Contains Non-Binding Recommendations 
185 
 
Guidance for Industry 
 
Target Animal Safety for Veterinary  
Pharmaceutical Products 
VICH GL43
 
 
This VICH document supersedes FDA Guidance for Industry #33, Target Animal 
Safety Guidelines for New Animal Drugs dated 6/89. 
For questions regarding this document, contact Steven Vaughn, Center for Veterinary Medicine, 
(HFV-100), Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855, 240-
276-8300, e-mail:  steven.vaughn@fda.hhs.gov.  
Comments and suggestions regarding the document should be submitted to Division of Dockets 
Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, 
Rockville, MD  20852. Submit electronic comments to http://www.regulations.gov.  All 
comments should be identified with the Docket No. FDA-2007-D-0430 (formerly Docket No. 
2007D-0166). 
Additional copies of this guidance document may be requested from the Communications Staff 
(HFV-12), Center for Veterinary Medicine, Food and Drug Administration, 7519 Standish Place, 
Rockville, MD  20855, and may be viewed on the Internet at 
http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/d
efault.htm.  
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
April 24, 2009 
       
                                            
VICH GL 43 (TARGET ANIMAL SAFETY) - PHARMACEUTICALS 
July 2008 
For implementation at Step 7 
 
 
 
 
 
T A S V
ARGET  NIMAL  AFETY FOR  ETERINARY 
P P  
HARMACEUTICAL  RODUCTS
 
 
 
 
Adopted at Step 7 of the VICH Process 
by the VICH Steering Committee 
in July 2008 
for implementation in July 2009 
 
 
 
 
THIS GUIDELINE HAS BEEN DEVELOPED BY THE APPROPRIATE VICH EXPERT WORKING GROUP AND 
HAS BEEN SUBJECT TO CONSULTATION BY THE PARTIES, IN ACCORDANCE WITH THE VICH PROCESS.  
AT STEP 7 OF THE PROCESS THE FINAL DRAFT IS RECOMMENDED FOR ADOPTION TO THE 
REGULATORY BODIES OF THE EUROPEAN UNION, JAPAN , AND USA. 
   
Contains Non-Binding Recommendations 
 
 
TABLE OF CONTENTS 
 
1.  INTRODUCTION..............................................................................................................................................4 
1.1  OBJECTIVE....................................................................................................................................................4 
1.2    BACKGROUND...............................................................................................................................................4 
1.3    SCOPE...........................................................................................................................................................5 
2.  MARGIN OF SAFETY STUDIES....................................................................................................................5 
2.1  STANDARDS..................................................................................................................................................6 
2.2  ANIMALS......................................................................................................................................................6 
2.3  ROUTE OF ADMINISTRATION.........................................................................................................................7 
2.4  DOSE, FREQUENCY, AND DURATION OF ADMINISTRATION...........................................................................7 
2.5  STUDY DESIGN.............................................................................................................................................8 
2.6  VARIABLES...................................................................................................................................................9 
2.6.1  PHYSICAL EXAMINATIONS AND OBSERVATIONS...........................................................................................9 
2.6.2  CLINICAL PATHOLOGY TESTS (HEMATOLOGY, BLOOD CHEMISTRY, URINALYSIS)....................................10 
2.6.3  NECROPSY AND HISTOPATHOLOGY EXAMINATIONS...................................................................................11 
2.7  STATISTICAL ANALYSIS..............................................................................................................................12 
2.8  STUDY REPORTS.........................................................................................................................................14 
3.  OTHER LABORATORY SAFETY STUDY DESIGNS...............................................................................14 
3.1  INJECTION SITE SAFETY STUDIES................................................................................................................14 
3.2  ADMINISTRATION SITE SAFETY STUDIES FOR DERMALLY APPLIED TOPICAL PRODUCT.............................14 
3.3  REPRODUCTIVE SAFETY STUDIES...............................................................................................................15 
3.4  MAMMARY GLAND SAFETY STUDIES.........................................................................................................16 
4.  TARGET ANIMAL SAFETY DATA FROM FIELD STUDIES.................................................................17 
5.  RISK ASSESSMENT IN ANIMAL SAFETY EVALUATION....................................................................17 
6.  GLOSSARY......................................................................................................................................................18 
 
 
 
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This guidance represents the Food and Drug Administrations (FDAs) current thinking on this 
topic.  It does not create or confer any rights for or on any person and does not operate to bind 
FDA  or  the  public.    You  can  use  an  alternative  approach  if  the  approach  satisfies  the 
requirements of the applicable statutes and regulations.  If you want to discuss an alternative 
approach, contact the FDA staff responsible for implementing this guidance.  If you cannot 
identify the appropriate FDA staff, call the appropriate number listed on the title page of this 
guidance. 
 
 
1.  INTRODUCTION 
 
Data from target animal safety (TAS) studies are required for registration of veterinary products1 
in the regions participating in the VICH.  International harmonization of standards for essential 
TAS studies will facilitate adequacy of data and minimize the need to perform separate studies 
for  regulatory  authorities  of  different  countries.  Appropriate  international  standards  should 
reduce research and development costs by minimizing repetition of similar studies in each 
region.  Animal welfare should benefit because fewer animals may be needed.  This VICH TAS 
guidance has been developed as a harmonized standard to aid in development of mutually 
acceptable TAS studies for relevant governmental regulatory bodies. The use of this VICH 
guidance to support registration of a product for local distribution only is strongly encouraged 
but is up to the discretion of the local regulatory authority. 
 
FDAs guidance documents, including this guidance, 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. 
 
1.1  Objective 
The  purpose  of  this  harmonized  guidance  is  to  provide  recommendations  regarding  TAS 
evaluation for regulatory submission of an Investigational Veterinary Pharmaceutical Product 
(IVPP),  which  is  appropriate  for  determining  the  safety  of  an  IVPP  in  the  target  animal, 
including identification of target organs, where possible, and confirmation of margin of safety, 
using the minimum number of animals appropriate for the studies. 
 
1.2   Background 
The  VICH  was  initiated  to  develop  internationally  harmonized  guidance  that  outlines 
recommendations for meeting regulatory requirements for registration of an IVPP in the regions 
                                                 
1 See 21 U.S.C.  360b(b)(1); 21 U.S.C.  360b(d)(1)(A); 21 CFR 514.1(b)(8). 
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participating  in  the  program.    By  their  nature,  guidance  documents  cannot  address  all 
possibilities.  The TAS Expert Working Group has developed the general principles included in 
this guidance document to aid in the development and conduct of TAS studies and to support the 
monitoring of potential adverse events in field studies.  For more specific recommendations, 
review of the study protocol by the relevant regulatory authorities prior to the initiation of the 
study is encouraged, where review is available.   
   
If in a particular circumstance an alternative approach is deemed more fitting, preparation of a 
reasoned explanation by the sponsor and discussion with the regulatory authorities is encouraged 
before work is initiated. 
 
1.3   Scope 
This guidance document is intended to cover TAS evaluation for any IVPP used in the following 
species:    bovine,  ovine,  caprine,  feline,  canine,  porcine,  equine,  and  poultry  (chickens  and 
turkeys). The recommendations in this guidance may not be appropriate for registration by 
national or regional authorities of products for use in minor species or minor uses. The guidance 
does not provide information for the design of TAS studies in other species, including aquatic 
animals. For other species and for minors uses, TAS studies should be designed following 
national or regional guidance.  
 
This guidance contributes to the international harmonization of methods used for evaluation of 
IVPPs.  The guidance is provided to aid sponsors in preparing and conducting TAS studies under 
laboratory  conditions  and  in  the  field.  All  recommendations  in  this  document  may  not  be 
necessary  for  every  IVPP.    For  other  IVPPs,  additional  information  not  specified  in  this 
document may be important to show target animal safety.  
 
 
2.  MARGIN OF SAFETY STUDIES 
 
The aim of TAS studies is to provide information on the safety of an IVPP in the intended 
species under the proposed conditions of use.  The margin of safety study is indispensable in the 
approval of an IVPP.2  Furthermore, adverse effects associated with overdoses and increased 
duration of administration of the IVPP should be identified, if possible.  Dose confirmation and 
field studies conducted to confirm the effectiveness of the IVPP provide further information on 
safety in the target species. Depending on the known or suspected properties of the IVPP, it may 
be necessary to conduct additional toxicologic or specialized tests. 
 
The margin of safety may be documented if the study includes both the recommended dose and 
overdoses, given for the proposed and longer treatment periods.  The selection of dose and 
overdose levels and durations of treatment should always be justified by the sponsor, taking into 
account the proposed use of the product and the known pharmacologic and toxicologic properties 
of the active pharmaceutical ingredient (API). Where usage or formulation involves a greater risk 
or consequence of overdose, then a separate study, or inclusion of a higher dose level in the 
                                                 
2 See 21 U.S.C.  360b(b)(1); 21 U.S.C.  360b(d)(1)(A); 21 CFR 514.1(b)(8) 
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margin of safety study, is recommended for the IVPP.  This may include cases where a dose 
calculation error may be likely, such as a mistake in decimal point identification during addition 
to feed. 
 
The design of the TAS evaluation and the prediction of potential adverse effects that may occur 
in the target species should be assisted by reference to data including published literature and 
preliminary studies, including pharmacokinetics, pharmacodynamics and toxicology  from target 
and non-target laboratory animal studies. The specific information used to evaluate the safety of 
an IVPP depends on factors such as proposed usage regimen and dose, type of drug, chemistry 
and manufacturing considerations, claims, previous use history of similar products, and animal 
species including class and breed.  Appropriate observations, physical examinations, clinical 
pathology tests (hematology, blood chemistry, urinalysis, fecal analysis, etc.), necropsy and 
histopathology should be conducted to identify possible adverse effects of the IVPP.   
 
Margin  of  safety  studies  are  generally  required  for  new  salts  or  formulations  of  an  API.3 
Exceptions should be justified, for example, on the basis of known toxicology and target animal 
safety  profiles  for  the  API,  widespread  clinical  use  of  existing  products,  and/or  where  the 
systemic or local exposure (as applicable) of the new product is proven to be equivalent to or less 
than that of the existing product. 
 
If  systemic  exposure  to  the  API  is  negligible,  and  based  on  pre-existing  knowledge  in 
pharmacology and toxicology there is no safety concern, then the margin of safety study may not 
be needed. This should, however, be justified by the sponsor, and a safety study at the site of 
administration (see 3.1. to 3.4.) is recommended. 
2.1  Standards  
Margin of safety and other laboratory safety studies must be performed in conformity with the 
principles of Good Laboratory Practices (GLP).4  The concepts of current Good Manufacturing 
Practices (cGMP) must be applied to the IVPP as appropriate for new animal products intended 
for investigational use.5 
 
2.2 Animals   
Healthy animals, representative of the species and class in which the IVPP will be used, should 
generally be used in TAS studies. The age of animals should be considered carefully; if the 
product is intended for use in young, immature animals, then the animals in the TAS studies 
should generally be the youngest age for which product approval is sought. Otherwise, healthy 
young mature animals should be used. Additional studies may be needed for potentially sensitive 
subpopulations, if such groups have been identified in the intended target population. 
 
                                                 
3 See 21 U.S.C.  360b(b)(1); 21 U.S.C.  360b(d)(1)(A); 21 CFR 510.3(i) 
4 See 21 CFR Part 58. 
5 See 21 U.S.C.  351(a)(2)(B); 21 CFR Parts 210 and 211. 
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Acclimatization of the animals to the study conditions is recommended.  Treated and control 
animals  should  be  managed  identically  and  prophylactic  treatments  completed  before  the 
baseline period of the study, where possible.  Use of concurrent therapy with other products 
during the study may make it more difficult to identify safety concerns due to the IVPP and is 
not recommended.  Studies should be carefully planned to provide adequate information while 
minimizing the number of animals used.  Housing and husbandry should be adequate for the 
purpose of the study, as well as conform to local animal welfare regulations. Environmental 
conditions, diet, and water should be controlled throughout the study as appropriate to the 
species, physiological state, and age. It is recommended that quality and composition of diet and 
water are monitored throughout the study.   Reduction or elimination of suffering during the 
study is essential.  Euthanasia and necropsy of moribund animals is recommended. 
 
2.3 Route of Administration  
The IVPP to be evaluated should be the product intended  to  be  marketed.    If  the  market 
formulation is not used, comparative (bridging) studies may be necessary, e.g., the relevance of 
TAS data for one formulation of an IVPP to another formulation can be demonstrated by the use 
of bioequivalence or other data between the two formulations.  The IVPP should be evaluated by 
comparison to a placebo (e.g., saline) or untreated control.  The formulation details, generic or 
trade name, and batch number should be documented. Details of preparation, handling and 
storage of the IVPP should be documented and the product should be used in accordance with 
the study protocol.  The site of administration is to be identified.  Dosing should follow the use 
conditions suggested in the proposed labeling.  If food affects API bioavailability, animals 
should be fed or fasted before administration to provide the highest likelihood of showing 
adverse effects.  If volume or palatability becomes a limiting factor for higher dose levels, 
alternative  techniques  (for  example,  multiple  sites,  gavage,  or  increased  frequency  of 
administration) may be considered.  If multiple routes of administration are proposed by the 
sponsor, the route that is most likely to cause adverse effects should be selected as the basis for 
safety studies.  Additional studies on local tolerance (see 3.1. or 3.2.) should be conducted, as 
appropriate. 
 
2.4 Dose, Frequency, and Duration of Administration  
The general design for margin of safety studies uses multiples of the proposed use dose and 
duration of administration of the IVPP.  Specific dose, frequency, and duration combinations for 
use in TAS studies should be selected and justified based on the pharmacology and toxicology of 
the IVPP.   
 
Unless  otherwise  justified  by  the  pharmacologic-toxicologic  properties  of  the  API  and  the 
proposed use of the product, the design of the margin of safety study should include a negative 
control, the highest recommended dose level (1X), and two multiples of this use dose (in most 
cases three times (3X) and five times (5X)) for a period of time in excess of the recommended 
maximum duration of use.  The highest recommended dose level (1X) is defined as the highest 
actual dose that will be stated on the proposed product label. The highest dose may be for the 
lowest body weight animal in a weight range for which a fixed mass of the API in a discrete 
dosage form (e.g., unit dose volume, tablet combination etc.) is recommended.  In some regions, 
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alternative designs based on the pharmacology and toxicology may be acceptable, for example, 
the administration of the product in excess of the recommended maximum duration of treatment 
given only in the highest recommended dose level (1X). Regardless of design, a negative control 
should always be included.   
 
In general, it is recommended that each group be treated for at least 3 times the proposed 
duration up to a maximum of 90 days (for example, for a proposed single use IVPP, treatments 
should  be  administered  for  3  consecutive  intervals  as  determined  by  the  pharmacologic 
characteristics of the IVPP; or for a proposed daily dosing for 7 days, treatments should be given 
for 21 consecutive days).  If short-term, intermittent use is intended, treatments should be 
administered 3 times at the recommended interval (for example, proposed weekly treatments 
should be administered for 3 consecutive weeks).  Where product use is expected to exceed 3 
consecutive months in individual animals then, depending on pharmacology and toxicology, 
longer duration studies may be recommended up to 6 months or longer if appropriate (e.g., where 
drug accumulation may increase over time or where duration of drug activity following a single 
dose exceeds 2 months).   
 
2.5 Study Design 
The most important techniques for avoiding bias in studies are randomization and masking 
(blinding).  A  randomization  plan  should  be  used  to  allocate  animals  to  treatment  groups.  
Blocking may be used to control, as far as possible, the distribution of the one or two most 
important factors, such as sex, age, stage of lactation or body weight to ensure balance between 
treatments. 
 
Target animal safety studies typically include relatively small numbers of experimental units 
(generally 8 per treatment) and assess large numbers of variables.  Both males (4 per treatment) 
and females (4 per treatment) should be included unless the product is only intended for use in 
one sex. Medical, animal welfare, and statistical considerations are generally used to determine 
the total number of animals used to evaluate potential safety concerns. When interim sacrifice or 
withdrawals of animals for other reasons are anticipated, the number of experimental units 
should be increased accordingly. Although there is strong interest in the null hypothesis of no 
difference  between  treatments,  study  design  constraints  limit  the  statistical  power  and 
discriminatory ability of these studies.  Under these conditions, statistical analysis alone may not 
detect  potential  adverse  effects  and  thus  provide  assurance  of  safety.    Results  should  be 
evaluated  and  interpreted  based  on  a  combination  of  medical,  toxicologic,  and  statistical 
principles with consideration of biological significance and plausibility.   
 
Where group housing is needed to provide appropriate animal welfare and allow for adequate 
experimental  conditions,  certain  variables,  such  as  diarrhea,  vomiting,  or  feed  or  water 
consumption may be difficult to measure on an individual animal basis.  In addition, even 
measurements that can be accurately made for individual animals from the same cage or pen may 
be influenced by the presence of the other animals in the group.  For example, presence of a 
dominant animal in a pen may be a contributing factor in weight loss among other animals in the 
pen.  Potential influence of group housing should be taken into consideration in interpreting drug 
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effects from a study, even if statistical analyses are not used.  Failure to consider this information 
might lead to incorrect attribution of effect to the IVPP. 
 
The planned times for measuring each variable should be described in the study protocol.  Often 
this schedule includes daily observations of animals throughout the study period, with more 
detailed measurements at several time points, including the beginning and end of the study.  
Pretreatment measurements should be made to identify baseline levels. Measurement at time 
points during the proposed label duration of IVPP use may help characterize the time course of 
potential safety issues.  Equally-spaced measurement intervals may facilitate statistical modeling.  
For  longer  studies,  data  collection  may  be  planned  in  designated  phases,  with  different 
frequencies of data collection. 
 
Data should be collected in a manner that minimizes bias.  For example, when examinations are 
needed on a subgroup from each treatment, animals should be randomly designated before study 
initiation.  Personnel collecting data, including gross post mortem results, should be masked to 
treatment. Histopathology data should be evaluated by recognized procedures (e.g., Crissman et 
al., Toxicologic Pathology, 32(1), 126-131, 2004). 
 
2.6 Variables 
Many  variables  are  measured  during  an  animal  safety  study.  The  types  of  observations, 
examinations and tests for safety depend on the nature of the IVPP, proposed use, target animal, 
and potential for adverse effects.  In general, there are four types of variables that should be 
considered  in  margin  of  safety  studies:  physical  examinations  and  observations,  clinical 
pathology tests, necropsy, and histopathology examinations.  In addition, other types of variables 
may be considered, such as toxicokinetic evaluation of drug exposure (e.g., sparse sampling 
around the expected peak and trough concentration times).  However, care should be taken to 
minimize the number of additional sampling points to avoid interference with primary safety 
endpoints.   
 
2.6.1   Physical Examinations and Observations 
A detailed physical examination by qualified personnel (generally a veterinarian) should be 
conducted at several time points during the study, including the beginning and end.  Baseline 
observations of other variables should be made by qualified personnel at the beginning of the 
study.  Observations relating to general health and behavior by trained personnel (not generally a 
veterinarian) should be recorded on all animals daily, seven days a week, or at pre-determined 
intervals appropriate for the purpose of the study, during the entire period.  Food and water 
consumption should be monitored at appropriate intervals.  Body weights should be measured at 
the beginning, end, and several other appropriate times. 
 
Generally, the following should be considered and measured depending on the nature of the 
IVPP and the intended population: 
 
General Physical Examination (these generally should be done by a veterinarian) 
Nervous system 
Ocular system 
Musculoskeletal system  Integumentary system 
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Cardiovascular system  Respiratory system  
Reproductive system  Urinary system  
Lymphatic system  Gastrointestinal system  
Behavior   
 
Specific Examination of Injection/Application Site (semi-quantitative or quantitative 
assessment should be used wherever possible) 
Appearance (e.g., erythema, eschar formation, hair 
Swelling 
loss, scaling, pigmentation)  
Pain  Heat 
 
Observations (these should be done by appropriately trained staff) 
Water intake 
Feed intake 
Weight  Behavior 
Body temperature  Signs of illness 
Feces (consistency, color and mucus, blood)   
 
2.6.2  Clinical Pathology Tests (Hematology, Blood Chemistry, Urinalysis) 
Hematology, blood chemistry, and urinalysis should be conducted at several points during the 
study,  including  at  the  beginning  and  end  of  the  study.    Other  specialty  tests  to  monitor 
appropriate physiologic parameters may be appropriate, depending on the IVPP.  A standardized 
feeding schedule prior to sample collection should be followed. Tests should be conducted on all 
animals or, where group size is greater than 8 (e.g., poultry), on subsets of animals that were 
selected for testing by a random process carried out at the beginning of the study. These tests are 
subject to influence by the conditions under which the samples are collected such as feeding or 
fasting, and sedation or anesthesia, and therefore it is critical that samples are collected in the 
same manner from concurrent negative control and treatment groups of animals. Blood samples 
from multiple animals should not be pooled.  For animals showing adverse events, additional 
clinical pathology and other diagnostic tests to determine the etiology may be appropriate.  
Collection of clinical pathology data twice during the pre-treatment stage can be helpful in 
providing  reliable  baseline  data  for  interpretation  of  study  results.    Measured  variables, 
depending on the nature of the IVPP and the intended population, may include (units should be 
appropriate for regions): 
 
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Hematology    
Erythrocytes:   Leukocytes:  
Total counts; and if applicable, reticulocyte  Total and differential counts 
count 
Packed cell volume (PCV)   Mean corpuscular volume  (MCV) 
Mean  corpuscular  hemoglobin  (MCH)  and  Hemoglobin 
Mean corpuscular hemoglobin concentration  
(MCHC)  
Prothrombin time  Platelet count 
Activated partial thromboplastin time  Buccal mucosal bleeding time 
Whole blood clotting time  Fibrinogen 
Acute phase protein    
 
Blood Chemistry    
Sodium      Urea nitrogen 
Potassium    Creatinine     
Chloride     Alanine aminotransferase  (ALT)  
Calcium     Aspartate aminotransferase  (AST)  
Phosphate      Lactate dehydrogenase  (LDH)  
Magnesium     Gamma-glutamyltransferase (GGT) 
Total protein     Alkaline phosphatase (AP)  
Albumin    Creatine kinase  (CK)  
Globulin    Total bile acids  
Glucose    Cholesterol    
Amylase    
 
 Urinalysis   
Color  Protein   
pH   Ketone bodies   
Specific gravity (e.g., by refractometer)  Bilirubin   
Glucose      Urobilinogen   
Microscopic examination of sediment (crystals, casts, RBCs, WBCs, epithelial cells) 
 
2.6.3  Necropsy and Histopathology Examinations 
Tissues  from  all  dose  groups  should  be  examined  grossly  and  preserved  for  microscopic 
evaluation.  Gross and microscopic examination of tissues of animals in all dose groups is 
recommended for IVPPs containing a new API, due to the small number of animals used and a 
general lack of other safety information in the target species. For other products, at a minimum, 
tissues from all animals in the negative control and highest dose groups should be examined 
microscopically (for recommended procedures, see Crissman et al., Toxicologic Pathology, 32 
(1), 126-131, 2004). If lesions are found in any tissue from the highest dose group, then samples 
from tissues in animals in the next lowest dose group of the IVPP should be examined until a no-
observable-adverse-effect level is determined by microscopy. In addition, all animals showing 
systemic clinical signs or abnormal findings in clinical pathology should normally be examined 
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grossly and microscopically.  Where the toxicity of the IVPP is anticipated to be relatively high, 
or where there is already information on toxicity from previous studies, different necropsy 
schemes may be recommended, to include gross and microscopic examinations for all animals or 
for subsets of animals that were selected for testing by a random process carried out at the 
beginning of the study. For an IVPP with a well documented broad margin of safety, including 
but not limited to comparative pharmacokinetic and comparative metabolism data, post-mortem 
examination may not be necessary in the absence of systemic clinical signs or abnormal findings 
in clinical pathology, based on appropriate justification and pre-specified in the study protocol. 
 
Organ weights, where appropriate, and gross lesions should be recorded.  The organs selected for 
gross  and  microscopic  examination  will  depend  upon  animal  species  and  target  tissues. 
Histopathology  should  be  conducted  on  organs/tissues,  with  particular  attention  given  to 
organs/tissues showing macroscopic lesions, including at the injection site, where applicable.  
Generally,  it  is  recommended  that  the  following  organs/tissues  be  considered  in  gross  and 
microscopic examinations, as appropriate:  
 
Organs/tissues considered for gross and microscopic examination: 
Pituitary gland  Brain  Bone and marrow 
Thyroid gland  Spinal cord  Marrow smear 
Parathyroid gland  Eyes  Spleen 
Adrenal gland  Lung  Stomach 
Pancreas  Muscle   Duodenum 
Ovaries  Mammary gland  Jejunum 
Uterus  Liver  Ileum 
Testes  Gall bladder  Colon 
Prostate  Kidneys  Cecum 
Epididymis  Urinary bladder  Thymus 
Heart  Lymph nodes  Injection site: e.g., muscle, subcutaneous tissue 
Crop  Proventriculus  Bursa of Fabricius 
Ventriculus  Skin    
 
2.7 Statistical Analysis   
In most studies, the safety implications are best addressed by applying descriptive statistical 
methods to the data. Tables and descriptive text are common methods of data summarization; 
however, it is also valuable to make use of graphical presentations in which patterns of adverse 
events are displayed both within treatments and within individual animals. Selection of the 
general form for a statistical model and the factors to be included in the model will depend on the 
nature of the response variable being analyzed and the study design.  Regardless of the methods 
chosen, the process and steps used to conduct any statistical evaluations should be described. The 
outcomes of the data analysis should be clearly presented to facilitate evaluation of potential 
safety concerns.  The terminology and methods of presentation should be chosen to clarify the 
results and expedite interpretation. 
 
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Tables may be used to present the data from individual animals and summary statistics from 
treatments.  For quantitative variables, useful descriptive statistics include the number of animals 
in each treatment, median, mean, standard deviation, maximum, minimum, and the number and 
percentage  of  cases  with  values  falling  outside  a  recognized  reference  range.    For  some 
quantitative variables, categorization of animals with values that fall within different ranges may 
help to identify patterns.  For qualitative variables, useful descriptive statistics include the total 
number of animals evaluated and the number and percentage of experimental units within each 
response category.  Other events, such as adverse events, mortalities, and early terminations may 
also be tabulated. 
 
Graphs may be very helpful in depicting the data and identifying potential safety concerns, 
including  possible  dose  trends,  time-related  patterns,  and  values  that  fall  outside  reference 
ranges.    Plots,  that  show  responses  over  time,  both  within  treatment  groups  and  within 
individuals, may illustrate consistency of responses between animals, or sex, age, or dose levels. 
These graphs may show trends or time-related patterns in adverse effects of treatment. 
 
Statistical models should represent the study design.  The individual animal may be considered 
to  be  the  experimental  unit  when  each  animal  is  penned  individually  or  animals  from  all 
treatments groups are mixed in a single pen.  When animals that are penned together are all 
assigned to the same treatment, the pen is typically the experimental unit.  If housing unit, 
environmental conditions, sex, or pretreatment covariates differ between experimental units, 
these factors should be balanced in the design and accounted for in the analysis, as appropriate.  
A  useful  approach  is  to  include  the  fewest  number  of  terms  that  adequately  represent  the 
underlying  process  that  generated  the  safety  data,  and  to  represent  the  longitudinal  nature 
(repeated measurements) of the design (if applicable).  Choice of model form should be driven 
by the nature of the response variable being analyzed. The potential impact of any missing data 
on the results should be considered. 
 
Analysis results from statistical modeling include statements of significance levels for terms 
included in the model.  The calculation of p-values is sometimes useful either as an aid to 
evaluating a specific difference of interest, or as a flagging device applied to a large number of 
safety variables to highlight differences worth further attention.  This is particularly useful for 
clinical  pathology  data,  which  otherwise  can  be  difficult  to  summarize  appropriately.  It  is 
recommended that clinical pathology data be subjected to both a quantitative analysis, e.g., 
evaluation of treatment means, and a qualitative analysis where counts of numbers above or 
below  certain  thresholds  are  calculated.  While  p-values  are  one  indication  of  a  substantial 
difference that should receive clinical appraisal, the small size of safety studies makes it crucial 
that clinical judgment be used to evaluate all differences, irrespective of the p-value observed.  A 
statistically  significant  test  does  not  necessarily  indicate  the  presence  of  a  safety  concern.  
Similarly, a non-significant test does not necessarily indicate the absence of a safety concern. 
Statistical adjustments for multiplicity can be counterproductive for considerations of safety; the 
importance and plausibility of results will depend on prior knowledge of the pharmacology of the 
IVPP and this evaluation should be made by clinicians or scientists with appropriate experience 
and training in interpreting the biological relevance of the results. 
 
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2.8 Study Reports 
A study report is a document describing the objectives, material and methods, any amendments 
or deviations from the protocol, results (including individual animal data, data summaries, and 
any analyses), and conclusions of a TAS study. In some regions, additional provision of raw data 
may be required.6 
 
 
3.  OTHER LABORATORY SAFETY STUDY DESIGNS 
 
Additional safety studies may be appropriate for a particular IVPP, depending on the conditions 
of use and the characteristics of the IVPP.  Such studies may be combined with the margin of 
safety evaluation and, in food producing animals, residue studies.  These specialized studies 
should be designed according to the general principles outlined below and follow any official 
local guidance. It is recommended that the specific study plans be determined by communication 
between the sponsor and the local regulatory agency. 
 
3.1  Injection Site Safety Studies   
The basic study design should consider dose (1X), duration, route(s), vehicle, and maximum 
volume of the injection, generally using 8 animals per group.  The study should include a saline 
control of the same volume as the complete and final formulation of the IVPP. In the case of 
non-liquid IVPP, an alternative suitable negative control should be used. The location and timing 
of each injection should be specifically noted to facilitate determination of time to resolution.  
The study should consider site lesions that may be produced by administration by syringe or 
other applicator by intravascular, intradermal, intramuscular and/or subcutaneous routes.  If 
intravascular administration is the only route proposed, consideration should be given to the 
effects of extravascular administration of the IVPP.  For formulations where intravascular use is 
not intended and there is a potential risk associated with unintended intravascular injection (e.g., 
certain subcutaneous injections in the ear), the safety in the event of intravascular injection 
should be considered. 
 
Evaluation of safety data from injection site studies may include the following: 
-  Clinical signs including changes in behavior or locomotion. 
-  Appearance, inflammation, edema, or other changes at the injection site.  
-      Measuring Creatine kinase and aspartate transaminase levels. 
-  Gross pathology and histopathology of lesions at appropriate times. 
 
If there is inflammation at the injection site that has not resolved on visual examination or by 
palpation  by  the  end  of  the  planned  study,  then  the  time  required  for  return  to  clinically 
acceptable resolution at the injection site should be determined. Where clinical signs indicative 
of injection site effects are evident, it may be necessary to conduct histopathology of the lesions. 
 
3.2 Administration Site Safety Studies for Dermally Applied Topical Product 
 
                                                 
6 See 21 CFR 514.1(b)(8). 
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Local adverse reactions to a topically applied IVPP should be evaluated, generally in 8 animals 
per group, at the dosage proposed on the label unless the pharmacology and toxicology of the 
product warrants multiples of dose and/or duration. For a systemically absorbed topical IVPP, it 
is recommended that evaluation of topical administration sites be included in studies of systemic 
TAS outcomes. In general, the site should be examined for swelling, pain, heat, erythema, and 
other clinical signs.  Changes in animal movement or behavior should be noted.  If there is 
inflammation or other clinical signs at the topical application site that have not resolved on visual 
examination or by palpation by the end of the planned study, then the time required for return to 
clinically  acceptable  resolution  at  the  topical  application  site  should  be  determined.  Where 
clinical signs indicative of administration site effects are evident, it may be necessary to conduct 
histopathology of the lesions. 
 
Oral dosing, generally at the maximum proposed dose of a topical formulation, is recommended 
to examine the safety of the IVPP if accidental ingestion is likely to occur after treatment (e.g., 
by licking). If, based on pharmacology and toxicology, there are no safety concerns regarding 
oral exposure, it may be appropriate to dispense with this study.  
 
3.3  Reproductive Safety Studies 
Reproductive safety studies are required for systemically absorbed API intended for use in 
breeding animals.7  The goal of reproductive safety studies is to identify any adverse effects of 
the IVPP on male or female reproduction or on offspring viability.  These studies generally focus 
on reproductive variables, although safety data on other body systems may be collected. These 
studies do not usually extend to considering viability of offspring beyond the post-natal period, 
unless there is specific evidence of possible effects on, for example, sexual maturity, based on 
pharmacology and toxicology of the API.   
  
Healthy,  intact,  reproductively-sound  males  and  females  should  be  selected  that  are 
representative of the species, age, and class in which the drug will be used.  In general, it is 
recommended that 8 animals per sex be included per treatment. Males and females may be 
evaluated in the same or separate studies. Dose, route, frequency, and duration of administration 
should be selected and justified based on the pharmacology and toxicology of the IVPP and 
intended use, and should ensure continuous exposure throughout the study interval.  Generally, 
males should be treated with a negative control and 3X doses of the IVPP throughout at least one 
spermatogenic cycle.  Generally, females should be treated with a negative control and 3X doses 
of the IVPP prior to breeding (covering the follicular phase until conception), throughout the 
gestation period (including embryonic phase, fetal phase, and natal phase), and after parturition 
for an appropriate time covering the post-natal period that is sufficient to assess the initial 
development and locomotor function of the offspring.  It is not necessary to administer the IVPP 
at or around parturition, unless the IVPP is specifically indicated for use at that time. 
 
Reproductive safety studies should evaluate, as appropriate: 
-  In the male: spermatogenesis, semen quality and mating behavior. 
                                                 
7 See 21 U.S.C.  360b(b)(1); 21 U.S.C.  360b(d)(1)(A); 21 CFR 514.1(b)(8) 
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-  In  the  female:  estrous  cycle,  mating  behavior,  conception  rates,  length  of  gestation, 
parturition, and lactation. 
-  In the offspring from treated males and/or females: developmental toxicology (including 
teratogenicity, fetotoxicity), fetal development, number of offspring, viability and growth, 
health and development to weaning. 
-  In poultry: also, egg weight, shell thickness, number of eggs laid by a hen, egg fertility, 
hatchability, and chick viability. 
 
Ideally, reproductive safety studies are conducted in the target species; however, data obtained 
from  reproductive  studies  in  laboratory  animals  may  be  considered,  provided  that  the 
pharmacokinetic profiles of the API are comparable in laboratory animals and in all species in 
which the IVPP is intended for use.  Depending on the results of such evaluation, appropriate 
information should be included on the labeling.  However, if reproductive safety studies have not 
been conducted in the target species, labeling should reflect this and state that safety has not been 
determined in breeding, pregnant, or lactating animals or their offspring. 
 
3.4  Mammary Gland Safety Studies  
Mammary gland safety studies should be conducted to evaluate the safety of IVPPs intended for 
intramammary use in lactating or non-lactating animals.  For these studies, animals should be 
free of subclinical or clinical mastitis.  The IVPP should be administered as one dose to each 
teat. The conditions of use, dose (1X), and frequency of administration should be those proposed 
on the label.  Alternatives should be justified by the sponsor.   
 
It is recommended that safety evaluation of an IVPP intended for use in lactating females include 
objective  evaluation  of  acute  inflammatory  effects  in  early  to  mid-lactation  animals  (data 
collected in conformity with the principles of GLP8). It is recommended that safety evaluation of 
an IVPP intended for use in non-lactating animals include both an objective evaluation of acute 
inflammatory effects in lactating animals (data collected in conformity with the principles of 
GLP9) and clinical evaluation of chronic inflammatory effects in non-lactating animals (data 
collected in conformity with the principles of GCP or GLP10). 
   
For both lactating and non-lactating claims, it is preferred that a one-group, comparative design 
be used to evaluate similarity of values from pre-treatment and post-treatment periods within 
each animal. A two-group design may also be used, with treated animals compared to negative 
control animals. In general, 8 lactating animals, including 4 in their first lactation, should be 
assigned to each treatment in either study design for both lactating and non-lactating claims.   
 
For all study animals, physical examination, including palpation, should be done to determine 
swelling, erythema, pain, or heat.  For either study design in lactating animals (both lactating and 
non-lactating claims), data on all relevant variables associated with tissue irritation and milk 
                                                 
8 See 21 CFR Part 58. 
9 Id. 
10 Id. 
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production  should  be  collected  pre-treatment,  at  treatment,  and  post-treatment.    The  post-
treatment monitoring period should be defined a priori based on the anticipated time for values 
to  return  to  pre-treatment  values.    Samples  for  quantitative  somatic  cell  count  (SCC)  and 
bacterial  culture  should  be  collected  from  each  teat  prior  to  milking.    Daily  milk  yield, 
composition (e.g., fat, protein, lactose, and non-fat solids), and appearance should be recorded.  
Key  variables  for  safety  assessment  generally  include  signs  of  mammary  gland  irritation, 
elevated SCC, and changed milk production.  The presence of very high post-treatment SCC or 
prolonged SCC elevation following treatment may not be acceptable and should be explained by 
the sponsor.  
 
 
4.    TARGET ANIMAL SAFETY DATA FROM FIELD STUDIES  
 
Field studies intended to evaluate effectiveness of an IVPP also provide essential TAS data under 
conditions of intended use.  These studies should be conducted in accordance with the principles 
of GCP.   
 
Field studies are typically conducted under conditions representative of the target population and 
provide an evaluation of potential adverse effects at the intended use dosage in a much larger 
number  of  animals.    Field  studies  use  the  target  population  which,  if  applicable,  includes 
diseased animals. Where disease and husbandry are similar between regions, international data 
may be used for field studies, as long as a minimum proportion of the data acceptable to the 
region is generated within the region where approval is being sought.  Including a relatively large 
number of animals in the study improves the ability to detect relatively low frequency adverse 
events.  Animals should be representative of the age range, class, breed, and sex for which the 
IVPP is intended. The study should be designed with an appropriate control group.  In each 
study,  health  observations  should  be  performed  by  appropriate  masked  (blinded)  personnel 
before, during, and after treatment, with specific evaluation of potential adverse effects (e.g., 
physical examination and clinical pathology tests). The appropriate variables for evaluation may 
be based on results of pharmacodynamic studies in laboratory animals or studies in the target 
species.  Adverse events should be reported and determination of causality for the adverse event 
attempted. 
 
 
5.   RISK ASSESSMENT IN ANIMAL SAFETY EVALUATION  
 
For some IVPPs, laboratory and field safety data may not alone provide sufficient information to 
determine if an acceptable safety profile exists in relation to IVPP benefits.  In these instances, 
risk assessment methodologies may provide a means to supplement or augment TAS evaluation.  
Risk assessment uses the available body of evidence to weigh the severity of an adverse effect 
(harm), the potential of reversibility, and the probability that it will occur. 
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6.  GLOSSARY 
 
Active Pharmaceutical Ingredient (API): Any substance or mixture of substances intended to 
be used in the manufacture of a drug (medicinal) product and that, when used in the production 
of a drug, becomes an active ingredient of the drug product. Such substances are intended to 
furnish  pharmacological  activity  or  other  direct  effect  in  the  diagnosis,  cure,  mitigation, 
treatment, or prevention of disease or to affect the structure and function of the body. 
 
Adverse Effect: Adverse event suspected to be related to an IVPP. 
 
Adverse Event:  Any observation in animals that is unfavorable and unintended and occurs after 
the use of an IVPP, whether or not considered to be product related. 
 
Baseline  Data:  Information  collected  after  the  acclimatization  period  and  before  the 
administration of an IVPP.   
 
Breeding Animal:  Any animal that is actively breeding, intended for breeding, or pregnant.  
 
Class: Subset of target animal species which is characterized by factors such as reproductive 
status and/or use (dairy vs. beef, broiler vs. layer). 
 
Current Good Manufacturing Practices (cGMP):  The part of a quality system which ensures 
that products are consistently produced and controlled to the quality standards which they are 
represented to possess. 
 
Experimental  Unit:    The  smallest  independent  grouping  of  animals  that  could  receive  a 
different  treatment  during  the  study,  given  the  methods  of  allocation  and  treatment 
administration. 
  
Good Clinical Practices (GCP):  A standard for the design, conduct, monitoring, recording, 
auditing, analysis, and reporting of clinical studies. Adherence to the standard provides assurance 
that the data and reported results are complete, correct and accurate, that welfare of the study 
animals and the safety of the study personnel involved in the study are ensured, and that the 
environment and the human and animal food chains are protected. 
 
Good Laboratory Practices (GLP):  A standard for the design, conduct, monitoring, recording, 
auditing, analysis, and reporting of non-clinical studies. Adherence to the standard provides 
assurance that the data and reported results are complete, correct and accurate, that welfare of the 
study animals and the safety of the study personnel involved in the study are ensured, and that 
the environment and the human and animal food chains are protected. 
 
Investigational Veterinary Pharmaceutical Product (IVPP):  Any pharmaceutical form of, or 
any animal feed containing one or more active pharmaceutical ingredients (API) being evaluated 
in  a  clinical  or  non-clinical  study,  to  investigate  any  protective,  therapeutic,  diagnostic,  or 
physiological effect when administered or applied to an animal. 
 
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Margin of Safety Study:  Well-controlled study designed to show if an IVPP is safe for the 
intended species. 
 
Masking/Blinding:  A procedure to reduce potential study bias in which designated study 
personnel are kept uninformed of the treatment assignment(s). 
 
Negative Control: Study animals that either receive a placebo or are untreated. 
 
Reference range (clinical pathology or blood chemistry): The range of usual values found in 
healthy animals of a given class. 
 
Target Animal:  The specific animal species, class, and breed identified as the animal for which 
the IVPP is intended for use.  
 
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