.9 91.9 94.0% 80% 7

‐ 14

Acute Toxicants

Cholecalciferol Pellets 86.9 14.2 14.0% 20% 8

Control (5 mice, chow only)

None n/a 206.4 n/a n/a 0%

‐

*Animals euthanized at end of study are not included.

‡Reported negative consumption is scored as zero.

The Cholecalciferol bait tested was accepted poorly and killed only one of 5 subjects (20%)

Pitt, et al (2011), report recent data on the effectiveness of certain rodenticide bait products that were

registered in the U.S. at the time of the research on wild

‐ type roof rats, Polynesian rats ( Rattus exulans ),

and house mice caught in the Hilo, Hawaii, area in places with no known “use of rodenticides in recent21 years”. At the time that the study was run, there were only two rodenticide baits available in Hawaii for

conservation uses. Both of those products

– a pelleted formulation and a bait

‐ block product

– contained

Diphacinone as the active ingredient. Zinc Phosphide products were registered in Hawaii for certain

agricultural and public heath uses. The Pitt, et al (2011), trials involved choice

‐ test procedures similar to

those used by Witmer (2007a, b), but some no

‐ choice exposure tests also were included in their study.

Like Witmer, Pitt, et al (2011), used > 80% mortality in test groups as the standard for considering a bait

to have been effective under test conditions.

Subjects were given 3 or more days from the time of capture to acclimate to laboratory conditions

before bait tests began. The trials with 3 days of bait exposure included a commercial laboratory

rodent diet as an alternative feed. The bait

‐ exposure period was followed by 10 days of observation and

monitoring with the laboratory diet continuing to be provided. Baits that failed to reach the 80%

criterion in the trials for one or more species with 3 days of bait exposure were used in choice tests with

the same species with the bait

‐ exposure period extended to 7 days. Baits that did not reach the 80%

mortality criterion for one or more species in the 7

‐ day choice tests subsequently were offered, no

‐

choice, for 7 days to additional subjects from the same species. Test groups consisted of 10 animals

each for the 3

‐ day choice tests and 5 animals each for the 7

‐ day choice and no

‐ choice trials. Post

‐

exposure monitoring periods were 10 days long for each of the exposure scenarios.

Table 4 summarizes the results of the Pitt, et al (2011), 3

‐ day trials involving roof rats. Of the 9

commercial products tested, the 3 second

‐ generation anticoagulant baits were the only ones that met

or exceeded Pitt’s 80% mortality criterion in the 3

‐ day choice test. Except for the Warfarin pellets and

the two Zinc Phosphide formulations, survivors in these trials accepted the baits approximately as well

as did the rats that died.22 Table 4. Results reported by Pitt, et al (2011), with roof rats ( Rattus rattus ) in laboratory choice efficacy

trials with 3 days of bait exposure and 10 days of post

‐ exposure monitoring

Active

Ingredient Bait

Form Animal Fates

(n) Mean

Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death

First

‐ generation Anticoagulants

Diphacinone Pellets Died (4) 25.6 72.3% 40% 7

‐ 11

Survived (6) 22.5 60.3%

Chlorophacinone Pellets Died (5) 27.5 81.5% 50% 4

‐ 7

Survived (5) 26.1 72.6%

Warfarin Pellets Died (2) 31.4 57.0% 20% 4

‐ 6

Survived (8) 12.3 23.4%

Second

‐ generation Anticoagulants

Brodifacoum Pellets Died (9) 35.1 77.2% 90% 4

‐ 9

Survived (1) 39.9 84.4%

Difethialone Pellets Died (9) 31.5 74.8% 90% 4

‐ 10

Survived (1) 30.3 88.3%

Bromadiolone Pellets Died (9) 34.7 81.5% 90% 4

‐ 13

Survived (1) 26.7 68.0%

Acute Toxicants

Zinc Phosphide Oats Died (1) 2.2 18.1% 10% 1

Survived (9) 1.8 5.6%

Zinc Phosphide Pellets Died (2) 2.6 23.4% 20% 2

Survived (8) 2.4 8.2%

Bromethalin Pellets Died (2)* 5.8 37.0% 20%* 3

Survived (8) 4.6 33.6%

Reference (laboratory diet only)

None n/a Survived n/a n/a 0%

‐

*These figures include one rat that was euthanized after having been found in a “moribund” state.

With the bait

‐ exposure period lengthened to 7 days in the choice

‐ test design, the Chlorophacinone,

Warfarin, and Zinc Phosphide oats reached or exceeded the 80% mortality criterion (Table 5). The

Chlorophacinone bait killed all 5 roof rats which were exposed to it in the 7

‐ day test.

The 3 toxic baits that did not achieve 80% mortality in the Pitt, et al (2011), 7

‐ day choice trials with roof

rats were used in their 7

‐ day no

‐ choice test. For each of those baits, 100% mortality was reported for

the no

‐ choice condition, with deaths occurring in 6

‐ 10 days for the Diphacinone bait, in 1

‐ 5 days for Zinc

Phosphide pellets, and in 1

‐ 2 days for the Bromethalin bait.23 Table 5. Results reported by Pitt, et al (2011), with roof rats ( Rattus rattus ) in laboratory choice efficacy

trials with 7 days of bait exposure and 10 days of post

‐ exposure monitoring

Active

Ingredient Bait

Form Animal Fates

(n) Mean

Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death

First

‐ generation Anticoagulants

Diphacinone Pellets Died (3) 71.6 74.1% 60% 4

‐ 7

Survived (2) 5.5 4.6%

Chlorophacinone Pellets Died (5) 69.1 92.1% 100% 5

‐ 9

Warfarin Pellets Died (4) 33.9 56.5% 80% 4

‐ 8

Survived (1) 34.5 36.1%

Acute Toxicants

Zinc Phosphide Oats Died (4) 3.0 18.5% 80% 1

‐ 2

Survived (1) 4.8 4.5%

Zinc Phosphide Pellets Died (1) 3.6 7.1% 20% 4

Survived (4) 3.6 4.0%

Bromethalin Pellets Died (3) 8.6 29.2% 60% 2

‐ 3

Survived (2) 8.5 10.9%

Reference (laboratory diet only)

None n/a Survived (10) n/a n/a 0%

‐

Table 6 summarizes the results of the Pitt, et al (2011), trials with 3

‐ days of choice exposure of

Polynesian rats to the same 9 baits that were tested with roof rats. One of the baits made with first

‐

generation anticoagulants (Chlorophacinone), one of the baits made with second

‐ generation

anticoagulants (Difethialone), and the Bromethalin bait reached or exceeded the 80% mortality

criterion. The other 6 baits were used in subsequent choice trials with a 7

‐ day bait

‐ exposure period. For

the Warfarin, Brodifacoum, Difethialone, Zinc Phosphide, and Bromethalin products, acceptance scores

for survivors were much lower than those for victims. Thus, survival might have resulted from low

palatability rather than insensitivity to the poison.

In the choice trial with a 7

‐ day bait

‐ exposure period, the Brodifacoum and Bromadiolone baits killed all 5

Polynesian rats exposed to them, exceeding the 80% mortality criterion (Table 7.).

On a percent

‐ mortality basis, the Diphacinone and Warfarin baits did slightly, but not significantly,

better with a 7

‐ day exposure period than they did with a 3

‐ day choice exposure period, while the

opposite was true for the two Zinc Phosphide formulations. The mean acceptance figure (18.0%) for

survivors of the Diphacinone bait was much lower than that (73.9%) for its victims.24 Table 6. Results reported by Pitt, et al (2011), with Polynesian rats ( Rattus exulans ) in laboratory choice

efficacy trials with 3 days of bait exposure and 10 days of post

‐ exposure monitoring

Active

Ingredient Bait

Form Animal Fates

(n) Mean

Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death

First

‐ generation Anticoagulants

Diphacinone Pellets Died (2) 11.8 53.6% 20% 6

Survived (8) 9.9 49.9%

Chlorophacinone Pellets Died (8) 17.2 85.4% 80% 3

‐ 7

Survived (2) 17.1 80.5%

Warfarin Pellets Died (3) 14.7 61.0% 30% 4

‐ 8

Survived (7) 4.2 12.7%

Second

‐ generation Anticoagulants

Brodifacoum Pellets Died (7) 15.7 64.1% 70% 4

‐ 8

Survived (3) 1.8 12.4%

Difethialone Pellets Died (9) 13.3 71.1% 90% 3

‐ 9

Survived (1) 1.4 10.5%

Bromadiolone Pellets Died (7) 16.4 67.5% 70% 4

‐ 8

Survived (3) 9.0 34.0%

Acute Toxicants

Zinc Phosphide Oats Died (7) 1.6 36.5% 70% 1

‐ 3

Survived (3) 2.4 11.4%

Zinc Phosphide Pellets Died (2) 2.2 42.9% 20% 1

Survived (8) 2.9 14.6%

Bromethalin Pellets Died (8) 3.1 48.6% 80% 1

‐ 3

Survived (2) 1.2 7.5%

Reference (15 rats, laboratory diet only)

None n/a Survived (15) n/a n/a 0%

‐

Three of these baits met or exceeded the 80% mortality criterion in the 7

‐ day no

‐ choice test. With the

Diphacinone bait, all 5 rats were dead within 5

‐ 8 days. With the Warfarin bait, 4 of 5 rats died within 4

‐

6 days. With the Zinc Phosphide oat bait, 4 of 5 rats died in 1

‐ 7 days, although the mean consumption

figure of 2.1 g for the victims does not rule out malnutrition as a contributing factor to the deaths of

these animals. Three of 5 subjects (60%) died after being exposed to the Zinc Phosphide pelleted bait,

no

‐ choice, for 7 straight days. The two survivors averaged 2.4 g of calculated bait consumption,

whereas the three victims averaged 1.8 g.25 Table 7. Results reported by Pitt, et al (2011), with Polynesian rats ( Rattus exulans ) in laboratory choice

efficacy trials with 7 days of bait exposure and 10 days of post

‐ exposure monitoring.

Active

Ingredient Bait

Form Animal Fates

(n) Mean

Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death

First

‐ generation Anticoagulants

Diphacinone Pellets Died (2) 25.5 73.9% 40% 3

‐ 6

Survived (3) 7.5 18.0%

Warfarin Pellets Died (3) 14.9 34.4% 60% 6

‐ 8

Survived (2) 10.9 28.1%

Second

‐ generation Anticoagulants

Brodifacoum Pellets Died (5) 28.1 83.2% 100% 4

‐ 9

Bromadiolone Pellets Died (5) 22.9 80.1% 100% 3

‐ 10

Acute Toxicants

Zinc Phosphide Oats Died (3) 1.8 19.9% 60% 2

‐ 4

Survived (2) 2.4 5.1%

Zinc Phosphide Pellets Died (0)

‐     ‐ 0%

‐

Survived (5) 3.5 5.7%

Reference (laboratory diet only)

None n/a Survived (10) n/a n/a 0%

‐

Table 8 summarizes the results reported by Pitt, et al (2011), for 3 days of exposure of house mice to

one of 9 rodenticide baits with laboratory diet as an alternative, followed by 10 days of post

‐ exposure

monitoring while the survivors were maintained on laboratory diet. All 3 acute rodenticide baits and all

3 baits which contained second

‐ generation anticoagulants met or exceeded the 80% mortality criterion

under the conditions of testing. None of the 3 first

‐ generation anticoagulant baits killed more than 5 of

10 test

‐ group mice.

The 3 first

‐ generation anticoagulant baits subsequently were evaluated in choice trials in which the bait

‐

exposure period was set at 7 days. Under these conditions, the Chlorophacinone bait killed all 5 (100%)

of test

‐ group mice; but the other two baits killed only one mouse each (Table 9).26 Table 8. Results reported by Pitt, et al (2011), with house mice ( Mus musculus ) in laboratory choice

efficacy trials with 3 days of bait exposure and 10 days of post

‐ exposure monitoring.

First

‐ generation Anticoagulants

Active

Ingredient Bait

Form Animal Fates

(n) Mean

Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death

Diphacinone Pellets Died (3) 11.0 84.0% 30% 3

‐ 4

Survived (7) 9.7 64.3%

Chlorophacinone Pellets Died (5) 8.6 85.2% 50% 3

‐ 5

Survived (5) 9.6 84.9%

Warfarin Pellets Died (4) 6.7 67.1% 40% 4

‐ 6

Survived (6) 7.7 68.0%

Second

‐ generation Anticoagulants

Brodifacoum Pellets Died (10) 9.5 77.4% 100% 5

‐ 11

Difethialone Pellets Died (8) 9.4 85.6% 80% 5

‐ 13

Survived (2) 10.4 86.1%

Bromadiolone Pellets Died (9) 9.8 76.1% 90% 4

‐ 12

Survived (1) 1.3 10.7%

Acute Toxicants

Zinc Phosphide Oats Died (10) 0.4 63.7% 100% 1

‐ 10

Zinc Phosphide Pellets Died (9) 1.6 48.9% 90% 1

‐ 10

Survived (1) 2.0 25.4%

Bromethalin Pellets Died (10) 1.0 63.7% 100% 1

‐ 3

Reference (15 mice, laboratory diet only)

None n/a Survived (15) n/a n/a 0%

‐

Table 9. Results reported by Pitt, et al (2011), with house mice ( Mus musculus ) in laboratory choice

efficacy trials with 7 days of bait exposure and 10 days of post

‐ exposure monitoring.

First

‐ generation Anticoagulants

Active

Ingredient Bait

Form Animal Fates

(n) Mean

Grams Bait

Consumed Percent Bait

Acceptance Percent

Mortality Days to

Death

Diphacinone Pellets Died (1) 13.0 82.9% 25% 8

Survived (3)* 7.9 38.5%

Chlorophacinone Pellets Died (5) 11.6 68.3% 100% 8

‐ 9

Warfarin Pellets Died (1) 12.6 72.4% 20% 7

Survived (4) 16.4 77.1%

Reference (laboratory diet only)

None n/a Survived (5) n/a n/a 0%

‐

*A mouse in this group “was accidentally killed”, thereby reducing the total number of subjects to 4.27 When offered no

‐ choice to wild

‐ type house mice, the Diphacinone and Warfarin baits both killed 2

(40%) of the 5 test

‐ group mice (in 5

‐ 8 days for the Warfarin bait and in 6 days for the Diphacinone bait).

For both of these products, the mean cumulative amounts of bait consumed over the seven days were

approximately equal for victims and survivors (16.4 g for victims and 16.3 g for survivors of the

Diphacinone bait; 19.0 g for victims and 19.9 g for victims of the Warfarin baits. As Pitt (e

‐ mail

communication of August 16, 2011) reports that all baits used in his trials were assayed and found to be

“within limits” for active ingredient concentration, mouse survival despite ample bait consumption

cannot be attributed to under

‐ formulation.

III. Possible Effects of Resistance to First

‐ generation Anticoagulants on Rodent Control in the U.S.

Does anticoagulant resistance render first

‐ generation anticoagulant baits ineffective in the U.S.?

Would the RMD’s limiting of the selection of rodenticide compounds in consumer outlets lead to

greater selection for resistance to first

‐ generation anticoagulants in local populations of Norway rats,

roof rats, and/or house mice?

There is no definitive answer at this time to the first part of this question. Significant problems with

efficacy of first

‐ generation anticoagulants would occur only in areas where resistant commensal rodents

occur. The RMD’s limiting of the selection of rodenticide compounds in consumer outlets could lead to

greater selection for resistance to first

‐ generation anticoagulants locally only where consumers began to

use baits containing first

‐ generation anticoagulants largely exclusively to control commensal rodents

and only if there were rodents with resistance conferring alleles in the populations that were baited. It

would take many users behaving similarly and somewhat in concert to exert sufficient selective pressure

to create a “pocket” of resistant animals over an area wider than that comprised by one to several

residences. Where consumers used baits that did not contain anticoagulants or alternated use of first

‐

generation anticoagulant baits with other toxic baits and/or trapping, selection for resistance to first

‐

generation anticoagulants would not occur or would be diluted by the use of additional methods, to the

extent that such methods were effective.

With few exceptions, the incidence of resistance to anticoagulant rodenticides in populations of

commensal rodents has not been monitored in the U.S. over the past 30 years. Between 1971 and the

early 1980’s, Norway rats and house mice resistant to Warfarin were found in many of the localities that

were researched in that regard (e.g., Frantz and Padula, 1980; Ashton and Jackson, 1984).

Although resistance might be suspected, poor control following use of anticoagulant baits can have

other causes (e.g., Timm, 1994a), including a bait

‐ exposure period of insufficient duration, failure to use

enough bait, failure to replenish bait faithfully, insufficient numbers of bait placements, bait placements

made “too far apart”, failure to treat a wide enough area to limit immigration. See also Drummond and

Rennison (1973).

As discussed above, Pitt, et al (2011) and Witmer (2007a) report problems with killing wild

‐ type house

mice with certain Diphacinone and Warfarin baits, and to a lesser extent, with Chlorophacinone in bait

form. The baits tested by these researchers by and large were accepted well in dietary choice trials and

consumed in species

‐ typical amounts in no

‐ choice trials. Consequently, there seem to have been28 problems with the toxicities of those rodenticide compounds to house mice from the populations that

were sampled. It is not clear at this point whether those results were due to selection for genetically

mediated anticoagulant resistance or to limited sensitivity of wild

‐ type house mice, in general, to those

compounds. (See prior discussion under III.D.). Pitt, et al (2011) also report that roof rats and

Polynesian rats trapped on the island of Hawaii were relatively insensitive to the Diphacinone and

Warfarin baits that they used in their research. These species and house mice were more vulnerable to

the baits made with second

‐ generation anticoagulants than to those containing first

‐ generation

anticoagulants, especially Warfarin and Diphacinone (Pitt, et al

, 2011). The wild

‐ type Norway rats from

Fort Collins, CO, used in Witmer’s (2007b) trials were vulnerable to all of the anticoagulant baits that he

tested.

In general, any lethal pesticide would be expected to be most effective against the individuals within the

pest population that were highly sensitive to it and to be less effective against the individuals that were

less susceptible, for any reason. If no individuals within the population were sufficiently invulnerable to

the pesticide to enable them to survive the treatment and reproduce subsequently, there would be no

opportunity to select for genetically mediated traits that serve to reduce sensitivity to the pesticide.

Where there are reproductively viable survivors, selection for resistance can occur. The extent to which

selection occurs can be affected by the degree of effectiveness of the pesticide to vulnerable individuals

that is achieved by the application and the scope of the application. Large

‐ scale applications that

remove virtually all vulnerable individuals from the pest population would exert strong selective

pressure favoring resistant individuals (if present), especially if there were a genetic basis for the

resistance that involved a single dominant allele that was not otherwise deleterious. 9 With localized

control efforts, survivors would be more likely to mate with immigrating conspecifics which might or

might not possess resistance

‐ conferring alleles. Shifting to a lethal pesticide that is not cross

‐ resistant to

the one previously used would be expected to remove individuals resistant to the first compound but

would not directly select against such organisms. The effectiveness of the second agent selected, and

the appropriateness of the procedures used to apply it, would affect the extent to which the residual

rodent population was controlled by it.

Labels for rodenticide baits registered in the U.S. bear directions for where to place baits and how much

bait to use at each placement location. Those directions are based upon field studies conducted with

the active ingredient used in the bait formulation. If followed, those directions are expected to result in

intelligent selection of bait placement locations and effective control of the rodent population, absent

confounding factors (e.g., nearby availability of attractive alternative feed source that the user cannot

remove, presence of a source of immigrant rodents that the user cannot control, genetic or behavioral

resistance to the rodenticide or bait formulation within the local population, etc.). As discussed above,

the bait formulations registered in the U.S. have been shown to be sufficiently palatable and lethal in

laboratory trials that there is reason to expect that their composition is not likely to be the limiting

factor in the success of the operation, absent resistance.

9 If several alleles were involved, there likely would be more genetic heterogeneity among the survivors.29 When commensal rodenticides are used in private residences by consumers, the population potentially

affected by the treatment often will be highly localized. The degree to which that population is

controlled will be affected by the vulnerability of the population as a whole to the product selected; the

user’s selection of control method(s); the user’s diligence in following the label’s application directions,

including timely bait replenishments and selection of baiting locations; as well as the other potential

confounding factors mentioned previously. A user who obtains incomplete control using a first

‐

generation anticoagulant could be selecting locally for resistant individuals, especially if the bait selected

and the baiting techniques employed resulted in removal of all rodents within the targeted population

that were highly susceptible to the active ingredient. If so, the user would have several alternatives,

including switching to a registered commensal rodenticide bait made with a toxicant (e.g., Bromethalin)

that is not an anticoagulant, attempting to trap or repel the rodent population, or, at greater expense

(Cook and Hill, 2011), hiring a professional pest control applicator to deploy a bait made with a second

‐

generation anticoagulant rodenticide.

Mechanical control methods remove individuals, presumably including animals resistant to some

anticoagulants, but might not control the entire population. Widespread use of acute rodenticides was

abandoned ~60 years ago in favor of anticoagulants as the compounds of choice for controlling

commensal rodenticides; but Bromethalin and Cholecalciferol, which are not anticoagulants,

subsequently were registered. Baits made from the “non

‐ anticoagulants” subject to the RMD, especially

Zinc Phosphide and Cholecalciferol, yielded varying results with house mice and other species in the

trials reported by Witmer (2007a, b) and Pitt, et al (2011). The Bromethalin baits used in those studies

met their 80% mortality criterion in choice tests for all of the species tested except for roof rats.

If residential rodent problems are relatively isolated, any selection for resistance resulting from a

“consumer’s” use of a first

‐ generation anticoagulant also would be localized, although resistant

survivors would be likely to reproduce, absent alternative effective control measures. If a larger area, a

city block for example, were infested and all residents attempted lethal rodent control using

appropriately placed first

‐ generation anticoagulant baits, the likelihood of there being selection for

resistant individuals in the treated population theoretically would increase, especially if highly palatable

first

‐ generation anticoagulant baits were used and were applied appropriately, such that all individuals

in the target species had access to the baits. 10 Whether “consumers” acting individually would

coordinate their baiting efforts and apply the products so as to maximize selective pressure is debatable,

but use of baits that for one reason or another are not effective against all individuals of the targeted

species would mean that the pest problem remains.

10 Within a neighborhood, selection of rodenticide baits offered at local stores might be limited to the one

‐ to

‐

several products that the merchants were persuaded to stock. Under such a scenario, neighbors simultaneously

attempting to poison rodents could wind up using the same product or active ingredient. However, Heiberg (2009)

reports essentially no differences in incidence of Bromadiolone

‐ resistant Norway rats collected from Danish sewer

systems in 3 municipalities with far different histories of use of 0.005% Bromadiolone bait blocks to control sewer

rats. As the most aggressive of those treatment histories consisted of using one 300

‐ g bait block per manhole per

year (in selected areas”), there likely were times when no attractive anticoagulant bait was available at points

within the sewer systems that were treated.30 Jackson and Ashton (1992) suggested that pockets of anticoagulant resistance emerged primarily in

urban areas in the U.S. and in rural areas in the U.K., respectively, because those localities were where

government

‐ sponsored rodent control programs, exerting strong selective pressures through sustained

and relatively broad use of anticoagulants, were concentrated in those countries. Concurrent use of

similar compounds by others living in the same areas would have contributed to the selective pressure,

however.

It has been speculated that a return to widespread use of Warfarin in the U.S. would lead to “rapid

selection” for alleles that confer resistance as such alleles are likely to remain present despite use of

second

‐ generation anticoagulants in the U.S. over the past 30 years (Kaukeinen and Prescott, 2007).

Thus far, there have been no Warfarin

‐ containing bait products that have been brought into conformity

with the RMD’s specifications for products offered at consumer outlets.

A. History of Anticoagulant Resistance

Warfarin was the first anticoagulant rodenticide to be developed, with early research being conducted

in the 1940’s (Hayes and Gaines, 1950; Link, 1959) and Warfarin products being registered as pesticides

in the U.S. as early as 1950 (Dykstra, 1951). Registrations for several other compounds now classed as

“first

‐ generation anticoagulants” were registered over the ensuing decade.

Initial field trials and operational applications of Warfarin were more successful compared to what

typically had been observed with other rodenticides (e.g., Alpha Naphthyl Thiourea, Red Squill, and Zinc

Phosphide) 11 that were available at the time (cf. Hayes and Gaines, 1950; Emlen and Stokes, 1947). The

relative safety of Warfarin over many of the acute agents also was viewed as a significant plus (e.g.,

Hayes and Gaines, 1950). Unlike most other rodenticides, anticoagulants have a true antidote, Vitamin

K 1

. The slow action and the known exposures to children or pets afford sufficient time to administer the

antidote to save lives. Rodents that ingested anticoagulant baits did not develop true bait shyness

(Hayes and Gaines, 1950), although the repeated feedings typically needed to kill the animal were less

likely to occur if the bait formulation was not palatable to rodents (e.g., Palmateer and McCann, 1976).

By the end of the 1950’s, the first evidence consistent with resistance to Warfarin and Diphacinone was

detected in Norway rats in Scotland (Boyle, 1960). 12 Over the next dozen years or so, pockets of

resistance to Warfarin and other anticoagulants were detected in Norway rat populations elsewhere in

the United Kingdom and throughout the world, including in the U.S. (Jackson, et al

, 1975a). Resistance

to first generation anticoagulants also was detected in house mice (Dodsworth, 1961

– cited by Brooks,

1973) and roof rats (Greaves, et al

, 1973).

In the U.S., the first evidence of Warfarin resistance in Norway rats was found in Johnson County, NC

(Jackson and Kaukeinen, 1972). Over the next several years, under a Federally funded surveillance

program, these researchers had detected resistant individuals in many other areas and concluded that

11 Except for Zinc Phosphide, all U.S. registrations of these active ingredients were canceled by 1992 (Jacobs, 1992).

12 Warfarin resistance also has been noted in humans, rendering Warfarin therapy ineffective for some people

(e.g., Watzka, et al, 2011).31 “resistant rat populations” occurred at 18 sites, 16 of which were urban areas and 2 of which were rural.

By the early 1980’s, screenings of commensal rodent populations had detected animals resistant to

Warfarin in additional localities. Frantz and Padula (1980) reported detection of Warfarin resistance in

Norway rats in more than 30 sampled areas in the continental U.S. plus Puerto Rico, with the incidence

of resistance being 10% or greater in many places. The incidence was especially high (43.5

‐ 76.2%) in

samples from the Chicago, IL, area in 1978 and 1979. The occurrence and expansion of “pockets” of

resistance in Norway rats was attributed to the presence of genetically resistant animals and selection

favoring them due to extensive, long

‐ term, and “indiscriminant usage” of anticoagulant rodenticides

without alternation with acute rodenticides and with insufficient sanitation (Jackson, et al

, 1975a,b).

Jackson, et al (1975b) reported resistance in house mice and roof rats from sites in California. Ashton

and Jackson (1984) reported that they and other researchers had detected house mice resistant to

Warfarin in 21 localities in the U.S. and in 3 places in 2 cities in Canada prior to the end of 1982. The

mice classed as resistant reportedly had survived 21 days of being fed a diet that was 0.025% Warfarin.

By the end of the Federal surveillance program, 98 sites in the U.S. had been investigated for

anticoagulant resistance in Norway rats. Of these 45 were identified as having resistance, meaning that

at least 5% or a minimum of 3 rats from a tested sample of at least 10 animals survived the screening

process used to determine resistance (Jackson, et al

, 1985). 13 Resistance in roof rats was found at 4 of 13

sites investigated. Resistant house mice were found in 14 of the 15 sites summarized by Jackson, et al

(1985). These authors suspected that Warfarin

‐ resistant populations of house mice were “widespread

throughout the U.S.”

The compounds that came to be known as “second

‐ generation anticoagulants” (Marsh, et al

, 1980)

were developed expressly for the purpose of finding agents which could control commensal rodents

resistant to first

‐ generation anticoagulants without sacrificing the delayed onset of symptoms and

seemingly related relative inability to induce bait shyness (Hadler and Shadbolt, 1975). Hadler and

Shadbolt synthesized new variants of 4

‐ hydroxycoumarin molecules, with one of their creations later

being called “Brodifacoum” (Dubock and Kaukeinen, 1978).

Dubock and Kaukeinen (1978) cite and present data adapted from Hadler and Shadbolt (1975) regarding

resistance factors which were calculated based upon elevations of Prothrombin ED 50 values of

homozygous resistant Norway rats vs. Wistar rats. Such factors were calculated for Brodifacoum (1.3),

13 Under this surveillance program, Norway rats were identified as Warfarin

‐ resistant if they survived 6 consecutive

days of no

‐ choice feeding on a diet that was 0.005% Warfarin. Roof rats were screened using 12 days of no

‐ choice

feeding on a diet that was 0.025% Warfarin. House mice were screened using 21 days of no

‐ choice feeding on a

diet that was 0.025% Warfarin. The concentrations selected for screening these species for resistance reflect

differences among them in sensitivity of susceptible animals to Warfarin and were consistent with the

recommendations of the World Health Organization at the time that the surveillance program was initiated (WHO,

1970). A protocol developed in the U.S. called for a 6

‐ day initial screening of subjects with one rodenticide

(0.005% Warfarin, in this case) from the 4

‐ hydroxycoumarin group of anticoagulants followed by a 30

‐ day

cleansing period, a second 6

‐ day screening period with an anticoagulant rodenticide from indandione group

(0.005% Pival, in this case) and another 30

‐ day cleansing period, followed by a choice test with the bait being

tested for effectiveness against “anticoagulant

‐ resistant” Norway rats (Peardon, et al

, 1979).32 Difenacoum (1.9), Coumatetralyl (14.2), Chlorophacinone (>90.9), Diphacinone (>227.3), and Warfarin

“(S

‐ )” (>166.7). Dubock and Kaukeinen (1978) also reported results of trials in which groups of

“Homozygous (Welsh) resistant” Norway rats were fed rodenticide bait preparations no

‐ choice for 10

days. Mortalities were 0 of 5 rats in the groups fed Warfarin at 0.025% and 0.020%, 0 of 5 in the group

fed Diphacinone at 0.025%, and 1 of 5 in the group fed 0.025% Chlorophacinone. No rats survived in

similar trials using Difenacoum at 0.025% (5 rats tested), 0.01% (5), 0.005% (10), 0.002% (10) and 0.001

(10), and Brodifacoum at 0.002% (5), 0.001% (5), and 0.0005% (5).

Soon after second

‐ generation anticoagulants came into common use, reports of resistance to

Difenacoum and Bromadiolone surfaced (e.g., Lund, 1984). Lund (1984) reported some tolerance to

Brodifacoum in house mice trapped in Sweden, but there was 100% mortality in test groups when mice

from this population were exposed (no

‐ choice) to bait formulated at 0.005% Brodifacoum for 3 days.

These discoveries were foreshadowed by the occasional survivors of laboratory feeding trials of second

‐

generation anticoagulants using commensal rodents established or believed to be Warfarin

‐ resistant

(e.g. Rowe and Bradfield, 1976).

Regarding resistant Norway rats in the U.S., Dubock and Kaukeinen (1978) reported mortality of all 7

subjects fed “TALON” after having survived a 6

‐ day no

‐ choice feeding exposure to Warfarin

(presumably) plus a 21

‐ day “cleansing” period, while none of 7 rats died after being fed 50 ppm

Warfarin or 50 ppm of Pindone as the post

‐ cleansing rodenticide. Peardon, et al (1979) reported that

none of 88 Norway rats collected from Johnson County, NC, that had survived a 6

‐ day no

‐ choice

exposure to 0.005% Warfarin died following a 6

‐ day no

‐ choice exposure to the indandione anticoagulant

Pival (Pindone), again suggesting cross

‐ resistance between these two rodenticides. 14

B. Mechanisms and Genetics of Anticoagulant Resistance

It became apparent to researchers early on that this resistance was a heritable trait (Greaves, 1972),

being antagonistic to Vitamin K in some way and, in some cases, behaving much, but not absolutely, like

a single dominant gene at one locus. It also became clear that the nature of resistance varied from place

to place. Warfarin

‐ resistant rodents collected from some places in the United Kingdom (UK) were cross

‐

resistant to a broader array of other anticoagulant rodenticide compounds than were rodents of the

same species collected from other regions. Patterns of anticoagulant resistance were named for the

regions from which rodents exhibiting them first were identified (discussed by Pelz, et al

, 2005; Rost, et

al

, 2009; Buckle, et al

, 2010).

Although anticoagulant resistant rodents collected from some areas have elevated dietary requirements

for substances containing Vitamin K (Greaves, 1972, MacNicoll and Gill, 1993), whatever deleterious

effects that condition might have on individuals apparently is insufficient to eliminate resistance

‐

conferring alleles from rodent populations in the absence of use of anticoagulant rodenticides (e.g.,

Jackson and Ashton, 1992; Heiberg, et al

, 2003). Where anticoagulant rodenticides are used, mutations

14 In a subsequent laboratory choice efficacy trial consistent with OPP Protocol 1.209, 18 of the 20 survivors of

Warfarin and Pival challenges were killed by the acute rodenticide Vacor (Peardon, et al

, 1979).33 favoring resistance to such compounds are inferred to “confer a tremendous selective advantage” (Pelz,

et al

, 2005).

Over the past 50+ years, much has been learned about the genetics of resistance to anticoagulant

rodenticides in rodents. Most of this progress has occurred outside of the U.S., as funding for rodent

surveillance, resistance testing, and related research in the U.S. was all but halted in the early 1980’s.

Advances in gene mapping have identified the VKORC1 locus (for “Vitamin K epoxide reductase complex

subunit 1”) as a site where mutations affect susceptibility to anticoagulants. The geographically named

types of resistance in the UK were found to be associated with different variations at this locus (Pelz, et

al

, 2005; Buckle, et al

, 2010). Variations in VKORC1 protein do not fully explain differences among

resistant populations in Vitamin K requirements or why some rodents resistant to first

‐ generation

anticoagulants also are resistant to some second

‐ generation anticoagulants (Pelz, et al

, 2005).

Until procedures for in vitro screening for resistance to anticoagulants (e.g., Kohn, et al

, 2000) were

developed, resistant individuals had to be identified through bioassay procedures which involved

feeding them for up to 28 days on a diet which included Warfarin (or another first

‐ generation

compound) and maintaining them for a cleansing period of several additional weeks on a diet free of

any rodenticide (e.g., WHO, 1970; Greaves, 1973; Jackson, et al

, 1985). Survivors were considered to be

resistant, but breeding and further screening was needed to isolate individuals who were homozygous

for the resistance

‐ conferring allele from heterozygotes (e.g., Jackson, et al

, 1975b). Animals that

survived no

‐ choice exposure to Warfarin plus the post

‐ exposure cleansing period could then be

challenged with other rodenticides (e.g., second

‐ generation anticoagulants) to assess susceptibility to

those compounds. In the U.S., such challenges were conducted via laboratory choice

‐ feeding trials

similar to those used to screen new bait formulations for efficacy (e.g., Protocols 1.203 and 1.204).

Depending upon the species tested, claims for effectiveness against Warfarin

‐ resistant Norway rats or

house mice were accepted if the test results met the product performance criterion of > 90% mortality

(and the test subjects were rodents that either had been trapped in the U.S. or were descendants of

such animals).

Pelz, et al (2005) listed 14 countries in which anticoagulant resistance has been reported in at least one

commensal rodent species. Along with Denmark, France, Germany, and the United Kingdom, the U.S.

was one of 5 nations in which resistance had been reported in Norway rats, roof rats, and house mice.

These nations also are among those where, at one time or another, there have been significant research

efforts devoted to surveillance for Warfarin resistance. Pelz, et al (2005) indicate that the VKORC1 gene

‐‐ terminology from human clinical studies

‐‐ corresponds to the Rw gene in Norway rats and the War

gene in house mice. 15 They conclude that Tyr139 is part of the Warfarin binding site but that mutations

15 Watzka, et al (2011) found 3 previously reported 13 “novel” and 3 previously known mutants on the VKORC1

gene, including one at the Tyr139 position, in 23 human patients who had exhibited resistance to oral

anticoagulant therapy. They report that a total of 24 VKORC1 mutants are known in humans. Mutations are

named according to position on a chromosome and the nature of the related amino acid substitution. For

example, “Tyr139Ser” means that what should be tyrosine at position 139 is replaced by serine.34 elsewhere on VKORC1 also might be involved in resistance, as might other factors. 16 They observe that

the elevated dietary need for Vitamin K seen in some resistant populations (e.g., Hampshire) is not seen

in all of them. They also state that resistance to second

‐ generation anticoagulants cannot be fully

explained by variability in VKORC1 sequence data. There might be “secondary or modifier genes”

involved.

Kohn and Pelz (1999) report apparent cross

‐ resistance probably due to the same allele for Warfarin and

Bromadiolone in rats collected in the Munsterland area of Germany. Their pool of subjects was cross

‐

bread from one wild

‐ type male with lab rats. They sampled tissue from 74 animals (37 males: 37

females) from F7 generation, with back

‐ crosses and allowed for “10% residual heterozygosity” in the

samples. Females were less susceptible (biochemically) than males to Bromadiolone. A slight trend in

same direction was seen for responses to Warfarin. In other research, Kohn, et al (2000), found lower

than expected incidences of heterozygosity for resistance to Warfarin and Warfarin + Bromadiolone in

Norway rats collected from several high

‐ resistance areas near Münster, suggesting to the authors that

homozygous resistant individuals were favored in the populations from which the rats were trapped.

Heiberg, et al (2003), suggest reduced fitness for homozygotes. These researchers report hemorrhagic

deaths among captive, presumably homozygous Bromadiolone

‐ resistant, Norway rats and their

descendants, collected in Denmark and maintained on a diet low in Vitamin K in the absence of

exposure to anticoagulant rodenticides.

Rost, et al (2009), identify sequences of VKORC1 in tissue samples from Norway rats and house mice

collected from locations in Africa, Asia, Europe, North America, and South America. They found a total

of 23 distinct mutations and polymorphisms, including 5 previously described, at various positions on

VKORC1 in samples from every continent but Africa. Some of the mutations are known to cause amino

acid substitutions, while others appear to be “silent mutations”. In vitro studies indicated that many of

the mutations were associated with reduced Vitamin K

‐ epoxide reductase activity, but Warfarin

administration reduced this activity greatly under the conditions of testing, except for the substitutions

at position 139 on VKORC1 (Tyr139Cys, Tyr139Phe, Tyr139Ser in rats and Tyr139Cys in house mice).

Limited effects of Warfarin administration would be expected in samples from Warfarin

‐ resistant

animals. Rodents with mutations at position 139 also have been shown to be resistant to Warfarin in in

vivo trials. Rost, et al (2009), suggest that Tyr139 is part of the binding site for Warfarin.

Rost, et al (2009) report “Arg35Pro” VKORC1 mutations associated with amino acid substitutions in

Norway rats collected from Chicago, where much earlier surveillance had indicated a high incidence of

Warfarin resistance (e.g., Frantz and Padula, 1980). The “silent” mutation “Ile82Ile” also was found in

Chicago rats. In a sample of 3 rats from Santa Cruz, CA, Rost, et al (2009) report the VKORC1 mutation

“Ile90Leu” as being associated with amino acid substitutions plus a total of 5 silent mutations. Based

upon their own research, Diaz, et al (2010), suggest that the rats collected from Santa Cruz, CA, for the

studies reported by Rost, et al (2009), were roof rats rather than Norway rats. In vitro studies of the

“Arg35Pro” (Chicago) rats showed that Vitamin K 2,3

‐ epoxide reductase (VKOR) activity was only about

one third that in wild

‐ type rats but that Warfarin greatly reduced VKOR activity. The “Ile90Leu” rats had

16 See also Hodroge, et al (2011).35 nearly normal VKOR activity which was greatly reduced by Warfarin administration under the conditions

of testing.

Ishizuka, et al (2007) used, as Warfarin

‐ resistant roof rats, animals live

‐ trapped in Tokyo that survived a

diet that was 0.025% Warfarin for one month. Their non

‐ resistant rats were collected from another

location or selected from a “closed colony” of captive roof rats. Warfarin was metabolized more rapidly

by resistant roof rats than by non

‐ resistant rats, based upon levels remaining in plasma 60 minutes after

administration. Evidence of Warfarin metabolism in liver was greater in the resistant rats than in the

Warfarin

‐ sensitive rats. Resistant rats also had higher rates of testosterone metabolism. The authors

concluded that the prevalence in resistant roof rats of one or more forms of cytochrome P450 that “are

known to metabolize warfarin” was responsible for clearing Warfarin out of their systems quickly. The

testosterone finding and some others that they reported were consistent with this hypothesis as those

processes also are P450

‐ dependent. The resistant rats had considerably elevated levels of the CYP3A

subfamily of P450 and slightly elevated levels of CYP2C11 compared to susceptible rats.

Ishizuka, et al (2007), fed small groups (n=3 or 4 animals) of Warfarin

‐ resistant roof rats Warfarin at

0.025%, SKF

‐ 525A at 0.05%, SKF

‐ 525A at 0.10%, Warfarin at 0.025% plus SKF

‐ 525A at 0.05%, and

Warfarin at 0.025% plus SKF

‐ 525A at 0.10%. The feeding period apparently lasted 12 days. All rats

survived in all groups except for 2 of 4 rats in each of the groups fed Warfarin plus SKF

‐ 525A. The last

mortality occurred on day 14. These researchers did not detect a “mutation at position 139 in VKORC1

in liver of resistant roof rat.” The authors suggested that

elevation of the ability to detoxify foreign chemicals due to the induction of enzyme

expression or genetic polymorphism in wild roof rats may adapt them to a rodenticide

‐

contaminated environment.

Wallin, et al (2001) suggest that the protein calumenin suppresses VKOR activity in livers of Warfarin

‐

resistant rats but also limits the effects that Warfarin can exert on residual VKOR activity.

These reports suggest that factors other than amino acid substitutions VKORC1 can affect the

susceptibility of commensal rodents to anticoagulant rodenticides.

IV. Effects of Bait Form on the Effectiveness of Commensal Rodenticides

Does bait form affect the efficacy of baits? Are bait block formulations inherently less effective

against commensal rodents than are small

‐ particle baits, including pelleted or meal formulations?

Bait form can play a role in uptake, especially in initial bait acceptance. For an item to be fed upon, the

rodent must perceive it as food. Various species of rodents and other vertebrates may be predisposed

to perceive certain types of items as foods, and their selecting items to eat may be affected further by

prior dietary experiences and other factors (e.g., Galef, 1998). Commensal rodents are omnivores.

History has shown that they are attracted to and to consume a wide variety of plant and animal matter,

including a wide variety of substances used to bait traps and many forms and formulations of

rodenticide baits.36 As noted above, active ingredients first registered to control commensal rodents in the U.S. since the

1972 amendments to FIFRA were passed have been laboratory

‐ and field

‐ tested for effectiveness

against targeted species. Pelleted baits typically were used in the field trials conducted to support initial

registration. Additional bait forms, including paraffinized bait blocks, subsequently were registered

based upon the results of laboratory efficacy trials, often conducted with laboratory strains of the

Norway rat and house mouse. Bait formulations for all active ingredients subject to the RMD are

screened for efficacy and concluded to be sufficiently effective before being accepted for registration.

A. Efficacy Data Comparing Bait Blocks with Other Bait Forms

O’Connor, et al (2000) and others have noted that there has been little in the way of controlled research

into the relative effectiveness of different bait forms (e.g., pellets vs. bait blocks) under field conditions.

As previously mentioned, paraffinized bait blocks were developed to provide greater durability and

resistance to the effects of moisture on the integrity of baits used in sewers and other wet or damp

areas. In deference to an assumption that bait blocks would be less palatable than meal or pelleted bait

forms, the U.S. efficacy criteria for paraffinized bait blocks containing anticoagulant rodenticides

originally were set at 80% mortality and 25% bait acceptance vs. the standard rat and mouse challenge

diet described above. Those criteria were lower than the 90% mortality and 33% acceptance criteria

established for other dry anticoagulant baits.

Palmateer (1974) summarized results of 747 laboratory screening tests run from 1968 through early

1974 in which albino laboratory (Norway) rats were tested using samples of anticoagulant baits

collected in the market surveillance program initiated by the U.S. Department of Agriculture and

continued, from 1970 through mid 1982, after EPA took over the lead responsibility for regulating

pesticides in the U.S. The bait

‐ exposure phase of these trials was set at 15

‐ days, although 100%

mortality occurred within that time period in many instances. During the 5

‐ day bait

‐ exposure phase of

the trial, EPA’s standard rat and mouse challenge diet was offered as the alternative feed. All of the

baits used in these trials contained first

‐ generation anticoagulants.

The baits used in the tests reported by Palmateer (1974) contained Diphacinone (typically if not always

at 0.005%), Fumarin (0.025%), Pindone (0.025%), Warfarin (0.025%) or Warfarin (0.025%) plus

Sulfaquinoxaline (also at 0.025%) 17

. The trials included 6 tests in which the bait form was classed as

whole grain, 387 tests with meal baits, 287 tests with pelleted baits, and 67 tests with “Wax Blocks”.

Pooled across all trials, 92% of the subjects exposed to these baits died in an average of 8.4 days, with

composite bait acceptance averaging 21.4%. Except for the few whole

‐ grain baits tested, the results for

the 3 bait forms were very similar, with the bait blocks involved in these trials falling between the meal

baits and pelleted baits in mean acceptance score (Table 10).

17 The combination of Warfarin and Sulfaquinoxaline was called “Prolin”. Prolin, Fumarin, and Pival baits no longer

are registered in the U.S.37 Table 10. Results of laboratory screening trials of first

‐ generation anticoagulant baits (adapted

from Palmateer, 1974).

______________________________________________________________________________

BAIT FORM NO. OF MEAN % MEAN DAYS MEAN BAIT

TESTS MORTALITY TO DEATH ACCEPTANCE

______________________________________________________________________________

Whole grains 6 100% 6.6 days 25.1%

Meal baits 387 91.0% 8.5 days 20.0%

Pelleted baits 287 92.5% 8.2 days 23.2%

Wax blocks 67 92% 8.7 days 21.7%

______________________________________________________________________________

In the trials summarized by Palmateer (1974), only 10.9% of the trials with meal baits, 13.9% of those

with pelleted baits, and 10.5% of those with wax blocks yielded bait acceptance scores of 33% or higher

plus mortality scores equal to or exceeding 90%. Most of the products collected in market sampling

through late 1973 failed to meet one or both performance criteria then in effect for meal, pelleted and

whole

‐ grain anticoagulant baits. 18 Failures usually were due to problems with bait acceptance.

Anticoagulant baits registered since then have been shown to meet the applicable criteria in efficacy

trials conducted prior to registration. Such baits include bait

‐ block products labeled for structural uses

(e.g., “in and around buildings”), rather than or in addition to uses in sewers and other wet or damp

areas.

O’Connor and Eason (2000) note that “it is generally assumed that some loss of palatability is to be

expected” with bait blocks relative to pelleted formulations, but the only report that they cite (Buckle,

1994, cited by O’Connor and Eason, 2000) is claimed to have shown equivalent efficacy for “wax blocks”

and “cereal baits containing the same toxicants”.

Kaukeinen and Buckle (1992) report that laboratory strains of the Norway rat and house mouse

accepted 3 Brodifacoum bait formulations (“STANDARD PELLETS WITH BITREX”, “MINIPELLETS WITH

BITREX”, and “WEATHERBLOK WITH BITREX”) at 38

‐ 77% of total consumption in choice tests with EPA’s

standard rat

‐ and

‐ mouse challenge diet with the bait

‐ exposure periods limited to 3 days and the post

‐

exposure monitoring period lengthened. In all of the 10 tests, the acceptance figure obtained exceeded

the 33% criterion set for the types of studies run. For laboratory mice, the two lowest composite

acceptance scores (51% and 40%) were obtained for the replicated groups tested with “WEATHERBLOK

WITH BITREX”

– a 0.005% Brodifacoum 20

‐ g bait block product

. The two replicates in which house mice

were tested using “STANDARD PELLETS WITH BITREX” yielded acceptance figures of 54% and 69%.

18 The tests run by Palmateer (1974) were conducted primarily for enforcement purposes, with additional samples

often being collected and tested for baits that failed to meet criteria in initial rounds of testing. The lack of

randomness for follow

‐ up testing would have reduced the mean acceptance scores when results of all tests within

a bait

‐ form category were pooled.38 Composite acceptance for the group of laboratory mice tested with the “MINIPELLETS WITH BITREX”

product was 77%. For laboratory rats, composite acceptance was 51% and 55% for the two groups

tested with “STANDARD PELLETS WITH BITREX”, 44% for the group tested with “MINIPELLETS WITH

BITREX”, and 38% and 43% for the groups tested with “WEATHERBLOK WITH BITREX”. Although the

acceptance scores were somewhat to quite a bit higher in these trials for the pelleted baits than for the

bait block, all of these figures are consistent with baits that would be expected to compete well with

other food sources and to control targeted species, assuming appropriate baiting practices and limited

confounding factors.

In other laboratory trials (“4 day choice tests”) reported by Kaukeinen and Buckle (1992), “BLOCKS”

containing Brodifacoum but not the bittering agent Denatonium Benzoate were accepted by wild

‐ type

Norway rats at 51% of total calculated feed intake, at 43% by albino rats, at 46% wild

‐ type house mice,

at 50% by albino house mice, and at 37% by wild

‐ type roof rats. In similar tests, “PELLETS” lacking

Denatonium Benzoate were accepted at 41% by wild

‐ type Norway rats, at 42% by albino rats, at 42%

wild

‐ type house mice, and at 52% by albino house mice. BLOCKS” containing Brodifacoum plus

Denatonium Benzoate at 0.001% were accepted by wild

‐ type Norway rats at 57% of total calculated

feed intake, at 49% by albino rats, at 49% wild

‐ type house mice, at 50% by albino house mice, and at

67% by wild

‐ type roof rats. In similar tests, Brodifacoum “PELLETS” containing Denatonium Benzoate

were accepted at 59% by wild

‐ type Norway rats, at 58% by albino rats, at 58% wild

‐ type house mice, and

at 48% by albino house mice. With these baits, acceptance of baits of different forms varied relatively

little within test organisms; and all reported acceptance figures were high enough to suggest that all of

the baits would be effective if used appropriately to control wild

‐ type commensal rodents.

Product

‐ specific efficacy data submitted to and reviewed by EPA over its 41 years of existence have

indicated that bait block formulations that are sufficiently palatable and lethal to laboratory rats and

mice to meet or exceed the Agency’s criteria have been produced for all of the active ingredients

directly affected by the RMD except for Zinc Phosphide, for which there are no bait

‐ block products

registered.

B. Selection of Bait Form for Controlling Commensal Rodent Populations

Those providing guidance on rodent control strategies sometimes include advice on selecting bait forms.

The New York City Department of Health and Mental Hygiene Pest Control Services “Best Practice

Procedure NO. 1” (included in Corrigan, [2011a]) notes that “Technically, each of the rodenticide

formulations (pellets, packets, blocks, meals, seeds) can provide good rat control.” However, Corrigan

(2011a, b,) recommends using (secured) bait blocks in bait

‐ station applications and pellets for treating

Norway rat burrows. Corrigan (2006) also has encouraged professional pest control applicators to use

ready

‐ to

‐ use bait stations containing bait blocks for controlling house mice. Based on their results with

captive wild

‐ type Norway rats, Quy, et al (2003), note that, as bait blocks do not resemble natural food

sources for rodents, it might take some time and prior experience with bait blocks before the animals

will feed readily on blocks wired in place in bait stations.

Timm (1994a) recommends using baits that resemble the items that house mice in the population to be

controlled have been eating, noting that grain and pelleted baits often have been effective but

observing that pelleted baits may be moved by the mice to other places, including some where the bait

becomes exposed to more nontarget species than it was in its original placement. Due to their being

“easily manipulated by mice”, pelleted baits are attractive to them. Timm (1994a) also observes that39 house mice readily accept “extruded bait blocks”, but “accept paraffin bait blocks less readily than loose

or pelleted grain baits.” 19

Rodents can relocate bait pellets from locations of placement to more accessible areas (e.g., Corrigan,

2001); and birds may do the same, even with bait blocks (O’Meara, 2008). Small (e.g., 20

‐ g) bait blocks,

or pieces of them, also may be relocated by Norway rats (Quy, et al

, 2003). Norway rats removed from

bait stations nearly all of the unsecured 15

‐ g bait blocks made available to them during the early stages

of the Hawea Island, NZ, eradication project (Taylor and Thomas, 1989).

As they may weigh nearly as much to much more than house mice, bait blocks may be difficult for them

to move. Commensal rats might have difficulty in moving large bait blocks, especially if they are wedged

or otherwise secured in the bait compartments of bait stations. Thus, use of bait blocks rather than

pelleted baits in bait stations has been recommended (e.g., Corrigan, 2011b), although for reasons of

safety and bait stewardship rather than of relative efficacy.

Loven and Williams (2010) recommend using placepacks or bait blocks for baiting burrows and bait

blocks for applications in areas where pellets might spoil. They add that pellets “are more appropriate

for areas (of poultry facilities) that require many bait placements or when using container baiting

methods”. They add that

bulk quantities of loose pellet baits or meal baits are a more economical formulation

... for

large scale baiting compared to using hundreds of the packet baits (although bulk baits do not

provide the convenience of packet baits or bait blocks).

They do not discuss relative palatability of blocks and pellets, but say that pellet and meal formulations

“are about equally attractive to rodents” with meal baits tending “to spoil more rapidly than pellet

baits.”

The New York City Department of Mental Health and Hygiene (NYC, 2004) recommends using loose

pellets, rather than placepacks or bait blocks, when baiting burrows because pellets placed well down

burrow systems are unlikely to be pushed out of them, unless the treated burrows are subsequently

plugged (e.g., with crumpled paper, dirt, and other debris).

Whisson (1996) also notes that pellets are susceptible to being moved to locations from which they are

not readily recovered or where the likelihood of exposure to nontarget organisms is increased. She adds

that “wax and extruded blocks “are generally less readily accepted by rodents” (than pellets), but allows

that bait preferences vary among species and individuals within species. Whisson (1996) adds that

“selection of the appropriate toxicant and formulation (i.e., grain, pelleted, or wax block)”; as well as

bait placement, are important.

Anonymous (2000) addresses bait selection from the standpoints of efficacy and relocation by rodents.

Whether rats will eat a bait or not depends both on what they are used to eating and on their individual

preferences. Within a wild rat population, baits highly palatable to some individuals may not appeal to

19 From appearances, it can be judged that bait blocks currently being marketed in the U.S. typically are of the

extruded type, whereas the “wax blocks” that commonly were produced many years ago had the appearance of

having been formed in molds. Extrusion does not mean that bait blocks lack paraffin, but it could mean that grain

and other flavor components of the blocks are more likely to be partially exposed at the outer surface than with

mold

‐ formed bait blocks. The bait

‐ block screened by Palmateer (1974) for efficacy using Wistar rats likely included

extruded and mold

‐ formed products.40 others. It is not practicable to test a range of bait bases before a treatment begins and with ready

‐ made

baits there is no alternative but to use the product supplied by the manufacturer.... [p. 16]

The efficiency of a rodenticide treatment is greatly enhanced if rats start to eat the bait as

soon as it is laid. Although rats appear to eat anything fancy, they are essentially seedeaters,

hence grain

‐ sized particles are ideal. Larger food items, such as blocks, may be less readily

recognized as being edible and may cause a delay in bait uptake. Moreover, as such items take

some time to eat, rats may prefer to eat them somewhere else, rather than risk attack from

other rats. Thus, wax blocks and place

‐ packs (sachets) are particularly prone to being carried

away. To counteract bait transfer, commercially available bait boxes may have a rod inside

them for holding blocks to encourage a rat to eat them in situ

, but this is not always successful.

Some rats, unable to remove a secured block will break it into smaller pieces and carry each

piece away separately or ignore it altogether. Rats may also carry away whole

‐ wheat baits.

[pp. 18

‐ 19]

Writing specifically about using bait stations, Vantassel, et al (2006), state

The best approach is to use extruded bait blocks that can be anchored inside the bait station

to prevent them from being removed. Loose grain and pelleted baits are often packaged in

small “place packs” that can be placed intact into bait stations. Use caution with loose and

pelleted grain formulations of toxicants, as rodents may relocate them to unsafe areas.

Again, the advice given relates more to the safety of the baiting operation than to its efficacy.

V. Possible Effects on Rodent Control of Limiting Applications to Bait Stations

Are baits ineffective or significantly less efficient in controlling commensal rodents when the

formulations are applied in bait stations rather than in the open?

As discussed below, use of protective bait stations (“tamper

‐ proof” or “tamper

‐ resistant”) has been

recommended or required in the U.S. since the 1960’s whenever anticoagulant baits and baits

containing rodenticides used to control commensal rodents wherever the bait placements otherwise

would be accessible to children, pets, domestic animals, and nontarget wildlife. Over that period of

time, therefore, it has been considered to be bad practice and/or misuse of a pesticide to place

commensal rodenticide baits in the open in places where children and nontarget animals might

encounter them. Protective bait stations reduce accessibility of rodenticide baits to nontarget species

that are larger than the targeted rodents. Under some circumstances, commensal rodents, especially

Norway rats, may not enter suitably placed tamper

‐ resistant bait stations for one to several days after

deployment. Once entered, tamper

‐ resistant bait stations provide commensal rodents with shelter and

a place to feed.

A. Protective Rodenticide Bait Stations and U.S. Policies Pertaining to Them

A bait station is any structure used to contain a bait at its place of deployment. Depending on their

design features, bait stations also may protect baits from disturbance by wind, precipitation, from some41 human activities, and from some nontarget species. The extent to which a rodenticide bait station

inhibits or delays feeding upon bait by targeted species can be affected by the behavioral tendencies of

the species being targeted, the design of the bait station, the location in which it is placed, the

attractiveness of the bait within it, the nature of the rodent infestation, the extent to which other food

sources can be limited during the bait application period, and other factors.

A bait station may consist only of an open dish or of a more elaborate structure. An open dish clearly is

not a protective bait station. If placed in a location to which targeted rodents would be the only

vertebrates (other than adult humans) with access, an open dish could be considered a “protected” bait

station. A “protective” bait station serves by itself to exclude nontarget vertebrates of concern from the

bait compartment, creating a “protected” placement for the bait.

Development of (at least somewhat) protective bait stations goes back at least as far as the early 20 th

century (Kaukeinen, 2007a). Barnett (1946) provided instructions for making a protective station, the

“P3” design. 20 Hayes and Gaines (1950) illustrate and discuss use of a secured bait station that would

have been at least semi

‐ protective. A design for a protective bait station was illustrated in the Center

for Disease Control’s rodent control manual at least as far back as the mid 1950’s (Bjornson and Wright,

1956). Specialty bait stations have been designed to admit targeted rodent species while behaviorally

excluding other types of rodents that are desired to be protected (Erickson, et al

, 1990; Whisson, 1998).

Barnett (1946), whose term for protective bait station was “container”, noted that “Baiting in containers

is primarily a safety measure.” As the rodenticides available in Britain at the time were acute toxicants

which lacked true antidotes, safety was a very important consideration. Although only Zinc Phosphide

of those agents currently is registered in the U.S. for controlling commensal rodents, the anticoagulants

and other rodenticides that are now registered are toxic to vertebrate animals in general and often

require several weeks of continuous use to control commensal rodent populations. Consequently,

safety in bait placement remains an important concern.

Of the rodenticide compounds addressed by the RMD, only the anticoagulants have a true antidote; and

the potential for effective use of Vitamin K 1 is limited to circumstances in which the exposure incident is

discovered in some way and the exposed person or animal can be brought to treatment and maintained

there until the organism has recovered. As free

‐ ranging wildlife seldom receive veterinary care, the

existence of an antidote is of little value to them, although Vitamin K occurs naturally in some plants.

To use anticoagulant rodenticides effectively, it is necessary to deploy relatively large quantities of bait

(4 oz. to 1 lb. per placement for commensal rats) at multiple locations for weeks at a time so as to

provide all targeted rodents present ample opportunity to access the bait. The baits used also must be

sufficiently palatable to draw rodents away from their established food sources and to sustain feeding.

Therefore, baits are formulated to contain feed and flavor items (including sweeteners) that also are

attractive to animals other than rodents.

20 “P3” stood for “Protected Poison Point”.42 The U.S. Department of Agriculture (USDA), which regulated pesticides in the U.S. from the passage of

FIFRA in 1947 until the creation of the EPA in 1970, was faced with the dilemma of whether to limit use

of anticoagulants to protected locations only or to create a policy that would afford isolation of baits

from nontarget species while allowing their deployment at a wider variety of sites where rodent control

was needed. USDA decided to require (or strongly recommend) use of super

‐ protective bait stations

‐‐

called “tamper

‐ proof bait boxes”

‐‐ when commensal rodenticide baits were to be used in “areas

accessible to children, pets, domestic animals and wildlife” (Jacobs, 1990a). Beginning in the 1960’s

statements to that effect were put on labels for registered rodenticide baits, often in the

“ ENVIRONMENTAL HAZARDS ” section. EPA's Office of Pesticide Programs (OPP) continued to require

such statements after assuming primary authority for regulating pesticides in the U.S. In the mid 1970’s,

OPP drafted a list of criteria for “tamper

‐ proof bait boxes".

Following receipt of several inquiries and allegations concerning unsafe use of rodenticide baits and the

inadequacy of bait stations then being offered for sale, OPP issued Pesticide Registration (PR) Notice 83

‐

5 pertaining to “Tamper

‐ proof bait boxes” (Johnson, 1983). PR Notice 83

‐ 5 expressed OPP’s

dissatisfaction with the ways in which rodenticide baits reportedly were being used, including

deployments in bait stations made of cardboard or thin plastic in places where existing labels called for

use of “tamper

‐ proof bait boxes”. PR 83

‐ 5 also announced OPP’s intention to host public hearings on

matters related to rodenticide bait stations and appropriate use of rodenticide baits and to issue a

subsequent notice outlining new or amended policies as determined to be warranted.

Sessions of public hearings were held on November 4, 1983, in Arlington, VA, and on March 5, 1984, in

Sacramento, CA. Major findings from the public hearings and related activities were summarized in a

report by the project officer (Jacobs, 1990a) and in a publication (Jacobs, 1990b). In the form of PR

Notice 94

‐ 7, EPA issued a revised statement of policies regarding rodenticide bait stations and labeling

for rodenticide baits (Johnson, 1994).

PR Notice 94

‐ 7 prescribes label text regarding bait stations and other aspects of appropriate use and

storage of rodenticide baits. That notice also includes a revised list of criteria for protective rodenticide

bait stations, the terminology for which was changed from “tamper

‐ proof bait boxes” to “tamper

‐

resistant bait stations”. 21 The latter change was the one most consistently requested by those who

commented orally and/or in writing to EPA concerning its policies on rodenticide bait stations (Jacobs,

1990a). In general, respondents felt that the expression “tamper

‐ proof” meant that any compromising

of bait protection, under whatever circumstance, could be regarded as evidence of misuse and that

“tamper

‐ resistant” more accurately described the degree of protection provided by the best of the bait

stations then on the market or that could be expected to be constructed by applicators at reasonable

cost (see also Jackson and Ashton, 1992). Due to the change from the essentially self

‐ defining

expression “tamper

‐ proof” to the broader “tamper

‐ resistant”, label text pertaining to the use of bait

21 Prior to the issuance of PR Notice 94

‐ 7, similar bait

‐ protection text was prescribed for Warfarin baits in the RED

issued in 1991 for that chemical. Proposed bait

‐ protection statements were offered for public comment in drafts

of the documents that became the Warfarin RED (EPA, 1991) and PR Notice 94

‐ 7.43 stations was expanded somewhat to covey the essence of EPA’s criteria for tamper

‐ resistant bait

stations directly to users of rodenticide baits.

The 8 criteria for “tamper

‐ resistant bait stations” listed in PR Notice 94

‐ 7 primarily address design

features intended to isolate the baits from children and nontarget animals that are larger than the

rodents for which the stations are designed. Stations consistent with the 8 criteria include designs that

can be made by the applicator or purchased from a commercial source. As long as the commercial

products are not sold with toxic bait packaged in or with them, these (empty) bait stations are

considered to be pesticide application equipment and, as such, are not regulated directly under FIFRA.

Labels for registered commensal rodenticide baits direct that products be applied according to certain

standards. Users of such products are bound by label requirements, including those that pertain to

tamper

‐ resistant bait stations. Thus, the onus is on those who apply the baits to use a tamper

‐ resistant

bait station rather than on the manufacturers of bait stations to produce only tamper

‐ resistant units.

Labels for many rodenticide bait products do not require use of tamper

‐ resistant bait stations for

placements that otherwise are protected from children and nontarget animals. Under such

circumstances, bait stations that are not tamper

‐ resistant may be used legally.

A ready

‐ to

‐ use bait station is a bait station is sold with bait already in it and/or provided in the same

retail package. Combining bait with a bait station results in the creation of a pesticide product. Thus,

ready

‐ to

‐ use bait

‐ station products created to meet the criteria of Tier 1, 2, 3, or 4, as described in the

RMD, must be registered under FIFRA as pesticide products. To qualify as a Tier 1, Tier 2, or Tier 3 unit,

each ready

‐ to

‐ use bait station design must be tested and found to meet the applicable requirements for

bait protection. Since the RMD was issued in 2008, ready

‐ to

‐ use bait station products consistent with

the stipulations of the RMD have been registered. Most such products are designed to target house

mice.

Irrespective of its protective qualities, a bait station is only as useful as its ability to bait targeted species

effectively. Overemphasis on designing bait stations to isolate bait from nontarget species could result

in building the targeted rodents out of the units as well.

B. Neophobia and Latency to Enter Rodenticide Bait Stations

A bait introduced into an area infested by commensal rodents might go untouched by the targeted

species for one to several nights, until individuals have approached it and identified it as a potential food

source. New objects also are approached cautiously. Norway rats are notoriously neophobic, especially

in areas where traps and poisons of various types have been used to combat them and many

generations of their ancestors. When a new bait in a new bait station are simultaneously introduced in

an infested area, latency to approach and enter the station may be additive to whatever reluctance

there might be to approach the new food itself (Kaukeinen, 1986). Writing on the use of acute

rodenticide baits in bait stations, Barnett (1946) advised installing the bait stations 10 days in advance of

prebaiting (with nontoxic material resembling the bait to be used later), and then extending the

prebaiting period from 4 to 5 days.44 As palatable and durable anticoagulant baits essentially serve as their own prebaits, the prebaiting step

can be skipped when such products are used. 22 When “empty” bait stations are obtained, they can be

placed in the rodent

‐ infested area for several days to allow rodents time to become familiar with the

new objects before bait is introduced. Such an approach would not be possible with ready

‐ to

‐ use bait

stations, especially those of the single

‐ use (not refillable) type, and is not suggested on the labels for

such products.

Rodents immigrating to an area where baits already have been deployed would be encountering an

entirely new environment. Consequently, any baited bait station found there would be no newer to

them than other features of the area and might be entered readily as a source of cover.

When placed into an area infested with rodents, bait stations and the bait in them are new objects,

which may elicit neophobic responses from targeted rodents (e.g., Cowan and Barnett, 1975).

The time needed to overcome inherent reluctance to enter and feed from bait stations varies from

design to design and from species to species (Palmateer, 1982; Kaukeinen, 1986; 2007b).

Palmateer (1982) tested 9 designs of rat

‐ sized bait stations and 2 mouse

‐ sized designs with wild

‐ type

Norway rats and house mice, respectively. He also tested the mouse

‐ sized units with Swiss

‐ Webster

strain laboratory mice. The stations tested included protective units available at the time as well as non

‐

protective but covered designs. The rat trials were conducted in outdoor colony pens, while the mouse

trials were tested in “bull pen” enclosures. Within 0 to 8 days over the 15

‐ day test period, rats took the

food from all but one of the bait station designs at essentially the same rate that they previously had

accepted the food from an open crock. Rats accepted bait well immediately from an internally baffled

design and several others. 23 Wild

‐ type house mice and Swiss

‐ Webster mice performed similarly with the

designs to which they were exposed, readily entering the tamper

‐ resistant unit and shredding the

cardboard design to the point where it was rendered unusable after 4 days.

Kaukeinen (1986) exposed captive wild

‐ type Norway rats to 9 different designs of protective bait

stations in indoor trials to assess how readily the rats would enter the units and feed from them. He

reported that the designs tested included some that rats entered and fed from on the day of

deployment, some that were fed from within several days, and some for which feeding did not begin for

a week or more. Kaukeinen (2007b) later noted that the stations with more complex internal baffling

tended to be the ones for which it took longer for Norway rats to enter and feed. With house mice, he

observed that entry to bait stations was rapid and that placement in active areas was the key to

successful control of house mice using bait stations.

22 Some bait manufacturers have made placebos for the toxic baits available for surveillance use. Placebo baits also

could be used to condition rodents to enter and feed from bait stations, and to assess the potential for use of

stations by nontarget organisms, prior to the application of toxic bait.

23 Hayes and Gaines (1950) noted that, although Norway rats readily entered the bait stations used in their field

trials, there had been “difficulty” on other trials “in getting rats to enter bait stations containing baffles so there

was no direct path through the station.”45 Based upon observations of free

‐ ranging and captive wild

‐ type Norway rats, Quy, et al (2003) suggested

that bait stations used in the control of this species should be designed to provide sufficient room for

simultaneous occupation by small groups of rats.

Spurr, et al (2006), tested the responses of wild

‐ type Norway rats to 4 bait station designs (including a

commercial design available in the U.S.). The rats were live

‐ trapped in New Zealand, given > 18 days to

acclimate to captivity and tested individually with the bait stations in outdoor enclosures. The station

designs all had been used in NZ Department of Conservation rodent control projects, and the specific

units tested had themselves been used in such programs. Stations were loaded with a non

‐ toxic bait

that essentially was a placebo for the Pestoff® bait block often used in rodent eradication projects

conducted on New Zealand’s off

‐ shore islands. Each of the 24 rats (9 males, 15 females) was tested

once, without an acclimation period in the test enclosure, with each of the 4 stations, which were

presented one at a time. The order in which individual rats were exposed to the various bait station

designs was randomized. Three enclosures, total, were used in the trials. Exposure periods began at ~4

PM and concluded with the recapture of the rats at ~9 AM. In addition to the placebo bait, rats had

access to “Normal laboratory rat food (rat and mouse pellets)

... scattered around the pens.” Rat

behavior during test periods was recorded via videotape. Amounts of bait removed from stations were

calculated via weigh

‐ back.

Of the bait station designs tested, the “Wooden motel” (basically a lidded wooden box with corrugated

pipes extending the rodent entrances a short distance beyond the sides of the box) and the Bell

Protecta® station were approached more rapidly (mean times being ~20 minutes) than were the “White

plastic bottle” or the “Yellow plastic pipe” (which was the type of station used in the successful rat

eradication on Breaksea island). First approaches to bait station designs reportedly were not

significantly affected by prior exposures to any type of bait station. Mean times to first entry of bait

stations were much longer, reportedly being on the order of 6

‐ 8 hours for the Protecta®, the motel, and

the yellow pipe, and nearly 30 hours for the bottle. Latency to enter a bait station was lowered

significantly if it was not the first unit to which the rat was exposed. The proportion of rats entering the

bait station at all was ~0.8 for the Protecta®, nearly 0.9 for the motel and the yellow pipe, but only ~0.45

for the bottle. The proportion of rats entering a bait station was significantly affected by whether they

had prior exposure to another bait station, with prior exposure to the motel apparently being the

experience most likely to encourage entry to a different unit presented subsequently. The wooden

motel had much longer initial stays than did any of the other designs. Mean duration of initial stay was

about 70 seconds in the motel vs. ~10 seconds for the other 3 designs. (Due to the authors’

presentation of results as histograms rather than in tabular form, numbers are given here as

approximations gleaned from examining the graphs.). The mean number of entries to the bottle was <1,

which was less by far than the mean numbers for the other designs (~7.5 for the Protecta®, ~8.5 for the

motel, and ~6 for the pipe. Prior exposure to any bait stations was associated with an increased

likelihood of station entry in a subsequent test. The mean total duration of all station entries during the

17

‐ h period was ~85 seconds for the motel, ~10 seconds for the pipe, and <10 seconds for the Protecta®

and for the bottle.46 Regardless of station design, however, the rats ate little bait from them (overall mean = 1.33 g/night,

with only 7 of 96 (7%) test nights resulting in > 5 g of bait removal. The greatest amount of calculated

consumption was 21 g. Mean amounts of bait consumed by rats that entered stations were ~1.8 g/night

for the Protecta® and the motel, ~2.,2 g/night for the bottle, and ~0.6 g/night for the pipe. The

proportions of rats entering stations that consumed bait were ~0.41 for the Protecta®, 0.67 for the

motel, 0.36 for the bottle, and 0.75 for the pipe, although those feeding from the pipe ate very little

bait. Rats removed bait blocks from bait stations that lacked securing rods on 13 exposure

‐ nights.

As the purpose of their testing was to identify a bait station design to use for post

‐ eradication

surveillance, Spurr, et al (2006) suggest that the motel or the yellow pipe

– the units with the highest

proportions of rats that entered having eaten bait

– would do better than the others for this purpose.

However, they also note that mean amount of placebo bait consumed (1.33 g) would not provide an

LD 50 dosage for a 400

‐ g Norway rat whether the bait was 0.002% Brodifacoum (such as the Pestoff®

block) or 0.005% Brodifacoum ( a la Weatherblok and other commercial baits). The amounts needed for

an LD 50 dosage for a 400

‐ g Norway rat would be 2.2 g for a 0.005% Brodifacoum formulation or 5.4 g for

a 0.002% Brodifacoum bait (calculated from data compiled by Timm, 1994c, from other sources).

Smaller rats would reach an LD 50 dosage with less consumption, but an LD 50 dosage would leave half of

the rats consuming it alive, not to mention the survival of rats that did not enter the station. Spurr, et al

(2006), also cautioned that different results might have been obtained if similar trials were run with

other species (e.g., roof rats, Polynesian rats).

C. Bait Stations vs. Placepacks and “non

‐ bait station baiting”

As its title implies, “The Control of Rats with Rodenticides: a Complete Guide to Best Practice”

(Anonymous, 2002) discusses procedures for controlling rats with rodenticides (not in the U.S.). This

document notes that neophobia is a real phenomenon but one that can be situation

‐ specific. In

relatively stable environments, neophobia can last a relatively long time, presumably because new

objects are easier for rodents to notice. In situations in which items are often moved about and

replaced, a new bait station might not be as likely to be singled out as strange. The report states that “a

new bait container” is apt to be avoided longer than a new food, especially “if bait is placed directly on

the ground”. As noted above, USDA and EPA decided long ago to discourage and prohibit the latter

baiting practice in areas to which children, pets, and other nontarget animals have access.

Vantassel, et al (2006), state that “Baits stations used in rodent control programs increase both the

effectiveness and safety of rodent baits. They go on to list the following advantages of using bait

stations:

• Bait stations are useful because they:

• protect bait from moisture and dust;

• provide a protected place for rodents to feed, allowing them to feel more secure and

consume more bait;

• keep nontarget species, including pets, livestock, wildlife, and children away from toxic baits;47 • allow bait to be placed in otherwise difficult locations given weather or potential hazards to

nontarget animals;

• help prevent accidental spillage; and

• offer the applicator easy access to bait, making it easier to determine the amount of bait

consumed by rodents, and to refill.

Vantassel, et al (2006), discuss users’ building their own stations, mentioning nailed lean

‐ boards on the

one hand and stating “It never hurts, however, to be more restrictive than the label requires” on the

other. They also state, without a relevant citation, that “Research has shown that mice prefer to enter a

low

‐ profile station, but the precise reason for this behavior is unknown.” They also discuss appropriate

placement locations (“between the rodents’ food supply and their shelter”; “near burrows, against

walls, and along their travel routes”); spacing of stations (< 12 feet apart for mice, 15

‐ 50 feet for rats);

and latency to enter stations (“up to two weeks for rats to enter recently placed bait stations”). They

recommend against perimeter fence

‐ line baiting (as it “may endanger nontarget species, while not

substantially increasing target species control”) except that stations “may be necessary along the

perimeter when structures are within 25 yards of thick vegetative covers and there is a large rodent

population.” They also recommend daily checking of units initially followed by 1

‐ 2

‐ wk intervals, with

replenishment of spoiled or contaminated bait.

Securing rods have been added to bait stations keep intact bait blocks or placepacks from falling out of

stations that are tipped or moved from place to place. This arrangement has been encouraged and

adopted primarily as a safety feature, although it also is said to limit feeding on bait by invertebrates

and to extend the effective life of bait by protecting it from water that may come into bait stations

(Corrigan, 1998). However, rats prefer to move bait out of stations to burrows or other locations and

will do so even with small bait blocks (e.g., Taylor and Thomas, 1989, 15

‐ g blocks). Securing blocks

within bait stations inhibits such behavior and requires rats to begin working on the blocks while they

are in the units. Rats would be able to carry off chunks of bait blocks that they managed to break off

and pelleted baits removed from placepacks. However, less bait is likely to be carried off when blocks

initially are secured. Whether securing blocks means that less bait is ingested or that the bait made

available is shared more equitably among rats visiting the stations is largely unstudied, although Quy, et

al (2003) suggest that some “training” may be needed to influence rats to ingest bait secured in stations

rather than to cache it first or to lift it so as to consume it in the station in a more typical feeding

posture.

VI. Effects of Bittering Agents on the Effectiveness of Commensal Rodenticide Baits

Would potential deterrents (e.g., bittering agents) added to baits to reduce the severity of incidents of

exposure of young children to rodenticide baits also adversely affect the performance of the

rodenticides against targeted species?

Whether the presence of a bittering agent or other feeding deterrent in rodenticide baits at levels that

commensal rodents will accept in laboratory trials translates into rodent control operations that are not

adversely affected by the agent and whether such baits would be rejected by children under conditions48 of actual use are important questions. 24 Among possible bittering agents, only the bittering agent

Denatonium Benzoate has been researched to the point where there is sufficient information available

to merit further discussion. This section addresses the effects of Denatonium Benzoate on the

effectiveness of commensal rodenticide baits.

As discussed further below, EPA has received data showing that many bait formulations that include

Denatonium Benzoate at low concentrations have met the applicable performance criteria in laboratory

choice

‐ feeding studies of the types used to screen new bait formulations of registered active ingredients

for effectiveness. Those studies, by and large, were conducted with laboratory strains of the Norway rat

and house mouse. There remain some questions about how well baits containing Denatonium Benzoate

perform in actual use situations, but some data consistent with effective use of them have been

reported.

A. Rationale for Adding Bittering Agents to Rodenticide Baits.

If there were a substance that rodents would readily accept in baits which humans and other nontarget

species found to be so objectionable that a single taste would cause them to reject the material

immediately, there would be little need to be concerned about where and how rodenticide baits are

used. No such material has been identified, and none is likely to be considering rodents’ olfactory and

gustatory abilities and their behavioral defense mechanisms against being poisoned. Denatonium

Benzoate tastes bitter to humans and, in that context, has been claimed to have applications in human

safety, including the off

‐ flavoring of toxic products. Denatonium Benzoate is used to “denature”

ethanol 25

, has been added to soaps and antifreeze, and is the only bittering agent that registrants have

specifically identified on labels as being used in commensal rodenticide bait products registered in the

U.S. Formulations for such bait products typically have been required to be screened for efficacy in

laboratory trials before the formulations have been registered.

B. Data on the Performance of Baits Containing Denatonium Benzoate

When adding Denatonium Benzoate to rodenticide baits first was proposed to OPP's Registration

Division (RD) in 1989, it had not been conclusively demonstrated whether the substance would have its

intended effect in deterring the extent of consumption of baits by children or any adverse effect on the

effectiveness of the baits in controlling rodents. Much of the history of bait development to that point

had been concerned with finding suitable attractants and masking agents to offset “off” flavors

associated with active ingredients, including some anticoagulants as well as many of the acute

compounds used historically as commensal rodenticides (Jacobs, 2000).

24 In the Rodenticide Cluster RED, EPA included a “Phase 1” requirement to reformulate rodenticide baits

registered for use in and around human residences to include a bittering agent (see also Jacobs, 2000). EPA

subsequently abandoned that requirement, but the decision to abandon was subjected to a court challenge in

which EPA’s decision was reversed and remanded to the Agency for further consideration.

25 The “Denat” portion of the chemical name “Denatonium” is based on this use.49 Rodenticide bait formulations containing Denatonium Benzoate were not accepted for registration by

EPA until RD had reviewed data from laboratory efficacy tests indicating that the new formulations met

applicable criteria for product performance, typically at least 33% acceptance and at least 90% mortality

among test

‐ group subjects (see prior discussions of performance criteria and Jacobs, 2000). Kaukeinen

and Buckle (1992) published data (summarized in IV.A. above) consistent with their thesis that adding

Denatonium Benzoate at 10 ppm would not adversely affect the efficacy of the "TALON" line of

Brodifacoum baits. In their paper, Kaukeinen and Buckle (1992) summarized the results of laboratory

efficacy studies that EPA had accepted for some of the first rodenticide baits containing Denatonium

Benzoate that were registered in the U.S. and presented information suggesting that baits with and

without that agent in them had performed similarly in field trials.

Although baits containing Denatonium Benzoate have been reported to perform at or above applicable

criteria, such results do not absolutely rule out the emergence of product performance problems.

Performance problems may include rejection of baits containing Denatonium Benzoate by some

individual rodents or rodent populations and/or selection for such rejection by eliminating animals that

readily accept baits containing that substance but not killing those that reject it.

Kaukeinen and Buckle (1992) reported on both the efficacy and human safety side of the question

broadly posed at the beginning of this section but gave special emphasis to Denatonium Benzoate

(Bitrex® 26 ). They reported that adult humans tended to find objectionable placebo pelleted and block

baits treated with Denatonium Benzoate at a concentration (0.001% or 10 ppm) that laboratory strains

and wild

‐ types of the Norway rat and house mouse, and wild

‐ type roof rats, accepted well in 0.005% (50

ppm) Brodifacoum baits. Roof rats’ acceptance of a bait

‐ block product formulated with Denatonium

Benzoate at 0.001% was much higher than that for a (presumably) similar product without the bittering

agent. Among strains of the Norway rat and house mouse, differences in acceptance between baits

containing or lacking Denatonium Benzoate went in both directions and were smaller than that seen

with roof rats.

Kaukeinen and Buckle (1992) also reported that 0.005% Brodifacoum baits containing Denatonium

Benzoate at 0.001% had been used successfully in a field trial conducted against “warfarin

‐ resistant

Norway rats” in the U.K. During a 4

‐ day exposure period on a hog farm in North Carolina, Kaukeinen

and Buckle (1992) report that a pelleted bait containing Denatonium Benzoate at 0.001% was consumed

in amounts essentially equal to those for a pelleted bait lacking the bittering agent.

Kaukeinen and Buckle (1992) state that perception of the bitterness of Denatonium Benzoate is affected

by the presence of other substances, suggesting that as an explanation for its being used at

concentrations of 10 ppm, 20 ppm (0.002%), and 50 ppm (0.005%) in various rodenticide baits that were

registered in the U.S. at the time their report was prepared. They also cautioned that

... rodenticides (or any pesticide or potential toxicant) containing Bitrex retain the same

toxicity to nontarget species, and must be used in the same careful way, according to good

26 MacFarlan Smith Ltd., Edinburgh, Scotland. References to trade names do not imply endorsement by the U.S.

Government.50 practices and label directions. The public and safety groups must be educated that such

additives are not ‘pesticide panaceas,’ or simple and quick product modifications. Additive

inclusion can require extensive research by manufacturers and others (including testing

required for modification of product registrations) encompassing, but not limited to,

determinations ensuring additives do not interfere with product efficacy and stability, and that

they do offer some increased product aversion in nontarget animals.

Veitch (2002) reported successful eradication of Polynesian (Pacific) rats ( Rattus exulans ) from Fanal

Island, NZ, using a 20

‐ ppm (0.002%) Brodifacoum pelleted bait that also contained Denatonium

Benzoate (concentration not indicated). However, Veitch (2002) also reported results of a laboratory

trial in which captive animals of the same species accepted the same bait formulation poorly relative to

20

‐ ppm Brodifacoum bait that lacked Denatonium Benzoate but was identical otherwise. Wild

‐ caught

R. exulans (which also occur in Hawaii and various U.S. Pacific territories) offered a choice between the

two baits reportedly consumed averages of 2.9 g of the version containing Denatonium Benzoate and

9.7 g of the bait that lacked that bittering agent. On average, those rats preferred the bait without

Denatonium Benzoate by a ratio of more than 3:1. Veitch (2002) reported considerable variation in

responses among individual subjects.

Veitch (2002) added that those responsible for treating Fanal Island were not aware at the time of

application that the bait used contained Denatonium Benzoate. Rather than structural applications

involving bait stations, the Fanal Island treatment was made via aerial broadcasting of 825 kg of bait to

the 73

‐ hectare island, a rate equivalent to about 10 lbs of bait per acre. Summarizing this baiting

program, Veitch wrote

The success of this operation, despite adverse conditions and an unattractive bait, is evidence

of the effectiveness of aerial application of brodifacoum baits for rat eradication from islands.

Nevertheless, it is prudent that future operations be managed with such variables in their

favour.

Also concerned with eradicating rats from New Zealand islands, Cromarty, et al (2002), expressed similar

sentiments.

Bitrex is added as a safety precaution to make some commercial rat bait less attractive to

children, by making it taste bitter. In laboratory efficacy tests involving bitrex in ICI

rodenticidal formulations with albino rats and mice (20 animal groups, 3 day choice tests)

some rats did not eat sufficient bait with bitrex in it to be killed (i.e., 3 out of 60 rats were not

killed) (Kaukeinen and Buckle 1992). These tests are required as part of USA registration

studies (EPA protocols). The ICI rodenticidal formulations all passed the EPA minimum test

criteria of at least 90% kill, and led to the EPA's statement in their letter to ICI of 29 March

1990, that "The efficacy tests submitted for (bitrex

‐ containing brodifacoum products) are

acceptable" (Kaukeinen and Buckle 1992). The test result is acceptable for a control operation.

However, for an eradication operation such information suggests a risk that should not be51 taken

– it is not acceptable to have a percentage of the population not eating sufficient bait to

be killed. On uninhabited islands closed to the public there is no benefit to including Bitrex.

MacFadden (2002) was of the same opinion and accurately noted that the reported survivors in the EPA

‐

type tests were 3 of 100 albino Norway rats and 2 of 100 albino house mice. McClelland (2002)

reported a personal communication from MacFadden to the effect that 2 of 4 attempted rat

eradications using a bait containing Denatonium Benzoate were unsuccessful.

Howald, et al (2005), observed that a Brodifacoum bait used as the primary control agent in an

unsuccessful attempt to eradicate roof rats from Palmyra Atoll (Line Islands, U.S. Territory) contained

Bitrex. The Bromethalin bait reportedly used to a much lesser extent in the same program also

contained Denatonium Benzoate. Howald, et al (2005), also reported that the Palmyra program had a

variety of organizational and strategic shortcomings. It is not clear which of the various problematic

factors identified, or others, were responsible for the ultimate failure to fatally poison every last roof rat

on Palmyra Atoll in the 2001

‐ 2003 eradication effort.

The attitude toward baits containing Denatonium Benzoate held by those engaged in island eradication

programs may be shared by many of those engaged in operational rodent control at mainland sites (e.g.,

the Chicago Bureau of Rodent Control, Howard, 1999). Whether or not the bait containing Denatonium

Benzoate worked in past baiting programs, there is concern that its presence might compromise the

success of the next operation. However, many of the rodenticide bait products used in operational

baiting programs have contained Denatonium Benzoate. 27

Selective removal of rodents that tolerate Denatonium Benzoate could lead to the development of local

populations of rats or mice that cannot be controlled with baits that contain the substance. Due to the

lack of relevant research on any other agent with a bitter taste, requiring that all baits contain a

bittering agent essentially would be requiring that they all contain Denatonium Benzoate. Applications

of baits containing Denatonium Benzoate might cull out rodents that accepted baits treated with that

agent, with residual populations being comprised of individuals that did not plus the offspring of those

behaviorally resistant survivors. If a rodent population consists largely of individuals that will not accept

a particular substance, requiring that substance to be in all baits would mean that all baits could fail at

the same time with respect to controlling that population. The experience reported with a Bitrex

‐

containing bait reported by the Chicago Bureau of Rodent Control (BRC) was consistent with local

27 As part of a survey of pesticide use in public schools, public parks, and public housing developments in Albany,

Buffalo, New York City, Syracuse, and Yonkers, NY, Surgan, et al (2002) obtained information on the names,

registration numbers, and amounts of pesticide used at the participating facilities over one

‐ year periods (calendar

year 2000 for parks, 2000 for housing developments, except in Buffalo which reported for 2001, 2000

‐ 2001 school

year for schools). According to their labels and formulations of record at the time, eight of the 15 rodenticide bait

products used at those sites contained Denatonium Benzoate. Those products reportedly were used at 21 sites

with amounts applied totaling over 340 lbs. Seven of the products used, at a total of 19 sites, were bait blocks.

Those products accounted for most of the total amount of use of baits that contained Denatonium Benzoate.

Seven baits lacking a bittering agent reportedly were used at 9 sites with the total amount applied being nearly

1200 lbs. Federal registrations for at least 3 of the 7 products that lacked a bittering agent have been canceled

since 2000, including a bait block product applied at two sites for a total of 615 lbs.52 selection for animals that would not accept Denatonium Benzoate as well as with the Bureau's inference

that the bait was rejected by rats upon its first presentation. 28

Although selection for rejection of Denatonium Benzoate has not yet been demonstrated

experimentally for any commensal rodent species, that it could happen as a result of rodent control

operations is plausible and consistent with other phenomena that have developed over generations

where rodent control efforts have occurred (e.g., anticoagulant resistance, extreme neophobia). In

theory, any heritable trait is likely to be selected for under conditions that impart an advantage to the

survival and/or reproductive success of individuals possessing it.

Depending upon local circumstances, commensal rodents may be very selective regarding what they will

eat or be willing to ingest practically anything (e.g., Richter, 1968; Barnett, 1946). The relative

availability of alternative feeds to the rodenticide bait being used will vary from one rodent control

project to another depending upon local circumstances and the extent to which removal or isolation of

alternate sources of feed for rodents is feasible. As rodents are removed from the local environment,

the abundance of alternative feed per rodent may increase, making it more difficult to control the last

few individuals with a bait of marginal palatability. 29

To summarize, it has been reported that Denatonium Benzoate has little or no deleterious effects on

bait acceptance and bait performance under use conditions (Kaukeinen and Buckle, 1992), but such

assessments are not accepted universally. See also Corrigan (2006b).

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Services, Fort Collins, CO, 12 pp.62 APPENDIX I

List of Cited EPA Protocols for Laboratory Efficacy Studies of Commensal Rodenticide Baits

Standard Norway Rat/Roof Rat Anticoagulant Dry Bait Laboratory Test Method, OPP Designation 1.203

(2

‐ 25

‐ 74), revision of 6

‐ 18

‐ 91, 4 pp.

Standard House Mouse Anticoagulant Dry Bait Laboratory Test Method, OPP Designation 1.204 (2

‐ 25

‐

74), revision of 6

‐ 18

‐ 91, 5 pp.

Standard Norway Rat/Roof Rat Acute Dry Bait Laboratory Test Method, OPP Designation 1.209, revision

of 10

‐ 8

‐ 91, 5 pp.

Standard House Mouse Acute Dry Bait Laboratory Test Method, OPP Designation 1.204 (2

‐ 25

‐ 75),

revision of 10

‐ 18

‐ 91, 5 pp.

Standard Norway Rat/Roof Rat Anticoagulant Wax Block and Wax Pellet Laboratory Test Method, OPP

Designation 1.213, revision of 12

‐ 2

‐ 90, 4 pp.

Standard House Mouse Dry Anticoagulant Wax Block and Wax Pellet Laboratory Test Method, OPP

Designation 1.214 (1

‐ 1

‐ 75), revision of 12

‐ 2

‐ 90, 5 pp.