Microbiological Considerations for 
Antimicrobial Agents Used in Food 
Applications: Guidance for Industry 
Additional copies are available from: 
Office of Food Additive Safety 
Center for Food Safety and Applied 
Nutrition 
Food and Drug Administration 
5001 Campus Drive 
College Park, MD  20740 
(Tel) 240-402-1200  
http://www.fda.gov/guidance.html 
You may submit written or electronic comments regarding this guidance at any time. 
Submit written comments on the guidance to the Dockets Management Staff (HFA-
305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 
20852.  Submit electronic comments to http://www.regulations.gov.  
All comments should be identified with the title of the guidance 
document. 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Food Safety and Applied Nutrition 
September 2021  Table of Contents 
I. Introduction
II. Questions and Answers
A. What is an antimicrobial agent?
B. What is FDAs statutory authority with respect to antimicrobial
agents used in food or in contact with food?
C. Which other Federal agencies have authority over certain uses of
antimicrobial agents in food applications?
D. Why does FDA request microbiological data on the use of an
antimicrobial agent?
E. What types of data and information should be included in a
submission to FDA to demonstrate that the use of an
antimicrobial agent is safe and achieves the intended technical
effect?
F. What factors should be taken into consideration when
developing a protocol to show that the use of an antimicrobial
agent is safe and achieves the intended technical effect?
G. How should data be generated to demonstrate that the use of an
antimicrobial agent achieves the intended technical effect?
H. Does FDA recommend any specific experimental methods to
perform a safety assessment?
I. Does FDA have a performance standard for the use of a new
antimicrobial agent?
J. What information may be helpful to provide to FDA to
demonstrate that a source of radiation achieves the intended
technical effect as an antimicrobial treatment for food?
2  K. What safety information should I provide to FDA if my
antimicrobial agent is derived from a microorganism or is a
microorganism?
3   Microbiological Considerations for 
Antimicrobial Agents Used in 
Food Applications: Guidance for 
Industry1  
This guidance represents the current thinking of the Food and Drug Administration (FDA 
or we) 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
This guidance will assist submitters of food additive petitions (FAPs), food contact 
notifications (FCNs), generally recognized as safe (GRAS) notices (GRNs), and threshold 
of regulation (TOR) exemption requests in providing data to demonstrate that an 
antimicrobial agent (defined in Section II.A, below) achieves its intended technical effect 
in or on food or food contact articles.  Also, this guidance discusses microbiological data 
that may be necessary to demonstrate that an antimicrobial agent is safe for the intended 
use and achieves its intended effect.  This guidance replaces a guidance previously issued 
as Microbiological Considerations for Antimicrobial Food Additive Submissions 
(September 2007; Revised June 2008) by expanding the scope to include GRNs, by using 
more consistent terminology, and by eliminating portions of the guidance that reiterate 
information about regulatory processes addressed in regulations or other guidance 
documents. 
Given the complexity and variety of antimicrobial agents and the diverse conditions of use, 
no single document can anticipate and address all microbiological issues.  Therefore, this 
guidance is intended to answer common questions associated with microbiological data 
needed to establish that an antimicrobial agent accomplishes the intended technical effect 
and is safe for use in or on food or food contact articles.  We recommend that you discuss 
any proposed experimental protocols with FDA before initiating them to ensure that the 
1 This guidance has been prepared by the Office of Food Additive Safety in the Center for Food Safety and 
Applied Nutrition at the U.S. Food and Drug Administration.   
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data will address our safety questions, including those related to achieving the intended 
technical effect.2  In addition, we recommend that you meet with us early during 
development of your products to avoid studies that may be unnecessary or that may fail to 
address all issues. 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe our 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 FDA guidances means that something is suggested or 
recommended, but not required. 
 
 
II.  Questions and Answers  
 
A.  What is an antimicrobial agent? 
 
The term antimicrobial agent, as used in this guidance, refers to a substance (including 
other microorganisms) or a source of radiation used to control microorganisms such as 
bacteria, viruses, fungi, protozoa, or other microorganisms in or on food or food contact 
articles. 
 
B.  What is FDAs statutory authority with respect to antimicrobial 
agents used in food or in contact with food? 
 
FDA regulates the uses of antimicrobial agents in food applications, including use in food 
contact materials, under the Federal Food, Drug, and Cosmetic Act (FD&C Act).  You can 
find details on resources for participating in FDAs pre-market submission programs for 
substances used in food applications in Table 1.  We encourage submitters to consult with 
us to determine which program is appropriate for the substance, its intended conditions of 
use, and the types of available information supporting its safe use. 
 
Table 1. Resources for Premarket Submissions 
Type of Submission  Applicable Regulations  Regulatory Guidance 
Documents 
Food Additive Petition  21 CFR 171.1  Guidance for Industry: 
Questions and Answers 
About the Food Additive or 
Color Additive Petition 
Process (April 2011) 
 
Guidance for Industry: 
Antimicrobial Food 
Additives (July 1999) 
 
2 We, us, and our refers to FDA.  You, I, and my refer to the responsible person (developer, 
sponsor, or proponent) who conducts a food safety evaluation and submits such information to FDA. 
 
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Type of Submission  Applicable Regulations  Regulatory Guidance 
Documents 
Food Contact Notification  21 CFR part 170  Subpart  Guidance for Industry: 
D  Preparation of Food Contact 
Notifications 
(Administrative) (May 
2002)
 
GRAS Notice  21 CFR part 170  Subpart  Guidance for Industry: 
E  Frequently Asked Questions 
About GRAS for 
Substances Intended for Use 
in Human or Animal Food 
(October 2016)
 
Threshold of Regulation   21 CFR 170.39  Guidance for Industry: 
Submitting Requests under 
21 CFR 170.39 Threshold 
of Regulation for 
Substances Used in Food-
Contact Articles (April 
2005) 
 
 
C.  Which other Federal agencies have authority over certain uses of antimicrobial 
agents in food applications? 
 
For details about regulatory authority over uses of antimicrobial agents in food 
applications, see our Guidance for Industry entitled Antimicrobial Food Additives (July 
1999).  Depending on the proposed conditions of use, an antimicrobial agent may also be a 
pesticide under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).  As 
such, it may be subject to registration as a pesticide by the U.S. Environmental Protection 
Agency as well as regulation by FDA. 
 
The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) is 
responsible for determining the suitability of the use of food ingredients used in the 
production of meat,3 poultry, or certain egg products.  FSIS derives its authority to regulate 
the suitability of food ingredients from the Federal Meat Inspection Act,4 the Poultry 
Products Inspection Act, and the Egg Products Inspection Act.  According to FSIS, 
suitability relates to the effectiveness of the substance in performing the intended technical 
purpose of use, at the lowest level necessary, and the assurance that the conditions of use 
will not result in an adulterated product or one that misleads consumers.5  FDA and FSIS 
 
3 Including Siluriformes fish, see footnote 4. 
4 As a result of amendments to the Federal Meat Inspection Act in 2008 and 2014, as of March 1, 2016, FSIS 
has jurisdiction over all wild-caught and farm-raised Siluriformes fish that are harvested and sold for human 
food in the United States. This includes Siluriformes fish and fish products that are imported into the United 
States.  
5 Guidance on the Procedures for Joint FSIS and Food and Drug Administration (FDA), Approval of 
Ingredients and Sources of Radiation Used in the Production of Meat and Poultry Products, U.S. Department 
of Agriculture, Docket No. 00-022N, E. coli O157:H7 Contamination of Beef Products. 
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currently have a Memorandum of Understanding (MOU) establishing each agencys 
responsibilities in the evaluation of food ingredients and sources of radiation used in the 
production of meat, poultry, and egg products.  Under this MOU, we collaborate with FSIS 
on the review of antimicrobial agents intended for use in the production of these products. 
 
D.  Why does FDA request microbiological data on the use of an antimicrobial 
agent? 
 
Microbiological data address certain issues related to the use of an antimicrobial agent, 
regardless of the regulatory status of the substance.  The legal bases for such requests are 
described below. 
 
  Under section 409(c)(3)(B) of the FD&C Act, a food additive regulation will not be 
o
established if an evaluation of the data shows that the proposed use of the additive 
would promote deception of the consumer in violation of this Act or would 
otherwise result in adulteration or in misbranding of food within the meaning of 
this Act.  Here, the microbiological data may be used to demonstrate that an 
antimicrobial agent does not promote consumer deception, such as making a food 
product appear to be fresher or of greater value. 
  Under section 409(c)(4) of the FD&C Act, if a tolerance limitation is required in 
o
order to assure that the proposed use of an additive will be safe, the Secretary shall 
not fix such a tolerance limitation at a level higher than he finds reasonably 
required to accomplish the physical or other technical effect for which such 
additive is intended; and, shall not establish a regulation for such proposed use if he 
finds upon a fair evaluation of the data before him that such data do not establish 
that such use would accomplish the intended physical or other technical effect. 
  When a tolerance limitation is required, the microbiological data may be used to 
o
demonstrate that the use of an antimicrobial agent achieves the intended technical 
effect and that the maximum intended use level is not higher than the level 
reasonably required to achieve this effect. 
  Microbiological data may be needed to supplement the safety assessment of an 
o
antimicrobial agent that is a food additive under section 409(c)(3)(A) of the FD&C 
Act, which states that No such regulation shall issue if a fair evaluation of the data 
before the Secretary fails to establish that the proposed use of the food additive, 
under the conditions of use to be specified in the regulation, will be safe... 
Circumstances when microbiological data may be used to supplement the safety 
assessment are described in Items F, J, and K below. 
  Section 409(b)(2)(C) of the FD&C Act requires that an FAP contain all relevant 
o
data bearing on the physical or other technical effect the additive is intended to 
produce and the quantity of such additive required to produce such effect. 
Therefore, a petition for a food additive that is used as an antimicrobial agent 
requires data on the intended technical effect. 
  21 CFR 170.230(d)6 on GRAS Notices states that when necessary to demonstrate 
o
 
6 In the preamble to the GRAS Final Rule, Under XIII. Comments on Part 2 of a GRN: Identity, Method of 
Manufacture, Specifications, and Physical or Technical Effect (Substances Generally Recognized as Safe, 
Final Rule 81 FR 54960 at 55001, August 17, 2016), D. Data and Information Bearing on the Physical or 
Other Technical Effect of the Notified Substance, we stated, An example of when such data and information 
would be relevant to safety is when the intended use of the notified substance is as an antimicrobial agent. 
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safety, Part 2 of a GRAS Notice must include: relevant data and information 
bearing on the physical or other technical effect the notified substance is intended 
to produce, including the quantity of the notified substance required to produce 
such effect.  
 
E.  What types of data and information should be included in a submission to FDA 
to demonstrate that the use of an antimicrobial agent achieves the intended 
technical effect? 
 
To demonstrate that the use of an antimicrobial agent achieves the intended technical effect 
and that the proposed use level is the minimum level necessary to accomplish the intended 
technical effect, we recommend that you provide the following (at a minimum): 
 
  The chemical or biological identity of the antimicrobial agent, whichever is 
o
appropriate; 
  A detailed description of the intended antimicrobial effect and identification of 
o
individual or groups of targeted microbes, if appropriate; 
  A description of the conditions of use and any limitations on conditions of use, e.g.: 
o
  types of foods or other substances for treatment; 
o
  proposed use level or range; 
o
  temperature range of use; 
o
  method of application, such as spraying, dipping or fumigating, when 
o
applicable; 
  post-processing steps (e.g., potable water rinse, cooking by the consumer); 
o
  Antimicrobial effect data, including methods used and results;7 
o
  Directions, recommendations, and suggestions regarding the proposed use, and for 
o
FAPs, a sample of the label proposed for the food additive and any labeling that will 
be required on the finished food because of the use of the food additive; and 
  For an FAP, wording for the proposed regulation or changes to an existing 
o
regulation.  This proposed language will clarify our understanding of the 
petitioners intent. 
 
F.  What factors should be taken into consideration when developing an 
experimental protocol to show that the use of an antimicrobial agent is safe and 
achieves the intended technical effect? 
 
You should consider certain factors when developing an experimental protocol to 
show that the use of an antimicrobial agent achieves the intended technical effect. 
These are described below: 
 
Simulate, to the extent feasible, the intended conditions of use of the 
o 
antimicrobial agent; 
Use samples of the relevant matrix (e.g., food, process water, food packaging) 
o 
treated under the intended conditions of use, and include appropriate controls; 
Consider the typical organisms expected to be observed and the conditions under 
o 
which they would be present in the food.  For example, the commonly consumed 
 
7 For GRAS notices, generally available methods are typically used to generate antimicrobial effect data. 
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portions of meat (beef, lamb, pork) and poultry are generally considered 
uncontaminated with pathogens prior to slaughter.  Contamination with enteric 
pathogens may occur because of improper slaughtering techniques and 
subsequent processing; therefore, enteric pathogens are the organisms of concern 
for meat and poultry and should be the subjects of any technical effect 
experiments.  (NOTE: We recommend that you consult with FSIS regarding the 
development of a protocol where the intended use includes the treatment of 
USDA-regulated products); 
Consider the pertinent microorganisms that may be present in a food.  For 
o 
purposes of this guidance, the pertinent microorganism is the most resistant 
microorganism of public health concern that may occur in a food.8  The protocol 
should include the pertinent microorganism(s) that the antimicrobial agent is 
intended to target, especially those associated with a given food (e.g., Salmonella 
spp. and Campylobacter jejuni for poultry, Escherichia coli for beef, Vibrio spp. 
for seafood, Salmonella spp. for juices, Salmonella spp. and Cronobacter 
sakazakii for infant formulas, etc.).  The pertinent microorganism(s) vary among 
different food types; 
Consider indigenous vs. introduced food pathogens.  Seafood, fruits, and 
o 
vegetables may have indigenous food pathogens present at harvest; other 
pathogens may be introduced during harvest or processing.  We recommend that 
notifiers/petitioners consider whether their antimicrobial agent is equally effective 
against both types of pathogens;  
Consider the value of spoilage. Organoleptic changes caused by spoilage 
o   
organisms are indicators that consumers may use to gauge the freshness of meat 
and poultry, seafood, and produce, and indirectly, their safety.  As such, an 
antimicrobial agent that preferentially eliminates spoilage organisms over 
pathogens might allow pathogens to proliferate while suppressing spoilage 
organisms and their effects.  Under such conditions, a consumers senses may not 
reliably identify or inform an individual about spoiled and contaminated products, 
and the use of an antimicrobial agent under those conditions might not be safe.  
We recommend submitters provide data on the effects of their antimicrobial agent 
on specific pathogens as well as general (spoilage) populations for comparison; 
typically, aerobic plate counts are sufficient to characterize general populations; 
Consider unintended consequences.  Experimental design should consider and 
o 
address whether the use of the antimicrobial agent may result in unintended 
consequences.  For example, an antimicrobial agent may change the 
microbiological profile of food such that it suppresses one group of pathogenic 
microorganisms while allowing others to proliferate, thereby creating a potential 
health problem.  
 
G.  How should data be generated to demonstrate that the use of an 
antimicrobial agent achieves the intended technical effect? 
 
We recommend that all protocols designed to demonstrate the intended technical 
effect(s) of the use of an antimicrobial agent:  
 
 
8  See, e.g., 21 CFR 120.24. 
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  Use a direct method to enumerate microorganisms, such as plate counting or 
o
visual microscopic counts, as opposed to measuring biomass; evaluate the 
reduction (or suppression) of microorganisms affected by the treatment; 
  When available, use enumeration methods that capture damaged or stressed 
o
microbial cells that survive the antimicrobial treatment; 
  For all data, collect and analyze replicate samples and describe the 
o
variability of the data; and 
  Use statistical analyses to compare data from the treated samples to data 
o
from controls. 
 
We recommend that submitters consult with FDA in designing experimental protocols 
prior to the initiation of any experiments.  It is our experience that these consultations 
lead to consensus between FDA and the submitter prior to conducting experiments.  We 
believe these consultations result in data that are more likely to address FDAs concerns, 
leading to a more efficient regulatory process. 
 
H.  Does FDA recommend any specific experimental methods to perform a safety 
assessment? 
 
We recognize that you can address relevant safety issues for an antimicrobial agent in 
multiple ways.  However, we recommend several resources specific to microbiological 
methods.  FDA publishes an online version of the Bacteriological Analytical Manual 
which presents useful laboratory procedures (used by FDA labs) for microbiological 
analyses of foods and cosmetics.  Furthermore, FDA maintains a website with links to 
related resources on Microbiological Methods.  Also, FSIS maintains a Microbiology 
Laboratory Guidebook of current protocols for analytical tests FSIS uses in USDA 
regulated products; these test procedures may be useful in developing protocols to assess 
technical effect.  However, a submission will not be treated differently simply because 
alternative protocols were used. 
 
I.  Does FDA have a performance standard for the use of a new antimicrobial agent? 
 
We do not have a performance standard requirement for the efficacy of an antimicrobial 
agent for use in or on food or food contact articles in general; however, data are needed to 
demonstrate that an antimicrobial agent achieves its intended technical effect and that the 
intended use level is the minimum level necessary to achieve the intended technical effect. 
Therefore, efficacy data should show a quantifiable reduction of target microbes in the 
samples treated with the antimicrobial agent when compared to corresponding negative 
controls (e.g., the treatment absent the active antimicrobial agent) and that the intended use 
level is the minimum level necessary to achieve such a reduction.  However, certain 
regulations may specify performance standards for some types of food (e.g., reductions as 
specified in the Juice Hazard Analysis and Critical Control Point regulation (21 CFR 
120.24)). 
 
   
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J.  What information may be helpful to provide to FDA to demonstrate that a 
source of radiation achieves the intended technical effect as an antimicrobial 
treatment for food? 
 
In addition to the data and information typically submitted in an FAP, a petition for the use 
of a source of radiation as an antimicrobial treatment for food must include additional 
information related to antimicrobial effects (21 CFR 171.1(c)).  Examples of this 
information include: 
 
  Information describing pertinent pathogens and other microorganisms of 
o
concern in the targeted foods; this includes the occurrence and levels of 
pathogenic and non-pathogenic microorganisms; and 
  Information describing the effects of the proposed irradiation on 
o
microorganisms in or on the targeted food, including the radiation sensitivity of 
the microbes of concern, how the change in microbial population could affect 
and permit the outgrowth of pathogens without competing spoilage organisms, 
and the comparison of the microbiological safety of the irradiated food to the 
untreated food.  For example, the pathogenic spore-forming bacterium 
Clostridium botulinum producing type E toxin is associated with coastal water 
fish in certain areas.  If a petitioner wished to irradiate fish that may be 
contaminated with C. botulinum, they would need to demonstrate that naturally 
occurring spoilage organisms would be able to outcompete the pathogen and 
spoil the fish before toxin production by C. botulinum occurs in the irradiated 
fish. 
 
K.  What safety information should I provide to FDA if my antimicrobial agent is 
derived from a microorganism or is a microorganism? 
 
If an antimicrobial agent is derived from a microorganism or is itself a 
microorganism, we recommend the following: 
 
  Describe the biological identity of the specific strain of microorganism to be used for 
o
production.  If the strain has been genetically altered, regardless of method, describe 
how the strain was derived, including information about the biological sources 
contributing genetic material to the production strain, focusing largely on the safety of 
the production strain or the microbe itself; 
  Describe the quality control procedures used during production, including how you 
o
ensure pure cultures of the production organism and the procedures you follow when 
contamination is observed in the starter cultures or during production; 
  Describe the methods used to verify that the microorganism does not produce 
o
clinically relevant antibiotics or reasons why this would not be needed.  The 
production microorganism should not produce clinically relevant antibiotics in 
preparations of the antimicrobial agent; 
  Demonstrate that the production strain is nonpathogenic and does not express toxins, 
o
or if you use a pathogenic or toxigenic strain, demonstrate that toxins are not present 
in the final food product at a toxicologically relevant level; and, 
  Describe the methods and relevant quality control procedures to ensure that no 
o
viable cells of the production strain are found in the product when those cells present 
a safety concern.  
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We understand that for some newly developed antimicrobial agents, considerations may 
differ.  We will consider whether the information you provide in antimicrobial agent 
submissions establish the safety of the intended use on a case-by-case basis. 
 
 
 
 
 
The document above supersedes a previous guidance issued in June 2008. 
 
 
 
   
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