Contains Non-Binding Recommendations 
 
206 
 
Guidance for Industry 
 
Studies to Evaluate the Metabolism and Residue Kinetics of 
Veterinary Drugs in Food-Producing Animals: Comparative 
Metabolism Studies in Laboratory Animals 
 
VICH GL47 
 
 
Submit comments on this guidance at any time.  Submit written comments to the Division of 
Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Room 
1061,  Rockville,  MD  20852.    Submit  electronic  comments  on  the  guidance  at 
http://www.regulations.gov  All written comments should be identified with the Docket No. FDA-
2010-D-8229.  
For  further  information  regarding  this  document,  contact  Julia  Oriani,  Center  for  Veterinary 
Medicine,  (HFV-151),  Food  and  Drug  Administration,  7500  Standish  Place,  Rockville,  MD 
20855, 240-276-8204, e-mail:julia.oriani@fda.hhs.gov 
Additional copies of this draft guidance document may be requested from the Communications 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  either 
http://www.fda.gov/AnimalVeterinary/default.htm or http://www.regulations.gov.   
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
September 15, 2011 
          Contains Non-Binding Recommendations 
 
 
VICH GL 47 (MRK)  METABOLISM AND RESIDUE KINETICS 
February 2011 
 
For Implementation at Step 7 - Final
 
 
 
S M
TUDIES TO EVALUATE THE  ETABOLISM 
R K V
AND  ESIDUE  INETICS OF  ETERINARY 
D F - A :
RUGS IN  OOD PRODUCING  NIMALS  
L A C
ABORATORY  NIMAL  OMPARATIVE 
M S  
ETABOLISM  TUDIES 
 
 
Adopted at Step 7 of the VICH Process 
by the VICH Steering Committee 
in February 2011  
for implementation in February 2012 
 
 
This Guidance has been developed by the appropriate VICH Expert Working Group and is subject to 
consultation by the parties, in accordance with the VICH Process.  At Step 7 of the Process the final draft 
will be recommended for adoption to the regulatory bodies of the European Union, Japan and the USA. 
 
 
 
 
 
GL47 -  2           Contains Non-Binding Recommendations 
 
 
Table of Contents 
 
1.  INTRODUCTION 
 
1.1.      Objective of the guidance 
1.2.      Background  
 
2.  GUIDANCE 
 
2.1.  Purpose 
2.2.  Scope 
2.3.  Comparative Metabolism Studies In Laboratory Animals  
2.3.1.  Test Materials 
2.3.1.1.  Drug  
2.3.1.2.  Radiolabeled Drug 
2.3.1.3.  Analytical Standards 
2.3.2.  In Vitro Test Systems 
2.3.3. In Vivo Test Systems 
2.3.3.1.  Animals  
2.3.3.2.  Animal Handling 
2.3.3.3.   Number of Animals      
2.3.3.4.  Drug Formulation 
2.3.3.5.  Route of Administration 
2.3.3.6.  Dosing 
2.3.3.7.  Animal Euthanasia 
2.3.3.8.  Sample Collection  
2.3.3.9.  Determination of Total Radioactivity 
    2.3.4. Separation and Comparison of Metabolites 
2.3.4.1.  Analytical Method 
2.3.4.2.  Extent of Characterization/Major Metabolites 
2.3.4.3.  Nonextractable Metabolites 
3. GLOSSARY 
 
GL47 -  3           Contains Non-Binding Recommendations 
 
 
STUDIES TO EVALUATE THE METABOLISM AND RESIDUE KINETICS OF VETERINARY DRUGS IN 
FOOD-PRODUCING ANIMALS: COMPARATIVE METABOLISM STUDIES IN LABORATORY ANIMALS 
(VICH GL47) 
 
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 appropriate FDA staff.  If you 
cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this 
guidance. 
 
1.  INTRODUCTION 
1.1.  Objective of the guidance 
The objective of this guidance is to provide recommendations for internationally harmonized 
procedures to identify the metabolites of veterinary drugs produced by laboratory animals.  
The purpose of the comparative metabolism studies is to compare the metabolites of the 
laboratory animals used for toxicological testing to the residues of the veterinary drugs in 
edible tissues of food-producing animals, in order to determine if the laboratory animals used 
for toxicological testing have been exposed to the metabolites that humans can be exposed to 
as residues in products of food-producing animal origin. 
1.2.  Background 
This  guidance  is  one  of  a  series  developed  to  facilitate  the  mutual  acceptance  by 
national/regional  regulators  of  residue  chemistry  data  for  veterinary  drugs  used  in  food-
producing  animals.    This  guidance  was  prepared  after  consideration  of  the  current 
national/regional requirements and recommendations for evaluating veterinary drug residues in 
the European Union, Japan, United States, Australia, New Zealand and Canada. 
 
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 Agencys guidances means that something is suggested or 
recommended, but not required. 
 
 
GL47 -  4           Contains Non-Binding Recommendations 
 
2.  GUIDANCE 
2.1.  Purpose 
The human food safety evaluation of veterinary drug residues helps ensure that food derived 
from treated food-producing animals is safe for human consumption.  As part of the data 
collection  process,  studies  should  be  conducted  to  characterize  the  metabolites  to  which 
laboratory animals are auto-exposed during the toxicological testing of the veterinary drug.  
The purpose of these studies is to determine whether the metabolites that people will consume 
from  tissues  of  target  food-producing  animals  are  also  produced  by  metabolism  in  the 
laboratory animals used for the safety testing.  It is understood that, if the laboratory animals 
produce substantially similar metabolites as those produced by the food-producing animal, the 
laboratory animals will have been auto-exposed to the metabolites that humans will consume 
from tissues of treated food-producing animals. Auto-exposure of metabolites will ordinarily 
be  taken  as  evidence  that  the  safety  of  metabolites  has  been  adequately  assessed  in  the 
toxicology studies. 
2.2.  Scope 
Demonstration of metabolites from the laboratory animal can be generally accomplished in 
one or more in vitro studies or in an in vivo study.  
Use of one or more in vitro laboratory animal metabolism studies (e.g., laboratory animal liver 
slice metabolism) for comparison to the metabolism in the food-producing animal can be 
conducted to demonstrate that the relevant laboratory animal produces the metabolites that are 
found as residues in the edible tissues of the target food-producing animal.  Conducting in 
vitro studies can avoid the use of in vivo laboratory animal studies, can reduce the number of 
animals that are euthanized, and can reduce the cost of comparative metabolism studies.  If the 
in vitro or in vivo studies do not demonstrate the metabolites produced by the target food-
producing animal, the sponsor should address by other means the relevance to consumer 
safety of the food-producing animal metabolites.   
Laboratory animal in vitro and in vivo metabolism studies are most often accomplished using 
radiolabeled drugs.  These studies are capable of monitoring all of the drug-derived residues 
resulting from the administration of test material (note: generally only the major metabolites 
should  be  identified).    This  guidance,  therefore, recommends procedures for metabolism 
studies conducted with radiolabeled drugs.  However, alternative approaches (i.e., not using 
radiolabeled drugs) to characterize the metabolites in laboratory animals can be suitable when 
the metabolites produced by the target food-producing animal as residues in edible tissues are 
readily identified in urine or tissues of the laboratory animals by chemical means.   
Generally auto-exposure has been adequately demonstrated if laboratory animals produce 
each of the major metabolites of the residue that people will consume from edible tissues of 
treated food-producing animals.  Qualitative information on the metabolites in laboratory 
animals should be reported.  Quantification of the metabolites found in urine, fluids or tissues 
of laboratory animals is not generally an objective of the comparative metabolism studies.  
Generally only the major metabolites found as residues in the food-producing animal should 
be identified in the laboratory animals.  Metabolites observed in laboratory animals that are 
GL47 -  5           Contains Non-Binding Recommendations 
 
not observed in the food-producing animal are not relevant to the objective of assuring that 
the laboratory animals are auto-exposed to the residue metabolites that humans will consume.   
Comparative metabolism studies should be conducted in compliance with applicable Good 
Laboratory Practice (GLP).  
2.3.  Comparative Metabolism Studies in Laboratory Animals  
2.3.1.  Test Materials 
2.3.1.1. Drug  
The chemical identity (including, for example, the common name, chemical name, CAS-
number, structure, stereochemistry and molecular weight) and purity of the drug substance 
should be described.  The test drug should be representative of the active ingredient to be used 
in the commercial formulation.   
2.3.1.2. Radiolabeled Drug 
The position(s) of the radiolabel should be indicated.  The characteristics of the radiolabeled 
drug used in comparative metabolism studies should meet the specifications identified in the 
guidance  VICH  GL  46  Studies  to  Evaluate  the  Metabolism  and  Residue  Kinetics  of 
Veterinary Drugs in Food-producing Animals: Metabolism Study to Determine the Quantity 
and Identify the Nature of Residues in the target food-producing animal regarding: a) the 
nature of the radiolabel, b) the site of the label in the test molecule, and c) the purity and 
specific activity of the radiolabeled drug.   
2.3.1.3. Analytical Standards 
Analytical standards should be available for the parent drug and, if possible, for metabolites 
known or expected to exist, for use in the chromatographic comparison of drug metabolites.  
The metabolites can be isolated from tissues generated in the target food-producing animal 
metabolism study (VICH GL 46).   
 
2.3.2.  In vitro Test Systems 
Single or multiple in vitro metabolism test studies can be used as an alternative for the in vivo 
comparative metabolism studies.   
The laboratory animal species used in the comparative metabolism study should preferably be 
the same species (and for rodents the same strain) as was used in the pivotal study for 
determining the toxicological acceptable daily intake (ADI) of the veterinary drug. In case 
another species is used, the choice of species should be justified in terms of relevance.  The 
source of the animals, their weights, health status, ages and gender should be reported.   
Various  test  systems  have  been  published  and  are  widely  used.    In  vitro  systems  for 
comparative metabolism studies include primary hepatocytes, liver microsomes, the S9 sub-
cellular fraction, cytosol, liver slices and whole cell lines.  Protocols for these in vitro studies 
GL47 -  6           Contains Non-Binding Recommendations 
 
have not yet been standardized (e.g., OECD), therefore some strengths and weaknesses of each 
of these systems are discussed below: 
-  Primary (fresh or cryopreserved) hepatocytes:   Primary hepatocytes are liver cells that are 
useful  in  evaluating  phase  I  and  phase  II  metabolism  and  moreover  have  the  added 
advantage of taking membrane transport effects into account.  These hepatocytes can be 
prepared in suspension, monolayer culture or sandwich cultures.  The sandwich cultures 
have the advantage of maintenance of enzyme activities for a longer duration of time.  If 
the food-producing animal residue metabolites are demonstrated in a primary hepatocytes 
system, then comparative metabolism has generally been demonstrated. Use of a primary 
hepatocytes-based system can be complimentary to one or more of the other in vitro 
systems to demonstrate the metabolism for a laboratory animal species. 
-  Liver microsomes:  Liver microsomes include most of activities of cytochrome P450 
(CYP)  and  flavin-containing  monooxygenase  (FMO)  systems  for  evaluating  phase  I 
metabolism, along with uridine diphosphate-glucuronosyl-transferase (UDPGT) for phase 
II glucuronidation.  If the food-producing animal residue metabolites are demonstrated in a 
liver microsome system, then comparative metabolism has generally been demonstrated.  
Use of a liver microsome-based system can be complimentary to one or more of the other 
in vitro systems to demonstrate the metabolism for a laboratory animal species. 
-  S9 sub-cellular fraction: The S9 sub-cellular fraction contains the same phase I and phase 
II  enzymes  present  in  liver  microsomes  as  well  as  additional  systems  such  as 
sulfotranferases and N-acetyltransferases.   The S9 sub-cellular fraction is suitable for 
evaluating  phase  I  and  II  metabolism  or  phase  I  metabolism  followed  by  phase  II 
conjugation.  If the food-producing animal residue metabolites are demonstrated in a S9 
sub-cellular  fraction  system,  then  comparative  metabolism  has  generally  been 
demonstrated. Use of a S9 sub-cellular fraction-based system can be complimentary to one 
or more of the other in vitro systems to demonstrate the metabolism for a laboratory animal 
species. 
-  Cytosol:    This  represents  the  supernatant  fraction  remaining  following  microsomal 
centrifugation.    It  contains  some  of  the  phase  II  conjugation  systems  but  otherwise 
represents  a  relatively  incomplete  matrix  for  metabolic  work.    In  general,  the  use  of 
cytosolic systems alone is unlikely to provide a complete comparative metabolism profile, 
but if the food-producing animal residue metabolites are demonstrated in a cytosol system, 
then comparative metabolism has generally been demonstrated. Use of a cytosol-based 
system can be complimentary to one or more of the other in vitro systems to demonstrate 
the metabolism for a laboratory animal species.  
-  Liver slices:  the use of whole liver slices for metabolism research is possible, however, the 
liver cell viability and corresponding enzyme activities decrease rather rapidly compared 
with the other alternatives.  The conduct of comparative metabolism studies using liver 
slice methodology should not be used unless cell viability and enzyme activity can be 
demonstrated.    However,  if  the  food-producing  animal  residue  metabolites  are 
demonstrated in a liver-slice system, then comparative metabolism has generally been 
demonstrated. Use of a liver-slice-based system can be complimentary to one or more of 
the other in vitro systems to demonstrate the metabolism for a laboratory animal species. 
GL47 -  7           Contains Non-Binding Recommendations 
 
-  Whole cell lines:  Use of whole cell lines is not currently recommended because the 
enzymatic  activity  is  generally  low.    However,  if  the  food-producing  animal  residue 
metabolites are demonstrated in a whole cell line system, then comparative metabolism has 
generally been demonstrated. Use of a whole cell line-based system can be complimentary 
to one or more of the other in vitro systems to demonstrate the metabolism for a laboratory 
animal species. 
   
It  is  generally  possible  that  only  one  of  these  specific  in  vitro  options  could  be  used  for 
demonstration  of  comparative  metabolism.    However,  if  the  target-species  metabolic  profile 
includes evidence of both phase I and phase II biotransformation,  the Sponsor should consider 
investigating multiple options (e.g. microsomes and S9) to reproduce the complete metabolic 
profile. 
Although many variations in test conditions have been reported in the literature, the following 
represents some general guidance for conduct of in vitro comparative metabolism studies: 
  Test molecules are usually incubated in the in vitro system at 37 oC. 
  The concentrations of target molecules are typically lower than 100M. 
  The incubation time is dependent upon the rate of metabolism of the target molecules 
and should be adjusted accordingly. 
  Cofactors of phase I and II metabolism are scientifically necessary for incubation of liver 
microsomes  and  S9,  such  as  NADPH  (NADPH  regeneration  system)  for  phase  I 
metabolism, UDPGA for glucuronidation, PAPS for sulfation. 
 
When more standardized in vitro system metabolism study protocols become available, the 
general guidance above can be replaced according to the standardized protocols.   
 
2.3.3.  In Vivo Test Systems 
2.3.3.1.  Animals 
The laboratory animal species used in the comparative metabolism study should preferably be 
the  same  species  (and  for  rodents  the  same  strain)  as  was  used  in  the  pivotal  study  for 
determining the toxicological acceptable daily intake (ADI) of the veterinary drug. In case 
another species is used, the choice of species should be justified in terms of relevance.  The 
source of the animals, their weights, health status, ages and gender should be provided.   
2.3.3.2.  Animal Handling 
Animals should be allowed adequate time to acclimatize.  Normal laboratory animal caretaking 
practices should be applied. (Note: metabolism cage housing can be used).   
Animals  should  be  healthy  and,  preferably,  should  not  have  been  previously  medicated. 
However, it is recognized that animals might have received biological vaccinations or prior 
treatment, for example with anthelmintics.  An appropriate wash-out time should be observed for 
the animals prior to enrollment in the actual trial.  Animals should have a known history of 
medication.    
GL47 -  8           Contains Non-Binding Recommendations 
 
Animal caretaking practices and disposal of animals and tissues from animals should be in 
compliance with all applicable national and regional laws and regulations.  
2.3.3.3.  Number of Animals 
Enough animals should be treated with the drug in the comparative metabolism study to provide 
enough composited tissue or excreta for analysis.  The samples of like material from different 
animals can be composited for a single analysis.  There is no minimum number of animals for a 
comparative metabolism study; however, four animals of each gender are often used (but less can 
be used) to assure there is enough sample material.  Demonstration of comparative metabolism is 
not generally conducted in each gender; therefore, the samples of like material can be pooled 
(without regard to gender) to increase the likelihood of demonstrating the metabolites of interest 
when gender differences in metabolic ratios might exist.   
2.3.3.4.  Drug Formulation  
The drug formulation, method of dose preparation, and stability of the drug in the formulation 
during the treatment period should be described.  It is not critical that the formulation used in the 
comparative metabolism studies is the same as the commercial product.     
2.3.3.5.  Route of Administration 
The drug should be administered orally.  Gavage or bolus dosing can be used to ensure that 
animals receive the complete dose and to minimize environmental concerns.   
2.3.3.6.  Dosing  
The dose should be high enough to result in concentrations of metabolites in excreta or tissues 
for comparison.  The dose should be administered daily for enough time that the drug undergoes  
all relevant metabolic events, including those associated with enzyme induction.  Normally, 
administration for five days is used unless there are data to show a longer time of administration 
can better demonstrate the formation of the metabolites of interest.  Doses near the minimum 
toxic dose can be used to generate high concentrations of the metabolites of interest in tissues 
and urine but lower doses can be used.  
2.3.3.7.  Animal Euthanasia 
Animals should be humanely euthanatized.  Chemical euthanasia can be used unless it will 
interfere with analysis of the metabolites of interest.  
Animals should be euthanized for metabolite analysis at a single time point, usually 2-4 hours 
after the last dose of the test substance.  Multiple days of dosing provides the presence of 
metabolites resulting from sequential metabolism of the parent drug over time, and therefore 
additional euthanasia time points are not called for. 
GL47 -  9           Contains Non-Binding Recommendations 
 
2.3.3.8.  Sample Collection  
Before euthanasia, urine, feces, and blood can be collected for analysis.  The samples should be 
analyzed  immediately  or  stored  frozen  (unless  freezing  causes  a  stability  problem  for  the 
metabolites  of  interest)  until  analysis.    Freezing  of  the  samples  is  to  reduce  microbial 
metabolism from altering the metabolic profile.  If the samples are stored after collection, the 
sponsor should ensure that the radiolabeled compound remains intact throughout the storage 
period.   
Following  euthanasia,  samples  of  tissues  can  be  collected.    The  tissue  samples  should  be 
analyzed  immediately  or  stored  frozen  (unless  freezing  causes  a  stability  problem  for  the 
metabolites  of  interest)  until  analysis.    Freezing  of  the  samples  is  to  reduce  microbial 
metabolism from altering the metabolic profile.  If the samples are stored after collection, the 
sponsor should ensure that the radiolabeled compound remains intact throughout the storage 
period.   
Comparative metabolism can be demonstrated with one or more excreta or tissues.  Samples that 
are typically taken for qualitative metabolite analysis can include blood/blood fractions, excreta, 
liver, bile, kidney, fat or other tissues.  Enough tissue of each type should be taken from each 
animal for analysis or for pooling from more than one animal for analysis.   
2.3.3.9.  Determination of Total Radioactivity 
Determination of total radioactivity in samples and accounting for the mass balance of the 
radioactivity are not normally conducted for the in vivo comparative metabolism studies.  When 
total radioactivity is to be determined, the procedures presented in VICH GL 46 should be 
followed.   
 
2.3.4.  Separation and Comparison of Metabolites  
Commonly  available  analytical  technology,  including,  for  example,  high  performance  liquid 
chromatography,  thin  layer  chromatography,  gas  chromatography,  and  mass  spectrometry,  are 
typically used for the separation of the total residue into its components and comparison of the drug-
derived residues. 
2.3.4.1.  Analytical Methods 
Similar procedures for chromatography and chemical characterization as those employed in VICH GL 
46  should  be  used  in  the  in  vivo  comparative  metabolism  studies  in  laboratory  animals.  Those 
methods can also be useful for in vitro investigations, although the sample preparation would be 
different. A description of the analytical methods should be provided as described in VICH GL 46.  
The repeatability of retention times for the analytical method should be demonstrated.   
2.3.4.2.  Extent of Characterization/Major Metabolites 
Characterization  and  structural  identification  of  the  metabolites  and  demonstration  of  the  tissue 
extraction efficiency during the comparative metabolism study are not normally conducted when the 
GL47 -  10           Contains Non-Binding Recommendations 
 
comparison of the chromatographic retention time(s) demonstrate the presence of the metabolites of 
interest in the laboratory animal.   
2.3.4.3.  Nonextractable Metabolites 
Characterization  of  nonextractable  metabolites  in  comparative  metabolism  studies  in  laboratory 
animals  is  normally  not  performed.    Characterization  of  the  covalently  bound  metabolites  of  a 
veterinary drug in laboratory animals should be performed only when the nonextractable residue 
contains a metabolite of interest that is not present in enough quantity for characterization in the 
easily extractable portion.  In this case, the procedures identified in VICH GL 46 should be followed.   
 
GL47 -  11           Contains Non-Binding Recommendations 
 
3.  GLOSSARY 
 
The following definitions apply for the purposes of this document: 
Acceptable daily intake (ADI) of a chemical is the daily intake which, during an entire 
lifetime, appears to be without appreciable risk to the health of the consumer. The ADI most 
often will be set on the basis of the drugs toxicological, microbiological or pharmacological 
properties. It is usually expressed in micrograms or milligrams of the chemical per kilogram of 
body weight. 
Major Metabolites are those comprising  100 g/kg or  10% of the total residue in a 
sample collected from the target animal species in the metabolism study (VICH GL 46).  
Metabolism for purposes of this guidance,  is the  sum total of all physical and chemical 
processes that occur within an organism in response to a veterinary drug.  It includes uptake 
and  distribution  of  the  drug  within  the  body,  changes  to  the  drug  (biodegradation),  and 
elimination of drugs and their metabolites.  
Metabolites of interest refers to the veterinary drug (parent) and its metabolites that were 
demonstrated in the edible tissues of the food-producing animal and have relevance to the 
toxicological ADI established for the veterinary drug.  
Nonextractable residues are residues that are not readily extractable from tissues using mild 
aqueous or organic extraction conditions.  These residues arise from (a) incorporation of 
residues of the drug into endogenous compounds, (b) chemical reaction of the parent drug or 
its metabolites with macromolecules or (c) physical encapsulation or integration of radioactive 
residues into tissue matrices. 
Residue means the veterinary drug (parent) and/or its metabolites. 
 
GL47 -  12