#159 
 
Studies to Evaluate the Safety of 
Residues of Veterinary Drugs in 
Human Food:  General Approach to 
Establish a Microbiological ADI 
 
VICH GL36(R) 
 
Guidance for Industry 
 
This revision has updated the volume of the human colon.  
 
Submit comments on this guidance at any time.  Submit electronic comments to 
https://www.regulations.gov.  Submit written comments to the Dockets Management Staff 
(HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD  
20852.  All comments should be identified with the docket number FDA-2003-D-0433. 
 
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 
https://www.regulations.gov. 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine (CVM) 
January 2022 Contains Nonbinding Recommendations 
 
VICH GL36(R2) (SAFETY  MICROBIOLOGICAL ADI) 
February 2019 
For Implementation at Step 7  
 
 
 
 
S E S
TUDIES TO  VALUATE THE  AFETY OF 
R V D
ESIDUES OF  ETERINARY  RUGS IN 
H F : 
UMAN  OOD
G A E
ENERAL  PPROACH TO  STABLISH A 
M ADI 
ICROBIOLOGICAL 
 
 
 
 
Adopted at Step 7 of the VICH Process 
by the VICH Steering Committee 
in February 2019  
for implementation in August 2020 
 
 
 
This Guidance has been developed by the appropriate VICH Expert Working Group and is 
subject to consultation by the parties, in accordance with the VICH Process.  At Step 7 of the 
Process the final draft has been recommended for adoption to the regulatory bodies of the 
European Union, Japan, and the USA. 
 
 
 
 
 
 
2    Contains Nonbinding Recommendations 
 
Table of Contents 
 
1.  INTRODUCTION................................................................................................................... 5 
1.1.  Objectives of the guidance............................................................................................. 5 
1.2.  Background .................................................................................................................... 5 
1.3.  Scope of the guidance..................................................................................................... 7 
2.  GUIDANCE ............................................................................................................................. 7 
2.1.  Steps in determining the need for a microbiological ADI .......................................... 7 
2.2.   Recommendations for determining NOAECs and NOAELs for the   endpoints     
of concern ........................................................................................................................ 8 
2.2.1.   Disruption of the colonization barrier ............................................................. 8 
2.2.1.1.   Detection of colonization barrier disruption .................................... 8 
2.2.1.2.   Test systems and study design ........................................................... 9 
2.2.1.2.1.   In vitro tests ....................................................................... 9 
2.2.1.2.2.   In vivo tests ........................................................................ 9 
2.2.2.   Increase in the population(s) of resistant bacteria in the human colon       
(as defined in section 1.2. Background) .......................................................... 10 
2.2.2.1.   Detection of changes in the population of resistant bacteria ........ 10 
2.2.2.2.   Test systems and study design ......................................................... 10 
2.2.2.2.1.   In vitro tests ..................................................................... 10 
2.2.2.2.2.   In vivo tests ...................................................................... 11 
2.3.   General recommendations .......................................................................................... 11 
2.4.  Derivation of a microbiological ADI .......................................................................... 12 
2.4.1.   Disruption of the colonization barrier ........................................................... 12 
2.4.1.1.   Derivation of an ADI from in vitro data ......................................... 12 
2.4.1.2.   Derivation of an ADI from in vivo data .......................................... 13 
2.4.2.   Increase in the population(s) of resistant bacteria ........................................ 13 
2.4.2.1.   Derivation of an ADI from in vitro data ......................................... 13 
2.4.2.2.   Derivation of an ADI from in vivo data .......................................... 13 
3.  GLOSSARY........................................................................................................................... 13 
4.  REFERENCES ...................................................................................................................... 17 
APPENDIX A .............................................................................................................................. 18 
1.   Experimental Conditions ............................................................................................... 18 
3    Contains Nonbinding Recommendations 
2.   Inoculum .......................................................................................................................... 18 
3.   Study duration ................................................................................................................. 18 
APPENDIX B .............................................................................................................................. 19 
APPENDIX C .............................................................................................................................. 21 
1.  Calculation of MICcalc ..................................................................................................... 21 
2.  Example Calculation ....................................................................................................... 21 
APPENDIX D .............................................................................................................................. 23 
1.  Introduction ..................................................................................................................... 23 
2.  Examples of Test Systems for the Assessment of the Fraction of Oral Dose  
Available to Microorganisms ......................................................................................... 23 
3.  Methodological Aspects of Test Systems....................................................................... 25 
4.  Description of How Test Systems Could Be Used in Determining the Fraction          
of Oral Dose Available to Microorganisms .................................................................. 26 
4    Contains Nonbinding Recommendations 
Studies to Evaluate the Safety of Residues of Veterinary Drugs in 
Human Food:  General Approach to Establish a Microbiological 
ADI 
 
Guidance for Industry 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic.  It does not establish any rights for any person and is not binding on FDA 
or the public.  You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff 
responsible for this guidance as listed on the title page. 
 
1.  INTRODUCTION 
1.1.  Objectives of the guidance 
A variety of toxicological evaluations can be performed to establish the safety of veterinary drug 
residues in human food.  An issue that needs to be addressed for veterinary antimicrobial drugs is 
the safety of their residues on the human intestinal flora.  The objectives of this guidance are (1) 
to outline the recommended steps in determining the need for establishing a microbiological 
acceptable daily intake (ADI); (2) to recommend test systems and methods for determining no-
observable adverse effect concentrations (NOAECs) and no-observable adverse effect levels 
(NOAELs) for the endpoints of health concern; and (3) to recommend a procedure to derive a 
microbiological ADI.  It is recognized that different tests may be useful. The experience gained 
with the recommended tests may result in future modifications to this guidance and its 
recommendations. 
 
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. 
1.2.  Background 
The intestinal flora plays an important role in maintaining and protecting the health of 
individuals.  This flora provides important functions to the host such as (1) metabolizing 
endogenous and exogenous compounds and dietary components; (2) producing compounds that 
are later absorbed; and (3) protecting against the invasion and colonization by pathogenic 
microorganisms. 
 
5    Contains Nonbinding Recommendations 
Ingested antimicrobial drugs can potentially alter the ecology of the intestinal flora.  They may 
reach the colon due to incomplete absorption or may be absorbed, circulated and then excreted 
via bile or secreted through the intestinal mucosa. 
 
It is recommended that the microbiological endpoints of current public health concern that 
should be considered when establishing a microbiological ADI are: 
  
Disruption of the colonization barrier: The colonization barrier is a function of the normal 
intestinal flora that limits colonization of the colon by exogenous microorganisms, as 
well as overgrowth of indigenous, potentially pathogenic microorganisms.  The capacity 
of some antimicrobial drugs to disrupt this barrier is well established and known to have 
human health consequences. 
 
Increase of the population(s) of resistant bacteria: For the purposes of this guidance, 
resistance is defined as the increase of the population(s) of bacteria in the intestinal tract 
that is (are) insensitive to the test drug or other antimicrobial drugs.  This effect may be 
due either to the acquisition of resistance by organisms which were previously sensitive 
or to a relative increase in the proportion of organisms that are already less sensitive to 
the drug. 
 
An extensive literature review did not reveal reports of human health effects (e.g., 
prolonged antimicrobial therapy, prolonged hospital stay, predisposition to infection, 
treatment failure, etc.) that occur as a result of changes in the proportion of antimicrobial 
resistant bacteria in the normal human intestinal flora.  However, based on the 
understanding of microbial ecology, such effects cannot be excluded. 
 
Although the effect of antimicrobial residues in food on the human intestinal flora has been a 
concern for many years, a harmonized approach to determine the threshold dose that might 
adversely disturb the flora has not been established.  International regulatory bodies have used a 
formula-based approach for determining microbiological ADIs for antimicrobial drugs.  These 
formulae take into consideration relevant data including minimum inhibitory concentration 
(MIC) data against human intestinal bacteria.  Due to the complexity of the intestinal flora, 
uncertainty factors have been traditionally included in the formula.  However, the use of 
uncertainty factors results in conservative estimates and it is recommended that more relevant 
test systems be developed that allow a more realistic estimate of a microbiological ADI, possibly 
without the use of these factors. 
 
The present revised guidance is an attempt to address the complexity of the human intestinal 
flora and reduce uncertainty when determining microbiological ADIs.  The guidance 
recommends a process for determining if a microbiological ADI is appropriate and discusses test 
systems that take into account the complexity of the human intestinal flora.  These test systems 
could be used for addressing the effects of antimicrobial drug residues on human intestinal flora 
for regulatory purposes. 
 
Since further research is needed to confirm the reliability and validity of all test systems 
discussed in this guidance (see Appendix A), this guidance does not recommend any one 
6    Contains Nonbinding Recommendations 
particular system for use in establishing an ADI.  Instead, this guidance provides 
recommendations for a harmonized approach to establish a microbiological ADI and offers 
test options rather than specifying a testing regimen. 
   
1.3.   Scope of the guidance 
This document provides guidance for assessing the human food safety of residues from 
veterinary antimicrobial drugs with regard to effects on the human intestinal flora.  However, it 
does not limit the choice of studies that may be performed to establish the safety of residues in 
human food with respect to adverse effects on human intestinal flora.  This guidance does not 
preclude the possibility of alternative approaches that may offer an equivalent assurance of 
safety, including scientifically based reasons as to why microbiological testing may not be 
appropriate. 
 
2.  GUIDANCE 
If a drug intended for use in food-producing animals has antimicrobial activity, the safety of its 
residues should be addressed with respect to the human intestinal flora.  Derivation of a 
microbiological ADI is only recommended if residues reach the human colon and remain 
microbiologically active. 
 
2.1.  Steps in determining the need for a microbiological ADI 
When determining the need for a microbiological ADI, the following sequence of steps is 
recommended.  The data may be obtained experimentally or from other appropriate sources such 
as scientific literature. 
 
Step 1.  Are residues of the drug, and (or) its metabolites, microbiologically active against 
representatives of the human intestinal flora? 
 
  Recommended data: 
-  MIC data, obtained by standard test methods, from the following relevant genera of 
intestinal bacteria (E. coli, and species of Bacteroides, Bifidobacterium, Clostridium, 
Enterococcus, Eubacterium (Collinsella), Fusobacterium, Lactobacillus, 
Peptostreptococcus/Peptococcus). 
-  It is recognized that the understanding of the relative importance of these 
microorganisms is incomplete and that the taxonomic status of these organisms can 
change.  The selection of organisms should take into account current scientific 
knowledge. 
  
  If no information is available, it is recommended to assume that the compound and (or) its 
metabolites are microbiologically active. 
 
Step 2.  Do residues enter the human colon? 
 
7    Contains Nonbinding Recommendations 
  Recommended data: 
-  Absorption, distribution, metabolism, excretion (ADME), bioavailability, or similar 
data may provide information on the percentage of the ingested residue that enters the 
colon. 
-  If no information is available in humans, it is recommended to use appropriate animal 
data.  If there is no available information, it is recommended to assume that 100% of 
the ingested residue enters the colon. 
 
Step 3.  Do the residues entering the human colon remain microbiologically active? 
 
  Recommended data: 
-  Data demonstrating loss of microbiological activity from in vitro inactivation studies 
of the drug incubated with feces or data from in vivo studies evaluating the drugs 
microbiological activity in feces or colon content of animals. 
 
If the answer to any of questions in steps 1, 2, or 3 is no, then the ADI should not be based 
on microbiological endpoints and the remaining steps are not recommended. 
 
Step 4.  Assess whether there is any scientific justification to eliminate testing for either one 
or both endpoints of concern.    It is recommended that available information regarding 
colonization barrier disruption and resistance emergence for the drug be considered.  If a 
decision cannot be made based on the available information, both endpoints should be 
examined. 
 
Step 5.  Determine the NOAECs/NOAELs for the endpoint(s) of concern as established in 
step 4.  The most appropriate NOAEC/NOAEL should be used to determine the 
microbiological ADI. 
 
2.2.      Recommendations for determining NOAECs and NOAELs for the endpoints of 
concern 
2.2.1.      Disruption of the colonization barrier 
2.2.1.1.      Detection of colonization barrier disruption 
Changes in bacterial populations are indirect indicators of potential disruption of the colonization 
barrier.  These changes can be monitored by various enumeration techniques in a variety of test 
systems.  A more direct indicator of barrier disruption is the colonization or overgrowth of an 
intestinal ecosystem by a pathogen.  In vivo test systems or complex in vitro test systems (e.g., 
fed-batch, continuous, or semi-continuous culture systems) have the potential to evaluate barrier 
disruption as evidenced by colonization of a challenge organism added to the test system. 
 
Challenge organisms (e.g., Salmonella, Clostridium) should be insensitive to the test drug.  
Inoculation schemes with the challenge organisms should take into account the timing of the 
8    Contains Nonbinding Recommendations 
challenge relative to drug treatment, the number of organisms per challenge dose, and the 
number of times that the test system is challenged. 
 
2.2.1.2.      Test systems and study design 
2.2.1.2.1.      In vitro tests 
The use of MICs to assess the potential for a drug to disrupt the colonization barrier does not 
take into account the complexity of the human intestinal flora.  Therefore, the MIC  of the most 
50
relevant genus/genera for which the drug is active (see section 2.1. Steps in determining the need 
for a microbiological ADI) results in a conservative estimate of a NOAEC for disruption of the 
colonization barrier.  The NOAEC estimate is conservative because, among other reasons, the 
inoculum density is orders of magnitude lower than the bacterial population in the intestinal tract 
(Ref. 1).  Therefore, the NOAEC may be considered as an option to establish an ADI.  The 
isolates should be obtained from multiple healthy individuals and include a minimum of 10 
isolates from each of the genera listed in section 2.1.   
 
Each MIC test of a pure culture of a relevant isolate provides data for a single strain of a species. 
Other in vitro test systems provide information for hundreds of bacterial species (>108 bacterial 
cells/g) for each fecal inoculum.  Each inoculum can be tested in replicate to determine treatment 
effects.  Based on all the above, in vitro systems using fecal batch cultures are inherently more 
robust and relevant than the MIC test system. 
 
Other test systems discussed below, which model the intestinal flora, may result in a more 
appropriate NOAEC and possibly a higher ADI. 
 
Fecal slurries provide a simple test system to derive a NOAEC for disruption of the colonization 
barrier following short-term exposure to the drug and may be appropriate for dose-titration 
studies.  The slurries can be monitored for changes in bacterial populations and the production of 
short chain fatty acids.  These two response variables, when monitored together, can be used as 
indirect indicators of barrier disruption.  The NOAEC derived from this test system may prove to 
be a conservative estimate of barrier disruption. 
Semi-continuous, continuous and fed-batch cultures of fecal inocula may be appropriate to 
evaluate disruption of the colonization barrier following prolonged exposure to the drug.  
However, exploratory work using continuous and semi-continuous cultures has given various 
NOAECs for barrier disruption because of differences in protocols.  As a consequence, it is 
recommended that study designs take into account the issues raised in Appendix A. 
In the case of fecal slurries, semi-continuous and continuous cultures, and fed-batch cultures of 
fecal inocula, there are unresolved issues such as the impact of fecal inocula (individual variation 
and gender), dilution rate, duration of drug exposure, and reproducibility of the tests. 
2.2.1.2.2.      In vivo tests 
In vivo test systems using human flora-associated (HFA) and conventional laboratory animals 
may be suitable for the assessment of disruption of the colonization barrier.  Compared to 
9    Contains Nonbinding Recommendations 
conventional laboratory animals, the intestinal flora of HFA animals possesses greater similarity 
to the human intestinal flora, both in terms of the range of bacterial populations and metabolic 
activity.  However, the intestinal flora derived from humans may not be stable in the HFA 
animals.  The relative importance of the stability of the implanted flora and the specific 
composition of the flora is unknown.  For technical reasons, the conventional laboratory animal 
can be tested in higher numbers, which allows a more robust statistical analysis of the results. 
 
It is recommended that study design take into account factors such as animal species, gender, 
inoculum variability among donors, number of animals per group, diet, randomization of 
treatment groups, minimization/elimination of coprophagy, housing of animals within an 
isolator, cross contamination within the isolator and route of drug administration (e.g., gavage, 
drinking water).  It is recommended that germ-free animals be inoculated in sequence, first with 
a Bacteroides fragilis strain, followed by the fecal inoculum. 
  
2.2.2.      Increase in the population(s) of resistant bacteria in the human colon (as defined 
in section 1.2. Background) 
The guidance below highlights the considerations that should be taken into account when 
addressing this endpoint. 
 
2.2.2.1.      Detection of changes in the population of resistant bacteria 
It is recommended that studies to evaluate the emergence of resistance take into account the 
organisms of concern in the intestinal tract and the documented resistance mechanisms to the 
drug class.  Preliminary information regarding the prevalence of resistance in the human 
intestinal flora, such as daily variation within individuals and the variation among individuals 
can be useful in developing criteria for evaluating resistance emergence.  MIC distributions of 
sensitive and known resistant organisms of concern can provide a basis to determine what drug 
concentration should be used in the selective agar media to enumerate resistant organisms in the 
fecal samples.  Since drug activity against an organism can vary with test conditions, the MIC of 
the organism growing on selective medium should be compared to the MIC determined by 
standard methods (e.g., Clinical and Laboratory Standards Institute [CLSI] (Ref. 2, 3)).  Changes 
in the proportions of resistant organisms during pre-treatment, treatment and post-treatment 
periods can be evaluated by enumeration techniques on media with and without the antimicrobial 
drug, applying phenotypic and molecular methodologies. 
 
Changes in antimicrobial resistance can be influenced by factors other than drug exposure (e.g., 
animal stress) which should be taken into consideration in animal test systems. 
 
2.2.2.2.      Test systems and study design 
2.2.2.2.1.      In vitro tests 
The duration of exposure required for resistance to develop in a population of bacteria can be 
dependent on the drug, the nature of the resistance mechanisms, and how it evolves in nature 
(e.g., by gene transfer between cells, by gene mutations).  For these reasons, acute studies of pure 
10    Contains Nonbinding Recommendations 
cultures to assess the endpoint are not recommended.  Therefore, MIC tests are not 
recommended to be used to determine a NOAEC for increases in resistant populations. 
 
Defined cultures may provide useful information to determine the potential for a resistant 
population to emerge due to mutation in an isolate and/or gene transfer among isolates.  
However, these test systems are not designed to evaluate changes in resistant populations and are 
not recommended. 
 
Test systems using short-term exposure of fecal slurries to a drug are not recommended for 
resistance emergence testing because the duration of the test is inadequate to assess changes in 
resistant populations. 
   
Continuous and semi-continuous cultures and fed-batch cultures of fecal inocula provide a means 
to evaluate long-term exposure of bacteria to the drug.  Refer to Appendix A for issues that are 
recommended to be addressed regarding study conduct and data evaluation. 
 
2.2.2.2.2.      In vivo tests 
Changes in resistant populations can be assessed in HFA-rodents.  General study design and 
supporting protocol should follow the recommendations stated in section 2.2.1.2.2. In vivo tests.  
The test system supports a complex flora and would be a source of genetic resistance 
determinants.  The system accommodates more replication than the continuous or semi-
continuous culture systems, but less than fed-batch cultures.  The variability of the HFA-rodent 
test has not been assessed; however it is useful for identifying gender differences.  There are also 
advantages to conducting resistance studies in conventional laboratory animals. 
 
HFA-rodents and conventional animals provide means to evaluate the potential for resistance 
emergence following long-term exposure of bacteria to the drug.  Refer to Appendix A for issues 
that are recommended to be addressed for study conduct and data evaluation. 
 
2.3.   General recommendations 
  It is recommended that fecal samples or bacterial isolates from human donors be obtained 
from healthy subjects with no known exposure to antimicrobial agents for at least 3 months. 
  In the case of in vivo tests, it is recommended that the test species selected for testing should 
allow for (1) maximum independent replication; (2) sufficient quantity of feces to be 
collected for analyses; and (3) minimal coprophagy.  Evaluation of both genders should be 
considered unless data demonstrate that only one gender is appropriate. 
  Statistical issues should be addressed when designing studies of antimicrobial residues (see 
Appendix B). 
  It is recommended that the pre-validation and validation process, such as that being 
developed by OECD since 1996 (Ref. 4), be considered for subsequent validation of test 
systems to assess the effects of antimicrobial drugs on human intestinal flora.  The process 
should be adapted and modified for this use depending on the test system being validated. 
11    Contains Nonbinding Recommendations 
  Study designs should take into account unresolved issues of the effects of storage and 
incubation conditions on fecal inocula. 
2.4.  Derivation of a microbiological ADI 
When more than one value can be determined for the microbiological ADI, in accordance with 
the methods discussed below, the most appropriate value (relevant to humans) should be used. 
 
2.4.1.      Disruption of the colonization barrier 
2.4.1.1.      Derivation of an ADI from in vitro data 
If the endpoint of concern is disruption of the colonization barrier, the ADI may be derived from 
MIC data, fecal slurries, semi-continuous, continuous, and fed-batch culture test systems. 
 
ADI derived from MIC data: 
     
 
MIC : The MIC  is derived from the lower 90% confidence limit for the mean MIC  of the 
calc calc 50
relevant genera for which the drug is active, as described in Appendix C. 
   
ADI derived from other in vitro test systems:  
                           
NOAEC: It is recommended that the NOAEC derived from the lower 90% confidence limit for 
the mean NOAEC from in vitro systems be used to account for the variability of the data.  
Therefore, in this formula uncertainty factors are not generally needed to determine the 
microbiological ADI. 
 
Volume of colon content: The 500 mL value was estimated from the result of three-dimensional 
abdominal magnetic resonance imaging measurement (Ref. 5). 
 
Fraction of an oral dose available for microorganisms:  It is recommended that the fraction of an 
oral dose available for colonic microorganisms be based on in vivo measurements for the drug 
administered orally.  Alternatively, if sufficient data are available, the fraction of the dose 
available for colonic microorganisms can be calculated as 1 minus the fraction (of an oral dose) 
excreted in urine.  Human data are encouraged, but in its absence, non-ruminant animal data are 
recommended.  In the absence of data to the contrary, it should be assumed that metabolites have 
antimicrobial activity equal to the parent compound.  The fraction may be lowered if the 
12    Contains Nonbinding Recommendations 
applicant provides quantitative in vitro or in vivo data to show that the drug is inactivated during 
transit through the intestine. 
 
2.4.1.2.     Derivation of an ADI from in vivo data 
The microbiological ADI is the NOAEL divided by the uncertainty factor.   
Uncertainty factors for in vivo studies should be assigned as appropriate, taking into 
consideration the class of compound, the protocol, numbers of donors, and sensitivity of the 
measured outcome variables.   
 
2.4.2.      Increase in the population(s) of resistant bacteria 
2.4.2.1.  Derivation of an ADI from in vitro data 
If the endpoint of concern is an increase in the population(s) of resistant bacteria, NOAECs 
derived from semi-continuous, continuous, and fed-batch culture test systems may be used to 
establish a microbiological ADI.  
                                 
NOAEC: It is recommended that the NOAEC derived from the lower 90% confidence limit for 
the mean NOAEC from in vitro systems be used to account for the variability of the data.  
Therefore, in this formula uncertainty factors are not generally needed to determine the 
microbiological ADI.  However, where there are concerns arising from inadequacies in the 
quality or quantity of in vitro data used in determining the NOAEC, the incorporation of an 
uncertainty factor may be warranted. 
 
2.4.2.2.      Derivation of an ADI from in vivo data 
The microbiological ADI is the NOAEL divided by the uncertainty factor.   
 
Uncertainty factors for in vivo studies should be assigned as appropriate, taking into 
consideration the class of compound, the protocol, numbers of donors, and sensitivity of the 
measured outcome variables.   
 
3.  GLOSSARY  
The glossary includes terminology referred to in the Appendices as well as in the text. 
 
Acceptable Daily Intake  An estimate of the amount of a substance, expressed on a 
(ADI)  body weight basis, that can be ingested daily over a lifetime 
without appreciable risk to human health. 
13    Contains Nonbinding Recommendations 
Antimicrobial Activity  The effect of an antimicrobial agent on a bacterial 
population. 
Antimicrobial Agent  A drug substance that is either biologically derived or 
chemically produced with antimicrobial activity as its major 
effect. 
Balanced Design  A statistical design is balanced if each combination of 
values or levels of all factors in the design (treatment 
factors, factors of interest such as gender, or blocking 
factors) have the same number of experimental units or 
replicates.  A partially balanced design is not balanced, but 
combinations of treatments and other factors occur in a 
regular way such that the analysis remains relatively simple. 
Batch Culture  A culture where neither substrate nor waste products are 
removed until completion of incubation, normally incubated 
for short periods, generally up to 24 hours. 
Blocking Factor  An experimental factor whose values or levels define groups 
of experimental units that are similar or that can be expected 
to respond in a similar manner.  Systematic variation among 
blocks can be removed from the estimate of error in the 
statistical analysis, resulting in greater precision.  An 
example is a cage containing several animals, which are the 
experimental units, or an isolator containing several cages. 
Challenge organism  An organism added experimentally to a test system to 
evaluate colonization barrier disruption. 
Colonization  The establishment of microorganisms in the intestinal tract. 
Colonization Barrier  A function of the normal intestinal flora that limits 
colonization of the colon by exogenous microorganisms, as 
well as overgrowth of indigenous, potentially pathogenic 
microorganisms. 
Complete Design  A statistical design is complete if all combinations of factors 
or groups in the design have at least one observation.  An 
incomplete design is one in which no observations are made 
for some combinations of factors. 
Continuous Culture  A culture maintaining continuous growth of microorganisms 
by the simultaneous supply of nutrient and removal of spent 
medium, maintaining a constant microbial load within a 
fixed incubation volume. 
Conventional Laboratory  A laboratory animal with its natural indigenous intestinal 
Animal  flora. 
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Coprophagy  The ingestion of feces. 
Defined Culture  A microbial culture in which all microbial species are 
known. 
Dilution (Flow) Rate  The rate of supply and removal of medium from a 
continuous culture system.  Dilution rate controls the 
microbial growth rate within a continuous culture system. 
Donor (Fecal) Inocula   Fecal flora obtained from human volunteers and used to 
inoculate the test system.  Fecal flora is considered to be 
equivalent to the intestinal flora. 
Drug Residue  The drug, including all derivatives, metabolites and 
degradation products that persists in or on food. 
Experimental Unit  The standard subject to which a treatment is applied and a 
measurement is made.  Examples include a whole animal or 
a specific organ or tissue, a cage containing several animals, 
a cell culture. 
Factorial Design  An experimental design that involves combinations of a 
number of factors, including a treatment factor, each having 
two or more values or levels.  Other factors may include 
stratification (e.g., gender) or blocking factors (e.g., cage).  
Typically, the outcome variable is measured on a number of 
experimental units at each combination of levels of the 
various factors.  The statistical analysis of the data involves 
a multifactorial analysis of variance. 
Fecal Slurry  Human feces or fecal solids minimally diluted in anaerobic 
buffer. 
Fed-Batch Culture  A batch culture fed continuously or semi-continuously with 
nutrient medium.  Portions of the fed batch culture can be 
withdrawn at pre-determined intervals.  A constant culture 
volume is not maintained. 
Human Flora-Associated  A germ-free host animal implanted with human fecal (HFA) 
Animal flora. 
Interaction Effect  Treatment effects that are modified by the presence of other 
factors.  For example, the effect of a treatment may be 
greater or less in males than females, or may change over 
time. 
Intestinal Flora  The normal microbial flora of the colon. 
15    Contains Nonbinding Recommendations 
Minimum Inhibitory  The lowest concentration of an antimicrobial compound that 
Concentration (MIC)  inhibits growth of the test organism as determined by 
standardized test procedures. 
MIC50  The concentration of an antimicrobial compound at which 
50% of the tested isolates within a relevant genus are 
inhibited. 
Microbiological ADI  An ADI established on the basis of microbiological data. 
No-Observable Adverse  The highest concentration that was not observed to cause 
Effect Concentration  any adverse effect in a particular study. 
(NOAEC) 
No-Observable Adverse  The highest administered dose that was not observed to 
Effect Level (NOAEL)   cause any adverse effect in a particular study. 
Outcome Variable  A specific parameter measured in an experiment.  Specific 
outcome variables should be defined as part of the protocol, 
and are the measurements actually made in the study. 
Semi-continuous  A culture where substrate and/or waste products are added 
Culture  and/or removed in a semi-continuous manner maintaining a 
fixed incubation volume. 
Short Chain Fatty Acid  The volatile fatty acids that are produced by the intestinal 
flora.  The principal acids are acetic, propionic and butyric. 
Solid Phase  The particulate matter in an in vitro test system. 
Systematic Variation  Factors that affect outcome variables.  Such variation is 
systematic in the sense that it represents an effect that is 
reliably present.  Systematic variation is distinguished from 
random variation, which is not predictable.  Systematic 
variation may be caused by factors that are of interest, such 
as gender, or by factors such as the particular isolator, which 
are not. 
Test System  A method used to determine the effects of antimicrobial 
residues on the human intestinal flora. 
Uncertainty Factor  A correction factor that takes into account the characteristics 
of the test data as described in sections 2.4.1.2. Derivation 
of an ADI from in vivo data, 2.4.2.1. Derivation of an ADI 
from in vitro data, and 2.4.2.2. Derivation of an ADI from in 
vivo data. 
 
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4.  REFERENCES 
1.  Cerniglia, C.E., and Kotarski, S.  1999.  Evaluation of Veterinary Drug Residues in Food 
for Their Potential to Affect Human Intestinal Microflora.  Regulatory Toxicology and 
Pharmacology. 29, 238-261. 
 
2.  National Committee for Clinical Laboratory Standards (NCCLS).  2004.  Methods for 
Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Anaerobically; 
Approved Standard  Sixth Edition.  NCCLS document M11-A6.  NCCLS, 940 West 
Valley Road, Suite 1400, Wayne, PA, USA.  (Note: In 2005, the organizations name 
changed to Clinical and Laboratory Standards Institute.) 
 
3.  National Committee for Clinical Laboratory Standards (NCCLS).  2003.  Methods for 
Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; 
Approved Standard  Sixth Edition.  NCCLS document M7-A6.  NCCLS, 940 West 
Valley Road, Suite 1400, Wayne, PA, USA.  (Note: In 2005, the organizations name 
changed to Clinical and Laboratory Standards Institute.) 
 
4.  OECD. 2001.  Series of Testing and Assessment No. 34, Environment, Health and Safety 
Publications.  Draft Guidance Document on the Development, Validation and Regulatory 
Acceptance of New and Updated Internationally Acceptable Test Methods and Hazard 
Assessment.  Organization for Economic Cooperation & Development, Paris. 
 
5.  Pritchard SE, Marciani L, Garsed KC, Hoad CL, Thongborisute W, Roberts E, Gowland 
PA, Spiller RC. Fasting and postprandial volumes of the undisturbed colon: normal 
values and changes in diarrhea-predominant irritable bowel syndrome measured using 
serial MRI. Neurogastroenterol Motil. 2014 Jan;26(1):124-30. doi: 10.1111/nmo.12243. 
Epub 2013 Oct 17. PMID: 24131490; PMCID: PMC3995006. 
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APPENDIX A 
Issues that Should be Investigated in Developing Test Systems and Data Interpretation 
 
1.  Experimental Conditions 
Data generated for continuous flow, semi-continuous flow and fed-batch studies will be affected 
by the growth conditions (e.g., growth medium, pH, dilution rate).  Different bacterial species 
may have different growth rates under the experimental conditions used for the test system.  If 
the dilution rate of the culture exceeds the growth rate of a bacterial species, then this species 
ultimately will be eliminated from the test culture.  The test system should be designed to 
maximize the retention of the different bacteria and maintain the complexity of the initial 
inoculum. 
 
Test antimicrobial agents can affect growth rates of various bacterial groups.  This may lead to 
loss of components of the mixed culture by a reduction of growth rate below that of the dilution 
rate used in the test system, which might cause some components of the flora to be washed out of 
the culture.  This may be minimized by developing test conditions with lower dilution rates. 
 
Antimicrobial susceptibility is influenced by the physical condition of the exposed organisms, 
which will be influenced by the growth conditions used in the test system.  Based on the above, 
further work is needed to determine the impact of different growth conditions on the NOAECs 
derived for colonization barrier disruption and the increase in the population of resistant bacteria. 
 
A number of factors should be considered in protocols for in vivo test systems.  For example, 
cross-contamination is a major issue when performing animal studies within a germ-free isolator.  
The protocol should be designed to minimize cross-contamination. 
 
2.  Inoculum 
The composition of the intestinal flora may vary among individuals with respect to bacterial 
groups and resistant organisms.  The bacterial populations are relatively stable within a single 
individual, but this is not necessarily the case for resistant bacterial groups. 
 
Multiple donors should be used to account for differences in flora between individuals.  Pooled 
inocula do not account for differences in flora between individuals.  Therefore, test systems that 
use fecal inocula obtained from individual donors are preferred to determine the effect of 
antimicrobial residues on the intestinal flora.  In addition, the composition of the donor inocula 
should be taken into account when interpreting study results. 
 
3.  Study duration 
The optimum incubation time to monitor for changes in bacterial populations in fecal batch 
cultures needs to be determined.  Likewise, in the case of complex long-term in vitro or in vivo 
test systems, it is important to determine the period during which the integrity and complexity of 
the intestinal flora remains stable and representative of the intestinal flora. 
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APPENDIX B 
Recommended Statistical Issues to be Considered When Designing Studies of 
Antimicrobial Residues 
 
Two broad endpoints of current public health concern were identified, disruption of the 
colonization barrier and increases in population(s) of resistant bacteria.  The experimental design 
must depend on which of these is to be addressed and should take account of the particular 
outcome variables.  It is recommended that a design paradigm for these test systems involves 
choice of the test system, application of treatments and follow-up of the system over time.  The 
choice of test system depends on the characteristics of the human intestinal tract that is 
represented by the test system.  Since the MIC tests are simple in design, many of the issues 
discussed below do not apply to this method. 
 
The experimental unit is a central component of the study design.  For an in vivo test system, for 
example, the unit may be an individual animal or an entire cage.  If cages are grouped within 
isolators, some or all of the treatments to different cages within each isolator can be applied.  In 
this case the isolator becomes a blocking factor, since cages within the same isolator would be 
expected to respond in a similar fashion.  The use of blocking factors is an important tool for 
reducing systematic variation.  A related question is whether there are other systematic factors 
such as gender that should be included, that is, whether a factorial design should be used.  If 
there are multiple factors, then the design involves choices of what combinations of these should 
be included.  It is important that this be done in such a way that the resulting design is balanced.  
In a complete, balanced design, all combinations are represented, and occur the same number of 
times. It is also possible to have incomplete designs, as well as various kinds of partial balance.  
The analysis of variance may be recommended for such designs; these designs can be useful 
when, for example, experimental resources are limited.  An example of an incomplete design is 
the standard two period cross-over design. 
 
It should be decided how the treatments are applied to the experimental units.  In some cases a 
two-stage treatment, involving a drug treatment and a bacterial challenge, may be recommended.  
There should be at least three antimicrobial treatment groups in addition to appropriate control 
groups.  The choice of antimicrobial treatment levels depends on the desired range of doses, but 
should cover both effect and no-effect levels.  The duration and the method of drug 
administration depends on the test system.  An important aspect of some studies is the evolution 
of effects over time, and repeated measurement of outcome variables may be recommended.  
Common issues are the timing and spacing of the measurements and bias caused by missing data. 
 
Control of random variation due to biological variability and to measurement error depends on 
the number of experimental units and number of samples.  This number can be determined from 
previous knowledge of the test system and outcome variables, either from past experience or 
through a sample size computation, which should be employed where possible.  Sufficient 
replication should be included to allow precise measurement of treatment effects and appropriate 
interaction effects, e.g., treatment effects that change over time.  In some studies, it may be 
important to examine such interaction effects as part of the statistical analysis.  Another type of 
replication is the pooling of fecal samples from animals in a single cage or the pooling of fecal 
19    Contains Nonbinding Recommendations 
samples from different donors.  In both cases, we have the benefits of averaging, but not the 
ability to estimate variability among replicates.  Pooling may obscure individual effects (of 
treatment and/or inoculum), and thus its use should be considered in terms of study objectives. 
20    Contains Nonbinding Recommendations 
 
APPENDIX C 
1.  Calculation of MICcalc 
The MIC  is derived from the lower 90% confidence limit for the mean MIC  of the most 
CALC 50
relevant genera for which the drug is active.  The lower 90% confidence limit is calculated using 
log transformed data.  Thus, the mean and standard deviation are calculated using the log 
transformed MIC  values.  This also implies that the lower 90% confidence limit needs to be 
50
back-transformed to obtain the correct value.  The formula for the confidence limit is: 
 
 
 
where:  Mean MIC  is the mean of the log transformed MIC  values, 
50 50
Std Dev is the standard deviation of the log transformed MIC values, 
50 
n is the number of MIC  values used in the calculations, 
50
t  is the 90th percentile from a central t-distribution with df degrees of freedom, and 
0.10,df
df = n-1. 
 
Examine the MIC  of relevant genera (see section 2.1. Steps in determining the need for a 
50
microbiological ADI).  The MIC  is based on a summary value of those genera which are not 
calc
inherently resistant to the compound.  Thus, the MIC is based on MIC  of those genera for 
calc  50
which the compound is active.  Ensure that all MIC  values are not characterized as </=, so 
50
they may be used in the calculation of the MIC . 
calc
 
2.  Example Calculation 
Any base log transformation of MIC  values can be used.  However, if 2-fold dilutions of drug 
50
are used in the MIC testing procedure, a base 2 log transformation conveniently will provide 
integer values for the calculation.  In the following example, the MIC values were transformed 
50 
as follows: 
 
 
 
 
 
 
 
 
 
 
 
 
 
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22    Contains Nonbinding Recommendations 
 
APPENDIX D 
Supplement to Section 2. Guidance Regarding the Determination of the Fraction of Oral 
Dose Available to Microorganisms 
 
1.  Introduction 
VICH GL 36 has been implemented since 2005.  Having gained experience in working with the 
guidance, regulators from all VICH regions agreed that additional guidance and clarity were 
needed regarding in vivo and in vitro testing methods to determine the fraction of oral dose 
available to microorganisms. 
 
This Appendix is based on review of new data, scientific literature, and information from 
disclosed sponsor submissions. 
 
This Appendix contains three sections: a table of examples of test systems for the assessment of 
the fraction of oral dose available to microorganisms, general considerations regarding 
methodological aspects of the implementation of these test systems, and a description of how the 
test systems could be used in determining the fraction of oral dose available to microorganisms. 
 
2.  Examples of Test Systems for the Assessment of the Fraction of Oral Dose Available to 
Microorganisms 
Various in vitro and in vivo test systems could be used separately and in combination to 
determine the fraction of oral dose available to microorganisms.  The table below provides 
examples of such test systems, the type of data generated and considerations relevant to their use. 
 
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24    Contains Nonbinding Recommendations 
3.  Methodological Aspects of Test Systems 
This section provides general considerations regarding the experimental conditions used in 
designing and conducting studies to determine the fraction of oral dose available to 
microorganisms. 
 
a)  Dose and concentration of drug: 
  Dose and drug concentration range to be used in the test systems and the experimental 
objective should be justified. 
  Dose and drug concentrations for testing should include levels that are expected with 
residue ingestion, as well as higher levels. 
b)  Fecal parameters: 
  Source and number of fecal samples: 
  Donors should be healthy with no known exposure to antimicrobial agents for at 
o
least 3 months before fecal collection (see section 2.3. General recommendations 
of the guidance). 
  Variability among donors (e.g., age, sex, diet) is inherent, and the implications of 
o
donor variability for experimental design should be taken into account. The 
number of fecal donors should be based on the experimental objective, and a 
minimum of six donors are recommended (Figure 1).  
  It is recommended that fresh samples (first motion of the day) should be 
o
processed within the day of collection.  Anaerobic storage for up to 72 hours at 
refrigerator temperatures is acceptable. 
  Physical characterization of fecal samples (e.g., fecal viscosity, water content, pH, 
and solid content) is recommended.  This information may be useful in interpreting 
variability in subsequent study results. 
  Fecal concentrations: 
  At least one fecal concentration should be considered. A 25% fecal preparation (1 
o
part fecal sample + 3 parts diluent) is recommended as representative of colon 
contents. 
  Diluent used to prepare fecal slurries: 
  The chemical components used in diluting fecal material should be standardized 
o
to minimize variability. 
  An anaerobic buffer that is based on minimal salts should be used. 
o
  Fecal incubation: 
  Consider an initial experiment using a minimum of two donor samples to 
o
determine an appropriate protocol.  This should include a relevant range of 
residue concentrations, incubation time and sampling at multiple time points, so 
as to enable kinetic calculations. 
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  The data for a minimum of six donors should be used for the final determination 
o
of the fraction of oral dose available to microorganisms. 
  Use of non-sterile or sterile fecal samples: 
  Consider the impact of sterilization of feces on drug binding to fecal suspensions 
o
in initial studies using a chemical assay. 
  Non-sterilized feces should be used where possible when conducting in vitro 
o
drug-binding/inactivation studies.  Small differences between binding to non-
sterilized and sterilized fecal suspensions may allow further studies to be based on 
sterilized feces only. 
c)  Methods to quantitatively determine the fraction of the microbiologically active drug 
available to microorganisms:   
  While either microbiological or chemical assays may be used in these experiments, 
justification of the specific type of assay should be provided.  If chemical assays are 
used, they should be bridged to the microbiological activity. 
  The strain of the indicator bacterial species will depend on the spectrum of activity of 
the drug. 
  The sensitivity and reproducibility of the assays should be considered. 
  Study controls should be considered according to the test system used. 
d)  Reversibility of observed drug binding: 
  A time course approach is recommended which will reveal possible reversibility of 
drug binding. 
  Further work to define the mechanism of binding is not essential for the purpose of 
establishing the fraction of oral dose available to microorganisms. 
4.  Description of How Test Systems Could Be Used in Determining the Fraction of Oral 
Dose Available to Microorganisms 
In vivo and in vitro approaches, using different test systems considered applicable to determine 
the fraction of oral dose available to microorganisms, were identified and reviewed.  Conceptual 
approaches of their application in deriving this fraction are outlined below and illustrated in 
Figure 1.   
 
APPROACH 1:  In vivo test systems.  Animals dosed with the drug, followed by one of the 
following options: 
  Option A:  chemical extraction and analysis of the intestinal content and (or) feces to 
determine the total drug concentration, is used to establish the fraction of oral dose 
available to microorganisms.  
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  Option B: both chemical and microbiological activity assays of the intestinal content and 
(or) feces of dosed animals is used to establish the fraction of oral dose available to 
microorganisms. 
APPROACH 2. In vitro test systems.  This approach comprises two steps (Phase A and B) 
using in vitro fecal slurry test systems (see Figure 1).  Phase A is an initial experiment, with fecal 
samples from two donors, used to identify the incubation times and relevant range of added drug 
concentrations sampling at multiple time points.  This phase includes both chemical and 
microbiological assays.  Phase B is conducted based on results from Phase A with samples from 
four additional donors and uses microbiological assays.  The data for all six donors are used for 
the final determination of the fraction of oral dose available to microorganisms. 
 
APPROACH 3:  Approach 1[Option A] + Approach 2.  This approach combines both in vivo 
studies and in vitro studies. 
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Figure 1. Schematic Representation of Test Systems to Determine the Fraction of Oral Dose 
Available to Microorganisms 
 
 
 
28