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Guidance for Industry

ASSESSMENT OF THE EFFECTS OF ANTIMICROBIAL DRUG RESIDUES FROM FOOD OF
ANIMAL ORIGIN ON THE HUMAN INTESTINAL FLORA

**Final Guidance**

This guidance discusses a recommended pathway approach for assessing the
effects of antimicrobial drug residues in food on the human intestinal
flora. This document supercedes the current guidance #52, Guideline for
Microbiological Testing of Antimicrobial Drug Residues in Food,
published in January of 1996.

Comments and suggestions regarding this document should be sent to the
Division of Dockets Management (HFA-305), Food and Drug Administration,
5630 Fishers Lane, Room 1061, Rockville, MD 20852. All comments should
be identified with the Docket No. 93D-0398. Submit electronic comments
at <http://www.fda.gov/dockets/ecomments>.

For questions regarding this document, contact Haydée Fernández,
Division of Human Food Safety (HFV-153), Center for Veterinary Medicine,
Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855,
301-827-6981, E-mail: <afernand@cvm.fda.gov>.

Additional copies of this final guidance may be requested from the
Communications Staff (HFV-12), Center for Veterinary Medicine, Food and
Drug Administration, 7519 Standish Place, Rockville, MD 20855 and may be
viewed on the Internet at <http://www.fda.gov/cvm>*.*

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**U.S. Department of Health and Human Services**

**Food and Drug Administration**

**Center for Veterinary Medicine**

**February 18, 2004**

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## **TABLE OF CONTENTS**

######## I. INTRODUCTION ........................................................................3

II. **PATHWAY APPROACH FOR ADDRESSING THE EFFECTS OF**

**ANTIMICROBIAL DRUG RESIDUES FROM FOOD OF ANIMAL**

**ORIGIN ON THE HUMAN INTESTINAL FLORA ..............................4**

######## III. REFERENCES .............................................................................8 

ASSESSMENT OF THE EFFECTS OF ANTIMICROBIAL DRUG RESIDUES FROM FOOD OF
ANIMAL ORIGIN ON THE HUMAN INTESTINAL FLORA[^1]

**I. INTRODUCTION**

The human intestinal flora is a balanced ecosystem that is very
important in maintaining an individual's health. Although this system is
generally stable, clinical studies have shown that therapeutic doses of
antimicrobials may change the balance. ^(1,2,3)^ The type or extent of
change in the system will depend on the spectrum of action of the
antimicrobial drug, its dose, and the length of an individual's exposure
to the drug. The lowest concentration of any antimicrobial drug that can
affect the intestinal flora is not clear. However, studies in *in vitro*
(continuous or semi-continuous flow culture systems) and *in vivo* human
flora-associated rodent (rodents implanted with human fecal flora) test
systems and in human volunteers have shown that low levels of
antimicrobial drugs are capable of altering different parameters of the
intestinal flora depending on the spectrum of action and concentration
of drug. ^(4,\ 5,\ 2,\ 3)^

The main adverse effects of antimicrobial drugs on the human intestinal
flora are selection of resistant bacteria and disruption of the
colonization barrier (or barrier effect) of the resident intestinal
flora. Colonization barrier or barrier effect is the "limiting action"
of the normal flora on colonization of the bowel by exogenous or
indigenous potentially pathogenic microorganisms. ^(6)^ Other effects,
such as alteration of the metabolic activity of the flora, may be
important, also.

Regulators and sponsors of new animal drugs have an interest in
establishing relevant and validated methods for determining the effects
of microbiologically active animal drug residues on the human intestinal
flora. Any such effects should be assessed in the human food safety
evaluation of new animal drugs intended for use in food-producing
animals. Among the *in vitro* and *in vivo* approaches currently used to
study the effect of antimicrobial drugs on the human intestinal flora
are quantitative *in vitro* antimicrobial drug susceptibility testing,
static batch cultures, semi-continuous and continuous flow culture
systems, simulated gut models, human volunteers, conventional animals,
gnotobiotic rodents, and human flora-associated rodents.

CVM has decided to modify Guidance No.52 "Microbiological Testing of
Antimicrobial Drug Residues in Food" published in January of 1996 ^(7)^
based on information made available after that time concerning the
effects of low doses of different classes of antimicrobial drugs on the
human intestinal flora. Therefore, CVM is recommending that sponsors use
a "pathway approach" (described below) for addressing the human food
safety of antimicrobial drug residues rather than the approach described
in the 1996 version of the guidance. The scientific rationale for this
decision and the FDA/CVM regulatory history on this issue are provided
in a separate document available to the public at the Division of
Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers
Lane, rm. 1061, Rockville, MD 20852. ("History and Scientific Issues
Related to Guidance #52")

The pathway approach presented in this guidance document represents a
general approach for assessing the microbiological safety of
antimicrobial drug residues in food. If further microbiological studies
are warranted for determining the Acceptable Daily Intake (ADI) for a
new animal antimicrobial drug, the sponsor of that drug is encouraged to
contact the Center to discuss the appropriate test systems and protocols
for the studies.

CVM is aware that the International Cooperation on Harmonisation of
Technical Requirements for Registration of Veterinary Medicinal Products
(VICH) is currently drafting a related guideline and that this guidance
may be superceded at a later date by a guidance document published by
the VICH.

**FDA's guidance documents, including this guidance, do not establish
legally enforceable responsibilities. Instead, guidances describe the
Agency's 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. **PATHWAY APPROACH FOR ADDRESSING THE EFFECTS OF**

**ANTIMICROBIAL DRUG RESIDUES FROM FOOD OF ANIMAL**

**ORIGIN ON THE HUMAN INTESTINAL FLORA**

The conditions and rationale for addressing the microbiological safety
of antimicrobial drug residues in food are simplified in a chart at the
end of this section.

The microbiological safety of antimicrobial drug residues in food is an
important issue that should be addressed by the sponsor of a new animal
drug. An assessment of the safety of antimicrobial drug residues in food
should be part of the human food safety component of new animal drug
applications for antimicrobial drugs. If these residues have no
antimicrobial activity against representatives of the human intestinal
flora (*E. coli*, and species of *Bacteroides, Bifidobacterium,
Clostridium, Enterococcus, Eubacterium (Collinsella), Fusobacterium,
Lactobacillus, Peptostreptococcus/Peptococcus*), an ADI should be
calculated based on traditional toxicology studies. However, if the
residues have antimicrobial activity, the sponsor should address the
potential availability of these microbiologically active residues in the
human colon. It should be assumed that the human colon would be exposed
to all residues present in the edible tissues, unless the sponsor can
demonstrate through reference to controlled experimentation in humans or
animals (e.g., pharmacokinetic studies, bioavailability studies, etc.)
that some or all of the residues have no potential to enter the colon.

If it is determined that microbiologically active residues can enter the
colon, the sponsor should assess the potential of these residues to
select for resistant bacteria, disrupt the protective barrier effect
provided by the intestinal flora, or otherwise alter the balance of
intestinal flora. The sponsor may attempt to demonstrate that the
residues are metabolized rapidly to microbiologically inactive compounds
or are rapidly bound to intestinal contents and rendered
microbiologically unavailable in the human colon. Alternatively, if the
antimicrobial residues are not metabolized or bound such that they are
microbiologically inactive, the sponsor should determine if these
residues would cause adverse effects on the intestinal flora. This could
be done using appropriate human data, if available, or data from *in
vitro* or *in vivo* test systems. The adverse effects of human health
concern that should be considered are the potential of these residues to
select for resistant bacteria, disrupt the protective barrier effect
provided by the intestinal flora, or otherwise alter the balance of
intestinal flora. If no information is available, the sponsor should
perform studies using an *in vitro* or an *in vivo* test system to
determine the endpoint(s) of human health concern.

The sponsor may wish first to perform preliminary studies such as batch
cultures with fecal suspensions or an *in vivo* preliminary study to
determine which microbiological endpoint is suspected to be altered by
the drug. The sponsor may also choose to perform a definitive study
using an *in vitro* or an *in vivo* test system to study the effect of
the drug on the endpoints of human health concern and determine the
no-observable effect concentrations/no-observable effect levels
(NOECs/NOELs) for these endpoints. However, if the endpoint(s) of
concern have been determined, definitive studies using *in vitro* or *in
vivo* test systems should be performed to determine the NOEC/NOEL for
the drug on the chosen endpoint(s).

If disruption of the colonization barrier is the endpoint of concern,
either *in vitro* (e.g., continuous or semi-continuous culture systems)
or *in vivo* test systems (e.g., human flora-associated rodent test
systems) are preferable for determining a NOEL for this endpoint, as
opposed to *in vitro* antimicrobial susceptibility testing to generate
minimum inhibitory concentration (MIC) data. This is because these
systems have the potential to better approximate the effects of
microbial interactions and high bacterial densities than MIC data.
However, the sponsor could use MIC data as an option to estimate a
conservative microbiological ADI. MIC data indirectly assess changes in
bacterial populations. If the antibiotic concentration is below the
levels that inhibit cell growth, it could be assumed that the bacteria
responsible for the barrier effect would not be affected. Vice versa, if
bacteria are not allowed to grow due to low concentrations of
antimicrobials, an unbalanced flora could allow the establishment of a
pathogen that is not sensitive to the antibiotic in study.

The ADI derived from MIC data is conservative because the inoculum
density used for testing is orders of magnitude lower than the bacterial
population of the colon. In addition, the growth conditions in MIC
testing (growth medium, pH, lack of fecal solids, lack of microbial
interactions and drug metabolism, etc.) minimize the potential of drug
inactivation. If MIC testing is used to derive an ADI, the median MIC
obtained by standard methods such as those of the National Committee for
Clinical Laboratory Standards (NCCLS) should be used to determine a
NOEC. It is recommended that at least 10 isolates from each of the most
representative bacteria listed on pages 4 and 5 of this guidance be
obtained from healthy human volunteers.

Although the use of MIC data may be an option to derive an ADI, CVM does
not encourage the use of this data for determining the NOEC for
disruption of the colonization barrier of the human intestinal flora.
Quantitative *in vitro* determinations of antimicrobial susceptibility
do not reflect or account for factors such as bacterial population
density, pH, intestinal growth conditions, bacterial metabolism,
bacterial antagonism, or other factors of relevance to the human colonic
flora. A more appropriate NOEC may be obtained through other test
systems that better model the intestinal flora.

If the endpoint of concern is the selection of resistant bacterial
strains, the sponsor should conduct *in* *vitro* or *in vivo* studies in
test systems to determine a NOEL for this endpoint. Quantitative *in
vitro* determinations of antimicrobial susceptibility, leading to the
generation of MIC data that is coupled to the effects generated in the
test system(s), should be an element of this analysis.

PATHWAY APPROACH FOR ADDRESSING THE EFFECTS OF ANTIMICROBIAL DRUG
RESIDUES FROM FOOD OF ANIMAL ORIGIN ON THE HUMAN INTESTINAL FLORA

> **Yes**

**No**

**No No**

> **Yes**

**No**

> **Yes**

**No**

> **Yes**

### 

**III.** **REFERENCES**

1.  Carman, R.J., Van Tassell, M.S., and Wilkins, T.D. The Normal
    Intestinal Microflora: Ecology, Variability and Stability.
    Veterinary and Human Toxicology 35 (Supplement 1): 11-14, 1993.

2.  Edlund, C. and Nord, C.E. Effect of Quinolones on Intestinal
    Ecology. Drugs 58 (Supplement 2): 65-70, 1999.

3.  Finegold, S.M., Mathisen, G.E., and George, W.L. Changes in Human
    Intestinal Flora Related to the Administration of Antimicrobial
    Agents. In: *Human Intestinal Microflora in Health and Disease*.
    Academic Press, Inc. ISBN 0-12-341280-3. pp 355-429, 1983.

4.  Gorbach, S.L. Perturbation of Intestinal Microflora. Veterinary and
    Human Toxicology 35(Supplement 1): 15-23, 1993.

5.  Heimdahl, A., Kager, L., and Nord, C.E. Changes in the Oropharyngeal
    and Colon Microflora in Relation to Antimicrobial Concentrations in
    Saliva and Feces. Scand. J. Infect. Dis. Supplement 44: 52-58, 1985.

6.  Vollaard, E.J. and Clasener, H.A.L. Minireview. Colonization
    Resistance. Antimicrob. Agents Chemother. 38 (3): 409-414, 1994.

7.  FDA/CVM. Guidance. Microbiological Testing of Antimicrobial Drug
    Residues in Food. January, 1996.

[^1]: This guidance has been prepared by the Office of New Animal Drug
    Evaluation in the Center for Veterinary Medicine at the Food and
    Drug Administration.