SAP Review of Proposed Guidelines for Efficacy Testing of Topically Applied Pesticides Used Against Certain Ectoparasitic Pests on Pets (June 11-14, 2019)

FIFRA SAP Peer Review
EPA Response

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))a
    iii. Mosquitoes and biting flies (section (l))
    iv. Insecticidal shampoo products (section (m))
    v. Environmental conditions (section (n))

Panel Recommendations:
1
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 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).  PG18-19
The Agency acknowledges the SAP comment; however, the required pest species for testing will not be included in the guideline as they will be included in a product performance rule that will publish separately.
2
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.  PG 19
The guideline was updated to reflect that if the fly species desired for testing do not have lab colonies, they may be obtained from wild populations.
3
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.  PG 19
The outcrossing recommendations listed in the tick and mosquito sections was deleted.
4
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.  PG 19
Based on the response to SAP comment #3, the outbreeding recommendation will no longer be included.
5
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.  PG 19
The specification of "pathogen-free" has been revised to "free of vector-borne pathogens" to be more specific.
6
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.  PG 19
The guideline currently already states to sedate dogs and cats for mosquito and biting fly tests. Based on comments from the public that some sedations do not diminish pain, the Agency added a statement to include the use of pain relievers.
7
The Panel also noted several issues that need clarification relative to the welfare of the pet animals. 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.  PG 19
The guideline has been revised to include the use of cones/E-collars, however specifying only during the periods between infestation and comb out counts, so that most of the study days the animals can groom.
8
The Panel also noted several issues that need clarification relative to the welfare of the pet animals. 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.  PG 19
EPA did not revise the guideline based on this comment because this topic is outlined adequately under section "v. Housing of test animals."
9
The Panel also identified some specific, detailed recommendations regarding clarification of terms  --  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).  PG 20
The counts in the guideline are allowed up to 48 hours (72 hours for the first time point). Therefore, to gain a kills or controls claim, the pests should be dead by 48 hours after exposure. No changes to the guideline are necessary.
10
The Panel also identified some specific, detailed recommendations regarding clarification of terms  --  Several terms needed clearer definitions:
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.  PG 20
Based on information and comments provided by the SAP and public, the Agency removed the term "engorged" in the guideline and the endpoint of "engorgement" was changed to "live ticks on treated animal" vs. "live ticks on control animal" by the specified time points. This is also discussed in response to question 14.

With respect to adding the tick species required for testing, this question was responded to in comment #1.
11
The Panel also identified some specific, detailed recommendations regarding clarification of terms  --  Several terms needed clearer definitions:
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.  PG 20
The guideline was clarified to include that retention rates of individual animals will be used to qualify an animal into the study, however, during the study, retention rates are based on group means.

In regard to the "clarification as to which species of fleas should be considered" the guideline already states that Ctenocephalides felis (cat fleas) are the species to be used.
12
The Panel also identified some specific, detailed recommendations regarding clarification of terms  --  Several terms needed clearer definitions:
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.  PG 20
The definition of a negative control was included in section (d)(1)viii.b of the guideline. However, it was also added to the definition section, with the revision of deleting "except for studies testing shampoo products."

13
The Panel also identified some specific, detailed recommendations regarding clarification of terms  --  Several terms needed clearer definitions:
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.  PG 20
Definitions have been added or clarified as suggested.
14
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  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.  PG 20
Currently, for general mortality tests for fleas, the endpoint is counting live fleas on the treated animals vs. the control animals.

Per SAP recommendations for "speed of kills claims," the guideline has been revised and will no longer recommend counting dead fleas. The guideline will be revised to align with the EMA guideline, which allows testing for claims in less than 48 hours using the same methods already provided at the same thresholds.
15
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
The guidelines should clarify whether the group means or counts on individual animals meet the minimum retention rate (adequacy of infestation).  PG 20
The guideline has been updated to clarify that individual pest counts should be used to qualify each animal to the study. However, once the study has begun, the group means are used to identify the minimum retention rates in the control groups.
16
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 20
Since determining the sex of fleas is difficult and unnecessary for testing, the sex ratio recommendation has been deleted. This revision is consistent with the European guideline.
17
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 21
The timing of the infestations and pest removals are all outlined in the guideline per the pest or endpoint desired.

The guideline was revised by deleting the "engorged" tick endpoint per SAP and public input and changing the end point to "live ticks on treated" vs. "live ticks on control."
18
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 21
Based on input from the SAP and citations, the guideline was revised to:

"Mosquitoes should not be blood fed prior to testing but can be provided sugar water up until trials. However, 4-24 hours of sugar water starvation is recommended (Franc and Bouhsira 2009, Tiawsirisup et al. 2007)."

"Biting flies should be starved with justification for the amount of time chosen."

19
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 21
Based on SAP and public comments, hand counting options for fleas was removed from the guideline since they are considered unreliable. Hand counting options will remain for ticks, which move differently than fleas and are therefore easier to hand count.

The guideline was revised to allow comb counts for fleas at earlier time points.  The fleas removed by comb counts should be counted and then replaced onto the animal with the stipulation that the technician avoid the treated area (so not to mechanically spread the solution). Controls should be handled in the same manner.
20
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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).  PG 21
The current end point for mortality testing for both fleas and ticks and the revised endpoint for repellency for ticks is "live pests on treated animal" vs. "live pests on control animals." Therefore, the previously stated endpoint of blood feeding or engorgement for ticks was removed as the SAP pointed out that it was impractical, and ticks would not have been engorged (and possibly not even attached) by 48 hours. There is no blood feeding endpoint for fleas.
21
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 21
The guideline was revised to "ticks should be placed on the host animals' bodies away from treatment sites."
22
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 21
The guideline was revised to clarify that at the final comb count for an infestation period, all ticks should be combed out and removed.
23
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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).  PG 21
Based on the SAP comment, the guideline has been revised to state that, "Ticks should be of similar age post-molt to the adult life stage."
24
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 21
The dosing used in the studies are based on mg/kg of the animal for efficacy. Companion animal safety studies are also required for pesticide registration but are not relevant for this efficacy guideline. No changes to the guideline are necessary.
25
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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 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.  PG 22
The guideline has been revised to: "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, or for 30 days after the longest claimed period for products with efficacy claims lasting longer than 3 months. One exception is for products that are known to have very short acting periods and no residual efficacy, such as pills that kill fleas on pets within 24 hours. Products such as these may be given at least 1 week prior to the initiation of testing to be sure they have been cleared completely from the animals' bodies."

26
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --  Procedural Concerns
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.  PG 22
No specific recommendation was provided by the SAP here; the overall issue will be responded to under Charge Question 7.
27
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures  --
Procedural Concerns
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.  PG 22
Based on SAP and public comments, the following statement has been added to the guideline: "For infestations using painful or aggressively biting mosquitoes or biting flies, the animals should be administered pain medications or a drug that has proven analgesic and sedative properties."

28
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures (including new protocols and emerging techniques)  --
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).  PG 22
EPA has included a statement in the guideline indicating that protocols may be submitted for new approach methodologies (NAMs), and that protocols proposing new approach methods, especially those methods which do not rely on utilizing vertebrate animals for testing, should be submitted to EPA for review before the study begins.
29
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures (including new protocols and emerging techniques)  --
Concerns with New Protocols and Emerging Techniques
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).  PG 22
The term "in vivo" has been revised to specify testing "utilizing vertebrate animals" in the guideline.
30
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures (including new protocols and emerging techniques)  --
Concerns with New Protocols and Emerging Techniques
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.  PG 22
EPA is not including a database at this time but will consider this as in-vitro methods are developed.
31
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures (including new protocols and emerging techniques)  --
Concerns with New Protocols and Emerging Techniques
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.  PG 22
EPA acknowledges that field tests have strengths but will not include field testing for these products in the guideline as they are conducted with client-owned pets and not ideal for testing as previously submitted field studies have not yielded useful data. Issues include inadequate or inconsistent pest pressure, lack of appropriate controls, and inaccurate pest counting by owners. However, protocols for field studies may be submitted if desired.
32
The Panel also identified some specific, detailed recommendations regarding clarification of .... procedures (including new protocols and emerging techniques)  --
Concerns with New Protocols and Emerging Techniques
Some Panel members suggested that an unbalanced study design be considered when statistically 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.  PG 22-23
EPA added a sentence in the protocol development section to indicate that novel methods to reduce animal numbers may be considered on a case by case basis. Reducing the number of control animals is a scenario that could be considered; however, at this time EPA does not have evidence to support a recommendation for a study design with a reduced number of control animals.

Public Recommendations (docket ID # in parentheses):
33
"Recommend adding definitions for "negative control treatment" and "placebo control"." (0008)
The definition of a negative control was included in section (d)(1)viii.b of the guideline. However, it will also be added to the definition section, with the revision of deleting "except for studies testing shampoo products."
A definition of placebo control is no longer necessary as the recommendation to use a placebo will be removed from the guideline.
34
"Challenge/infestation/exposure (c)(1)  -  suggest adding definition to include "or close to" (the most important being the successful infestation in the control group) as long as it is representative of what happens in nature. For example, most ticks will infest dogs by climbing up from the ground or grass. If ticks are applied on the dog, there is a risk to place the pest where the product was applied." (0008)
For mortality testing, the guideline has been revised to indicate that fleas and ticks should be placed on the host animals' bodies away from treatment sites.

For repellency testing, the guideline has been revised to "...placed inside a cage but not on the animal" to test if the tick moves onto the pet.
35
"Comb out (c)(2) statement on removal should include live, moribund and dead" (0008)
The guideline has been revised adding "moribund" to this statement.
36
"Control/residual (c)(3) states that treated animals should be tested at least 24 hours after application. Timing should match the proposed registrant's desired label claim (i.e., if the target label claim is efficacy after 8 hrs., testing at that time interval should be acceptable). Suggest removing time restriction." (0008)
The guideline is defining here what a residual product is (one that works longer than 24 hours). Time points shorter than that would be for "kills" claims, but not "controls" claims. No change to the guideline is necessary.
37
"Hand count (c)(5) The statement "Live pests are not removed during a hand count" is true for ticks, not for fleas. For example. with studies on fleas, the parasite is counted and then replaced on the animal. Recommend removing or adding "generally not removed."" (0008)
The guideline was revised to remove the hand counting option for fleas and therefore this comment will not be addressed.
38
"Knocked down (c)(7) Unclear how this definition differs from moribund. Recommend adding additional detail to clearly differentiate between both terms.... Moribund (c)(9) Unclear how this definition differs from Knocked down. Recommend adding additional detail to clearly differentiate between both terms." (0008)
The guideline was revised to define the term knockdown in terms of "the action of rendering an arthropod incapable of coordinated movement or unable to right itself following exposure to a pesticide product." All other uses have been deleted.
39

"Lowest labeled dose (c)(8) The definition appears to be overly prescriptive for guidelines. There is concern that there will be limitations upon the final packaging (pipette vs. syringe) and that the study will not evaluate the full product. What about our actual claims, will it be revised (lost) if we do have min dose studies for the relevant parasites? Recommend revising to address these concerns." (0008)
The definition for the lowest labeled dose is to determine and test the most conservative dosing on the product label and apply that dose to all animals in treatment group. It will have no bearing on the final packaging or application method (pipette vs. syringe) or the formulation. Claims will be based on efficacy achieved with the lowest labeled dose, which is how efficacy testing is conducted for all product types. No change to the guideline is necessary.
40
"Should other companion animals such as rabbits and ferrets be considered under this guideline as well?" (0026)
Other companion animals such as rabbits and ferrets are rarely tested for efficacy claims. If a company would like to add them to a label, they may discuss with the Agency or submit a protocol. No changes to the guideline are necessary.
41
"Specific values for efficacy were not provided (e.g. >=90% corrected treatment mortality). We would like for efficacy thresholds to be included in the document" (0008)
Specific thresholds for efficacy will be included in the upcoming product performance rule.
42
"i Test materials and treatments :: Ambient temperature and humidity are not defined. Is there an acceptable temperature range?" (0008)
The comment seems to be in reference to the following statement in the guideline: "Test materials should be stored at ambient temperature and humidity for at least one day before use." Since the study conductor will have the information for and be responsible for how to handle the product, it is not necessary to add that into the guideline.
43
"Housing of test animals. :: Group housing of animals of the same treatment groups during periods of non-infestation should not be a requirement. Cross-contamination could occur for spot-on or collar applications. Further, collars could be removed and/or consumed by other animals when group housed in a laboratory situation. In addition to effects on treatments, compatibility issues could develop between the animals within the housing group (e.g., fighting, antisocial behavior) presenting a concern for animal welfare. Group housing also allows bias into the study design. Standard environmental enrichment programs provide consideration to animal welfare aspects related to individual housing." (0008)
The guideline indicates that animals "may" be housed together and is therefore at the discretion of the study director. No changes are necessary for the guideline.
44
"v. Selection and allocation of animals :: Body weight could also be considered for allocation (before pest attractiveness), especially if using a prescribed label dose." (0008)
Since the dosing for testing of products (with the exception of pet collars) is on a mg/kg basis and exact for each dog's weight, there is no need to consider body weights for allocation. Pest attractiveness is the most relevant factor. No change to the guideline is necessary.
45
"vii. Test organisms. If pests are to be certified as free of disease-causing pathogens, it should be outlined as to by whom and what certification is acceptable. Without these specifications, the requirement should be omitted. Certification of pathogen free colonies is a difficult unit to measure (e.g., who does it, how often, when, what does the certification look like, how long is the certification valid). Is EPA aware of any instance where established colonies of arthropods have been diagnosed with a pathogen? Certification is an unnecessary hardship with little apparent benefit. Recommend removing requirement." (0008)
The recommendation for pests to be "certified disease-free" was removed from the guideline.
46
"viii. Representative sampling. c. Positive control  -  Recommend changing "should not be used" to "is not recommended". There may be cases where a positive control is necessary" (0008)
A positive control is not warranted for any of the tests, however the guideline will be revised to read "a positive control is not recommended."
47
Re: disease free: "Does this refer only to zoonotic pathogens? How should strains be "certified?" Other regulatory agencies, such as FDA Center for Veterinary Medicine, do not require certification. Such a requirement may be excessive and impractical in terms of cost for some CROs. Additionally, AHI recommends wild-caught ticks be allowed to be used in studies. It will not be possible to certify wild-caught ticks... as wild-caught ticks more closely represent current circulating parasites... Also, some parasites such as Ixodes scapularis has a life cycle of 2 years. How would one get them to be parasite free?" (0026)
Based on numerous SAP and public comments, the specification of "pathogen-free" has been revised to "free of vector-borne pathogens" to be more specific.
48
"AHI recommends that the decision to use untreated controls vs placebo controls should be flexible and made based on the type of product, where the study is conducted, study design, etc. Ideally the guideline would allow for either." (0026)
Variation of the study designs described in the guideline may be warranted based on the reasons presented by the commenter; it is recommended that before such an experiment commences, the Agency be contacted to discuss the protocol deviations or that a  protocol be submitted for approval. No changes to the guideline are necessary.
49
"Does EPA want to review all efficacy protocols or only those associated with novel testing methods and when testing new species?" (0026)
The guidelines are being published to outline established testing methods for certain typical tests and claims. If a company would like to run different types of tests or use novel testing methods, they have the option to submit a protocol to gain written approval or comments, if desired, but submitting protocols prior to testing are not typically required.
50
"Amendments typically cover any change to an activity or task that has not started yet, rather than the initiation of the study itself. (As an example, tick counts on Day 14 could be amended between the start of the study and before Day 14. Documenting such changes by amendment is important as it indicates that the change was planned prior to occurrence rather than as a deviation, which implies that the change was not planned but occurred without being planned" (0026)
All amendments and deviations to the study are to be documented and included in the final report.
51
"Test animals. A general requirement that animals should not be treated with any substance that could affect the results of the study for at least 3 months prior to initiating the study is excessive. Standard hygiene and husbandry practices following a study include bathing and cleaning the environment where the previously treated animals were housed. The "quarantine" period should be changed to 30 days to allow any animal that might be still be contaminated to be identified during the prequalification challenge. Prequalification will show whether an animal still has remnants of a previous treatment. It is the responsibility of the registrant, with due knowledge of their active and formulation, to require a longer wash-out period if required." (0008)

 See response to comment #25 above.
52
Re: Section (g)): "Is this level of detail needed in the final report? It can be documented in the raw data but details of how the parasites are reared and maintained would be cumbersome in a final report." (0026)

Yes, these basic items should be included in the final report as they are important information to assess the validity of the test and are not considered cumbersome as they are already included in many studies submitted already. No changes to the guideline are necessary.

53
"Why are animal body weights required post study completion? Change in body weight is not typically a study variable for an ectoparasite efficacy study." (0026)

It is standard to include some basic health evaluations in efficacy studies such as body weight pre- and post-study. No change to guideline is necessary.
54
Re: Section (g)): "Does this imply that the study protocol and amendments and deviations need to be appended to the final study report? As other agencies do not require this, it will be cumbersome to do this. It would be more flexible and effective to include these documents in the submission as stand-alone documents instead of incorporated into the final report." (0026)

Yes, all amendments and deviations should be appended in the final study report and should not be in a standalone document. This would ensure that all parts of the study would be easily associated with the study and trackable. This is not considered cumbersome and is typically currently done.
55
Re: field tests: "Is this section referring to, for example, using a European field trial to support a US-EPA registration? Does the EPA have statutory authority to approve a field trial in companion animals? If yes, is the review of the protocol going to be under a PRIA action?" (0026)
It is unclear what specific statement in the guideline this comment is referring to as there are no field test sections in the guideline. The only discussion about field testing is:

(2) laboratory tesing "...Field tests are not a preferred method to test efficacy of pet products but on occasion can be used to supplement laboratory data or to support specific claims; field studies should have protocols submitted to the agency for approval prior to commencing the experiment."

If field tests are submitted, they should be submitted in a similar manner of all other studies for review.
56
Re: not treating with any substance: "This statement may be overly restrictive. The prohibition is likely not necessary provided that the pre-treatment infestation used for allocation reveals adequate retention rate(s). For example, this statement would preclude the use of animals that had been treated with nitenpyram to clear fleas; this product has a label claim for only 24 hours and would be cleared from animal's system within 1 week. On the other hand, as part of inclusion criteria, animals should not have been treated with any substance that could affect the results for at least 3 months (90 Days). This is important as we now have long acting products in the market." (0026)
 See response to comment #25.
57
"Application method and exposure to fleas. The proposed 50:50 ratio of male to female fleas is unrealistic, as this would be extremely time consuming and subject the fleas to additional handling stress. The normal sex ratio of a colony should be used." (0008)
The proposed 50:50 ratio of male to female fleas was deleted from the guideline, allowing the normal sex ratio of a colony to be used instead. This is consistent with the European guideline.
58
"In addition, the proposal to have them "evenly distributed over the host animal" is unrealistic. The fleas should be infested onto the animal in a manner to avoid direct exposure to the treatment site...  As in the flea section, the proposal to have them "evenly distributed over the host animal" is unrealistic. Ticks should be infested onto the animal in a manner to avoid direct exposure to the treatment site." (0008)
Neither the flea nor tick sections were worded this way. However, the guideline has been revised to indicate that ticks should be placed on the host animals' bodies away from treatment sites.
59
"Also, 60% of fleas emerging is high. Consider changing to 50% or less. This will be corrected for by using Abbot's." (0008)
The 60% emergence will be maintained since >60% emergence is typically observed in submitted studies (most are even >70%). Any lower than this would indicate another issue with the study.

60
"Hand counts are not appropriate for fleas and would result in inaccurate data due to the mobility of adult fleas through the haircoat and the fact that they are not going to be attached like ticks." (0008)
The option of hand counting fleas was deleted from the guideline based on SAP and public comments.
61
"IGRs There is no need to separate these into two groups, even when both endpoints are being measured. Larvae that eclose can be counted, recorded and transferred to larval rearing media from which adult emergence can then be counted and recorded. In some cases, 50 or even 25 eggs can be difficult to obtain because some IGR's can inhibit egg production in exposed female fleas" (0008)
The guideline is already worded to allow either way, depending on if both endpoints are to be tested and how the test will be set up. The guideline reads: "Collected eggs can be divided into two groups to determine both larval hatch and adult emergence rates or can be combined into a single group if only one of the endpoints is being measured. At least 50 eggs total per animal should be collected so that both the larval hatch group and the adult emergence group each have a minimum of 25 eggs per animal in both the treated and control group at each time point for statistical comparisons between treated and control animals."

62
"In both the flea and tick sections I dont see the scientific need of change in preforming dead tray counts. None of the calculations presented use this collected data... 2nd you need to more carefully consider the need for collecting the dead flea and tick data. Specifically the dead pan counts. Because you can't guarantee collection of all the dead this data will be  inaccurate. Also currently I don't see the dead data is actually not used in any of the calculations. So why do the extra unnecessary work." (0011, 0032)
Dead pest counts and tray counts have been removed from the guideline for all test methods. See response to comment #14 on the changes to "speed of kill" claim testing, which previously used dead pest numbers in calculations.
63
"In section j(4)(i) I was surprised to once again see reference to hand counts for short time points. My understanding from the past was that hand counts were deemed to inaccurate for fleas. In my lab we have been successfully using comb and replace count for several years." (0011)
Hand counts for fleas have been removed based on SAP and public comments.
64
"In section j(4)(ii) for IGR hatch rates counting at 96 hours after collections is appropriate, however counting again at day 7 could be problematic. The hatched larvae will cannibalize each other within that time without a food source provided. Adding a food source or moving them on to rearing media greatly complicates the counting process. Again, on the adult emergence count doing two counts (35&42) is unnecessary. Procedure in my lab during the day 35 count includes dissecting any intact pupae and any normal fully formed fleas are counted as emerged." (0011)
We have re-reviewed the addition of a second egg count. Based on the literature, it seems in most situations that even if egg hatch was delayed by the IGR exposure, they would not mature, therefore the second count would not be necessary, and it was deleted.
65
"Treating with only the IGR component of the formulation is an interesting concept and this option is appreciated. However, could this be included as an option rather than a requirement? Other regulatory agencies may not accept this which could lead to repeating studies for different geographies." (0026)
The guideline has been revised to clarify that in evaluating efficacy in relation to some IGR claims, it may be possible to use the  end-use product in a study; however, EPA expects obtaining sufficient numbers of viable eggs to assess larval emergence and support a claim like prevents emergence of biting adults is likely to be difficult in studies conducted on a formulation containing an effective adulticide component and in these cases, the IGR only component should be tested.
66
"Why is computer-generated randomization mandatory? Other manual methods would work equally well." (0026)
The "computer-generated" part of the randomization statement has been deleted.
67
"Fleas are not typically sexed prior to infestation onto animals. Sexing fleas requires anesthetizing the fleas and examining them under a microscope, which can damage the fleas and decrease their viability. As both sexes infest and feed on the Guidance published by the European Medicines Agency (EMEA) is 50% retention rate for fleas on dog and cats. AHI requests EPA explain the rationale for the proposed 40/60 split." (0026)
The guideline previously indicated that a 50:50 ratio of male to female fleas be used (not a 60:40), however the guideline has been revised to delete that recommendation based on SAP and public comments.
68
"Since some a.i. (active ingredients) take time to spread, registrants can now measure efficacy at 72 h for earlier time points and 48 h for later time points. These are different standards at different time points. AHI recommends EPA consider offering greater flexibility for when the last test count should be recorded to account for the variability in a.i." (0026)
If a product is meant to last <30 days and it takes longer than 48 hours to kill/control/repel a pest (after the initial 3 days allowed to spread and start working), then efficacy of the product is not considered adequate for protecting animals from these pests. No change to the guideline is necessary.
69
Re: ticks. "Test species. Outcrossing laboratory colonies with field populations every 6 years is not appropriate. The idea behind having an established laboratory colony is to have a "reference" colony to which things can be compared back to over long periods of time. The outcrossing of field populations has the potential to introduce pathogens which would deem the colony unacceptable for use. Additionally, it poses the risk of introducing an arthropod pathogen that could destroy the entire colony. If claims to control resistant populations are sought, separate tests should be conducted on arthropods with known resistance to the active ingredient (or mode of action) to support such claims." (0008)
Outcrossing recommendations were deleted from the guideline based on SAP and public comments.
70
"Test species. Although current efficacy guidance 3 different species for general tick claims, the protocol defines no more than testing 2 species at same time on same animal. HCPA recommends reconciling this discrepancy and applying appropriate scientific foundation." (0008)
The guideline recommends testing two species at a time to help decrease the total number of studies run, and therefore reducing the number of animals used in testing, since previously each tick was typically run as a separate study. All 3 representative species however should be tested to gain a general tick claim. Therefore, two total studies should be run to test all 3 representative species. However, it is common for registrants to test additional tick species and suggesting them all run 2 at a time will cut down on the total number of tests. No change to the guideline is necessary.
71
"Recommend including additional detail to ensure consistent counting... Do they want us to count dead fleas on the tray/cage and the animal compared to live fleas on the control animal? What is the threshold for starts killing claim (90%)?" (0008)
These sections were revised based on public comments; see response to comments #14 & 62 for more information.

The threshold for efficacy is 90% and will be listed in the upcoming product performance rule.
72
"Table 2 change in infestation day to Day -1 vs. current Day -2" (0008)

Based on SAP recommendations, the guideline has revised the first tick infestation day from -1 to day -2 in Table 2 (in the tick section). The comment has this in reverse.
73
Re: ticks. "Repellency testing. Cage substrate reference is confusing. Is a light-colored substrate or carpet the only material that can be used? If so, there should be some definition on what type of carpet, as pile height and fiber density, etc., can be quite variable and may result in substantially different outcomes. It is recommended that carpet be eliminated from use as a cage substrate." (0008)
The guideline was updated to read:
"For each infestation/re-infestation, line individual pet carriers with a light-colored material, such as a light colored felt sheet. For each tick challenge, animals should be placed in the cage overnight or during a 12-hour dark period and 50 (+- 5) unfed and similarly aged adult ticks of each species should be placed inside the cage away from the animal."
74
"Mortality. For some treatments, i.e. collars, 72 hours after the initial infestation my not be long enough for the treatment to release and disperse across the surface of the body, which would not result in over 90% reduction. More importantly, some active ingredients are known to be slower acting against ticks. Knowing this, 72- hour pest counts should be an accepted endpoint for each re-infestation challenge." (0008)
If a product that is meant to last <30 days takes longer than 48 hours to kill/control/repel a pest (after the initial 3 days allowed to spread and start working), then efficacy of the product is not considered adequate for  protecting animals from these pests. For products lasting over 30 days, longer initial efficacy starting points may be justified.

For all product types, it is recommended that efficacy be determined for each re-infestation at 48 hours to demonstrate that the product provides continuous adequate protection of the animal.
75
"Tick removal by forceps should be added to comb counts as an accepted method for final tick counts, or "other appropriate means". (0008)
The guideline never prescribed a way to physically remove the ticks, only how to do the final counts, and therefore forceps could be used. No change to the guideline is necessary.
76
"(4)(i) Adult mortality counts can be made 72 hrs. after initial and 48 hrs. after residual (currently using 24 and 48 hrs.) Change could mean less efficacious products or existing products could reformulation of existing products to meet the reduced standard." (0008)
The standards in the guideline are based on what is consistently seen in studies and the standards that have been used for several years. If products work quicker than this, they may indicate so on the label.
77
"In both the flea and tick sections I dont see the scientific need of change in preforming dead tray counts. None of the calculations presented use this collected data. Most specifically the second paragraph of section K(6) is completely contradictive of itself stating to use the dead status ticks in the calculation but the last sentence defines survival as the no acaricidal effect which is the Live categories. Then the calculation presented uses survival or live only. Even in a tray collection system not all dead ticks are going to be recovered. This number cannot be accurately used to calculate the efficacy. Finally, moving the animals to special collection cages and isolating them for these infestation collection periods could be considered an unnecessary stressor simply for a meaningless visual confirmation of yes there are a few dead parasites" (0011)
See response to comments #14 & 62.

In response to the comment about moving the animals to special collection cages and isolating them during infestations, this is to prevent pests from moving from one animal to another between the time of infestation to comb out/final counts.
78
"In section
n k(3)(ii) for short time tick repellency, it is more scientifically accurate to real world application if ticks are not placed directly on the animal. Procedure should include infestation of the chamber floor and allow the ticks to naturally find and infest the animal as is procedure in the Dumont et al 2015 paper." (0011)
Based on SAP and public comments, the tick repellency section was revised to have ticks not applied directly to the animal, but rather in the same environment (i.e., in a pet carrier) and left overnight or in a 12-hour dark cycle. Repellency will then be assessed by whether the ticks are found on the host or not.
79
"Request that the EPA justify the need for three tick species and which species are required. As multiple tick species carry a variety of infectious agents, it seems more rationale to allow Sponsors the flexibility to choose the species that they feel most necessary to control." (0026)
The three recommended tick species for testing were chosen based on the potential for carrying the most common diseases and for host specificity (e.g., ticks that infest cats are different than ticks that infest dogs). The species will be published in the upcoming Product Performance Rule.
80
"AHI agrees with the infestation methods suggested in the document. However, we note that some sites are moving away from the use of an infestation chamber because of animal welfare reasons." (0026)
The guideline does not mention the use of infestation chambers, rather that the animals be individually caged during the infestation period which can represent many options.  Section 2 of the mosquito and biting fly section states "...animals should be individually caged and exposed to 25  -  100 adults... Cages should be sized to comfortably house the sedated animal; typical cages are approximately 24 inches x 24 inches x 24 inches, but larger animals may need larger cages (Meyer et al. 2003, Ross et al. 1997)." No change to the guideline is necessary.
81
"why are the pre-treatment infestations restricted to occurring by Day -5? We suggest that the latest time point for pre-treatment infestations should be decided by the Sponsor to allow flexibility in scheduling studies." (0026)
The guideline was revised to add the word "approximately" to allow more flexibility.
82
Re: using sticky tape to prevent escape: "This is a task that is up to the study conductor to determine  -  and should not be mandated by the guidelines." (0026)
The guideline has been revised to "may be used."
83
Re: timing of infestation: "The initial infestation (listed as Day -1) should be allowed to be on Day -1 or Day -2. Day -2 is required by the EMA guidance. Allowing that in EPA studies will have no impact on the study outcomes but will allow studies to support registration in both geographies" (0026)
Based on SAP and public comments, and based on biology of the pests, the first day of infestation for fleas should be day -1 and for ticks should be day -2. The guideline was revised for ticks.
84
"The required use of heavy-gauge plastic pet carriers is too specific for a guideline. The guideline should allow flexibility in the enclosures used for tick exposures" (0026)
The Agency agrees with this comment and the guideline has been revised.
85
"In addition, the time period of 1 hour is not sufficient to allow proper evaluations for repellency or blood feeding. Circadian rhythms are important to mosquito feeding biology and, depending on time of day, the prime feeding time may be missed. The minimum of 60% of insects taking a blood meal is extremely high. This is highly dependent upon species and host preference. Some species or genera that are required to be tested do not prefer to feed on some species of companion animals and, in some cases, on mammals in general. This minimum percentage should be eliminated, with efficacy demonstrated where the average number of mosquitoes taking a blood meal in the control group is statistically significantly different that the average number taking a blood meal in the treated group." (0008)
If the time of day is important to get valuable results from a test, then it would be up to the study director and laboratory to test during optimal times. The guidelines do not prescribe all times best for testing.

The minimum of 60% blood feeding is based on historical data submitted to the Agency as well as statistical confidence needed to make claims. If arthropod behavior is not adequate to ensure the desired effect is real, then the label claim would not be substantiated.

EPA does not use statistical significance between the control and treatment groups as threshold for achieving efficacy.  Instead EPA determines a product to be efficacious when it achieves a minimum of 90% mortality to the treated pest.

No changes to the guideline are necessary.

86
"Exposure to mosquitoes. Should "containing 150 (+- 10)" be containing 150 (+- 15)? If the proposal is for 50 (+- 5) and 100 (+- 10), this should translate to 150 (+- 15)" (0008)
The guideline will be revised to reflect this comment.
87
"The proposal that "if more than 10% of insects in a treated group blood-fed prior to death, mortality should not be considered a viable endpoint" is unreasonable. Dead should be dead. This puts EPA regulated products at a disadvantage when compared to some FDA regulated products that are systemic and require feeding to affect an arthropod population. As FDA-regulated, these products would support a claim, but the EPA product would not, even though it results in the death of the mosquito. It is not clear the intent of excluding insects that have died which ultimately skews the data set. Some active ingredients are slower in speed of kill than others, but if the insects die within the test period it should be counted as dead." (0009)
Section (m)(4) i. of the guideline now reads:

Mortality. Mortality may be considered a viable endpoint for a study if it occurs prior to the insect taking a blood meal. Mosquitoes and biting flies that take a blood meal are not repelled and can transmit agents that may cause disease; therefore, if more than 10% of mosquitoes blood fed, mortality should not be considered a relevant endpoint.

The EPA does not regulate systemic products, and thus, the intent of EPA registered products is contact toxicity and/or repellency, unlike for systemic products which require the mosquito to bite the animal for an effect to be seen. Thus, the purpose of topically applied, non-systemic products, is to repel and/or kill the mosquito.  Because claims preventing disease transmission are not made on EPA registered products, bites from mosquitoes which could transmit pathogens would not be providing the implied protection for the treated animal.

88
"For mosquitos in section l, I am worried that the 60% blood feeding rate is to lofty a goal to target. In my lab I typically see about 40% blood feeding on controls. However, I dont sedate the animals on my current protocols. This non sedation could possibly account for the reduction that I see. I was not able to access all the scientific references listed in this section to verify that other researchers are meeting the new recommend amounts. Is this value supported by known science or is there a more appropriate value that should be used." (0012)
The minimum of 60% blood feeding is based on historical data submitted to the Agency as well as statistical confidence needed to make claims. If arthropod behavior is not adequate to ensure the desired effect is real, then the label claim would not be substantiated. Lowering the feeding to 50% would decrease confidence in the statistics, which would result in increasing the number of animals necessary for the tests.

No changes to the guideline are necessary.
89
Re: % bloodfeeding for inclusion: "AHI suggests that 50% would be appropriate because it would ensure adequate infestation but also reduce animal usage. Requiring 60% may lead to more animals being included in the studies to ensure an adequate number of dogs have the adequate infestation level." (0026)
See response to comment #88 above.

No changes to the guideline are necessary.

90
"Request that the EPA justify the need for three mosquito species and which species are required. As multiple mosquito species have been shown to transit heartworms, it seems more rationale to allow Sponsors the flexibility to choose the species that they feel most necessary to control." (0026)
The representative species have been chosen to represent the different genera that transmit diseases of public health importance. These will be included in the upcoming Product Performance Rule.

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 Recommendations:
91
2a. 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 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.  PG 23-24
EPA added language to the guideline encouraging the submission of protocols for NAMs; many computer-based models can be considered NAMs.
92
2a. 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.  PG 24
EPA added language to the guideline encouraging the submission of protocols for NAMs; many computer-based models can be considered NAMs.
93
2a. 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.  PG 24
EPA is not incorporating any specific guidance for NAM testing into the guideline, however, to prepare for future opportunities for developing NAMs and reduce animal testing a statement was added to encourage submission of NAM protocols.
94
2a. 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 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.  PG 24-25
EPA added language to the guideline encouraging the submission of protocols for NAMs.
95
2a. 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).  PG 25
See response to comment #28.
96
2a. 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.  PG 25
See response to comment #28.
97
2a. 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.  PG 25
EPA appreciates the comment and will continue efforts to refine animal testing to use fewer animals when possible.
98
2a. 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.  PG 25
These panel members recommended the Agency continue to apply computational NAMs that include Monte Carlo simulations [underline added by EnDyna for emphasis] for justifying a reduction in the required number of vertebrate animals to six per treatment group.  PG 26
They also suggested adding text to the guideline document that details these approaches and encourages manufacturers to apply computational technologies to reduce animal usage.  PG 26
EPA added language to the guideline encouraging the submission of protocols for NAMs, many computer-based models can be considered NAMs.
99
2a. 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.  PG 25
They also recommended exploring Markov Chain Monte Carlo Simulations, [underline added by EnDyna for emphasis] which consider covariance and other uncertainties, and coupling simulations with Bayesian approaches [underline added by EnDyna for emphasis] when new scientific data are available.  PG 26
They also suggested adding text to the guideline document that details these approaches and encourages manufacturers to apply computational technologies to reduce animal usage.  PG 26
See response to comment #98.
100
2a. 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 nonvertebrate 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."  PG 26
EPA does accept non-animal testing for spray-on type products that only make "kills" claims and do not make residual claims. However, the residual assays conducted on paper, etc., do not sufficiently replicate residual activity on an animal so until it is demonstrated these assays could be used in place of animal testing, EPA does not intend to accept them for residual efficacy claims.
101
2a.  .....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.  PG 26
The Agency does not currently have any data to provide evidence that rodents would be able to represent cats or dogs in these tests nor is there evidence that cats and dogs would be able to represent each other. No changes to the guideline are necessary.

102
2b. 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 due to a provoked inflammatory reaction than the flea itself, while ticks may go unnoticed in some cases.  PG 26
The prequalification stage of the animals is intended to help address and rule out any issues with an animal's behavior. Any non-standard test should address any potential behavioral issues in the proposed protocol.
103
2b. The Panel proposed that the test arthropods be in the environment of cats and dogs for only a short period of time.  PG 27
The guideline outlines the timing between infestation of arthropods and removal/counts. No changes to the guideline are necessary.
104
2b. 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.  PG 27
See response to comment #28.

Public Recommendations (docket ID # in parentheses):
105
The following article was provided to the Agency for consideration: "Itching for change: Embracing modern flea and tick product development. Please contact the EPA Docket Center, Public Reading Room to view this document." (0009)
EPA has read and considered the article. The only current actionable recommendation for this guideline is that test parasites be fed on artificial membrane feeding systems.  As the guideline is currently written, arthropod colonies are not required to be fed on animals, thus in vitro feeding is acceptable to the Agency.
106
2a. "Similarly, before being included in the guidelines, the ability to extrapolate efficacy data from dogs to cats, or vice-versa, should be analyzed. The data would show whether a product that is effective in one species can predict efficacy in the other species, and, therefore, whether testing on one species could be waived. Waiving testing for one species would be in line with EMA guidance, which permits efficacy data from tests on dogs to be extrapolated to cats when scientifically justified and one dose confirmation study in cats for each parasite species on the label is conducted (CVMP, 2016)." (0019)
EPA does not have specific methods or modeling to extrapolate efficacy data from one species to another and thus has not provided methods for this in the guideline.
107
2a. "The guidelines do not sufficiently address the use of field studies to reduce and replace testing on animals in laboratories. In the updated draft guidelines, field studies are described as not "preferred" by the agency and only used to supplement laboratory data or for specific claims on occasion (OCSPP 810.3300(i)(ii)). However, justification for largely excluding the use of field studies is not provided. Due to ethical considerations, diligence must be given to incorporating the use of field studies to replace animal use in laboratories for testing ectoparasiticides. In addition, robust clinical studies are more indicative of efficacy than the artificial conditions created in laboratory tests. Unlike laboratory tests, field studies can assess efficacy against natural infestations under a wide range of conditions, from differences in physical and behavioral characteristics of the animals to environmental differences (Marchiondo, 2007). The One Health Company (www.theonehealthcompany.com/), for example, designs and conducts multicentric clinical efficacy trials and has the expertise to assist with the coordination of efficacy trials across veterinary clinics in order to run multi-arm studies using real-time analytics to compare naturally non-treated controls, positive controls, and a new treatment." (0019)
Field studies may have inadequate or inconsistent pest pressure, lack of appropriate controls, and inaccurate pest counting. They are often conducted using endpoints which do not support claims commonly made by pesticide companies which fall under EPA's regulatory authority (e.g., severity of flea allergy dermatitis).  In addition, field studies are also not conducted in ways that allow different claimed effects (e.g., repellency vs. mortality) to be differentiated. Thus, although field studies can sometimes provide useful information, they do not typically directly support the types of efficacy claims made on pesticidal products and EPA recommends laboratory studies be conducted instead of field studies. However, protocols for field studies may be submitted if desired.

108
2a. "While we believe that demonstrating effectiveness in each target parasite is important. Nevertheless, in selected cases and with appropriate justification, the use of foreign data on a particular parasite could minimize the need for testing in dogs and cats." (0026)

Studies are not required to be conducted within the United States; studies conducted outside the U.S. are commonly submitted. The EPA may accept studies conducted in another country, so long as the data support the product use directions and claims.

109
"HCPA is supportive of new approach methodologies (NAMs) that reduce the number of vertebrate animals needed for testing, where possible, while ensuring confidence in test results" (0008)
See response to comment #28.
110
"We do not agree with the statement that, "at this time no reliable non-animal alternatives are available to avoid the use of animals for efficacy testing of fleas, ticks, mosquitoes and biting flies." In our view, that conclusion is premature since the agency has not sufficiently considered available methods and how they might be adapted. For example, some of the established skin models and skin sensitization systems already used in toxicity testing may provide potential for reducing or replacing animal use in this case. Further, EPA has a unique opportunity to advance the field by engaging with test method developers to determine how the requirements of those making regulatory decisions can be met. It may be that adaptations can be made without too much effort, thereby preventing significant animal suffering. "We urge the agancy and the panel to consider the following: :: In consultation with experts in the field of alternative methods, performing a thorough and formal review of animal replacement technologies that have relevance or potential usefulness, including computational methods; :: Field testing or clinical studies using animals who could benefit from treatment; :: Information cited and discussed in Groff, K., & Bishop, P. (2017). Itching for change: Embracing modern flea and tick product development. Regulatory Toxicology and Pharmacology (6 July online) (http://dx.doi.org/10.1016/j.yrtph.2017.07.002)" (0010)
We appreciate the comment and citation. For discussion of the relevance of the citation please see response to comment #105. For discussion on NAMs, please see response to comment #28 and #91.
111
"Test methods to replace animal use in efficacy testing of ectoparasites on animal companions have been available for decades and have been submitted to regulatory agencies. For example, data submission packages to the EPA and the European Medicines Agency (EMA) Committee for Medicinal Products for Veterinary Use (CVMP) have included results from in vitro efficacy tests in which fleas and ticks were directly exposed to products in a petri dish (EPA, 2010; CVMP, 2006) and from in vitro studies on adulticidal activity of a product on fleas using an artificial membrane system (CVMP, 2011). Artificial membrane systems, which allow parasites to naturally attach and feed on blood or media through a membrane, are described in the literature, and they can be used to evaluate the efficacy of both oral and topical ectoparasiticides that are added to the blood in the in vitro system or applied to the membranes (Krober and Guerin, 2007a, b, c; Kuhnert et al., 1995; Andrade et al., 2014; Kuhnert, 1996; de Moura et al., 1997; Tajeri and Razmi, 2011; Bohme et al., 2014;Wade and Georgi, 1988; Li et al., 2015; Banks et al., 2000; Williams et al., 2014; Meola et al., 2000).  Artificial membrane systems offer advantages over in vivo methods, including greater control and standardization, such as the ability to control components of the blood meal, quantify dose effects of products or pathogen concentration in the blood, eliminate variability caused by animal grooming, and avoid complications from host-arthropod-pathogen interactions (Musyoki et al., 2004; Waladde et al., 1996; Macaluso et al., 2001; Krober and Guerin, 2007b; Kuhnert, 1996; Bonnet and Liu, 2012; Wade and Georgi, 1988). Further, variability between animals can confound the interpretation of results from studies in which fleas or ticks are fed using in vivo methods, an issue in vitro methods can overcome by using blood or media from the same source to evaluate controls and test compounds (Kuhnert, 1996). Researchers have successfully demonstrated the efficacy of parasite control products using artificial membrane systems (for review, see Groff and Bishop, 2017). For example, Li et al. (2015), Krober and Guerin (2007b), and Kuhnert et al. (1995) used silicone membrane systems to confirm the efficacy of commercial ectoparasiticides against Amblyomma americanum adults, Ixodes ricinus adults, and Amblyomma hebraeum nymphs; and Williams et al. (2014) used a parafilm membrane system to demonstrate that a commercial flea product controlled flea reproduction. The SAP should advise the EPA to convene an expert working group or workshop to develop a plan to incorporate non-animal methods in OCSPP 810.3300. The goal of these discussions would be to devise an approach to evaluate the efficacy of ectoparasite products using a combination of in silico models, in vitro tests, and field studies. Further, we appreciate that the updated guidelines note that the EPA is open to reviewing protocols proposing novel testing methods (OCSPP 810.3300(e)), and language included in the guidelines should encourage companies to propose methods that reduce and replace animal testing. Expedited review of such protocols will facilitate the adoption of non-animal test methods. To ensure timely review, we propose the addition of a practicable timeline to OCSPP 810.3300(e)." (0019)
EPA is aware of many of these types of tests (e.g., membrane feeding tests), and these tests have strengths especially in confirming that an active ingredient has a specific effect and controlling for dose. However, there are insufficient data to show that these tests adequately evaluate residual efficacy of products applied to animal skin. Residual efficacy can be affected by a myriad of factors including degradation, movement across skin, and exposure to elements. As mentioned previously, EPA is open to protocols and studies assessing the methods simultaneously. Review timeframes for submitted protocols can be found under the appropriate categories for protocol submission in PRIA.
112
"The potential expansion of the use of in-vitro techniques to demonstrate contact activity of ectoparasiticides against fleas and ticks could be considered as means to decrease the use of dogs and cats in research. Several well developed and validated methods exist that are currently used by many companies in early development stages. These include impregnated filter paper tests for ticks or coated glass surfaces for fleas and ticks." (0026)
See response to comment #28.
113
"Animals are used to rear ectoparasites as well as assess product efficacy. These animals are immobilized and areas of skin are shaved to allow parasites to feed undisturbed. During efficacy testing dogs and cats are literally infested with fleas and ticks. Use of animals in these ways leads to obvious adverse effects, including stress, anemia, unrelieved itching, and constant pain and discomfort. NAMs that replace animals in these situations already exist. Substantial research has been carried out over the last twenty years on use of artificial membrane systems, which essentially act as skin and allow fleas or ticks to attach and feed on blood or synthetic media through the membrane. These systems have been used with varying degrees of success to rear and test products on fleas and ticks. They have also been employed in studies of the transmission of disease by these vectors. Recent work has demonstrated the utility of these systems in feeding and maintaining mosquitoes as well. However, further research and development is needed to optimize and standardize artificial membrane systems if they are to be used for regulatory purposes. We recommend that government should make funding available to support work on non-animal methods of rearing fleas and ticks and testing ectoparasiticides. Additionally, the ectoparasite product industry should play a role by dedicating a portion of profits to development of these systems. Researchers, regulators, and industry need to collaborate on identification of specific research needs, as well as optimization and standardization of protocols using in vitro systems for efficacy testing and rearing of different species and life stages. This could be accomplished through a series of workshops, which the Humane Society would be happy to help organize. We look forward to further dialog with EPA on this subject. Thank you." (0027)

As the guideline was initially drafted, EPA does not require arthropod colonies to be fed on animals for use in testing, nor is EPA making this recommendation for the final guideline. EPA will consider ways (e.g., workshops, etc.)  to promote the development of NAMs for efficacy testing.
114
"During the process of bringing a new ectoparasiticide to market, animals are used in three areas: toxicity testing, rearing of ectoparasites (includes breeding and feeding) for later use in efficacy testing, and efficacy testing itself. A recent paper published in Regulatory Toxicology and Pharmacology, Itching for change: Embracing modern flea and tick product development (Groff and Bishop 2017), gives a comprehensive overview of both current and alternative systems of feeding/rearing and efficacy testing. The paper's main findings are summarized here. To rear large numbers of parasites for later use in efficacy testing, animals are often immobilized with areas of skin shaved to allow fleas and ticks to feed undisturbed. For efficacy testing, dogs and cats are repeatedly infested with 100+ ticks or fleas for up to a month or more. Use of animals in these ways leads to obvious adverse effects, including stress, anemia, unrelieved itching, and constant pain and discomfort. Alternative feeding systems for rearing and efficacy testing have been developed and used with varying degrees of success. Artificial membranes which essentially act as skin, allow fleas or ticks to attach and feed on blood (typically obtained from a slaughterhouse) or synthetic media through the membrane. The membranes can be modified to accommodate different species and life stages, and they permit researchers to directly observe attachment to the membrane, feeding, reproductive output, and mortality. The membranes may be made from silicone, parafilm, or animal-derived skins. The cat flea Ctenocephalides felis (C. felis), the most common flea species infesting cats and dogs, has been successfully reared using in vitro parafilm membrane systems, with survival rates equal to fleas fed on cats, although reproductive rates were lower. For efficacy assessment of oral and topical products that act through the blood, these in vitro systems provide greater control and standardization, such as the ability to accurately quantify doses, eliminate variability caused by animals grooming, and control components of the blood meal. Variability among test animals, which can confound the interpretation of results, can be overcome by using in vitro methods. In addition, they offer cost savings when compared to the use of animals in test systems. Researchers have successfully demonstrated the efficacy of tick control products using silicone membranes with comparable data from paired in vitro/in vivo studies (e.g., Krober et al. 2007a, 2007b; Fourie et al. 2013; Li et al. 2015). In vitro parafilm membrane systems also have been used successfully for determining the efficacy of products to control C. felis. Other studies have demonstrated the viability of the parafilm membrane system by using it to study the transmission of viruses and bacteria by fleas, including the length of infectivity and mode of transmission (e.g., transmission from blood to fleas, from fleas to blood, through flea excrement, and from fleas to their progeny) (e.g., Vobis et al. 2003; Bouhsira et al. 2013, Kernif et al 2014). The same disease transmission capabilities and routes of infection were captured as in the in vivo studies. A recent study by Tajeri et al. (2016) of Theileria lestoquardi infection by ticks found that while in this case an in vitro system using silicone membrane was largely unsuccessful, high rates of attachment and feeding were observed using an in vitro mouse skin system. Alternative systems are not without challenges, however. Multiple variables affect the success of membrane feeding, including the choice of host species blood used in the in vitro system, preservation of the blood, the addition of supplements to the blood, and attachment stimuli. Hard ticks have complex feeding requirements, depending on a range of olfactory, mechanical, and host-contact stimuli conditions. In addition, the use of anticoagulants, antibiotics, and antifungals in the blood may affect success of tick feeding. Also, meals have to be long-lasting, as hard ticks feed for up to two weeks at a time depending on life stage. To overcome some of these obstacles, researchers have experimented with ways to improve feeding and rearing success. For instance, Krull et al. (2017) showed that the ixodid tick, Dermacentor reticulatus, experienced increases in both the engorgement mass and fecundity when fed at an increased CO2 level. Similarly, membrane systems have been improved to increase successful use by fleas. One researcher demonstrated the importance of providing hair as a substrate for survival and reproduction of the cat flea (Pullen and Meola 1995), while another found that human blood can be used successfully as a blood meal and has a relatively long shelf life (Kernif et al. 2015). While further studies are needed to identify a complete set of optimal conditions for fleas, current parafilm membrane systems are suitable for rearing fleas for ectoparasiticide testing and, with regulatory approval, for efficacy testing. An alternative system has been recently reported for use with mosquitoes. Luo (2014) describes a novel multiple membrane blood‐feeding system for mosquitoes that can be used for the study and routine maintenance of Aedes aegypti L. and Aedes albopictus Skuse, which require a meal of vertebrate blood to produce eggs. This blood‐feeding system uses cattle collagen sausage‐ casing membrane to facilitate feeding and adds adenine nucleotides to the blood to stimulate the feeding response. The efficiency of this blood‐feeding system was compared to a live mice blood source. The Ae. aegypti fed on pig whole blood had 89.7% (without ATP) and 90.7% (with ATP) blood‐feeding rates, which were not significantly different from the mice‐fed ones (98.0%). The Ae. albopictus fed on pig whole blood (with ATP) had a success rate of 84.4%, which was significantly different (better) from the mice‐fed mosquitoes (51.1%). The feeding rates did not differ between sausage‐casing membrane and Parafilm‐M(R). The survival rate, fecundity, pupation, and pupal emergence rates of Aedes females fed on pig whole blood were not significantly different from the mice‐fed ones. While a significant amount of research has been conducted in this area, further work is needed in development and standardization of artificial membrane systems to rear ectoparasites and for use in regulatory efficacy testing. In addition, to eventually transition to completely non-animal systems, development of appropriate synthetic feeding media that could replace the use of animal blood is needed. Groff and Bishop (2016) lay out a series of steps that can be taken for this to occur as summarized below: * Governments should make additional funding available for research on non-animal methods of rearing fleas and ticks and testing ectoparasiticides. * A portion of the profits from the ectoparasite product industry should be dedicated to research and development of these systems. A series of workshops should be convened to bring together researchers, regulators, and industry to collaborate on identification of specific research needs, as well as optimization and standardization of protocols using in vitro systems for efficacy testing and rearing of different species and life stages. * Studies comparing results of in vivo and in vitro systems should be conducted to increase acceptance by regulators. * Once accepted protocols are developed, regulatory agencies must adapt their guidelines to allow and encourage the use of these alternative systems. * To encourage acceptance and uptake of these methods, governments should provide incentives for companies that use in vitro systems to rear parasites and test products whenever possible. The Groff and Bishop paper is included with these comments for the SAP's perusal. HSUS and HSLF feel strongly that this area of testing is ripe for change and that systems currently exist that could be used successfully to replace some or all of the animals used for these purposes. Significant progress could be made by convening a series of workshops that that focuses on optimization and standardization of these approaches for regulatory use." (0028)
See response to comments #28 and #113.
115
"The draft guidelines recommend a heavy ectoparasite burden. Animals experience obvious physical and emotional adverse effects from infestations with 100 fleas or 50 ticks, which is well in excess of infestations typically present on companion animals. As a reference, a study for a flea control product notes that a cat, who was artificially infested with 50 fleas throughout the test, died from anemia due to severe flea infestation. Animals in field studies are considered infested and included in studies when they have just three to ten ectoparasites. We recommend further discussion around the increase in parasite burden in the draft guidelines." (0031)
EPA understands the concern with parasite load. Parasite loads as high as those recommended would be on the high end of parasite loads that might be observed on companion animals. However, reducing parasite load typically results in a higher number of test animals because arthropod behavior can be difficult to account for.
116
"The recommendation to conduct testing for all species of ticks as well as fleas, if included on the proposed label, results in the use of a large number of dogs and cats (OCSPP 810.3300(d)(1)(vii), OCSPP 810.3300(i)(2), (OCSPP 810.3300(k)(1)). A comparison of efficacy data for different species of ticks and fleas must be conducted before requiring such a large number of animals to be used. This   evaluation would determine whether one species of tick or flea is the most difficult to control, and therefore, if a product controls that species, testing on other species can be avoided. This analysis would consider how the modes of action of different active ingredients may affect the results. The EPA OPP has undertaken similar retrospective analyses in order to inform testing guidelines (e.g., Hilton et.al., 2019)." (0019)

EPA does not have information on which species is the most conservative and thus is not including that in this guideline.  However, EPA is open to an analysis and comparison of susceptibility of the different species for future testing.
117
"We support the exclusion of a positive control (OCSPP 810.3300(d)(1)(viii)(b)) and the clear language that the EPA uses in the draft guideline. The use of animals in negative control groups should be reconsidered in the draft guideline as these tests are particularly painful to the animals used (OCSPP 810.3300(d)(1)(i)). Instead, for example, the efficacy of a product can be determined by the percent reduction in number of fleas and ticks on animals from pre- to post-dosing without the use of negative control groups, as is common in field studies (Karadzovska et al., 2017; Cavalleri et al., 2017a, b; Rohdich et al., 2014; Meadows et al., 2017; Navarro et al., 2016). The SAP should advise the EPA to conduct an evaluation of the use of control groups. Kramer and Font (2017) note that "[c]ontrol groups...offer a relatively untapped potential to reduce sample size without sacrificing statistical power" and conclude that "the number of current control subjects can be reduced by more than half by including historical controls in the analyses. In other experimental scenarios, control groups may be unnecessary." They describe how the results of prior experiments can help to determine the need for control groups and write that "[w]hen similar experiments are performed repeatedly in the same laboratory, using the same standard research and husbandry protocols and the same animals...there will often be scope for the incorporation of historical controls"" (0019)
At this time, the EPA does not have any information on validated methodologies to  generally support this across study types, however the Agency is open to considering a reduced number of animals in the control group on a case-by-case basis with justification provided prior to study commencement.
118
"The draft guidelines state that end-use formulations should be tested (OCSPP 810.3300(d)(1)(ii)), but do not specify testing requirements for substantially similar formulations. The EPA and FDA have guidelines to minimize animal testing using existing information and bridging to similar products (e.g., EPA, 1998; FDA, 2001)." (0019)
When formulations are considered substantially similar, EPA allows bridging of data between the two products, similar to what is done with toxicity data.
119
"Dogs and cats are social animals whose suffering is exacerbated when isolated. At a minimum, the guidelines should require enrichment for when animals are housed alone. Further, the draft guidelines stipulate that animals "should" be housed individually during infestations, but only that they "may" be housed together during other time periods (OCSPP 810.3300(d)(1)(v)). Considering the social needs of dogs and cats, the guidelines should stipulate that when not infested with parasites, animals "should" be socially housed." (0019)
EPA has incorporated both suggested revisions.
120
"(2) Laboratory testing What about "ex vivo" studies such as the ones using dog's hair sampled from treated or untreated animals at different time point. This is much more ethical especially when many different species must be tested and when biting is painful (for example, stomoxys flies). Recommend that" (0008; note that the sentence just ends)
See response to comment #28.

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 Recommendations:
121
3a. 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.  PG 27
Specific mite testing guidance will not be added to the guideline at this time. Currently, EPA has received very few studies for mites over the last 15 years and therefore these studies are not common enough, nor have a study design that produces reliably consistent data to establish standardized testing. Registrants seeking mite claims are encouraged to submit protocols for review. The Agency will use the information provided here by the SAP to guide discussions that may arise about methods for mites.

For soft ear ticks, currently data do not need to be submitted to support labeling claims; however, registrants are required to have data on file supporting all pests if they are listed on the label.
122
3a. 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).  PG 27

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).  PG 30
See response to comment #121.
123
3a. 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 be tested. A similar testing approach as suggested for Otodectes cynotis (ear mites) could be employed for trials using subadult O. megnini.  PG 27-28
See response to comment #121.
124
3b. 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.  PG 28

Overall Summary
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.  PG 30
See response to comment #121.
125
3b. 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.  PG 28

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.  PG 28
See response to comment #121.
126
3b. 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.  PG 28

 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 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.  PG 28-29
See response to comment #121.
127
3b. 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.  PG 28

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.  PG 29
See response to comment #121.
128
3b. 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.  PG 28

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."  PG 29
See response to comment #121.
129
3b. 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 cm[2]."  PG 30
See response to comment #121.
130
3b. Additional Methodological Issues:
Several other issues were raised by the Panel:
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).  PG 30
See response to comment #121.
131
3b. Additional Methodological Issues:
Several other issues were raised by the Panel:
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.  PG 30

Overall Summary
With respect to methodology, the Panel recommended that ...... EPA state that non-vertebrate studies would be considered as an acceptable alternative for scabies tests.  PG 31
See response to comment #121.
132
3b. Additional Methodological Issues:
Several other issues were raised by the Panel:
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.  PG 30

Overall Summary
With respect to methodology, the Panel recommended that EPA consider that efficacy studies involving client-owned pets be conducted at clinics with volunteer patients, .....  PG 31
See response to comment #121.

Public Recommendations (docket ID # in parentheses):
133
"Additionally, models of artificial infection of pets with mites exist and are validated. These have been previously used in support of product registration with other regulatory agencies, such as FDA and EMA. Whilst these rely on the use of animals like cats and dogs to demonstrate efficacy, recent developments in genotyping can help demonstrate similarities of various mite species. This may allow for using studies across geographies and therefore reducing the overall number of studies." (0035)
See response to comment #121.
134
"Do the guidelines incorporate the responsibilities outlined in Memorandum of Understanding Between the Environmental Protection Agency and The United States Department of Health, Education and Welfare Food and Drug Administration which incorporates ear mites (225-73-80102)?" (0008)

EPA has registered products with claims to control ear mites, however, this guideline is not intended to clarify the responsibilities outlined in the MOU. In addition, we have not included mites in the guideline due to reasons discussed in comment #121.

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 Recommendations:
135
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.  PG 31
Thank you for the agreement on sample sizes. The sample sizes provided were based on power analyses using previously submitted studies which fit into the proposed testing methods and reflect our efficacy standards.
136
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 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.  PG 32
The Agency appreciates the feedback about sample size. The tick and stable fly models have been re-run with to explore lowering the number of host animals or pests used. For tick testing, the number of animals (cat or dog) used per treatment group was lowered from 14 to 11.

Currently, most studies run a positive control group and the groups average 8 animals per group regardless of pest. Therefore, tick studies are on average run with 8 animals x 3 groups (= 24 dogs) and have never indicated issues with counting space in labs or technician fatigue. In this guideline we are specifically stating not to run positive controls, as well as encouraging the utilization of multiple pests where possible, to lower the total number of tests required for registration, which would in turn lower the total number of animals to be tested to gain basic registration.

Public Recommendations (docket ID # in parentheses):
137
"HCPA believes that 6-10 animals should be sufficient and is concerned with the practicality of additional animals. There also appears to be an inconsistent approach applied between different invertebrates." (0008)
The minimum number of animals indicated by EPA is based on a power analysis conducted using the study design and control retentions of pests on the different animals (e.g., dog vs. cat) and for each different pest (e.g., biting flies vs. fleas). These sample sizes will ensure that data submitted are adequate to show that if a product reaches the 90% efficacy threshold, it is effective.
138
"AHI supports the principles described in the WAAVP guidelines, where a minimum sample size of 6 animals per group is acceptable. A requirement to demonstrate statistical difference in the primary outcome of efficacy  -  such as number of live fleas or ticks  -  is appropriate." (0026)
See response to comment #137.
139
Re: flea test: "What is the scientific justification of requiring seven animals in studies using cats and six animals for studies using dogs? Power would be calculated by the registrant. This is usually less than six animals per group. The conventional six animals per group is an arbitrary number... What is the justification for requiring 8 animals per group for biting fly studies but only six per group for mosquito studies? Six per group is traditionally required. If Sponsors wish to include more to reduce risk of not having adequate infestations, this should be their option, rather than a requirement." (0026)
See response to comment #137.
140
"The statistical modeling that the EPA has conducted serves as an underpinning to many of the recommended changes in requirements for the guideline. It is this methodology that has led to increased sample sizes (up to 14 animals per group required in tick studies). But the statistical modeling is based on assumptions of pest retention that are questionable. Testimony has been provided by Dr. Everett, for instance, that the assumption/requirement of 60% blood feeding of mosquitoes in controls is not seen in his laboratory (as a CRO). If the assumption is lowered to more reasonable values, the size of the treatment group must increase to maintain the same level of statistical power. The assumptions made by the EPA are at the high end of our experience in the conduct of these studies, and are not representative values." (0033)
See response to comment #137.
141
"Selection and allocation of test animals. Why are 14 animals the minimum number to be used for tick studies when only 6 or 7 are the minimum for flea studies? This is a significant difference that is not justified. Recommend a consistent, science-based and statistically sound value." (0008)
See response to comment #137.
142
"In section k(2) the new minimum of 14 animals seems way overkill to me. For ticks on cats it is a little more understandable as those studies can be very difficult with ticks not really liking cats and cats possibly being groomers. We have run studies with 12-15 cats to ensure that we get the minimum of 6 that meet the 25% hold rate. Now if you insist on a minimum of 14 per group meet the minimum hold rate will we have to run 25-30 per group. Again, raising my question on group average or individual animal requirement. For ticks on dogs the idea of 14 animals being needed is absolutely ridiculous. Ticks like dogs and dogs are typically not good at grooming ticks off. My lab typically runs 8 dogs per group and havent had a tick infestation study failure in many years. I would suggest repeating your powers calculations using real data provided from past submitted studies." (0011)
The power calculations were run using data from past submitted studies and have been rerun for the tick models to account for some of the factors in the guideline and changes based on SAP comments. The number of animals recommended for tests for ticks have been reduced from 14 to 11.

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))