#3 
General Principles for Evaluating the 
Human Food Safety of New Animal 
Drugs Used in Food-Producing Animals 
Guidance for Industry 
This version of the guidance replaces the version made available April 2018.  This revision 
references a recently published guidance (GFI #257 VICH GL57) and a revised guidance (GFI 
#159 (VICH GL36(R2)). 
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 FDA2005D0155. 
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 https://www.fda.gov/animal-
veterinary, https://www.fda.gov/regulatory-information/search-fda-guidance-documents, or 
http://www.regulations.gov. 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
May 2022  Contains Nonbinding Recommendations 
 
Table of Contents 
 
 
I.  Introduction and Scope ...................................................................................................... 4 
II.  Legal Authority ................................................................................................................. 5 
III.  Overview of the Human Food Safety Evaluation ............................................................. 5 
IV.  Microbial Food Safety (Antimicrobial Resistance) ........................................................... 6 
V.  Toxicology ......................................................................................................................... 7 
A.  Introduction .............................................................................................................. 7 
B.  Generally Recommended Testing Approach ........................................................... 8 
C.  Evaluation of Non-traditional New Animal Drugs .................................................. 9 
D.  Evaluation of Endogenous Sex Steroid Hormones - Permitted Incremental 
Increases ................................................................................................................. 10 
E.  Additional or Specialized Toxicological Studies ................................................... 10 
F.  Establishing the Acceptable Daily Intake (ADI), Acute Reference Dose (ARfD),     
and Safe Concentrations ......................................................................................... 11 
1.  Determination of the Toxicological Acceptable Daily Intake ....................... 11 
2.  Effects of Antimicrobial Drug Residues on the Human Intestinal Flora - 
Determining the Microbiological Acceptable Daily Intake (mADI) ............ 12 
3.  Establishing the Final ADI ............................................................................ 12 
4.  Determining the Acute Reference Dose (ARfD) .......................................... 13 
5.  Partitioning of the ADI .................................................................................. 13 
6.  Food Consumption Values for Edible Tissues .............................................. 14 
7.  Calculation of the Safe Concentrations ......................................................... 14 
a.  Safe concentrations for total residues in edible tissues ........................ 14 
b.  Safe concentration for total residues in muscle injection sites ............. 15 
VI.  Residue Chemistry .......................................................................................................... 17 
A.  Introduction ............................................................................................................ 17 
B.  Total Residue and Metabolism Study .................................................................... 17 
C.  Comparative Metabolism Study ............................................................................. 18 
D.  Selection of Target Tissue and Marker Residue .................................................... 18 
E.  Assignment of Tolerance ........................................................................................ 19 
F.  Tissue Residue Depletion Study ............................................................................. 20 
G.  Analytical Methods for Residues ........................................................................... 21 
1.  Introduction ................................................................................................... 21 
2  Contains Nonbinding Recommendations 
2.  Evaluation Criteria ........................................................................................ 21 
a.  Determinative Procedure ...................................................................... 22 
(1)  Calibration Range and Linearity ................................................. 22 
(2)  Bias (Systematic Error) ............................................................... 22 
(3)  Precision and Accuracy ............................................................... 22 
(4)  Limit of Detection (LOD) and Lower Limit of Quantitation 
(LLOQ) ........................................................................................ 23 
(5)  Robustness and Ruggedness ........................................................ 23 
(6)  Specificity and Selectivity ........................................................... 23 
(7)  Parallelism ................................................................................... 24 
(8)  Stability ....................................................................................... 24 
(9)  System Suitability ........................................................................ 24 
(10)  Additional Criteria ....................................................................... 24 
(11)  Confirmatory Procedure .............................................................. 25 
(12)  Adaption of a Previously Validated Method ............................... 25 
3.  Specific Data Needed .................................................................................... 26 
a.  Sponsor's Submission ........................................................................... 26 
b.  Inter-laboratory Study .......................................................................... 27 
H.  Establishing a Withdrawal Period .......................................................................... 28 
I.  Statistical Analysis of the Residue Data ................................................................. 29 
1.  Theory ........................................................................................................... 29 
2.  Statistical Software:  Residues in Tissue and Milk ....................................... 32 
VII.  Carcinogenic Compounds ............................................................................................... 32 
VIII.  References ....................................................................................................................... 33 
3  Contains Nonbinding Recommendations 
 
General Principles for Evaluating the Human Food Safety 
of New Animal Drugs Used in Food-Producing Animals 
 
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. 
 
I.  Introduction and Scope 
This guidance describes the type of information that the Food and Drug Administrations 
(FDAs) Center for Veterinary Medicine (CVM) recommends sponsors provide to address the 
human food safety of new animal drugs used in food-producing animals. 
 
The human food safety evaluation of new animal drugs used in food-producing animals helps 
ensure that food derived from treated animals is safe for human consumption.  Sponsors are 
required to furnish to CVM scientific data or information necessary to demonstrate that residues 
of the new animal drug in the edible tissues of treated animals are safe (see section 512(b)(1) of 
the Federal Food, Drug, and Cosmetic Act (the FD&C Act)).  In general, studies conducted to 
provide this information must be conducted in accordance with FDAs Good Laboratory Practice 
(GLP) regulations.  See Title 21, Code of Federal Regulations, part 58 (21 CFR part 58). 
 
FDA conducts a human food safety assessment of new animal drugs for use in food-producing 
animals through hazard identification, hazard characterization, exposure assessment, and 
mitigation of human exposure to drug residues in food derived from treated animals.  CVM 
developed this guidance to inform sponsors of the scientific data and/or information that may 
provide an acceptable basis to determine that the residue of a new animal drug in or on food, 
when consumed, presents a reasonable certainty of no harm to humans.  This guidance describes 
a recommended approach for providing human food safety scientific data and/or information.  
CVM acknowledges that alternate approaches also may be appropriate and encourages sponsors 
to discuss with CVM whether an alternate approach may be appropriate for specific new animal 
drugs. 
 
This guidance provides, in one document, an overview of the overall process for the human food 
safety evaluation of new animal drugs used in food-producing animals, including: 
  Determining an acceptable daily intake (ADI); 
  Calculating safe concentrations; 
  Assignment of a tolerance; 
  Calculation of a withdrawal period and a milk discard time; and 
4  Contains Nonbinding Recommendations 
  Evaluation of carcinogenic compounds 
 
It is intended to be used in conjunction with other CVM Guidance for Industry (GFI) documents 
that are cited in this guidance or listed on FDAs website.1 
 
The contents of this document do not have the force and effect of law and are not meant to bind 
the public in any way, unless specifically incorporated into a contract.  This document is 
intended only to provide clarity to the public regarding existing requirements under the law.  
FDAs guidance documents 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. 
 
II.  Legal Authority 
FDA is required by sections 512(d)(1)(A), (B), (D), and (F) of the Federal Food, Drug, and 
Cosmetic Act (the FD&C Act) to determine whether the edible tissues derived from treated 
animals are safe for human consumption.  The pertinent regulations implementing these statutory 
provisions are found at 21 CFR parts 514 and 556. 
 
Section 512(d)(2) of the FDA&C Act explicitly provides that, in determining whether a new 
animal drug is safe, FDA considers the probable consumption of such drug and of any substance 
formed in or on food because of the use of such drug. 
 
III.  Overview of the Human Food Safety Evaluation 
FDAs evaluation of human food safety may include hazard identification, hazard 
characterization, exposure assessment, and exposure mitigation.  The assessment is conducted 
from the perspectives of toxicology, residue chemistry, and microbial food safety as outlined in 
the following flow diagram (Figure 1). 
 
 
1 All FDA guidances mentioned in this document are available at https://www.fda.gov/regulatory-
information/search-fda-guidance-documents. 
5  Contains Nonbinding Recommendations 
 
 
 
 
Figure 1: Flow Diagram Illustrating the General Approach for the Human Food Safety 
Evaluation.  The dashed lines indicate that the component applies to new animal drugs with 
antimicrobial activity.  ADI means Acceptable Daily Intake.  ARfD means Acute Reference 
Dose. 
 
IV.  Microbial Food Safety (Antimicrobial Resistance) 
The Agency routinely evaluates the ability of antimicrobial new animal drugs to exert pressure 
towards the emergence or selection of antimicrobial resistant bacteria of human health concern. 
 
In GFI #152, Evaluating the Safety of Antimicrobial New Animal Drugs with Regard to Their 
Microbiological Effects on Bacteria of Human Health Concern, CVM outlines two potential 
approaches to address the risk associated with new animal drugs possessing antibacterial activity: 
1) a hazard characterization, and 2) a complete, qualitative antimicrobial resistance risk 
assessment. 
 
Sponsors electing to use these approaches should first characterize the hazard and the conditions 
that influence the occurrence of that hazard.  CVM recommends sponsors submit the hazard 
characterization as a distinct, stand-alone document, separate from the complete, qualitative risk 
assessment.  This hazard characterization will enable CVM to advise the sponsor about the 
information that should be included in a complete, qualitative risk assessment.  It also may 
6  Contains Nonbinding Recommendations 
enable CVM to determine that completion of a complete, qualitative risk assessment is not 
necessary for certain new animal drugs possessing antibacterial activity. 
 
Concerns outlined in GFI #152 may need to be addressed if the antimicrobial new animal drug, 
including the drugs metabolites, are: 
  regularly considered to have properties that would exert pressure towards the emergence 
or selection of resistant bacteria of public health concern; 
  used to treat zoonotic gastroenteritis or other bacterial diseases in humans; 
  under development to treat bacterial diseases in humans; or 
  indicated for a bacterial disease in food-producing animals (i.e., indication is instead 
antifungal, antiprotozoal, anthelminthic, etc.). 
 
Contact the Director, Division of Human Food Safety, for further guidance should you think 
these conditions are attributable to your new animal drug product. 
 
V.  Toxicology 
A.  Introduction 
For new animal drugs used in food-producing animals, CVM is generally concerned with 
both intermittent and chronic exposure of people to relatively low concentrations of 
residues.  The toxicity studies described in this guidance document are a standard set of 
studies designed to evaluate the oral toxicity of new animal drug residues to humans who 
may be exposed to these residues through the consumption of food derived from animals 
treated with the new animal drug.  CVM recognizes, however, that this standard 
toxicological testing paradigm may not always be the most appropriate approach for such 
an evaluation.  CVM considers alternative approaches to address toxicological concerns 
when such approaches are scientifically justified.  For such approaches, CVMs 
recommendations for the type and duration of toxicological studies and other information 
needed to demonstrate human food safety are specific to each new animal drug. 
 
In making its recommendations to address toxicological concerns, CVM considers, for 
each new animal drug: 
  Physiochemical properties 
  Proposed use 
  Mode of action 
  Probable amounts and patterns of human exposure to the residues under the 
proposed conditions of use 
  Possible biological effects of the residues as deduced through structure-activity 
comparisons; and 
  Observed effects when new animal drugs (or their metabolites(s)) are tested in 
toxicological and biological model systems. 
7  Contains Nonbinding Recommendations 
This information can be used to identify a more targeted approach to the design of 
individual toxicology studies, such as a recommendation to include alternative 
toxicological endpoints in specific studies.  It can also lead to modifications to the overall 
toxicological testing strategy for a particular new animal drug, such as recommending 
that a non-traditional toxicology study be conducted to help understand the results of 
traditional studies, or recommending that one or more standard toxicology studies may 
not be needed to determine the human food safety for a particular new animal drug. 
 
The purpose of toxicological studies is to define the biological effect(s) of the new animal 
drug and to establish the threshold dose for the effect(s).  CVM typically requests testing 
of the new animal drug.  In addition to the information regarding the new animal drug, 
separate information on the toxicology of a metabolite(s) and/or excipient(s) may be 
considered necessary to assess human food safety. 
 
B.  Generally Recommended Testing Approach 
One recommended testing approach to assure the safety of human food derived from 
animals treated with new animal drugs is outlined in GFI #149 (VICH GL33), Studies to 
Evaluate the Safety of Residues of Veterinary Drugs in Human Food:  General Approach 
to Testing.  However, CVM recommends that sponsors discuss their proposed approach 
with CVM for determining human food safety prior to conducting studies.  GFI #149 
references additional human food safety toxicology guidance documents adopted from 
the corresponding International Cooperation on Harmonisation of Technical 
Requirements for Registration of Veterinary Medicinal Products (VICH) guidelines (GL), 
as listed in Table 1. 
 
Table 1:  Guidance Documents for Toxicology Studies for the Human Food Safety 
Evaluation 
Title  GFI #  VICH GL # 
Studies to Evaluate the Safety of Residues of Veterinary  147  31 
Drugs in Human Food:  Repeat-Dose (90-Day) Toxicity 
Testing 
Studies to Evaluate the Safety of Residues of Veterinary  160  37 
Drugs in Human Food:  Repeat-Dose (Chronic) Toxicity 
Testing 
Studies to Evaluate the Safety of Residues of Veterinary  148  32 
Drugs in Human Food:  Developmental Toxicity Testing 
Safety Studies for Veterinary Drug Residues in Human Food:   115  22 
Reproduction Toxicity Testing 
Studies to Evaluate the Safety of Residues of Veterinary  116  23(R) 
Drugs in Human Food:  Genotoxicity Testing 
Studies to Evaluate the Safety of Residues of Veterinary  141  28 
Drugs in Human Food:  Carcinogenicity Testing 
Studies to Evaluate the Safety of Residues of Veterinary  159  36(R2) 
Drugs in Human Food:  General Approach to Establish a 
Microbiological ADI 
8  Contains Nonbinding Recommendations 
Sponsors should consult the referenced guidance documents if they intend to design and 
conduct repeat-dose (90-day) subchronic toxicity studies, repeat-dose chronic toxicity 
studies, developmental toxicity studies, reproduction toxicity studies, genotoxicity 
studies, carcinogenicity studies or evaluate the effects of antimicrobial residues on the 
human intestinal flora.  The studies and approaches described in these guidance 
documents recommend standard testing protocols that are likely to satisfy the toxicology 
requirements for the human food safety evaluation of a wide range of new animal drugs.  
Alternative, scientifically justified protocols may also be appropriate for some new 
animal drugs. 
 
The specific recommendations to demonstrate safety with reasonable certainty of no 
harm with respect to potential residues in the edible tissues of animals that humans may 
consume are determined on a case-by-case basis.  In all cases, sponsors should contact 
CVM to discuss the types of studies recommended to demonstrate human food safety of a 
particular new animal drug, the details of any human food safety experimental protocol, 
or to receive additional information for any other questions they have about the details of 
a particular study. 
 
C.  Evaluation of Non-traditional New Animal Drugs 
CVM recognizes that the traditional toxicological testing paradigm described in guidance 
documents is often the most appropriate approach for new animal drugs that are small 
molecules, but may not be the most appropriate approach to evaluate the safety to 
humans from the consumption of food derived from animals that have been treated with 
other, non-traditional new animal drugs.  For non-traditional new animal drugs, CVM 
considers appropriate alternative approaches to address toxicological concerns.  When 
evaluating such new animal drugs, CVM adopts a case-by-case approach that is often 
based on their unique biological and chemical properties or mode of action.  Examples of 
non-traditional new animal drugs that could be evaluated by an alternative 
toxicological approach are biotechnology-derived new animal drugs, including some 
enzymes, fusion proteins, synthetic hormones, and antibodies.2 
 
Sponsors of such non-traditional new animal drugs for use in food-producing animals 
should provide sufficient information to demonstrate that the residues are safe for human 
consumers.  Scientific rationale should be provided to justify alternative approaches for 
evaluation.  Because the approaches are non-standard, it is recommended that the sponsor 
contact CVM early in the development process to discuss approaches and specific studies 
to address toxicological concerns for these non-traditional new animal drugs for use in 
food-producing animals. 
 
 
2 This document does not cover intentional genomic alterations in animals as discussed in GFI #187, Regulation of 
Intentionally Altered Genomic DNA in Animals (Draft Revised).  The document also does not apply to altered 
genomic DNA that meets the definition of a veterinary biologic and is regulated by the Animal and Plant Health 
Inspection Service (APHIS) of the United States Department of Agriculture (USDA).  21 CFR 510.4. 
9  Contains Nonbinding Recommendations 
D.  Evaluation of Endogenous Sex Steroid Hormones - Permitted Incremental 
Increases 
For endogenous sex steroid hormones, also known as gonadal steroids, CVM usually 
establishes incremental increases rather than an Acceptable Daily Intake (ADI) or 
tolerance.  This is because endogenous steroid hormones produced mainly by the ovaries 
(estrogen, progesterone) and testes (testosterone), throughout the lifetime of humans and 
mammals are required for the proper physiological functions, such as the growth or 
function of the reproductive organs or the development of secondary sex characteristics.  
Therefore, because of the existence of these naturally occurring hormones in humans and 
food-producing animals (e.g., cattle), the human consumer is exposed throughout his/her 
lifetime to rather large quantities of these steroid hormones through his/her own daily 
production and to much lesser quantities from untreated food-producing animals. 
 
FDA has concluded that no physiological effect could be expected in consumers eating 
meat containing additional amount of the hormone that is less than or equal to 1% of the 
amount produced by the human daily.  Using the daily production values in pre-pubertal 
boys and girls, FDA calculated a permitted increased exposure above the amount 
naturally present in untreated target animals (1% of the daily production values) [see 
21 CFR 556.240 for estradiol, 21 CFR 556.540 for progesterone, and 21 CFR 556.710 for 
testosterone]. 
 
The sponsor using a fully validated analytical method should demonstrate that the 
compound is safe under the proposed conditions of use and that the concentration of 
residue of the endogenous sex steroid in treated food-producing animals is such that the 
actual increase in exposure of people will not exceed the permitted incremental increases. 
As such, a fully validated analytical method using a contemporaneous gender-matched 
untreated control for every test is used. The baseline addresses endogenous steroid 
hormones that contribute to the overall and unavoidable exposure associated with eating 
animal-derived foods. 
 
E.  Additional or Specialized Toxicological Studies 
Occasionally, on a case-by-case basis, CVM will recommend that sponsors conduct 
additional or specialized toxicological studies to address unanticipated or unusual 
toxicological or pharmacological findings from the initial set of recommended toxicology 
studies.  Additional studies may be requested to address a particular toxicity concern 
(e.g., pharmacological effects, immunotoxicity, neurotoxicity, carcinogenicity).  
Specialized studies performed to understand the mode of action of the drug and used in 
interpretation of, or the assessment of the relevance of the data may also be 
recommended. 
10  Contains Nonbinding Recommendations 
 
F.  Establishing the Acceptable Daily Intake (ADI), Acute Reference Dose (ARfD), 
and Safe Concentrations 
1.  Determination of the Toxicological Acceptable Daily Intake 
The safety of residues in foods is usually addressed through calculation of an 
acceptable daily intake (ADI).  The ADI is the daily intake which, during up to an 
entire life of a human, appears to be without adverse effects or harm to the health of a 
consumer.  The toxicological ADI is based on the new animal drugs toxicological or 
pharmacological properties as determined through recommended toxicology studies 
and other applicable scientific information and reflects the uncertainties with the 
information.  It is usually expressed in micrograms or milligrams of the new animal 
drug per kilogram of body weight per day (g/kg bw/day) or (mg/kg bw/day).  CVM 
uses a weight of evidence approach for this hazard characterization, which means that 
all data from available studies and other applicable scientific information are 
considered. 
 
Traditional toxicology studies are designed to determine if the new animal drug 
produces an adverse effect(s) in a biological test system and to identify the highest 
dose of the new animal drug that produces no observable effect(s), which may be a 
no-observed-effect level (NOEL) / a no-observed-adverse-effect level (NOAEL) or a 
benchmark dose (BMD).3  The BMD is the dose associated with a specified low 
incidence of response, generally in the range of 1 to 10%, of a health effect, or a dose 
associated with a specified measure or change in a biological effect.  Because it is 
often difficult to determine whether a particular observed effect in an animal 
toxicology study is adverse to humans or not, it is generally assumed that such effects 
are adverse, and that a new animal drugs NOEL is equivalent to its NOAEL. 
 
When establishing the ADI, the toxicities of the new animal drug and of its 
metabolite(s) are considered with all available information.  For non-carcinogenic 
endpoints, the NOEL/NOAEL or the BMDL (lower one-sided confidence limit of the 
BMD) from the most appropriate toxicological study will be used as the point of 
departure (POD) for calculating the toxicological ADI for the total residues of the 
new animal drug.  The toxicological ADI is derived by dividing the POD by an 
appropriate safety factor, using the following formula: 
 
 
 
3 See Benchmark Dose Software (BMDS) available from the US Environmental Protection Agency 
[https://www.epa.gov/bmds] and PROAST available from the National Institute for Public Health and the 
Environment of the Netherlands (RIVM) 
[http://www.rivm.nl/en/Documents_and_publications/Scientific/Models/PROAST]. 
11  Contains Nonbinding Recommendations 
The safety factor used in this calculation reflects uncertainties associated with the 
extrapolation of data and information from toxicology studies to humans, including 
extrapolation of long-term, chronic effects from laboratory studies with shorter-term 
exposures, extrapolation of animal data to humans, and variability in sensitivity to the 
toxicity of the new animal drug among humans.  Generally, the safety factor consists 
of multiples of 10, with each factor representing a specific uncertainty inherent in the 
available data.  For example, CVM has historically applied a safety factor of 100 for 
an ADI based on the NOEL/NOAEL from a chronic toxicity study (10-fold for 
extrapolating animal data to humans and 10-fold for variability in sensitivity to the 
toxicity of the new animal drug among humans).  Thus, the overall safety factor used 
in the calculation of a toxicological ADI is dependent on the interplay among many 
factors, including the type of study from which the NOEL/NOAEL was derived, the 
species of animal used in the study, and the endpoint(s) observed in the study.  The 
toxicological ADI is expressed with the appropriate number of significant figures as 
justified by the underlying scientific information used in these calculations (usually 
one significant figure).  This avoids any inference of inappropriate precision, and is 
consistent with mathematically acceptable rounding procedures. 
 
For compounds of carcinogenic concern used in food-producing animals, see section 
VII. Carcinogenic Compounds. 
 
2.  Effects of Antimicrobial Drug Residues on the Human Intestinal Flora - 
Determining the Microbiological Acceptable Daily Intake (mADI) 
FDA GFI #159 (VICH GL36(R2), Studies to Evaluate the Safety of Residues of 
Veterinary Drugs in Human Food:  General Approach to Establish a Microbiological 
ADI, aims to ensure that residues of antimicrobial drugs in the edible tissues of 
food-producing animals are safe for humans to consume.  It is recommended that the 
harmonized step-wise approach be used to determine if residues of an antimicrobial 
new animal drug reach the human colon and remain active, affect intestinal bacteria, 
and whether a determination of an mADI is necessary. 
 
The microbiological endpoints that should be considered when establishing an mADI 
are 1) the disruption of the colonization barrier, and/or 2) an increase of the 
population(s) of antibacterial resistant bacteria.  The final mADI for total residues in 
edible tissues is the lesser value of these two microbiological endpoints. 
 
3.  Establishing the Final ADI 
Antimicrobial new animal drugs may be assigned both a microbiological ADI and 
toxicological ADI.  The final ADI is the more appropriate of these two ADIs, which 
is usually the lower of the toxicological and microbiological ADIs.  In some 
instances, CVM may not establish an ADI for a new animal drug if it does not present 
a hazard to human health.  In these situations, CVM will conclude that an ADI is not 
needed for the new animal drug. 
12  Contains Nonbinding Recommendations 
 
4.  Determining the Acute Reference Dose (ARfD) 
There is the potential for some new animal drugs to cause acute toxicity to the human 
consumer following consumption of a single meal or consumption of food over a 
single day.  Some toxicological tests or information may suggest the need for a value 
in addition to the ADI to account for new animal drugs that cause acute toxicity.  In 
these cases, the ADI is not the appropriate value for quantifying the dose above which 
exposure from a single meal or over a single day can produce acute adverse effects; 
instead, determining an acute reference dose (ARfD)4 is the most appropriate 
approach. 
 
The ARfD is an estimate of the amount of residues, expressed on a body weight basis, 
which can be ingested in a period of 24 hours or less without adverse effects or harm 
to the health of the human consumer.  The ARfD is expressed with the appropriate 
number of significant figures, as justified by the underlying scientific information 
used in such calculations (usually one significant figure). 
 
Consult GFI #232 (VICH GL54), Studies to Evaluate the Safety of Residues of 
Veterinary Drugs in Human Food:  General Approach to Establish an Acute 
Reference Dose (ARfD), for additional information on the derivation of the ARfD 
and implementation of this approach for new animal drugs that can produce acute 
adverse effects.  The need for an ARfD is determined on a case-by-case basis.  If 
studies to determine an ARfD are needed, sponsors should contact CVM to discuss 
the type(s) of study(ies) and the experimental protocol(s) for those studies prior to 
conducting any study. 
 
5.  Partitioning of the ADI 
Partitioning refers to allocating portions of the ADI across edible tissues when 
consumption of those tissues may contribute to the total human exposure to a new 
animal drug and/or its residues.  In this situation, the ADI is partitioned across 
applicable edible tissues based on the expected use of the new animal drug and the 
food consumption values for each tissue (discussed below).  Edible tissues include 
muscle, liver, kidney, fat, or skin with fat in natural proportions (as appropriate), 
whole eggs, whole milk, and honey.  Skin with fat in natural proportions is the edible 
tissue for swine and poultry; however, in this guidance we refer to fat for all species 
as the edible tissue for simplicity. 
 
For some products (e.g., products not used in lactating dairy cows or laying hens), the 
ADI can be allocated to edible muscle, liver, kidney, or fat without partitioning.  The 
partitioning of the ADI across edible tissues is based on the requested allocation by 
the new animal drugs sponsor and on the information of the residues in edible 
 
4 Prior to GFI #232 (VICH GL54), FDA referred to this value as the Acceptable Single-Dose Intake (ASDI). 
13  Contains Nonbinding Recommendations 
tissues.  For example, for products approved in lactating dairy cows or for products 
approved in lactating dairy cows and other meat-producing species, a part of the ADI 
would be reserved for milk.  The remaining part of the ADI would be partitioned 
among the animals edible tissues, as described above.  Sponsors should consider the 
need to partition the ADI early in the development plan for their product to account 
for anticipated needs in other animal species or edible tissues. 
 
6.  Food Consumption Values for Edible Tissues 
When estimating the daily consumption for edible tissues, CVM assumes that, when 
an individual consumes a full portion of any edible tissue (e.g., muscle, liver, kidney, 
or fat) from one species, that individual would not consume a full portion of the any 
other edible tissue from any another species on the same day. 
 
The default consumption values (quantity consumed per day) for edible tissues are 
listed in Table 2.  These food consumption values are used for all applicable species. 
 
Table 2:  Food Consumption Values 
 
Edible Tissue  Quantity Consumed per day 
Muscle  300 g 
Liver  100 g 
Kidney  50 g 
Fat or Skin/Fat5  50 g 
Milk  1500 mL 
Eggs  100 g 
Honey  20 g 
 
CVM regulates milk, eggs, and honey as independent food commodities; this means 
that CVM assumes these edible tissues are consumed in addition to the consumption 
of muscle, liver, kidney, or fat.  Therefore, CVM assumes that a person consumes a 
full portion of milk, a full portion of eggs, and a full portion of honey in addition to a 
full portion of one of the other edible tissues each day. 
 
7.  Calculation of the Safe Concentrations 
a.  Safe concentrations for total residues in edible tissues 
The safe concentration is the amount of total residue of a new animal drug that 
can be consumed from each edible tissue every day for up to the lifetime of a 
human without exposing the human to residues in excess of the ADI.  The 
calculation of each edible tissues safe concentration reflects the partition of the 
ADI described above (section V.F.5. Partitioning of the ADI).  CVM calculates 
 
5 Skin/Fat refers to skin that includes fat in natural proportions.  See section V.F.5. Partitioning of the ADI. 
14  Contains Nonbinding Recommendations 
the safe concentration for each edible tissue using the ADI, or the portion of the 
partitioned ADI among meat, milk, eggs, or honey when applicable, the weight in 
kilograms (kg) of an average adult human body (approximated at 60 kg),6 and a 
conservative estimate of the amount of the edible tissue eaten per day in grams 
(see Table 2).  The safe concentration of total residues of the new animal drug in 
each edible tissue is calculated using the following formula: 
 
 
For example, the safe concentration for milk equals the percentage of the full ADI 
reserved for milk (g/kg bw/day) times 60 kg divided by 1500 mL/day (i.e., 
1500 g/day).  Safe concentrations for other tissues are calculated in a similar 
manner. 
 
b.  Safe concentration for total residues in muscle injection sites 
Human consumption of all or part of a new animal drug residue in a single meal is 
a relatively rare event, likely limited to occasional consumption of a site of 
injection from which the new animal drug did not yet distribute to other tissues in 
the animal.  In order to ensure human food safety for this possible acute exposure, 
the safe concentration of residues in the injection site tissue is calculated for 
injectable new animal drug products.  Typically, injection site residues should be 
less than 10x the muscle safe concentration, or the injection site safe 
concentration calculated using the ARfD. 
 
  When no specific acute toxicological concern has been identified, the safe 
concentration for injection sites is estimated by multiplying the safe 
concentration for muscle by a factor of 10. 
 
  When a specific acute toxicological concern has been identified, the safe 
concentration for the muscle injection site is calculated using the ARfD as 
follows: 
 
 
CVM applies the muscle consumption value for injection site consumption 
(300 g) to determine the safe concentration for injection sites for most new animal 
drugs.  Thus, a conservative estimate of the amount of the injection site tissue 
consumed in a single meal is estimated to be 300 g/day. 
 
 
6 FDA recognizes that some humans weigh less than 60 kg.  These individuals are accommodated by the built-in 
safety factors used to determine an ADI. 
15  Contains Nonbinding Recommendations 
CVM may accept an alternative consumption value or approach for calculating 
the safe concentration with appropriate scientific justification.
16  Contains Nonbinding Recommendations 
 
VI.  Residue Chemistry 
A.  Introduction 
The purpose of the residue chemistry studies is to assess the quantity and nature of 
residues in tissues derived from animals treated with new animal drugs.  Sponsors should 
consult the referenced guidance documents below in Table 3 prior to design and conduct 
of metabolism studies, residue depletion studies, or analytical method validation. 
 
The sponsor should present the results of each study in a format that will facilitate CVM 
review and should include individual animal data from analyses of each sample derived 
from treated animals. 
 
Table 3:  Guidance Documents for Residue Chemistry Studies for the Human Food 
Safety Evaluation 
 
Title  GFI #  VICH GL # 
Studies to Evaluate the Metabolism and Residue Kinetics of  205  46 
Veterinary Drugs in Food-Producing Animals:  Metabolism 
Study to Determine the Quantity and Identify the Nature of 
Residues 
Studies to Evaluate the Metabolism and Residue Kinetics of  206  47 
Veterinary Drugs in Food-Producing Animals:  
Comparative Metabolism Studies in Laboratory Animals to 
Determine the Quantity and Identify the Nature of Residues 
Studies to Evaluate the Metabolism and Residue Kinetics of  207  48(R) 
Veterinary Drugs in Food-Producing Animals:  Marker 
Residue Depletion Studies to Establish Product Withdrawal 
Periods 
Studies to Evaluate the Metabolism and Residue Kinetics of  208  49(R) 
Veterinary Drugs in Food-Producing Animals:  Validation 
of Analytical Methods used in Residue Depletion Studies 
Studies to Evaluate the Metabolism and Residue Kinetics of  243  56 
Veterinary Drugs in Food-Producing Species: Study Design 
Recommendations for Residue Studies in Honey for 
Establishing MRLs and Withdrawal Periods 
Studies to Evaluate the Metabolism and Residue Kinetics of  257  57 
Veterinary Drugs in Food-Producing Species: Marker 
Residue Depletion Studies to Establish Product Withdrawal 
Periods in Aquatic Species 
 
B.  Total Residue and Metabolism Study 
The objective of the total residue and metabolism study is to develop information on the 
amount, persistence, and chemical nature of the total residues, and the metabolic fate of 
the new animal drug in the treated target animals.  The recommended study design for a 
17  Contains Nonbinding Recommendations 
total residue and metabolism study is described in 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. 
 
While GFI #205 describes administering the dose to achieve steady state in edible tissues 
for continuously administered new animal drugs, the dosing period for laying hens and 
the sampling timepoints for eggs are not provided.  For new animal drugs intended for 
continuous use in laying hens, a 12-day dosing regimen after steady state has been 
reached should be used to approximate the time required for complete development of the 
yolk. For continuous use new animal drugs, recommended sampling timepoints for eggs 
are from 2 days before cessation of treatment through 2 days after cessation of treatment. 
For new animal drugs administered to laying hens on a noncontinuous basis, we 
recommend consulting with CVM about the appropriate dosing and sampling periods. 
 
The sponsor may use tissues from the total residue depletion study to determine the 
metabolic fate of the new animal drug.  If the tissues are stored frozen for long periods, 
CVM asks that the sponsor to demonstrate that the metabolites are stable in the frozen 
state, such as by demonstrating that metabolite profiles do not change. 
 
C.  Comparative Metabolism Study 
The purpose of the comparative metabolism study is to determine whether the 
metabolites to which people consuming tissues of treated animals will be exposed are 
also produced by metabolism in the laboratory animals used for the toxicological testing 
(i.e., autoexposure).  Autoexposure, in the context of the current document, refers to 
exposure of the test animal to the administered test article, its metabolites and/or 
breakdown products.  The test animal serves as a surrogate for the human consumer.  The 
recommended study design for a comparative metabolism study is described in 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 to Determine the Quantity and Identify the Nature of Residues. 
 
CVM may ask for separate toxicological studies on a metabolite if it is not tested through 
autoexposure and it is likely to have toxicological potency significantly greater than the 
parent new animal drug.  CVM would use the information obtained from target animals 
on the concentration, persistence, and chemical structure or characterization of that 
metabolite to determine whether separate toxicological testing is desirable. 
 
D.  Selection of Target Tissue and Marker Residue 
The marker residue refers to the residue whose concentration is in a known relationship 
to the concentration of total residue in an edible tissue.  The target tissue refers to the 
edible tissue selected to monitor for residues in the target animal.  The total residue and 
metabolism data should be presented so that it is possible to determine the marker 
residue, the target tissue, and the marker residue to total residue ratio (M:T ratio).  The 
target tissue is usually, but not necessarily, the last tissue in which residues deplete to the 
18  Contains Nonbinding Recommendations 
permitted concentration.  The target tissue and marker residue are selected so that the 
absence of marker residue above the tolerance would confirm that each edible tissue has a 
concentration of total residue at or below its safe concentration.  Typically, for terrestrial 
animals, the target tissue is liver or kidney, but it could also be fat.  Although residues 
persist in injection sites from certain formulations, injection site is not an appropriate 
target tissue because it is not always identifiable at the slaughter plant. 
 
To facilitate clearance of nontarget tissues into the food supply, CVM recommends that 
sponsors provide total residue and metabolism data that would allow the determination of 
the M:T ratio and the marker residue in a nontarget tissue (often muscle or fat for 
terrestrial animals) for purposes of assigning a nontarget tissue tolerance value. 
 
When a new animal drug is intended for use in milk- or egg-producing animals, milk or 
eggs may be a target tissue in addition to one tissue selected by CVM to monitor for 
residues in the edible carcass.  Milk or eggs may be a target tissue because milk or eggs 
enter the food supply independently.  In these cases, it may be necessary to identify a 
marker residue for milk or eggs and one additional edible carcass tissue.  The marker 
residue in milk or eggs may be different from the marker residue selected for the target 
tissue representing the edible carcass, where appropriate. 
 
Honey is the target tissue for drugs used in honey bees and will have its own marker 
residue. 
 
E.  Assignment of Tolerance 
Once a final ADI, safe concentration, target tissue, and marker residue are selected, a 
tolerance for the new animal drug can be determined.  The tolerance is the maximum 
concentration of a marker residue, or other residue indicated for monitoring, that can 
legally remain in a specific edible tissue of a treated animal.7  The tolerance is 
determined by examining depletion data consisting of total residue concentrations 
(typically from radiolabel studies as described in GFI #205) and marker residue 
concentrations measured by the proposed analytical method.  Tolerances for new animal 
drugs approved for use in food-producing animals are listed in 21 CFR part 556. 
 
CVM recommends that sponsors provide data that will allow assignment of a tolerance in 
one or more edible tissues of the target animal.  CVM recommends providing data for 
establishing a tolerance in the target tissue as well as other edible tissues, as needed.  A 
practicable method is required to be submitted by the sponsor when a tolerance is 
assigned to ensure the safe use of the new animal drug (21 CFR 514.1(b)(7)). 
 
CVM may assign the tolerance by harmonizing with previously established international 
safety numbers (MRLs, maximum residue limits).  Tolerances and MRLs are both 
numbers that describe the limits of residues.  However, tolerances and MRLs are not 
 
7 21 CFR 556.1 
19  Contains Nonbinding Recommendations 
derived in the same manner and are used for different purposes.  At times, it may be 
appropriate to harmonize the tolerance with an already established MRL. 
For injectable products, there may be a need to address safety of residues in injection site 
tissue.  Typically, injection site residues should be less than 1) 10x the muscle safe 
concentration, or 2) the injection site safe concentration calculated using the ARfD (see 
section V.F.7.b. Calculation of the Safe Concentrations above) at the timepoint at which 
total residues at the injection site are below the safe concentration. 
 
For certain injectable products, residues at the injection site may persist far longer than 
residues in other edible tissues, including the target tissue. Safety of injection site 
residues may be addressed by lowering the target tissue tolerance with a resultant 
extension of the withdrawal period to ensure that injection site residues are less than the 
injection site safety number.  One procedure that may be used to address safety of 
residues in injection site tissue is to do the following: 
1.  Determine the concentration of the marker residue in the target tissue at the 
timepoint at which total residues at the injection site are below the safe 
concentration. 
2.  Assign a tolerance for the marker residue in the target tissue at that timepoint. 
3.  Confirm that the analytical method performs at the target tissue tolerance. 
4.  Conduct a tissue residue study to assign the withdrawal period based on the 
new target tissue tolerance. 
5.  Confirm that residues at the injection site at the new withdrawal period are less 
than the injection site safety number. 
 
F.  Tissue Residue Depletion Study 
The purpose of the tissue residue depletion study is to provide data to demonstrate the 
depletion of the marker residue post-treatment to below the tolerance and to provide data 
suitable for calculating a withdrawal period or milk discard time.  The recommended 
study design for a tissue residue depletion study is described in GFI #207 (VICH 
GL48(R)), Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary 
Drugs in Food-Producing Animals:  Marker Residue Depletion Studies to Establish 
Product Withdrawal Periods. 
 
Ideally, tissues from treated animals should be collected to provide residue data above the 
tolerance at a minimum of two sampling timepoints and residue data below the tolerance 
(but above the LOQ) at a minimum of one sampling timepoint.  The purpose for 
providing residue data below the tolerance is to avoid excessive extrapolation of the 
linear regression to the proposed withdrawal period. 
 
Depletion studies in laying hens should be designed to provide residue data to 
demonstrate that the tolerance is compatible with zero-day withdrawal.  Layers should be 
treated with the new animal drug as proposed on the labeling.  For continuous use new 
animal drugs, recommended sampling timepoints for eggs are from 2 days before 
20  Contains Nonbinding Recommendations 
cessation of treatment through 2 days after cessation of treatment.  For new animal drugs 
administered to laying hens on a noncontinuous basis, we recommend consulting with 
CVM about the appropriate dosing and sampling periods. 
 
G. Analytical Methods for Residues 
1.  Introduction 
Sponsors must provide a practicable method for analyzing tissue residues as 
described in 21 CFR 514.1(b)(7) except when it is not reasonable to expect the new 
animal drug to become a component of food at concentrations considered unsafe.  For 
purposes of this guidance, a practicable method means an analytical method that 
meets the criteria in 21 CFR 514.1(b)(7).  Typically, the analytical method is 
developed to analyze the marker residue in the target tissue.  The sponsor usually 
develops a method for use in residue chemistry studies. 
 
A method for analyzing the marker residue in the target tissue consists of two 
procedures: the determinative (quantitative) and confirmatory procedures.  The 
determinative and confirmatory procedures may be combined into a single procedure 
as instrumentation allows. 
 
2.  Evaluation Criteria 
Criteria for an analytical method for use in residue chemistry studies are outlined in 
GFI #208 (VICH GL 49(R)), Validation of Analytical Methods Used in Residue 
Depletion Studies.  
 
Criteria for the determinative and confirmatory method are described in this section.  
The method should have the ability to be transferred easily between laboratories and 
performed by competent analysts unfamiliar with the method.  The process of 
obtaining the acceptance of the method by CVM typically consists of up to three 
steps.  It is strongly recommended that the presumptive tolerance be available prior to 
completing the following three steps to ensure that the method performance meets 
regulatory needs: 
1.  The sponsor develops and validates the method. 
2.  CVM reviews the sponsors validation data to determine that the method 
satisfies the performance criteria (a)(1) through (12) below).  In most cases, a 
meeting dedicated to the analytical method may be held with the sponsor to 
determine the suitability of the method for an inter-laboratory study (method 
transfer trial). 
3. The inter-laboratory study (section VI.G.3.b. Inter-laboratory Study of this 
guidance) is conducted to determine whether the method performs as claimed 
and thus can be accepted as a practicable method. 
 
21  Contains Nonbinding Recommendations 
a.  Determinative Procedure 
The determinative procedure quantifies the amount of new animal drug residue 
present in the tested matrices.  The sponsors validation data are evaluated by 
CVM based on the following performance criteria: 
 
(1) Calibration Range and Linearity 
CVM recommends the use of solvent-based calibration standards.  However, 
CVM also will accept standards prepared in extracted matrix provided there is 
acceptable justification for the use of a matrix standard curve that includes an 
isotopically labeled internal standard.  CVM recommends that the linearity of 
the non-weighted calibration curves range from approximately 30-250% of 
the tolerance.  The concentrations of the solvent-based standards for the 
calibration curve should extend beyond the equivalent tissue fortification 
concentrations because CVM allows recoveries to range from 60-110% for 
tolerance <0.1 ppm, or 80-110% for tolerance 0.1 ppm.  If a weighting factor 
is used for the calibration curve, justification for weighting should be 
provided. 
 
(2) Bias (Systematic Error) 
Bias, or systematic error, is the difference of the measured value from the true, 
assigned, or accepted value.  Bias generally is expressed as the percent 
difference between the measured mean concentration and the nominal 
concentration. 
 
Recovery refers to the percentage of the measured analyte concentration 
compared to the known concentration of a spiked sample.  Recovery also may 
be measured for specific parts of a method such as an extraction step 
(extraction recovery).  Method recovery is assessed using control tissue 
fortified with the proposed marker residue.  Assuming acceptable precision, an 
average recovery of 80-110% should be obtained when the designated 
concentration of marker residue (tolerance) is 0.1 ppm or greater.  An average 
method recovery of 60-110% is acceptable when the designated concentration 
of marker residue (tolerance) is less than 0.1 ppm.  Higher recoveries (greater 
than 110%) may indicate a lack of specificity. 
 
(3) Precision and Accuracy 
Precision is a measure of the variability of repetitive measurements.  
Contributions to variability from numerous sources affect precision, but the 
major components are those from different laboratories (reproducibility) and 
those from within a laboratory (repeatability).  Precision is usually expressed 
as a standard deviation or coefficient of variation (relative standard deviation).  
CVM recommends expressing precision as the coefficient of variation (CV) 
because it is relatively constant over a considerable concentration range. 
22  Contains Nonbinding Recommendations 
 
The within-laboratory CV should not exceed 10% where the designated 
concentration of marker residue (tolerance) is greater than or equal to 
0.1 ppm.  Where the designated concentration of marker residue (tolerance) is 
less than 0.1 ppm, the within-laboratory CV should not exceed 20%. 
 
Accuracy is defined as the closeness of agreement between the measured 
value and an accepted reference value.  CVM will evaluate accuracy of the 
method based on both bias and precision (repeatability). 
 
(4) Limit of Detection (LOD) and Lower Limit of Quantitation (LLOQ) 
The method should be capable of reliable detection and measurement of 
concentrations around the tolerance of the analyte (drug marker residue).  In 
order to estimate the LOD signal, the standard deviation of the signal 
(analytical response) from control samples is multiplied by a factor related to 
the acceptable probability of false positive and false negative (refer to 
GFI #208, Annex 2).  A factor of 3.3 is commonly used to estimate the LOD 
signal.  The factor of 3.3 corresponds to a 5% probability of both false 
positive and false negative.  In order to estimate the LLOQ signal, the 
standard deviation of the signal from control samples may be multiplied by a 
factor of 10.  For a calibration-based method, the standard error of the 
calibration curve intercept is an acceptable substitute for the standard 
deviation of the response from control samples.  The respective LOD and 
LLOQ signals are then converted to concentrations using the slope of the 
calibration function. 
 
(5) Robustness and Ruggedness 
The final version of the method should be optimized for robustness and 
ruggedness based on experimental data.  For example, robustness of a LC-
MS/MS method is demonstrated by using different lots of Solid Phase 
Extraction (SPE) and High-Performance Liquid Chromatography (HPLC) 
columns.  Ruggedness is a measure of the transferability of the method 
between operators, laboratories, instrumental platforms, or long intervals of 
time.  Examples of demonstrating ruggedness of a LC-MS/MS method may 
include the use of different MS platforms from multiple manufacturers and 
different LC systems.  Ruggedness is assessed by performing the analyses on 
identical samples.  The method should be performed by a competent analyst 
who was not involved in the development of the method in order to verify the 
adequacy of the method's description and to identify critical points. 
 
(6) Specificity and Selectivity 
Specificity is the ability of the method to respond only to the substance being 
measured, irrespective of other substances and materials present in the matrix.  
Selectivity of the analytical method is the extent to which substances affect 
23  Contains Nonbinding Recommendations 
the determination of the marker compound of interest according to the 
procedure.  The method should demonstrate the absence of the following 
aspects of interference: 
  Matrix Interference:  No interference from the control tissue greater 
than 10% of the tolerance in the retention time window of the analyte. 
  Interference from other new animal drugs:  No interference from other 
compounds of similar chemical structure approved for use in the same 
target species. 
  Carryover:  Method should be free of carryover during or between runs.  
No interference from carryover peak greater than 10% of the tolerance. 
 
(7) Parallelism 
Parallelism is the ability to dilute samples with analyte concentrations above 
the upper limit of quantitation (ULOQ).  The sponsor should provide data to 
demonstrate that using control matrix extract to dilute extracts of samples with 
initial concentration above ULOQ does not compromise the accuracy and 
precision of the method following re-analysis of the diluted sample. 
 
(8) Stability 
Data should be provided in support of the following aspects of stability of the 
analyte (marker residue): 
  Stability of stock solutions of standards of the analyte and internal 
standard. 
  Stability of analyte in the matrix (short-term stability, long-term 
stability, and freeze-thaw stability). 
  Stability of the analyte in extracts. 
 
(9) System Suitability 
CVM recommends that the method protocol include system suitability criteria 
that will evaluate instrument performance prior to an analytical run.  In 
particular, to aid the transferability of the method between the different LC-
MS platforms in government laboratories, CVM recommends that the sponsor 
include in their system suitability criteria the requirement that the CV for the 
ratios of analyte area to internal standard peak area for the suitability solutions 
should be below 6%. 
 
(10) Additional Criteria 
The method should: 
(a) Utilize commercially available reagents, supplies, instruments (except 
that new or unusual reagents or standards may be used but it would be 
24  Contains Nonbinding Recommendations 
the responsibility of the sponsor to ensure these are available in the 
future upon request); 
(b) Be capable of being performed by competent analysts; 
(c) Be capable of being completed within reasonable time periods 
consistent with regulatory objectives (usually no more than 48 hours of 
total elapsed time); 
(d) Not need unique instrumentation, large quantities of solvents, reagents, 
and supplies which would render the method economically impractical; 
and 
(e) Be capable of being performed safely. 
 
(11) Confirmatory Procedure 
The confirmatory procedure unequivocally identifies the marker residue 
present in the tested matrices. 
 
As with the determinative procedure, the performance criteria for the 
confirmatory procedure are evaluated using tolerance-related concentrations.  
CVM recommends the use of solvent standards for comparison with sample 
extracts during the confirmation procedure.  Whenever possible, the 
confirmatory procedure should be performed on the same sample extracts 
used for the determinative portion of the method.  Details of the performance 
criteria for a confirmatory procedure for a marker residue are described in GFI 
#118, Mass Spectrometry for Confirmation of Identity of Animal Drug 
Residues. 
 
(12) Adaption of a Previously Validated Method 
Instead of developing and validating a new method for the marker residue, the 
sponsor may elect to adapt a method that was validated in a government 
laboratory (e.g., FDA/ORA, USDA/FSIS), or a method designated as an 
Official Method by the Association of Analytical Chemists (AOAC).  
Typically, an AOAC method is not validated at the tolerance while the 
practicable method is validated around the tolerance.  When adapting a 
previously validated method, the sponsor will need to provide only partial 
validation data to demonstrate that the method can be satisfactorily performed 
in the sponsors laboratory.  CVM recommends that, before developing and 
validating a new method for the marker residue, the sponsor should determine 
if there is a previously validated government laboratory or AOAC method that 
they can adapt.  Adaption of an FDA or AOAC validated method may reduce 
the extent and expense of the method transfer trial study that is part of the 
method approval process. 
 
25  Contains Nonbinding Recommendations 
3.  Specific Data Needed 
a.  Sponsor's Submission 
In the development of a method, sponsors should collect data from three types of 
samples:  (a) control target tissue from untreated animals; (b) fortified target 
tissue containing known concentrations of the marker residue added to the sample 
of control target tissue; and (c) incurred target tissue from animals of the target 
species that have been treated with the new animal drug. 
 
In presenting a submission for evaluation of a proposed method, the sponsor 
should provide: 
  A complete description of the method (method SOP), including sampling, 
preparation of analytical samples, storage conditions, reagents, 
instrumentation, standards, identification of critical steps, and stopping 
points. 
  Quality control criteria necessary to verify and maintain method 
performance. 
  A typical solvent standard curve prepared from marker residue of known 
purity (or matrix matched curve that includes an isotopically labeled 
internal standard). 
  Data derived from control, fortified, and incurred tissue samples showing 
that the method meets the specificity, precision, and bias attributes. 
  Relevant worksheets, calculations, statistical analyses, chromatograms, 
etc., from the analyses of control, fortified, and incurred tissue samples. 
  Using the proposed determinative procedure of the method, results of 
analyses of the following samples (as a minimum): 
5 control tissues 
5 control tissues fortified with marker residue at 0.5X tolerance 
5 control tissues fortified with marker residue at 1X tolerance 
5 control tissues fortified with marker residue at 2X tolerance 
5 tissues containing biologically incurred marker residue at 
approximately its tolerance 
  Using the proposed confirmatory procedure, results of analyses to verify 
the identity of the marker residue with the following samples (as a 
minimum): 
5 control tissues to ensure the absence of false positives 
26  Contains Nonbinding Recommendations 
5 control tissues fortified with marker residue at tolerance 
5 tissues containing biologically incurred marker residue at 
approximately its tolerance 
 
b.  Inter-laboratory Study 
After the method has passed validation data have been reviewed and accepted 
(section VI.G.2. Evaluation Criteria, step 2, above), FDA may conduct an inter-
laboratory method transfer trial of the proposed method.  The inter-laboratory 
study, also termed a method transfer trial, is designed to validate the 
reproducibility (ruggedness) component of the precision of the method.  The 
method will be tested in one FDA laboratory, the sponsors reference laboratory 
that developed and validated the procedure, and two independent testing 
laboratories selected by the sponsor.  One USDA laboratory may optionally 
participate in the method transfer trial.  The method transfer trial is conducted 
using target tissues supplied by the sponsor.  In addition to the sponsors reference 
laboratory in which the method was developed, the method transfer trial 
participants are the independent laboratories and the FDA laboratory, that are 
naive laboratories with no previous experience with the method.  Only the FDA 
laboratory conducts a validation of the confirmatory procedure.  To evaluate the 
performance of the determinative procedure, the data from the naive laboratories 
are compared to the data obtained in the sponsors reference laboratory.  During 
the method transfer trial, the results for each day of analysis in the sponsors 
reference and testing laboratories are sent to the sponsor and to the CVM method 
transfer trial coordinator for review.  The results from the FDA laboratory are sent 
only to the CVM method transfer trial coordinator.  The inter-laboratory study is 
supervised by CVM.  Adaption of an FDA or AOAC validated method may 
reduce the size of the method transfer trial study. 
 
Each of the three laboratories taking part in the method transfer trial will analyze 
the following samples by the determinative procedure: 
5 control tissues 
5 control tissues fortified with marker residue at 0.5X tolerance 
5 control tissues fortified with marker residue at 1X tolerance  
5 control tissues fortified with marker residue at 2X tolerance 
5 tissues containing biologically incurred marker residue at each of two 
concentrations at approximately 0.5X and 1X, respectively. 
 
The samples analyzed by the participating laboratories are blinded and 
randomized.  Both batch sequence and individual sample analysis within an 
analytical batch are randomized. 
 
The confirmatory procedure is performed in the FDA laboratory on the same 
extracts used for the determinative portion of the method. 
 
27  Contains Nonbinding Recommendations 
If the sponsor elects to adapt a method that was developed in a government 
laboratory, the sponsor may elect to conduct a collaborative study with that 
government laboratory by providing the aforementioned tissues to the government 
laboratory (single lab validation (SLV)).  If the sponsor elects to adapt a method 
that is being validated, or already designated as an Official Method by the 
AOAC, then the sponsor may have the option to conduct a collaborative study 
with the AOAC following validation experiments recommended by CVM. 
 
H. Establishing a Withdrawal Period 
The withdrawal period or the milk discard time is the interval between the time of the last 
administration of a new animal drug and the time when the animal can be safely 
slaughtered for food or the milk can be safely consumed.  When a tissue withdrawal 
period or milk discard time is assigned pursuant to an approved application, neither 
tissues nor milk collected during treatment and during the withdrawal period or milk 
discard time are safe and, as a result, cannot be used for human consumption.  See section 
512 of the FD&C Act [21 U.S.C. 360b].  For convenience, this guidance will use the 
withdrawal period to refer to both the withdrawal period and the milk discard time. 
 
This guidance describes a procedure for establishing a withdrawal period that is based on 
a statistical tolerance limit procedure (De Gryze et al., 2007).  Tolerance limits explicitly 
account for the degree of variation in the background population and the size of the 
sample of measurements used.  When determining the withdrawal period, CVM uses the 
99th percentile tolerance limit with a 95% confidence (ITRC 2013).  The withdrawal 
period is determined when the tolerance limit on the residue concentration is at or below 
the permitted concentration.  A tolerance limit provides an interval within which a given 
percentile of the population lies, with a given confidence that the interval in fact contains 
that percentile of the population.  This means that 99% or more of tissue or milk residue 
samples at and beyond the withdrawal period (or discard time) are expected to fall below 
the tolerance. 
 
The statistical program using R to calculate the withdrawal period and examples 
(vignettes) are available upon request by contacting AskCVM@fda.hhs.gov. 
 
If the calculated withdrawal period is a fraction of a day or milking, CVM will establish 
the withdrawal period as the next day or milking. 
 
A zero-day withdrawal period usually will be assigned when total residues from a 1X 
study are below 12 the safe concentrations or when total residues from a 1.5X study are 
below 1X the safe concentrations.  Typically, drugs that qualify for zero-day withdrawal 
are assigned a tolerance and an analytical method for monitoring compliance with that 
tolerance should be provided.  CVM recommends that the sponsor provide residue 
depletion data to ensure that the tolerance assignment is consistent with a zero-day 
withdrawal period determined from the radiolabeled study.  A single timepoint residue 
depletion study may be conducted to demonstrate that the marker residue is less than the 
tolerance at a zero-day withdrawal period. 
 
28  Contains Nonbinding Recommendations 
I.  Statistical Analysis of the Residue Data 
1.  Theory 
The depletion of new animal drug residues in an animal is assumed to follow 
exponential elimination kinetics.  This means that the concentration of residues as a 
function of time after stopping treatment of a drug can be described as the sum of one 
or more exponential terms (Equation 1). 
 
 
where: 
C is the concentration at time t; 
t
C  is the extrapolated concentration at time=0 and the ith exponential 
0,i
term; and 
 is the rate constant corresponding to the ith exponential term. 
i
 
CVM makes a simplifying assumption for analysis of residue data that the depletion 
curve, during the phase of the depletion closest to the established tolerance, can be 
represented by a single exponential equation.  This assumption enables the use of 
ordinary least squares (OLS) regression methods to estimate the intercept (b ) and 
0
slope (b ) of log-linear depletion curves in either groups or individual animals 
1
(Equation 2).8  The intercept and slope of the line are used to estimate the time at 
which the concentration in tissue or milk achieves the target tolerance concentration.  
The straight-line fit of concentration data relies on natural logarithm transformation 
of the dependent variable (Equation 2). 
 
  ln(y) = ln(b ) b x 
i 0 1 i
where: 
ln( ) = is the natural log of the regression-estimated concentration at x ; 
i i
x = the ith sampling period (in units of time) for either tissue or milk 
i
samples; 
b  = regression-estimated intercept of the elimination curve; and 
0
b  = the estimate of the slope (regression coefficient) from the OLS 
1
regression. 
 
Several measures of statistical confidence are used to describe the possible range of 
values given the variability among the observations.  These confidence limits include 
the confidence limits on the regression line and the confidence limits for a new 
estimate of concentration (taken as ln( )) given a new value of x (also known as 
prediction limits Myers, 2000), and the tolerance limits for the new estimates of 
concentration.  The tolerance limits de syycribe an interval in which a specified 
 
8 A variety of notation is used to describe the estimates of intercept and slope (intercept, slope) from linear 
regression including (b ,b ), (a,b) and (b,m). 
0 1
29  Contains Nonbinding Recommendations 
proportion (P) of the sampled population is expected to fall given the current 
observations (De Gryze et al., 2007).9  If the true regression parameters for the 
intercept and slope (, ) are known, then the tolerance limits and confidence limits 
0 1
on the new estimate of concentration are the same.  Because the true regression 
parameters are seldom known and must be estimated from the observations, the 
tolerance and prediction (confidence) limits differ.  Tolerance intervals may be either 
two-sided (for which the estimated parameter is expected to lie between a lower and 
upper limiting value) or one-sided, (in which the estimated parameter is expected to 
lie above or below a specified limit).  CVM uses a one-sided tolerance limit to set an 
upper limit on the concentration of residue in tissue or milk. 
 
CVM uses a 99th percentile tolerance with 95% confidence as an upper limit of 
residue concentration in either tissue or milk.  In general terms, this means that a new 
individual value of concentration, randomly sampled from animals at the proposed 
withdrawal or discard period, is expected to have only a 1% chance of exceeding the 
established tolerance limit.  The purpose of the regression error (variance) analysis is 
to calculate uncertainty (e.g., y  s) in the estimated concentration near the proposed 
tissue withdrawal period or milk discard time.  CVM solves for a factor, k, that 
adjusts the regression-estimated error (s) for the 99th percentile tolerance with 95% 
confidence.  In the form of a linear equation (y = b + mx) the residue calculation to 
estimate y from a new value of x  (Equation 3): 
0
 
 
 
The modifying factor, k, is derived from the noncentral t-distribution, and it is a 
function of degrees of freedom, the distance from the expected value of  , the 
desired proportion of coverage for future values (P=0.99) and the confidence level for 
the difference ( ).  The noncentral t-distribution can be obtainelnd( fyyro)m 
tables of the noncentral t-distribution (Owen 1968) or statistical software. 
  1=0.95
CVM solves Equation 3 for the value of x = T, the tissue withdrawal period or milk 
discard time.  Given simultaneous unknown values in Equation 3, the value of x, such 
that concentration estimated at x = T satisfies the 99/95 tolerance condition, is found 
iteratively either by hand or computer program.  The general location of the tolerance 
limit, the withdrawal period or discard time (T) and the various OLS regression 
results are shown in Figure 2. 
 
9 See De Gryze, S; Langhans, I; Vandebroek, M (2007) for a discussion of tolerance intervals from linear regression. 
30  Contains Nonbinding Recommendations 
 
 
Figure 2.  Hypothetical Residue Depletion Curve and 
Withdrawal Period Estimate. 
A least squares fit of the data (circles) is shown as a decreasing, 
straight and small-dashed line through the data.  The 95% 
prediction bands and the 99th percentile tolerance for the upper 
95% confidence limit are shown.  If the regulatory limit is the 
value of concentration marked by the asterix (*=2 units), then 
the regression analysis predicts that the withdrawal period (T*) 
is about 12.5 sampling period units determined by where the 
tolerance band crosses the desired limit.  Note that the tolerance 
band is not drawn to scale for ease of presentation:  the 
tolerance generally lies much closer to the upper 95% 
prediction band. 
 
Calculation of the tolerance from the linear regression data relies on first calculating the 
amount of variation among the observations (samples).  The sources of variation 
include both the within-animal variation as a function of the sampling time, and 
variation from the difference among the animals.  Laboratory analytical variability also 
contributes to overall variability.  The assumptions for OLS regression include that the 
samples are independent from each other; the residue assays are independent from 
each other and from the animal in question; the depletion of the concentration of 
residue is log-linear with time; and the measured log(concentration)s of residue are 
distributed normally and have a constant variation over the time periods.  The validity 
of the assumptions for an experiment can be examined using analysis of variance for 
regression and various diagnostic statistics (Draper and Smith 1998; Myers 2000).  
Generally, statistical software or spreadsheets with statistical functions can be used to 
calculate the intercept, slope, and regression diagnostics. 
31  Contains Nonbinding Recommendations 
2.  Statistical Software:  Residues in Tissue and Milk 
Most commercial, off-the-shelf statistical programs include tools for ordinary linear 
regression (OLS), generalized linear models (GLM) and analysis of variance (AOV).  
Commercial spreadsheet software generally includes OLS and AOV functions.  
Open-source software, such as R statistical software and Python Scipy, is freely 
available and has extensive libraries of packages for OLS, GLM, and AOV (R Core 
Team, 2013; Python, 2015).  Although the formulae used in regression and AOV 
might appear complex, they all derive from elementary sample statistics that can be 
calculated manually or using built-in software functions.  CVM has developed 
statistical programs for fitting tissue and milk residue data according to the rationale 
discussed in this guidance and in Martinez et al. (2000).  The programs (in R or 
SAS) are available freely upon request as open-source programs and without any 
expressed warranties for their use. 
 
In contrast to carcasses, which are individually sampled by USDA in the residue 
monitoring program, milk from an entire dairy herd is pooled in the bulk tank before 
sampling in a compliance program.  Therefore, the statistical procedure for 
calculating the milk discard time must contain a term for the expected number of 
cows contributing milk to the pooled sample.  To approximate the size of a small 
dairy operation, CVM uses 10 for that value. 
 
Alternative methods of analysis for residues are well known, and alternatives are 
recommended by the European Union and others (FAO, 2003).  The approach 
described in this guidance was discussed in detail as described in Martinez, et al., 
2000.  CVM believes that the default methods described here are adequate for setting 
operational withdrawal periods or milk discard times.  Essentially, CVM addresses 
the milk discard time analysis more as decision analysis than as a search for the best 
statistical fit of the entire residue depletion curve.  The latter is focused on statistical 
rationale for the optimal statistical fit of all the data while CVM methods seek a 
reasonable estimate of the time at which the concentration falls below a 99th 
percentile tolerance. 
 
VII.  Carcinogenic Compounds 
FDA cannot approve a compound for use as a new animal drug in food-producing animals when 
the compound or any of its metabolites has been found to cause cancer in animals or humans, 
unless:  1) the compound will not adversely affect the animals for which it is intended, and 2) no 
residue of the compound will be found by approved regulatory methods in any edible tissues (see 
section 512(d)(1)(I)) of the FD&C Act).  The regulations in 21 CFR part 500, subpart E entitled 
Regulation of Carcinogenic Compounds Used in Food-Producing Animals ( 500.80 through 
500.92) describe the requirements for demonstrating that no residue of the new animal drug will 
be found by an approved regulatory method in any edible tissues of or in any foods yielded from 
the animal.  Contact CVM for additional information regarding carcinogenic compounds used in 
food-producing animals.
32  Contains Nonbinding Recommendations 
 
VIII.  References 
1.  De Gryze, S., Langhans, I., and Vandebroek, M., Using the correct intervals for prediction: 
A tutorial on tolerance intervals for ordinary least-squares regression. Chemometrics and 
Intelligent Laboratory Systems, 87:147-154, 2007. 
 
2.  ITRC (Interstate Technology & Regulatory Council). Groundwater Statistics and Monitoring 
Compliance, Statistical Tools for the Project Life Cycle.  GSMC-1. Washington, DC: 
Interstate Technology & Regulatory Council, Groundwater Statistics and Monitoring 
Compliance Team.  http://www.itrcweb.org/gsmc-1/, 2013. 
 
3.  Myers, R.H., Classical and Modern Regression with Applications, Second Ed., Pacific 
Grove, CA: Duxbury Press, 2000. 
 
4.  Owen, D.B., A Survey of Properties and Applications of the Non-central t-Distribution. 
Technometrics, 10:445, 1968. 
 
5.  Draper, N.R. and Smith, H., Applied Regression Analysis, Third Edition, Wiley, New York, 
1998 
 
6.  R Core Team, R: A language and environment for statistical computing. R Foundation for 
Statistical Computing, Vienna, Austria.  https://www.r-project.org/, 2013. 
 
7.  Python  https://www.python.org/about/, 2015. 
 
8.  Martinez, M., Friedlander, L., Condon, R., Meneses, J., ORangers, J., Weber, N. and Miller, 
M. Response to criticisms of the US FDA parametric approach for withdrawal time 
estimation:  rebuttal and comparison to the nonparametric method proposed by Concordet 
and Toutain. J. Vet. Pharmacol. Therap. 23: 21-35, 2000. 
 
9.  FAO (Food and Agriculture Organization of the United Nations), Software-based workbook 
for statistical evaluation of residue depletion data for veterinary drugs, 2003.  
http://www.fao.org/food/food-safety-quality/scientific-advice/jecfa/guidelines0/residue-
depletion/en/ 
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