Product Development 

Under the 

Animal Rule 

Guidance for Industry 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
Center for Biologics Evaluation and Research (CBER) 
 
 
October 2015 

Animal Rule
 
    
Product Development 

Under the 

Animal Rule 

Guidance for Industry 

 
Additional copies are available from: 
 
Office of Communications,  Division of  Drug Information 
  
Center for Drug Evaluation  and Research 

Food and  Drug Administration 
 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor 

Silver Spring, MD 20993-0002 
  
Phone: 855-543-3784 or 301-796-3400;  Fax: 301-431-6353 

Email: druginfo@fda.hhs.gov 
  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm  
 
or  
 
Office of Communication, Outreach and Development  

Center for Biologics Evaluation and Research 
 
 Food and Drug  Administration 
 
10903 New Hampshire Ave., Bldg. 71, Room 3128 

Silver Spring, MD 20993-0002 

Phone: 800-835-4709 or 240-402-8010 

Email: ocod@fda.hhs.gov 
 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm     
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
Center for Biologics Evaluation and Research (CBER) 
 
 
October 2015 

Animal Rule 
 
  Contains Nonbinding Recommendations 
TABLE OF CONTENTS 
 
 
 
I.   INTRODUCTION............................................................................................................. 1
  
II.   THE ANIMAL RULE ...................................................................................................... 2 
 
III.   REGULATORY CONSIDERATIONS .......................................................................... 7 
 
A.   Drug Development Plan ................................................................................................................ 7
  
B.   Access to Investigational Drugs During a Public Health Emergency ....................................... 9 
 
C.   Communications With FDA........................................................................................................ 10 
 
D.   Animal Model Qualification Program ....................................................................................... 10 
 
IV.   ANIMAL STUDIES  GENERAL EXPECTATIONS ............................................... 12 
 
A.   Animals Used in Investigations................................................................................................... 12 
 
B.   Study Conduct .............................................................................................................................. 13 
 
C.   Types of Animal Care Interventions .......................................................................................... 14 
 
D.   The Study Report......................................................................................................................... 14 
 
E.   Submission of the Study Report and Data ................................................................................. 15
  
V.   ESSENTIAL ELEMENTS OF AN ANIMAL MODEL.............................................. 16 
 
A.   Elements Related to the Etiologic or Challenge Agent-Induced Disease or Condition ......... 17 
 
1.  Characteristics of the Etiologic or Challenge Agent That Influence the Disease or Condition .... 17
  
a.   The Challenge Agent............................................................................................................... 17 
	
b.   Pathophysiological Mechanisms of Toxicity  or Virulence ..................................................... 18 
	
c.   Route of Exposure................................................................................................................... 18 
	
d.   Dose and Quantification of Exposure  ..................................................................................... 19 
	
2.  Host Susceptibility and Response .................................................................................................. 19 
 
3.  Natural History of the Disease or Condition  Pathophysiological Comparability ...................... 20 
 
a.   Time to Onset .......................................................................................................................... 21 
	
b.   Progression.............................................................................................................................. 21 
	
c.   Manifestations......................................................................................................................... 22 
	
4.  Trigger for Intervention ................................................................................................................. 22 
 
B.   Elements Related to the Investigational Drug and the Selection of an Effective Dose in 

Humans.................................................................................................................................................. 23 
 
1.  The Investigational Drug ............................................................................................................... 23 
 
a.   Mechanism of Action .............................................................................................................. 23 
	
b.   Drug Class............................................................................................................................... 24 
	
c.   Dosage Form and Route of Administration ............................................................................ 24 
	
2.  Selection of an Effective Dose in Humans ..................................................................................... 24 
 
a.   PK and PD Information to Be Obtained in Animals and Humans .......................................... 25 
	
b.   PK/PD Considerations for Human Dose Selection ................................................................. 26 
	
VI.   DESIGN CONSIDERATIONS FOR THE ADEQUATE AND WELL-
CONTROLLED EFFICACY STUDIES IN ANIMALS ......................................................... 30 
 
A.   General Principles........................................................................................................................ 31 
 
  Contains Nonbinding Recommendations 
B.   Dose Selection in Animals ........................................................................................................... 33 
 
VII.   CONSIDERATIONS FOR PREVENTIVE VACCINES AND FOR CELLULAR 

AND GENE THERAPIES ......................................................................................................... 35 
 
A.   Vaccines ........................................................................................................................................ 35 
 
B.   Cellular and Gene Therapies ...................................................................................................... 36 
 
1.  Cellular Therapy Products  ............................................................................................................ 36 
 
2.  Gene Therapy Products ................................................................................................................. 37 
 
VIII.   HUMAN SAFETY INFORMATION ........................................................................... 38 
 
IX.   CHECKLIST OF ESSENTIAL ELEMENTS OF AN ANIMAL MODEL .............. 41
  
X.   CHECKLIST OF ELEMENTS OF AN ADEQUATE AND WELL-CONTROLLED 

ANIMAL EFFICACY STUDY PROTOCOL.......................................................................... 42 
 
APPENDIX A:  GENERAL PRINCIPLES FOR THE CARE AND USE OF ANIMALS IN 

BIOMEDICAL RESEARCH ..................................................................................................... 43 
 
APPENDIX B: TYPES OF ANIMAL CARE INTERVENTIONS ....................................... 46 
 
APPENDIX C: GENERAL EXPECTATIONS FOR NATURAL HISTORY STUDIES... 48 
 
APPENDIX D: ACRONYMS AND ABBREVIATIONS ....................................................... 49 
 
  Contains Nonbinding Recommendations 
Product Development Under the Animal Rule 

1
 
Guidance for Industry  
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic. It does not create 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 office responsible for this guidance as listed on the title 
page. 
 
 
 
I.  INTRODUCTION  
 
This guidance2 provides information and recommendations on drug and biological product3  
development when human efficacy studies are not ethical or  feasible.  The regulations that set 
forth the pathway for approval4  of these products under 21 CFR 314.600 through 314.650 
(drugs) or 21 CFR 601.90 through 601.95 (biological  products) are commonly referred to as the 
Animal Rule. 
 
This guidance does not address the following topics: 
  
	   The chemistry, manufacturing, and controls or nonclinical pharmacology and toxicology 
studies necessary for drug development 
	   Issues related to initial proof-of-concept studies  
	   The details of study design and conduct for drug-specific animal efficacy studies or for 
human pharmacokinetic (PK) and/or safety studies 
	   Drug development in specific populations (e.g., pediatrics, geriatrics, and pregnant 
women)5  
	   The development of combination products 
                                                 
1 This  guidance has been  prepared by the Center for Drug Evaluation and Research (CDER) in cooperation with the 
Center for Biologics Evaluation and Research  (CBER) at the Food and Drug Administration.  The Office of  Good  
Clinical Practice and the  Office of  Counterterrorism  and  Emerging  Threats also  provided  input.  
2  This guidance finalizes the 2014 revised draft guidance for industry Product Development Under the Animal Rule, 
which replaced the 2009  draft  guidance for industry  Animal Models    Essential Elements to Address Efficacy Under 
the Animal Rule. 
3  As used  in  this  guidance, all references to  drugs include human drugs, therapeutic  biological products, cellular and 
gene  therapies,  and vaccines,  unless  otherwise specified. 
4  As used  in  this  guidance, the term  approval  refers to  approval or licensure. 
5  The Animal Rule applies equally to  adult and  pediatric populations. 
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	 Contains Nonbinding Recommendations 
 	  Requirements for procurement of medical countermeasures by the federal government 
(e.g., Strategic National Stockpile (SNS)6) 
 	  The development of animal models for other purposes, such as for assessment of 

toxicology 

 
Information on FDA guidances is available on FDAs Web page.7  In addition, FDA guidances 
related to medical countermeasures for chemical, biological, radiological, and nuclear (CBRN) 
agents can be  accessed through FDAs Medical Countermeasures Initiative (MCMi) Web page.8  
 
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.  THE ANIMAL RULE 
 
FDA's regulations concerning the approval of new drugs when human efficacy studies are not 
ethical9  and field trials are not feasible are codified in 21 CFR 314.600 through 314.650 for 
drugs and 21 CFR 601.90 through 601.95 for biological products.  Approval under the Animal 
Rule can be pursued only if human efficacy studies cannot be  conducted because the conduct of 
such trials is  unethical and field trials after an accidental or deliberate exposure are not feasible.  
The Animal Rule does not apply to drugs that can be approved for the proposed indication 
based on efficacy standards described elsewhere in FDAs regulations . . . .10    
 
The Animal Rule states that for drugs developed to ameliorate or prevent serious or life-
threatening conditions caused by exposure to lethal or permanently disabling toxic substances, 
when human efficacy studies are not ethical and field trials are not feasible, FDA may grant 
marketing approval based on adequate and well-controlled animal efficacy studies when the 
results of those studies establish that the drug is reasonably likely to produce clinical  benefit in 
humans.  Drugs evaluated for efficacy under the Animal Rule should be evaluated for safety 
under the existing requirements for establishing the safety of new drugs.  The Animal Rule states 
                                                 
6  Sponsors  should  discuss issues related  to  the SNS with  the Department  of  Health  and  Human  Services/Biomedical 
Advanced Research and Development Authority (HHS/BARDA) and the Centers for Disease Control and 
Prevention (CDC).  
7  FDA guidances are updated periodically.  The most recent versions are available at 
http://www.fda.gov/RegulatoryInformation/Guidances/default.htm. 
8 The MCMi  Web page is available at  
http://www.fda.gov/emergencypreparedness/medicalcountermeasures/default.htm. 
9 As described in the Scope of  the Animal Rule,  . . . it would be unethical  to deliberately expose healthy human 
volunteers to a lethal or permanently disabling toxic biological, chemical, radiological, or  nuclear substance . . . .   
See 21  CFR 314.600  for  drugs and  21  CFR 601.90  for  biological products. 
10  See 21  CFR 314.600  for  drugs and  21  CFR  601.90  for  biological products. 
  2
	 Contains Nonbinding Recommendations 
that FDA will rely on evidence from animal studies to provide substantial evidence11  of 
effectiveness only when all of the following four criteria are met: 
 
1.		  There is a reasonably well-understood pathophysiological mechanism of the toxicity of 
the substance and its prevention or  substantial reduction by the product; 
 
2.		  The effect is demonstrated in more than one  animal species expected to react with a 
response predictive for humans, unless the effect is demonstrated in a single animal 
species that represents a sufficiently well-characterized animal model for predicting the 
response in humans; 
 
3.		  The animal study endpoint is clearly related to  the desired benefit in humans, generally 
the enhancement of survival or prevention of major morbidity; and 
 
4.		  The data or information on the kinetics and pharmacodynamics of the product or other 
relevant data or information, in animals and humans, allows selection of an effective dose 
in humans.12  
 
If  all of  these criteria are  met, it is reasonable to  expect the effectiveness of the drug in animals to 
be a reliable indicator of its effectiveness in humans.  
 
The use of the Animal Rule as a regulatory pathway to approval is intended for drugs developed 
to ameliorate or prevent serious or life-threatening conditions caused by chemical, biological, 
radiological, or nuclear substances  regardless of whether the substances are considered potential 
threat agents for deliberate exposure (e.g., nerve agent, Bacillus anthracis) or threats to 
individuals health from  accidental exposure (e.g., emerging infectious pathogens, snake venom, 
industrial chemicals), provided that  human efficacy studies are not  ethical and field trials to 
study effectiveness of the drug are not feasible. 
 
FDA will determine whether the previously noted  criteria have  been met and the Animal Rule 
can be used.  In general, the determination of whether it is ethical to conduct deliberate exposure 
studies in humans is not difficult; however, the determination  that human field trials are not 
feasible may be challenging.  The feasibility issues to be considered will depend on the disease 
or condition to be studied and may change over time.  For example, there may be circumstances 
that affect the feasibility of  planning and conducting human field trials for the disease or 
condition, such as (1) a low prevalence and/or incidence, (2) an unpredictable incidence rate 
from  year to  year, (3) an inability to predict geographic locations where outbreaks may occur, (4) 
occurrences limited to areas lacking critical infrastructure, and/or (5) occurrences limited to areas 
                                                 
11  The  term  substantial evidence has been  defined previously in section 505(d)  of the Federal Food, Drug, and 
Cosmetic Act (FD&C Act) (21  U.S.C. 355(d)) as follows:   . . . evidence  consisting of  adequate and well-controlled 
investigations, including  clinical investigations, by  experts qualified  by  scientific training  and experience to  evaluate 
the effectiveness of the drug involved, on the basis of  which it could fairly and responsibly be concluded by such  
experts that the drug  will have  the effect it purports or is  represented  to have under the conditions  of use prescribed, 
recommended, or suggested in the labeling or proposed labeling thereof. 
12  See 21  CFR 314.610(a)  for  drugs and  21  CFR 601.91(a)  for  biological products. 
  3
	 Contains Nonbinding Recommendations 
in which there is some extraordinary  threat  to subject or investigator safety.  However, the 
international collaboration in response to  the 20142015 Ebola epidemic in West Africa 
highlights the fact that  determination  of  infeasibility of  clinical  trials can change over time.  
Sponsors should provide FDA with a clear rationale to support the use of the Animal Rule for the 
development of their drug  before proceeding with drug development. 
 
With regard to establishing evidence of efficacy, the Animal Rule states:  In assessing the 
sufficiency of animal data, the agency may take into account other data, including human data, 
available to the agency.13   Although data from  different types of studies (e.g., in vitro studies, 
other types of animal studies, human studies) may be supportive, adequate and well-controlled 
animal efficacy studies are required for approval under the Animal Rule.   
 
Supportive human data can include clinical efficacy  data either from  available human data for 
the same  indication (as was the case for the 2006 approval under the Animal Rule of Cyanokit 
(hydroxocobalamin) for the treatment of known or suspected cyanide poisoning), or from  
available human data for a relevant non-Animal Rule-based indication (as was the case for the 
2012 approval under the Animal Rule of Levaquin (levofloxacin) for the treatment of pneumonic 
and septicemic plague caused by Yersinia pestis). 
 
For Cyanokit (hydroxocobalamin), efficacy was established in a dog model of cyanide poisoning 
that FDA considered sufficiently well-characterized for predicting the response in humans.  
Existing human data from open-label, uncontrolled studies using hydroxocobalamin to treat 
cyanide poisoning from smoke inhalation, cyanide ingestion, or cyanide inhalation provided 
additional support for its approval. 
 
For Levaquin (levofloxacin), efficacy was established in an African green monkey model of 
pneumonic plague that FDA considered sufficiently well-characterized for predicting the 
response in humans.  Existing human data from  its prior approvals for other respiratory 
infections (i.e., nosocomial and community-acquired pneumonias) provided additional support 
for its likely  effectiveness in the treatment of pneumonic and septicemic plague.   
 
When human efficacy data from  a relevant indication may support the approval of the Animal 
Rule-based indication, FDA encourages sponsors to  evaluate the drug in an indication for which 
obtaining human data is possible under  a traditional regulatory  pathway.14  In addition to the 
previously discussed Levaquin (levofloxacin) example, another example of when human efficacy 
data obtained in a related human disease or condition may support the determination of  efficacy 
for the Animal Rule-based indication is when the drug targets a pathway in the 
pathophysiological cascade that is common to both the disease or condition intended for 
evaluation under the Animal Rule and a disease or condition for which clinical trials are ethical 
and feasible.  
                                                 
13  See 21  CFR 314.610(a)  for  drugs and  21  CFR 601.91(a)  for  biological products.   
14  As stated in the preamble to the final rule New Drug and Biological  Drug Products; Evidence Needed  to 
Demonstrate Effectiveness of New Drugs  When Human Efficacy Studies Are  Not Ethical or Feasible  (the final 
rule) (67 FR  37988 at  37990  footnote  2, May 31, 2002),  . . . with anti-infective drug products, it would usually be 
expected  that human data on safety and effectiveness for other indications may be available.  
  4
	 Contains Nonbinding Recommendations 
 
The Animal Rule specifies that the choice of  species for the adequate and well-controlled 
efficacy studies must be appropriate with regard to the disease or condition of interest and the 
investigational drug.15   There is no requirement for the use of  a specific species.  For each animal 
species selected by sponsors, the sponsors should provide scientific justification that the animal 
species exhibits key characteristics of  the human disease or condition when the animal is 
exposed to the challenge agent.16   In addition, the species should be selected based on an 
understanding of the drugs mechanism of action, such that the drugs effect in the animal 
species is expected to be predictive of  its effect in humans, and to allow extrapolation from the 
animal data to the selection of an effective dose and regimen for humans. 
 
The number of animal species necessary to support approval of  a drug under the Animal Rule 
depends on the nature and clinical significance of any differences between the animal models17  
and humans with regard to the essential elements as described in section V.  Sponsors should 
provide data or information to demonstrate that each animal model reflects key aspects of the 
pathophysiology of the human disease or condition of interest and that the response to the 
investigational drug in each animal model is likely to predict the response in humans.  
 
FDA will evaluate the suitability of a proposed animal model on a case-by-case basis.  
Generally, the efficacy of the drug should be demonstrated in more than one animal species 
expected to react with a response predictive for humans.  In certain circumstances, studies in 
more than two species may be necessary to model the relevant aspects of the human disease or 
condition and response to the investigational drug.  If the effect  is demonstrated in a single 
species that represents a sufficiently well-characterized animal model18  for predicting the 
response in humans, then the Animal  Rule allows for approval based on substantial evidence of 
effectiveness demonstrated in studies  conducted in that species.  The acceptability of using a 
single animal species will require FDA review and agreement on the body of evidence 
supporting the adequacy of the model.  As discussed in the preamble to the final rule:19   
 
[The]  circumstances in which the agency will rely on evidence from  studies in 
one animal species to provide substantial evidence of the effectiveness of these 
products in humans would generally be limited to situations where the study 
model is sufficiently well-recognized so as  to render studies in multiple species 
                                                 
15  See 21  CFR 314.610(a)  for  drugs and  21  CFR 601.91(a)  for  biological products.   
16  As used  in  this  guidance, the term  challenge agent  refers to  the substance used  to  cause the disease or  condition in  
the animal studies, whereas the term etiologic agent  refers to the substance causing the disease or condition in  
humans.  
17  For the purpose of this guidance, an  animal model  is defined as  a specific combination of an animal species, 
challenge agent, and  route of  exposure that produces a disease process or  pathological condition  that in multiple 
important  aspects corresponds to  the human  disease or  condition  of interest. 
18  In  the preamble to  the final rule (67  FR 37988  at 37989, May 31, 2002), a well-characterized animal model was 
defined as meaning the model has been adequately evaluated for its responsiveness.  
19  See 67  FR 37988  at 37991, May 31, 2002. 
  5
	 Contains Nonbinding Recommendations 
unnecessary.  In addition, other human  data for the product could provide 
support for such approvals. 
 
When efficacy is demonstrated in a single study conducted in a sufficiently well-characterized 
animal model, it may be necessary to conduct a confirmatory efficacy study in that animal 
model.20  The confirmatory study ideally should be conducted at a different  laboratory, however, 
use of the same laboratory may be acceptable with justification.  Supportive human efficacy data 
may negate the need for a confirmatory study, as  was the case for Levaquin (levofloxacin) for 
pneumonic plague and Cyanokit (hydroxocobalamin) for cyanide poisoning. 
 
There may be situations in which the application of the Animal Rule requires a more complex 
development plan.  For example, variola virus (the  etiologic agent of smallpox) presents a unique 
challenge because humans are the only known natural host, no animal species has been found to 
have comparable susceptibility to variola virus, and naturally occurring smallpox has been 
eradicated.  Therefore, efficacy of investigational drugs developed to treat smallpox needs to be 
studied using other orthopoxviruses in relevant animal species (e.g., monkeypox in nonhuman 
primates, rabbitpox in rabbits, ectromelia in mice).  Depending on the strength of the animal 
studies and other supporting evidence, the efficacy findings from such studies may support 
approval of the drug against variola.  As with all animal efficacy studies, FDA strongly 
recommends  that in such situations, sponsors discuss the scientific approach under consideration 
with the review division before  initiating the animal studies.  
 
Approval of a drug under the Animal Rule imposes three additional requirements, which are 
summarized below (for greater detail, see 21 CFR 314.610(b)(1) through (3) for drugs and 21 
CFR 601.91(b)(1) through (3) for biological products): 
 
1.		  Postmarketing studies (e.g., field studies) to provide evaluation of safety and clinical 
benefit if circumstances arise in which a study would be feasible and ethical (i.e., in the 
event an emergency arises and the drug is used).  A plan or approach to conducting such 
a study must be included with the new drug application (NDA) or biologics license 
application (BLA). 
 
2.		  Restrictions to ensure safe use, if needed (e.g., restricting distribution to facilities or 
health care practitioners with  special training, requiring specified types of follow up, or 
imposing record keeping requirements). 
 
3.		  Information to be provided in  the labeling to patient recipients that explains that for 
ethical or feasibility reasons, the drugs approval was based on efficacy studies conducted 
in animals alone.  This drug labeling should also include all the other relevant 
information required by FDA at the time  of approval (e.g., directions for use, 
contraindications, a description of any reasonably  foreseeable risks, adverse reactions, 
                                                 
20  As stated in the preamble to the final rule,  . . . the  animal studies should be replicated or substantiated in each 
species as  needed to ensure credible results . . .  (67  FR 37988  at 37991, May 31, 2002). 
  6
	 Contains Nonbinding Recommendations 
anticipated benefits, and drug interactions). This information must be provided before 
administration or dispensing, if possible. 
 
Products approved under the Animal Rule are subject to postmarketing recordkeeping and safety 
reporting applicable to all approved drug and biological products.21   Information on withdrawal 
procedures, submission of promotional materials,  and termination of certain requirements for 
products approved under the Animal Rule is specified in the regulations.22  
 
 
III.  REGULATORY CONSIDERATIONS  
 
A.  Drug Development Plan 
 
Obtaining the body of evidence necessary to support approval of a drug using the Animal Rule is 
a complex and iterative process.  FDA strongly encourages sponsors to establish early and 
ongoing communications with the Agency.  Sponsors also may wish to seek input from public 
health officials and/or the military about the potential need for, and operational use of, the 
investigational drug and  discuss this with FDA.  Developing a drug development plan  will 
support the discussion of important issues, including, but not limited to, the following: 
 
 	  The proposed indication and whether a drug can be developed under the Animal Rule 
 	  The design of an animal  study as it relates to the anticipated clinical use of the drug 
during an incident   
 	  The development and/or selection of the animal models, including, when necessary, the 
design of the natural history studies 
 	  The results of the proof-of-concept studies 
 	  The proposed methods for selecting an effective dose and regimen in humans 
 	  The design of the adequate and well-controlled animal efficacy studies intended to 
provide the primary evidence of effectiveness of  the drug 
 	  The proposed approach for ensuring the quality and integrity of data23  
 	  The size and composition of the human safety database  
                                                 
21  See 21  CFR 314.630  for  drugs and  21  CFR  601.93  for  biological products. 
22  See 21  CFR 314.620, 21  CFR 314.640, and 21  CFR 314.650, respectively, for drugs and 21 CFR  601.92, 21 CFR  
601.94, and 21 CFR 601.95, respectively, for biological products.  
23  In  issuing  the Animal Rule, FDA stated that  . . . studies subject to this rule must be  conducted in  accordance 
with  preexisting requirements under the good laboratory  practices (21 CFR part  58) regulations . . .  (67 FR  37988  
at 37989, May 31, 2002).  The good  laboratory practice (GLP) regulations, however, were developed  as a quality 
system for nonclinical safety studies.  FDAs current expectations are described in section IV.B.  
  7
	 Contains Nonbinding Recommendations 
 	  Plans or approaches for conducting the required postmarketing studies (e.g., field studies) 
to demonstrate safety and clinical benefit when such studies are feasible and ethical24    
	   Timelines and/or milestones for FDA feedback or meetings 
	   Eligibility for expedited  development and review designation programs 
	   Additional issues critical to the sponsors funding agencies25  
 
Drug development is data-driven; any development plan should allow for modification or 
refinement as data are gathered and analyzed and projections or expectations change.  It is the 
sponsors responsibility to provide  complete and  accurate submissions.  Sponsors should explain  
any proposed deviations from  the recommendations expressed in this guidance.  The potential 
impact of these deviations on the drug development program should be discussed with FDA 
before the conduct of the relevant studies. 
 
FDA strongly recommends that sponsors obtain Agency concurrence on the design of the 
adequate and well-controlled animal efficacy studies because these substitute for the efficacy 
trials in humans (see sections VI  and X).  Sponsors should allow adequate time  for FDA review, 
comment, and agreement before initiating these studies to ensure that the study design is 
adequate to support the proposed indication.   
 
The protocols for animal efficacy studies intended to provide primary evidence of effectiveness 
are eligible for evaluation under special protocol assessment (SPA) provisions.26,27  Before 
submitting a Request for SPA, the sponsor should have FDA concurrence on the model proposed 
for use in the efficacy study (including, but not limited to, the species, the details of the challenge 
agent, the conditions of exposure) and the method that will be used to extrapolate from the 
animal data to select an effective dose and regimen in humans. 
 
Drugs developed under the Animal Rule may be eligible for two of the expedited development 
and review programs28  (fast track and priority review) or  other FDA programs, such as orphan 
drug designation.29  Sponsors requesting these designations should use established procedures.  
Drugs being developed under the Animal Rule do not meet the statutory requirement for 
                                                 
24  See 21  CFR 314.610(b)(1)  for drugs and  21  CFR 601.91(b)(1)  for  biological products. 
25  Product development plans required by  funding  agencies for medical countermeasures against CBRN agents may 
dictate certain proof-of-concept studies and an accelerated  timeline for efficacy studies in  animals.  The sponsors  
relationship with  its funding  agency is independent  of  its  relationship with  FDA.  
26 Section 505(b)(5)(B) of the  FD&C Act (as amended by the Pandemic and All-Hazards Preparedness 
Reauthorization Act of  2013,  Public Law 113-5) provides for the use of SPA provisions in the case where human 
efficacy studies are not ethical or feasible, of animal and any associated clinical trials which, in  combination, are 
intended  to  form  the primary basis of an effectiveness claim. 
27 For procedural information, see FDAs guidance for industry  Special Protocol  Assessment.    
28  See FDAs guidance for  industry Expedited Programs  for Serious ConditionsDrugs and  Biologics. 
29 For information on orphan drug designation, see the  Web page, available at  
http://www.fda.gov/ForIndustry/DevelopingProductsforRareDiseasesConditions/HowtoapplyforOrphanProductDesi 
gnation/ucm135122.htm. 
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	 Contains Nonbinding Recommendations 
breakthrough therapy designation, which specifically requires clinical evidence of a treatment 
effect.30  The Best Pharmaceuticals for Children Act (BPCA)31 and the Pediatric Research Equity 
Act of 2003 (PREA)32  may also apply to drugs developed under the Animal Rule. 
 
Sponsors should note that FDA may seek input from  advisory committees for various  issues 
related to the Animal Rule.  Issues for discussion can include whether the Animal Rule is the 
appropriate regulatory development pathway for drugs intended for a specific indication, 
concurrence on the animal model of a disease or  condition, the acceptability of the use of an 
animal model with a specific investigational drug, the design of adequate and well-controlled 
animal efficacy studies, and whether the data obtained support approval.  In some  instances, 
more than one advisory committee meeting may be warranted at different times in a single 
development program. 
 
B.  Access to Investigational Drugs During a Public Health Emergency  
 
Data collected from  animal efficacy studies may support the emergency use of drugs under an 
investigational new drug application (IND)33  or an emergency use authorization (EUA).34   
FDAs decision to allow emergency use of a drug under an IND or EUA will be made on a case-
by-case basis, taking into account  the anticipated or actual emergency, size of the affected 
population, data included in the submission, and risk-benefit analysis. A decision to allow 
emergency use of the drug under an IND or EUA, based on data submitted in support of either 
mechanism, should not be viewed as an FDA determination on approvability of the drug or a 
final drug development goal.  FDA emphasizes that drug development and systematic data 
collection should be continued to obtain the body of evidence to support drug approval and 
associated postmarketing requirements.  In the event of an unexpected increase or occurrence of 
                                                 
30 See section 506  of the FD&C Act  (21 U.S.C. 356) (as amended by the Food and Drug  Administration Safety and 
Innovation Act (FDASIA), Public Law 112-144).   
31 See Public Law 107-109.  
32 See Public Law 108-155.  
33 Expanded access for individual patients (including  for emergency  use), intermediate-size patient  populations, and 
large patient  populations (under a treatment IND or treatment protocol) are described in  21 CFR  312.300 through  
312.320.  Individual patient INDs, however, are not a feasible strategy  for a large-scale event requiring mass access 
to an investigational drug.  Sponsors anticipating multiple access requests for an  investigational drug should discuss 
proposals for IND protocols with FDA, including any  potential for clinical trial design that could provide safety and 
efficacy information as well as access.   FDA has issued  two draft guidances on expanded access.  When the 
guidance on  Expanded Access to Investigational Drugs for Treatment Use  Qs & As and  the guidance on   
Individual  Patient Expanded  Access Applications:  Form FDA 3926  are finalized, they will represent the Agencys 
current thinking  on  this topic. Additional information on expanded  access  can be found on FDAs  Expanded Access  
(Compassionate Use)  Web page, available at  
http://www.fda.gov/NewsEvents/PublicHealthFocus/ExpandedAccessCompassionateUse/default.htm. 
34  EUA  criteria  are described in FDAs guidance  Emergency Use Authorization of  Medical Products.  Additional 
information on EUA can be found  on FDAs Emergency  Use Authorization Web page, available at  
http://www.fda.gov/EmergencyPreparedness/Counterterrorism/MedicalCountermeasures/MCMLegalRegulatoryand 
PolicyFramework/ucm182568.htm. 
 
  9
	 Contains Nonbinding Recommendations 
a disease or condition, sponsors should discuss with FDA if field studies have become feasible 
and ethical (see related discussion in section II).   
 
C.  Communications With FDA 
 
Sponsors are encouraged to hold discussions with FDA in the early stages of a drug development 
program.  Sponsors unsure of the appropriate regulatory review division or office for their 
investigational drugs can inquire through the electronic mailbox,  CDER-CBER-
ARJurisdiction@fda.hhs.gov, provided by CDER and CBER for this sole purpose. 
 
Sponsors should consult Agency guidance regarding the process and expectations for formal 
meetings.35  The sponsor and review division should discuss the avenues and expectations for 
obtaining Agency concurrence on the design of the adequate and well-controlled animal efficacy 
studies and for addressing extenuating or unforeseen circumstances.  It is the sponsors 
responsibility to build sufficient time into the development plan to permit the review, discussion, 
and resolution of issues prior to  the initiation of relevant  studies.  FDA will try to accommodate 
the sponsor should unforeseen circumstances arise. 
 
Some of the drug development issues that should be the subject of meetings with FDA36  will 
differ from  those for drugs developed under other regulatory pathways.  Examples of issues for 
Animal Rule drug development discussions are listed in section III.A.  
 
D.  Animal Model Qualification Program 
 
The Animal Model Qualification Program (AMQP)37  is jointly supported by CDER and CBER 
to address the need for publicly  available animal models for use in drug development under the 
                                                 
35  See FDAs guidance for  industry Formal Meetings Between the FDA and Sponsors or Applicants  (Revision 1).   In  
the Federal Register  of March  11, 2015  (80  FR 12822), FDA  published  a notice announcing  the availability of  a 
draft guidance Formal  Meetings Between the FDA and  Sponsors or Applicants of PDUFA Products  (Revision  2).  
The revised draft guidance updates  the guidance for industry Formal Meetings Between the FDA and Sponsors or  
Applicants  (Revision  1)  and,  when  finalized, will represent  the Agencys current thinking  on  the topic.  
36  Section 565(d)  of  the FD&C  Act (21  U.S.C. 360bbb-4(d))  (as amended by the Pandemic and  All-Hazards 
Preparedness Reauthorization Act of  2013,  Public Law 113-5) provides that sponsors developing countermeasures 
under the Animal Rule  may  request and receive two meetings with FDA, one to discuss  proposed animal  model  
development activities and  the other prior to  initiating  pivotal animal studies.  These meetings  and  procedures for 
obtaining such  meetings are within the scope of FDAs guidance for industry  Formal Meetings Between the FDA 
and Sponsors or Applicants  and satisfy this  requirement (see the preceding footnote).  
37 The AMQP was established under FDAs Drug Development Tools (DDT) Qualification Programs (see FDAs 
guidance for industry and FDA staff Qualification Process for Drug Development Tools).   Additional  information 
about qualifying animal  models can be accessed through the Animal  Model Qualification Program  Web page, 
available at 
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugDevelopmentToolsQualificationProgram/ucm284078 
.htm. 
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	 Contains Nonbinding Recommendations 
Animal Rule.38  Through this program, animal models are evaluated and qualified for a specific 
context of use (COU) that describes the appropriate use and application of  the qualified animal 
model in drug development and regulatory review and specifies the details39 necessary to 
replicate the model.  Submitting a model for qualification is voluntary.  Approval under the 
Animal Rule does not require the use of a qualified model. 
 
Qualification is a regulatory conclusion40,41  that is not linked to a specific drug.  Qualification of 
an animal model through the AMQP  indicates that (1) FDA has concluded that a specific animal 
species, given a specific challenge agent by a specific route, produces a disease process or 
condition that in multiple important aspects corresponds to the human disease or condition of 
interest, and  (2) FDA has accepted the description of the models appropriate use in regulatory 
applications,  including the definition of  the parameters of  the disease or  condition that will be 
used as measures of quality control and quality  assurance when the model is used.  After the 
animal model is qualified, FDA does not have to  reevaluate this conclusion each time  this 
qualified model is used within the bounds of its  stated COU. 
 
Before using a qualified animal model of a disease or condition in an adequate and well-
controlled efficacy study,  the sponsor of an investigational drug  should establish that the model 
is a suitable test system  for the drug with regard to the drugs mechanism  of action and related 
host factors and the ability to extrapolate from  the animal data to select a dose and regimen in 
humans (see section V.B).  Similarly,  because animal models are qualified  without reference to a 
specific drug, the use of a qualified animal model does not ensure that the model will be found 
acceptable as a single animal species that represents a sufficiently well-characterized animal 
model for predicting the response in humans, as stated in the second criterion for drug approval 
under the Animal Rule.42   FDA may not accept evidence of effectiveness from  a single animal 
model (even if it is qualified) for an investigational drug unless FDA concludes there is sufficient 
evidence that the results generated in this model adequately predict the response to the drug in 
humans.  The regulatory decision to allow approval of a drug based on the use of an animal 
model in a single species will be made by the review division  on a case-by-case basis (see 
section II).  
 
                                                 
38  Qualification  of  an animal model is voluntary and  is limited  to animal models developed for the intended  purpose 
of supporting the development programs for multiple investigational  drugs for the same targeted  disease or  
condition.  A model developed  by  a sponsor of  an  investigational drug  for the intended  purpose of use in  the 
development program  of  that drug  alone will not be  eligible for qualification. 
39  These details include, but are not limited  to, the following:  characterization  of  the animals to  be  used, 
characterization and preparation of the challenge  agent, procedural information  for the challenge  agent exposure, 
identification of the primary and any secondary efficacy  endpoints, triggers for intervention, and ranges of values of 
key parameters of the disease or  condition  that will be  used  as measures of quality control and quality assurance 
when  the model is replicated. 
40  Woodcock, J, S Buckman, F Goodsaid, MK  Walton, I Zineh, 2011, Qualifying Biomarkers for Use in Drug  
Development: A US Food and Drug Administration Overview, Expert Opin  Med Diagn, 5(5):369-374. 
41  The qualification recommendation  for the animal model and  its COU will be  made  publicly available and  can  be  
referenced  by its FDA-assigned tracking  number for use in regulatory submissions.  
42  See 21  CFR 314.610(a)(2)  for  drugs  and 21  CFR 601.91(a)(2)  for  biological products. 
  11
	 Contains Nonbinding Recommendations 
Qualification is a regulatory conclusion; therefore, FDA recommends the use of the GLP 
regulations, to the extent practicable,  for the model-defining natural history studies43  submitted 
to support the qualification of an animal model. This will facilitate conduct of the study in a 
manner that ensures data quality and integrity.  These model-defining natural history studies will 
be subject to inspection by FDA to verify the quality and integrity of  the data (see section IV.B). 
 
 
IV.  ANIMAL STUDIES  GENERAL EXPECTATIONS 
 
The general expectations discussed in  this section apply to the Animal Rule-specific studies, i.e., 
the natural history studies that define  the animal model in which efficacy will be tested, the 
adequate and well-controlled animal efficacy studies intended to provide the primary evidence of 
effectiveness to support approval,44  and the PK and/or pharmacodynamic (PD) studies in animals 
used to select a dose and regimen in humans.  Specific recommendations for natural history 
studies are discussed in Appendix C.  Specific recommendations on design considerations for the 
adequate and well-controlled efficacy studies in animals are discussed in section VI and section 
X, with additional information for preventive vaccines and cellular and gene therapies in section 
VII.  Specific recommendations for the PK and/or PD studies used to select a dose and regimen 
for humans are discussed in section V.B.2. 
 
A.   Animals Used in Investigations 
 
For the Animal Rule-specific studies, the animals should be research naive.  Any prior research 
experience, even as a control animal, has the potential to cause stress and alter an animal's 
physiological responses.  The number  of animals should be determined to ensure scientifically 
valid results.   Well-designed experiments use a sufficient number of animals to achieve the 
scientific objective, include the necessary control groups, and incorporate appropriate statistical 
analyses.  FDA expects adequate representation of  both sexes in these studies.  The male/female 
composition of the study groups should be justified.   
 
Appropriately designed protocols generally control for age, body weight, current health status, 
and the physical environment of the test animals.  When defining the animal characteristics for 
the development of animal models and the subsequent efficacy studies using those models, 
sponsors should discuss with FDA the applicability of the animal models and the results obtained 
from  the efficacy studies to the diverse human population.  
 
Appropriate inclusion and exclusion criteria for the acceptance of the animals into the study 
should be prespecified and discussed with FDA before initiating the studies.  The information 
that should be provided for the characterization of individual animals used in the investigation is 
described in section IV.D. 
                                                 
43  In  the context of  animal model qualification, the model-defining natural  history studies  are the  animal studies  that 
establish  the ranges of  values of  key parameters of  the disease or condition that will be specified  in the COU for the 
qualified  model and that will be  used  as measures of quality control and  quality assurance when the model is 
replicated.  
44  See 21  CFR 314.610(a)  for  drugs and  21  CFR 601.91(a)  for  biological products. 
  12
	 Contains Nonbinding Recommendations 
 
B.   Study Conduct 
 
The adequate and well-controlled animal efficacy  studies and the PK and/or PD studies in 
animals used to select a dose and regimen in  humans should be conducted in a manner that 
ensures data  quality and integrity, as would be expected for the studies in humans submitted to 
establish effectiveness and any other studies needed to support the labeling of a drug approved 
under a traditional regulatory pathway.  There are no regulations that specifically address data 
quality and integrity issues for Animal Rule-specific studies.45  The GLP regulations46 were 
developed as a quality system for nonclinical safety studies.  Nonetheless, these regulations 
provide a framework (e.g., definitions, procedures, roles and responsibilities, and controls) for 
the conduct of nonclinical studies, and FDA considers them  to be a well-established and relevant 
system  for ensuring data quality and integrity for the adequate and well-controlled animal 
efficacy studies and the PK and/or  PD studies in animals used to select a dose and regimen in 
humans.  FDA, therefore, recommends the use of  the GLP regulations, to the extent practicable, 
to ensure the  quality and integrity of data from  these studies.47    
 
There may be justifiable limitations in the ability to apply the GLP regulations when conducting 
these studies, especially for those using challenge agents that require high containment facilities.  
Before initiating these studies, sponsors should identify aspects of the studies anticipated to be a 
challenge with regard to the GLP regulations and propose methods for adapting the studies to 
ensure the quality and integrity of the resulting data.  Sponsors should seek concurrence from  
FDA on the data quality and integrity  plan before the studies are initiated.  
 
The adequate and well-controlled animal efficacy  studies and the PK and/or PD studies in 
animals used to select a dose and regimen in humans serve as the basis for a regulatory action 
(e.g., approval) under the Animal Rule.  Thus, FDA has the authority to inspect these studies 
prior to taking an action.   Inspections will be conducted to verify  the quality and integrity of  the 
raw data, supporting documentation, facilities, equipment, and the results submitted to  FDA in 
the final report.  Quality  includes, but is not limited to, whether the study was conducted in 
accordance with the protocol, standard operating procedures, and applicable standards of 
research.  Integrity  includes, but is not limited to, the assurance that  the raw data and 
documentation are consistent with reported results.  FDA will verify that study personnel 
followed the  agreed-upon data quality and integrity plan.  Inspectional observations will be 
shared with the inspected entity and evaluated by the review division to determine the  impact of  
the observations on the acceptability of  the data to support drug approval.  
                                                 
45  In  issuing  the Animal Rule, FDA stated that  . . . studies subject to this rule must be  conducted in  accordance 
with  preexisting requirements under the good laboratory  practices (21 CFR part  58) regulations . . .  (67 FR  37988  
at 37989, May 31, 2002).  The good  laboratory practice (GLP) regulations, however, were developed  as a quality 
system  for nonclinical safety studies.  This section  describes FDAs current expectations. 
46  See 21  CFR part 58. 
47  In  addition,  FDA recommends  the use of  the GLP regulations, to  the extent  practicable, for the model-defining  
natural history studies  submitted  to  support the qualification  of  an animal model (see section  III.D).  Qualification  is 
a regulatory conclusion, and thus, these studies should  be  conducted in  a manner that ensures data quality and  
integrity.  
  13
	 Contains Nonbinding Recommendations 
 
Animal studies must comply with applicable laws and regulations as prescribed by the Animal 
Welfare Act48  and the Public Health Service Policy on Humane Care and Use of Laboratory 
Animals.49   All studies should comply with general principles for the care and use of animals in 
biomedical research (see Appendix A).  Sponsors should ensure that  adequate safety and security 
provisions are in place for all studies  when needed.  For example, for select agents and toxins, 
sponsors must adhere to the regulations found under 42 CFR part 73, and when applicable, 
sponsors should comply with standards on the use of biosafety level (BSL) laboratory facilities.50  
 
The investigational drug used in the adequate and well-controlled animal efficacy studies and the 
animal PK and/or PD studies used to select a dose and regimen in humans  ideally should be 
manufactured under current good manufacturing practice regulations.51  The investigational drug 
also should be as close as practicable to the to-be-marketed drug; any differences should be 
discussed with the review division before studies are initiated. 
 
C.  Types of Animal Care Interventions 
 
As used in this guidance, animal care interventions in animal studies are divided into three 
categories based on the rationale for their use:  (1) intervention as part of adequate veterinary 
care, (2) intervention as supportive care to mimic the human clinical scenario, and (3) 
intervention to permit the manifestation of the disease or condition of interest for the purpose of 
model development.  These categories of interventions are discussed individually in Appendix B.  
The potential effects of the interventions on the animal (e.g., toxicity, effects on the immune 
system) and on the pharmacokinetics, pharmacodynamics, and efficacy of the investigational 
drug should be considered in the design and interpretation of each study.  In addition, protocols 
for the adequate and well-controlled efficacy studies should include plans for addressing the 
impact of potential differences in care among animals. 
 
D.  The Study Report 
 
FDA expects that complete, final study reports will be submitted for the Animal Rule-specific 
studies.  Complete study reports should include, but are not limited to, the following: 
 
 	  The prospectively designed protocol (including the statistical analysis plan), all protocol 
amendments, and a description of all protocol deviations 
                                                 
48  See 7  U.S.C. 2131  et seq. 
49  U.S. Department  of Health  and Human  Services, National Institutes of Health, Office of Laboratory Animal  
Welfare, Public Health  Service Policy on  Humane  Care  and  Use of  Laboratory  Animals, Revised 2015, available 
at http://grants.nih.gov/grants/olaw/references/PHSPolicyLabAnimals.pdf, accessed on October 16, 2015. 
50 U.S. Department of  Health and Human Services, Public Health Service, Centers for Disease Control and 
Prevention,  and  National Institutes of  Health, 2010, Biosafety in Microbiological  and Biomedical Laboratories,  5th  
Edition, Atlanta, GA:  CDC.  
51  See 21  CFR parts 210  and 211. 
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	 Contains Nonbinding Recommendations 
 	  Detailed descriptions of the elements of the study design, including the characterization 
of the animals used in the study;52 information on the formulations and administration of 
the investigational drug and controls; and information on the characterization, 
preparation, and delivery of the challenge agent 
	   A comprehensive description of study procedures 
	   The results53  of each parameter or variable evaluated at each time  point in the study and  
any unscheduled medical intervention 
 	  The final audited54  study report that includes analyses and interpretation of the study data 
and explanation of any deviations from the agreed-upon plan for data quality and 
integrity  
 
Preliminary plans for collection, organization, format, and level of detail of study data should be 
discussed with the review division before conducting these studies.  Sponsors are encouraged to 
submit prototype versions of the study datasets prior to finalization of  datasets.  
 
E.  Submission of the Study Report and Data 
 
The submission of study data in a standardized electronic format supports data analysis and 
review and has therefore been encouraged by FDA.  Section 1136 of FDASIA55 requires that 
nonclinical and clinical  study data be submitted electronically to FDA in a format specified by 
guidance.  FDAs guidance for industry Providing Regulatory Submissions in Electronic Format 
 Standardized Study  Data56  specifies  that electronic submissions must be in a format that FDA 
can process,  review, and  archive and  describes the requirements, including the types of 
                                                 
52 The individual animal information should include, when appropriate, species; strains and substrains (when 
applicable); breed (when applicable); age;  sex; body weight; vendor source; origin  of the animal (to the extent  
known);  procedures for identification and individual  animal  identification;  physiological status  (e.g., adult, juvenile, 
lactating, and pregnant); data  collected during  routine husbandry  pre- and  post-protocol assignment, including pre-
study  health screen, health  records, and medications or therapies administered; and an adequate  description of  
housing and husbandry conditions.  For individual animal tracking purposes, a table that cross-references  the unique  
animal  identification  number for the study,  treatment allocation,  fate or  disposition,  and  chain  of custody should  be  
submitted.  For each animal assigned more than one  identification number during life, the  table also should include 
reference to all other identification numbers (e.g., a unique  animal number assigned by the source).   
53  These results should include group summary tabulations, line  listings  of the results for each  individual animal, 
copies of the individual animal case report forms (all veterinary medical records), and any other primary data  
necessary for the reconstruction of  key analyses and  evaluation  of  the study report. 
54  Final study  reports should  be  audited  for quality assurance in  accordance with  the data quality and  integrity plan  
to verify that the protocol, protocol amendments, and standard  operating procedures were followed by study  
personnel; the raw data were accurately recorded and support the findings and conclusions in  study reports; and any 
findings  identified  during  the audit as having the potential to  affect the quality and  integrity of  the data have  been  
addressed. 
55 See Public Law 112-144.  
56  This guidance implements the electronic submission  requirements of  section 745A(a) of the FD&C Act (21  
U.S.C. 379k-1(a)  (added by  section  1136  of  FDASIA, Public Law 112-144).  
  15
	 Contains Nonbinding Recommendations 
submissions to which these requirements apply57  and timetables for the implementation.  For 
studies initiated prior to the requirement date specified in the timetables, sponsors are strongly 
encouraged to submit study data in  a standardized electronic format.  Sponsors should consider 
the implementation of data standards and seek FDA feedback as early as possible during animal 
model and drug development, so that the data standards are included in the design, conduct, and 
analysis of studies.58  
 
The Electronic Common Technical Document (eCTD) is the standard format for regulatory 
submissions to CBER and CDER.59  The eCTD does not provide a specific location for the 
natural history or model characterization studies  conducted in animals and for the adequate and 
well-controlled animal efficacy studies.  For consistency, it is recommended that these studies 
be submitted to Module 4 (Nonclinical Study Reports), section 4.2.1.1 (Primary 
Pharmacodynamics).  This recommendation does not determine the disciplines of the primary 
reviewers for the studies; that decision is the purview of the FDA review division. 
 
 
V.  ESSENTIAL ELEMENTS OF AN ANIMAL MODEL 
 
The selection of an animal model for an efficacy study should  be based on  its adequacy as a 
model of key elements of the human disease or condition and  its suitability with regard  to the 
investigational drug.  Section V.A describes the elements related to the disease or  condition 
induced by the etiologic or challenge agent.60   It is the sponsors responsibility to provide, to the 
fullest extent possible, a documented summary of  the etiologic agent-induced human disease or  
condition and a detailed discussion that delineates how these data support selection of  the 
proposed animal model.  Evidence supporting the relevance of an animal model to a human 
disease or condition can be obtained from  various sources61 that provide adequate documentation 
of study quality.62  For example, data from literature or historical studies may support the use of 
an animal model when the reports include a level of  detail that  is sufficient to assess the 
appropriateness of the animal model.  The source, organization, format, and level of detail of the 
available study data should be discussed with  the review division before submitting the data.  
 
                                                 
57 An IND for a drug  or biological product intended to  progress to an  NDA  or BLA, respectively, is considered to  be 
a commercial IND and, therefore,  is subject to  these requirements. 
58 Information is available through FDAs Web page, Study  Data Standards Resources, available at  
http://www.fda.gov/ForIndustry/DataStandards/StudyDataStandards/default.htm. 
59  See FDAs guidance for  industry Providing Regulatory Submissions in Electronic Format  Certain Human 
Pharmaceutical Product Applications and Related Submissions  Using the eCTD Specifications  (Revision  3).   
60  As used  in  this  guidance, the term  etiologic agent  refers to the substance causing  the disease or condition  in 
humans.  The term  challenge agent  refers to the substance used  to  cause the disease or condition  in the animal 
studies.  
61  Comparable to  the sources of  clinical data described in  21 CFR 314.50(d)(5)(iv).  
62  Comparable to  the discussion  of  the documentation  of the quality of evidence described in  FDAs guidance for 
industry Providing Clinical Evidence of Effectiveness for Human  Drug  and Biological Products.   
  16
	 Contains Nonbinding Recommendations 
Section V.B describes elements related to the investigational drug and the  selection of  an 
effective dose in humans.  The sponsor should provide justification of the suitability of each 
model based on the investigational drugs mechanism  of action, dosage form, and route of 
administration, and the method proposed to extrapolate from  the animal data to select a dose and 
regimen in humans.  Issues related to animal model development for one or more investigational 
drugs that are to be developed for use in combination or concurrently are beyond the scope of 
this guidance and should be discussed with the review division. 
 
The following essential elements should be considered in the development and/or the selection of  
an animal model.63   Any element not achievable for an etiologic or challenge agent or drug under 
investigation should be discussed with FDA. 
 
A.  	 Elements Related to the Etiologic or Challenge Agent-Induced Disease or 
Condition 
 
1. 	 Characteristics of the Etiologic or Challenge Agent That Influence the Disease or 
Condition  
 
The characteristics of the specific etiologic or  challenge agent that influence the disease 
or condition under study include its pathophysiological mechanisms of toxicity or 
virulence, the route of exposure, and the dose and quantification of exposure.  These 
characteristics are discussed below.  
 
a. 	 The Challenge Agent 
 
The challenge agent used to establish the disease or condition in the animal 
studies generally should be the same  as the etiologic agent that causes the human 
disease or condition.  If the challenge agent is different from  the etiologic agent, 
the sponsor should provide justification for the use of that challenge agent.  The 
sponsor also should explain why, when used in the proposed animal model, the 
challenge agent should be considered suitable for establishing effectiveness of the 
investigational drug in humans against the intended etiologic agent.  For example, 
for an animal  efficacy study to support approval of a drug to treat the 
gastrointestinal subsyndrome of acute radiation syndrome (GI-ARS), a sponsor 
may not be able to predict the actual radiation exposure that would follow a 
nuclear detonation or the subsequent fallout.  In such  a case, the sponsor should 
provide a detailed explanation of the appropriateness of the type and dose of 
radiation used in the study and their relevance to the clinical situation.  
 
The selection of a biological challenge agent should be based on known virulence 
factors, using standardized, validated test methods, and the challenge agent used 
ideally should be of low passage history.  For plague studies conducted in 
animals, pigmented Y. pestis  strains are preferred because non-pigmented strains 
                                                 
63  See section IX for the associated Checklist of Essential Elements of  an Animal Model. 
  17
	 Contains Nonbinding Recommendations 
rarely cause disease.  Generally, bacterial and viral strains known to be associated 
with outbreaks of human disease should be used for the natural history and animal 
efficacy studies (e.g., Ebola Zaire virus isolated from  a human who died from  an 
infection during an outbreak should be used in the animal studies); however, there 
may be issues regarding differences in the strain or serotype  of the biological 
agent that will limit the relevance, or preclude the use, of data obtained to support 
the proposed clinical indication.  For example, there may be various strains of a 
bacterium that differ in the expression of virulence factors.  When an 
investigational drug targets a particular virulence factor or pathogenic mechanism  
associated with a particular virulence factor, effectiveness may be limited to 
strains that express that particular virulence factor, and an indication for all 
variants of that bacterium may not be warranted. 
 
The challenge agents and their preparations should be characterized in terms 
relevant for their category (i.e., biological, chemical, radiological, or nuclear).  
For biological agents, these terms should include passage history, method of 
preparation, and concentration.  For chemical agents, characteristics should 
include source and stated purity of the agent, dosing formulation, concentration, 
and stability  under the conditions  of use.  For radiation or nuclear challenges, the 
terms should include the type and source of radiation.  Such characterization 
facilitates comparison among studies. 
 
b.  Pathophysiological Mechanisms of Toxicity or Virulence 
 
The pathophysiological mechanisms  of toxicity or virulence of the challenge 
agent expressed in the animal model should be similar to those expressed by the 
etiologic agent in humans.  For a biological agent, the pathophysiological 
mechanisms of virulence are the pathogenic determinants of the microbe (i.e., its 
genetic, biochemical, or structural features  that  enable it to elicit disease in a 
host).  Examples of microbial pathogenic determinants include toxins, substances 
that promote invasion, substances that modulate inflammation, substances that 
cross-react with host tissues, and mechanisms to evade host defenses.  For a 
chemical agent, the mechanisms of toxicity can include receptor binding, 
inhibition of enzymes, and binding of intracellular components. For radiation, the 
mechanisms  of toxicity include DNA damage and the generation of free radicals.  
 
c.  Route of Exposure 
 
When the pathogenesis of the disease or  condition is dependent on the route of 
exposure to the challenge agent, the animal models should use the same route as 
that anticipated in humans.  For example, human infection with Y. pestis can  
occur through flea bite or inhalational exposure.  Exposure through a flea bite 
typically leads to development of bubonic plague, which can progress to 
septicemic plague, whereas inhalational exposure usually leads to development of 
pneumonic and septicemic plague.  Thus, an animal model of pneumonic plague 
should use an inhalational route of exposure to Y. pestis. 
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	 Contains Nonbinding Recommendations 
 
In cases when the challenge agent-induced disease or condition is not clearly tied 
to its route of  exposure, alternate routes of exposure may be acceptable.  If a 
sponsor proposes to use a route of exposure to the challenge agent in animals that 
is different from  that expected in humans, scientific justification should be 
provided.  If such an approach is under consideration, it should be discussed with 
FDA before initiation of the natural history and animal efficacy studies. 
 
Sponsors should discuss potential paths forward with FDA when trying to 
develop a drug for a disease or condition for which limited or no human data are 
available for the etiologic agent by the route of exposure in the proposed clinical 
indication.  
 
d.  Dose and Quantification of Exposure 
 
Ideally, the sponsor should use a challenge agent dose that produces a disease or 
condition in animals that corresponds to the expected extent and severity of the 
human disease or condition.  The dose of  the etiologic agent that causes the 
human disease or condition may not be known, or the exposure may not be fully 
quantifiable, as can be the case for the radiation exposure to humans following a 
nuclear incident.  In such a case, a sponsor developing a drug to treat the 
hematopoietic subsyndrome of acute radiation syndrome (H-ARS) should provide 
a detailed description of the methods of radiation exposure used in the animal 
studies, including type and source of radiation, dose and dose rate, whole versus 
partial body irradiation, and their relevance to the clinical situation. 
 
The method for the delivery of the challenge agent should be described in 
sufficient detail to permit  replication of test conditions.  Reliable quantification 
using a validated assay and reproducibility of the challenge agent dose should be 
demonstrated from model development through its use in the animal efficacy 
studies.  In general, the target dose and actual dose delivered to an individual 
animal should be expressed in absolute terms (e.g., colony forming units or 
plaque forming units for a biological agent, or the radiation dose expressed in 
gray), as well as in terms that indicate the toxicity or virulence of the challenge 
agent (e.g., the LD , which is the dose sufficient to  kill 50% of those exposed to 
50
the agent).  
 
2.   Host Susceptibility and Response 
 
The animal species chosen for model development should be susceptible to the challenge 
agent.  Also, if the host immune response is part of the pathogenesis of the disease or 
condition in humans, it should play a similar role in the animal  model.  FDA recognizes 
there may be susceptibility differences among species.  For example, an animal species 
used to study the efficacy of a treatment for H-ARS may require a threshold of radiation 
exposure to develop the subsyndrome that is different from  the threshold that is needed in 
humans.  If the thresholds in humans and in the animal model differ greatly, the 
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suitability of the animal model may be called into question and the model should be 
discussed with FDA.  The animal species may still be appropriate for study if the 
resulting disease or condition and the progression of the associated manifestations are 
similar in the animal species and humans.  The factors that determine differences in 
susceptibility to the agent should be described to the extent possible.  For example, when 
selecting an animal model to study the lethal effects of soman, an important consideration 
is the endogenous level of carboxylesterase in the selected animal species.  This enzyme  
has a detoxifying effect on soman.  Certain species are less susceptible to the effects of 
soman because of higher endogenous levels of this enzyme.64    
 
Animal species that are not susceptible to the etiologic agent may not  be suitable models 
for efficacy studies.  Other approaches to the accrual of relevant animal data may need to 
be explored (for an example,  see the discussion of the variola virus and human smallpox 
in section II). 
 
The response to the challenge agent (i.e., the resulting disease or condition) manifested 
by the animal species should be similar to the disease or condition seen in humans 
exposed to the etiologic agent with respect  to the proposed clinical indication.  For 
example, mustard gas typically produces extensive blistering to exposed human skin.  If 
the animal species evaluated does not have blistering as a prominent feature of exposure 
to mustard gas, it is unlikely that  this animal model will be acceptable to FDA for the 
development of a treatment for mustard gas-induced injury to  the skin.  Similarly, 
although mice are known to be susceptible to B. anthracis, the pathogenesis of the 
disease process in mice differs from that in humans.  Therefore, mice may not be 
appropriate models for anthrax efficacy studies.65   If the sponsor believes that such a 
model is supportive to the study of its investigational drug, a justification should be 
provided and the model should be discussed with  FDA before  proceeding.  
 
3.  Natural History of the Disease or Condition  Pathophysiological Comparability 
 
The general expectations for the design and conduct of animal natural history studies are 
described in Appendix C.  The natural history of the disease or condition in the selected 
animal species and in humans should be characterized and the similarities and differences 
compared and contrasted.  This information should be discussed with FDA before 
                                                 
64  Pretreatment with pyridostigmine bromide was shown to  decrease  the  lethality of soman in rhesus macaques and 
guinea pigs.  Pyridostigmine bromides protective effect was not consistently demonstrated in  other species tested 
because these  other species were  protected  from  soman by high levels  of endogenous carboxylesterase, an enzyme 
that detoxifies soman.   To  confirm  the theory for inter-species differences, a study was conducted in  rats pretreated  
with  a carboxylesterase inhibitor before exposure to  soman.  In these  carboxylesterase inhibitor-treated  rats, 
pretreatment  with  pyridostigmine  bromide demonstrated  decreased lethality to  soman,  compared  to  rats not 
pretreated  with pyridostigmine bromide.  These results were similar to  the survival benefit demonstrated  with  
pyridostigmine bromide in the rhesus macaques.  
65  Leffel, EK and  MLM Pitt, Characterization  of  New and  Advancement of  Existing  Animal Models of Bacillus  
anthracis Infection, in JR  Swearengen (ed.),  Biodefense Research  Methodology and Animal Models, Boca Raton, 
FL:  CRC Press, 2012, pp. 81-98.  
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	 Contains Nonbinding Recommendations 
initiation of the efficacy studies.  To facilitate these discussions, sponsors should provide 
an adequately documented summary of the etiologic agent-induced human disease or  
condition and a detailed discussion as to how these data support the selection of the 
animal model.  This information should include (but not be limited to) the following 
parameters: 
 
 	  Time from  exposure to onset of the manifestations of disease or injury 
 	  Time course and order of the progression of the manifestations 
 	  Manifestations (e.g., signs and symptoms, clinical and pathological features, 
laboratory parameters, extent of organ involvement, morbidity, and outcome)  
 
These parameters can be influenced by many factors, such as the type of etiologic or 
challenge agent, virulence or lethal potential of  the etiologic or  challenge agent, route of  
exposure, concentration, host factors (including immune status), and medical 
management  in humans versus animal care interventions.  Potential endpoints for 
evaluating efficacy should also be discussed.  Experimental parameters may need to be 
modified to create a disease or condition that more closely mimics that seen in humans, 
or the model may need to be tailored for the proposed clinical indication. 
 
It may not always be possible to compare the pathophysiology of the disease or condition 
in animal models to that in humans.  For some  diseases or conditions, relevant human 
data are not available, or the data are limited to references in the literature describing the 
end-stage pathology for symptomatic patients.  For example, the description of the 
pathophysiology of H-ARS has been derived mainly from the literature discussing 
accidental occurrences in which humans received variable exposures to radiation.  
 
a.  Time to Onset 
 
The time to onset of the disease or condition in animals should be reasonably 
similar to that in humans.  Factors such as route of exposure, level of exposure 
(e.g., dose, concentration), and species or strain of  the infective microorganism  
can influence time to onset and should be taken into consideration in model 
development. 
 
b.  Progression 
 
Ideally, the progression of the manifestations of the disease or condition 
(including the order of their presentation) in the selected animal models should be 
similar to that observed in humans; when different, it should allow time for 
identification of the disease or condition, intervention, and assessment of the 
outcome of treatment.  Demonstration of the effect of  the investigational drug 
may be more challenging when the time between onset and death is short.  For 
example, hamsters challenged with B. anthracis have such a rapid disease 
progression that this species is not useful for testing the efficacy  of drugs for the 
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	 Contains Nonbinding Recommendations 
treatment of anthrax in humans.  The route of exposure may affect the progression 
of the disease or condition, including the time  course. 
 
c.  Manifestations 
 
The manifestations of the disease or condition, including laboratory parameters, 
histopathology, gross pathology, and outcome (morbidity and/or mortality), and 
their known time courses should be compared between untreated animals and 
humans (e.g., historical information from  human cases).  Differences should be 
clearly noted and explained based on the understanding of the pathophysiological 
differences between the species, when possible.  Certain manifestations in humans 
(e.g., fever, shortness of breath) may be  difficult to discern in animals through 
clinical observation; therefore, a sponsor may need to use more refined 
techniques, such as telemetry, to evaluate affected animals.  Animals in the 
natural history studies and the efficacy studies should be observed with greater 
frequency over the entire course of the day than would be typical of most  animal 
studies used for toxicology evaluation.  The frequency of observations per day 
may vary over the course of the study, depending on the animal species and strain, 
the experimental conditions, and the mechanism  of disease or injury of the 
challenge agent. The observation frequency should be adequate to characterize 
the course of disease or condition and to define the desired treatment triggers and 
efficacy endpoints.  
 
Animals should be evaluated in the context of prospectively defined euthanasia 
criteria to ensure animal welfare.  Study results may be influenced by the 
euthanasia criteria used.  Sample integrity may be compromised if  the sample is 
not obtained prior to or immediately after death or euthanasia.  Study personnel 
should be blinded to exposure status and should follow the observation frequency 
paradigm and euthanasia criteria to minimize the possibility of unnecessary 
suffering of moribund animals and to reduce potential study bias as much as 
possible. 
 
4.  Trigger for Intervention  
 
A clearly defined trigger for intervention should be established for use in animal efficacy 
studies when needed (e.g., post-exposure prophylaxis and treatment indications).  The 
trigger for intervention should be identified based on the natural history studies.  For a 
post-exposure prophylaxis indication, a trigger for intervention should be defined to 
ensure drug administration within a reasonable time  frame after exposure to the challenge 
agent and prior to the onset of the disease or condition of interest.  The time frame should 
be justified with respect to administration of the drug to humans.  Animals cannot 
simulate the health-seeking behavior manifested by humans; therefore, a clearly defined 
trigger for intervention for a treatment indication  will ensure that treatment is not initiated 
until the disease or injury  process is established.  If  signs observed in the animal model 
closely resemble those in humans and are predictive for the disease, they may serve as the 
trigger for intervention.  
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	 Contains Nonbinding Recommendations 
 
In the absence of disease- or condition-defining manifestations, sponsors can propose a 
biomarker as a trigger for intervention, if  information can be provided that it relates to the 
pathophysiology of the disease or  condition.  The utility of the biomarker should be 
justified through an analysis that correlates  the time course of the appearance of the 
parameter in animals with the onset of the disease or condition in the animals.  The assay 
method and its performance characteristics for a biomarker used as a trigger for 
intervention in animal studies should be adequately described.  
 
Sponsors are encouraged to initiate early discussions with FDA regarding the utility of 
the chosen triggers for intervention, particularly when the manifestations of the disease or 
condition in the animals differ from  those in humans, or when a biomarker is used as a 
trigger for intervention.  
 
B. 	 Elements Related to the Investigational Drug and the Selection of an 
Effective Dose in Humans 
 
The concepts discussed in this section apply primarily to small molecule drugs and therapeutic 
proteins.  For information regarding preventive vaccines and cellular and gene therapies, consult 
sections VII.A and VII.B, respectively.  
 
1. 	 The Investigational Drug 
 
The characterization of the investigational drug with regard to identity, concentration, 
purity, composition, and stability is the same  under the Animal Rule as for any 
investigational drug developed under other regulatory pathways.  Additional elements of 
the investigational drug  that are important considerations for animal model selection 
include the mechanism  of action, drug class, dosage form, and route of administration. 
These elements are discussed below.  
 
a. 	 Mechanism  of Action 
 
Approval under the Animal Rule requires a reasonable understanding of the 
investigational drugs mechanism  of action with regard to its ability to prevent or 
substantially  reduce the toxic effects of the challenge agent.66  The sponsor should 
relate the mechanism  of action of the drug in the proposed animal species to the 
presumed mechanism of action in the human.  This information is critical to the 
selection of appropriate animal species in which to test the efficacy of the 
investigational drug and  the interpretation of  the results of those studies, in order 
for FDA to conclude that the drugs effect in the animal model is reasonably 
likely to be predictive of  the drugs effect in humans.67  
 
                                                 
66  See 21  CFR  314.610(a)(1)  for drugs;  21  CFR  601.91(a)(1)  for biological  products.  
67  See 21  CFR 314.610(a)  for  drugs; 21  CFR 601.91(a)  for  biological products. 
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	 Contains Nonbinding Recommendations 
An understanding of the mechanism of action of the investigational drug may help 
in the identification of specific safety or efficacy issues, the interpretation of 
findings in the proposed animal studies, and the identification  of additional 
studies that should be performed.  This understanding also may lead to the 
identification of a relevant biomarker for potential use in selecting a dose and 
regimen in humans (see section V.B.2.b. for further discussion). 
 
b.  Drug Class 
 
Information that is available about other drugs that are members of the same  
therapeutic class or  pharmacologic class as the investigational drug can be used to 
help identify  potential animal models.  This information also may help anticipate 
safety and efficacy issues  in the proposed animal model and in the projected 
human use. 
 
c.  Dosage Form and Route of Administration 
 
The suitability of the dosage form and route of administration with regard to the 
proposed indication should be considered in the development of the drug.  For 
example, an oral dosage form  may be  preferred for post-exposure prophylaxis for 
large populations while an intravenous dosage form may be more appropriate for 
seriously ill patients.  
 
To the extent practicable, drug administration in the animal and human studies 
should be comparable to the expected clinical use of the investigational drug (e.g., 
dosage form, route of administration, to-be-marketed formulation).  Comparative 
bioavailability information may be necessary to bridge pharmacokinetics across 
studies, for example, when changes in formulation occur during development.  If 
multiple dosage forms or routes of  administration are being developed, sponsors 
should discuss with the review  division the types of PK data that may be needed 
to support the approval of each. 
 
2.  Selection of an Effective Dose in Humans 
 
The Animal Rule requires that PK and PD data or information (or other relevant data or 
information) for the investigational drug68  be sufficient to permit the selection of a dose 
and regimen expected to be effective in humans.69   The methods used for selecting an 
effective human dose may differ based on factors including, but not limited to, the target 
of the investigational drug, prior human experience in related indications, and the 
availability of a relevant biomarker.  Several approaches to the selection of  an effective 
dose for humans are described in section V.B.2.b.   
                                                 
68  This section focuses on  the investigational drug  as the active moiety; however, active metabolites also  should  be  
considered  for the purposes of dose selection.   Issues pertaining  to  active metabolites are handled  on  a case-by-case 
basis and should  be discussed  with the review  division.  
69  See 21  CFR 314.610(a)(4)  for  drugs  and 21  CFR 601.91(a)(4)  for  biological products. 
  24
	 Contains Nonbinding Recommendations 
 
Agency concurrence on the animal model in which the efficacy of an investigational drug 
will be tested  will be contingent, in part, on the ability to extrapolate from  the animal data 
to select an effective dose and regimen in humans.  Sponsors are encouraged to initiate 
discussions with FDA on the proposed rationale  for human dose selection early in their 
drug development program.  Protocols for animal PK, PD, and efficacy studies should  
include adequate plans for assessment of PK and PD data for purposes of defining drug 
exposure and response characteristics. 
 
Issues related to dose selection for the adequate and well-controlled animal efficacy 
studies for drugs and therapeutic biological products are discussed in section VI.B; for 
vaccines, see section VII.A. 
 
a.  PK and PD Information to Be Obtained in Animals and Humans 
 
The absorption, distribution, metabolism, and excretion (ADME) of an 
investigational drug70,71  should be characterized in animals and humans.  In 
addition, protein binding characteristics and in vitro interaction potential (e.g., 
through inhibition, induction, or transporters) should be assessed.  As in a 
traditional drug development paradigm, it is important  to ascertain at an early 
stage of development whether a drug is eliminated primarily  by excretion of  the 
unchanged drug or by one or more routes of metabolism.72   If elimination of the 
investigational drug is dependent in part  on metabolism,  the metabolites should be 
identified and the metabolizing route(s) should be understood.73   This information 
will help identify potential interactions with medical products that are likely to be 
co-administered based on  the clinical  scenario and will help predict the 
consequences of metabolic differences among humans. 
 
Studies should be conducted in healthy animals74  and healthy human volunteers75  
to characterize the PK profile  of the drug in each following the administration of a 
                                                 
70 Biodistribution should  be studied for certain products that are not  biologically amenable to traditional  ADME  
measures, such  as cellular and gene therapies.   
71  Therapeutic biological products do  not  share the same  ADME pathways as small molecules.  The ADME 
characteristics of therapeutic biological products, including receptor-mediated clearance mechanisms  leading to 
nonlinear pharmacokinetics, should  be determined.  
72  Sponsors  should  discuss with the review  division  whether PK  information  in specific human  subpopulations  (e.g., 
renally and hepatically impaired) also should  be obtained.  
73  FDA has issued a draft  guidance on this topic.  When the guidance on  Drug  Interaction Studies  Study  Design, 
Data Analysis, Implications for Dosing, and  Labeling Recommendations  is finalized, it will represent the Agencys 
current thinking  on  this topic.  
74  The  healthy animals used in these studies should be  representative of those used  in the  efficacy studies with regard 
to key animal characteristics, such  as species/subspecies, country of  origin, source, age, sex, and weight range.  
75  PK assessments in healthy volunteers  may not be  possible for some  investigational drugs because of the nature of 
the drug, such as cellular therapies and gene therapies, or because of  an unfavorable safety  profile of  the drug.  In  
such cases, alternative plans should be discussed with the review division.  
  25
	 Contains Nonbinding Recommendations 
single dose and multiple  doses (if  applicable).  The assays used for measuring 
drug concentration in the appropriate body fluids should be validated.76  As in a 
traditional drug development program, clinical trials in healthy humans should 
evaluate safety and PK data over a range of doses.  Based on nonclinical and 
human data, sponsors should discuss the appropriate upper limit for human dosing 
with the review division, and this agreed-upon upper limit should be used to 
support final human dose selection (see section V.B.2.b for further discussion).  
The drug exposures associated with efficacy  in the adequate and well-controlled 
animal efficacy studies should be determined.  PK information from  affected 
animals77,78  should be compared to PK information from  healthy animals to 
determine whether the challenge agent-induced disease or  condition affects the 
pharmacokinetics of the investigational drug.  
  
The relationships between PK exposure parameters (e.g., area under the plasma  
concentration-time curve (AUC), peak plasma  concentration (Cmax), trough 
plasma  concentration (Cmin), and steady state plasma concentration (Css)) and 
PD parameters (e.g., efficacy endpoints and potential biomarkers) in the animal 
models should be determined over a range of at least three doses and the shape of 
the exposure-response (E/R) curves established in dose range-finding studies.  To 
the extent practicable, protocols for the adequate and well-controlled animal 
efficacy studies should include plans for PK and PD assessments to enable 
quantitative E/R analyses.  
 
When a biomarker is used as the basis for human dose selection, the assay method 
and performance characteristics for that biomarker should be described for the 
animal species and humans. 
 
b.   PK/PD Considerations for Human Dose Selection 
 
PK/PD information can be informative in a number of ways.  One approach to the 
selection of an effective dose for humans takes into account whether the effect of 
the investigational drug is mediated through its action on the etiologic or 
challenge agent, rather than on the host (e.g., antimicrobial drugs that target 
microbial pathogens or investigational drugs intended to bind or detoxify 
substances such as cyanide or neurotoxins).  In such circumstances, it may be 
possible to use data from  in vitro studies to estimate the target concentration or 
                                                 
76  See FDAs guidance for  industry Bioanalytical Method Validation.  In  the Federal Register of September 13,  
2013  (78  FR 56718), FDA published  a notice announcing  the availability of  a draft guidance for industry 
Bioanalytical Method Validation  (Revision  1).  This revised  draft guidance updates the guidance for industry 
Bioanalytical Method Validation  and, when finalized, will represent the Agencys current thinking  on  the topic.  
77  Affected animals are defined as those with the challenge agent-induced  disease or condition of interest  using the 
animal  models proposed  for the adequate  and well-controlled efficacy studies. 
78  If there are barriers to  performing  intensive PK sampling  in  affected animals, sparse sampling approaches can be 
used.  
  26
	 Contains Nonbinding Recommendations 
exposure of the investigational drug.79   The PK/PD parameters that correlate with 
activity of the drug should be identified in animal models.  This information can 
guide the selection of doses to be tested in adequate and well-controlled animal 
studies to evaluate efficacy.  Similar PK/PD parameters should be identified for 
humans to support human dose selection.   
 
For example, in the case of antimicrobial drugs, in vitro studies can be used to 
determine a PD characteristic such as susceptibility (e.g., minimum inhibitory 
concentration (MIC), which is a measure of drug-organism interaction).  
Nonclinical studies then can be used to identify potentially relevant PK/PD 
parameters (e.g., Cmax/MIC ratio, AUC/MIC  ratio, the time the concentration 
remains above the MIC (T>MIC)) that may correlate with an effective response.    
 
If the investigational drug has been used  in humans for other relevant indications, 
previously established human PK/PD information may guide dose selection for 
the animal efficacy studies, which in turn may support selection of the human 
dose for the proposed indication.  For example, existing human E/R data from  an 
antibacterial drug shown to be effective in pneumonia may guide the dose 
selection for the animal efficacy studies intended to support an indication for the 
treatment of pneumonic plague.  Efficacy  of the guided dose (e.g., the humanized 
animal dose) should then be evaluated in the animal model.  In some cases, 
animal studies may suggest that the human dose and regimen needed for the new 
indication are different from the human  dose and regimen used for other 
indications. 
 
Another approach for human dose selection may be through the identification and 
use of an appropriate biomarker.  The biomarker should be shown to be related to 
the mechanism by which the drug prevents or substantially reduces the etiologic 
or challenge agent-induced disease or condition and to the desired clinical 
outcome  (i.e., reduction in mortality or major morbidity).  In addition, there 
should be an ability to determine drug doses for humans that would result in 
biomarker levels in the desired range based on the biomarker levels associated 
with efficacy in the adequate and well-controlled animal studies.   
 
A common and challenging situation is one  in which the relationship between the 
drug exposure and effectiveness is established in animals, but there is no evidence 
of a relevant link (e.g., biomarker, AUC/MIC) that can predict an effective drug 
exposure in humans.  In this situation, it may be reasonable to assume that the E/R 
relationship80  in humans will be similar  to the E/R relationship in animals and use 
a conservative approach to human dose selection (discussed below), based on an 
                                                 
79 The extent to  which in vitro data may be relevant and useful varies; sponsors should discuss their supporting 
information with the review division.  
80  For the purpose of this guidance, the term  exposure-response relationship  is used  broadly to  include dose-
response relationship.  
  27
	 Contains Nonbinding Recommendations 
understanding of the E/R relationship in animals, the exposures associated with a 
fully effective dose in animals81  (see Figure 1), and exposures associated with the 
agreed-upon upper limit for human dosing.  This approach to human dose 
selection, based solely on comparing relevant exposure parameters (e.g., AUC, 
Cmax, Cmin, Css) between humans and animals, should be used only when there 
is no better alternative.  
 
Figure 1 	  A Representative Dose-Response Curve for Survival Based on 
Four Doses of an Investigational Drug Studied in a Well-
Characterized Animal Model 
 
 
 
As depicted in Figure 1, survival is increased (compared to placebo) following 
administration of Doses A, B, C, and D of  the investigational drug.  The results of 
the testing of  Dose D confirm  that Dose  C is a fully effective dose, because  
increasing the dose from C to D did not further increase survival.  Although it 
may seem  reasonable to use the exposures in animals resulting from  the 
administration of Dose C to serve as  the reference point for choosing the 
appropriate human exposure, when there is uncertainty as to whether the E/R 
relationship in humans is similar to the E/R relationship in animals, doses should 
be selected for humans that provide exposures that exceed those associated with 
the fully effective dose in animals, ideally by several-fold, if  the drugs safety 
profile allows such dosing.  To further minimize the possibility of sub-therapeutic 
exposures, human dose selection should also  take into account the variability of 
exposure parameters in humans and healthy and affected animals so that any low 
values of exposure in humans will be greater than  those associated with efficacy 
in animals. 
 
  
                                                 
81  In most cases, the animal species requiring the highest drug exposure  to demonstrate efficacy should be  the basis 
for choosing the human dose.  
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	 Contains Nonbinding Recommendations 
Figure 2  Comparisons of Animal and Human PK Data to Support the 
Selection of an Effective Dose in Humans 
 
 
 
 
 
In Figure 2, ranges of systemic drug concentration-versus-time profiles from  
human subjects following administration of three well-tolerated doses of an 
investigational drug are superimposed on the systemic concentration profiles from  
individual animals administered a fully effective dose.  Based on a comparison of 
the animal and human PK data, Dose 3 represents an ideal situation with the full 
range of human exposures exceeding the exposures for each animal administered 
the fully effective dose, both for Cmax and overall exposure.  If efficacy is not 
associated with the drugs Cmax, Dose 2 also  represents an ideal situation.   In the 
absence of scientific justification, Dose 1 is not acceptable because the full range 
of human exposures is not greater than the exposures associated with efficacy in 
animals. These situations are handled on a case-by-case basis and should be 
discussed with the review division.  
 
Interspecies differences in ADME should be considered when determining the 
human dose.  Differences in ADME between animals and humans may result in 
different systemic concentration-versus-time profiles among species,82,83  that may 
necessitate adjustments in the dose or regimen tested in the adequate and well-
controlled animal efficacy studies to  achieve concentration-versus-time profiles 
that are similar to the profile observed in humans.  Failure to account for 
                                                 
82  Deziel, MR, et  al., 2005, Effective Antimicrobial Regimens  for Use in  Humans  for Therapy of Bacillus anthracis  
Infections and Postexposure  Prophylaxis, Antimicrob Agents Chemother, 49(12):5099-5106. 
83  Kao, LM, et al., 2006, Pharmacokinetic Considerations and Efficacy of Levofloxacin in an  Inhalational Anthrax 
(Postexposure) Rhesus  Monkey Model, Antimicrob Agents Chemother, 50(11):3535-3542.  
  29
	 Contains Nonbinding Recommendations 
interspecies differences in pharmacokinetics may result in exposures in animals 
that are not achievable in humans, leading to the inability to select  an effective 
dose in humans (see section VI.B for additional discussion).  Differences in 
protein binding characteristics between animals and humans also should be 
considered, because only free drug, or the unbound fraction, is pharmacologically 
active.  If the protein binding characteristics in the selected species differ from  
those in humans, comparison of free drug exposures will be  relevant for dose 
selection.  
 
Although not discussed further in this document, quantitative methods, such as 
PK modeling, PK/PD modeling, physiologically-based pharmacokinetic (PBPK) 
modeling, or population modeling, can be used to support the extrapolation of 
exposures in animals to doses in humans.  The use of such methods should be 
discussed with the review division. 
 
Sponsors should consider PK interactions in humans of the investigational drug 
with medical products likely to be used concomitantly in the clinical scenario.  
The sponsor, with knowledge of the ADME of the investigational drug, should 
discuss with FDA other medical products that are likely to be co-administered 
based on the clinical scenario and develop a plan to address the potential for 
human PK interactions using in vitro and in vivo assessments, if warranted.  
Potential combinations that may affect the pharmacokinetics of either drug should 
be considered for interaction studies.  For example, if  the investigational drug is 
metabolized  via the cytochrome P450 system  (CYP450), the safety or efficacy of 
the investigational drug can be compromised by the concomitant use of CYP450 
inhibitors or inducers, and such drug-drug interactions should be evaluated.  In the 
case of therapeutic biological products, the design and conduct of relevant drug-
biologic interaction studies should be discussed with FDA with the overall goal of 
determining interactions with clinical  impact.  
 
When PD-based interactions (i.e., non-ADME based synergy or antagonism) with 
other drugs likely to be used in the anticipated clinical scenario have been 
identified, the sponsor should discuss with FDA the potential impact of these 
findings on the final human dose selection. For further discussion, see section 
VI.A, below. 
 
 
VI.  D	 ESIGN CONSIDERATIONS FOR THE ADEQUATE AND WELL-
CONTROLLED EFFICACY STUDIES IN ANIMALS  
 
These animal efficacy studies substitute for efficacy trials in humans, and therefore, the 
assessment of efficacy in animals should follow best practices for adequate and well-controlled 
human efficacy studies, with endpoints that demonstrate an important clinical benefit, generally 
the enhancement of survival or prevention of major morbidity.  When efficacy is demonstrated in 
a single study conducted in a sufficiently well-characterized animal model, FDA may require a 
  30
	 Contains Nonbinding Recommendations 
confirmatory animal efficacy study in that animal  model.84   The confirmatory study ideally 
should be conducted at a different laboratory; however, use of the same laboratory may be 
acceptable with justification.  Supportive human  efficacy data may negate the need for a 
confirmatory study.  Early discussions between the sponsor and FDA about key study design 
elements and study conduct are highly recommended.  Examples of key elements for discussion 
include the endpoints, proposed statistical analysis  plan, and data quality and integrity  plan (see 
the discussion on data quality and integrity in section IV.B).   Agreement on these issues should 
be reached before the initiation of studies.  
 
A.  General Principles  
 
Studies should be designed to mimic the ultimate clinical use of the investigational drug and to 
achieve meaningful outcomes similar to the effectiveness desired in humans.  The animal studies 
should not use surrogate endpoints85 as the sole evidence of efficacy.  It  is unlikely that  surrogate 
endpoints will be persuasive to FDA because the Animal Rule requires that the animal study 
endpoint (generally, the enhancement of survival or prevention of major morbidity) be clearly 
related to the clinical  benefit.86  Analyses of secondary endpoints may contribute to an 
understanding of the disease or condition and a characterization of the treatment effect. 
 
With rare exceptions, the adequate and well-controlled animal efficacy studies should evaluate 
the E/R relationship of the investigational drug, unless earlier studies have established the 
effective dose.  For further discussion of dose selection in the animals, see section VI.B.  The 
study duration is determined by the endpoint selected for the proposed indication.  The study 
duration should incorporate adequate follow-up time to observe for recurrence of disease or 
condition after stopping drug administration.  The route of administration of the investigational 
drug in animals should be the same as the route in humans, unless adequate justification is 
provided.  Different dosing regimens in animals and humans may be needed to provide 
comparable exposure to the drug. 
 
Animals of both sexes should be included and the male/female composition of the groups 
justified.  FDA recognizes that there are significant supply constraints on the use of adult animals 
of certain species.  The sponsor should discuss the age and the immune status of the animals used 
in efficacy studies, as compared to the intended human population.  Inclusion and exclusion 
criteria for the acceptance of the animals into the study should be appropriate and prespecified 
before initiating the studies.  
 
                                                 
84  As stated in the preamble to the final rule,  . . . the  animal studies should be replicated or substantiated in each 
species as  needed to ensure credible results . . .  (67  FR 37988  at 37991, May 31, 2002). 
85  In  this context,  the term  surrogate endpoint  refers to  a surrogate endpoint for efficacy, i.e., a drug-induced change 
in  a biomarker that is considered  reasonably likely to  predict clinical benefit of  the drug  and  that may be  used  in 
human clinical trials for a  development program  under the accelerated approval regulations (see  21 CFR  314.500 
through  314.560  for  drugs and 21  CFR 601.40  through  601.46 for biological products).   Surrogate endpoints for 
efficacy are  conceptually distinct from  humane endpoints.   Prospectively  defined, objective euthanasia criteria that 
are necessary to  address  animal welfare  are  based on the selected humane endpoints (see Appendix A). 
86  See 21  CFR  314.610(a)(3)  for drugs;  21  CFR  601.91(a)(3)  for biological  products.  
  31
	 Contains Nonbinding Recommendations 
The time course of observation should be optimized to assess the true treatment effect and to 
detect possible adverse effects.  Animals should be monitored frequently; the frequency of 
observation may vary over the course of the study depending on the actual mechanism  of disease 
or injury.  In  these studies that use mortality or  major morbidity as an endpoint, observation 
frequency should be sufficient to ensure animal  welfare and to minimize the potential loss or 
compromise  of data.   
 
Prospectively designed statistical analysis plans should be developed, incorporating the 
appropriate levels of statistical significance, including descriptions of the randomization 
procedures and methods to address missing data and, if applicable, outlying data.  Protection 
against bias is critical in the adequate and well-controlled animal efficacy studies, just  as it is in 
human trials.87   Studies should be randomized.  Variable  block randomization is one approach to 
minimize bias in these animal studies that are frequently small in size.  Euthanasia criteria should 
be prospectively specified and sponsors should provide a discussion of the potential effects of the 
criteria on the interpretation of results.  Studies should be blinded.  All personnel (e.g., 
investigators, veterinarians, animal caretakers, technicians) involved in making decisions 
regarding animal care interventions and/or euthanasia should  be blinded.  All personnel 
responsible for the collection, assessment, or interpretation of data obtained in the study 
(including but not limited to animals clinical signs; vital signs; laboratory tests; procedures; 
imaging studies; and necropsy, gross pathology, and histopathology data) should also be blinded.  
Any situation in which study personnel may become aware of treatment assignments should be 
discussed with FDA in advance because of  the potential for major effects on study 
interpretability.   
 
For almost any situation in which the  Animal Rule is used, there will be no basis for relying on a 
non-inferiority study to support effectiveness, and placebo-controlled animal studies should be 
used to demonstrate effectiveness.  Data obtained in the placebo-control group of the efficacy 
study should be compared with the data obtained in the natural history or  model characterization 
studies to substantiate the animal model.  For example, if animals in the placebo-control group 
do not exhibit morbidity or mortality similar to that  seen in the natural history studies,  this may 
reflect a problem  with preparation of the challenge agent that limits the ability to interpret 
outcomes in the active treatment arm(s) of the study. 
 
If a drug has already been approved for the same  indication and approval was based on the same  
animal species in which the investigational drug is being evaluated, the use of the approved drug 
in an active comparator arm,  in addition to the investigational drug and placebo arms, is 
encouraged and should be discussed with the review division.  The inclusion of the active 
comparator can be used to test for assay sensitivity (i.e., the ability of the study to differentiate an 
effective drug  from  an ineffective drug). 
 
Investigational drugs should be evaluated within  the context that reflects anticipated clinical use.  
The need for supportive care should be discussed with the review division early in drug 
                                                 
87  See 21  CFR 314.126(b)(5)  for drugs and  21  CFR 601.25(d)(2)  for  biological products.  
  32
	 Contains Nonbinding Recommendations 
development (see Appendix B).88   When supportive care is used, the study should show that the 
investigational drug with supportive care is superior to placebo with supportive care.  When  
incorporated  into a study, supportive care should be administered  either to all animals on a set 
schedule or to individual animals according to prospectively defined triggers, based on 
preliminary studies or available literature.  When supportive care will  be administered to 
individual animals based on prospectively defined triggers, the statistical plan should take into 
account the potential impact on the efficacy endpoint of differing supportive care among 
animals.  The potential effects of the supportive care on the animal and on the pharmacokinetics 
and/or pharmacodynamics of the investigational drug should be considered in the design and 
interpretation of the study. 
 
In addition, the sponsor, in consultation with FDA,  should consider other drugs that are likely to 
be used in combination with the investigational drug in the clinical scenario and evaluate 
whether the activity of either the investigational drug or the concomitant medication, when used 
in combination, is affected by PD-based interactions (i.e., non-ADME based synergy or 
antagonism)  and develop a plan to address the potential for such interactions.  For example, it 
should be known whether the use of an anthrax antitoxin monoclonal antibody will have an 
effect on the activity of the antimicrobial drugs used for the treatment of disseminated anthrax, or 
whether the use of a drug that prevents replication of  the target organism, resulting in a 
diminished immune response, may decrease the efficacy of a vaccine against that organism. 
 
A checklist of elements of an adequate and well-controlled animal efficacy study protocol is 
provided in section X.  In general, FDA should have the opportunity to  review information on 
the proposed clinical indication, animal model, and method to be used to translate the effective 
exposures in animals to a dose and regimen in humans prior to detailed discussions regarding the 
design of a specific adequate and well-controlled efficacy study in animals.  The design  of an 
animal efficacy study should incorporate the principles discussed in sections IV and V.  
Protocols for these studies can be submitted with  a request  for review under the SPA provisions 
(see section III.A).  
 
B.   Dose Selection in Animals  
 
The selection of  the doses of  the investigational drug89,90  to be studied in the adequate and well-
controlled animal efficacy studies should be based on an understanding of the E/R relationship in 
the proposed animal model.  Dose range-finding studies should include at least three adequately 
spaced doses to help define the shape of the E/R curve, including establishing a fully effective 
dose (see Figure 1 in section V.B.2.b), that is, a dose that produces the largest effect the 
investigational drug can have. To identify a fully effective dose, it is almost always necessary to 
                                                 
88  When it is anticipated that supportive care will be used  in the adequate  and well-controlled animal efficacy 
studies, the assessment of similar supportive care in model development, including the natural  history studies used  
to define the model, should be discussed with the review  division.  
89  This discussion  assumes that the investigational drug  is the active moiety.  Issues related  to  active metabolites are 
handled on a case-by-case basis and should be discussed with the review  division.  
90  For information on preventive vaccine dose  selection, see section VII.A.   
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	 Contains Nonbinding Recommendations 
have studied a higher dose and shown no added benefit.  For example, in Figure 1, the survival 
demonstrated with Dose D confirms that Dose C is a fully effective dose.  At least one of the 
doses evaluated in the adequate and well-controlled efficacy studies should be a fully effective 
dose. 
 
Prior to selecting doses for the efficacy studies, sponsors should understand the differences in 
ADME between humans and the selected animal species.  Differences in ADME between 
animals and humans may result in different systemic concentration-versus-time profiles between 
species.91,92   Failure to account for PK differences among species may result in exposures in 
animals that are not achievable in humans, leading to the inability to select  an effective dose in 
humans.  Some  differences in systemic concentration-versus-time profiles between animals and 
humans may necessitate adjustments of dose regimens studied in animal efficacy studies to 
achieve concentration-versus-time profiles that are similar to the profile observed in humans.  
This is known as humanization of dose regimens  and it is  illustrated in  Figure 3.  
 
Figure 3 	 An Example of a Humanized Dose and Regimen for 
Evaluation in an Animal Model of Disease93    
 
 
 
In this example, the shapes of the animal and human exposure profiles following once daily 
dosing are not comparable because the half-life of  the drug in animals is much shorter than in 
                                                 
91  Deziel, MR, et  al., 2005, Effective Antimicrobial Regimens  for Use in  Humans  for Therapy of Bacillus anthracis  
Infections and Postexposure  Prophylaxis, Antimicrob Agents Chemother, 49(12):5099-5106. 
92  Kao, LM, et al., 2006, Pharmacokinetic Considerations and Efficacy of Levofloxacin in an  Inhalational Anthrax 
(Postexposure) Rhesus  Monkey Model, Antimicrob Agents Chemother, 50(11):3535-3542.  
93 Adapted from  Bergman, KL, 2009, The Animal Rule and  Emerging Infections:  The Role of Clinical  
Pharmacology  in  Determining  an  Effective Dose, Clin Pharmacol Ther, 86(3):328-331.   
  34
	 Contains Nonbinding Recommendations 
humans.  The dose regimen in animals is manipulated  to achieve an exposure profile that is more 
similar in shape to that of  humans.  Adjusting the dose regimen used in animal studies based on 
differences in pharmacokinetics allows an improved comparison of exposures between animals 
and humans and, thus, provides greater confidence in selecting an effective dose in humans.  
 
 
VII.  C	 ONSIDERATIONS FOR PREVENTIVE VACCINES AND FOR CELLULAR 
AND GENE THERAPIES   
 
Although the overall principles of this guidance are applicable to vaccines94 and to cellular and 
gene therapy products, additional considerations in the design of the animal efficacy studies exist 
because of the biological nature of these products.  This section describes general considerations 
for study design and selection of relevant animal  species for the adequate and well-controlled 
animal efficacy studies specific to vaccines and to cellular and gene therapy products.  Before 
conducting an adequate and well-controlled animal efficacy study, FDA recommends that a 
sponsor request a meeting to discuss the details of the animal  model(s) and study design, 
including the rationale and methods  that will be used to extrapolate from a dose level(s) that 
shows substantial benefit in the animal studies to the final human dose and regimen.  
 
A.  V	 accines 
 
FDA will rely on animal efficacy data for approval  of vaccines using the Animal Rule only when 
the animal study endpoint is clearly related to the desired benefit in humans, generally the 
enhancement of survival or the prevention of major morbidity.  To estimate efficacy of vaccines 
in humans using the Animal Rule, the vaccine  dose chosen for adequate and well-controlled 
animal efficacy studies should elicit an immune response in animals reflective of that in humans.  
Using pilot and proof-of-concept studies, a relationship should be established between the 
vaccine dose and the desired immune response,  depending upon the study endpoint.  The dose, 
route of immunization, and schedule may be different in the animal and human studies if the 
relevant immune response is similar, and adequate justification is made. 
 
Sponsors should develop an approach for bridging animal responses to humans by careful 
selection of appropriate immune markers.  Sponsors should accumulate as much immune 
response data as possible in their animal model(s), sufficient to characterize the immune 
response that is associated with the desired outcome of disease prevention.  Such data can then 
be used to establish the vaccine dose and immunization schedule in humans needed to induce 
analogous immune responses.  Sponsors should discuss with FDA their choice of an immune 
marker, which will depend upon the product and the animal model selected for these studies.  
 
A single immune marker in an animal model may not reflect the spectrum of protective immune 
responses generated by humans.  For example, for  certain intracellular pathogens, animal models 
should be selected that demonstrate the induction of a protective antibody response as well as 
novel cellular immune response markers similar to humans.  The choice of animal species should 
                                                 
94  Cancer vaccines and therapeutic vaccines for non-infectious diseases are outside the  scope of this guidance. 
  35
	 Contains Nonbinding Recommendations 
be made based on consultation with experts, review  of  the literature, discussions at  scientific 
workshops and meetings, and discussions with FDA. 
 
The challenge agent used in animal studies with  vaccine products should  be relevant to the 
human disease.  When the etiologic agents host range prevents the development of an 
acceptable animal model, studies should be conducted in animal models with closely related 
challenge strains, assuming that cross strain immune markers, such as cross reacting neutralizing 
antibodies, allow bridging to the human immune response.  Ideally, the animal model(s) should 
show pathophysiology, progression of disease, symptoms, and host immune response similar to 
that observed in humans.  Achieving this may call for optimization of  the animal models in pilot 
and proof-of-concept studies using variable doses of the challenge agent to allow evaluation of 
the products effectiveness and interpretation of the study endpoints in the adequate and well-
controlled animal efficacy study(ies).  Ideally, the route of exposure should reflect the 
anticipated route of human exposure (especially if the route of exposure significantly affects the 
pathophysiology, onset, and progression of disease).  However, when the natural route of 
exposure is not known or cannot be  replicated in a model, animal studies to demonstrate 
protective immune responses using other routes of exposure should be considered and discussed 
with FDA.  Appropriate animal efficacy studies should be designed to provide information about 
the duration of protection afforded by the vaccine. 
 
Sponsors should seek and carefully  consider guidance from  public  health officials and experts 
concerning the intended use of the vaccine product.  Either pre- or post-exposure prophylaxis 
clinical indications, or both, may be desired depending upon public health needs.  Important 
immunization parameters, including the optimal dose, the schedule, and the desired time  and 
duration of protection, may differ depending upon the indication.  Studies supporting post-
exposure use may be more technically challenging to design, depending upon the animal model.  
Vaccines used in a post-exposure scenario are expected to be  given as soon as exposure is 
recognized.  Therefore, post-exposure studies in animals should be designed to administer the 
vaccine at a time point considered relevant to exposure in humans and induce an immune 
response that can be extrapolated to humans and suggest clinical benefit.  Data derived from  pre-
exposure prophylaxis studies could support the design of post-exposure animal studies, 
especially with regard to the kinetics and peak of the immune response.  Sponsors should 
evaluate the possible concomitant use and resulting influence of therapeutic drugs and 
antimicrobial drugs on effectiveness of the product when designing studies of vaccines intended 
for use in post-exposure scenarios. 
 
B.  Cellular and Gene Therapies  
 
1.  Cellular Therapy Products 
 
The selection of relevant animal species for evaluation of a cellular therapy product 
should include consideration of the host animals response to the product, including 
inflammatory responses, innate and acquired immune responses, and interactions of the 
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	 Contains Nonbinding Recommendations 
cells with the host (direct and indirect biological responsiveness).95  In addition, in vivo 
cell fate following delivery using the clinical route of administration should be 
characterized in each species.  Cell fate includes cell distribution to target and non-target 
sites, survival/engraftment, differentiation and integration, phenotype, and proliferation.  
Administration of the cellular therapy product to healthy animals will not likely  result in 
data representative of cell fate in humans.  For example, in GI-ARS, cell turnover and 
mitotic rate will affect cell fate; thus, the response of  the crypt cells to the cellular therapy 
pre- and post-radiation exposure will not be the same.  In addition, if  the cellular therapy 
product is delivered in combination with a matrix and/or scaffold or in an 
immunoisolation device, the biodegradation profile of these constructs should also be 
characterized. 
 
If the cell fate, cell function, and/or host response to the cells in the animal species differs 
greatly from what is known or predicted in humans, administration of a well-
characterized analogous cellular product in the animal studies may be considered.96  The 
use of an analogous cellular product in an animal efficacy study is predicated on the 
ability to identify, harvest, and characterize (e.g., phenotyping and potency) a similar cell 
population in the animal species used for testing.  Production of the analogous cellular 
product should meet the same standards as those applied to production of the final human 
cellular therapy product.  Sponsors are encouraged to initiate discussions with FDA early 
in product development for guidance on the animal models and the potential use of an 
analogous cellular product prior to initiating the adequate  and well-controlled efficacy 
studies. 
 
2.  Gene Therapy Products 
 
The selection of relevant animal species for evaluation of a gene therapy product should 
include consideration of the host animals response to the clinical vector, the expressed 
transgene, and/or the genetically modified cells.97  Vector-specific issues include 
determining (1) the permissiveness and/or susceptibility of various  animal species to 
infection and replication by the viral vector; (2) whether an immune or inflammatory 
response develops against the vector and if so, the effect of the response on the in vivo 
expression and persistence of the vector; (3) whether an immune response develops 
against vector positive cells; and (4) whether preexisting immunity to the vector  exists in 
the animals.  
 
Transgene-specific issues include determining (1) the pharmacological response of the 
species to the expressed transgene; (2) whether an immune or inflammatory response to 
                                                 
95 For a more comprehensive discussion  of the overall  principles for the cellular and gene therapy  products, refer to  
FDAs guidance  for  industry  Preclinical Assessment of Investigational Cellular and Gene Therapy Products.  
96  As  used  in  this  guidance,  analogous cellular products  are defined as cellular products  derived from  the animal 
species used  for testing that are analogs of the ultimate clinical product in  phenotype  and biologic activity. 
97 For a more comprehensive discussion  of the overall  principles for the cellular and gene therapy  products, refer to  
FDAs guidance  for  industry  Preclinical Assessment of Investigational Cellular and Gene Therapy Products. 
  37
	 Contains Nonbinding Recommendations 
the expressed transgene and/or protein develops; and (3) if an immune or inflammatory 
response does develop, the effect of the response on the in vivo expression levels, 
persistence, and functionality of the expressed transgene and/or protein in the animal 
species.  If these transgene-specific factors significantly differ in the animal species from  
what is known or predicted in human cells and tissues, administration of the clinical 
vector modified to express an analogous transgene can be considered.98  In such 
instances, product characterization comparison between the intended clinical construct 
and the animal homolog should be provided. 
 
Issues related to genetically modified cells include (1) the sensitivity of  the species to the 
biological actions of the modified cells and (2) the considerations conveyed in section 
VII.B.1. 
 
 
VIII.  HUMAN SAFETY INFORMATION 
 
The Animal Rule neither replaces the need, nor establishes special requirements, for an  adequate 
human safety database for drug development.  The expectation is that  drugs will be evaluated 
for safety under preexisting requirements for establishing the safety of new drug and biological 
products.99   FDA anticipates that the nonclinical and clinical safety development programs will 
proceed in a manner similar to that of  drugs developed under traditional regulatory pathways.  
Some  of the general principles include the following: 
 
	   Nonclinical toxicology, safety pharmacology, and PK studies (when relevant) should be 
conducted to provide adequate safety data to support the initiation of human trials. 
	   Risk-benefit assessment and ethical considerations must guide the design of human trials 
at each phase of development.100  The regulatory and ethical complexities of establishing 
the necessary safety database should be discussed with the review division, preferably 
early in the drug development program. 
	   The size and composition of the human safety database should be consistent with the 
proposed use of the drug. 
	   The adverse event grading scale should be appropriate for the population to be studied 
(e.g., healthy adult human volunteers101).  
                                                 
98  As used  in  this guidance,  an analogous transgene  is defined as  a transgene derived from  the animal species used  
for testing  that is an  analog  of  the human-derived transgene in  the clinical vector. 
99  See 67  FR 37988  at 37989, May 31, 2002. 
100  See Protection of Human  Subjects regulations at 21  CFR  part 50  and  Institutional Review Boards  regulations  at 
21  CFR part 56. 
101  The  principles  expressed in the following  FDA  guidance for  industry  may  be useful:   Toxicity Grading  Scale for 
Healthy Adult and  Adolescent Volunteers Enrolled in Preventive Vaccine  Clinical Trials.  
  38
	 Contains Nonbinding Recommendations 
   Safety signals identified from  animal studies or human trials should be characterized, and 
if necessary, specific study design elements should be incorporated into the proposed 
nonclinical and/or clinical protocols to prevent or mitigate toxicity in future studies.  
 
The evolving safety profile of the drug may necessitate changes in the clinical development 
program.  When evaluating the available human and animal data at important steps during drug 
development, sponsors should determine whether the program remains on a suitable path to 
achieve an adequate human safety database and consult with FDA if necessary. 
 
The size and composition of the human safety database necessary to support drug approval 
depend on issues such as the proposed indication, the drugs toxicity, and/or the extent of FDAs 
experience with a particular drug class.  For a drug intended for the treatment of a specified life-
threatening disease or condition, greater known risks or greater uncertainty about undefined risks 
may be acceptable when the drug offers a clear benefit for those patients.   A database of at least 
300 individuals will be needed for a 95% confidence interval to rule out a 1% rate of a specific 
adverse reaction (e.g., liver failure) if that specific adverse reaction did not occur in the 
population studied.  In contrast, drugs intended for prophylaxis in large numbers of healthy 
persons with variable or unclear risk of disease or injury may require a safety database in the 
thousands to  support a favorable risk-benefit assessment because little if any toxicity risk or 
undefined risks will be acceptable in this population.  The numbers suggested above for the size 
of the safety database refer to individuals exposed to the drug using the proposed route of 
administration, dosage form, and formulation, and, at a minimum,  the proposed dose, regimen, 
and duration. 
 
For some drugs, there may be existing relevant human safety data.  If the drug of interest is 
already approved, some  of the existing safety data  may be relevant to the proposed Animal Rule 
indication.  For example, the safety information used to support the pneumonic and septicemic 
plague indications for Levaquin (levofloxacin) was obtained from the large safety database from  
its other approved indications.  Similarly, if the drug of interest is in development for another 
indication, accrued safety data may be relevant for the proposed Animal Rule indication. 
 
When an adequate safety database does not exist and human safety studies are needed, the risks 
should be carefully considered because the potential for benefit to individual human subjects 
participating in studies of drugs being developed under the Animal Rule is remote (i.e., the 
human subjects have no predicted prospect of  exposure to the etiologic agent).  Even a 
compelling need for a drug (e.g., an intentional release of a lethal or permanently disabling 
chemical, biological, radiologic, or nuclear substance) does not justify exposing study subjects to 
risks greater than those acceptable for other drug development programs.  For drugs with only 
minor anticipated risks, studies in competent, appropriately consented adults are considered 
reasonable.102   If concerns about safety and/or relevance limit the extent or usefulness of studies 
in healthy adult volunteers, sponsors should explore alternative approaches to contribute to the 
aggregate safety database and discuss them with  FDA.  In some  circumstances, studies can be 
                                                 
102  As  stated  in 21  CFR 56.111(a)(2), Risks  to subjects are  reasonable in  relation  to anticipated  benefits, if  any, to 
subjects,  and the importance  of  the knowledge that may be expected  to  result. 
  39
	 Contains Nonbinding Recommendations 
conducted in existing patient  populations for whom development of the drug may fill a need 
(even if the population is different from the intended target population). 
 
Some  safety concerns may not become  apparent  until the drug is used in  the general population 
during an actual event.  Examples include the potential for drug-drug interactions (e.g., a colony-
stimulating factor and another drug that modifies the host immune system) and adverse 
interactions between the drug and a disease (preexisting or  agent-induced).   Such adverse 
interactions reinforce the  critical need for postmarketing studies.103    
 
Animal models used to demonstrate efficacy may not predict specific interactions of the agent-
induced disease or condition and the investigational drug in humans.  If adverse findings occur 
only when the investigational drug is tested in  challenge agent-affected animals, further 
investigation may be warranted to determine the pathophysiological mechanism  for the 
unexpected toxicity and its relevance to the risk assessment for the intended human population. 
 
                                                 
103  Postmarketing studies to provide evaluation of safety and efficacy in  the event an  emergency arises  and the 
product is  used are required  under the Animal Rule when such studies are  feasible and ethical.  A plan or approach  
for  conducting such  trials must be  included with  the NDA  or BLA  (for  greater  detail, see 21  CFR 314.610(b)(1) for 
drugs and  21  CFR  601.91(b)(1)  for  biological  products).  
  40
	 Contains Nonbinding Recommendations 
 
IX.  CHECKLIST OF ESSENTIAL ELEMENTS OF AN ANIMAL MODEL  
 
The following checklist provides a list of data elements (and the corresponding sections within 
this guidance) for consideration when developing an animal model.  The purpose of this 
checklist is to remind sponsors of the need to compare the data elements for the selected animal 
species to what is known about the human disease or condition in their submissions to FDA.  
Sponsors should note and explain any differences and indicate whether they expect these 
differences to have an impact on the interpretability of  the data.  
 
 )
s  
( n
DATA ELEMENTS (Corresponding Sections Within the Guidance)  al a
m m
i u
n H
A
ELEMENTS RELATED TO THE  ETIOLOGIC OR CHALLENGE  AGENT-INDUCED DISEASE OR 
CONDITION     
CHARACTERISTICS OF THE ETIOLOGIC OR CHALLENGE AGENT   
 The Challenge Agent (V.A.1.a)      
  Pathophysiological Mechanisms of Toxicity or Virulence (V.A.1.b)      
 Route of Exposure (V.A.1.c)      
  Dose and Quantification of Exposure (V.A.1.d)      
HOST SUSCEPTIBILITY AND RESPONSE (V.A.2)      
NATURAL HISTORY OF THE DISEASE OR CONDITION - PATHOPHYSIOLOGICAL 
COMPARABILITY  
 Time to Onset (V.A.3.a)     
 Progression (V.A.3.b)     
 Manifestations (V.A.3.c)     
TRIGGER FOR INTERVENTION (V.A.4)      
ELEMENTS RELATED TO THE  INVESTIGATIONAL  DRUG AND THE SELECTION OF AN EFFECTIVE   
DOSE IN HUMANS 
 THE INVESTIGATIONAL  DRUG  
 Mechanism of Action (V.B.1.a)     
 Drug Class (V.B.1.b)     
 Dosage Form and Route of  Administration (V.B.1.c)     
SELECTION OF  AN EFFECTIVE  DOSE IN HUMANS  () 
 PK and PD Information to Be Obtained in Animals and Humans (V.B.2.a)     
  PK/PD Considerations for Human Dose Selection (V.B.2.b)     
() For information on vaccine dose  selection, see section VII.A. 
  41
	 Contains Nonbinding Recommendations 
 
X. 	 CHECKLIST OF ELEMENTS OF AN ADEQUATE AND WELL-CONTROLLED 
ANIMAL EFFICACY STUDY PROTOCOL 
 
This checklist is included to remind sponsors of the information that should be included in their 
adequate and well-controlled animal efficacy study protocols.  For further information, refer to 
section VI. 
 
PROTOCOL CONSIDERATIONS 
   Indication to Be Studied 
   Agency Concurrence on the Details of the Animal  Model 
   Comparability of  the Study Design to  the Clinical Scenario  
 
 d
e  d 
b e
STUDY DESIGN ELEMENTS   ri fi
sc sti
  e u
D J
   Controls      
   Size of Study Groups and Male/Female Composition of Groups      
   Animal Characteristics ()  (e.g., species, age, weight, source of animals)     
   Inclusion and Exclusion Criteria for Acceptance Into Study      
   Dose, Route of Exposure, and Preparation of the Challenge Agent     
   Trigger for Intervention      
   Dose, Regimen, and Route of Administration of the Investigational Drug     
   Randomization      
   Blinding     
   Statistical Plan     
   Endpoints     
   Euthanasia Criteria      
   Observation Frequency and Schedule     
   Animal Care Interventions      
   Plan for Ensuring the Quality and Integrity of  the  Data     
() See section IV.D for further description.  
  42
	 Contains Nonbinding Recommendations 
 
APPENDIX A:  GENERAL PRINCIPLES FOR THE CARE AND USE OF ANIMALS IN 
BIOMEDICAL RESEARCH  
 
Animal studies must comply with applicable laws and regulations as prescribed by the Animal 
Welfare Act104  and the Public Health Service Policy on Humane Care and Use of Laboratory 
Animals.105  FDA endorses the principles of replacement, reduction, and refinement of the use 
of animals in biomedical research.106  
 
The following statements summarize general principles for the care and use of animals in 
biomedical research based on the animal welfare references listed at the end of this Appendix: 
 
1. 		 All persons involved in the use of animals in biomedical research should be appropriately 
qualified for and experienced in conducting procedures on living animals. 
 
2. 		 The living conditions of animals should be appropriate for the species and contribute to 
their health and comfort. 
 
3. 		 Unless otherwise established, procedures that cause pain or distress in human beings 
should be considered to cause pain or distress in animals.   For such procedures, the 
following practices should be observed, unless there is compelling scientific reason 
precluding such practices: 
 
a. 		 Appropriate sedation, analgesia, or anesthesia should be used during and/or 
following procedures that may cause more  than momentary or  slight pain or 
distress.  
 
b. 		 Humane endpoints that do not jeopardize the scientific objectives of the study 
should be established to prevent animals from  suffering unrelieved pain or 
distress.107   Humane endpoints are the earliest indicators of severe distress, severe 
pain, suffering or impending death observed in an  experimental animal.108   
Predetermined humane endpoints are used to develop objective euthanasia 
                                                 
104  See 7  U.S.C. 2131  et seq. 
105  U.S. Department  of Health  and Human  Services, National Institutes of Health, Office of Laboratory Animal  
Welfare, Public Health  Service Policy on  Humane  Care  and  Use of  Laboratory  Animals, Revised 2015, available 
at http://grants.nih.gov/grants/olaw/references/PHSPolicyLabAnimals.pdf, accessed on October 16, 2015. 
106  Russell, WMS and  RL Burch, 1959, The Principles of Humane  Experimental  Technique, London:  Methuen and  
Co. Ltd.  [Reissued:   1992, Universities Federation  for Animal Welfare, Herts, UK]. 
107  Humane endpoints are conceptually distinct  from  surrogate endpoints  for efficacy.  Surrogate endpoints for 
efficacy, discussed in  section VI.A, should not be  used as  the sole evidence of efficacy  in the  adequate and well-
controlled animal efficacy studies.  
108  Organisation for Economic Co-operation  and  Development, 2000, Guidance Document  on  the Recognition, 
Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation,  
ENV/JM/MONO(2000)7,  OECD,  Paris, France.   
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criteria.  Research necessitating endpoints for which pain and distress are not 
alleviated needs to be justified to, and  approved by, the Institutional Animal Care 
and Use Committee (IACUC).109  
 
c. 		 Animals experiencing severe or chronic pain or distress that cannot be relieved 
should be euthanized painlessly. The appropriate use of euthanasia criteria is 
beneficial to  the animal because unnecessary terminal distress is eliminated or 
significantly reduced.  Also, it benefits the research effort because experimental 
goals can be met more consistently.  Data collected after the development of 
severe physiologic derangements may not be useful or may be misleading for 
some purposes.  Also, tissues that might otherwise be lost can be collected for 
postmortem analysis.  Prospectively defined criteria for euthanasia should be 
included in protocol development.  The criteria should be predictive of imminent 
death or specific moribund conditions and should be defined in objective terms 
that are relevant to the specific experiment. 
 
d. 		 For studies in which major morbidity  or mortality  are expected, observation 
frequency should be increased around the expected time of major morbidity or 
death to prevent animals  from  experiencing unrelieved pain or distress and also to 
minimize the potential compromise or loss of data. 
 
4. 		 Adequate veterinary oversight and care provided by a qualified veterinarian, as defined 
by the Animal Welfare Act, and involvement of the IACUC must be in place to ensure 
humane care and use of animals.110,111,112  During protocol development, the attending 
veterinarian, or the veterinarian he or she designates, and the IACUC should play an 
active role in providing advice on humane endpoints and adequate veterinary care 
necessary to  ensure the humane needs of animals are met and are compatible with the 
scientific requirements of the study, as determined through discussion with the principal 
investigator. 
 
Animal welfare references include: 
 
 	  The Animal Welfare Act113  
 	  Guide for the Care and Use of Laboratory Animals, 8th  edition114  
                                                 
109  See 9  CFR 2.31(d)(1)(iv)(A).  
110  See 7  U.S.C. 2131  et seq. 
111  See Health  Research Extension Act of  1985, Public Law  99-158.  
112  See 9  CFR 2.31  and 9 CFR 2.33. 
113  See 7  U.S.C. 2131  et seq. 
114  National Research Council (US) Committee for the Update  of  the Guide for  the Care  and  Use  of Laboratory  
Animals, 2011, Guide for the Care and Use of  Laboratory Animals, 8th  edition,  Washington,  DC:  National 
Academies Press (US). 
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   Public Health Service Policy on Humane Care and Use of Laboratory Animals115   
   U.S. Government Principles for the Utilization and Care of  Vertebrate Animals Used 
in Testing, Research and Training116   
   AVMA Guidelines for the Euthanasia of Animals, 2013 edition117   
   Recognition  and Alleviation of  Pain in Laboratory Animals118   
                                                 
115  U.S. Department  of Health  and Human  Services, National Institutes of Health, Office of Laboratory Animal  
Welfare, Public Health  Service Policy on  Humane  Care  and  Use of  Laboratory  Animals, Revised 2015, available 
at http://grants.nih.gov/grants/olaw/references/PHSPolicyLabAnimals.pdf, accessed on October 16, 2015. 
116  See 50  FR 20864, May 20, 1985. 
117  American Veterinary Medical Association, AVMA Guidelines for the Euthanasia of Animals:  2013  Edition, 
2013,  available at  https://www.avma.org/KB/Policies/Documents/euthanasia.pdf, accessed on October 16, 2015.  
118  National Research Council (US) Committee on  Recognition and  Alleviation  of  Pain in  Laboratory Animals, 
2009, Recognition and Alleviation of Pain in  Laboratory Animals, Washington, DC:  National Academies Press 
(US).   
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APPENDIX B:  TYPES OF ANIMAL CARE INTERVENTIONS 
 
 
As described in this guidance, animal care interventions incorporated into animal studies are 
divided into three categories based on  the rationale for their use:  (1) intervention as part of 
adequate veterinary care,  (2) intervention as  supportive care to mimic the human clinical 
scenario, and (3) intervention to permit the manifestation of the disease or condition for the 
purpose of model development.  These categories of interventions are discussed here: 
 
Intervention as part of adequate veterinary care:  Animal studies must comply with applicable 
laws and regulations as prescribed by  the Animal Welfare Act119  and the Public Health Service 
Policy on Humane Care and Use of Laboratory Animals.120   In addition, all studies should 
comply with general principles for the care and use of animals in biomedical research (see 
Appendix A for details).  Compliance with these laws and general principles ensures that 
adequate veterinary care is provided, such that animals experiencing more than momentary or 
slight pain or distress are provided relief through appropriate analgesia, treatment, or, when 
prospectively defined criteria are met, euthanasia.  Exceptions to this standard are permitted only 
when scientifically justified and approved by the IACUC. The standards for adequate veterinary 
care also include treatment of unexpected events, such as injury or the development of an 
unrelated disease.  An example of an intervention that is considered part of adequate veterinary 
care is the administration of analgesics in a study assessing the effects of an investigational drug 
on vesicant-induced effects on the skin. 
 
Intervention as supportive care to mimic the human clinical scenario:  Supportive care, as 
defined in this document, is needed only to mimic, to the extent possible, the human clinical 
scenario.  In  general, it is relevant only for efficacy studies designed to support treatment of the 
disease or condition and the natural history studies on which the animal model is based.  Animal 
supportive care can range from  minimal intervention (particularly in the case of small rodents) to 
comprehensive medical support; however, it is not necessarily  equal to patient care in a human 
clinical setting and in many cases may be significantly less intensive.  The ability to provide 
certain types of supportive care can be species dependent (e.g., the ability to provide blood 
transfusions in a nonhuman primate model versus a rodent model).  When included in an animal 
efficacy study, supportive care ideally should reflect the intended conditions of use of the 
investigational drug.  It also should  reflect the intended types of medical intervention and the 
timing of the availability of medical intervention  expected in the human clinical or incident 
setting.  The anticipated supportive care should be adapted, as appropriate, from  the standard of 
human clinical practice to the animal species used, such as modifying the doses, route of 
administration, or the specific medical products administered. 
 
                                                 
119  See 7  U.S.C. 2131  et seq. 
120  U.S. Department  of Health  and Human  Services, National Institutes of Health, Office of Laboratory Animal  
Welfare, Public Health  Service Policy on  Humane  Care  and  Use of  Laboratory  Animals, Revised 2015, available 
at http://grants.nih.gov/grants/olaw/references/PHSPolicyLabAnimals.pdf, accessed on October 16, 2015. 
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When supportive care is administered to the animals as part of the design  of the efficacy study, 
the study should show that the investigational drug with  supportive care is superior to placebo 
with supportive care.  When incorporated into a study, supportive care should be administered 
either to all animals on a set schedule or to individual animals  according to prospectively defined 
triggers, based on preliminary studies or available literature.  When supportive care will be 
administered to individual animals based on prospectively defined triggers, the statistical plan 
should take  into account the potential impact on the efficacy endpoint of differing supportive 
care among animals.  The potential effects of the supportive care on the animal and on the 
pharmacokinetics and/or pharmacodynamics of the investigational drug should be considered in 
the design and interpretation of the study.  
 
Intervention to permit the manifestation of the disease or condition for the purpose of model 
development:  To study certain diseases or conditions, interventions are needed to permit the 
manifestation of the disease or condition of interest.  Interventions used in  this way are essential 
parts of the model development.  For example, to  establish a  model of  GI-ARS, it is necessary to  
attenuate the potentially lethal effects of H-ARS that occur before, or concomitantly with, GI-
ARS. The interventions used to attenuate the H-ARS (e.g., partial bone marrow shielding during 
irradiation or bone marrow transplantation) are considered to be components of model 
development.  
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APPENDIX C:  GENERAL EXPECTATIONS FOR NATURAL HISTORY STUDIES  
 
Natural history  studies  are studies in which animals are exposed to a challenge agent and 
monitored to gain an understanding of the development and progression of the resulting disease 
or condition, including parameters such as manifestations (e.g., signs, clinical and pathological 
features, laboratory parameters, extent of organ involvement, morbidity, and outcome), the time 
from  exposure to manifestation onset, time course and order of manifestation progression, and 
severity.   Ideally, natural history studies should be prospectively designed,121 adequately 
controlled, well-documented, and statistically powered to demonstrate the anticipated morbidity 
or mortality.   In addition,  the studies should include a statistical analysis of  potential treatment 
triggers or critical determinants of disease or condition such as signs, endpoints, or biomarkers.  
Challenge dose standardization should occur before, or as part of, the natural history study.  
 
In general, natural history studies should include randomized concurrent controls (i.e., 
unchallenged control animals) to reduce experimental bias (e.g., age- and sex-matched controls, 
or controlling for the effect of vehicle on the respiratory tract of experimental animals in aerosol  
challenge models).  Blinding should be used, to the extent possible,  to reduce investigator bias.  
Observation times and/or frequencies should be specified in the study protocol and should be 
based on available information and/or preliminary studies.  The frequency of observation should 
be adequate to characterize the course of disease or injury and to define the desired endpoints 
and treatment triggers.  The frequency of observation may vary over the course of the study, 
depending on the actual mechanism of disease or injury.  Observation frequency should be 
increased around the expected time of  major morbidity or death to ensure animal welfare and to 
minimize the potential loss or compromise of data.  Findings from  the natural history studies 
should be substantiated through replication of the study or a demonstration of results consistent 
with other relevant studies.  For example, the median survival at a relevant  time point and time to 
the development of neutropenia following exposure to a specified dose of whole body radiation 
should be similar for irradiated rhesus macaques in the natural history studies and in the control 
groups for the associated efficacy studies.  
 
The natural history studies should be adequate in design, conduct, and reporting.  These studies, 
designated for drug development under the Animal Rule, will be subject to inspection and audit 
by FDA to verify the reliability of the data.  The expectations for data quality and integrity for 
model-defining natural history studies submitted for qualification are discussed in section IV.B. 
 
The general expectations with regard to the animals used in  the investigation, study conduct, the 
study report, and the submission of the study report and data are discussed in section IV. 
                                                 
121  When it is anticipated that supportive care  will be used  in the adequate  and well-controlled animal efficacy 
studies, the assessment of similar supportive care in model development, including the natural  history studies used  
to define the model, should be discussed with the review  division (see section VI.A and Appendix B).  
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APPENDIX D:  ACRONYMS AND ABBREVIATIONS 
 
ADME     Absorption, distribution, metabolism, and excretion 
AMQP     Animal Model Qualification Program 
AUC      Area under the plasma  concentration-time curve 
BARDA    Biomedical Advanced Research and Development Authority 
BLA      Biologics license application 
BPCA     Best Pharmaceuticals for Children Act 
BSL      Biosafety level 
CBER     Center for Biologics  Evaluation and Research 
CBRN     Chemical, biological, radiological, and nuclear 
CDC      Centers for Disease Control and Prevention  
CDER     Center for Drug Evaluation and Research  
CFR      Code of Federal Regulations 
Cmax      Maximum  (peak) plasma concentration 
Cmin      Minimum  (trough) plasma concentration 
Css      Steady-state plasma concentration  
COU      Context of use 
eCTD      Electronic common technical document 
CYP450    Cytochrome P450 
DDT      Drug development tools 
E/R      Exposure-response 
EUA      Emergency use authorization 
FDA      U.S. Food and Drug Administration  
FDASIA    Food and Drug Administration Safety and Innovation Act 
FD&C Act    Federal Food, Drug, and Cosmetic Act  
FR      Federal Register 
GI-ARS     Gastrointestinal subsyndrome of acute radiation syndrome   
GLP      Good laboratory practice 
H-ARS    Hematopoietic subsyndrome  of acute radiation syndrome  
HHS      Department of Health and Human Services  
IACUC    Institutional Animal Care and Use Committee  
IND      Investigational new drug application 
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LD       Lethal dose sufficient to kill 50% of those exposed to the agent 
50 
MCMi     Medical Countermeasures Initiative 
MIC      Minimum  inhibitory concentration  
NDA      New drug application 
PBPK      Physiologically-based pharmacokinetic 
PD      Pharmacodynamic  
PK      Pharmacokinetic 
PREA     Pediatric Research Equity Act of 2003 
SPA      Special protocol assessment  
SNS      Strategic National Stockpile 
USC      United States Code 
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