CVM GFI #276 Effectiveness of Anthelmintics: Specific Recommendations for Products Proposed for the Prevention of Heartworm Disease in Dogs

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

#276
Effectiveness of Anthelmintics: Specific
Recommendations for Products Proposed
for the Prevention of Heartworm Disease
in Dogs
Guidance for Industry
Submit comments on this guidance at any time. Submit electronic comments to
https://www.regulations.gov. Submit written comments to the Dockets Management Staff
(HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD
20852. All comments should be identified with the docket number FDA-2022-D-2899.
For further information regarding this document, contact AskCVM@fda.hhs.gov.
Additional copies of this guidance document may be requested from the Policy and Regulations
Staff (HFV-6), Center for Veterinary Medicine, Food and Drug Administration, 7500 Standish
Place, Rockville MD 20855, and may be viewed on the Internet at https://www.fda.gov/animal-
veterinary, https://www.fda.gov/regulatory-information/search-fda-guidance-documents, or
http://www.regulations.gov.
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Veterinary Medicine (CVM)
June 2024

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Table of Contents
I. 	Introduction .......................................................................................................................... 1
II. 	Recommended Approach to Demonstrate Substantial Evidence of Effectiveness......... 1
III. 	Laboratory Dose Confirmation Studies ............................................................................. 2
A. Number of Studies.......................................................................................................... 2
B.	
D. immitis Isolates .......................................................................................................... 3
C. Number of Dogs per Investigational Group ................................................................ 3
D. Number of L3 Injected for Induced Infection ............................................................. 3
E. Pre-existing Heartworm Infection ................................................................................ 3
F. 	Assessment of Effectiveness .......................................................................................... 3
1. 	Parasite Counts ........................................................................................................ 3
2. 	Criteria to Demonstrate Effectiveness ................................................................... 4
G. Pharmacokinetic Evaluation ......................................................................................... 4
IV. 	Field Effectiveness Study..................................................................................................... 4
A. Study Duration ............................................................................................................... 5
B. Enrollment ...................................................................................................................... 5
C. Location of Study Sites .................................................................................................. 6
D. Assessment of Effectiveness .......................................................................................... 6
E. Active Control Group .................................................................................................... 6
F. 	Number of Evaluable Cases .......................................................................................... 7
G. Pharmacokinetic Evaluation ......................................................................................... 7

Contains Nonbinding Recommendations
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Effectiveness of Anthelmintics: Specific Recommendations for
Products Proposed for the Prevention of Heartworm Disease in
Dogs
Guidance for Industry
This guidance represents the current thinking of the Food and Drug Administration (FDA or
Agency) on this topic. It does not establish any rights for any person and is not binding on FDA
or the public. You can use an alternative approach if it satisfies the requirements of the
applicable statutes and regulations. To discuss an alternative approach, contact the FDA staff
responsible for this guidance as listed on the title page.
I. 	Introduction
This guidance provides recommendations for the evaluation of effectiveness for drugs indicated
for the prevention of heartworm disease caused by Dirofilaria immitis in dogs. These
recommendations should be read in conjunction with Guidance for Industry (GFI) #90 (VICH
GL7), “Effectiveness of Anthelmintics: General Requirements,”1 and GFI #111 (VICH GL19),
“Effectiveness of Anthelmintics: Specific Recommendations for Canines,”2 and are intended to
provide additional detail to elements of study design and interpretation under the
recommendations laid out in these guidances.
In general, FDA’s guidance documents do not establish legally enforceable responsibilities.
Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only
as recommendations, unless specific regulatory or statutory requirements are cited. The use of
the word should in Agency guidances means that something is suggested or recommended, but
not required.
The recommendations in this document are based on current technology and veterinary
epidemiology, including available diagnostic methodologies. Individuals pursuing approval of
drugs indicated for the prevention of heartworm disease in dogs are encouraged to discuss
deviations from these recommendations with FDA, especially as advances in veterinary medicine
related to heartworm disease, including disease epidemiology, isolate characterization, or
diagnostic testing are identified.
II. 	Recommended Approach to Demonstrate Substantial Evidence of Effectiveness
The recommended approach to demonstrate substantial evidence of effectiveness of an
investigational new animal drug intended for the prevention of heartworm disease in dogs is for
1 https://www.fda.gov/media/70349/download (August 2022). When final, this guidance will represent FDA’s
current thinking on this topic.
2 https://www.fda.gov/media/70091/download (August 2022). When final, this guidance will represent FDA’s
current thinking on this topic.

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the sponsor to conduct two laboratory dose confirmation studies and one multi-site field
effectiveness study in accordance with the principles of Good Clinical Practice (GCP) as
described in GFI #85 (VICH GL9), “Good Clinical Practice.’’3 Laboratory dose confirmation
studies are experimentally-induced infection studies that provide known exposure to infectious
Dirofilaria immitis (D. immitis) larvae (L3) due to contemporaneous experimental infection of
the same number of L3 to negative control dogs and dogs administered the investigational new
animal drug. Laboratory dose confirmation studies also allow for quantitative evaluation of
outcome by determining the presence of adult worms as well as the individual worm burden in
each dog.
The field effectiveness study evaluates the effectiveness of the investigational new animal drug
under actual conditions of use in client-owned dogs and with the current enzootic status,
ecologic, and genetic factors affecting heartworm disease in dogs in each location.
FDA’s recommended approach considers the laboratory dose confirmation studies and the field
effectiveness study together when evaluating if substantial evidence of effectiveness (21 CFR
514.4) and inferential value to the target population have been met. FDA has provided certain
study design elements in the recommendations for the laboratory dose confirmation studies in
section III. Laboratory Dose Confirmation Studies (e.g., percent effectiveness threshold,
susceptibility characterization of isolates) based on the assumption that the conduct of the field
effectiveness study will incorporate the recommendations listed in section IV. Field Effectiveness
Studies.
III. 	Laboratory Dose Confirmation Studies
The laboratory dose confirmation studies are intended to confirm effectiveness of the
investigational new animal drug under laboratory conditions.4 The studies should evaluate
effectiveness of a defined minimum labeled dose (mg/kg), dosage interval, and number of doses.
Timing of study procedures (infection, initiation of dosing) should be justified based on the
specific properties of the investigational new animal drug.
A. Number of Studies
FDA recommends sponsors conduct two induced laboratory dose confirmation studies,
each conducted at different laboratory facilities, led by different independent investigators,
and using recent isolates of D. immitis from two separate geographic locations in the
United States.5 For products applied topically, one of the two laboratory dose confirmation
studies should include evaluation of the effect of bathing and water immersion.
3 https://www.fda.gov/media/70333/download (May 2001)
4 For additional information about the recommended design and conduct of laboratory dose confirmation studies,
refer to GFI #90 (VICH GL7) and GFI #111 (VICH GL19).
5 Locations which are sufficiently far enough apart that organisms isolated from those areas are likely to represent
distinct populations.

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B. D. immitis Isolates
Laboratory dose confirmation studies should be conducted with D. immitis isolates that are
established from current D. immitis populations circulating in the United States and that
were established for experimental use within 5 years of study initiation. The two isolates
should be from two separate geographic locations, and at least one of the two isolates
should be from a highly endemic area, typically the southeastern United States6 or the
Mississippi Delta Region.7 Isolates used in studies for investigational new animal drugs
may, at the sponsor’s discretion, be characterized for susceptibility in accordance with the
principles stated in GFI #90 (VICH GL7 regarding field isolate characterization prior to
conduct of the laboratory dose confirmation studies.
C. Number of Dogs per Investigational Group
The chosen sample size should support the ethical principles of replacement, reduction, and
refinement,8 and the goal of using the minimum number of animals necessary to generate
data to demonstrate substantial evidence of effectiveness.
D. Number of L3 Injected for Induced Infection
FDA recommends that 50 infective D. immitis L3 larvae be injected subcutaneously per
dog to introduce consistent and adequate infections without overburdening the negative
control group dogs.
E. Pre-existing Heartworm Infection
FDA recommends testing for pre-existing (pre-study) heartworm infection at baseline and
at approximately 120 days (4 months) after the day of experimental infection with D.
immitis L3 larvae. Dogs determined to have pre-existing infections should be removed
from the effectiveness evaluation.
F. 	Assessment of Effectiveness
1. 	Parasite Counts
The pivotal effectiveness variable is the D. immitis worm count (combined live and
dead) at necropsy. Necropsy for D. immitis worm counts may occur as early as 150
days after infection with the D. immitis L3 larvae. Individuals conducting worm counts
6 FDA considers the southeastern United States to include the states of Alabama, Arkansas, Florida, Georgia,
Louisiana, Mississippi, North Carolina, South Carolina, and Tennessee.
7 FDA considers the Mississippi Delta Region to be the area covered by the Delta Regional Authority. Available
online at https://dra.gov/about-dra/map-room/ (Accessed February 20, 2024).
8 Russell WMS, Burch, RL (1959). The Principles of Humane Experimental Technique. London: Methuen & Co.
Special edition published by Universities Federation for Animal Welfare, 1992. Available online at:
https://caat.jhsph.edu/russell-and-burchs-principles-of-humane-experimental-techniques/ (Accessed February 20,
2024).

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should have sufficient training and expertise to perform the counts and should be
masked to treatment group.
Heartworm fragments, if any, should be counted as follows: Worm fragments
containing a head and worm fragments containing a tail are counted separately. The
greater of the two counts (number of fragments containing a head or number of
fragments containing a tail) are included in the total worm count for effectiveness
calculations. Fragments without heads or tails are not included in the worm count if
fragments containing heads or tails are found. If only fragments without heads or tails
are found, the fragments are collectively considered to represent one worm for
inclusion in the effectiveness calculations.
2. 	Criteria to Demonstrate Effectiveness
In addition to having an adequate infection in the negative control group and a
statistically significant difference in D. immitis worm count in the investigational new
animal drug group compared to the negative control group, investigational new animal
drugs should demonstrate at least 99.5 percent effectiveness and no more than one dog
in the investigational new animal drug group should have one or more worms identified
at necropsy. The geometric group means estimated from the statistical analysis of log-
transformed worm counts should be used. Study design features (e.g., blocks, cohorts)
should be included in the statistical model for the analysis.
Percent effectiveness = (M c - M t ) /M c x 100
Where:
M c = geometric mean back-transformed from the Least Squares mean of the control
group, and
M t = geometric mean back-transformed from the Least Squares mean of the
investigational new animal drug group.
G. Pharmacokinetic Evaluation
FDA recommends the collection of pharmacokinetic data during the dose confirmation
studies to verify dose exposure.
IV. 	Field Effectiveness Study
The field effectiveness study should be a multi-site study conducted with investigators in various
geographic regions of the continental United States with endemic heartworm disease. FDA’s
recommendations for specific study design elements are intended to increase the confidence that
exposure to infective D. immitis larvae, as well as a variety of isolates, occurred during the
evaluation period for the investigational new animal drug and that the evaluation of effectiveness
is assessed appropriately.

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A. Study Duration
Because of the length of the heartworm life cycle and the delay in the ability of diagnostic
tests (heartworm antigen and microfilaria tests) to identify infection with adult heartworms
after exposure to D. immitis larvae, the effectiveness of the investigational new animal drug
is ultimately evaluated for only a portion of the total study duration (the effectiveness
evaluation window).9
FDA recommends that for products intended for monthly administration, the effectiveness
evaluation window be a minimum of 6 months following the first dose. For products with
a duration of effectiveness greater than 6 months, the effectiveness evaluation window
should be the entire duration of effectiveness intended from one dose administration (for
example, 12 months for a product to be administered every 12 months). The study should
continue for an additional 240 days after the end of the effectiveness evaluation window to
allow for the development and detection of adult heartworms (based on currently available
testing methodologies) and collection of safety data (for example, a total study duration of
at least 420 days for a product intended for monthly administration and at least 605 days
for a product intended for yearly administration).
Longer study durations may be appropriate to address potential safety concerns identified
during product development. Dogs should remain on the same drug product (either
investigational new animal drug or active control) throughout the study.
B. Enrollment
The timing of enrollment should result in the investigational new animal drug being
evaluated during the time of year that maximizes the likelihood of exposure to mosquitos
infected with D. immitis. In the Northern Hemisphere, peak transmission is considered to
occur in July and August;10 therefore, unless the peak transmission season for a specific
geographic location differs, the effectiveness evaluation window for studies conducted in
the continental United States should include July and August. For example, in the case of
an oral product intended for monthly administration,11 for all dogs to be treated with the
investigational new animal drug, or active control, during the effectiveness evaluation
window of July and August, the enrollment period for a field effectiveness study should be
such that first dose administration occurs between April 1 and June 30.
9 The field safety of the investigational new animal drug is evaluated for the entire study.
10 American Heartworm Society (AHS). Current Canine Guidelines for the Prevention, Diagnosis, and Management
of Heartworm (Dirofilaria immitis) Infection in Dogs. Available online at
https://d3ft8sckhnqim2.cloudfront.net/images/pdf/AHS_Canine_Guidelines_11_13_20.pdf?1605556516 (Accessed
June 10, 2024).
11 In this example, the drug acts through the killing of larval stages with which the dog was infected within the prior
30 days.

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C. Location of Study Sites
To evaluate the effectiveness of the investigational new animal drug in highly endemic
portions of the United States, the majority (> 50 percent) of evaluable cases should be from
the southeastern United States,12 eastern/central Oklahoma, eastern/central Texas, or the
Mississippi Delta Region.13 No more than 40 percent of evaluable cases should be
provided by one site.
D. Assessment of Effectiveness
Effectiveness and detection of pre-existing infection should be determined by commercial
adult heartworm antigen testing and microfilaria detection testing by a concentration
method (e.g., modified Knotts). FDA recommends that heartworm tests (antigen and
microfilaria detection testing) be performed at baseline (prior to Day 0, the day of the first
dose), at appropriate intervals to detect preexisting infection, and on the predetermined
pivotal effectiveness study day based on the characteristics of the investigational new
animal drug.
For an individual dog to be considered a success, the individual dog should not test
heartworm antigen- or microfilaria-positive up to and including Day 120 (indicating a pre-
existing infection), at any subsequent interim timepoints, and on the predetermined pivotal
effectiveness day. Dogs testing positive between Day 120 and Day 240 should be
evaluated on a case-by-case basis.
For an investigational new animal drug to be considered effective, the lower bound of the
95 percent two-sided confidence interval for the percent success for all evaluable cases in
the investigational new animal drug group should be no lower than 98.0 percent. The
confidence interval should be computed using exact methods for binomial proportions.14
E. Active Control Group
To facilitate randomization and masking, and minimize bias in data collection,15 the study
should include an active control group that is administered an FDA-approved product16 for
12 See footnote 6 on page 3.
13 See footnote 7 on page 3.
14 Clopper, C. J. and Pearson, E. S. (1934) “The use of confidence or fiducial limits illustrated in the case of the
Binomial,” Biometrika, 26, 404 -413.
15 Masking is intended to minimize the potential biases resulting from differences in management, treatment, or
assessment of subjects, or interpretation of results, that could arise as a result of subject or investigator knowledge of
the assigned treatment. (From GFI, “E 10 Choice of Control Group and Related Issues in Clinical Trials,”
(https://www.fda.gov/media/71349/download).
16 Products subject to an approved new animal drug application (NADA) or abbreviated new animal drug
application (ANADA) for the prevention of heartworm disease in dogs.

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the prevention of heartworm disease in dogs.17 The ratio of cases allocated to the
investigational new animal drug group to the active control group should be 1:1 or greater
with a minimum of 50 control group animals. If possible, the active control should contain
the same active ingredient and route of administration as the investigational new animal
drug. The active control group is not used in the statistical evaluation of effectiveness.
F. 	Number of Evaluable Cases
The field effectiveness study should be large enough to provide independent substantiation
of evidence and inferential value to the target population. The number of evaluable cases
administered the investigational new animal drug should be at least 300 dogs. This number
of minimum evaluable cases increases the confidence that exposure to D. immitis-infected
mosquitos occurred and that dogs were exposed to a variety of D. immitis isolates during
the study. In addition, sponsors should consider the proportion of likely treatment
successes and the corresponding lower bound of the 95 percent confidence interval when
considering the target sample size.
G. Pharmacokinetic Evaluation
FDA encourages the collection of pharmacokinetic data during the field effectiveness study
to verify dose exposure.
17 To avoid the unnecessary risk of developing heartworm disease in client-owned dogs, the study should not include
a negative control group.