#207 
 
Guidance for Industry 
 
Studies to Evaluate the Metabolism and  
Residue Kinetics of Veterinary Drugs In 
 Food-Producing Animals:  Marker Residue Depletion 
Studies to Establish Product Withdrawal Periods 
 
VICH GL48(R) 
 
Submit comments on this guidance at any time.  Submit  electronic comments on the guidance to 
http://www.regulations.gov.   Submit written comments to the Division  of Dockets Management 
(HFA-305), Food and Drug Administration,  5630 Fishers Lane, Room 1061, Rockville,  MD  
20852.  All written comments should be identified  with the Docket No. FDA-2010-D-0166.  
 
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, 
240-402-0788,  email:   julia.oriani@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,  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 
March 2015 
    Contains Non-Binding Recommendations 
VICH GL 48 (R) (MRK)  METABOLISM AND RESIDUE KINETCIS 
February 2015 
Revision at Step 9 
For implementation at Step 7  Final - Corrected 
 
 
 
 
 
 
 
STUDIES TO EVALUATE THE METABOLISM 
AND RESIDUE KINETICS OF VETERINARY 
DRUGS IN FOOD-PRODUCING ANIMALS: 
MARKER RESIDUE DEPLETION STUDIES 
TO ESTABLISH PRODUCT WITHDRAWAL 
PERIODS 
     
 
 
 
 
 
 
Revision at step 9 
 
Adopted at Step 7 of the VICH Process by the VICH Steering Committee in January 2015 
for implementation by January 2016. 
 
 
 
 
This Guidance has been developed by the appropriate VICH Expert Working Group. At Step 7 of the 
 
Process the final draft is recommended for adoption to the regulatory bodies of the European Union, 
 
  Japan and the USA. 
 
 
   
2  Contains Non-Binding Recommendations 
Table of Contents 
 
1. INTRODUCTION ............................................................................................................. 4 
1.1. OBJECTIVE OF GUIDANCE ................................................................................................ 4 
1.2. BACKGROUND................................................................................................................ 4 
2. GUIDANCE ....................................................................................................................... 4 
2.1. PURPOSE........................................................................................................................ 4 
2.2 SCOPE ............................................................................................................................ 5 
2.3. MARKER RESIDUE DEPLETION STUDIES ........................................................................... 5 
2.3.1. Test Article ............................................................................................................ 5 
2.3.2. Animals and Animal Husbandry ............................................................................ 5 
2.3.2.1. Intramammary Studies ..................................................................................... 5 
2.3.2.2. Other Parameters.............................................................................................. 6 
2.3.3. Number of animals for the study ............................................................................ 6 
2.3.3.1. Cattle, pigs and sheep for tissue residue studies ................................................. 6 
2.3.3.2. Dairy animals for milk residue studies .............................................................. 6 
2.3.3.3. Poultry............................................................................................................. 6 
2.3.4. Dosing and Route of Administration ...................................................................... 7 
2.3.4.1. General guidance ............................................................................................. 7 
2.3.4.2. Considerations for products intended for intramammary administration ............. 7 
2.3.4.3. Considerations for products intended for multiple routes of administration......... 7 
2.3.4.4. Considerations for Use of Multiple Injection Sites per Animal ........................... 8 
2.3.5. Animal Euthanasia ................................................................................................ 8 
2.3.6. Sampling ............................................................................................................... 8 
2.3.6.1. General Considerations .................................................................................... 8 
2.3.6.2. Injection Sites .................................................................................................10 
2.3.6.3. Other Considerations.......................................................................................11 
2.3.6.4. Milk Sampling ................................................................................................11 
2.3.6.5. Egg Sampling .................................................................................................11 
2.3.7. Recommendations for Products Proposed for a 0-Day Tissue Withdrawal Period or 
a 0-Day (nil) Milk Discard Time.................................................................................... 11 
2.3.7.1 Zero-Day Tissue Withdrawal Time Study Design..............................................12 
2.3.7.2 Zero-Day (nil) Milk Discard Time Study Design...............................................12 
2.4. ANALYTICAL METHOD FOR ASSAY OF MARKER RESIDUE ................................................14 
3.  GLOSSARY .................................................................................................................... 15 
 
   
3  Contains Non-Binding Recommendations 
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 represents the Food and Drug Administrations (FDA or Agency) 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 using the contact 
information on the title page of this guidance. 
 
 
 
1. INTRODUCTION 
 
1.1. Objective of guidance 
As part of the approval process for veterinary medicinal products in food-producing  animals, 
national/regional  regulatory authorities require data from marker residue depletion  studies in 
order to establish appropriate withdrawal periods in edible tissues including  meat, milk and eggs.  
The objective of this guidance is to provide study design recommendations  which will facilitate 
the universal acceptance of the generated residue depletion data to fulfill  the national/regional 
requirements. 
 
1.2. Background 
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.  This guidance was prepared 
after consideration  of the current national/regional  requirements and recommendations  for 
evaluating veterinary drug residues in the VICH regions. 
 
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. 
 
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. 
 
4  Contains Non-Binding Recommendations 
2.2 Scope 
The intent is that one residue depletion  study (per species), conducted within any VICH region, 
would satisfy the data recommendations for establishment of appropriate withdrawal periods for 
a specific product in food-producing  animals. 
 
The guidance encompasses the most common species, namely cattle, pig, sheep and poultry; 
however, the principles  of this guidance can also be applied to related species not mentioned in 
this core group (e.g., cattle vs. all ruminants).  The guidance does not provide study design 
recommendations for fish or honey bees (as producers of honey). 
 
Studies should be conducted in conformity  with the applicable  principles  of Good Laboratory 
Practice (GLP). 
 
2.3. Marker Residue Depletion Studies 
 
2.3.1. Test Article 
The test article used for the study should be representative of the commercial formulation.   Use 
of final 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. 
 
2.3.2. Animals and Animal Husbandry 
Ordinarily,  one marker residue depletion study (for tissues) should be performed in swine, sheep 
and poultry.  For cattle, a single study in ruminating  beef cattle could be applied to dairy cattle 
(or vice versa).  However, because of differences in ruminant and pre-ruminant physiology, 
separate studies are recommended when the target species encompasses both adult and pre-
ruminating  animals.  A separate study should be performed to demonstrate the residue depletion 
profile in milk of dairy animals or in eggs produced by laying hens. 
 
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.   In the latter case, an appropriate wash-out time 
should be observed for the animals prior to enrollment in the actual trial.  Study animals should 
be representative of the commercial breeds and representative of the target animal population 
that will be treated. The source of the animals, their weights, health status, ages and sex should 
be reported. 
 
Animals should be allowed adequate time to acclimatize and normal husbandry practices should 
be applied to the extent possible.  The feed and water supplied  to the animals should be free from 
other drugs and/or contaminants and adequate environmental  conditions  should be ensured to be 
consistent with animal welfare, in accordance with applicable  national and regional regulations. 
 
2.3.2.1. Intramammary Studies 
For studies with intra-mammary products, all animals should have visibly  healthy udders free 
from effects from chronic mastitis.  For pre-parturition  studies, pregnant animals with a predicted 
parturition  date should be introduced into the study facility well in advance of study enrollment. 
5  Contains Non-Binding Recommendations 
2.3.2.2. Other Parameters 
The marker residue depletion  study should take into account all factors that might contribute to 
the variability  of residue levels in animal commodities  in the planning  and conduct of trials.  The 
intent here is that these other factors (e.g., animal breeds, physical maturity, etc) be considered 
within the pool of animals to be included in the marker residue depletion study without 
warranting an increase in the number of animals as recommended in 2.3.3.  For example, if a 
milk residue depletion  study recommends 20 animals, all other factors should be represented 
within the 20 initially  selected animals (not an additional  20 animals representing each other 
factor). 
 
2.3.3. Number of animals for the study 
The number of animals used should be large enough to allow a meaningful  assessment of the 
data.  From a statistical point of view, residue data from a minimum  of 16 animals with four 
animals being euthanized at four appropriately distributed  time intervals are recommended.  
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 for related 
analytical methods testing.  The following  section provides a general recommendation  for 
numbers of animals to be included  in the study design. 
 
2.3.3.1. Cattle, pigs and sheep for tissue residue studies 
At least 4 (evenly mixed as per sex) per each slaughter time are recommended.  The suggested 
bodyweight  ranges are ~40 to 80 kg for swine, ~40 to 60 kg for sheep and ~250 to 400 kg for 
beef cattle.  Consistent with Section 2.3.2, non-lactating dairy cows could also be used for these 
tissue residue studies. 
 
2.3.3.2. Dairy animals for milk residue studies 
For lactating animal studies, at least 20 animals, randomly  selected from a herd where all 
lactation stages are represented, are recommended.  High yielding  animals at an early lactation 
stage and low yielding  animals at a late lactating stage should be included in the group of 
animals but specific numbers of each are not called for. 
 
For pre-parturition  (i.e. dry-cow) studies, a minimum  of 20 animals is recommended.  The study 
should include randomly selected cows representative of commercial dairy practices. 
 
2.3.3.3. Poultry 
A sufficient number of birds should be used to obtain at least 6 samples at each slaughter time 
for tissue residue studies. 
 
For egg residue studies, a sufficient number of birds should be used to collect (10) or more eggs 
at each interval time point. 
   
6  Contains Non-Binding Recommendations 
2.3.4. Dosing and Route of Administration 
 
2.3.4.1. General guidance 
Animal treatment should be consistent with the intended product label including,  for injectable 
products, the location and injection  method.  For multiple  treatments, the injections  should be 
given alternately between left and right sides of the animal. 
 
The highest intended treatment dose should be administered for the maximum  intended duration.  
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 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. 
 
2.3.4.2. Considerations for products intended for intramammary administration 
Drug products intended for intra-mammary administration  either for lactating animals or for  
pre-parturition  (i.e., dry cow treatment) studies should be given to all quarters (i.e., normally  four 
quarters in bovine).  Although,  it is unlikely  that all quarters will be treated with an intra-
mammary product during commercial practice, for residue studies this study design represents a 
worst-case scenario. 
 
For pre-parturition  (i.e., dry cow treatment) studies, the test article should be administered after 
the last milking  (dry-off) and consistent with the desired pre-calving interval.  It is recognized 
that these (dry-cow) studies comprise both a milk residue depletion phase (post-calving)  as well 
as a decision on a desired pre-calving treatment interval.  In order to reduce variability  in the 
residue data, the pre-calving period should be tightly  controlled  and the study should be designed 
such that a sufficient number of animals give birth in a limited  time interval (i.e., the differences 
between dry periods among animals within an experiment should be kept as small as 
possible).  For instance, to target a pre-calving treatment interval of 30 days, data should be 
collected from at least 20 cows calving between, for example, 20-30 days after treatment.  For a 
pre-calving treatment interval of 60 days, data should be collected for at least 20 cows calving 
between, for example, 40-60 days after treatment.  This can be accomplished by drying-off and 
infusing  animals with the test formulation  based on the expected calving date. 
 
2.3.4.3. Considerations for products intended for multiple routes of administration. 
If the drug product is intended to be administered via more than one parenteral route 
(intramuscular (IM), subcutaneous (SC) or intravenous (IV)), a separate marker residue depletion 
study for each route of administration  should be provided.  If the withdrawal period is clearly 
defined by depletion  of residues from the injection  site following  SC or IM dosing,  a separate 
intravenous residue study (at the same dose) is not recommended provided the same withdrawal 
period (for SC or IM) can be applied to the IV route.  
 
A single marker residue depletion  study can be conducted for drug formulations  containing  the 
same active substance but which are applied via different dermal routes (e.g., dipping,  spray or 
pour-on).  However, the methodology  used in the study should represent delivery of the highest 
possible dose and this should be appropriately justified.   The consequence of this approach is that 
7  Contains Non-Binding Recommendations 
the same withdrawal time would be applied to all approved dermal application  routes.  Separate 
residue studies are recommended if differentiation  among these routes of administration  is 
desired. 
 
2.3.4.4. Considerations for Use of Multiple Injection Sites per Animal 
Where the withdrawal period will clearly be determined by residue depletion at the site of 
injection,  the Sponsor generally has the option of collecting  data from two injection  sites per 
animal (and using the data from both sites in a determination  of the withdrawal time).  This 
practice can have a positive impact on study design with respect to animal welfare by reducing 
animal numbers.  An example of where this approach is applicable is described below: 
 
  For a product that utilizes  only a single injection,  treatment can be given on the right side of 
the neck on Day 0 and then on the left side of the neck on Day 4.  Euthanasia on Day 7 
following  the final treatment would provide depletion data at 7-days (left injection  site (IJS)) 
and 11 days (right IJS) withdrawal.  In this case, however, collection  and assay of the other 
tissues would not be warranted since the product was administered contrary to the label (two 
injections  vs. one injection)  and residues could be excessively elevated.  Such a dosing 
regimen is designed specifically  for determination  of injection  site residue depletion. 
 
2.3.5. Animal Euthanasia 
Animals should be euthanized using commercially  applicable  procedures, making certain to 
observe appropriate exsanguination  times.  Chemical euthanasia can be used unless it will 
interfere with the analysis of the marker residue. 
 
2.3.6. Sampling 
 
2.3.6.1. General Considerations 
Following  euthanasia, edible tissue samples in sufficient amounts should be collected, trimmed 
of extraneous tissue, weighed and divided into aliquots.  If the analysis can not 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 the time of assay. 
 
The tissue sampling  protocol encompasses two sections; (1) those tissues that are recommended 
in support of registration or approval, as applicable,  in all VICH regions and (2) additional 
tissues that can be collected to address specific national/regional  consumption  habits and/or legal 
concerns.  Table 1 indicates the recommended samples for collection  for all VICH regions. Table 
2 indicates the recommended additional  samples for collection. 
 
For purposes of this guidance, one of the additional  tissues from Table 2 (per species) should be 
selected for assay, based on the results of the total residue (TRR) study.  This would typically  be 
the additional  tissue with the highest residues or the slowest depletion  rate.  It is important to 
emphasize that collection  of only one additional  tissue is recommended.  For example, if the 
TRR study indicates that cattle heart has the slowest depletion  rate, that additional  tissue should 
be selected for assay in the marker residue depletion  study, but cattle small intestine marker 
residue data are not recommended.   Similarly,  if poultry gizzard has the highest residues, assays 
8  Contains Non-Binding Recommendations 
of poultry heart are not recommended.  If no TRR data are available for the additional  tissues, it 
is suggested that the Sponsor discuss with the appropriate national/regional  authorities how best 
to conduct the marker residue depletion  study to satisfy the specific national/regional 
consumption  habits and/or legal concerns, if any. 
 
Table 1.  Sample Collection from Animals in the Marker Residue Depletion Study (All 
Regions) 
Edible  Species / Sample Description 
Tissue  Cattle / Sheep  Swine  Poultry 
Type 
Muscle  Loin  Loin  Breast 
Injection  Core of muscle tissue  Core of muscle tissue   Collect sample from 
Site  ~500 g  ~500 g  entire site of injection, 
Muscle  10 cm diameter x 6 cm  10 cm diameter x 6 cm  e.g., chicken whole neck, 
deep for IM;  deep for IM;  whole breast or whole leg. 
15 cm diameter x 2.5 cm  15 cm diameter x 2.5 cm  Larger birds, not to 
deep for SC  deep for SC  exceed 500 g 
Liver  Cross-section of lobes  Cross-section of lobes  Entire 
Kidney  Composite from  Composite from combined  Composite from combined 
combined kidneys  kidneys  kidneys 
Fat  Peri-renal  NA  NA 
Skin/Fat  NA  Skin with fat in natural  Skin with fat in natural 
proportions  proportions 
Milk  Whole milk  NA  NA 
Eggs  NA  NA  Composite from combined 
white and yolk 
NA:  not applicable 
 
Table 2.  Additional Tissues that can be Collected to Address Specific National/Regional 
Consumption and/or Legal Concerns in the Marker Residue Depletion Study 
Edible  Species / Sample Description 
Tissue Type 
Cattle / Sheep  Swine  Poultry 
Gizzard  NA  NA  Entire 
Heart  Cross-section  Cross-section  Entire 
Small  Composite,  rinsed of content  Composite,  rinsed of content  NA 
Intestine 
Other  Composite  Composite  Composite  Composite 
Tissues 
NA:  not applicable 
   
9  Contains Non-Binding Recommendations 
2.3.6.2. Injection Sites 
For parenteral preparations (IM or SC), residue depletion  data from the injection  site(s) should 
be included.  Injection site residues are local residues (i.e. those that do not arise via the systemic 
circulation)  that might or might not remain localized  at the site of administration.   As such, it is 
important for the Sponsor to develop appropriate quality control sampling procedures which 
ensure that the collected tissue actually encompasses the injection  site.  Any approach taken by 
the Sponsor should be justified  on a case-by-case basis, taking into account the data available 
and the formulation  characteristics.  The following  methodologies  should be considered, 
however, this list is not to be considered comprehensive. Regardless of the option selected, the 
primary core sample should target 500 g  20%. 
 
  Collection  of an additional  ring sample (300 g  20%) around the primary core sample (500 g 
 20%).  These tissue amounts would generally not apply to small animals that do not allow 
sampling  of 500 g.  For these situations,  the optimum  sampling  strategy should be defined on 
a case by case basis and should be justified.   However, collection  of two samples (a core and 
surrounding  sample) remains appropriate. 
 
  Collection  of an elliptical  (or other appropriate shape) sample along the injection  track and/or 
the site of irritation.   The Sponsor should provide evidence that this method correctly targets 
the injection  site residues, such as with accompanying  photographs of the site(s) of sampling. 
 
  Provide data on the migration  potential of injection  site residues based on information 
obtained from the TRR study.  For example, a circular core (or elliptical)  sample would be 
taken along the injection  track and/or site of irritation  as well as several adjacent samples for 
TRR comparisons.  If this protocol demonstrates an appropriate sample collection  technique, 
only the primary sample should be collected during the marker residue depletion study.  It 
might be constructive to include an additional  time point (i.e., at a longer withdrawal time) in 
this study. 
 
  Provide data on the migration  potential of injection  site residues based on information 
obtained from a target animal safety study (i.e. pathological  examinations  of the physical 
injection  site). 
 
  Conduct one of the above study designs using a colored dye to provide a visual assessment of 
the migration  potential of injection  site residues. 
 
Samples should be collected from the last injection  site (or sites), as appropriate (see Section 
2.3.4.4, Considerations for Use of Multiple  Injection  Sites per Animal).  In the case of products 
requiring  multiple  injections,  the study design should be such that the last injection  site will 
occur on the side of the animal receiving the higher number of injections.   When a circular cores 
sample is indicated, collection  of the injection  site muscle tissue (from large animals) should be 
centered on the point of injection  and consistent with the recommendations shown in Table 1. 
   
10  Contains Non-Binding Recommendations 
2.3.6.3. Other Considerations 
  For formulations  that are able to leave local residues, such as dermal pour-on products, 
samples of relevant tissues (e.g., muscle, subcutaneous fat or skin/fat from the application 
site) should be harvested for analysis (in addition  to those specified in Table 1). 
 
  For clarity, if two or more of the tissues are assayed as composite tissues such as skin plus fat 
in natural proportions  (pig and poultry),  it is not recommended to assay separate samples of 
skin and fat. 
  Muscle samples can be obtained from skeletal (striated) muscles that include   intramuscular 
fat in natural proportion. 
 
2.3.6.4. Milk Sampling 
Milk samples should be obtained from all animals at appropriate intervals.  Based on information 
obtained with respect to global  commercial dairy management practices, 12 hr collection 
intervals represent the most common milking  frequency.  Variations to this practice occur within 
a range of 6 hr (4X per day) to 24 hr (1X per day) and the selection of the specific collection 
interval should be justified  by the Sponsor.  Four-quarter composite samples should be collected 
from individual  cows at each time point.  For multiple  dosed products used in dairy animals, 
samples should be taken after the last treatment which should be administered  following 
complete milk-out  of the udder.  For products that may qualify for a 0-day (nil) milk discard 
time, samples should also be collected during treatment.  There is no standard number of 
sampling  times.  Milk collections  should continue until  the residues fall below the appropriate 
reference point (e.g., MRL, tolerance, LOQ, etc) as determined by the chemical properties of the 
drug product. 
 
Although beyond the scope of this guidance, the Sponsor might be requested to assess residues in 
calves fed milk (including  colostrum) from treated adults (i.e., mothers), if these animals are 
intended for human consumption  (such as for veal calves). 
 
2.3.6.5. Egg Sampling 
Egg samples should be obtained from 10 or more laying hens at every laying time point during 
the medication period and after the final medication.   Egg samples should be collected after the 
period necessary to complete egg yolk development,  which is usually up to 12 days.  Egg white 
and yolk should be combined for analysis. 
 
2.3.7. Recommendations for Products Proposed for a 0-Day Tissue Withdrawal Period or a 
0-Day (nil) Milk Discard Time 
For products administered as one treatment or as several treatments (i.e., daily for 3-5 days), or 
for continuous  use products in which residues have reached steady state, a single time point 
study may be sufficient to qualify for 0-day tissue withdrawal period or a limited  time point 
study may be sufficient to qualify for a 0-day (nil) milk discard time.  The Sponsor should 
provide justification  for use of a single (tissues) or limited  (milk)  time point study design.  
Considerations  may include (1) the availability  and relevance to the final commercial product of 
a VICH GL 46 compliant  study where the total residue depletion  characteristics of the drug have 
been adequately described and/or (2) reference to information  in the public  domain (e.g. 
regulatory summaries or the general scientific literature).  If such information  is available, then a 
11  Contains Non-Binding Recommendations 
single (tissues) or limited  (milk) time point study conducted with the specified minimum  number 
of animals is recommended to demonstrate acceptability of 0-day tissue withdrawal or nil milk 
discard time. 
 
2.3.7.1 Zero-Day Tissue Withdrawal Time Study Design 
 
  Poultry    12 birds (to provide at least 6 individual  samples for assay) 
 
  Large Animals:   6 animals 
 
The sampling  time chosen for this study should be consistent with the peak concentrations 
observed during the total residue depletion  study, a minimum  transit time (practical zero 
withdrawal; e.g., not less than 3 hr) and a maximum time that would still qualify  as 0-day 
withdrawal (e.g.,  12 hr). The increased animal numbers from that recommended in Section 
2.3.3 is generally appropriate for the single time point. 
 
2.3.7.2 Zero-Day (nil) Milk Discard Time Study Design 
Sampling  times should be consistent with peak concentrations and be consistent with commercial 
dairy practices.  It is recognized that local and regional differences may exist with respect to 
number of milk collections  per day from treated animals and that this may vary from as short as 
every 6 hours (4X per day) to every 24 hr (1X per day).  On the other hand, milk collections 
every 2 hr (i.e. 12X per day) would not occur commercially.   A recommendation  that studies be 
conducted to take into account all potential milk dosing and sampling  schemes was judged to be 
inconsistent with the principles  of VICH and the objective of one common study design.  It was 
also recognized that the vast majority of historical data have been generated using 2X per day 
milk collections.    Nevertheless, consideration  of only the 2X/day practice was judged to be 
insufficient  to address global regulatory concerns when alternate milking  frequencies may be 
employed,  especially where a 0-day (nil) withdrawal classification  is being pursued.  As such, 
the following  study design is recommended for drugs that may qualify for a zero-day withdrawal 
period (nil discard time) including  no discard of milk during treatment. 
 
  A minimum  of 16 animals is recommended which should be subdivided  into three groups 
(Group 1:  n=3, Group 2:  n=3, Group 3:  n=10). 
 
  All animals should receive treatment as soon as possible after the morning  (or evening) 
milkout  consistent with the vast majority of global  commercial husbandry practices and 
when the animals are most readily available for handling. 
 
  For products to be administered  as multiple  treatments (e.g. once daily for 3-5 days), milk 
should be collected during treatment at appropriate intervals justified  by the Sponsor. 
 
  Group 1 animals (minimum  of n=3) should be fully milked out approximately  6 hours 
after the final (or cessation of) treatment and then milked  out again at approximately  12 
hr after final (or cessation of) treatment. 
 
12  Contains Non-Binding Recommendations 
  Group 2 animals (minimum  of n=3) should be fully milked out at approximately  8 hours 
after the final (or cessation of) treatment and then milked  out again at approximately  12 
hr after final (or cessation of) treatment. 
 
  Group 3 animals (minimum  of n=10) should be fully  milked out at approximately  12 hr 
after final (or cessation of) treatment. 
 
  All animals should be fully milked out at approximately  24 hr and at subsequent 
approximately  12 hour intervals to confirm that milk residues do not increase. 
 
 
   
13  Contains Non-Binding Recommendations 
Milk collection  at 12 hr after dose administration  represents the maximum sampling  interval 
which may still qualify for a 0-day (nil) withdrawal period designation. 
 
While a 0-day milk withdrawal designation  could possibly  be achieved based solely on this study 
design (provided sufficient information  was available to determine Cmax), it is strongly 
recommended that at least 4 additional  samples be collected following  the 12 hr sampling  time to 
confirm that residue concentrations do not increase (as would not be expected for a 0-day 
withdrawal designated product).  As milk studies do not call for terminal euthanasia following 
sample collection,  compliance with this recommendation is straightforward.  The recommended 
sampling  interval for these additional  collections  is approximately  12 hr or at some alternative 
interval as justified  by the Sponsor. 
 
If the depletion study is conducted as described above (to include Groups 1-3), drug 
concentrations which remain below the appropriate reference point (e.g., MRL, tolerance) at 
collection  times  12 hr after the final (or cessation of) treatment, would qualify for a 0-day (nil) 
milk discard designation  based on appropriate regional data analysis procedures.  Milk could be 
taken for human consumption  at any time after treatment (e.g. at 4 hr, 8 hr, 10 hr, etc).  Note that 
if the study design does not include the intermediate sampling  points at 4 hr and 8 hr (Groups 1-
2), and sampling  occurs only at 12 hr (Group 3), milk could be taken for human consumption 
after 12 hr only, while milk  collected prior to 12 hr would need to be discarded. 
 
The study design outlined  in this section is not appropriate where a withdrawal time other than 0-
days is anticipated.  For products anticipated to have a finite withdrawal time (greater than 0-
days), a minimum  of 20 animals is required as indicated in Section 2.3.3.2. 
 
Alternative study designs for demonstration  of 0-day withdrawal may be appropriate if justified 
by the Sponsor. 
 
2.4. 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 in the edible tissues and where 
applicable,  in milk and eggs.  The method(s) should be capable of reliably  determining 
concentrations of marker residue which encompass the appropriate reference point (i.e., MRL / 
Tolerance) for the respective tissues or products. 
 
The parameters to be included in the method validation  are fully discussed in the VICH GL 49, 
Studies to Evaluate the Metabolism  and Residue Kinetics of Veterinary Drugs in Food 
Producing Animals:   Validation  of Analytical Methods Used in Residue Depletion Studies. 
   
14  Contains Non-Binding Recommendations 
3.  GLOSSARY 
 
The following  definitions  are applied for 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. 
 
Edible tissues are tissues of animal origin  that can enter the food chain and include but are not 
limited  to muscle, injection  site muscle, liver, kidney,  fat, skin with fat in natural proportions, 
whole eggs and whole milk. 
 
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. 
 
Practical zero withdrawal is representative of the shortest time interval between administration  of 
the last dose of the drug (e.g. at the farm) and slaughter (including  transport from the farm). 
 
Pre-ruminant is defined as immature cattle (including  dairy breeds) lacking a functional  rumen 
and intended for meat production. They are recognized as a separate class from suckling  calves 
because of their handling,  housing,  and proximity  to slaughter. 
 
Residue means the veterinary drug (parent) and/or its metabolites. 
 
Residue of concern refers to the total amount of residues that have relevance to the ADI 
established for the veterinary drug. 
 
Total residue of a drug in edible tissues is the sum of the veterinary drug (parent) and all 
metabolites as determined in radiolabeled  studies or other equivalent studies. 
 
Zero-day withdrawal refers to a label indication  that allows entry of edible tissues into the food 
chain without regard to the time of last drug administration. 
 
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