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Guideline No. 159

Guidance for Industry: Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: General Approach to Establish a Microbiological ADI - VICH GL-36, Final Guidance

Revised August 30, 2006

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
FOOD AND DRUG ADMINISTRATION
CENTER FOR VETERINARY MEDICINE

Guidance for Industry
Studies to Evaluate the Safety of Residues of
Veterinary Drugs in Human Food:
General Approach to Establish a Microbiological ADI
VICH GL-36

FINAL GUIDANCE

(This version of the guidance replaces the version that was made available in July 27, 2006. This guidance document has been revised to add a sentence that had been inadvertently omitted in Section 2.4.1.1. (This guidance replaces CVM’s Guidance for Industry # 52 entitled “Assessment of the Effects of Antimicrobial Drug Residues from Food of Animal Origin on the Human Intestinal Flora” dated February 18, 2004.)

Comments and suggestions regarding the document should be submitted to Division of Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD  20852. Submit electronic comments to http://www.fda.gov/dockets/ecomments.   All comments should be identified with the Docket No. 2003D-0474. 

For questions regarding this document, contact the Division of Human Food Safety, Center for Veterinary Medicine, (HFV-150), Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855, 301-594-1626.

Additional copies of this guidance document 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.

 

 

VICH GL36 (SAFETY)
May 2004
For implementation at Step 7 - Final

STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF VETERINARY DRUGS IN HUMAN FOOD:
GENERAL APPROACH TO ESTABLISH A MICROBIOLOGICAL ADI

Recommended for implementation by May 2005
by the VICH SC at its meeting held in May 2004

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 is recommended for adoption to the regulatory bodies of the European Union, Japan and USA.

STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF VETERINARY DRUGS IN HUMAN FOOD:

GENERAL APPROACH TO ESTABLISH A MICROBIOLOGICAL ADI

 

1. INTRODUCTION

1.1.  Objectives of the guidance
1.2.  Background
1.3.  Scope of the guidance

2. GUIDANCE

2.1.   Recommended Steps in determining the need for a 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
2.2.1.2. Test systems and study design   

2.2.1.2.1. In vitro tests
2.2.1.2.2. In vivo tests

2.2.2.  Increase in the population(s) of resistant bacteria in the human colon

2.2.2.1. Detection of changes in the population of resistant bacteria
2.2.2.2. Test systems and study design

2.2.2.2.1. In vitro tests
2.2.2.2.2. In vivo tests

2.3.  General recommendations
2.4.  Recommended Procedures for Derivation of a microbiological ADI

2.4.1. Disruption of the colonization barrier

2.4.1.1. Derivation of an ADI from in vitro data
2.4.1.2. Derivation of an ADI from in vivo data

2.4.2. Increase in the population(s) of resistant bacteria

2.4.2.1. Derivation of an ADI from in vitro data
2.4.2.2. Derivation of an ADI from in vivo data

3. GLOSSARY

4. REFERENCES

APPENDIX  A

APPENDIX  B

APPENDIX  C

STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF
VETERINARY DRUGS IN HUMAN FOOD:
GENERAL APPROACH TO ESTABLISH A MICROBIOLOGICAL ADI

This final guidance represents the agency’s current thinking on the subject matter.  It does not create or confer any rights for or on any person and does not operate to bind FDA or the public.  You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations.  If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance 


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 should 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.

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.

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.    

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.

Despite an extensive literature review, we found no reports examining effects on length of antimicrobial therapy, length of hospital stays, predisposition to infection, treatment failure, or other impacts on human health resulting from changes in the proportion of antimicrobial resistant bacteria in the normal human intestinal flora.  However, we believe that such effects are likely, based on what is known about the role of microbial ecology in human health.

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.

The present 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 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. Recommended 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?

           - 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. 

Step 2.  Do residues enter the human colon?

Step 3.  Do the residues entering the human colon remain microbiologically active?

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.  Is there 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 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 MIC50 of the most relevant genus/genera for which the drug is active (see Section 2.1.) 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 tract1.  Therefore, the NOAEC may be considered as an option to establish an ADI.  If the NOAEC is used to establish an ADI, it is recommended that the isolates be obtained from multiple healthy individuals, and should 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 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.)

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 where available (e.g., National Committee for Clinical Laboratory Standards [NCCLS]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 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.

Tests 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 2.2.1.2.2.  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

    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 19964, 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.

2.4. Recommended Procedures for 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:



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. 

Mass of colon content: The 220 g value is based on the colon content measured from accident victims.

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 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.

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 Animal: A laboratory animal with its natural indigenous intestinal flora.

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 (HFA) Animal: A germ-free host animal: implanted with human fecal 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.

Minimum Inhibitory Concentration (MIC): The lowest concentration of an antimicrobial compound that 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 Effect Concentration (NOAEC):  The highest concentration that was not observed to cause any  adverse effect in a particular study.
 
No-Observable Adverse Effect Level (NOAEL):  The highest administered dose that was not observed to cause any adverse effect in a particular study.
 
Outcome Variable: A specific parameter measured in an experiment and defined as part of the protocol.  They are the measurements actually made in the study.

Semi-continuous Culture: A culture where substrate and/or waste products are added
 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 2.4.1.2., 2.4.2.1. and 2.4.2.2.    


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.

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.

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.

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, the period during which the integrity and complexity of the intestinal flora remains stable and representative of the intestinal flora should be determined. 

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 should 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 involve 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, such interaction effects should be examined 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 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.

APPENDIX C

Calculation of MICcalc

The MICcalc is derived from the lower 90% confidence limit for the mean MIC50 of the most 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 MIC50 values.  This also implies that the lower 90% confidence limit needs to be back-transformed to obtain the correct value. The formula for the confidence limit is:


where: Mean MIC50 is the mean of the log transformed MIC50 values,
  Std Dev is the standard deviation of the log transformed MIC50 values,
  n is the number of MIC50 values used in the calculations,
t0.10,df is the 90th percentile from a central t-distribution with df degrees of freedom, and df = n-1.


Examine the MIC50 of relevant genera (see Section 2.1).  The MICcalc is based on a summary value of those genera which are not inherently resistant to the compound.  Thus the MICcalc is based on MIC50 of those genera for which the compound is active.  Ensure that all MIC50 values are not characterized as “</=”, so they may be used in the calculation of the MICcalc.

Example Calculation

Any base log transformation of MIC50 values can be used.  However, if 2-fold dilutions of drug 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 MIC50 values were transformed as follows: 


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horizonal rule

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