#276 
 
Effectiveness of Anthelmintics:  Specific 
Recommendations for Products Proposed 
for the Prevention of Heartworm Disease 
in Dogs  
 
Guidance for Industry 
 
Draft Guidance 
 
This guidance document is being distributed for comment purposes only. 
 
Submit comments on this draft guidance by the date provided in the Federal Register notice 
announcing the availability of the draft guidance.  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 docket number FDA-2022-D-2899. 
 
For further information regarding this document, contact Steven Fleischer, Center for Veterinary 
Medicine (HFV-110), Food and Drug Administration, 7500 Standish Place, Rockville MD 
20855, 240-402-0809, email: steven.fleischer@fda.hhs.gov. 
 
Additional copies of this draft guidance document may be requested from the Policy and 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) 
November 2022
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
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 .......................................................................................................... 2 
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.  Pilot Study Considerations ............................................................................................ 4 
H.  Pharmacokinetic Evaluation ......................................................................................... 4 
IV.  Field Effectiveness Study ..................................................................................................... 4 
A.  Study Duration ................................................................................................................ 4 
B.  Enrollment ....................................................................................................................... 5 
C.  Location of Study Sites ................................................................................................... 5 
D.  Assessment of Effectiveness ........................................................................................... 5 
E.  Active Control Group ..................................................................................................... 6 
F.  Number of Evaluable Cases ........................................................................................... 6 
G.  Pharmacokinetic Evaluation ......................................................................................... 6 
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  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
Effectiveness of Anthelmintics: Specific Recommendations for 
Products Proposed for the Prevention of Heartworm Disease in 
Dogs 
 
Draft Guidance for Industry 
 
This draft guidance, when finalized, will represent 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 draft guidance, if finalized, provides recommendations for the effectiveness evaluation of 
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, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys 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. 
 
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 
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 
 
1 https://www.fda.gov/media/70349/download 
2 https://www.fda.gov/media/70091/download 
3 https://www.fda.gov/media/70333/download 
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  Contains Nonbinding Recommendations 
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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.   
 
FDAs 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.  We have 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 you will conduct a field 
effectiveness study that incorporates 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.  For products applied topically, one of the two laboratory dose confirmation studies 
should include evaluation of the effect of bathing/water immersion. 
 
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 isolated from the field within 5 years of study initiation.  The two isolates should be 
from two different geographic locations, and at least one of the two isolates should be from 
a highly endemic area, typically the southeastern United States5 or the Mississippi Delta 
Region.6  Isolates used in studies for investigational new animal drugs may, at the sponsors 
 
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 We consider the southeastern United States to include the states of Alabama, Arkansas, Florida, Georgia, 
Louisiana, Mississippi, North Carolina, South Carolina, and Tennessee. 
6 We consider 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 November 16, 2022) 
2    Contains Nonbinding Recommendations 
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discretion, be characterized for susceptibility against FDA-approved products7 at the 
approved dosages, 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 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 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. 
 
 
7 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. 
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: 
http://altweb.jhsph.edu/pubs/books/humane_exp/het-toc.  (Accessed November 16, 2022) 
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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 -M) /M  x 100 
c t c
Where: 
M  = geometric mean back-transformed from the Least Squares mean of the control 
c
group, and 
M = geometric mean back-transformed from the Least Squares mean of the 
t
investigational new animal drug group. 
 
G. Pilot Study Considerations 
D. immitis isolates used in pilot laboratory dose confirmation studies should not be used in 
the pivotal laboratory dose confirmation studies. 
 
H. 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.  FDAs 
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. 
 
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   
 
9 The field safety of the investigational new animal drug is evaluated for the entire study. 
4    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
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 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, 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. 
 
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,11 eastern/central Oklahoma, eastern/central Texas, or the 
Mississippi Delta Region.12  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 
 
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 
November 16, 2022) 
11 See footnote 5 on page 2. 
12 See footnote 6 on page 2.  
5    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
microfilaria detection testing) should 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 240 days after first dose 
administration, at any subsequent interim timepoints, and on the predetermined pivotal 
effectiveness day.  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.13 
 
E.  Active Control Group 
To minimize bias in data collection, the study should include an active control group that is 
administered an FDA-approved product14 for the prevention of heartworm disease in 
dogs.15  The ratio of cases allocated to the investigational new animal drug group to the 
active control group should be 1:1 or greater.  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 recommends the collection of pharmacokinetic data during the field effectiveness 
study to verify dose exposure. 
 
13 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. 
14 See footnote7 on page 3. 
15 To avoid the unnecessary risk of developing heartworm disease in client-owned dogs, the study should not include 
a negative control group. 
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