CVM GFI #206 (VICH GL47) Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs in Food-Producing Animals: Comparative Metabolism Studies in Laboratory Animals

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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Additional copies of this draft guidance document may be requested from the Communications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 R ESIDUE K INETICS
February 2011
For Implementation at Step 7 - Final
S TUDIES TO EVALUATE THE 	M ETABOLISM
AND 	R ESIDUE 	K INETICS OF 	V ETERINARY
D RUGS IN 	F OOD - PRODUCING 	A NIMALS :
L ABORATORY 	A NIMAL 	C OMPARATIVE
M ETABOLISM 	S 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.
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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
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STUDIES TO EVALUATE THE M ETABOLISM AND RESIDUE K INETICS OF VETERINARY DRUGS IN
FOOD-PRODUCING ANIMALS : COMPARATIVE M ETABOLISM STUDIES IN LABORATORY ANIMALS
(VICH GL47)
This guidance represents the Food and Drug Administration’s (FDA’s) 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.
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should
be viewed only as recommendations, unless specific regulatory or statutory requirements are
cited. 	The use of the word should in Agency’s guidances means that something is suggested or
recommended, but not required.
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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
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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 sex 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
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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.
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- 	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 o C.
• 	The concentrations of target molecules are typically lower than 100μM.
• 	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 sex 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.
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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 sex 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 sex; therefore, the samples of like material can be pooled
(without regard to sex) to increase the likelihood of demonstrating the metabolites of interest
when sex 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.
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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
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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.
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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 drug’s 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 μg/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.