#264 
 
Standardized Medicated Feed Assay 
Limits 
 
Guidance for Industry 
 
Draft Guidance 
 
This guidance document is being distributed for comment purposes only. 
 
Submit comments on this draft guidance by the dated provided in the Federal Register notice 
announcing the availability of the draft guidance. 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 docket number FDA-2019-D-5664. 
 
For further information regarding this document, contact Katie Ciesienski, Center for Veterinary 
Medicine (HFV-141), Food and Drug Administration, 7500 Standish Place, Rockville MD 
20855, 240-402-0676, email: katie.ciesienski@fda.hhs.gov.  
 
Additional copies of this draft guidance document may be requested from the Policy and 
Regulations Staff (HFV-6), Center for Veterinary Medicine, Food and Drug Administration, 
7500 Standish Place, Rockville MD 20855, and may be viewed on the Internet at either 
https://www.fda.gov/animal-veterinary or https://www.regulations.gov. 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine (CVM) 
February 2020    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
Standardized Medicated Feed Assay Limits 
 
 
Draft Guidance for Industry
 
This draft guidance, when finalized, will represent the current thinking of the Food and Drug 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  
 
CVM establishes assay limits for drugs in medicated feeds (i.e., Type B and Type C medicated 
feeds) as part of the approval process for Type A medicated articles.  The term assay limits 
refers to how much the measured amount of drug can deviate from the labeled amount of drug.  
The assay limits for Type B and Type C medicated feeds are specified as a range and published 
in 21 CFR 558.4(d).  When a medicated feed assay value falls within this range using a 
validated method accepted by CVM, it indicates that the measured drug level in medicated feed 
is consistent with the labeled drug level.  The assay limits are not intended to permit a feed 
manufacturer to add more or less than the labeled amount of drug to the medicated feed.  Assay 
limits are used pre-approval to ensure that medicated feeds in Target Animal Safety (TAS), 
Effectiveness (EFF), Chemistry, Manufacturing, and Controls (CMC), Bioequivalence (BE), and 
Human Food Safety (HFS) residue chemistry studies contain the appropriate amount of drug, and 
post-approval for compliance and customer service purposes. 
 
This guidance recommends a standardized set of assay limits for medicated feeds.  Standardized 
medicated feed assay limits allow predictability in the review process as sponsors can determine 
early in the drug development process what assay limits they should expect to meet for 
medicated feeds used in TAS, EFF, CMC, and HFS residue chemistry studies.  The 
implementation of standardized medicated feed assay limits does not lower or otherwise change 
the current standards for safety and effectiveness and does not change the expectation regarding 
medicated feed assay methods, medicated feed assay method validation, and method transfer 
studies (see Guidance for Industry (GFI) #135, Validation of Analytical Procedures for Type C 
Medicated Feeds,1 GFI #136, Protocols for the Conduct of Method Transfer Studies for Type 
C Medicated Feed Assay Methods,2 and GFI #137, Analytical Methods Description for Type 
C Medicated Feeds3).  CVM will not require any additional medicated feed studies beyond 
those already required in the CMC technical section due to the implementation of standardized 
                                                 
1 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-135-validation-analytical-
procedures-type-c-medicated-feeds 
2 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-136-protocols-conduct-
method-transfer-studies-type-c-medicated-feed-assay-methods  
3 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-137-analytical-methods-
description-type-c-medicated-feeds  
2    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
medicated feed assay limits.  CVM does not intend to tighten any previously approved medicated 
feed assay limits but would be willing to consider widening previously approved medicated feed 
assay limits to match the assay limits in this guidance at the request of the pioneer Type A 
medicated article sponsor.  Sponsors should request a meeting with CVM if they wish to discuss 
widening previously established medicated feed assay limits.  Sponsors of generic Type A 
medicated articles may not initiate a change to widen the medicated feed assay limits and should 
follow the assay limits listed in 21 CFR 558.4(d).  CVM would also be willing to discuss 
alternate medicated feed assay limits with sponsors on a case-by-case basis (e.g., for medicated 
feeds with drug inclusion rates below 5 ppm4), but this type of discussion should occur early in 
the drug development process, based on the sponsor's knowledge of their drug, assay 
methodology, and proposed indications.  
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
the word should in Agency guidances means that something is suggested or recommended, but 
not required. 
 
II.  Background  
 
Medicated feed assay limits are based on Permissible Analytical Variability (PAV).  Previously, 
CVM used an equation for PAV that quantified the variability due to the analytical method and 
adjusted for the other variations that have not been quantified.5  
 
PAV = (2 x %CV) + 5% 
 
In this equation, %CV = (standard deviation/mean) x 100% and was derived from the feed assay 
method transfer study results for fortified feed samples according to GFI #136; 5% was added to 
account for the sources of variability that are not quantified.  The following equation was used to 
calculate the assay limits:  
 
Assay Limits = % Recovery  PAV, 
 
where % Recovery was obtained from the feed assay method transfer study results for fortified 
feed samples (see GFI #136).  PAV values typically ranged from 15% to 20%; however, CVM 
has required tighter assay limits and accepted wider assay limits for some medicated feeds on a 
case-specific basis.  A major limitation to this approach was that medicated feed assay limits 
were not finalized until the CMC technical section was complete.  In many cases, significant 
work on safety and effectiveness had already been conducted prior to establishing the medicated 
feed assay limits, and that work had to be reassessed if medicated feeds used in those studies 
assayed outside of the finalized medicated feed assay limits.  
 
                                                 
4 5 ppm is equivalent to 4.5 g/ton. 
5 Association of American Feed Control Officials (AAFCO) Official Publication 1985, pp 88  90. 
3    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
III. Standardized Assay Limits for Medicated Feeds
This guidance recommends a standardized set of PAVs for medicated feeds, based on feed type 
and drug inclusion rate.  For the purpose of determining PAVs, medicated feeds are categorized, 
based on similarity, into two main types: 1) swine, poultry, and minor avian; and 2) cattle and 
minor ruminant.  In general, swine, poultry, and minor avian feeds are similar with respect to 
their flow and handling characteristics, including particle size, particle size distribution, moisture 
content, and composition; thus, it is reasonable to assign the same standardized PAVs to the 
medicated feeds for these species.6  Likewise for cattle and minor ruminant feeds.  Due to the 
use of unique manufacturing processes and lack of historical data, the PAV for other species 
(e.g., fish, exotic animals) will be determined on a case-by-case basis.  PAVs for each feed type 
will vary, as shown below, depending on the drug inclusion rate. 
PAVs for Medicated Feeds7 
PAV  PAV  PAV 
Species 
< 25 ppm  25 - 500 ppm  >500 ppm
Swine, poultry, minor avian  20%  15%  15% 
Cattle, minor ruminant  25%  20%  15% 
Standardized medicated feed assay limits are calculated by adding and subtracting the standard 
PAV from 100%, unless the sponsor has data to show that the typical recovery is not 100%, in 
which case the sponsor could propose that the assay limits be centered on the average percent 
recovery.  If a Type A medicated article is intended for use in multiple species or is used across 
medicated feed inclusion rate ranges that cross 25 or 500 ppm, there could be more than one set 
of approved medicated feed assay limits for that product. 
For free-choice medicated feeds,8 CVM suggests using the same PAVs listed in the table above; 
however, sponsors may meet with CVM on a case-by-case basis to discuss alternate PAVs for 
these types of products. 
The details of how CVM calculated the standardized PAVs for medicated feeds are provided as 
an appendix to this guidance. 
IV. Appendix: CVMs Calculation of Standardized PAVs for Medicated Feeds
The standardized PAVs are obtained from two contributing factors: analytical method variability 
and mixing/manufacturing variability,  
6 Chiba, L. Animal Nutrition Handbook, Section 12: Poultry Nutrition and Feeding, 2014. 
7 25 and 500 ppm are equivalent to 22.7 and 453.6 g/ton, respectively. 
8 Free-choice medicated feeds contain one or more animal drugs and are placed in feeding and grazing areas but are 
not intended to be fully consumed at a single feeding.  These include medicated blocks, mineral mixes, and liquid 
feeds (see 21 CFR 510.455). 
4  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
PAV = Analytical Method Variability + Mixing/Manufacturing Variability. 
 
Analytical method variability, as a percent, is calculated using the Horwitz equation for predicted 
interlaboratory variability ( )9 as follows: 
H
 
  = 2C-0.15, 
H
  
where C is the concentration of the drug in the medicated feed expressed as a dimensionless 
fraction (e.g., 1 g/g, C=10-6 g/g). In the Horwitz equation, the predicted interlaboratory 
variability is the overall measure of analytical variability, including the intralaboratory 
component.  
 
The mixing/manufacturing variability represents the variability in the uniformity of a batch of 
medicated feed.  The uniformity of a batch of medicated feed is generally assessed by assaying 
samples across the batch and calculating the variability of the assay results.  Therefore, the 
uniformity variability includes the analytical variability associated with the method used to assay 
the samples (i.e., the intralaboratory analytical variability).  Because the amount of total 
analytical variability, both inter- and intra-laboratory variability, is accounted for using the 
Horwitz equation, the intralaboratory analytical variability is subtracted from the uniformity 
variability to obtain the mixing/manufacturing variability, as shown in the equation below.  
 
Mixing/Manufacturing Variability = Uniformity Variability  Intralaboratory Analytical Variability. 
 
Historically, an intralaboratory analytical method variability, %CV, of 5% was applied to drug 
inclusion levels of greater than 10 ppm and a %CV of 7.5% was applied to drug inclusion levels 
of 10 ppm or less.  These %CV values and the 10 ppm drug inclusion cut-off for analytical 
variability were established using medicated feed assay data from sponsors of New Animal Drug 
Applications (NADAs) for Type A medicated articles and the values published in GFI #135.  
More recently, in an effort to reevaluate the drug inclusion cut-off for analytical variability, 
CVM analyzed blinded medicated feed assay data provided by NADA sponsors for cattle, 
poultry, and swine medicated feeds and determined that for this guidance, the drug inclusion cut-
off will be set at 25 ppm.10  For ease of use, an intralaboratory analytical method variability 
value of 8% is applied to medicated feed concentrations less than 25 ppm and a value of 5% is 
applied to medicated feeds with concentrations of 25 ppm or greater.  It is noted that the assigned 
intralaboratory analytical method variability is in line with Horwitz study results, which found 
that intralaboratory variability is usually one-half to two-thirds the predicted interlaboratory 
                                                 
9 Vander Heyden, Y., Smeyers-Verbeke, J., Massart, D., Benchmarking for Analytical Methods: The Horwitz 
Curve, LCGC Europe, Vol. 18, Issue 10, pp 528  531, October 2005. 
10 CVM evaluated the data by applying the standardized medicated feed assay limits in this guidance at drug 
inclusion cut-off values of 10, 25, 50, and 100 ppm.  The proportion of data that fell within the assay limits did not 
meaningfully increase as the drug inclusion cut-off values increased from 25  100 ppm.  Therefore, 25 ppm was 
selected as the drug inclusion cut-off. 
5    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
variability ( ).11, 12  At low concentrations, such as 5 ppm, the intralaboratory analytical method 
H
variability can be calculated as 2    = 8%, and at the drug inclusion cut-off of 25 ppm the 
3 H  
intralaboratory analytical method variability can be calculated as 12   = 5%. 
H .
 
For uniformity variability, it is generally accepted that a %CV for uniformity across the batch of 
10 - 15% is considered good, while a %CV of 15 - 20% is regarded as fair.13  A uniformity 
variability of 15% is applied to swine, poultry, and minor avian medicated feeds less than 25 
ppm and 10% uniformity variability is applied to swine, poultry, and minor avian medicated 
feeds greater than or equal to 25 ppm.  It is known that mixing drugs in cattle feed 
homogenously is difficult due to the nature of the feed (e.g., particle size, feed ingredients).  
Therefore, a uniformity variability of 20%, 15%, and 10% is applied to medicated cattle and 
minor ruminant feeds at concentrations of less than 25 ppm, 25 - 500 ppm, and greater than 500 
ppm, respectively, where 500 ppm could be either a concentrated Type C or Type B medicated 
feed.  
  
Standardized PAVs are calculated as follows: 
 
PAV = 2C-0.15 + (Uniformity Variability  Intralaboratory Analytical Variability), 
 
where, 
 
C = medicated feed concentration as a dimensionless fraction (e.g., 1 g/g, C=10-6 g/g) 
 
Uniformity Variability =  
 
Medicated  Medicated  Medicated 
Feed  Feed  Feed 
Species 
Concentration  Concentration  Concentration 
< 25 ppm  25 - 500 ppm  > 500 ppm 
Swine, poultry, minor avian  15%  10%  10% 
Cattle, minor ruminant  20%  15%  10% 
 
Intralaboratory Analytical Variability =  
 
Medicated Feed Concentration  Intralaboratory variability 
< 25 ppm  8% 
 25 ppm  5% 
 
                                                 
11 See footnote 9. 
12 Horwitz, W. Evaluation of Analytical Methods Used for Regulation of Foods and Drugs. Analytical Chemistry, 
Vol. 54, No. 1, pp 67  76, January 1982. 
13 Herman, T., Behnke, K. Testing Mixer Performance. MF1172. Kansas State University Agricultural Experiment 
Station and Cooperative Extension Service Bulletin, Manhattan, KS: Kansas State University, October 1994.  
6    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
The recommended PAVs are derived as shown below, where 5 ppm is considered a low drug 
inclusion concentration, 25 ppm is the drug inclusion cut-off, and 500 ppm could be either a 
concentrated Type C or Type B medicated feed.  
 
For swine, poultry, minor avian: 
 
5 ppm: PAV = 2(0.000005)-0.15 + (15% - 8%) = 12% + 7% = 19%  
25 ppm: PAV = 2(0.000025)-0.15 + (10% - 5%) = 10% + 5% = 15% 
500 ppm: PAV = 2(0.0005)-0.15 + (10% - 5%) = 6% + 5% = 11% 
 
For cattle, minor ruminant: 
 
5 ppm: PAV = 2(0.000005)-0.15 + (20% - 8%) = 12% + 12% = 24%  
25 ppm: PAV = 2(0.000025)-0.15 + (15% - 5%) = 10% + 10% = 20% 
500 ppm: PAV = 2(0.0005)-0.15 + (10% - 5%) = 6% + 5% = 11% 
 
For ease of use, 19%, 24%, and 11% are rounded up to the nearest 5%. The rounded PAVs are 
shown in section III. Standardized Assay Limits for Medicated Feeds.  
 
 
 
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