cc:

Eric Bohnenblust Alexandra Dunn Cheryl Dunton Michael Goodis Arnold Layne Anna Lowit Autumn Metzger Jennifer Saunders

OPP Docket

FIFRA Scientific Advisory Panel: Robert E. Chapin, PhD Joseph Shaw, PhD Sonya K. Sobrian, PhD Clifford P. Weisel, PhD Raymond S.H. Yang, PhD

FQPA Science Review Board Members: Arthur Appel, PhD Michael J. Daniels, ScD Marion Ehrich, PhD Jerome Hogsette, PhD Eric Kwok, PhD Lisa Murphy, VMD Weste Osbrink, PhD Michael K. Rust, PhD Jeffrey G Scott, PhD Keith Shockley, PhD Daniel E. Snyder, DVM, PhD Larisa Vredevoe, PhD

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FIFRA Scientific Advisory Panel Meeting Minutes and Final Report No. 2019-02

Peer Review on EPA Office of Pesticide Programs’ Proposed Guidelines for Efficacy Testing of Topically Applied Pesticides Used Against Certain Ectoparasitic Pests on Pets

June 11-14, 2019 FIFRA Scientific Advisory Panel Meeting

Held at

U.S. Environmental Protection Agency Conference Center Lobby Level One Potomac Yard (South Bldg.) 2777 S. Crystal Drive, Arlington, VA 22202

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4 NOTICE

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) is a federal advisory committee operating in accordance with the Federal Advisory Committee Act and established under the provisions of FIFRA as amended by the Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides advice, information, and recommendations to the U.S. Environmental Protection Agency (EPA or Agency) Administrator on pesticides and pesticide-related issues regarding the impact of regulatory actions on health and the environment. The SAP serves as a primary scientific peer review mechanism of the EPA, Office of Pesticide Programs (OPP), and is structured to provide balanced expert assessment of pesticide and pesticide-related matters facing the Agency. The FQPA Science Review Board members serve the FIFRA SAP on an ad hoc basis to assist in reviews conducted by the FIFRA SAP. The meeting minutes and final report are provided as part of the activities of the FIFRA SAP.

The FIFRA SAP carefully considered all information provided and presented by the Agency, as well as information presented by the public. The minutes represent the views and recommendations of the FIFRA SAP and do not necessarily represent the views and policies of the Agency, nor of other agencies in the Executive Branch of the federal government. The mention of trade names or commercial products does not constitute an endorsement or recommendation for use.

The meeting minutes and final report do not create nor confer legal rights nor impose legally binding requirements on the EPA or any other party. The meeting minutes and final report of the June 11-14, 2019, FIFRA SAP meeting represent the SAP’s consideration and review of scientific issues associated with the “Proposed Guidelines for Efficacy Testing of Topically Applied Pesticides Used Against Certain Ectoparasitic Pests on Pets.” Steven Knott, MS, FIFRA SAP Executive Secretary, reviewed the minutes and final report. Robert E. Chapin, PhD, FIFRA SAP Chair, and Suhair Shallal, PhD, Designated Federal Official, certified the minutes and final report that is publicly available on the SAP website at http://www.epa.gov/sap under the heading of “Meetings” and in the public e-docket, Docket No. EPA-HQ-OPP-2019-0161, accessible through the docket portal: http://www.regulations.gov. Further information about FIFRA SAP reports and activities can be obtained from its website at http://www.epa.gov/sap. Interested persons are invited to contact Steven Knott, MS, FIFRA SAP Executive Secretary, via e-mail at [email protected].

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TABLE OF CONTENTS

NOTICE ...... 5 LIST OF ACRONYMS AND ABBREVIATIONS ...... 10 INTRODUCTION ...... 11 PUBLIC COMMENTERS ...... 11 EXECUTIVE SUMMARY ...... 12 DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE ...... 18 LITERATURE CITED ...... 72

6 signatures redacted signatures redacted Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel Meeting June 11-14, 2019

Peer Review of Proposed Guidelines for Efficacy Testing of Topically Applied Pesticides Used Against Certain Ectoparasitic Pests on Pets

PARTICIPANTS FIFRA SAP Chair

Robert E. Chapin, PhD, Independent Consultant, Preston, Connecticut

Designated Federal Official

Suhair Shallal, PhD, Environmental Scientist/Designated Federal Official US. Environmental Protection Agency, Washington, District of Columbia 202-564-2057, [email protected]

FIFRA Scientific Advisory Panel Members

Joseph R. Shaw, PhD, Associate Professor, School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana

Sonya K. Sobrian, PhD, Associate Professor, Howard University College of Medicine, Howard University, Washington, District of Columbia

Clifford P. Weisel, PhD, Professor, Rutgers University, Piscataway, New Jersey

Raymond S.H. Yang, PhD, Professor (Emeritus), College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado

FQPA Science Review Board Members

Arthur Appel, PhD, Professor of Entomology, Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama

Michael J. Daniels, ScD, Professor, Chair, and Andrew Banks Family Endowed Chair, Department of Statistics, University of Florida, Gainesville, Florida

Marion Ehrich, PhD, DABT, Professor of Pharmacology/Toxicology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia

Jerome Hogsette, PhD, Lead Scientist and Research Entomologist, Center for Medical, Agricultural and Veterinary Entomology, U. S. Department of Agriculture, Gainesville, Florida

8 Eric S. C. Kwok, PhD, DABT, Senior Toxicologist, Exposure Assessment Section and Human Exposure & Health Effect Modeling Section, California Department of Pesticide Regulation, Sacramento, California

Lisa Murphy, VMD, DABT, Associate Professor of Toxicology, School of Veterinary Medicine University of Pennsylvania, Kennett Square, Pennsylvania

Weste Osbrink, PhD, Research Entomologist, U.S. Department of Agriculture-Agricultural Research Service, Knipling-Bushland U.S. Livestock Insect Research Lab, Kerrville, Texas

Michael K. Rust, PhD, Distinguished Professor of Entomology and the Graduate Division, University of California Riverside, Riverside, California

Jeffrey G. Scott, PhD, Professor of Pesticide Toxicology, Cornell University, Ithaca, New York

Keith Shockley, PhD, Staff Scientist, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Daniel E. Snyder DVM, PhD, Dipl ACVM, EVPC, Owner and Managing Principal, Daniel E. Snyder DVM, PhD Consulting, LLC, Indianapolis, Indiana

Larisa Vredevoe, PhD, Professor, Biological Sciences Department, California Polytechnic State University, San Luis Obispo, California

9 LIST OF ACRONYMS AND ABBREVIATIONS

ACRONYMS DESCRIPTION a.i. Active ingredient ECHA European Chemicals Agency ELISA Enzyme-Linked Immunosorbent Assay EMEA European Medicines Agency EPA or Agency U.S. Environmental Protection Agency FDA Food and Drug Administration FFDCA Federal Food, Drug and Cosmetics Act FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FQPS Food Quality Protection Act GLMM Generalized Linear Mixed Model ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods NAMS New Approach Methodologies OCSPP Office of Chemical Safety and Pollution Prevention OPP Office of Pesticide Programs OPPTS Office of Prevention, Pesticides and Toxic Substances PBPK Physiologically Based Pharmacokinetic PCR Polymerase-Chain Reaction SAP Scientific Advisory Panel WAAVP World Association for the Advancement of Veterinary Parasitology

10 INTRODUCTION

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) completed its peer review of the set of scientific issues being considered by the U.S. Environmental Protection Agency (EPA or Agency) regarding a new proposed guideline for efficacy testing of topically applied pesticides used against certain ectoparasitic pests on pets. The meeting minutes and final report of the June 11-14, 2019 FIFRA SAP meeting represent the SAP’s consideration and review of scientific issues associated with this topic. Advanced notice of the meeting was published in the Federal Register on April 15, 2019.

The review was conducted in an open public meeting held in Arlington, Virginia. The Agency position paper, Charge Questions, and related documents in support of the SAP meeting are posted in the public e-docket at http://www.regulations.gov (ID: EPA-HQ-OPP-2019-0161). Dr. Robert E. Chapin chaired the meeting. Dr. Suhair Shallal served as the Designated Federal Official.

In preparing these meeting minutes and final report, the Panel carefully considered all information provided and presented by the Agency presenters, as well as information presented by public commenters. The meeting minutes and final report address the information provided and presented at the meeting, especially the Panel response to the Agency Charge Questions.

U.S. EPA presentations were provided during the FIFRA SAP meeting by the following (listed in order of presentation):

• Introduction - Product Performance Data Requirements and the Importance of Efficacy Testing Guidance- Michael L. Goodis, Director, Registration Division, Office of Pesticide Programs, EPA • A Background and Introduction to Proposed Methods for Efficacy Testing of Treatments Topically Applied to Pets to Control Certain Invertebrate Ectoparasitic Pests – Jennifer Saunders, PhD, Registration Division, Office of Pesticide Programs, EPA • Treatments Topically Applied to Pets to Control Certain Invertebrate Ectoparasitic Pests – Eric Bohnenblust, PhD, Biopesticides and Pollution Prevention Division, and Autumn Metzger, MS, Registration Division, Office of Pesticide Programs, EPA

PUBLIC COMMENTERS

Oral statements were presented by: • Rachel Cumberbatch, Director, International and Regulatory Affairs, Animal Health Institute • Nina Wertan, Program Manager for Animal Research Issues, Humane Society of the United States (HSUS) • Katherine Groff, Research Associate, People for the Ethical Treatment of Animals (PETA) • William Russell Everett, President/Laboratory Director, BerTek Inc.

11 EXECUTIVE SUMMARY

The United States Environmental Protection Agency’s (EPA) Office of Pesticide Programs (OPP) is updating their proposed guideline for efficacy testing of topically applied pesticides used against certain ectoparasitic pests on pets. This guideline is one of a series of test guidelines established by the Office of Chemical Safety and Pollution Prevention (OCSPP) [formerly the Office of Prevention, Pesticides and Toxic Substances (OPPTS) prior to April 22, 2010], for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and Cosmetics Act (FFDCA) (21 U.S.C. 346a). The OCSPP test guidelines serve as a compendium of accepted scientific methodologies for research intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA.

The Agency has multiple guidelines intended to assist in the development of appropriate protocols to test product efficacy. EPA Product Performance Test Guideline OPPTS 810.3300 Treatments to Control Pests of Humans and Pets was published in March 1998. To increase clarity and consistency in efficacy testing and to include current scientific standards, the Agency is updating and revising this product performance guideline.

The proposed guideline applies to products in any topically applied formulation, such as a spray, spot-on, collar, shampoo, or dust, if intended to be directly applied to pets for a pesticidal purpose such as to kill, repel, or control ticks, fleas, mosquitoes, and biting flies. The proposed guideline does not apply to those products exempt from FIFRA Registration under 40 CFR 152.25, products applied to humans or livestock, or product performance testing described in other Agency guidelines. In addition to guidance for testing efficacy against fleas, ticks, mosquitoes, and biting flies, the proposed guideline also includes testing methods for evaluating efficacy under simulated environmental conditions.

The Agency sought advice and recommendations from the FIFRA SAP on scientific issues associated with the proposed revised guideline. The FIFRA SAP was charged with providing recommendations to the Agency in updating the current guidelines for efficacy testing of topically applied pesticides used against certain ectoparasitic pests on pets. As part of the SAP, EPA is soliciting comments from the Panel on approaches that may, in the future, support the replacement or reduction of animal use in efficacy testing of ectoparasitic pests on pets. The FIFRA SAP addressed twenty-four Charge Questions and provided the following summary of the major conclusions and recommendations that are detailed in the report.

General Comments

The Panel commended the Agency for their work in updating Test Guideline OCSPP 810.3300 and supported the Agency’s desire to reduce the number of vertebrate animals required for testing and minimize the potential adverse effects on animals that are needed for testing. The methods and goals provided in the draft guidelines are generally consistent with past practice and incorporate current state of the art, validated methodologies for testing the efficacy of pesticidal pet products. While the proposed guidelines represent a major advancement over the previous

12 version, there are some areas that require clarification, revision or both, and several terms that need clearer definitions.

For example, the Panel recommended that the mix of scientific names (genus species) and common names be corrected, and a concise definition of repellency similar to that found in the European guidelines be provided. The term “engorgement” also needs to be revised in this guideline as the EMEA and WAAVP guidelines are not being used correctly. The Panel therefore recommended that all instances of “engorgement” in this proposed guideline should instead be changed to “partially fed.” The Panel also found that there were several issues that need clarification relative to the welfare of the vertebrate animals, including, repeated sedation, exposure to multiple pests and the duration of exposures.

New Alternative Methodologies (NAMs)

The Panel recognized that the goal of the guidelines is to provide appropriate protocols that are deemed acceptable for regulatory purposes based on published literature and previous studies. Therefore, many on the Panel agreed that new alternative methodologies (NAMs) for efficacy testing do not currently exist and, therefore, are not currently recommended. However, the Panel noted that due to the extended time period that guidelines are often in place before being modified, a statement on how a new protocol and/or technique may be implemented, beyond saying that it will be reviewed by the Agency, would be useful. The Agency should provide guidance on how NAMs protocols and the results from NAMs should be assessed so that they could eventually be used for testing products. For these reasons, the Panel’s recommendation that NAMs be assessed via direct comparison to the animal tests they are attempting to replace becomes critical. At this time, the Panel recommended that the Agency also consider computational NAMs that include Monte Carlo simulations to justify a reduction in the required number of vertebrate animals to six. They also suggested exploring Markov Chain Monte Carlo simulations, which consider covariance and uncertainties, and coupling simulations with Bayesian approaches when new scientific data are available. Currently, there is a lack of the necessary data (e.g., retention, feeding and engorgement, sampling, etc.) to make these simulations valuable.

Sample Size Determination

The 1998 OPPTS 810.3300 guidelines required at least 6 animals per group for testing, while preferring 10 animals per group. The EPA document titled, “Sample Size for Pet Product Studies” states that historically the EPA has recommended 6 animals per group, but often received studies with 2-12 animals per study group. The Panel recommended that the EPA provide more historical data describing the number of animals per study group and determine how often each study design (by group size) was deemed to be acceptable; such information could help to inform these new guidelines. The Panel recommended that power simulations be further explored to determine whether design modifications could be used to decrease the number of vertebrate animals in tick studies (and potentially in flea, mosquito and biting fly studies). The Panel also recommended that the impact of the values of each of the parameters on the power and sample size in the simulations should be assessed carefully.

13 The Panel recommended that the Agency should provide further documentation of the validity of the parameter values in the simulations based on historical information regarding host-pest interaction from the literature or studies submitted to EPA in the past. The simulations should be conducted using lower numbers of pests, particularly for biting flies and fleas, and improved estimates of retention rates, to assess the implication on power and sample size (i.e., the number of animals needed). For multiple species studies, when making an assumption of no interaction, the lack of interaction should be carefully documented from the literature. Otherwise, the power and sample size simulations and their validity would be suspect in the presence of interactions (unless such interactions were included in the simulations).

One of the Panel’s major concerns regarding the proposed Generalized Linear Mixed Model (GLMM) was the widespread lack of model convergence. The lack of convergence represents a major limitation for using the GLMM reliably for power and sample size calculations. Further, an analyst outside the Agency may also have difficulty with GLMM model convergence when performing their own analysis of these data. Suggestions for resolving the convergence issue include evaluating more accurate estimation methods, analyzing data with only the random animal effect and fitting the model separately by each of the “true efficacy of treatment” levels (0.95, 0.925, 0.90, and 0.85) instead of including “day” as an additional random effect in the model (fewer random effects should lead to fewer convergence problems). More accurate estimation methods include the Laplace approximation, quadrature, and “exact” maximization of true marginal likelihood using Monte Carlo methods. Alternative statistical methods based on Bayesian approaches or the “Rao-Scott” approach should also be considered.

Given the importance of the blood-fed/retention proportions and the numbers of arthropod ectoparasites per host animal, the Panel recommended that the Agency verify these values based on information obtained from the open scientific literature, registrant submitted studies (since the inception of the 1998 testing guideline), and experts who routinely work with these ectoparasites.

Efficacy Testing for Ear Mites

Protocols used in testing for ear mites (Otodectes cynotis) and sarcoptic mange mites (Sarcoptes scabiei) should be included in the OCSPP 810.3300 Guidelines. The Panel recommended that in addition to ear mites (O. cynotis) and sarcoptic mange mites (S. scabiei), Demodex mites (Demodex spp.) should be included in the guidelines. Examples of possible test methods for evaluating efficacy against each of these 3 species for on-animal treatments are available in the scientific literature. Test methods include both clinical trials and laboratory studies primarily in pets with natural infestations. With respect to methodology, the Panel recommended that EPA consider that efficacy studies involving client-owned pets be conducted at veterinary clinics on volunteer vertebrate animals. The Panel also suggests that EPA consider non-vertebrate studies as acceptable alternatives for scabies tests.

Efficacy Testing for Fleas, Ticks and Mosquitoes/biting flies

Techniques used to determine blood feeding in cat fleas include Drabkin’s technique to determine the quantity of blood consumed by adult cat fleas. This method also should be applicable to flies. The Panel suggested that some of the more modern methods also be used to detect the presence of

14 blood in an insect to confirm if blood feeding has occurred. For example, confirmation of a consumed blood meal could be performed using an enzyme-linked immunosorbent assay (ELISA) or a real-time Polymerase Chain Reaction (PCR) analysis of blood feeding in fleas which has been shown to be 10,000-fold more sensitive than the Drabkin technique.

The Panel noted that in efficacy testing for Ixodes spp. ticks, both ratios of 50:50 and 90:10 (females:males) would be acceptable, although lower numbers of males could affect female feeding success if mating opportunities are compromised. For Dermacentor, Amblyomma, and Rhipicephalus spp. which are metastriate ticks, mating occurs only on the host, and both females and males feed. Male metastriate ticks consume substantially less blood than females, but unlike the prostriate Ixodes spp., they do take some blood. Using either ratio of ticks could lead to up to 50 metastriate ticks attaching. Placement of a 50:50 ratio females:males for both groups of ticks would likely result in fewer adverse effects to the host during trials than 90:10, although not much difference would be seen if ticks are removed at 48 hours after infestation from the host. With the metastriate ticks, males must attach and feed before they mate so there would be no statistical advantage to skewing the ratio of females to males; therefore, the Panel recommended a 50:50 ratio. The Panel also suggested that Dermacentor spp. should be considered, as this is a common tick found on U.S. pets, in particular, D. variabilis on dogs.

The Panel concluded that the determination of engorgement is not a feasible data collection point. The term “engorgement” needs to be revised in the guidelines as the EMEA and WAAVP guidelines are not being used correctly. Adult tick counts are generally performed 48 hours after each infestation time point, but initiation of blood feeding during this period will not be readily apparent as very little blood is consumed within this time frame. It is impossible to accurately visualize any increase in size (plumpness) over this short period. This is even difficult if individual ticks are weighed (milligrams).

In addition, the proposed guidelines specify using pathogen-free ticks; however, a more specific definition of this is needed. The Panel is concerned that combining 2 tick species could facilitate dermal or sub-dermal pathogen exchange that might adversely affect test results. The Panel recommended allowing simultaneous testing of 1 flea + 1 tick species only, except when the tick is Amblyomma maculatum. The Panel has also provided the EPA with further information about tick species that have been used to co-infest animals without interactions so as to allow for accurate data collection. One advantage of testing multiple tick species on the same host is this approach could decrease the number of vertebrate animals to be used.

The Panel agreed that simultaneous testing with three mosquito species should not affect test results. The mosquitoes do not compete for feeding sites and do not otherwise interact in ways to alter results expected with a single species. The proposed methods look reasonable, but the Panel suggested that ways to reduce the number of recommended vertebrate animals should be considered. The proposed guideline states that a total of 150 mosquitoes/flies are needed if the testing involves three species concurrently (i.e., 50 per mosquito/fly species), but 100 mosquitoes/flies are needed if the testing involves only one species. The Panel noted that there is no basis for increasing the number of test insects from 50 to 100 when only a single species is used.

15 Rather than periodic outcrossing of mosquitoes, the Panel suggested testing a standard strain (insecticide resistant or susceptible) and comparing the treated versus untreated groups. Believing that there will be widespread differences between strains assumes facts not in evidence. Outcrossing with uncharacterized field-collected individuals could introduce pathogens, as well as, resistance to ectoparasiticidal compounds that are being evaluated. Furthermore, every time a strain is outcrossed, the strain changes and tests could yield non-reproducible results, a major drawback to any manipulations of the strain used for testing.

The Panel had some concerns about the general approaches proposed for testing the efficacy of products for biting flies. For flies known to exhibit aggressive behavior and (or) inflict a painful bite (e.g., stable flies), the Panel was concerned that efficacy testing conducted with 50-100 flies would cause adverse effects on the vertebrate animals involved. The Panel recommended that the guidelines also address the duration of each experimental exposure. Many of the Panel member’s concerns about animal welfare related to both sedation and exposure to biting flies could be addressed by limiting the exposure time. The Panel recognized that it may be necessary to sedate dogs and cats for short time periods when they are infested with particularly aggressive biting insects, such as stable flies. Since repeated sedation can be harmful to a vertebrate animal, more specifics are needed regarding how it can be accomplished safely. If starved insects are used, this should cause more rapid feeding on the test vertebrate, and sedation time and exposure to the insects might thus be reduced. Additionally, the effects of the pesticidal agents on pet animals can vary, e.g. small breed dogs may be more adversely affected than larger dogs. The number of exposures should adequately reflect the actual claims of the products. The exact number of exposures necessary will depend on the types of products, the claims, and how the Agency will evaluate submitted data.

The efficacy or effectiveness of treatment is appropriately determined by comparing the proportion of pests killed by the treatment (i.e., proportion of living in the control group - proportion living in the treatment group) over the proportion of pests living in the control group. The Panel recommended that the guideline should use different approaches to count fleas and ticks remaining on the vertebrate animals. A fine-toothed comb should be used to recover fleas, whereas ticks still attached to the vertebrate animal may be additionally discovered by carefully palpating the host animal.

For efficacy testing of collars, the Panel recommended that the EPA request that the registrant defend why they are cutting off excess collar material above and beyond what is needed to make contact with the neck region of the treated pet regardless of neck and body size (surface area). Additionally, the registrant should clearly indicate to the EPA reviewers how the active ingredient (a.i.) is being released from the collar matrix, including distribution and/or release kinetics of the a.i.

The Panel concluded that looking for repellency before assessing mortality is not needed. Blood feeding should be assessed in all recovered insects and this should drive the label claim (use pattern). The Panel did not agree with the assumption that the proposed endpoints for mortality are secondary to repellency.

16 Insecticidal Shampoos

Even non-pesticidal soaps may have a mortality factor against fleas and ticks. A shampoo can cause mortality in fleas, ticks and other arthropods by breaking the surface tension of the water. This allows water to enter the spiracles and interfere with oxygen exchange and could enhance the effect of the a.i. in a pesticidal shampoo. Therefore, the Panel recommended that the guidelines should specify that the appropriate control to compare against the pesticidal shampoo is the shampoo without the active pesticidal ingredient. A completely untreated control (i.e., received no shampooing treatment) is not an appropriate control for the efficacy determination of a pesticide in a pesticidal/medicated shampoo product because it does not control for the short-term pesticidal effect of the shampoo “inert” ingredients.

The Panel recommended that the guidelines specify that reinfestation should occur 24 hours after each exposure to water or bathing (e.g., bathing at day 6 with reinfestation at day 7, bathing at day 13 with reinfestation at day 14, etc.). In this way the animal will be dry prior to the reinfestation, even for vertebrate animals with thick hair coats.

Assessing live arthropods in the bathing tub is an adequate and practical endpoint for determining mortality. The Panel was not aware of any alternative methods that are adequate for determining mortality of fleas and ticks from shampoo products. However, the Panel recommended that the Agency guidelines be open to reviewing and including alternative methods for determining mortality of pests for shampoo products as these new alternative methods proceed through development and become available.

Environmental Conditions

The Panel recommended that if the current approach is used for assessing efficacy after exposure to sunlight, that the host be exposed to sunlight for two hours per day. This would potentially offer some standardization, although variation in weather may make such a requirement difficult or impossible to achieve. It would be of some additional value if the daily weather (average daily temperature, high temp, low temp, sunshine, rain, etc.) was also recorded. While such information may not alter the approval process, these variables would help identify differences that might occur between studies. Alternatively, the Panel noted that field studies at clinics could be very useful; if issues such as pet breed, hair length, sunlight, bathing, water exposure, etc. are important, then field studies will likely reveal them.

Given the difficulty in measuring the amount of sunlight the vertebrate animal is exposed to, the Panel recommended consideration of non-vertebrate (i.e., in vitro) testing of treated fur. The treated fur could be exposed to sunlight and the amount of residue of the active ingredient could be measured after different days or weeks of exposure to sunlight.

17 DETAILED PANEL DELIBERATIONS AND RESPONSE TO CHARGE QUESTIONS

GENERAL METHODS

Charge Question 1.

1) The proposed guideline describes test methods for evaluating the efficacy of a variety of pesticidal pet products to control fleas, ticks, mosquitoes, and biting flies on pets. Please discuss whether, given the objectives and the types of products being evaluated, the test methods described in the following sections are appropriate to evaluate the efficacy based on the pesticide labeling claims made, e.g. relating to repellency, mortality, and residual control: i. Fleas (section (j)) ii. Ticks (section (k)) iii. Mosquitoes and biting flies (section (l)) iv. Insecticidal shampoo products (section (m)) v. Environmental conditions (section (n))

Panel Response 1:

The Panel observed that the proposed guidelines represent a major advancement over the previous version by providing specific guidance for acceptable methodologies for testing the efficacy of pesticidal pet products, the rationale for guidelines, a recognition that some flexibility is needed in study design across species, the desire to reduce the number of vertebrate subjects to be used, and that new techniques are becoming available that need to be evaluated for use within the regulatory realm. EPA conducted a statistical simulation to quantify the number of vertebrate subjects necessary to be tested to adequately evaluate a product’s claim by considering the pest type, what the pet product is to be used for and the duration of efficacy claim rather than attempting to apply a single set of conditions to all conditions. A more complete discussion of this is provided in response to Charge Questions 4, 5, and 9. Throughout the proposed guidelines, consideration is given to minimize the number of dogs or cats required for testing within accepted scientific considerations.

The methods and goals provided in the proposed guidelines are generally consistent with past practice and incorporate current state of the art, validated methodologies for testing the efficacy of pesticidal pet products. The proposed guidelines recognize the need to evaluate the actual label claims, and the efficacy studies are designed to test the products to meet the extent of those claims. The Panel commends EPA and emphasizes that it is important to verify the efficacy of the products across the conditions and time periods claimed by the companies that produce them.

The Panel concluded that the general approaches used for testing the efficacy of products against fleas and mosquitoes are appropriate.

The Panel concluded that the general approaches used for testing the efficacy of products for ticks are appropriate, though there are concerns about the number of dogs and cats that need to be tested. The Panel recommended that the proposed guidelines should be revised to provide specific

18 recommendations about which tick species should be used for testing on cats and dogs. This may lead to a reduction in the numbers of animals used for testing. Utilization of tick species that reliably feed on these animals can facilitate a) typical tick attachment and feeding behavior during trials, b) reduction in the use of vertebrate subjects by preventing wasted trials, and c) results that reflect real-world exposure scenarios for each animal. The Panel suggested the protocols should recommend using 2 to 3 species for testing from: Rhipicephalus sanguineus, Dermacentor variabilis/D. andersoni/D. occidentalis, Amblyomma americanum, A. maculatum, and Ixodes scapularis for dogs (Koch and Sauer, 1984), and among D. variabilis, A. americanum, and I. scapularis/Ixodes pacificus for cats (Coles and Dryden, 2014).

The SAP expressed concerns about the general approaches proposed for testing the efficacy of products for biting flies. It may not be practical to form and maintain stable colonies for some fly species (e.g., horse fly tests are typically conducted with wild populations and, it is hard to get a uniform age and gender split for black flies). The Panel recommended that EPA clearly specify when laboratory colonies of single species should be used and under which conditions using wild insects or a mixture of species is acceptable.

The Panel noted that one issue that needs more consideration across all pests is the request to regenerate colonies with wild species every three years. This may introduce pathogens and could substantially change the genetics of the colony, making it difficult to compare a study to historic data and across studies. The Panel also questioned whether a three-year time interval is appropriate and recommended the use of a well-defined strain would be best. However, if the Agency decided to provide more guidance on outbreeding, the Panel suggested that the frequency should be based on the life cycle of each pest type and the length of time needed for the colony to stabilize after new genetic material has been added. A frequency based on the number of generations required to replenish a colony rather than a specific number of years may be more appropriate. In the updated European Medicines Agency document (EMEA, 2016), a longer time frame of 6 years is suggested.

While the Panel recognized the importance of fleas, ticks, and mosquitoes used in studies to be pathogen free, it may not be practical for all species. In the proposed guideline, EPA needs to provide a clear plan on which pathogens should be eliminated and how they should be removed, as well as a verification or certification process to demonstrate that a colony is considered pathogen free. This may be particularly problematic if wild individuals are introduced into a colony as they may introduce new pathogens to the colony.

The Panel also noted several issues that need clarification relative to the welfare of the pet animals. The Panel recognized that it may be necessary to sedate dogs and cats for short time periods when they are infested with particularly aggressive biting insects, such as stable flies, but more specifics are needed regarding how that can be accomplished. This is discussed in more detail in the response to Charge Question 18. Allowing the pet animal to groom is important; however, since grooming can affect the outcome of pest counts, methods to document similar grooming behaviors in control and treatment groups should be provided. Details of animal welfare considerations from the perspective of housing individual or multiple animals for socialization, particularly for longer studies, should be provided as these can affect infestations and the outcome of pest counts.

19 The Panel also identified some specific, detailed recommendations regarding clarification of terms and procedures (including new protocols and emerging techniques).

Several terms needed clearer definitions:

The guideline’s definition for moribund insects reflects incapacitated arthropods that could be considered either alive or dead. The Panel suggested that moribund insects be held longer (24-48 hours) under observation to determine mortality vs. recovery. When doing so, the conditions under which they are being held should be appropriate for recovery (e.g. relative humidity and temperature).

The Panel noted that a clear definition of tick engorgement is needed, as there are different stages for engorgement. The time frame of 48 hours is insufficient for full engorgement of ticks to occur and engorgement to repletion is not the appropriate endpoint to use. Clarification is also needed as to which tick species is being used for testing. This is discussed in more detail in response to Charge Question 14.

The Panel also noted that a clear definition of what is considered to be an adequately infested vertebrate animal is needed; including, the number of pests that should be added and whether retention rates should be based on the group mean count or counts per individual vertebrate animal (i.e., dog or cat). There needs to be clarification as to which species of fleas should be considered. Some designations are too broad.

The Panel further noted that a clear definition of what is considered to be an untreated or negative control should be provided. A potential definition of an untreated control group would be those vertebrate animals which receive no treatment that will affect the number or status of pests with which they have been infested. This definition of untreated control would not fit for pets that have been shampooed (or water washed) with non-insecticide shampoo since, at least the process of shampooing will affect the number of pests attached. Perhaps that could be referred to as a vehicle control.

The Panel also recommended that simple definitions for the terms repel, knock down, and kill, should be provided. The Panel suggested that, where appropriate, the European Medicines Agency definitions (EMEA, 2016) be used.

Procedural Concerns

The Panel concluded that the counting of dead fleas is problematic, and it is better to count live fleas and utilize the difference between control and treatment groups. This is discussed in more detail in the response to Charge Question 22.

The guidelines should clarify whether the group means or counts on individual animals meet the minimum retention rate (adequacy of infestation).

The Panel recommended that the level of variation in the ratio of male to female pests used for animal infestations that is acceptable be presented. For some species of fleas, the Panel noted, it may not be possible to obtain an exact 50:50 split. A ± 10% variation seems appropriate.

20 The guidelines should clarify the timing of when infestations should occur, how to classify the feeding stage of ticks that are removed, and at what time point removal should take place. This may vary based on the goals of an individual study. This is discussed in more detail in the response to Charge Questions 14 and 15.

The guidelines should state that the arthropods should be starved or unfed before infestation, though mosquitoes cannot be starved for too long (maybe < 24 hours). Mosquitoes could be provided sugar water to keep them alive until they are used in test trials.

The Panel agreed that combing is the best way to determine flea counts. Thumb counts were not considered to be acceptable. Also, comb counts may not be the best way to fully count and remove ticks, since forceps are typically used during the tick removal process.

To identify and quantify the amount of blood taken up by fleas and ticks, the Panel proposed using qPCR as a tool (Fourie et al., 2014). For tick mortality testing, the placement of ticks “over the entire vertebrate animal” (as worded in the guideline) suggests that they may attach over the entire surface of the animal. Regardless of placement location, ticks will select preferred attachment sites on the vertebrate. Ticks tend to congregate in groups on the vertebrate in locations where they are harder to remove by grooming behaviors of the vertebrate (e.g., ears, head, back, between toes, and axilla for R. sanguineus) (Dantas-Torres and Otranto, 2011). The guidelines should instead suggest that ticks are placed on animals away from treatment sites and allowed to freely disperse for attachment.

The guidelines do not clearly define when the endpoint of the trials and removal of attached live/dead and unattached ticks should occur. Although the timeframes for checking are mentioned in the proposed guidelines, tick removal is not specified. The Panel recommended stating that tick checks and removal should occur at 48 hours post-infestation or post-treatment.

Regarding use of laboratory colonies of ticks for trials, the Panel agreed with the statement that “similarly aged” ticks should be used for infestations, and perhaps the importance of this should be more clearly emphasized in the guidelines. The age of ticks used in studies could dramatically affect mortality/repellency trial outcomes. Cohorts of adult ticks used in trials should be the same age post-molt to the adult life stage, within a range of time in which vigor is evident, to avoid age- related deterioration effects on trial outcomes. Physiological age of ticks can affect attachment, feeding, and susceptibility of ticks to pesticides (Heller-Haupt and Varma, 1982; Rupes et al., 1972, 1977; Uspensky and Ioffe-Uspensky, 2006).

The effects of the pesticidal active agents on pet animals can vary, e.g., small breed dogs may be more adversely affected than larger dogs. The Panel expressed concern that the weight/dose ranges may be too wide for some products (i.e., smaller animals get more active ingredient per pound). Attention should be paid to label claims to protect the animals, though the Panel recognized that the lowest labeled dose across all weight classes is being considered. This is discussed in more detail in response to Charge Question 8.

The guidelines proposed: “Animals should not be treated with any substance (e.g., pesticidal shampoo) that could affect the results of the study for at least 3 months prior to initiating the study.” The Panel suggested that the following wording from the EMEA 14 July 2016 guidelines

21 be used instead: “It should be ensured that included animals have not been treated with an ectoparasitic substance within a timeframe that might impact the study outcome.” The time period after a test animal is treated should reflect the products’ actual residual effectiveness on an animal rather than using a standard 3-month period. The Panel also suggested that the same restrictions should be made for other animals that have been housed with the test animals.

The Panel noted that it is important to consider the vertebrate animal’s welfare when exposing a dog or cat to multiple pests. This is discussed in more detail in the response to Charge Question 7.

The Panel recommended more detail be provided in the guidelines on the use of sedatives, i.e., the type of sedative (provide a suggested list of approved sedatives), dose by animal type-weight, frequency that it can be repeated on a single animal, and whether the pest will also be sedated. This is discussed in more detail in the response to Charge Question 18.

Concerns with New Protocols and Emerging Techniques

The Panel recognized that the goal of these guidelines is to provide appropriate protocols that are deemed acceptable for regulatory purposes based on the published literature and on previous studies. However, due to the extended time period that guidelines are often in place before being modified, a statement on how a new protocol and/or technique may be implemented beyond saying that it will be reviewed by the Agency would be useful. In particular, some specifics to judge if new approach methodologies (NAMs) can be used to satisfy regulatory requirements and how they can be submitted by organizations or manufacturers would be helpful. The Panel suggested that the Agency encourage product researchers to run non-vertebrate (in vitro) tests along with the approved traditional animal testing methods (in vivo) to determine if the same outcomes are obtained, to develop a database of acceptable in vitro assays. For example, if product testing on pests exposed to treated animal hair in vitro demonstrates similar results to in vivo tests in repellency assays, these may be considered as useful surrogates to reduce vertebrate animal testing for certain product applications (Stanneck et al., 2012a). Furthermore, as the EPA finalizes the proposed guidelines, the Panel recommended that the different types of tests be defined more clearly. Specifically, the Panel suggested the term non-vertebrate animal testing (NAT, NVT, or NVAT) for tests that do not use the host be substituted for in vitro as this term has different meanings in different disciplines (see also the response to Charge Question #10).

The database should include an understanding of the biological basis for selected in vitro tests and the conditions under which they are conducted, so that future users would have the same premise to follow. The issue of non-vertebrate testing is discussed in more detail in response to Charge Question 2.

Some Panel members suggested that field tests should be considered in judging the efficacy of applied pesticides. These have strengths in that they are potentially more representative of real- world conditions as they could include a greater diversity of pet animals and pests and may have reduced requirements for housing of animals. However, they may require more animals and veterinarians on site. The approaches described in EMEA 2016 guidelines could be used as a starting script if field testing is acceptable to the EPA.

Some Panel members suggested that an unbalanced study design be considered when statistically

22 appropriate: i.e., fewer animals in the control group compared to the number of animals in the treatment group, in order to reduce the overall number of animals tested. This could be appropriate when previous data have shown that the variance in the number of pests that remain on the control animals is small.

Charge Question 2

New approach methodologies (NAMs) is a broadly descriptive reference to any non-animal technology, methodology, approach, or combination thereof that can be used in this case for efficacy purposes. EPA seeks to reduce the number of vertebrate animals needed for testing, where possible, while ensuring confidence in test results.

a. For each of the following sections from the guideline, please discuss potential alternative methods, including ways to reduce or replace, the use of animals in testing: i Fleas (section (j)) ii Ticks (section (k)) iii Mosquitoes and biting flies (section (l)) iv Insecticidal shampoo products (section (m)) v. Environmental conditions (section (n)) b. Considering the importance of accurate animal behavior (e.g., grooming) during these tests and confidence in the results of these tests for protecting public health, for each of the following sections of the guideline please discuss methods for refining, e.g., reduce the pain and suffering, the use of animals. i. Fleas (section (j)) ii. Ticks (section (k)) iii. Mosquitoes and biting flies (section (l)) iv. Insecticidal shampoo products (section (m)) v. Environmental conditions (section (n))

Panel Response 2:

The Panel commended the Agency for their work updating Test Guideline OCSPP 810.3300 and was in universal agreement with the Agency position that when revising guidelines, it should, when appropriate, try to reduce the number of vertebrate animals required for testing and minimize the pain and suffering of animals that are needed for testing. To this end, new approach methodologies (NAMs), which refer to any non-animal technology, methodology, approach, or combination thereof that can be used in this case for efficacy purposes, offer potential solutions, and the Agency is not alone as there has been near global recognition of the need for NAMS to reduce the number of vertebrate animals used in chemical safety testing (ECHA, 2016 and ICCVAM, 2018). As a result, there has been a rapid increase in the development of alternative testing strategies.

a. Most Panel members suggested that the Agency should pursue an area that holds great promise for the development of NAMs for chemical safety testing, namely, predictive mathematical and computer-based models. These models can be used to predict biological effects by incorporating

23 data from chemical descriptors and do not involve laboratory experiments. However, due to variability in model performance, the reliability of in silico models should be confirmed for each application. Including biological information can improve predictive modeling. One source of such data is in vitro bioactivity from quantitative high throughput screening (HTS) assays, which simultaneously produces concentration-response profiles for thousands of test agents in a single experiment. For example, an important goal of the NRC report “Toxicity Testing in the 21st Century: A Vision and a Strategy” (2007) involved moving away from whole animal testing towards in vitro methods.

A good predictive model requires good training data across all classes of chemicals for relevant biological endpoints. Data gaps can lead to biased or inaccurate prediction (Choudhuri et al., 2018). The US Tox21 collaboration is a relatively data rich program with bioassay data and complementary in vivo data for hundreds of chemicals. Yet, despite the successes achieved in prioritization and application to regulatory decisions, the Tox21 approach has not yet removed the need for animal testing.

One Panel member completely disagreed with the Tox21 approach. This panelist stated that while in silico models might have predictive abilities for some systems (e.g., identifying new drugs), that is completely different from product efficacy testing, which is what the EPA testing protocols are about. This panelist also noted that FDA does not allow in silico models as the justification for a new drug use (even if the company discovered it using such a pipeline). FDA would still require product testing and EPA should require the same.

The majority of Panel members noted that while NAMs, like in vitro screening, are beginning to be used, they have not fully matured. For example, in toxicity testing HTS may be used to aid in hazard identification for prioritization of further testing that would thereby reduce animal testing, and safety evaluation of certain products (e.g., dermal, ocular, inhalation). While in vitro systems may offer certain advantages in experimental research settings by providing more control of experimental factors, the Agency seeks a testing strategy that is not a research agenda, but rather one that is fully developed. It requires NAMs that are currently available specifically for product testing for treatments topically applied to pets to control certain invertebrate ectoparasitic pests and that reliably provide “confidence in the results of these tests for protecting public health”.

The Panel recognized that the goal of the guidelines is to provide concrete protocols that are deemed acceptable for regulatory purposes based on the scientific literature and previous studies. Therefore, many on the Panel agreed that NAMs required for the efficacy testing in the proposed guidelines do not currently exist and, therefore, are not currently recommended.

However, the Panel recognized that guidelines are anticipated to have a long shelf-life, e.g., the current set of guidelines that these will replace are 20+ years old. Due to the extended time period that guidelines are often in place before being modified, it is likely that NAMs that sufficiently replicate the animal tests proposed in these guidelines will be developed within the anticipated lifespan of this document. The Panel noted that section (e) of the proposed guidelines allows for the introduction of novel testing methods, indicating that they should be submitted to the Agency for review prior to testing. While the purpose for including this allowance was to recognize that novel products may arise that require different methods, it seems reasonable that this could also

24 pertain to the introduction of NAMs. The Panel recommended adding specific language to this section that makes it clear that the development of NAMs is encouraged and that the Agency will accept these for review.

In addition, the Panel suggested adding language that specifies how a new protocol and technique may be implemented beyond saying that it will be reviewed by the Agency. In particular, the Panel recommended adding some specifics as to how NAMS will be deemed appropriate replacements for the proposed animal tests. One suggestion is to encourage product researchers to run non-vertebrate (in vitro) tests along with the approved traditional animal testing methods (in vivo) to determine if the same outcomes are obtained. For example, if product testing on pests exposed to treated animal hair in vitro demonstrates similar results to in vivo tests in repellency assays, these may be considered as a useful surrogate to reduce vertebrate animal testing for certain product applications (Stanneck et al., 2012a).

At least one Panel member viewed the tests in the proposed guidelines much like Phase II and Phase III clinical trials that the FDA requires for human and veterinary drugs. In this regard, the panelist agreed that NAMs are not currently recommended, and concluded that, as for drugs regulated by the FDA, to determine whether products work, testing of these products must be done on the patients themselves (i.e., using cats and dogs). The panelist noted that the numbers of test animals suggested by the Agency are far fewer than those for FDA clinical trials. The panelist also approved of the Agency’s use of refinement rather than replacement for animal testing, as the Agency has conducted power analyses to help decision-making on number of animals needed and is recommending that several test species be tested simultaneously on the same vertebrate animal, where possible.

The use of computational approaches to refine the test design with respect to the number of test animals employed was also noted by other Panel members and is discussed in detail in other charge questions. In accordance with the goals to refine, reduce and replace animal testing, the use of NAMs to reduce or replace vertebrate animals in testing is a very important research endeavor and a desirable goal for the future of EPA Product Performance Test guidelines. The full Panel agreed that computational NAMs to reduce animals could be used to justify modifications of parameter settings used for the power and sample size analysis to permit fewer vertebrate animals per group (e.g., permit a lower observed efficacy or lower precision). Such use of NAMs would complement animal testing without requiring that they model the full complexity of the vertebrate animal.

Several Panel members applauded the Agency on their use of Monte Carlo simulations in the accompanying document titled, “Sample Size for Pet Product Studies”. One Panel member noted that in using such simulations, as detailed in Portier and Kaplan (1989), they were able to enhance the robustness of experiments with limited sample size. Thomas et al. (1996), with limitation of a theoretical N=1 in their experiments, applied Monte Carlo Simulations to model probability distribution functions of physiologically based pharmacokinetic (PBPK) parameters to estimate means, medians, standard deviations, first and third quartiles, skewness, and kurtosis and obtained age- and chronic dosing related pharmacokinetic differences in mice in parallel with a two-year chronic toxicology study.

25 These panel members recommended the Agency continue to apply computational NAMs that include Monte Carlo simulations for justifying a reduction in the required number of vertebrate animals to six per treatment group. They also recommended exploring Markov Chain Monte Carlo Simulations, which consider covariance and other uncertainties, and coupling simulations with Bayesian approaches when new scientific data are available. They also suggested adding text to the guideline document that details these approaches and encourages manufacturers to apply computational technologies to reduce animal usage.

A few Panelists held a contrasting view regarding the readiness of NAMs. They did not think that the testing on animals is necessary or justified to evaluate the insecticidal activity of a product in all cases. They indicated that while new approaches were not sufficiently developed, there are existing alternative methods to evaluate spray on insecticides against mosquitoes and biting flies, and probably for fleas and ticks as well. They made the point that there are a multitude of insecticide resistance studies done on fleas, ticks, mosquitoes and biting flies that all use non- vertebrate testing methods. They concluded that the methods proposed in the guidelines, although realistic, are a very expensive residual bioassay. They recommended to the Agency that an alternative is to require that the proposed insecticide has activity in a residual assay that is on par with the insecticides that are already known to be highly effective. There are abundant technologies for conducting residual insecticide bioassays on substrates such as paper, glass, plastic, etc. (Ferrero et al., 2006; Deletre et al., 2016; Borges et al., 2019; Navarro et al., 2013; Saytal et al., 2019). While these technologies may not be appropriate to verify claims of persistence of product performance on a pet, they are more than appropriate for claims of “kills.”

The point made by some of the public comments that non-animal methods of testing are widely available is correct, although many of the examples they provided are not applicable. The public comments largely involved advancements in the use of non-animal blood meals for rearing of arthropod pests, which several members of the Panel noted does not work for rearing arthropod pests for efficacy testing because they often lose their ability and/or desire to ingest a blood meal. While these methods might prove useful in testing compounds that are orally administered to pets and circulating in the blood, they would not be appropriate for topically acting insecticides or acaricides.

Notwithstanding the flaw in this rationale, one Panel member indicated that there probably are non-vertebrate tests that could replace the use of cats and dogs in some of these studies. The panelist suggested that repellency of a material can be effectively evaluated without using animals. The panelist’s recommendation is to use laboratory choice and/or no-choice assays performed and compared against known standards that make claims of efficacy quite certain. The panelist further suggested that tests with cats and dogs could be replaced with tests that employ a model animal, such as a rat or mouse, which would cut the number of vertebrate animals needed for testing in half. The panelist concluded there is no reason to test both cats and dogs and at a minimum most tests should only be conducted in either dogs or cats, especially if the arthropod pest is found on both animals. b. Fleas, ticks, mosquitoes and biting flies commonly affect dogs and cats. The Panel concluded that one should consider the role of animal behavior in testing. For example, could it change due to the test product and/or the parasite? The scratching seen after a flea infestation is more likely

26 due to a provoked inflammatory reaction than the flea itself, while ticks may go unnoticed in some cases. The Panel proposed that the test arthropods be in the environment of cats and dogs for only a short period of time. In addition, the Agency proposes use of pathogen-free, laboratory raised test species, which would eliminate the potential for disease transmission.

While NAMs may be appropriate as screening tests for repellency, for when loss of efficacy occurs, for initial kill, and possibly for rearing pests, the Panel expressed concern regarding how to incorporate pet behavior, such as grooming, that can affect the arthropod counts into the NAMs test. The Agency should provide some guidance on how NAMs protocols and the results from NAMs could be assessed so that they could eventually be used for testing products. For these reasons, the Panel’s recommendation that NAMs be assessed via direct comparison to the animal tests they are attempting to replace becomes critical.

Charge Question 3

Should protocols for testing ear mites and Sarcoptic mange mites be included in the guideline?

a. Why or why not?

b. If so, please provide appropriate detailed test methods for evaluating the efficacy against these pests for on-animal treatments.

Panel Response 3: a. The Panel recommended that protocols for testing for ear mites (Otodectes cynotis) and Sarcoptic mange mites (Sarcoptes scabiei) should be included in the proposed guidelines. These are two of the most common mite pests found on dogs and cats, and ectoparasiticides such as fipronil and selamectin are used to treat fleas, ticks and mites. Ear mites and Sarcoptic mange mites can be important pests of companion animals and mange mites can be transferred to humans. Therefore, standardization of testing procedures for mites is essential for the development of multi-use products.

No rationale is provided by the Agency for the exclusion of Demodex mites (Demodex spp.) that are also one of the most common mite pests on dogs and cats. Generalized demodicosis is a severe condition that is difficult to control with currently approved therapies, and new treatments are always being sought. Tests with all three of these mite species and the effects of various topical preparations are in the literature, and some products currently have some or all of them included on their labels (Stanneck et al., 2012b).

A Panel member also suggested that testing protocols for the soft tick, Otobius megnini (spinose ear tick), should also be considered as it can be a pest of dogs and occasionally cats in the Western U.S. Testing protocols for this tick would differ from hard ticks as soft tick life cycles differ substantially from hard ticks. O. megnini is an ear canal parasite. Only larvae and nymphs are parasites. Adults are free living and lay eggs in the environment, so only subadult ticks would

27 be tested. A similar testing approach as suggested for Otodectes cynotis (ear mites) could be employed for trials using subadult O. megnini.

b. Protocols for testing the efficacy and/or safety of topically applied ectoparasiticides against all three species of mites are in the published literature. Some examples are listed below.

Demodectic Mange:

1. Becskei et al. (2018) describe a safety and efficacy, randomized, single-blind, multi-center clinical study, in which monthly oral doses of sarolaner was evaluated in comparison with weekly topical applications of imidacloprid plus moxidectin for the treatment of generalized demodicosis in client-owned dogs. Efficacy evaluation involved the number of live Demodex mites relative to pretreatment in five deep skin scrapings from each dog, and the proportion of dogs with no live mites on days 0, 30 and 60, and, if applicable, on days 90, 120, 150 and 180, in both treatment groups.

2. Fourie et al. (2019) describe a laboratory study that compared the efficacy of two topical spot-on medications, fluralaner or a combination of imidacloprid and moxidectin, against naturally acquired generalized demodicosis in dogs. Client-owned dogs were transferred to the study site and individually housed indoors. On Day 0, dogs in one group were treated once with fluralaner spot-on solution. Dogs in the other group were treated with the imidacloprid/moxidectin spot-on solution on 3 occasions (Days 0, 28 and 56) or weekly in severe cases. Mites were counted in skin scrapings and demodectic lesions were evaluated on each dog before treatment, and at 28-day intervals over the 12-week period. Deep skin scrapings were made from the same 5 sites on each dog at each examination.

Summary: Both clinical and laboratory-based studies have been used. Both protocols use a positive control; however, Becskei et al. (2018) used geometric means, while Fourie et al. (2019) used arithmetic means. Housing conditions differed markedly, which could impact socialization issues and contact with other pets with the same infestation. Natural infestations were used in both studies.

Sarcoptic Mange [Sarcoptes scabiei]:

1. Stanneck et al. (2012a) describe a laboratory study with naturally infested dogs. Ten (10) mixed breeds were used in a within subject design. On Days −2, 29, 60 and 90, skin scrapings (+/− 4 cm²) were taken from five places on the dog’s body likely to be infested with mites, and the number of mites in these scrapings were counted. The clinical signs and extent of lesions on each dog were assessed on the days on which scrapings were made. Success rate was defined as a dog that complied with all of the following conditions: no live mites, a complete resolution of the presence of papules and skin crusts and a > 90% improvement in body areas with hair loss by Day 90 after the collars had been fitted.

2. Fourie et al. (2019) describes a clinical trial in which 16 client-owned dogs with naturally acquired generalized demodicosis were randomly allocated to 1 of 2 study groups: on Day 0, dogs in 1 group were treated once with fluralaner spot-on solution. Dogs in the other group were

28 treated with the imidacloprid/moxidectin spot-on solution on 3 occasions (Days 0, 28 and 56) or weekly in severe cases. Mites were counted in skin scrapings and demodectic lesions were evaluated on each dog before treatment, and at 28-day intervals over the 12-week period. Deep skin scrapings were made from the same 5 sites on each dog at each examination.

Summary: Both a laboratory-based study and a clinical trial have been used; efficacy evaluations have been made using a between group (positive control) and within group design. Skin scrapings are used in both studies. Natural infestations were used in both studies.

Ear Mite (Otodectes cynotis)

1. In a laboratory study, Six et al. (2016) used 32 dogs with a previously induced Otodectes cynotis infestation (100 mites in each ear), in a randomized, complete block design to evaluate product efficacy. Total ear mite counts, range and percent efficacy relative to placebo control (not defined) were compared to dogs treated with either a single oral dose or two monthly doses of sarolaner. Counts of mites were made otoscopically on Day -4 and Day 30 and 60.

2. In a second laboratory study, Taenzler et al. (2018) employed an experimental infestation of O. cynotis in dogs, by harvesting mites by lavage from donor animals and transferring approximately 50 to 100 mites, depending on the intensity of infestation in donor animals, into each ear of the recipient animal. Animals used in the study were experimentally infested within one month prior to study start. To determine efficacy, an otoscopic examination of both ears from each animal was performed prior to treatment and at 14 and 28 days after treatment to determine the number of live mites. However, 28 days after treatment, animals were sedated, and both ears were flushed to determine the number of live mites (adults, larvae, nymphs).

Summary: These 2 laboratory-based studies described in this section evaluated the efficacy of chewable systemically acting drugs rather than topically applied acaricidal products. Both natural and experimentally induced (Six et al. 2016; Taenzler et al. 2018) infestations were used to determine product efficacy. Number of mites used for initial infestation varied from 50-100 between the studies. Primary assessment in both was the total number of live mite counts after treatment.

The methods used to determine the number of mites vary: Six et al. (2016) employed otoscopic counts at all time points. However, Taenzler et al. (2018) at the final evaluation (Day 28) flushed both ears of sedated dogs to determine the number of live mites, i.e., adults, larvae, nymphs.

Different Techniques to Count Ear Mites:

Bosco et al. (2019) states: “Assessment of O. cynotis infestation. Prior to enrollment, O. cynotis infestation was confirmed by direct or otoscopic examination of the external ear canal of both ears. On Days 28, 56, and 84 post-treatment, cats were sedated with dexmedetomidine hydrochloride (40 μg/kg IM), the ear ducts were filled with a solution of 0.9% sodium chloride (3 to 5 ml per ear canal) and the ears were massaged externally to displace the contents.”

Machadoa et al. (2018) states: “The diagnosis of otoacariasis was confirmed by bilateral videootoscopy, and by evaluating the presence of mites.”

29 Additional Methodological Issues:

Several other issues were raised by the Panel:

a. One involved the necessity of using 5 deep-skin scrapings in dogs with either type of mange to evaluate the number of mites. Becskei et al. (2016) describe the procedure:

“To count mites, deep skin scrapings were taken from at least four separate sites on each dog. If no mites were detected in the first four scrapings, additional scrapings were made until live mites were found or the maximum of ten scrapings was reached. Selected scraping sites were those that had the most severe or most likely evidence of current mite infestation. Scrapings were conducted to an approximately constant depth (to capillary bleeding) over an area of approximately 2.5 cm2.”

A Panel member suggested that evaluation of mite mortality could be done by dipping (Croft et al., 1982), drenching (Pap et al., 1997) or residual exposure (Pasay et al., 2008). b. Given that these mites tend to be localized and that long-term residual activity is unlikely to be needed, non-vertebrate testing would be a logical alternative for scabies.

Panel members indicated that it is possible to conduct scabies tests without vertebrate subjects using methods from the literature (Pasay et al., 2008; Pap et al., 1997) and recommended EPA state that non-vertebrate studies would be considered as an acceptable alternative.

The Panel also recommended that EPA consider that efficacy studies involving client-owned pets be conducted at clinics with volunteer patients. Cited above are several papers that outline procedures for testing product efficacy in three mite species, and the Panel recommended that EPA provide some latitude in the designs to be submitted.

There is also a paper that discusses the transfer of ear mites to other animals in the laboratory and testing them (Taenzler et al., 2018). While it is possible, the Panel noted the likelihood is that mites will have to be colonized from a wild source. Maybe this will stimulate some interest in researchers maintaining mite colonies.

Overall Summary

The Panel recommended that in addition to ear mites (Otodectes cynotis) and Sarcoptic mange mites (Sarcoptes scabiei) that Demodex mites (Demodex spp.) should be included in the proposed guidelines (Panahi et al., 2015).

Examples of possible test methods for evaluating efficacy against each of these 3 species for on- animal treatments are summarized from the literature. Test methods include both clinical trials and laboratory studies primarily in pets with natural infestation, although an experimental infestation study is cited for ear mites. In addition, several methodological issues that the Panel felt were germane to the evaluation of dependent variables and infestations are presented.

30 With respect to methodology, the Panel recommended that EPA consider that efficacy studies involving client-owned pets be conducted at clinics with volunteer patients, and that EPA state that non-vertebrate studies would be considered as an acceptable alternative for scabies tests.

Charge Question 4

For each section listed below, comment on the proposed sample sizes for vertebrate test animals (e.g., dogs, cats). EPA recommends these numbers based on the power vs. sample size analysis provided to ensure adequate power using the minimum number of vertebrate animals. Are these numbers of animals practical? a. Fleas (section (j)) b. Ticks (section (k)) c. Mosquitoes and biting flies (section (l))

Panel Response 4:

The 1998 OPPTS 810.3300 efficacy testing guidelines required at least 6 animals per group, while preferring 10 animals per group. The “Sample Size for Pet Product Studies” document states that historically the EPA has recommended 6 animals per group, but often received studies with 2-12 animals per study group (see pages 2-3 of the document “Sample Size for Pet Product Studies”). The Panel recommended that the EPA provide more historical data describing the number of animals per study group that was received and describe how often each study design (by group size) was deemed to be acceptable; such information could help to inform these new guidelines.

Aside from considering the historical data in more detail, the Panel agreed that the recommended group sizes in the proposed guidelines are in line with the EPA documentation of power vs. sample size that suggests statistical support for 6-14 vertebrate animals per group. The Panel also found that the sample sizes suggested by the EPA are generally consistent with the stated EPA historical practice requiring 6 animals per group (preferring 10 animals per group) and accepting studies with up to 12 animals per group.

The Panel viewed the number of vertebrate animals for the fleas and mosquitoes and biting flies as practical. The Panel had some concern about the number of animals recommended, particularly for the tick studies.

The Panel discussed a variety of logistical issues in the context of tick lab studies by having 14 treated and 14 control dogs. In order to have 28 dogs that are eligible to be enrolled in the study with adequate infestation levels (25% of infested tick species), there will likely be a need to start with 32 to 34 dogs. This would result in increased study costs in terms of the number of animals and per diems and goes against the 3R’s (i.e., Replacement, Reduction, and Refinement). It would also result in more ticks needed at the study site for each infestation. Other issues noted by the Panel include: more dogs to comb that often results in technician fatigue; more data to record for each dog if multiple tick classifications need to be recorded (dead attached, dead free, live attached, live free; moribund, etc); an inability of contract research labs to schedule multiple

31 studies on the same day/week; possibly reduced revenue for contract labs which are small businesses; contract labs needing to hire more technicians; and the inability to accommodate all study dogs into a single animal room (which would introduce another source of variability if more than one room is required).

The Panel noted that there are several design modifications that could potentially lessen these concerns. Given that these data are ’binomial’, the variance depends on the true rate. As such, the group with the more extreme (farther away from 1/2) rate should use fewer animals. The animals could still be blocked (matched), but instead of using matched pairs, there would be matched sets (e.g., 2 controls + 1 treated or vice versa). Such matching is common in human clinical trials and could simply be implemented here (it is not more complex than the current blocking approach). In addition, the modeling does not seem to include the blocking/matching effect. Inclusion of such terms could help further increase the power by reducing the relevant variance for testing. Another possible design would be a cross-over design (see e.g., Section 5.4 of Chow and Liu, 2008) which, if it could be implemented, would decrease the sample sizes by 50% (but would require each animal to participate in both the control and treatment arms).

In addition, the Panel noted that the parameters of the power vs. sample size analysis have a large influence on the sample size calculations. For instance, the number of animals (dogs or cats) could be reduced by requiring a lower observed efficacy, a greater true efficacy accompanying a 90% observed efficacy, a greater proportion of pests retained, or less precision.

The Panel recommended that power simulations be explored to determine whether design modifications, like those proposed above, could be used to decrease the number of vertebrate animals in tick studies (and potentially in flea, mosquito and biting fly studies). The Panel also recommended that the impact of the values of each of the parameters and criteria, on the power and sample size in the simulations should be assessed carefully.

Charge Question 5

For each section listed below, comment on the proposed numbers of different arthropod pest species. EPA recommends these numbers based on review of studies available in the literature studies submitted to EPA, and analysis of power vs. sample size. Are there ways to reduce the number of pests used per test? a. Fleas (section (j)) b. Ticks (section (k)) c. Mosquitoes and biting flies (section (l))

Panel Response 5:

Charge Question #5 focuses on means for reducing the number of pests used per animal per test. To facilitate the Panel’s discussion, the Table below summarizes the required number of pests per animal, extracted from pages 10, 12, and 17 in the proposed guideline.

32 Required Blood-fed Proposed Testing Number of Pests per Pest /Retention Proportions Guideline Section Animal (“base values”)

j Fleas 0.4-0.6 100

k Ticks 0.25 50

l Mosquitoes/Flies 0.6 100

Based on Tables A3.1 and A3.2 in Appendix III of the EPA supplemental document titled “Sample Size for Pet Product Studies,” the required blood-fed/retention proportions (referred to as “base values” hereafter) play an important role in generating the number of pest species per vertebrate animal presented in the proposed guideline (last column of the table above); these base values are likely determined by the biology of pest-host interactions. For example, pests that are "aggressive" feeders on the vertebrate host tested may have a much higher host retention proportion than pest species with lower host feeding success. Accordingly, one way of reducing the number of pests per animal for testing is to “modify” these base values based on the “observed" (i.e., historical) information on pest-host interactions. Despite the importance of these base values in terms of sample size and power determinations, the proposed guidelines provided minimal information on their sources and biological rationales. Hence, the Panel recommended that the Agency should provide support of these base values or reexamine these base values for potential modifications; the potential sources of historical information of host-pest interaction are open literature, e.g., Taenzler et al. (2016); studies submitted by the pesticide registrants (since the inception of the 1998 testing guideline); and professional judgment from experts of these ectoparasites.

In particular, the Panel recommended that the EPA provide a clear justification for the arthropod “% retained” for inclusion of a vertebrate animal in the study. Using a higher “% retained” would decrease the number of vertebrate animals needed.

The Panel further recommended that the EPA should carefully consider whether there are interactions between arthropod species when making sample size recommendations for scenarios in which multiple species are permitted to be tested on the same animal. Input should be sought from subject matter experts (biologists) on these possible interactions. For mosquitoes, two experts (personal communication with Dr. Dan Kline, USDA, and Dr. Bill Reisen, UC-Davis) anticipate no problems in testing multiple species simultaneously (see response to Charge Question #18).

The statistical models in the power vs. sample size calculations did not consider interactions between arthropod species (the species were treated in the models as not interacting with each other). Significant interactions among different pest species could result in reduced retention of each pest species (from that assumed in the power analysis) and potentially could also impact the effectiveness of the treatment (e.g., effective when single species but not for the same species when with other species [i.e., the multi-species scenario]). Thus, the Panel observed that interactions could have important implications on power (e.g., reduced power with reduced

33 retention) and could bias the estimated efficacy for a single species when assessed from a multi- species study.

In addition, for the larger numbers of pests per vertebrate animal, the Panel expressed concern about the logistics of counting (and subsequent errors).

Below, the Panel provided Specific guidance on each arthropod.

The use of 50 ticks per test was viewed as reasonable since decreasing the number of ticks per test would result in requiring more than the currently recommended 14 vertebrates per group (which was a concern – see response to Charge #4).

For mosquitoes, the rationale for using 50 per species when testing three species simultaneously, and 100 for individual species tests, is unclear. Unless there is a justification for the larger number for individual species tests, the Panel recommended 50 for the individual species tests, as well.

For biting flies, there was considerable concern about using 50 flies per test (in terms of animal safety and welfare). Twenty-five per test was viewed as a more humane number of pests.

The Panel had considerable discussion about fleas regarding the number of live fleas recovered in controls and flea retention. The literature is clear that visual thumb counting is vastly inferior to combing techniques. However, Dryden et al. (1994) provides a possible method by which the actual number of fleas could be estimated from thumb counts. This would be relevant to the earliest time studies in the guidelines. Dryden et al. (1994) also provides a study comparing counting procedures. Visual counting (six regions of a dog) was compared with a 10-minute count using a flea comb. After the counts the animals were sprayed with an alcohol based pyrethrin spray and combed for 10 minutes and the number of fleas counted. These animals were retained for 24 hours to determine if any additional fleas were counted. Area visual counting accounted for 21.5-28.0% of the fleas placed on the dog and combing counts provided 76.0-85.6 % of the fleas placed on dogs. After applying the pyrethrin sprays, 82.5-86.4% of the fleas were accounted for in the study at 24 hours. In a cross over study, Heckenberg et al. (1994) found that thumb counting found means of 8.8% and 7.7% on dogs infested with 50 and 100 fleas, respectively. Comb- counting provided 67.6% and 75.4 % of the fleas placed on the dogs. In another study, combing for 5, 10, and 15 minutes provided at least 81.5, 90.5, and 85.1% recovery of fleas placed on beagles 1 hour earlier (Zackson et al., 1995).

For ticks, the Panel recommended 50 ticks per test.

For mosquitoes, the Panel recommended 50 for the individual species tests.

For biting flies, the Panel recommended 25 biting flies per test; however, the impact on power and sample size would need to be assessed.

For fleas, the Panel recommended:

1) Only short-haired animals like beagles should be used in the tests. Issues regarding long- haired vertebrate animals and environmental issues like sunlight or rain could be addressed in field studies.

34 2) Comb counting should be used based on the technique from (Dryden et al. 2016).

3) If beagles are used in the studies, retention estimates needed for the models might be obtained from the study by Dryden et al. (1994).

4) The methodology used by Dryden et al. (1994) should be repeated with short haired cats. Instead of the alcohol-based pyrethrin spray, nitenpyram could be used on the cats to remove all fleas. The use of an Elizabethan collar should also be considered as it can prevent cat grooming behaviors so its value for increasing flea retention can be determined.

5) The Charge Question regarding the issue of counting fleas for “the earliest time (e.g., 8 hours, 12 hours, etc.) needs to be revisited when EPA revises the guidelines. All on-animal counts prior to the final count may be conducted using hand counts. Values can be estimated for live fleas on beagles using the Dryden approach (Dryden et al., 1994).

6) A similar study based on the approach of Dryden et al. (1994) with short-haired cats should also be conducted.

The Panel also provided the following, general recommendations:

The Agency should provide further documentation of the validity of the parameter values in the simulations based on historical information regarding host-pest interaction from the literature or studies submitted to the EPA in the past.

Simulations should be conducted using lower numbers of pests, particularly for biting flies and fleas, and improved estimates of retention rates, to assess the implication on power and sample size (i.e., the number of animals needed).

For multiple species studies, when making an assumption of no interaction, the lack of interaction should be carefully documented from the literature. Otherwise, the power vs. sample size simulations and their validity would be suspect in the presence of interactions.

Charge Question 6

Comment on the timing of exposing vertebrate test animals to fleas and ticks. a. Are the number of exposures for products with different durations of efficacy adequate for determining efficacy? Why or why not? b. If the number of exposures to pests can be decreased, please indicate specifically which exposures can be skipped. If efficacy should be evaluated at more time points, indicate when exposures should occur and for what product types (e.g., spot-ons, collars, and residual shampoos), and discuss the value provided by the additional time points.

35 Panel Response 6:

The Panel recommended that the number of exposures should adequately reflect the actual claims of the products. For example, if a product claims efficacy for 60 days, there should be data for at least 60 days. The exact number of exposures necessary will depend on the types of products, the claims, and how the Agency will evaluate the submitted data. If it is necessary to provide ≥ 90% reduction of fleas and ticks at the “longest labeled duration of efficacy” claim, then there is little need for the day 1 exposures. If the claim is “provides control (assume >90%) throughout a period of 60 days,” then some periodic exposures are appropriate. In addition, for products with claims of effectiveness over several months, acquired immunity to tick infestations may occur, leading to host-induced tick mortality (European Medicines Agency, 2016; Wada et al., 2010). Therefore, infestation frequencies should be reduced to the minimum necessary to test the duration of product efficacy.

However, it is unlikely that removing some exposure times would actually change the methodology or the numbers of vertebrate animals being treated because the investigators will still design the study to generate data necessary to show efficacy and residual performance. Exposure tests will be repeated until the treatment fails to reach the necessary control level. The main factor dictating the numbers of vertebrate animals to be tested are the findings from assessment provided in the EPA document entitled, “Sample Size for Pet Product Studies.”

a. For products with efficacy claims of > 7 days, there is no need to expose treated animals at 1 or 2 days to fleas and ticks, respectively. Again, the testing protocol should reflect the specific claims. For research purposes, testing for efficacy occurs at multiple time points up to and past the maximum claim. Laboratory bioassays without animals could be conducted to test for breakdown of the product. For tick mortality trials, the Panel suggested changing the infestation timing in Table 2 to be Day -2 for ticks (not Day -1), to give adequate time for attachment, as outlined in European Medicines Agency (2016). b. The Panel recommended that, depending on the specific product efficacy claims, at a minimum 14- and 21-day exposures could be removed for products with >30-day claims. The 1 or 2-day exposures could also be removed from >7-day claims depending on the EPA’s goals for having 1-day data. The >4-week infestation timing is excessive; weekly repeated infestations of animals with fleas or ticks for the >4-week endpoints are unnecessary. Re-infestation of hosts every 2-4 weeks for longer duration product trials is common practice (Stanneck et al., 2012a, Wengenmayer et al., 2014; Jones et al., 2015; European Medicines Agency, 2016). For the >4- week group, infestations on Days -2, 28 and then monthly should provide the same level of information as weekly infestations. Timing of application for tick repellency testing should be similar to mortality trials at the >4-week endpoint.

Charge Question 7

In the interest of reducing the use of vertebrate animals in testing, we have suggested in the guideline to allow testing of up to two species simultaneously for fleas and ticks (e.g., fleas and 1 tick species, 2 tick species). Are there any potential known interactions between multiple tick

36 species or ticks and fleas if on a single vertebrate animal at the same time which would require individual pest species be tested separately? If there are interactions, please describe the interactions and provide references.

Panel Response 7:

The Panel observed that testing multiple species on the same host could decrease the number of vertebrate animals to be used. While flea and tick co-infestations are commonly encountered on companion animals, this would make testing 1 flea + 1 tick species simultaneously acceptable and potentially even desirable. It is, however, unknown if increased grooming by cats in response to the presence of fleas might influence normal tick attachment. While retention is not expected to be altered by co-infestation with two different tick types, some species, such as Rhipicephalus sanguineus and Ixodes scapularis, may be very similar in body size and markings after being attached to a dog for 48-72 hours. This may make the efforts to separately identify and count these two tick species too slow and cumbersome. Dermacentor variabilis and Ambylomma maculatum (Gulf Coast tick) also share similar coloration. Additionally, A. maculatum is an aggressive species that causes painful, inflamed attachment sites. Thus, from an animal welfare standpoint, adding a second co-infested tick species with A. maculatum exposure would probably be excessively stressful to the test animal.

Regardless of the previous comments, when combining 1 flea + 1 tick or 2 tick species, would it be practically possible to adequately “tease out” (i.e., count) the effects of each of the two species from the same animal? And is it known if one species could potentially affect the survival rate of the other, thus influencing the experimental outcomes? If one of the two species used during a trial doesn’t meet the test criteria, the guidelines should address what should be done with these data for the other insect species.

When using more than one pest, the Panel recommended that the numbers of each species should be modified. Depending on the frequency of feedings and arthropod numbers used, the amount of blood consumed by a combination of ticks and fleas could be substantial, especially on control animals. Fewer ticks and fleas would need to be used on animals where multiple species are combined. There is also a potentially increased risk of inflammation at feeding sites that should be monitored. While 50 ticks total would likely be well-tolerated, doubling this to 100 ticks total to test 2 species may be excessive and infested animals should be monitored closely for any adverse reactions. In contrast, placement of 150 adult female mosquitoes (3 species x 50 mosquitoes each) will not likely lead to a risk of either undesirable mosquito-mosquito interactions or undue stress on the test animal.

Finally, the proposed guidelines specify using pathogen-free ticks; however, the Panel noted that a more specific definition of this is needed. There is some concern that combining 2 tick species could facilitate dermal or sub-dermal pathogen exchange that might adversely affect test results.

The Panel recommended allowing simultaneous testing of 1 flea + 1 tick species only, except when the tick is Amblyomma maculatum. The Panel has also provided the EPA with further information about tick species that have been used to co-infest animals without interactions that might hinder accurate data collection (see response to Charge Question # 15).

37

Charge Question 8

For pet collars, one of the typical methods of application requires cutting the collar to size and therefore the collar application rates are often dependent on neck size and not animal’s weight as for other pet products like spot-ons that have dosing labeled based on weight ranges. Because neck size may not correlate with body weight or surface area, there is uncertainty that testing with the formulated collar sized for the neck may not represent the most conservative situations encountered in the field (e.g., dog breeds with small necks but relatively big bodies).

• Please discuss how application rate should be addressed during testing of collars. Is cutting the formulated collar to neck size sufficient for testing, even though the typical breeds used in testing may not represent the most conservative ratio of neck size to body weight/surface area? Why or why not? If not, how should this issue be practically addressed during testing?

Panel Response 8:

The Panel found it unclear that the neck size to body weight/surface area or cutting off excess pieces of the collar after it is placed on the pet is having a negative impact on efficacy of the pests claimed on the label for each respective collar. There is limited peer reviewed scientific literature that discusses the efficacy of collars in controlling fleas or ticks.

The availability of collars meant for small versus large dogs (defined by weight of dog) would appear to be an effort to address this issue. Additionally, the active ingredients (a.i.) listed by % weight of a.i. on commercial collars is the same in collars for small and large dogs.

The registrant for the collar likely conducted initial efficacy testing to show that the collar delivers residual efficacy (flea and/or tick) for the desired length of time listed on the product label with any excess collar trimmed off prior to laboratory testing in both small and large dogs. Therefore, the excess collar that is cut off in pivotal studies mimics early testing and the desired length of residual flea/tick efficacy is confirmed, as well as, in clinical field studies conducted globally using dogs of different sizes and breeds (see Stanneck et al., 2012b).

Excerpt from Stanneck et al., 2012b: Seresto® (Bayer Animal Health), a new collar for dogs and cats, provides long term broad spectrum parasiticidal activity by combining the insecticidal properties of imidacloprid with the acaricidal properties of flumethrin. The collar matrix system ensures that both active ingredients are slowly and continuously released from the collar towards the animal thereby avoiding peak concentrations and ensuring that acaricidal/insecticidal concentrations of both active ingredients are present in the cat’s or dog’s hair coat during the entire efficacy period. The active ingredients spread from the site of direct contact over the entire skin surface of the treated animal.

The Panel recommended that the EPA request that the registrant defend why they are instruct users to cut off excess collar material not needed to make contact with the neck region of the treated pet regardless of neck and body size (surface area). Additionally, the registrant should

38 clearly indicate to the EPA reviewers how the a.i. is being released from the collar matrix, including distribution and/or release kinetics of the a.i (or multiple active ingredients).

There are two primary reasons for cutting off the excess formulated collar to the neck size. The first is that only the collar matrix that touches the skin/hair coat is going to release a.i. so any excess collar not touching the animal will not release and provide a.i to the animal so it’s not needed. Removing the excess reduces the potential for exposing the pet, the pet owner, and the pet’s surroundings to unnecessary amounts of a.i. The second reason is for vertebrate animal safety. Animal safety studies for the end-use collar formulation are tested with the excess collar cut off so that the animal is not exposed to excess amounts of the a.i. and to minimize potential accidental entanglement. Since some collars will break and detach as a safety feature, this may also lead to a potential health concern if the vertebrate animal ingests it.

Charge Question 9

Historically the Agency has requested studies for fleas, ticks, and mosquitoes on pets be conducted with 6 to 8 animals per treatment, although, the Agency has received studies with sample sizes ranging from 2 to 12 animals per treatment. Based on the statistical simulations provided in Appendix 1, EPA is proposing a minimum number of vertebrate animal subjects for each test type (i.e., pest) in the guideline. This number varies according to test type, ranging from 6 to 14, and is intended to achieve 80% statistical power (confidence) with a precision of 4 or 5%, depending on the specific methods employed for each pest. Please discuss and provide comment on:

a. The appropriateness of statistical methods to analyze the data and calculate percent efficacy and the associated 95% confidence interval. b. The appropriateness of statistical methods and simulations EPA has developed to estimate the power of the proposed design, and specifically to achieve an adequate estimate of precision around the estimated mortality/repellency in the treated group for each section below: i. Fleas (section (j)) ii. Ticks (section (k)) iii. Mosquitoes and biting flies (section (l)) c. The assumptions used to inform the simulations (e.g., minimum blood-feeding rate of mosquitoes on control test animals, number of pests per animal) for each section. Specifically, please discuss any information that can be used to update or clarify these assumptions, especially where doing so would increase power with a smaller sample size (i.e., using fewer animals). Where possible, indicate which assumptions can be changed for each pest species or type of test animal and provide value estimates. i. Fleas (section (j)) ii. Ticks (section (k)) iii. Mosquitoes and biting flies (section (l))

Panel Response 9:

Charge Question 9 focuses on the utilities of the proposed generalized linear mixed model (GLMM) developed by the Agency for (1) establishing a point estimate for the efficacy of pesticide product and its associated 95% confidence interval, and (2) designing studies in terms of

39 the number of arthropod ectoparasites per animal and the number of animals per test for assessing the efficacy of a pesticide product by power analysis based on pre-established performance criteria and assumptions. Overall the Panel considered the proposed GLMM method to be consistent with commonly accepted statistical principles for point estimation (i.e., of efficacy); however, two Panel members showed a preference for conventional data analytical methods (e.g., geometric and arithmetic means) (Pfister and Armstrong, 2016) for characterizing the product efficacy, and the public comment by Dr. Kenneth Portier (see docket # EPA-OPP-2019-0161- 0034) also suggested that the implementation and interpretation of the results of the proposed GLMM method may pose challenges to practitioners without advanced statistical training. Accordingly, given the importance of using valid methods (and their complexity), the Panel recommended that the Agency create generic or example “software,” a step-by-step guidance document, and fully worked-out examples for implementing the GLMM method. Also, the Panel agreed that power analysis using GLMM via simulation allows the determination of sample size for the efficacy testing of pesticide products; however, additional efforts remain to clarify the methodology and assumptions employed (please see below). One Panel member suggested that, as an addition to the power analysis, the Agency could address the sample size issue in terms of number of animals per test via in silico approaches such as conducting “experiments” on computers using Monte Carlo simulation or Markov Chain Monte Carlo simulation techniques integrated with biologically-based modeling (Portier and Kaplan, 1989; Thomas et al., 1996; Bernillon and Bois, 2000; EPA, 2010). Considering that the original publication of the 810.3300 Guidelines was in 1998, this Panel member considered that the wealth of information possessed by the Agency could be used to strengthen the probability distributions of the parameters needed for conducting the suggested computer simulations, and therefore, may substantially reduce the number of animals needed.

Proposed GLMM Method for Efficacy Determination

The Panel recommended expanding the justification of why day was included as a random effect in the GLMM scenarios. Also, the day random effects were assumed to be equally correlated in the proposed GLMM framework. The Panel recommended using a temporal correlation structure to model the correlation between different days, such as a decaying correlation as the difference between days increases (e.g., an autoregressive structure). The use of GLMM with a random animal intercept is essential to account for the non-independence of Bernoulli trials (i.e., the binary result on each arthropod) on an animal, and standard GLMs that do not account for correlations between counts within each animal should be avoided. Also, the random animal effects account for different animal characteristics (animals have different length, weight, grooming patterns, etc.). Other Panel suggestions for improving the proposed method included computing a population rate (as opposed to a rate conditional on the animal random effect) after fitting the GLMMs (in particular binominal GLMM with a logit link), the adoption of a Bayesian approach with priors to address issues such as uncertainty in “binomial” sample sizes and measurement error in counting (likely more of an issue of undercounting), and the application of data submitted by the registrants to validate the proposed GLMM.

In terms of efficacy estimation from the GLMM, the Panel realized that the approach to deriving a point estimate for efficacy and effectiveness is based on a metric/statistic that has been used for many years where the difference between treatment rate and control rate is normalized by the control rate. However, the uncertainty in these estimates was computed using an asymptotic

40 (large sample) standard error (based on the delta method) which may be problematic for small sample sizes. Accordingly, the Panel expressed concerns about the accuracy of this uncertainty calculation and the corresponding coverage of 95% interval estimates. Hence, the Panel recommended that the Agency consider alternative approaches to obtain improved characterization of the uncertainty and more accurate confidence intervals including bootstrapping or using an ’exact’ fully Bayesian approach.

One of the major concerns of the Panel regarding the proposed GLMM was the widespread lack of model convergence. The lack of convergence represents a major limitation for using the GLMM reliably for power versus sample size calculations. Further, an analyst outside the Agency may also have difficulty with GLMM model convergence when performing their own analysis of the data. To partially compensate for the problem of model convergence, the more proper binomial model (conditional on random effects) was replaced by a Poisson model (also conditional on random effects) in the simulations and a log pseudo-likelihood function was maximized (due to the true marginal likelihood not being able to be computed in closed form). However, the Panel noted that while the lack of convergence was not solved using this approach as the GLMM models also failed to converge in many cases, the convergence issue could be addressed by using more accurate estimation methods including the Laplace approximation and quadrature (both available in SAS) and “exact” maximization of true marginal likelihood using Monte Carlo methods (available in R). Furthermore, the choice between binomial and Poisson modeling approaches should be based on the appropriateness of the model framework for the type of data, and not on minimizing the lack of model convergence. In this case, the binomial modeling approach is more appropriate than the Poisson modeling approach. Accordingly, the Panel recommended the use of binomial modeling approach with a logit link function instead of the Poisson approach. Other Panel suggestions for resolving the convergence issue included analyzing the data with only the random animal effect and fitting the model separately by each of the “true efficacy of treatment” levels (0.95, 0.925, 0.90, and 0.85) instead of including day as an additional random effect in the model (fewer random effects should lead to fewer convergence problems).

To facilitate the implementation of the aforementioned suggestions, the Panel recommended consulting the reference by Diggle et al. (2002) and the following R packages (function performed in parentheses): MCMCglmm (fit Bayesian GLMM), glmm (fit GLMM using MC likelihood), bootstrap (bootstrapping), vglm (fit betabinominal), and Rjags (fit Bayesian GLMM with random effects distributions besides normal and complexities including measurement error etc.).

Power Analysis Using GLMMs

The Agency performed power versus sample size analysis based on selected parameter settings and criteria for sample size determination, including (a) a true efficacy of treatment equal to 92.5%, (b) an observed efficacy of 90.0%, (c) a precision of 4%-5%, (d) 80% power, and (e) the blood-fed proportion of arthropod ectoparasites on each animal. The Panel noted that the Agency’s analyses could be strengthened by including a detailed discussion of the rationale for the chosen settings. For example, animal group sizes could be reduced by requiring a greater true efficacy, less observed efficacy, or less precision. Also, increasing the number of arthropod ectoparasites per host animal will allow smaller sample sizes to be used. Guidance from subject

41 matter experts on the proportion of arthropod ectoparasites retained would be very valuable. One particularly challenging issue surrounds the declaration of “true efficacy” in the power calculations. In the simulations performed by the Agency, the true efficacy was set to 92.5%. The rationale for this true efficacy seems to be that setting the true efficacy to 90% with 5% precision and 80% power would result in required samples that are too large to be practical. Since setting a true efficacy equal to 92.5% in the power versus sample size calculation moves the Agency away from the previous standard of 90.0% efficacy, the Panel recommended discussing this constraint in detail. Also, in standard (more common) power calculations, the statistical power might refer to an 80% chance of rejecting the null hypothesis of no treatment effect in a study when there is a true non-null effect. The Agency employed a different approach of considering accuracy in the estimation of efficacy with a pre-specified level of precision (5%); the Panel observed that this approach is a lesser known, but still an acceptable form of a power calculation.

The Panel also realized that based on different parameter values in simulations (e.g., parameters change for different study designs in Appendix I of the “Sample Size for Pet Product Studies” document), the results of power analyses are highly variable with the power often larger than needed (which then results in a larger sample size than needed). The Panel recommended consulting subject matter experts to find other biologically plausible parameter settings that could lead to smaller sample sizes. For example, increasing the number of arthropod ectoparasites per animal would increase the study’s power and potentially lead to smaller required sample sizes. However, as discussed in response to Charge Question # 5, it appears to be neither practical nor humane to further increase the number of arthropod ectoparasites per animal beyond the level already specified in the Agency’s proposed guidelines (please refer to Charge Question #5 for exploring the options in reducing the number of animals used). Also, the Panel noted that alternative designs and (or) models could be explored to potentially decrease the number of animals needed or to address the problem of lack of GLMM model convergence. For example, the Rao-Scott approach (Rao and Scott, 1992) can be used to model correlated binary data exhibiting over dispersion and is computationally simpler than GLMMs; computer code for implementing the Rao-Scott approach are available in SAS and R (e.g., the raoscott() function in the aod package).

As mentioned previously, the GLMM proposed by the Agency has both animal and day as random effects. GLMM modeling often assumes that the random effects are normally distributed, and SAS only allows random effects to be normally distributed. However, in the “Sample Size for Pet Product Studies” document, the power versus sample size calculations evaluated cases in which the random effects were simulated using normal distributions or Weibull distributions. The Weibull distribution is flexible and can assume many different shapes depending on the shape and scale parameters. However, with small sample sizes, or small changes to the Weibull parameters, the shape of Weibull distribution can change substantially. This difference in shape could potentially alter the sample size calculations. Tables A1.4-2 and A1.4-3 show that the GLMM based results can differ considerably when assuming the random effects follow a normal distribution or Weibull distribution. Accordingly, the Panel recommended using only the normal random effect assumption for any final sample size determination based on SAS PROC GLIMMIX and to use other software (e.g., Rjags) for non-normally distributed random effects.

42 Appropriateness of the Assumptions Employed

Based on Tables A3.1 and A3.2 in Appendix III of the “Sample Size for Pet Product Studies” document, the blood-fed/retention proportions of 0.25-0.6 and the numbers of arthropod ectoparasites per animal of 50-100 were used by the Agency for performing the simulation exercises of sample size and power determinations. However, the Agency provided no information on their sources or biological rationales. Nevertheless, the blood-fed/retention proportions appear to be consistent with the literature values of >25% attachment rate needed for establishing an adequate tick infestation and >50% attachment rate needed for establishing an adequate flea infestation (Taenzler et al., 2016). Similarly, the numbers of arthropod ectoparasites per animal employed in the simulations are consistent with the efficacy studies involving ticks (50) (Taenzler et al., 2016), fleas (100) (Beugnet et al., 2011), and mosquitoes (100) (Meyer et al., 2003). Given the importance of the blood-fed/retention proportions and the numbers of arthropod ectoparasites per animal, the Panel recommended that the Agency verify these values based on information obtained from the open scientific literature, registrant submitted studies (since the inception of 1998 testing guideline), and experts of these ectoparasites. As stated above, if an ectoparasite retention proportion parameter setting that can be used is higher than the setting assumed in the current models, smaller sample sizes may be possible. Also, the “actual” retention proportion can help in a case-by-case basis for “flexibly” deciding the number of animals needed for the product efficacy testing. For example, the tick Rhipicephalus sanguineus has a high expected retention proportion on dogs, in comparison to Ixodes scapularis, with a typically low retention proportion on cats, suggesting that the number of dogs used for the product efficacy testing with R. sanguineus can be reduced. The data for ectoparasite retention proportions on individual animals may be limited in the published literature. However, the testing laboratories may already have historical ectoparasite retention data collected and available to the Agency upon request. Average retention rates can also be informative for use in reducing the number of animals used, and some sources do cite individual animal data that may be useful for the Agency’s consideration (Dryden et al., 2008; Halos et al., 2014; Dumont et al., 2015a, 2015b; Baker et al., 2016; Six et al., 2016; Vatta et al., 2019).

Editorial Comments

1. The use of “true efficacy” in the EPA document titled, “Sample Size for Pet Product Studies” is difficult to follow. For example, on page 7 (line 5 under the heading) the true efficacy is for sample size calculations was listed as “…true efficacy ≥ 90% with a precision of ≥ 5%...” But it appears that the sample sizes are based on true efficacies of ≥ 92.5% with a required efficacy of ≥ 90%, and a precision of 4 or 5%.

2. Is there a discrepancy between true blood fed proportion in control group of 0.45 in the table on page 7 and the listing of 0.40 under the “input parameters for the simulations” on page 8 of the document “Sample Size for Pet Studies”?

3. The numbers under “Details simulations” on page 37 that reads in part “P*(1-TE) and equal 0.0475, 0.045, 0.04, and 0.3.” It appears that “0.3” is an error and should be “0.03.”

The effort by the statisticians of the OPP to clarify the details of the math modeling and computer simulations in their work on SAS program of simulations (docket # EPA-HQ-OPP-2019-0161-

43 0003) on “Sample Size for Pet Product Studies,” were very helpful and appreciated. Given the fact that the “1000 sets of data” were created by Monte Carlo Simulations, the EPA are already “doing experiments on computers.” Therefore, the Panel further encouraged EPA colleagues to develop in silico approaches for the goal of reducing animal usage.

Charge Question 10

Please provide comments on the overall clarity, accuracy, and completeness of the draft pet product guidelines. Please provide any additional comments that highlight areas of the draft guidelines that may need to be clarified and note any critical topics that are missing. Please include references to published literature that could help improve the completeness and clarity of the draft guidelines.

Panel Response 10:

Given the importance of the blood-fed/retention proportions and the numbers of arthropod ectoparasites per animal, the Panel recommended that the Agency verify these values based on information obtained from the open scientific literature, registrant submitted studies (since the inception of 1998 testing guideline), and experts of these ectoparasites.

1. There is a mix of scientific names (genus species) and common names. The Panel recommended that the scientific name be used exclusively or at least at the first use.

2. The term “Biting fly species” is not entirely accurate, at least for the list of flies presented. For example, face flies are not biting flies. Thus, either they should be removed from the list or the term modified. If the term is modified, to include face flies, then house flies should also be included. The Panel recommended that the language be changed to “mosquitoes and stable flies” and that some language about “evaluation of other biting flies would be considered on a case-by- basis (contact EPA for guidance).”

3. The need for “outcrossing” is unclear and what is meant by this is not sufficiently documented.

• Outcrossing is commonly done when the investigator wants to retain a “field relevant trait”, such as a parasitoids ability to hunt for food. • Outcrossing is avoided for toxicology studies because responses to insecticides do not generally change and having a standard reference strain is desirable and makes reproducibility feasible. • Thus, for the types of assays in the proposed guidelines, The Panel suggested that outcrossing is unnecessary and even perhaps detrimental. Using a standard strain is more desirable. Outcrossing changes the strain in unpredictable ways, making reproducibility problematic. • The Panel suggested that outcrossing be removed from the proposed guidelines. • If outcrossing is to be retained in the protocol, then more details are needed as to what strain should be used for outcrossing, what methods should be employed (e.g., reciprocal

44 crosses?), how many individuals are to be used, etc. It seems that this level of insect husbandry is not needed for these protocols.

4. The design of the repellency tests are worth reconsidering. The current method is a no-choice assay where the pests are applied directly onto the animal, whereas, the purpose of the test is to determine if a product prevents pests from associating with an animal. The Panel noted that alternative protocols could include adjoining cages where the pests can move from one animal to another (choice) or releasing the pests into the cage and determining if they are repelled (willing to get on the animal or not) (no-choice test). The Panel recommended that repellency assays be done independently of mortality assays.

5. The procedure for the assessment of blood feeding is a bit vague. Most blood feeding arthropods can take a full blood meal or some partial bit of a blood meal. The current assay (“crushed on a light background”) suggests that even a tiny blood meal is counted. Is that desired or are people being asked to score the relative size of the blood meal taken (and if so, how)? The Panel recommended that blood feeding be determined by PCR so that the blood meal can be quantitated, and the species identified.

6. For shampoos, the Panel recommended that the formulation blank be used as the control because of the variability in shampoo products. Registrants will almost certainly have the formulation blank available to them.

7. Pests that have fallen off the animal can be hard to find. It is unlikely that 100% of the ticks applied can be recovered. The Panel noted that this presents some statistical challenges to dealing with the data. If 50 fleas are released and only 35 fleas are recovered, how will the statistical analysis be carried out?

8. The Panel recommended that the title of the guideline be changed from “control” to “efficacy”.

9. The Panel recommended that the suitability of a vertebrate animal to be used for the flea or tick tests be determined individually, but that the final control and treatment groups be compared as averages.

10. Although the guidelines address efficacy of new products, it appears that only pre- and post- treatment body weights are required. The same ingredients that kill ectoparasites can also cause a variety of neurological and physiological reactions in dogs/cats; scratching of bites can lead to inflammation and infections. Anemia has been reported in control dogs. The Panel recommended daily inspection of animals for health problems be conducted.

11. Page 7 of the proposed guidelines: the last sentence of d(3) Analyzing data, second to last sentence - “Software for analysis using GLMs or GLMMs is available in many widely sold statistical analysis packages.” In this sentence, the Agency appears to be endorsing the use of commercially available statistical software packages; however, there are open source software packages (e.g., R) available to perform the same task. The Panel recommended that the guidelines clarify when specific software is required and when specific software is only a suggestion.

12. Page 9 of the proposed guidelines: first sentence of j(2) Selection and allocation of test

45 animals – “The minimum number of qualified animals per group should be determined to achieve sufficient power in addressing the study objectives…..” The Agency already employed the power analysis technique to determine the minimum number of animals required. Perhaps, the purpose of the aforementioned statement is to address the situation when the number of animals employed is different from (i.e., less than) the recommended value? The Panel recommended that this concept needs to be clarified. The same comment also applies to k(2) (page 12) and l(2) (page 16).

13. Page 11 of the proposed guidelines: first sentence of j(5) Data Analysis: “If the study has only one measurement (one infestation) per animal, GLMs for binomial distribution data (with log link function), Poisson distribution data, or negative binomial distribution data should be used to estimate the survival or hatching rate of each group.” The Panel noted that this sentence is in contrast to the recommendation specified in section (d)(3) that techniques other than GLM or GLMM may also be used. Also, additional guidance on employing different distribution functions should be provided, e.g., betabinomial distribution for the “binomial” data exhibiting over dispersion etc. The same comment also applies to k(6) (page 15) and l(6) (page 18).

14. The Panel noted that the term “mode-of-action” as it appears on pages 14, 15, and 18 of the proposed guidelines needs to be defined in section (c). The “definition” of mode of action on page 15 (e.g., volatile repellent, toxicant) is not correct.

15. Page 4 of the proposed guidelines: (c) Definitions The definitions of “Knock down” and ‘Moribund” are very similar and difficult to distinguish. The Panel recommended they be clarified.

16. Page 6 of the proposed guidelines: d (1), v. Housing of Animals: the Panel recommended that the permissive “may” be replaced with “should” with respect to group housing of dogs and cats at times other than during infestation.

17. The Panel recommended that for analyzing and presenting data, the guidelines need to indicate that the arithmetic means must be used.

18. The Panel noted that in the tick efficacy section, EMA tick efficacy wording should be used with in-house revisions.

19. The Panel recommended inserting the phrase "adequately infested" when discussing minimum percent tick/flea retention thresholds.

20. The Panel recommended that the guidelines define the different types of tests discussed. Specifically, the Panel suggested the term non-vertebrate animal testing (NAT, NVT, or NVAT) for tests that do not use the host. The Panel recommended that the terms in vivo, in vitro and in situ be avoided because they have different meanings in different scientific circles (e.g. some would claim that a NAT was an in vitro test, while others would claim it is not). Thus, the guidelines should avoid terms that have non-equivalent synonyms in different scientific disciplines.

21. The Panel recommended that the guidelines should state how many laboratory efficacy studies are required for each parasite that is included on the final approved product label. For example,

46 FDA approved drugs require two separate pivotal dose confirmation efficacy studies for each specific parasite. Additionally, these two studies must be conducted at two different independent locations using different parasite isolates.

22. One Panel member indicated a desire to add a section on “The Use of Computational Technology” to encourage manufacturers to utilize such applications of computational technologies to reduce animal usage

23. Page 5: (d) (1) i. of the proposed guidelines: The guidelines should define in practice what the term “Negative control” should encompass.

24. There are some formatting issues (for example, Tables break across pages; citations that need to be separated by a hard paragraph).

25. It is not clear if the words ‘must’, ‘should’ and ‘may’ are always used appropriately in the guidelines. The Panel recommended that the use of these words be examined carefully.

26. Section (k)(5)I of the proposed guidelines: The Panel suggested that the Agency consider reducing the number of categories of ticks examined from six to four. Efficacy should be based on the number of attached/free live ticks in the control group versus number of attached/free live ticks in the a.i. treated group.

27. Section (k)(5)i top of page 15 of the proposed guidelines, The paragraph starting with “in situations:”

• “In situations demonstrating the product has an acaricidal effect only after the tick blood fed (engorged)”, is this a realistic outcome for a topical application? • The statement, “Dead ticks combined with live ticks to calculate mortality?” is confusing.

28. Section (k)(3)ii provides detailed information for repellency testing against ticks. The Panel recommended the equivalent information be provided for fleas if registrants also have repellency claims for fleas.

29. Table 1: The heading of column #2 should be changed to “Timing of infestations and re- infestations.”

30. Section (l)(1)ii: The Panel recommended that the species that should be tested needs to be clarified. This would alleviate the need to contact EPA when registrants want to do these tests. Clarity is needed to understand if each species must be tested if it goes on the label. Also, does the Agency want only some species (or 3 of five species) tested to give a “biting flies” label?

31. Section (l)(2): The Panel recommended that the phrase, “caged and exposed to 50-100 adults” be changed to “caged and exposed to 50-100 female adults” since blood meals are the end point for this bioassay.

32. Section (m)(3)ii: Delete “for testing mosquitoes” from the end of the last sentence. This is already stated.

47 33. Page 6: viii. c: The Panel noted that in some field studies, it might be necessary to use a positive control for animal welfare concerns; therefore, the statement that “a positive control should not be used” should be modified.

METHODS FOR TESTING AGAINST FLEAS (SECTION (J))

Charge Question 11

The proposed studies currently are not blinded but untreated controls are used.

a. Should the study be blinded and/or utilize an inert control (i.e., formulation minus active ingredient)? b. Why or why not?

Panel Response 11:

The Panel concluded that the answer to this Charge Question depends on whether it is essential to identify all of the active ingredients within the product or formulation and to determine their contribution as it relates to the label claims. If it is necessary to identify the effects of formulations, carriers, diluents, ingredients, adjuvants, etc. on performance, then it will be necessary to conduct studies with the blank formulations. If only determining the performance of a formulation at some designated end point (e.g., ≥ 90% kill) is the primary objective, then conducting studies with “so-called” inert controls is probably not necessary, nor is conducting a blinded study.

The Panel found that this question seems most relevant to products such as powders, shampoos, and dips in which carriers, adjuvants, emulsifiers, and other ingredients may in themselves have some lethal effects against fleas. For example, the product Avon® Skin So Soft is widely reported to have repellent activity against cat fleas (Fehrer and Halliwell, 1987). Contact with diluted household detergents and cleaning products is lethal to German cockroaches (Szumlas, 2002; Baldwin and Koehler, 2007). Components of soaps such as fatty acids (Sims et al., 2014) and linear alcohol ethoxylates (Sims and Appel, 2007) are toxic to German cockroaches. Aliphatic alcohols and mineral oil have been shown to be toxic to German cockroaches and combinations are synergistic (Sims and O’Brien, 2011). The Panel found that it is also likely that some of these potential ingredients may synergize conventional insecticides included in the formulated products and that these combinations will also be toxic to fleas.

To isolate and identify each of the compounds that may contribute to the toxicity of these formulations will require a considerable amount of testing. However, the Panel noted that it should be possible to conduct these studies without the use of vertebrate animals. Topical applications of serial dilutions of individual compounds and combinations of compounds in various formulations applied to adult fleas will provide data regarding their contact toxicity allowing for the determination of synergism, additivity or antagonism. Similarly, adult fleas confined to surfaces treated with serial dilutions of individual compounds and combinations of compounds in various formulations should permit the determination of residual activity and allow

48 for the determination of synergism, additivity or antagonism.

Charge Question 12

Repellency is typically determined by whether or not an insect takes a blood meal. Based on flea feeding behavior, repellency is not considered relevant for flea control and therefore is not included in the guideline. a. Please discuss whether repelling fleas is a viable endpoint. Why or why not? b. If repellency can be considered a viable endpoint, please provide detailed methods to test for repellency against fleas. c. Can repellency be determined by assessing the number of live fleas on treated vs. untreated animals? If so, can repellency based on counts of live fleas on treated vs. untreated animals be differentiated from mortality, which is also currently measured by counts of live fleas on treated vs. untreated animals? If the two endpoints can be differentiated please describe the methods, including specifically how and why the counts of live fleas on treated vs untreated animals differ for repellency and mortality, and provide literature references.

Panel Response 12:

The Panel found that the statement, “Repellency is typically determined by whether or not an insect takes a blood meal,” is not supported by the scientific literature. Repellents cause oriented movements away from the source. In relation to feeding, a better term might be a feeding inhibitor (Deletre et al., 2016). Even the European guidelines define repellent effect as “a product with a repellent effect will cause the parasite to avoid contact with a treated animal completely and/or to leave a host” (EMEA/CVMP/EWP/005/2000-Rev. 3). Flea repellents have never gained wide acceptance as a strategy to control fleas or protect vertebrate animals. Repellents have been better defined for tick control. Marchiondo et al. (2013) write, “Two types of repellency are defined: sensu stricto for repellency characterized by an irritant effect, causing the tick to move away from the treated animal or leading it to fall off soon after contact with the treated hair coat within 6–8 h and sensu lato for all other tick repellency (or expellency) up to 24 h. The first, repellency sensu stricto, may be attributed to the vapor phase of a compound or irritant effect through direct∼ contact (for example, oil of Citronella but also some synthetic pyrethroids),∼ while the second repellency (or expellency) causes inhibition of attachment or detachment of already attached ticks, (for example, some synthetic pyrethroids or amitraz).”

Pfister and Armstrong (2016) write, “Repellency refers to the action of a product that causes ectoparasites to avoid or leave the dog, or to fail to feed on the dog, i.e. the ability of the compound to prevent the parasite from attaching or migrating onto the dog. A similar concept is the “anti-feeding” effect, which refers to the ability of the compound to stop the parasite from taking a meal from the dog.” Herein lies the problem. The Panel recommends that the authors of the guidelines not confuse/conflate repellency or avoidance and anti-feeding properties. The Panel recommended that the Agency provide a concise definition of repellency similar to that found in the European guidelines.

49 a). The central premise regarding this Charge Question is based upon the following statement in the proposed guidelines section (j). “Based on flea feeding behavior, repellency should not be considered relevant for flea control (Pfister and Armstrong, 2016).” The Pfister and Armstrong (2016) paper is a review of two types of insecticides and this statement may be true for the systemic fluralaner and cutaneous delivered permethrin, but that does not mean it is true for all compounds.

Flea repellents have been reported in the literature. Chemicals were tested for repellency (prevention of feeding) with the flea Xenopsylla cheopis (Bar-Zeev and Gothilf, 1972). Unfed fleas were allowed to feed on treated skin surfaces of guinea pigs. The fleas were examined for blood feeding. Of the 538 compounds tested, four compounds were as effective as DEET. The repellency of essential oils was evaluated in a filter paper strip choice test with adult cat flea, Ctenocephalides felis. Thymol and trans-cinnamaldehyde were as repellent as 15% DEET (Su et al., 2013).

b and c). The Panel found that if there is a claim that a compound repels fleas, then repellency must be considered as the viable endpoint. The number of live fleas off host compared with the number of live fleas off host in control should provide a relative estimate of the repellency. If the compound also exhibits some contact toxicity, then both live and dead fleas may be present off the host.

Cats or dogs could be treated with the test compound. The treatments would be permitted to dry and spread in the pelage of the animal (12-24 hours). The treated animals would be held within cages constructed with Plexiglass to ensure that fleas don’t escape and are collected. Aliquots of fleas (25-50) would be placed on the fur or placed in the cage and allowed access to the treated and control animals. At 1 hour, the contents of the cage would be vacuumed, and the live and dead fleas trapped.

Repellency is not a widespread label claim of many topical products applied to cats and dogs. The Panel noted that EPA has some latitude to accept non-vertebrate studies to demonstrate repellency or request protocols for determining repellency studies on vertebrates.

METHODS FOR TESTING AGAINST TICKS (SECTION (K))

Charge Question 13

The guideline states that tick infestations should consist of a 50:50 ratio of male to female ticks for all species. The European guideline contains a clause that for Ixodes spp. infestations the sex ratio should consist of approximately 10% males: 90% females since males do not readily attach (Marchiondo et al., 2013). However, the scientific literature for Ixodes spp. appears to be split between using a 50:50 ratio or a 90:10 ratio. Please discuss the merits of each ratio:

a. Is one of the ratios better for testing Ixodes spp., or are they both appropriate? If one ratio is better than the other, please explain why. b. Is one of the ratios better for testing Rhipicephalus spp., or are they both appropriate? If one ratio is better than the other, please explain why. c. Is one of the ratios better for testing Amblyomma spp., or are they both appropriate? If one ratio

50 is better than the other, please explain why.

Panel Response 13:

a. The Panel noted that it appears that for testing Ixodes spp., both ratios 50:50 and 90:10 (females:males) would be appropriate. In general, placement of a somewhat balanced number of both females and male ticks on hosts is important because tick mating while on the host promotes feeding success by female ticks to engorgement (Akov, 1982; Wang et al., 1998; Donohue et al., 2009). The role of mating on female feeding during the very early feeding stages is unclear. Insufficient numbers of males that may survive during trials could possibly confound testing results by causing females to poorly feed due to lack of mating opportunities. So, a 50:50 ratio is probably best for all species tested to ensure normal mating behaviors.

The European guideline (European Medicines Agency, 2016) contains a clause that for Ixodes spp., there appears to be split between using a 50:50 ratio or a 90:10 ratio annotated below in the following references: Vatta et al. (2018), Becskei et al. (2017), EMEA (2016), Dumont et al. (2015), Fourie et al. (2015), Kuzner et al. (2013).

A reason some researchers prefer to use more female than male Ixodes spp. in testing is because male Ixodes spp. do not necessarily require a blood meal (attachment) to mate, which can skew the attachment rate statistics on untreated hosts, particularly when testing systemics.

Studies have shown that attachment rates of I. ricinus can be increased by using a higher number of ticks (60) and a lower number of males compared to females ( 25:75) as male I. ricinus attach only briefly or not at all (Kiszewski et al., 2001; Kuzner et al., 2013). However, Marchiondo et al. (2013) noted the presence of male I. ricinus is sufficient to stimulate∼ female tick attachments and to achieve greater attachment rates.

“The metastriate ticks, including Dermacentor, Amblyomma, and Rhipicephalus, invariably attain sexual maturity and mate solely on their hosts. The more primitive prostriate Ixodes spp. ticks; however, may copulate both in the absence of hosts and while the female engorges. These expanded opportunities for insemination complicate the mating systems of the Ixodes ricinus complex of species. In these ticks, autogenous spermatogenesis must precede host contact...” (Kiszewski et al., 2001).

In the U. S., Ixodes pacificus and I. scapularis mate on and off the host and adult males rarely attach to the host, taking little blood if they do. In general, the blood loss to a dog host is less than that taken by females and males of the other genera (Koch and Sauer, 1984). Placing 50% female and 50% male ticks (N = 50 ticks) challenges the host with 25 potentially attaching ticks, whereas 90% female to 10% male could lead up to 45 females attaching to the host. Especially if repeated infestations occur on the animal, or if more than one species of tick is used in a trial, the 50:50 ratio would cause less stress to control animals in particular. A 50:50 ratio would ensure adequate mating opportunities and normal feeding behaviors for females while being consistent with most previous studies for comparison (McCall et al., 2011). b. The Panel noted that a 50:50 ratio is best for testing Rhipicephalus spp. As indicated above, with the metastriate ticks, such as Rhipicephalus spp., the males must attach and feed before they

51 mate so there would be no statistical advantage to skewing the ratio of males to females away from 50:50.

Thus, for Dermacentor, Amblyomma, and Rhipicephalus, mating occurs only on the host, and both females and males feed. Males consume substantially less blood than females, but unlike Ixodes spp., they do take some blood. Using either ratio of ticks could lead to up to 50 ticks attaching. Placement of 50:50 would likely result in fewer adverse effects to the host during trials than 90:10, although not much difference would be seen if ticks are removed at 48 hours from the host.

c. The Panel noted that a 50:50 ratio is best for testing Amblyomma spp. As indicated above, with the metastriate ticks, such as Amblyomma spp., the males must attach and feed before they mate.

Although not included in the Charge Question, Dermacentor results should also be considered, as this is a common tick found on U.S. pets, in particular, D. variabilis on dogs.

Charge Question 14

The guideline states that ticks that feed to engorgement can transmit pathogens that may cause disease; therefore, mortality after blood-feeding to the point of engorgement should not be considered a viable endpoint. a. Is the determination of engorgement a feasible data collection point? Why or why not? b. If so, please provide detailed methods with references for determining engorgement. c. Can blood feeding be determined prior to engorgement and would this be a viable endpoint? If so, please describe methods for determining blood feeding prior to engorgement and provide any relevant references.

Panel Response 14: a. The Panel concluded that, no, the determination of engorgement is not a feasible data collection point. The Panel recommended that the term “engorgement” needs to be revised in the guidelines as the EMEA and WAAVP guidelines are not being used correctly. Adult tick counts are generally performed 48 hours after each infestation time point, but initiation of blood feeding during this period will not be readily apparent as very little blood is consumed within this time frame. It is impossible to accurately visualize any increase in size (plumpness) over this short period. This is even difficult if individual ticks are weighed (milligrams). All instances of “engorgement” in the guidelines should instead be changed to “partially fed.” If feeding to engorgement and drop off is to be mentioned, then the term “repletion” should be used. Throughout the guidelines, for clarity, it should be specified that all ticks found on the host (attached and unattached) are removed at 48 hours post-infestation, not just counted. This is in addition to off-host ticks that are also counted and collected.

The efficacy of systemically-acting acaricides depends on tick attachment and ingestion of the compound. Transmission of bacterial/protozoan pathogens by hard (ixodid) ticks typically starts

52 to take place between 36-72 hours after attachment, before substantial blood feeding occurs in adults. If the kill occurs prior to the 48 hours tick removal time point, this delay after attachment can allow a systemic acaricide to take effect prior to transmission of many pathogens (Uspensky and Ioffe-Uspensky, 2006; Wengengmayer et al., 2014). However, viruses such as Powassan fever virus could be transmitted within minutes to hours after attachment (Richards at al., 2017). b & c. The Panel found that engorgement status does not appear to be relevant to these revised guidelines. Researchers are assessing survival of partially fed ticks after 48 hours of exposure (contact) with a topically applied acaricide. As long as female ticks fail to survive to repletion and oviposition, the lifecycle is interrupted. As noted by the Panel in response to question 14a., all instances of “engorgement” in the guidelines should instead be changed to “partially fed.”

If blood feeding status and duration of attachment are desirable factors for protocol guidance, effective methods have been developed to determine these parameters. Blood consumption during the early feeding phase can be verified by PCR, as well as, identification of the host blood meal source (Che et al., 2015). However, depending on the sensitivity of the assay, it is also possible to detect remnant blood from the host on which the adult tick previously fed during the nymphal stage. The duration of tick attachment and phase of blood feeding could also be scored morphologically using tick engorgement indices based on comparisons of scutal and full body dimensions = “scutal index” (Yeh et al., 1995; Falco et al., 2018). The approximate timing (in days) for initiation of blood feeding by adults feeding on mammals has been established for Ixodes pacificus/scapularis, D. variabilis, A. americanum, and R. sanguineus. Prior to and during the early blood feeding phase, when other fluids are primarily being taken up by the feeding tick, the tick cuticle does not look substantially different in color and no inflation of the body size is evident. Physically, the tick cuticle begins to change color and the tick becomes inflated during the initiation of the rapid blood consumption phase. On the day this occurs, the change in appearance may be quite rapid over the course of several hours as the “big sip” commences (body typically turns a dark gray color as it inflates).

As an example, an adult Ixodes ricinus female takes ~7-8 days to feed to repletion. The slow feeding period occurs between ~1-5 days after attachment, during which the female ingests ~1/3 of the total blood meal. Between 24-72 hours very little blood is ingested. About 2/3 of host blood is consumed during the rapid engorgement phase (“big sip”), which occurs during the last 24-48 hours before the engorged tick drops off the host (~6-8 days post-attachment) (Figure 1, Franta et al., 2010; Sojka, 2015).

53

Figure 1 (Franta et al., 2010)

Charge Question 15

The endpoints for repellency and mortality contain numerous different categories which define how ticks should be recorded for each endpoint (e.g., repellency). a. Are there any additional categories which might be needed for the different endpoints (e.g., repellency)? Is the separate presentation of the endpoints clear? b. Can both repellency and mortality be assessed in a single study, or does each endpoint need to be assessed separately? Please explain.

Panel Response 15:

General Comments a. The Panel did not think there is a need for more categories; rather, the Panel recommended a reduction in the number of categories. While the multiple categories offer some potential increased resolution into the responses of the ticks to product testing, the number of categories would be unrealistic in practice.

54

Efficacy Endpoints

The proposed guidelines state: “Mortality should be determined by comparing dead, unengorged ticks on the animals and in trays/cages to the control group. Ticks that feed to engorgement can transmit agents that may cause disease; therefore, mortality after blood-feeding to the point of engorgement should not be considered a viable endpoint.”

The Panel suggested revising this to indicate that mortality at or before 48 hours would be a reasonable endpoint for mortality to prevent pathogen transmission, if that is the goal.

The proposed guidelines state: “For testing repellency against ticks, a repellent effect is a reaction by a tick to avoid contact with a treated animal. Since repelled ticks will move away from the treated animal and possibly be unrecovered, repellency should be determined indirectly by counting the number of live or dead ticks (attached or unattached) on the animal plus live or dead ticks in the cage that are engorged compared to the control animals. Moribund ticks should be considered alive. Other methods of evaluating repellency against ticks (e.g., hot foot, inhibition of attachment, disruption of attachment; Halos et al. 2012) may also be considered but should be justified.”

The Panel noted that the time frame is important here. If following EMEA guidelines (European Medicines Agency, 2016), the ticks should not attach to the host within a 24- hour time frame. Therefore, host and cage surroundings should be checked for ticks and numbers on and off the host need to be compared to control animals. Off-host live, or dead ticks could both be counted. Antifeedant could be considered as an additional endpoint. The number of live and unattached ticks would be a measurement of this.

Data collection and recording for ticks

For mortality trials, the mode of action of the product would dictate when the data is collected. Systemic products must be ingested by ticks for mortality to occur and could take several days to act based on the prolonged feeding period of ixodid ticks. The Panel noted that a time point of 48- 72 hours should be reasonable to capture tick mortality with systemic acaricides. For some acaricides that act much sooner, earlier measurements should be made. Comb counts should be specified to include tick removal, unless mortality measurements are being made for retained ticks over longer time points in studies.

For the tick mortality effect categories, as the Panel has noted previously, all instances of “engorged” should be removed and replaced with “partially fed.” The Panel observed that it seems like there are too many categories here to be truly useful. Reducing the number of categories to evaluate would simplify study interpretations and perhaps reduce the sample sizes of ticks and animals needing to be used. The simpler categories from the European Medicines Agency (EMA, 2016) would probably suffice for most study goals. Also, it seems questionable for moribund ticks to be counted as alive; it is probably more accurate to count them as dead because it is unlikely that they will adequately recover to the point of being capable of re-

55 attaching to a host. Holding moribund ticks off the host for at least 24 or up to 48 hours, under optimal temperature and humidity conditions for survival would allow the study to differentiate live from dead ticks unambiguously. For the tick repellency categories, the Panel agreed the data should typically be collected within 24 hours post-infestation, though sometimes ticks may take longer to attach to the host; up to 48 hours may be appropriate to evaluate repellency in some host-tick systems. Also, there are too many categories for tick scoring here. Within 24 hours, there is no blood acquisition by ticks, and so these categories are not accurate. Blood feeding as a factor for repellency should be removed when using short time frames.

In mortality testing studies, how can host immune response to ticks be differentiated from the effects of the product tested? The Panel noted that it may be important to provide guidance for individual variations that occur in host rejection of ticks due to an immune response vs. product induced mortality determinations during trials as an endpoint (European Medicines Agency, 2016; Wada et al. 2010). This may not be an issue for R. sanguineus, but it could be the case for other tick species tested, such as I. scapularis (Szabo and Bechara, 1999; Gebbia et al., 1995). It is possible to have large numbers of ticks attach to a host, over the 25% threshold, and rejection by the host may occur during feeding trials that is due to host immune response, not due to the product’s effects. Monitoring of host inflammation around feeding ticks would provide insight regarding product-induced mortality vs. immune-induced mortality. Animals with noted inflammation around bite sites should be removed from the study to prevent confounding of results. This could be of particular importance as animals are infested multiple times over the course of several months and may develop acquired immunity to tick feeding during later trials.

b. Optimally, mortality and repellency should be determined in different studies with different designs, because they are different outcomes and have the strong potential to bias the outcome. The current methods muddle the distinction between mortality and repellency. For example, if 50 ticks are put on an animal, and at 24 hours investigators find 10 dead ticks on the animal, 0 live ticks on the animal, and 40 moribund ticks in the cage, this could be scored as a repellent, when equally likely this was a slow acting acaricide without repellent activity. While some materials are both toxic and can act as a repellent, other materials may do one or the other. Thus, the Panel suggested that the design for repellency assays needs reconsideration, as discussed in more detail in the response to Charge Question #10. The Panel also recommended that for a repellency study, ticks should not be placed directly onto an animal. Instead, ticks should be placed in the local environment with the animal to allow ticks to naturally attach to the host; if there is a repellent effect, fewer ticks will be found on the animal (Dumont et al., 2015b).

METHODS FOR TESTING AGAINST MOSQUITOES OR BITING FLIES (SECTION (L))

Charge Question 16 Comment on the adequacy of outcrossing lab-reared colonies of mosquitoes every 3 years. a. Is three years an adequate timeframe to retain genetic diversity in lab-reared populations? Why or why not? b. If not, how often should lab-populations be outcrossed with wild-type mosquitoes? Please provide references if relevant.

56

Panel Response 16:

What is the goal of periodic outcrossing of mosquito (as well as other ectoparasite) colonies? The Panel observed that retaining genetic diversity is irrelevant for toxicology testing (unless for example, examining frequency of resistance alleles) and questionable for behavioral studies. Most insect species including mosquitoes do not die off with inbreeding. Mosquito colonies at the USDA laboratory in Gainesville, FL have never been outcrossed and have been used successfully for a variety of tests for years. If the goal is to maintain some part of a large population with relevant proportions of resistance alleles, then the Panel found that this protocol is not detailed enough. A recent paper by Ross et al. (2019) concludes that inbreeding depression occurs in this species due to low population size. Colonized mosquito populations suffer inbreeding depression and adaption to laboratory conditions. Laboratory environments are inherently artificial, and colonized mosquito populations experience an entirely different set of selective pressures compared to natural populations, which could lead to declines in reproduction (Bryant and Reed., 1999). Ross et al. (2017) suggested that while life history traits of Ae. aegypti do not change consistently with laboratory maintenance, traits where selective pressures are absent in the laboratory, such as flight ability, feeding behavior, and thermal tolerance might still be compromised.

Ross et al. (2019) concluded that variation in fitness and performance was dependent on colony size, but that most individual traits were unaffected, and patterns of adaptation were not consistent across populations. Performance of laboratory populations of Ae. aegypti maintained in a larger population increases. Rates of laboratory adaptation can be slowed or minimized by using more natural rearing environments such as maintaining large populations (400 vs. 100 in larger cages at a variable temperature and environments that are more complex.

The Panel agreed that many traits are lost in colonized insects shortly after they are selected for colony conditions. However, genetic conditions tend to stabilize. Adding new (uncharacterized) genetic material periodically would change the genetics of the colony so they are different than they were when the colony was used for previous studies. This could produce different results in certain types of tests.

In a study, Gillett (1967) describes changes in feeding behavior of wild and colonized species of mosquitoes: “When a blood-sucking insect 'bites’ a sensitized host it must, to survive, depart before the host is alerted by irritation that accompanies or precedes the 'immediate’ reaction. There is a safety period between initial salivary injection by the insect and onset of host irritation when the insect’s meal must be completed; immunity from host attack must, therefore, depend partly on the speed at which the insect can tap the blood supply and complete its meal, and partly on an adequate delay in the onset of host irritation. In mosquitoes, and other insects that depend on a blood meal for egg production, only those that complete the meal within the safety period can lay a full complement of eggs; the others will either be killed or injured by the host before egg development begins, or they will be disturbed before completion of the meal and so lay fewer eggs. Thus, fast feeders and those producing a delay in the onset of irritation will tend to lay more eggs, and these 2 properties will be maintained by natural selection, with the onset of host irritation acting as the main selection force. Experiments were made comparing a population of wild mosquitoes (Aedes africanus) with that of a colonized species (A. aegypti), which had been

57 protected from retaliatory action by the host during the previous 3 years. They showed that while the period between salivary injection and the onset of irritation was the same in both species (ca. 3 min), feeding was usually fast in the wild species, which had presumably been subjected to rigorous selection against slow feeding, but variable and often slow in the captive species, which had been reared for generations in the absence of this pressure. The results also indicated a slight compensatory delay in the onset of irritation following slow feeding in the wild mosquitoes; presumably a difference in antigenic properties of the saliva affects the speed at which the insect can take up blood and provides what appears to be a remarkable alternative pathway to successful feeding, and so to maximum egg output and thus to survival.”

Rather than periodic outcrossing, the Panel suggested testing a standard strain that is well-studied and has a history in the scientific literature and comparing the counts of live mosquitoes on treated versus untreated animals. Believing that there will be widespread differences between strains assumes facts not in evidence. Furthermore, every time a strain is outcrossed the strain changes and could result in non-reproducible results, a major drawback to any manipulations of the strain used for testing. In addition, outcrossing with uncharacterized field-collected individuals could introduce pathogens, as well as, resistance to ectoparasiticidal compounds.

If the Agency decides to include protocols regarding outbreeding in the updated guidelines, the Panel cautioned that it would be more appropriate to suggest outbreeding after every so many generations than to specify a time period (i.e., every 3 years). Several articles indicated that the simplest way to maintain the fitness of colonized mosquito populations, especially Ae. aegypti, is to cross laboratory colonies to an outbred population. Yeap et al. (2011) suggest a 2-year period for out-crossing, but a rationale was not readily discernable from the article.

With an estimated generation time of approximately 30 days for Ae. aegypti, (Sowilem et al., 2013), a 3-year outcrossing interval could result in >36 inbreed generations. However, Ross et al. (2019) established replicate populations of Ae. aegypti mosquitoes and maintained them in the laboratory for twelve generations at different census sizes [<100 individuals or 400 individuals]. At F5, life history traits were compared between replicated large populations and inbred lines. After two generations of brother-sister mating, the inbred lines had reduced fitness relative to the large populations, with substantial costs to larval survival and development time. Although the study design involved a comparison among replicates established from eggs from an ancestral population, the data suggest adaptive changes to laboratory conditions in the F5 generation. Therefore, if outbreeding was important, it should be done more frequently than on a 3-year basis.

Alternatively, Ross et al. (2017) describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which minimizes laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. They suggest outcrossing be repeated for at least three consecutive generations to produce colonies that are at least 87.5% similar, genetically, to the field population.

Charge Question 17

In the interest of reducing vertebrate animal testing, we propose simultaneous testing of up to three mosquito species on a single vertebrate animal.

58 a. Is simultaneous testing of three mosquito species feasible? Why or why not? b. If simultaneous testing of three mosquito species on a single vertebrate animal is feasible, then please comment if the proposed methods are adequate, and if there are alternative methods please provide detailed methods and references. c. If testing three species at once is not feasible, provide additional detailed methods and associated references to limit the number of vertebrate test animals necessary to complete efficacy testing against the three required mosquito species and maintain adequate statistical power.

Panel Response 17:

After consulting mosquito experts (e.g., Drs. Dan Kline and Bill Reisen, personal communication), the Panel determined that simultaneous testing with three mosquito species should not affect the test results. The mosquitoes do not compete for feeding sites and do not otherwise interact in ways to alter results expected with a single species. Proposed methods look reasonable, but it would be nice to reduce the number of recommended vertebrate animals used.

The proposed guideline recommends at least 6 vertebrate animals per group for assessing the efficacy of an insecticidal product. Also, the proposed guideline states that a total of 150 mosquitoes/flies are needed if the testing involves three species concurrently (i.e., 50 per mosquito/fly species), but 100 mosquitoes/flies are needed if the testing involves only one species. For an insecticidal product designed to kill, given the fact that the nervous system of the insect is the primary (universal) target and the endpoint of efficacy analysis is mortality, unless a biological reason(s) exists, each mosquito/fly species should be similarly affected by the product’s insecticidal active ingredient (e.g., permethrin); therefore, the Panel found that regardless of the number of species involved, the testing can be conducted with the same number of each species of mosquitoes/flies. It is noteworthy that the test size requirement of at least 6 vertebrate animals per group is identical to the existing testing guideline: U.S. EPA 1998 Product Performance Test Guidelines: OPPTS 810.3300 Treatments to Control Pests of Humans and Pets. Also, based on the sample size and power simulation analyses performed by the Agency, the results (table [last two rows] on page 7 of the PDF document titled “Sample Size for Pet Product Studies”) indicate that 6 vertebrate animals and 50-100 mosquitoes/flies per animal should provide the statistical power needed.

For flies known to exhibit aggressive behavior and (or) inflict a painful bite (e.g., stable flies), efficacy testing conducted with 50-100 flies may not be recommended because of the adverse effects on the vertebrate animals involved. The Table below summarizes the required number of pests per animal in the proposed guideline (page 17 of the PDF).

Proposed Required Blood-fed Number of Pests Efficacy Testing Pest /Retention Proportion per Animals Guideline (“base value”)

Section (l) Mosquitoes/Flies 0.6 100

59 Based on Tables A3.1 and A3.2 in Appendix III of the document titled “Sample Size for Pet Product Studies,” the required blood-fed/retention proportion (referred to as “base value” hereafter) forms the basis of computer simulations to generate the number of pests per animal presented in the proposed guideline (last column of the table above). Accordingly, one way of reducing the number of pests per vertebrate animal for testing is to modify the required blood- fed/retention proportion based on the “actual biology" of flies as well as expert recommendations. Using the “updated” base value(s), the possibility of reducing the number of pests per animals can be assessed using the proposed computer simulation techniques.

After an extensive discussion of this Charge Question, the Panel recommended the following: If three mosquito species are tested simultaneously, 50 mosquitoes of each species should be used. For flies known to exhibit aggressive behavior and (or) inflict a painful bite (e.g., stable flies), efficacy testing conducted with 50-100 flies may not be recommended because of the adverse effects on the vertebrate animals involved. Under natural conditions, stable flies rarely complete a blood meal on the first try because they are dislodged by the host because the bite is painful. The fly merely flies around the host and tries again. This continues until the fly is replete. When the fly is undisturbed, as it would be under laboratory testing conditions, the time to repletion averages 147 seconds, as documented Schofield and Torr (2002).

Charge Question 18

The proposed methods to test mosquitoes and biting flies indicate vertebrate test animals should be sedated prior to exposure to invertebrate pests. a. In the interest of minimizing stress of sedation on vertebrate test animals please discuss whether sedation is necessary. If sedation is not necessary, please provide detailed methods and references and discuss the strengths and weaknesses of the methods for conducting efficacy testing. b. If sedation is necessary, please provide methods to minimize the number of times vertebrate animals should be sedated.

Panel Response 18:

Because the efficacy of a product is being evaluated, a sedated animal allows this evaluation to be conducted without the effects of host defensive behaviors. An un-sedated animal confined in a 2 ft3 cage with 50-100 flies or mosquitoes feeding on it would be in constant motion. Pain associated with the bites of mosquitoes and stable flies would interfere with an accurate evaluation of product efficacy and the pain would be difficult for a test vertebrate (e.g., a dog) to endure for 1 hour. The Panel suggested reducing the number of fly exposure trials requiring animal sedation to every 2 weeks (Days 7, 28, etc.), when possible.

If starved insects are used, this should cause more rapid feeding on the test vertebrate, and sedation time and exposure to the insects might thus be reduced. Young (3 to 5 days old) stable flies are routinely starved for 4 hours before a test where feeding is involved. Stable flies will remain alive for 24 hours or more if given water on cotton ball or pads (Jones et al., 1992). Stable

60 flies will live several days if provided with a 10% sucrose solution or Gatorade (powder is easy to keep and store). Mosquitoes are not usually starved but maintained on water or 10% sucrose.

If one of the criteria for a successful test is the presence of blood in the insects’ gut, the Panel observed that small amounts of blood would be sufficient. Insects do not need to feed to repletion. Stable flies can fill up in an average of 147 seconds (Schofield and Torr, 2002). Some mosquitoes can fill up in about ≤ 3 minutes, if undisturbed.

Charge Question 19

Comment on the timing of exposing vertebrate test animals to mosquitoes and biting flies. a. Are the number of exposures for products with different durations of efficacy adequate for determining efficacy? Why or why not? b. If the number of exposures to pests can be decreased, please indicate specifically which exposures can be skipped. If efficacy should be evaluated at more time points, indicate when exposures should occur and for what product types (e.g., spot-ons, collars, and residual shampoos), and discuss the value provided by the additional time points in relation to the additional stress on test animals from additional sedations.

Panel Response 19:

a. The proposed guidelines variably specify that each test vertebrate animal should be exposed to 50-150 adult, pathogen-free mosquitoes or biting flies for one or more time points. As has been previously mentioned, the Panel questioned whether adequate numbers of all insect species listed in the proposed guidelines are readily available for this.

Furthermore, the Panel strongly recommended that the requirement for exposing vertebrate test animals to 50 stable flies be rewritten due to the extreme pain and distress caused by exposure to this aggressive biting species. Even decreasing the number of stable flies from 50 to 25 may still be excessive for an individual animal to tolerate for the proposed one-hour exposure period, let alone multiple exposures. The reduction in power and corresponding need for an increased number of vertebrate test animals caused by decreasing the pest number to 25 stable flies, and potentially even lower, should be further considered through modeling. The Panel was interested in seeing just how much decreasing the number of stable flies would increase the number of necessary test animals. Similarly, the number of control animals should be related to the expected degree of response. Since the control mortality would be expected to be very low in the case of stable flies, the Panel suggested that perhaps fewer control vertebrate animals could be used.

As previously raised by the Panel in discussing other charge questions, better definitions of “adequate infestation” of test animals, preferably specific to both pest and test animal species, would be another strategy for reducing overall sample sizes. The Panel also discussed specifying appropriate expected levels of blood feeding on control animals. The figure of 75% currently appears in the “Sample Size for Pet Product Studies” document (page 7) and the Panel found that it was reasonable to expect that this would be achieved quite quickly. This would be especially

61 true when using flies in their most active 3-5 days of age period and after adequately starving them for 3-4 hours prior to exposing them to the test animal. In both cases, better-refined definitions and study design recommendations may also increase the power of the resulting data and allow further reduction of vertebrate animal numbers.

b. If the registrant can reasonably expect that their product will last a full month (as a repellent and/or with insecticidal activity), the Panel found that it may be permissible to allow testing to occur at Day 7 to show the topically applied product has spread over the dog and then eliminate Day 14 exposures. Day 21 and Day 28 exposures should still be included. The Panel noted that this eliminates one time point (Day 14) and still proves month-long residual efficacy.

The Panel recommended that the guidelines address the duration of each experimental exposure. Many of the Panel’s concerns about animal welfare related to both sedation and exposure to biting flies could be addressed by limiting the exposure time. A 30-minute to one-hour exposure time for stable flies was suggested. The same exposure times should also be applied to the enrollment phase of these studies.

Charge Question 20

Comment on the methods for assessing mosquito and biting fly blood-feeding.

a. Are the proposed methods adequate to determine blood feeding for insects where blood feeding status cannot be visually observed through the abdomen? b. Are there other methods for determining if insects blood-feed? If so, please provide additional methods and references.

Panel Response 20:

a. The Charge Question seems to be asking if sampling half the live flies (where blood feeding cannot be determined by observation) with a blood smash test is acceptable. This question is subjective and depends on what level of risk is acceptable, particularly where transmission of pathogens is of concern. The remainder of the flies which are to be checked at 48 hours post treatment could be evaluated for blood using one of the more sensitive blood detecting techniques described below, at which time all the flies would be evaluated, thus eliminating uncertainty.

Of concern to the Panel was that EPA’s definition of “repellency” (has not imbibed blood) differs from the traditional entomological definition of repellency which is directed movement away from repellent. The proposed guidelines (i.e., on page 18) seem to confuse repellency and feeding inhibition. The guideline states:

“i. Repellent effect. Insects should be evaluated to determine if they took a blood meal. Insects that take a blood meal are not repelled. More conservative endpoints such as insect landing may also be considered but should be justified.”

62 “i. Repellency. To determine repellency only, immediately after removing animals from the cage all insects should be aspirated from the cage, knocked down (e.g., using CO2, or frozen), identified to species, crushed on a light background to assess for blood-feeding.”

The Panel noted that this does not conform to definitions of repellency put forward by several groups. Repellents cause oriented movements away from the source. In relation to feeding, a better term might be a feeding inhibitor (Deletre et al., 2016). For example, the European guidelines define repellent effect as “a product with a repellent effect will cause the parasite to avoid contact with a treated animal completely and/or to leave a host” (EMEA/CVMP/EWP/005/2000-Rev. 3).

Marchiondo et al. (2013) writes, “Two types of repellency are defined: “sensu stricto” for repellency characterized by an irritant effect, causing the tick to move away from the treated animal or leading it to fall off soon after contact with the treated hair coat within 6–8 hours and “sensu lato” for all other tick repellency (or expellency) up to 24 hours. The first, repellency sensu stricto, may be attributed to the vapor phase of a compound or irritant effect∼ through direct contact (e.g., oil of Citronella but also some synthetic pyrethroids),∼ while the second repellency (or expellency) causes inhibition of attachment or detachment of already attached ticks, (e.g., some synthetic pyrethroids or amitraz).”

Collection of unfed flies means either: 1) the flies simply didn’t feed during the test period, 2) the test material was a true repellent, or 3) the test material was a feeding inhibitor. b. The Panel noted that techniques used to determine blood feeding in cat fleas include Drabkin’s technique to determine the quantity of blood consumed by adult cat fleas (Rust et al., 2002) and should be applicable to flies. Several other procedures of determining blood are outlined in Marchiondo et al. (2013). A real-time PCR analysis of blood feeding in fleas was 10,000-fold more sensitive than the Drabkin technique (Wang et al., 2012). Wang et al. (2012) concludes, “The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of blood in single fleas, particularly immediately after colonization of the host, will provide a superior tool for studying flea-host interactions, flea-borne disease transmission, and flea control strategies.”

Charge Question 21

To reduce the amount of vertebrate animal testing, we propose to allow assessment of repellency and mortality within the same study by evaluating half of the insects for repellency before determining mortality. a. Discuss the viability of evaluating both endpoints in the same study by using a subset of the total mosquitoes to determine repellency and provide any additional methods that would determine both endpoints within the same study. b. The proposed endpoints for mortality are secondary to repellency (i.e., for mortality to be assessed repellency should be observed first) because a mosquito or fly that is not repelled may blood-feed and could potentially transmit pathogens that cause disease. Discuss the potential

63 value of assessing mosquito mortality that are not also repelled. Should mortality of mosquitoes that blood-feed be considered a viable endpoint to show efficacy of a repellent-product? Why or why not? If yes, provide specific rationale as to why.

Panel Response 21:

a. The Panel observed that if the timing of endpoint determinations is different, it seems reasonable to study both repellency and insecticidal activity against mosquitoes and biting flies during the same test. Repellency is immediate; knockdown/mortality occurs up to 48 h after exposure. One is, however, looking to see that none of the insects had a blood meal if the products are to be effective in either category.

As stated in the proposed guidelines (OCSPP 810.3300) Section (l) (4), efficacy endpoints, the “endpoints (e.g., blood feeding, mortality) should be selected with respect to the intended use pattern for the product and mode of action of the a.i.”

An active ingredient (a.i.) could have only: (a) a repellent effect; (b) a repellent and a toxic effect (e.g., permethrin); or only a (c) cidal effect (e.g., fipronil). Thus, the a.i.’s mode of action and its intended use pattern should drive the testing methodology and the efficacy endpoints.

The goal of insecticides/repellents applied topically to dogs/cats is to protect the animal from the biting adult stages of mosquitoes and biting flies and to interrupt blood feeding and thus significantly reduce the potential for disease transmission. Assessment of blood feeding is the critical thing to assess in live, knocked down, moribund and or dead insects after being exposed to a topically treated dog/cat. Thus, if based on blood feeding ≥90% of the insect species being assessed are alive and have no blood in them, then this a.i. has a mode of action that should be considered a repellent. If the a.i. has both a repellent and a mortality effect, then ≥90% of the recovered insect species whether they are alive, dead, or moribund should have no blood in them since the a.i. either repelled them and/or killed them before a blood meal could be taken.

Therefore, the Panel concluded that looking at subsets (e.g.-removing half of the insects) of the total number of mosquitoes and biting flies and looking for repellency before assessing mortality is not needed. Blood feeding should be assessed in all recovered insects and this should drive the label claim (use pattern). Depending on the extent of blood feeding the product can claim to be a repellent or an insecticide/repellent or a standalone insecticide that works so fast that insects that land on a treated dog/cat are knocked down or killed so quickly that they can’t obtain a blood meal before dying or becoming moribund and subsequently dying.

The Panel suggested that some of the automated methods be used to detect the presence of blood in an insect to confirm if blood feeding has occurred. For example, confirmation of a consumed blood meal could be performed using an enzyme-linked immunosorbent assay (ELISA) following the procedure described by Beier et al. (1988). There are likely other references that could be cited for these methods as well. b. The Panel noted that blood feeding should not occur if the test product is effective either as a repellant or as a knockdown/mortality agent. As noted in the Charge Question, repellency is primary and needs to be observed before mortality is determined. However, knowing if insects

64 die quickly could be important. If the biting insect is not repelled and blood feeding occurs, there is potential for disease transmission unless killed quickly. Therefore, even if mortality occurs after insects are on the test animals, mortality/knockdown could also be considered as a viable endpoint.

The Panel did not agree with the assumption that the proposed endpoints for mortality are secondary to repellency. As stated above, the mode of action of the a.i. is going to define the endpoint (and label use pattern) and the ultimate goal of the a.i. is to significantly reduce or totally inhibit any blood feeding by mosquitoes/biting flies exposed to a treated dog/cat. In this Charge Question, the Agency states that “a mosquito or fly that is not repelled may blood-feed and could potentially transmit pathogens that cause disease.” This may be true but if the a.i. has a predominant killing or mortality effect, the insect can be killed or incapacitated so quickly after landing on the host that blood feeding doesn’t occur.

To respond to the first bullet point in Charge Question (b),the Panel would again refer the EPA reviewers to the previous discussion points that blood feeding should be assessed in all recovered insects and this should drive the label claim (use pattern) as a repellant, and/or an insecticide/repellent or a standalone insecticide that works so fast that insects that land on a treated dog/cat are knocked down or killed so quickly that they can’t obtain a blood meal before dying or becoming moribund and subsequently dying.

To respond to the second bullet part of Charge Question (b), the Panel concluded this question would only apply to an a.i. with a mode of action that has both repellency and a mortality (toxic) effects. Under this scenario, if, based on blood feeding, ≥ 90% of the insect species being assessed are alive and/or dead (demonstrated mortality) and have no blood in them, then this a.i. has a dual mode of action (repellent combined with mortality effects, such as permethrin). Thus, if ≤ 10% of live/dead insects do have blood detected in them, then the 90% of insects that don’t have blood detected will be making a significant impact on reducing disease transmission and reducing irritation due to the biting process by the insects and potential immune reactions at the bite site. No product in a real-world biological system will consistently provide 100% efficacy, thus, the Panel observed that having a minimum threshold of ≥ 90% seems appropriate.

Additional Comments related to Assessing Efficacy in Mosquitoes and Biting Flies

One committee member suggested that the EPA should consider adding a new section to this part of the revised guidelines that assesses knockdown/mortality on topically treated dogs/cats, regardless of their blood feeding status. This would be analogous to efficacy assessments performed on treated dogs/cats infested with fleas and ticks where they ingest blood prior to being killed by the topically applied a.i. The primary concept is to kill these insects in their local environment which negatively affects reproduction and helps to interrupt the life cycle of the pest and reduces the overall population of the insects in the pet’s local environment and also potentially reduces human exposure to bites. It has been documented in the scientific literature that some mosquitoes do not move significant distances from where they breed, thus killing local populations of these insects can be just as important as preventing blood feeding. Since only small numbers of biting insects are actually carrying infectious agents, the impact of significantly reducing blood feeding and reducing transmission of infectious agents is minimal.

65 Charge Question 22

Efficacy testing is based on the specific label claims proposed for product marketing. Common claims are “kills,” “repels,” and “controls” (i.e., residual efficacy). For most pesticide products (e.g., direct sprays and residual applications) the typical endpoint to support claims of “kills” or “controls” is based on dead arthropods. To evaluate dead arthropods for shampoo products we propose pests should be shampooed in a tub with a screen over the drain to keep fleas and/or ticks in the tub. This may differentiate the pesticidal effect from the effect of pest removal, which could also occur by using a non-insecticidal shampoo. Discuss the value of using counts of dead arthropods vs. using counts of live arthropods on treated compared to untreated vertebrate animals to determine different endpoints (e.g., kills).

a. Is assessing live arthropods on treated vs. untreated animals an adequate and practical endpoint for determining mortality? Why or why not? b. Are there alternative methods for determining mortality for shampoo products? If so please provide the methods and supporting references.

Panel Response 22:

a. The Panel noted that the efficacy or effectiveness of treatment is appropriately determined by comparing the proportion of pests killed by the treatment (i.e., proportion of living in the control group - proportion living in the treatment group) with the proportion of pests living in the control group (Abbott, 1925). Determining the proportion of live pests in the control group, and the proportion of live pests in the treatment group, requires counts of living pests on each vertebrate animal and knowledge of the total number of pests applied to each vertebrate animal. Ideally, all the arthropods applied to the vertebrate animals would be counted and classified as live or dead to assess the proportion of live arthropods in the control or treated group. Counts of the dead arthropods would support the claim of killing for the tested product. However, it is unlikely that the total number of arthropod pests applied to each vertebrate animal will be recovered during the counting process. Fleas originally applied to a cat or dog may be ingested or hide within or upon a vertebrate animal. Accordingly, it has become standard practice to use Abbott’s formula with live arthropod counts.

In the proposed guidelines, approximately the same total number of arthropods is applied to each animal. It is reasonable to assume that the total number of pests on the vertebrate animal does not change appreciably between the time of application and the time of counting. If all the arthropods originally applied to the vertebrate animal are not recovered, the arthropod counts could be subject to measurement error in counting, which more likely would be an issue of undercounting. Bias often becomes apparent when more than 5%-10% of the data are missing. If the extent of undercounting varies considerably between the control and treated groups, or if the application of the treatment leads to very small counts in the treated group (e.g., 0 to 5 pests remain on the animal after application of treatment), the efficacy assessment could be biased. The Panel recommended that measurement error in counting should be investigated by the Agency or taken into account in the modeling framework as discussed in more detail in the response to Charge Question #9.

Given the discussion above, assessing live arthropods in the bathing tub is an adequate and

66 practical endpoint for determining mortality. However, the Panel noted that the practicality of this recommendation includes the considerations discussed below.

Tick placement for pesticidal shampoo products should follow the same suggestions previously made for mortality and repellency studies. Ticks should be placed on Day-2 (not Day-1) to permit adequate attachment time before the treatment is applied.

Any live pests found in the plugged tub should be added to the total live arthropod counts. The vertebrate animal should be allowed to fully dry for 24 hours before counting the remaining fleas or ticks. Fleas are, however, more likely to be washed off than ticks, while ticks are more likely than fleas to remain attached to a vertebrate animal and not removed by the act of shampooing. Therefore, the Panel recommended that the EPA guidelines should use different approaches to count the fleas and ticks remaining on the vertebrate animals. A fine-toothed comb should be used to recover fleas; ticks still attached to the vertebrate animal may be discovered by carefully palpating the animal.

The Panel noted that more specific guidelines may be needed for how to assess the mortality of fleas or ticks collected after being washed off or recovered from the vertebrate animal after the shampoo treatment as this can be a difficult endpoint to assess. The determination of tick mortality (whether the tick is attached or unattached to the vertebrate animal) can be evaluated by breathing on the pests and looking for movement, but there may be a delayed response.

Fleas and ticks may not die immediately after they are removed from a vertebrate animal, but these pests might die soon after being removed. The death of the removed arthropods may or may not be due to the pesticidal ingredient in the test product. The death of fleas and ticks after detachment from a vertebrate animal may occur because they no longer have access to blood or are not maintained in adequate environmental conditions. Fleas and ticks need appropriate levels of humidity and temperature to ensure that their mortality is not due to environmental factors alone. Conversely, the pest may be moribund and not recover due to the pesticidal treatment. The mortality of the recovered arthropods should be evaluated after the pest is removed from the vertebrate animal to determine if the pest will recover or die from treatment within a short timeframe. Due to the vulnerability of the arthropod while off the animal, the mortality of fleas should be evaluated within 24 hours. Because ticks might take up to 48 hours to recover, the mortality of ticks should be evaluated within a time window of 24-48 hours.

Soaps have a mortality factor against fleas and ticks. Shampoo may remove the waterproofing ability of the pest and make the arthropod more vulnerable to desiccation. Therefore, the Panel recommended that the guidelines should specify that the appropriate control to compare against the pesticidal shampoo is the shampoo without the active pesticidal ingredient. The use of a control shampoo without the active ingredient is needed to determine whether the pesticidal agent had a greater effect than that of the shampoo alone.

Each vertebrate animal should only be shampooed according to the product claims, and not continuously. Shampoos do not usually have long-term residual efficacy against fleas and ticks. A study by Beugnet et al. (2012) indicates that weekly shampooing of dogs reduced the number of fleas compared to controls, but the overall reduction in flea number never rose above 79.2%, which would not meet the EPA required efficacy of 90.0%. The Beugnet et al. (2012) study also

67 showed that missing a weekly shampoo resulted in a decrease in efficacy from 68.2% to 34.8%. To assess residual activity of the shampoo on the vertebrate animal, it is important to wet and bathe the vertebrate animal, and then allow the vertebrate animal to fully dry for 24 hours before re-infesting with fleas or ticks on the following day. Counts for fleas should be made on the day following re-infestation and counts for ticks should be made two days after re-infestation to confirm that the active ingredient has not been removed from the animal. The Panel recommended that the guidelines should specify that a vertebrate animal should only be infested when their fur is dry (not with wet fur). In addition, it is not practical to try to comb count a vertebrate animal with wet fur.

b. The Panel was not aware of any alternative methods that are adequate for determining mortality of fleas and ticks from shampoo products. However, the Panel recommended that the Agency guidelines be open to reviewing and including alternative methods for determining mortality of pests for shampoo products as these new alternative methods proceed through development and become available.

In addition, the Agency should review the process that the U.S. Food and Drug Administration (FDA) uses to assess and approve topical insecticidal products available as prescription and over- the-counter drugs used on people and on animals. The FDA has approved insecticides as topical lotions and shampoos for human use against lice and topical insecticidal solutions for pets. Some useful assistance could be found by reviewing FDA protocols for approval of these drugs.

INSECTICIDAL SHAMPOO PRODUCTS (SECTION (M))

Charge Question 23

Typically, efficacy testing for pet products is conducted with a treated group and an untreated control group. To determine efficacy of shampoo products, the mechanical removal of arthropods during the shampoo process should be considered. Efficacy of a product would therefore be considered in relation to a group treated with non-insecticidal shampoo. In this guideline we propose a non-pesticidal/non-medicated shampoo as the control when testing insecticidal shampoo products.

a. Please discuss the value of using a non-pesticidal/non-medicated shampoo as the control i. Does this provide valuable additional information about efficacy beyond an untreated control? ii. If so, please provide an appropriate methodology for the comparison of treatment vs. control groups, including references. b. Should an untreated control also be provided for comparison with the non-pesticidal shampoo? If so, please describe the most appropriate untreated control (e.g., water only, massaging the animal). What is the value of the untreated control and is the informational value of an additional treatment group offset by the increased number of vertebrate animals used for testing? c. If an untreated control group is included, how should efficacy be determined? Which control group (the non-insecticidal shampoo control or the untreated control) should be compared with the treated group to determine efficacy?

68

Panel Response 23:

Charge Question #23 focuses on the utility of non-pesticidal/non-medicated shampoo control for determining the efficacy of its pesticidal/medicated shampoo counterpart. Unlike the inert ingredient(s) in other (i.e., non-shampoo) pesticidal products, the ingredients of shampoo are not “truly inert.” Specifically, shampoo typically contains a variety of ingredients; these may include water, surfactants (surrounds dirt and oil so water can rinse them away), foaming agents (provide the "suds”), an acid (to balance pH), silicones (create smoothness and add shine), polyquaternium (like a fabric softener), panthenol (fatty alcohols and oils for hydration), fragrances, and preservatives. Since arthropod ectoparasites are relatively small and have large surface area to body weight ratios, anything that disrupts their waterproofing (epicuticular lipids, especially hydrocarbons) could cause desiccation and knockdown even in a relatively high humidity environment. Also, the mechanical action of shampooing can remove the ectoparasites from their hosts. In other words, the non-pesticidal/non-medicated shampoo may kill the ectoparasites or hamper their ability to adhere onto their hosts. In fact, non-pesticidal/non‐medicated shampoo was shown to reduce the flea count on dogs (Beugnet et al., 2011). However, in the same study by Beugnet et al. (2011), unlike the pesticidal/medicated shampoo employed, weekly shampooing was needed to maintain the flea count reduction (i.e., continuous control), and the efficacy of shampooing alone on the flea count reduction was shown to be below the performance standard (i.e., >90%) required by different regulatory agencies including the U.S. EPA. Hence, the value of a pesticide in a pesticidal/medicated shampoo product is to provide the continuous control (an important attribute of the product’s commercial viability) that cannot be achieved through “treatment” by shampooing only. Accordingly, the utilities of the shampoo control are to control for the short term (i.e., non-continuous) pesticidal action of shampoo “inert” ingredients, as well as to assess the efficacy of pesticidal/medicated shampoo for providing the continuous control. Following a lengthy discussion, the general consensus among most Panel members, except for one Panel member (please see the last paragraph), was that a non-pesticidal/non-medicated shampoo control (i.e., formulation blank) should be used for determining the efficacy of the corresponding pesticidal shampoo product.

As mentioned previously, a completely untreated control (i.e., host receiving no shampooing treatment) is not an appropriate control for the efficacy determination of pesticide in a pesticidal/medicated shampoo product because it does not control for the short-term pesticidal effect of shampoo “inert” ingredients. Similarly, control with water only or messaging “treatment” (i.e., placebos) provides no more information than the non-pesticidal/non‐medicated shampoo control (Taenzler et al., 2016). Accordingly, the inclusion of such untreated control(s) will only increase the number of animals used. Most Panel members concluded that for evaluating the performance of the pesticide in a pesticidal/medicated shampoo product, only two groups are needed: a treatment group with the pesticide added to the pesticidal/medicated shampoo product and a shampoo control group with the formulation blank. Because the data collected are counts, a nonparametric test such as Mann-Whitney rank-sum test can be used to compare the treatment versus the control groups (Glantz, 2012). In conclusion, the efficacy of pesticidal shampoo product should be determined relative to a non-pesticidal shampoo, or as close a formulation as possible to the candidate pesticidal shampoo.

As mentioned above, one Panel member, suggested that the decision on “what is the appropriate

69 treatment of control group?” depends upon the question(s) to be answered and the product claims; specifically, what is (1) the efficacy of the product as sold (shampoo with the pesticide added), (2) the efficacy of the addition of the pesticide to a non-medicated shampoo base, and (or) (3) the need to know the efficacy of each separately - the non-medicated shampoo base alone and the pesticide alone. To address these questions, this Panel member proposed the following experimental designs (in the same order as the questions raised) for the product efficacy testing: (1) the use of water as the control group and the shampoo containing the pesticide as the treatment group, (2) the use of base shampoo without the pesticide for the control group and the shampoo containing the pesticide as the treatment group, and (3) the use of a three group design: water, the base shampoo and the base shampoo containing the pesticide. The last may need additional animals since two effects are being tested.

SIMULATED ENVIRONMENTAL CONDITIONS (SUNSHINE, WATER EXPOSURE AND SHAMPOOING CLAIM) (SECTION (N))

Charge Question 24

Discuss whether the timing for infesting vertebrate test animals after water exposure are adequate.

a. Are the number of infestations presented in the guideline adequate to support bathing and water immersion claims? b. Discuss appropriate timing of infestations after vertebrate test animals are immersed in water. c. Are the methods for determining exposure to sunlight adequate? If not, please provide detailed methods for evaluating the effects of exposure to sunlight along with supporting references.

Panel Response 24:

The Panel recommended that the reinfestation be specified as 24 hours after each exposure to water or bathing (e.g., bathing at day 6 with reinfestation at day 7, bathing at day 13 with reinfestation at day 14, etc.). In this way the animal will be dry prior to the reinfestation, even for animals with thick coats of fur. The Panel recommended the use of a “non-medicated” shampoo as the control, because this represents a baseline of control that a pet owner could achieve on their own without a shampoo containing a pesticide. The Panel did not recommend the use of the additional water exposure control, as the value of such studies does not justify the number of vertebrate animals needed for this. a. Yes, the Panel found that the number of infestations presented in the guideline are adequate to support bathing and water immersion claims. b. The Panel recommended that the reinfestation be specified as 24 hours after treatment. In this way the vertebrate animal will be dry prior to the reinfestation, even for animals with thick coats of fur. c. It is difficult to state unequivocally if the methods for determining exposure to sunlight are adequate or not. The current protocol does not really clarify if the product will be stable in

70 sunlight. The vertebrate animals will be outside for part of the day but will be provided shade. Will the animals go into the sunlight? How long will they go into the sunlight? The UV index differs geographically, and this would potentially impact the results. Monitoring the sunlight each animal receives (using an on-collar monitor) would probably be necessary to standardize exposure and back up claims that a product continues to work after some specified amount of sunlight exposure. The Panel recommended that if the current approach is used, that the host be exposed to sunlight for two hours per day. This would potentially offer some standardization, although variation in weather may make such a regulation difficult or impossible to achieve. It would be of some additional value if the daily weather (average daily temperature, high temperature, low temperature, sunshine, rain, etc. was also recorded). While such information may not alter the approval process, these variables would help understand differences that might occur between studies.

The Panel noted that under the current proposed guidelines, vertebrate animals will spend prolonged periods outdoors and that some of them (i.e., the controls) could pick up additional or new pests while outdoors. As such, it would be valuable if the total number of pests were reported on the controls and treated animals at the end of the study, not just the number of pests that the animal was infested with. If the outdoor activity of the pets results in accumulation of additional pests of the same species that they were infested with, the number of control animals might need to be increased to get a better assessment of this.

The Panel also considered using artificial sunlight on caged animals. This has potential for standardization of the “sunlight” exposure but requires protection of the animal’s eyes and raises other animal welfare concerns. As such, the Panel did not recommend this approach.

The Panel also suggested that EPA consider claims for sunlight stability using a post-approval field study with client-owned dogs under real world conditions with natural pest infestations in different geographic areas of the US and with a significant portion of the enrolled dogs in the study that spend at least half of their time outdoors with exposure to sunlight (collar monitors to record sunlight would be beneficial).

Given the difficulty in understanding the amount of sunlight the animal is exposed to, the Panel recommended consideration of non-vertebrate testing of treated fur. The treated fur could be exposed to sunlight and the residue of the active ingredient determined after different days or weeks of exposure to sunlight.

Suggested editorial changes:

The Panel noted that in section n(1)(i) for products with efficacy claims of 2-4 weeks, animals should be bathed on Days 6 and 20 so infestations can occur on appropriate Days 7 and 21. The rationale is that the dogs that are wetted should be allowed to dry for ca. 24 hours prior to being re-infested the next day.

Section (n)(1)ii. The Panel suggested the following revision “fur and skin through submerging (except the head) or showering the animal”

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