#257 
Studies to Evaluate the Metabolism and Residue 
Kinetics of Veterinary Drugs in Food-Producing Species: 
Marker Residue Depletion Studies to Establish 
Product Withdrawal Periods in Aquatic Species 
Guidance for Industry 
VICH GL57 
Submit comments on this guidance at any time.  Submit electronic comments to 
https://www.regulations.gov/.  Submit written comments to the Dockets Management Staff 
(HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 
20852.  All comments should be identified with the docket number FDA-2016-D-4461. 
For further information regarding this document, contact AskCVM@fda.hhs.gov. 
Additional copies of this guidance document may be requested from the Policy and 
Regulations Staff (HFV-6), Center for Veterinary Medicine, Food and Drug Administration, 
7500 Standish Place, Rockville, MD 20855, and may be viewed on the Internet at either 
https://www.fda.gov/AnimalVeterinary/default.htm or https://www.regulations.gov/.
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
August 2019 Contains Nonbinding Recommendations 
 
VICH GL 57 (MRK) RESIDUES IN FISH 
For implementation at Step 7 
 
 
 
Studies to Evaluate the Metabolism 
and Residue Kinetics of Veterinary 
Drugs in Food-Producing Species: 
Marker Residue Depletion Studies to 
Establish Product Withdrawal Periods 
in Aquatic Species 
 
 
 
 
 
Adopted at Step 7 of the VICH Process by the VICH Steering Committee 
in March 2019 
 for implementation by February 2020 
 
 
 
 
 
 
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 is recommended for adoption to the regulatory bodies of the European 
Union, Japan, and USA. 
 
   
2  Contains Nonbinding Recommendations 
Table of Contents 
 
 
1.  INTRODUCTION ..........................................................................................................4 
2.  GUIDANCE ....................................................................................................................5 
2.1.  Purpose ...................................................................................................................5 
2.2.  Scope .......................................................................................................................5 
2.3.  Test Article .............................................................................................................5 
2.4.  Study Design ...........................................................................................................5 
2.4.1.  Animals ...............................................................................................................5 
2.4.2.  Critical Study Design Parameters ....................................................................6 
2.4.3.  Animal Husbandry.............................................................................................6 
2.4.4.  Single Species Claim ..........................................................................................7 
2.4.5.  Single Order Claim ............................................................................................8 
2.5.  Number of Animals for the Study ........................................................................9 
2.6.  Dosing and Route of Administration .................................................................10 
2.6.1.  General guidance .............................................................................................10 
2.6.2.  Immersion Treatment ......................................................................................10 
2.6.3.  In-feed Treatment ............................................................................................10 
2.6.4.  Injectable Treatment .......................................................................................10 
2.7.  Animal Euthanasia ..............................................................................................10 
2.8.  Sampling ...............................................................................................................10 
2.8.1.  General Considerations ...................................................................................10 
2.8.2.  Tissue Sampling ...............................................................................................11 
2.8.3.  Sampling of Eggs for Human Consumption from Treated Aquatic 
Species ...............................................................................................................12 
2.9.  Recommendations for Products Proposed for 0-Day Withdrawal Periods 
(Single Time-Point Studies) .................................................................................12 
2.10.  Analytical Method for Assay of Marker Residue .............................................12 
3.  GLOSSARY ..................................................................................................................13 
   
3  Contains Nonbinding Recommendations 
Studies to Evaluate the Metabolism and Residue Kinetics of 
Veterinary Drugs in Food-Producing Species:  Marker Residue Depletion 
Studies to Establish Product Withdrawal Periods in Aquatic Species 
 
Guidance for Industry 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic.  It does not establish any rights for any person and is not binding on 
FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations.  To discuss an alternative approach, contact the FDA 
staff responsible for this guidance as listed on the title page. 
 
1.  INTRODUCTION 
 
This guidance is one of a series developed to facilitate the mutual acceptance of residue 
chemistry data for veterinary drugs used in food-producing animals by national/regional 
regulators.  This guidance was prepared after consideration of the current national/regional 
requirements and recommendations for evaluating veterinary drug residues in the 
International Cooperation on Harmonisation of Technical Requirements for Registration of 
Veterinary Medicinal Products (VICH) regions. 
 
The objective of this guidance is to provide study design recommendations that will facilitate 
the universal acceptance of the generated residue depletion data to fulfill the national/regional 
requirements. 
 
This document is an extension to the parent residue guidance: CVM Guidance for Industry 
(GFI) #207/VICH GL48, Studies to Evaluate the Metabolism and Residue Kinetics of 
Veterinary Drugs in Food-producing Animals: Marker Residue Depletion Studies to Establish 
Product Withdrawal Periods.  This guidance, VICH GL57, provides recommendations on 
what should be included in a marker residue depletion study design for aquatic food-
producing species. 
 
Metabolism studies based on CVM GFI #205/VICH GL46, 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, can be 
used in aquatic food-producing species to identify a marker residue. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys current thinking on a topic and should be viewed 
only as recommendations, unless specific regulatory or statutory requirements are cited.  The 
use of the word should in Agency guidances means that something is suggested or 
recommended, but not required. 
   
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2.  GUIDANCE 
 
2.1.  Purpose 
 
Marker residue depletion studies for registration or approval, as applicable, of a new 
veterinary medicinal product in the intended species are recommended to: 
 
  demonstrate the depletion of the marker residue upon cessation of drug treatment to 
the regulatory safe level (e.g., maximum residue limit or tolerance). 
  generate data suitable for elaboration of appropriate withdrawal periods/withholding 
times to address consumer safety concerns. 
 
2.2.  Scope 
 
The intent is that a residue depletion study conducted according to the recommendations 
described in this guidance would satisfy the data requirements or recommendations for 
establishment of appropriate withdrawal periods in all VICH regions.  Conducting a depletion 
study under worst-case conditions provides data for calculating the withdrawal period.  It 
may be desirable to conduct an additional study or provide additional information to further 
define the withdrawal period under alternate management conditions or to adjust the 
withdrawal period based on the concept of degree days. 
 
The guidance encompasses food-producing aquatic species.  The principles of this guidance 
are also applicable to eggs from aquatic species for human consumption.  Studies should be 
conducted in conformity with the applicable principles of Good Laboratory Practice (GLP).  
The in vivo phase should be conducted under GLP conditions; if this is not practical, a 
justification for all deviations from GLP and the impact on the study results should be 
provided. 
 
2.3.  Test Article 
 
The test article used for the study should be representative of the commercial formulation.  
Use of final Good Manufacturing Practice (GMP) manufactured material (pilot scale or 
commercial scale) is the preferred source of test article; however, laboratory scale 
preparations characterized with respect to GLP could also be appropriate. 
 
2.4.  Study Design 
 
2.4.1.  Animals 
 
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, antimicrobials).  In the latter case, an appropriate wash-out time 
should be observed for the animals prior to enrollment in the actual study. 
 
Study animals should be representative of the commercial species and representative of the 
target animal population that will be treated.  The source of the animals, health status, 
age/development stage, and body weights should be reported.  The bodyweight ranges should 
be consistent with the intended product label for the proposed use.  If the product is intended 
to be used at various stages of development, then the study should be conducted in animals 
5  Contains Nonbinding Recommendations 
representing the highest development stage (the stage that has a metabolic state that is 
representative of market size). 
 
2.4.2.  Critical Study Design Parameters 
 
Critical residue depletion design parameters to address include water temperature, housing, 
and salinity.  The body temperature and hence absorption, metabolism, and excretion of 
aquatic species is driven by the surrounding water temperature.  Generally, the lower the 
water temperature the slower the depletion, but higher temperatures may result in higher 
absorption of drug.  Table 1 shows examples of critical design parameters.  The sponsor 
should investigate the effects of the critical design parameters and provide a study design that 
would result in the worst-case scenario for residues. Selection of the final design parameters 
should be justified and should be consistent with the proposed use of the product.  If more 
than one option from Table 1 is consistent with the proposed use, then the conditions that 
result in the worst-case scenario should be chosen. 
 
Table 1. Critical Study Design Parameters 
Critical Parameter  Options  Choice 
Water Temperature  High or Low within the test  Choose the temperature that 
animals recommended water  results in the worst-case 
temperature range  scenario for residues 
Salinity  Salt or Fresh Water  If applicable choose the one 
that results in the worst-case 
scenario for residues 
Housing  Recirculation or flow-through  If applicable choose the one 
or net pens  that results in the worst-case 
scenario for residues 
 
2.4.3.  Animal Husbandry 
 
Adequate environmental conditions should be ensured to be consistent with animal welfare, 
in accordance with applicable national and regional regulations.  Additionally, endemic 
pathogens or parasites should be controlled or eliminated so as to maintain the health of the 
test animals.  Animals should be allowed adequate time to acclimatize to surroundings, 
procedures, and stocking density.  Normal husbandry practices should be applied to the 
extent possible. 
 
2.4.3.1.  Housing 
 
The study should be conducted under commercial growing conditions or the housing should 
mimic that used in commercial growing conditions. 
 
Examples of possible housing are flow-through cages (free-swimming), racks (attached, e.g., 
oysters), net pens, recirculating water systems, and ponds.  The holding conditions should be 
suitable so as to prevent escape of test animals or entry of predators.  If more than one 
housing condition is used commercially, then the housing condition that potentially results in 
maximum tissue residues should be selected. 
 
 
 
6  Contains Nonbinding Recommendations 
2.4.3.2.  Feeding 
 
Animals should be given feed appropriate, in both quality and quantity, to their development 
stage and to ensure adequate nutrition and growth, as per commercial conditions.  An 
adequate number of animals (stocking density) should be present in the enclosure to ensure 
proper feeding behavior.  The feed supplied to the animals should be free from other drugs 
and/or contaminants. 
 
2.4.3.3.  Water Temperature 
 
Water temperature is critical to the residue depletion rate in animals whose body temperature 
is dictated by their environment.  However, it is recognized that deviations from the 
recommended water temperature ranges may occur during study conduct, because studies 
conducted under commercial conditions or over an extended duration are subject to natural 
fluctuations in water temperature. 
 
Water temperature should be recorded at least daily until the last animals are euthanized. 
 
2.4.3.4.  Water Quality Parameters 
 
Animals should be raised in water that has quality and quantity appropriate for their 
development stage as per commercial conditions. 
 
Water quality parameters that may be critical to study outcome should be monitored at a 
frequency appropriate to the study.  Contaminants known to be capable of interfering with the 
study should be monitored.  Water should be exchanged at a rate suitable to maintain health 
and welfare. 
 
2.4.3.5.  Animal Anesthesia 
 
Chemical anesthesia or sedation can be used for finfish in order to handle them for group 
allocation, treatment, and euthanasia.  Chemicals used for these processes should cause no 
interference in the assay for the marker residue. 
 
2.4.4.  Single Species Claim 
 
Selection of the final design parameters for the worst-case scenario should be justified (see 
section 2.4.2 Critical Study Design Parameters). 
 
2.4.4.1.  In-feed Treatment 
 
A claim for a single species can be supported by conducting a study in that species. In order 
to be accepted in VICH regions, studies should be conducted within the lowest range of 
temperatures in which in-feed treatment is administered under commercial settings. 
 
2.4.4.2.  Injectable Treatment 
 
A claim for a single species can be supported by conducting a study in that species. In order 
to be accepted in VICH regions, studies should be conducted within the lowest range of 
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temperatures in which the injection is administered under commercial settings unless a higher 
temperature is justified (see section 2.4.2. Critical Study Design Parameters). 
 
2.4.4.3.  Immersion 
 
A claim for a single species can be supported by conducting a study in that species in 
consideration of worst-case scenario parameters (see section 2.4.2. Critical Study Design 
Parameters). Immersion treatments may result in differential drug absorption at different 
water temperatures. Selection of the appropriate water temperature should be investigated and 
subsequently justified. Water quality parameters should be selected to ensure lack of 
interference with drug treatment. 
 
2.4.5.  Single Order Claim 
 
A claim for an order can be supported by conducting a study in a representative species. The 
resulting withdrawal period can then be applied to other species of the same order. However, 
residue data in a second species to confirm the withdrawal period are recommended. The 
representative species listed in Table 2 are the species that can be reared at recommended 
temperatures so that the data can be accepted by all regions or countries. However, the 
second species need not come from Table 2. 
 
Treatment parameters should be the same as described for a single species claim (see section 
2.4.4. Single Species Claim). 
 
The choice of representative species generally depends on critical residue depletion design 
parameters. Critical parameters include water temperature, salinity, and housing conditions. 
Selection of the final design parameters for the worst-case scenario should be justified. 
 
Table 2 shows recommended target water temperature ranges for the residue depletion studies 
using representative species for different orders of finfish and shrimp.  Representative species 
are chosen based on: 1) the species being either widely cultured in a certain region (or a 
country) or closely related to such a species, 2) residue depletion studies being able to be 
carried out at recommended water temperature range at which the species are cultured, and 3) 
the assumption that the representative species have similar metabolism to other species in the 
same order.  For immersion treatments the effect of temperature on residues should be 
considered (see section 2.4.4.3. Immersion). 
 
   
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Table 2. Representative Species and Recommended Water Temperature Range for 
Residue Depletion Study 
Recommended Water 
Order  Representative Species  Temperature Range (C) 
Salmoniformes1  Atlantic salmon (Salmo salar)  5-10
Coho salmon (Oncorhynchus kisutch) 
Rainbow trout (Oncorhynchus mykiss) 
Cypriniformes  Carp (Cyprinus carpio)  15-20
Common bream (Abramis brama) 
Perciformes1  European seabass (Dicentrarchus labrax)  15-20
Hybrid striped bass (Morone saxaltilis X 
Morone chrysops) 
Red sea bream (Pagrus major) 
Yellowtail (Seriola quinqueradiata) 
Walleye (Sander vitreus) 
Scorpaeniformes  Mebaru (Sebastes inermis/Sebastes  10-15
cheni/Sebastes ventricosus) 
Silurformes  Channel catfish (Ictalurus punctatus)  16-21
Mudfish (Clarias anguillaris) 
Osmeriformes  Ayu (Plecoglossus altivelis)  13-18
Anguilliformes  Eel (Anguilla japonica)  20-25
European eel (Anguilla anguilla) 
Pleuronectiformes  Bastard halibut (Paralichthus olivarceus)  15-20
Summer flounder (Paralichthys dentatus) 
Tetraodontiformes  Japanese pufferfish (Takifugu rubripes)  13-18
Acipenseriformes  Siberian sturgeon (Acipenser baerii)  14-19
Gadiformes  Atlantic cod (Gadus mohrua)  5-10
Shrimp or prawns in  Japanese tiger prawn (Penaeus japonicus)  18-23
the order of Decapoda  Whiteleg shrimp (Penaeus vannamei) 
1 Order contains fresh and salt water representative species that have different salinity requirements 
based on their life stages. 
2.5.  Number of Animals for the Study 
The number of animals used should be large enough to allow a meaningful assessment of the 
data.  Residue data from a minimum of 10 animals per time point are recommended.  For 
small finfish, shrimp, or mollusks, a composite sample of multiple animals may be used. In 
cases where a composite is critical, a sufficient number of animals should be collected in 
order to facilitate assessment of the marker residue. It is recommended that composite residue 
data from a minimum of 10 pools per time point be assessed. It is recommended that animals 
should be euthanized at a minimum of four appropriately distributed time intervals. Higher 
numbers of animals should be considered if the biological variability is anticipated to be 
substantial as the increased numbers might result in a better-defined withdrawal period. 
Control (non-treated) animals are not necessarily called for as part of the actual marker 
residue depletion study; however, sufficient amounts of control matrices should be available 
to provide material for related analytical method testing. 
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2.6.  Dosing and Route of Administration 
 
2.6.1.  General guidance 
 
Animal treatment should be consistent with the intended product label. 
 
At least the highest intended treatment dose should be administered for the maximum 
intended duration for the proposed product.  If an extended drug administration period is 
intended, duration of treatment sufficient to reach steady state in target tissue(s) can be used 
instead of the full length of the treatment.  The time to steady-state data are often obtained as 
part of the total residue study, see CVM GFI #205/VICH GL46. 
 
2.6.2.  Immersion Treatment 
 
Animals can be treated with the test article dissolved or suspended in water. 
 
2.6.3.  In-feed Treatment 
 
Animals may be treated by incorporation of the test article into the feed to deliver on a group 
basis standardized mg/kg body weight dose. Generally individual medication of aquatic 
species is not possible as they will not eat if confined singly, so dosing should be conducted 
on a group basis. Ideally animals should consume the medicated feed within a short period of 
feeding so that the test article does not leach into the water.  During the acclimation period 
tests should be conducted to determine the group feeding rate and body weights to ensure the 
target dose is administered. If feed remains and if it is possible, the uneaten feed should be 
collected and used to adjust the administered dose calculation. 
 
2.6.4.  Injectable Treatment 
 
Animals should be treated with an injectable product, by the intended route (such as 
intramuscular, intravenous, intraperitoneal, or intracardial) in accordance with the proposed 
label. The dose injected should be the maximum amount as per the proposed label. Animals 
may require anesthesia in order to be handled for the treatment. Individual animal weights 
should be reported. 
 
2.7.  Animal Euthanasia 
 
Animals should be euthanized using commercially applicable procedures, observing 
appropriate exsanguination times.  Chemical euthanasia can be used unless it will interfere 
with the analysis of the marker residue. 
 
2.8.  Sampling 
 
2.8.1.  General Considerations 
 
Following euthanasia, edible tissue samples in sufficient amounts should be collected, 
trimmed of extraneous material, weighed, and divided into aliquots (if appropriate).  If the 
analysis cannot be completed immediately, the samples should be stored under frozen 
conditions pending analysis.  If samples are stored after collection, the sponsor generally 
bears the responsibility for demonstrating residue stability through to the time of assay. 
10  Contains Nonbinding Recommendations 
 
2.8.2.  Tissue Sampling 
 
The tissue sampling protocol encompasses two sections; (1) those tissues that are 
recommended in support of registration or approval, as applicable, to all species in all VICH 
regions and (2) additional tissues that may be collected to address specific national/regional 
consumption habits and/or legal concerns. 
 
Table 3 indicates the recommended samples for collection for all VICH regions.  Table 4 
indicates the additional tissues that should be sampled to address specific national/regional 
consumption habits and/or legal concerns. 
 
In principle, for finfish, muscle including skin in natural proportions should be sampled for a 
single order claim.  For a single species claim for finfish, skin can be eliminated from 
samples if the skin of the particular species is not consumed in any VICH region. 
 
Table 3. Sample Collection from Animals in the Marker Residue Depletion Study 
(All VICH Regions) 
Aquaculture Species Edible Tissue Samples
Muscle including skin in natural proportions, which is the 
entire fillet with the overlying skin from one or both sides 
Finfish with edible skin 
of the fish (scales can be included or excluded based on 
consumption and practicality of removal). 
Finfish with inedible skin 
Muscle, which is the entire fillet from one or both sides of 
(Example: Channel catfish, 
the fish. 
threadsail filefish) 
Mollusks  Soft tissue excluding shell. 
Shrimp or prawns with hard 
Soft tissue including mid-intestinal gland, excluding shell. 
(inedible) shell 
Shrimp or prawns (during  The entire animal including the shell is considered as the 
molting) with soft (edible)  edible tissue.  The edible tissue for shrimp includes the 
shell mid-intestinal gland and shell.
 
The entire sample as defined above should be collected, homogenized, and then subsamples 
(if appropriate) taken from the homogenate. 
 
Table 4. Additional Tissues that can be Collected to Address Specific National/Regional 
Consumption and/or Legal Concerns in the Marker Residue Depletion Study 
Order  Edible Tissue Type 
Any orders of finfish  Either one additional tissue that has been shown to have the 
highest concentration or slowest depletion of residue among the 
tissues of visceral organs by previous residue studies, or the offal 
mixture of available liver, kidney, spleen, stomach, intestine, heart, 
ovary and testis. 
 
Samples in Table 3 and Table 4 should be collected separately from individual animals, but if 
the amount of samples collected from one animal is not sufficient for the assay of marker 
residue, composite samples from multiple animals may be appropriate. For composite 
samples at least 10 composite samples should be prepared at each sampling period. 
 
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2.8.3.  Sampling of Eggs for Human Consumption from Treated Aquatic Species 
 
Eggs should be collected from a minimum of 10 sexually mature individuals of the aquatic 
species.  Ten composite samples with an equal amount of eggs from each individual (collect 
sufficient sample for analysis) should be prepared for residue analysis. 
 
2.9.  Recommendations for Products Proposed for 0-Day Withdrawal Periods 
(Single Time-Point Studies) 
 
For products administered as one treatment or as several treatments (for example daily for 3-5 
days), or for continuous use products in which residues have reached steady state, a single 
time point study may qualify for 0-day withdrawal, provided that the absorption and 
depletion characteristics of the drug have been described, for example, as indicated in CVM 
GFI #205/VICH GL46, 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 (MRK). If such data are available, then a single time 
point study conducted with the specified minimum number of animals is recommended to 
demonstrate 0-day withdrawal. 
 
Number of animals recommended:  a minimum of 15 individuals or 15 composites. 
 
The sampling time chosen for this study should be consistent with the peak concentrations. 
 
Higher numbers from those recommended in Section 2.5 Number of Animals for the Study 
are generally appropriate for single time point determinations. 
 
2.10.  Analytical Method for Assay of Marker Residue 
 
The sponsor should submit a validated analytical method for the determination of the marker 
residue in samples generated from the residue depletion studies. The method(s) should be 
capable of reliably determining concentrations of marker residue which encompass the 
appropriate reference point (i.e., MRL (Maximum Residue Limit) / Tolerance) for the 
respective tissues or products. 
 
The parameters to be included in the method validation are fully discussed in CVM GFI 
#208/VICH GL49, Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary 
Drugs in Food Producing Animals: Validation of Analytical Methods Used in Residue 
Depletion Studies. 
   
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3.  GLOSSARY 
 
The following definitions are applied for purposes of this document. 
 
Aquatic species include finfish, crustaceans, and mollusks. 
 
Degree days means an expression of the withdrawal period where it is assumed that time 
multiplied by water temperature is constant. 
 
Marker residue is that residue whose concentration is in a known relationship to the 
concentration of total residue in an edible tissue. 
 
Maximum residue limit (MRL) is the maximum concentration of a veterinary drug residue 
that is legally permitted or recognized as acceptable in or on a food as set by a national or 
regional regulatory authority.  The term tolerance, used in some countries, can be, in many 
instances, synonymous with MRL. 
 
Residue means the veterinary drug (parent) and/or its metabolites. 
 
Shrimps and prawns belong to the family of Penaeidae.  This includes most of the shrimps 
or prawns cultured worldwide but exclude crabs, machrobrachium, lobsters, and crayfishes.  
Some regions use the term shrimp and some use the term prawns and these terms can be used 
interchangeably. 
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