#229 
 
Guidance for Industry 
 
Evaluating the Effectiveness of New Animal Drugs 
for the Reduction of Pathogenic 
Shiga Toxin-Producing E. coli in Cattle 
 
Submit comments on this guidance at any time.  Submit  electronic comments on the guidance to 
http://www.regulations.gov.   Submit written comments 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. FDA-2015-D-0235. 
 
For further information  regarding this document, contact Joshua R. Hayes, Center for Veterinary 
Medicine (HFV-133), Food and Drug Administration,  7500 Standish Place, Rockville,  MD 
20855, 240-402-0585,  e-mail:  joshua.hayes@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,  7519 Standish 
Place, Rockville,  MD 20855, and may be viewed on the Internet at either 
http://www.fda.gov/AnimalVeterinary/default.htm  or http://www.regulations.gov. 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
October 2015  Contains Non-Binding Recommendations 
Table of Contents 
 
 
I.  INTRODUCTION ...................................................................................................... 3 
II.  GENERAL CONSIDERATIONS .............................................................................. 4 
A.  Protocol Development ..................................................................................... 4 
B.  Animal Welfare Considerations ...................................................................... 5 
C.  Substantial Evidence of Effectiveness ............................................................. 5 
D.  Experimental Parameters ............................................................................... 6 
1.  Study Animals ............................................................................................ 6 
2.  Experimental Unit ....................................................................................... 6 
3.  Experimental Group .................................................................................... 6 
E.  Randomization ................................................................................................ 6 
F.  Masking........................................................................................................... 7 
G.  Personnel Training and Experience ................................................................ 7 
H.  Record Keeping............................................................................................... 7 
I.  Nutritional Content of Diets............................................................................ 8 
J.  Drug Assays..................................................................................................... 8 
1.  Assay Limits for Drugs in Feed ................................................................... 9 
2.  Sampling for Drug Assays ........................................................................... 9 
K.  Combination Approvals ................................................................................ 10 
III.  RECOMMENDED STUDY DESIGNS AND ANALYSES ...................................... 10 
A.  Variables for Evaluation of Effectiveness ..................................................... 11 
1.  Fecal Pen Prevalence ..................................................................................11 
2.  Fecal Pen Quantity .....................................................................................11 
3.  Screen for Induced Resistance ....................................................................12 
B.  Statistical Methods ........................................................................................ 12 
C.  Basis for Study Conclusions .......................................................................... 12 
   
2  Contains Non-Binding Recommendations 
Guidance for Industry 
 
Evaluating the Effectiveness of New Animal Drugs 
for the Reduction of Pathogenic Shiga Toxin-Producing E. coli in Cattle 
 
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. 
 
I.  INTRODUCTION 
 
This guidance provides recommendations to industry relating to study design and describes 
criteria that the Center for Veterinary Medicine (CVM) thinks are the most appropriate for the 
evaluation of the effectiveness of new animal drugs that are intended to reduce pathogenic Shiga 
toxin-producing Escherichia coli (STEC) in cattle. 
 
Section II discusses general considerations regarding the development of protocols, study 
conduct, animal welfare, substantial evidence of effectiveness, experimental parameters, 
nutritional  content of experimental diets, and the assessment of drug concentrations in 
experimental diets. 
 
Section III discusses the studies and analyses CVM recommends for sponsors to substantiate the 
effectiveness of pathogenic STEC reduction drugs. 
 
The guidance is not a comprehensive source of information  on conducting clinical  effectiveness 
studies. Alternative study designs for providing  substantial evidence of effectiveness may be 
acceptable. Sponsors should contact CVM to discuss their development plan prior to initiating 
any studies. Sponsors and clinical  investigators should consult the Code of Federal Regulations 
(21 CFR Parts 511 and 514) for information  on the proper shipment,  use, and disposition  of 
investigational  new animal drugs, as well as submission  of the results of clinical  investigations. 
 
This guidance does not address the evaluation  of human food safety with respect to microbial 
food safety and/or concerns related to antimicrobial  resistance.  CVM encourages sponsors to 
discuss any related concerns in their project plan with CVM as early as possible in the 
development process. 
 
FDAs guidance documents, including  this guidance, do not establish legally  enforceable 
responsibilities.   Instead, guidances describe the Agencys current thinking  on a topic and should 
be viewed only as recommendations,  unless specific regulatory or statutory requirements are 
cited.  The use of the word should in Agencys guidances means that something is suggested or 
recommended, but not required. 
   
3  Contains Non-Binding Recommendations 
II.  GENERAL CONSIDERATIONS 
 
A.  Protocol Development 
 
A protocol should be developed to specifically  describe the plan for conducting  an 
effectiveness study.  The protocol must include a clear statement of the study 
objective(s), state the hypothesis, describe the experimental design in detail, and include 
success, entrance, and exclusion criteria (21 CFR  514.117(b)(2),  (4)-(6)).  A protocol 
should be based upon sound scientific principles  and procedures.  The characteristics of 
an adequate and well-controlled  study are described in 21 CFR  514.117.  Sponsors 
should follow the format for writing protocols that is recommended in CVM Guidance 
for Industry No. 85: Good Clinical  Practices: VICH GL9, Final Guidance, 05/09/01, 
section 6 
(http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/Gu
idanceforIndustry/UCM052417.pdf). 
 
CVM recommends that sponsors submit protocols for review and concurrence before 
beginning  essential studies.1  CVMs concurrence with a protocol represents a 
fundamental agreement between CVM and the sponsor that we agree with the design, 
execution, and analyses proposed in the protocol.  CVM concurrence represents a 
commitment  that we will not later alter our perspectives on these issues unless public or 
animal health concerns appear that we did not recognize at the time of the protocol 
assessment.  Because this concurrence does not extend to any subsequent changes a 
sponsor may make to the protocol, sponsors may want to seek concurrence on the revised 
protocol if they make changes.  Protocol concurrence does not guarantee that the results 
of the study will support a particular finding  or approval of the new animal drug.2 
 
Sponsors may choose to submit either a master or a site-specific protocol.  Sponsors 
should identify which type of protocol they are submitting.   A master protocol provides 
general information  on principles  that apply to all study sites.  Master protocols provide 
any clinical  investigator  the details to conduct the entire study, including,  but not limited 
to the following:   test and control article specifications,  blocking  and randomization 
schemes, a description of the animal model, inclusion/exclusion  criteria, variables of 
interest, statistical analysis, treatment groups, schedule, and success criteria.  
Additionally,  a master protocol may be used as the basis from which more detailed, site-
specific protocols are developed and written. 
 
A site-specific protocol should contain information  present in a master protocol plus any 
detailed information  pertaining to the study site, including,  but not limited  to the 
following:   location of the study/studies,  personnel involved,  diet(s) to be fed, detailed 
facility diagrams showing pen locations,  location of feeders and waters, environmental 
conditions,  and standard operating procedures. 
                                              
1 See Guidance for Industry #215:  Target Animal Safety and Effectiveness Protocol Development and Submission, 
Final Guidance. 
2 Animal Drug User Fee Act Performance Goals and Procedures - 
http://www.fda.gov/ForIndustry/UserFees/AnimalDrugUserFeeActADUFA/ucm042936.htm. 
4  Contains Non-Binding Recommendations 
B.  Animal Welfare Considerations 
 
All studies using live animals that are conducted in the United States must conform to the 
requirements of the Animal Welfare Act (AWA), which is administered by the United 
States Department of Agriculture (USDA).  The USDA has issued policies and 
regulations on how to comply with the requirements of the AWA.  In addition,  many 
research institutions  that conduct research studies using live animals are also accredited 
by the Association for Assessment and Accreditation of Laboratory Animal Care 
(AAALAC). 
 
Farm animals used for biomedical  research (such as drug studies) fall under the purview 
of the AWA and USDA regulations.  Two publications:   the "Guide  for the Care and Use 
of Agricultural  Animals in Agricultural  Research and Teaching,"  published  by the 
Federation of Animal Science Societies (FASS), and the Guide for the Care and Use of 
Laboratory Animals, published  by the Institute for Laboratory Animal Research (ILAR), 
may assist researchers in the implementation  of the USDA regulations.  These guides 
provide information  on the appropriate handling,  housing,  care, treatment, and 
transportation of farm animals for nonagricultural  purposes and should be referenced 
when designing  studies to demonstrate the effectiveness of pathogenic STEC reduction 
drugs in cattle. 
 
C.  Substantial Evidence of Effectiveness 
 
Effectiveness must be demonstrated by substantial evidence consisting of one or more 
adequate and well-controlled  studies [21 U.S.C.  360b(d)(1)(E) and (d)(3); 21 CFR  
514.1(b)(8)(ii);  21 CFR  514.4(a); and 21 CFR  514.117]. 
 
CVM recognizes that non-specific drugs may have a similar effect on pathogenic STECs 
other than O157:H7, but only the major serotypes of concern to human health would be 
currently considered for the indication  (specifically,  O26, O45, O102, O111, O121, and 
O145).  You should first demonstrate the effectiveness of a drug against E. coli O157:H7 
if you wish to include any or all of these additional  serotypes.  CVM does not expect the 
criteria for including  non-O157 serotypes to be as rigorous as for E. coli O157:H7.  
Therefore, although a natural infection field study should be conducted for E. coli 
O157:H7, challenge model studies may be acceptable to demonstrate effectiveness for 
other pathogenic STECs. 
 
CVM recommends a multi-location  field study to establish the effectiveness of 
pathogenic STEC reduction drugs in cattle.  A multi-location  field study should be 
conducted at the appropriate number of sites to ensure that the two principle  objectives 
for the demonstration  of effectiveness, independent substantiation  and inferential  value, 
are met.  Power calculations can help determine the number of experimental units per 
site.  You should conduct the study using at least two different investigators for 
independent substantiation.   You should specifically  state in the protocol the number of 
study sites to be used. 
 
5  Contains Non-Binding Recommendations 
D.  Experimental Parameters 
 
1.  Study Animals 
 
Study cattle should be representative of those in US commercial 
production  in terms of class and weight prior to shipment to slaughter. 
 
You should enroll only healthy cattle in pivotal field studies.  Eligible 
groups of cattle may be screened for the presence of E. coli O157:H7 to 
ensure a useful prevalence in the group prior to enrollment,  but should 
reflect prevalences that are observed among naturally-infected  animals.  
Enrolling  only E. coli O157:H7-positive  cattle does not provide adequate 
inferential value in a field study. 
 
You should state in the protocol the criteria to be used to exclude animals 
or groups of animals from enrollment  during the study as well as under 
what circumstances that would cause a site, pen, or animal to be removed 
after the study has started. 
 
2.  Experimental  Unit 
 
The experimental unit should be the level which is evaluated as the 
primary variable or variable (typically  a pen or group).  Typically,  this is 
determined by the smallest unit to which the drug is intended to be 
administered.  In studies of pathogenic STEC reduction drugs that are 
administered to multiple  animals in or on feed or in water, the 
experimental unit is the pen or group and not the individual  animal. 
 
3.  Experimental  Group 
 
CVM recommends that the experimental groups include the treated 
group(s) receiving the drug at the proposed label dose(s) for the proposed 
frequency and duration and a control group.  CVM recommends that 
you use a placebo concurrent control (referred to as control in the 
remainder of this document) so that the control group cattle receive the 
same handling  and procedures as the treated group cattle.  Using a placebo 
may also help maintain the masking of the study cattle to their 
experimental group assignment. 
 
E.  Randomization 
 
Randomization  in a study design with multiple  levels of organization  usually includes at 
least two recommended steps.  Using the pen as the experimental unit, the first step 
should be to randomly assign cattle to pens.  The second step should be to randomly 
assign each pen to an experimental treatment group. 
 
6  Contains Non-Binding Recommendations 
Blocking  may be used to maintain balance across treatment groups.  Blocking  is 
recommended as a means to reduce variation associated with variables other than the 
experimental treatment within  a block so that the overall unexplained  variation will be 
reduced by accounting for variation among the blocks. 
 
If multiple  arrival groups or lots of cattle are to be enrolled at a given site, care should 
be taken to randomize cattle so that the cattle within  each lot are distributed  in a balanced 
manner across treatment groups in order to avoid any bias offered by a given lot.  As an 
example, pens can be filled with a certain number of cattle from each lot with the number 
proportional  to the size of each lot and then assigning  the individual  pens to a treatment 
group. 
 
The protocol should provide the following  information  concerning the process of 
randomization: 
 
  Identification  of all steps to be used in the process of randomization; 
  A description of the method(s) to be used to generate random assignments; 
  A description of the relationship  between the experimental unit, the process of 
randomization,  and other organizational  levels of the study design; and, 
  Identification  of any blocking  variables, restrictions to randomization,  and other 
variables that are part of the randomization  process. 
 
F.  Masking 
 
In order to minimize  bias, you should mask all personnel responsible for day-to-day 
management of the animals, including  those making and recording observations. You 
must describe masking procedures in the protocol and final study report (21 CFR  
514.117(b)(7)). Masking is an important design technique for avoiding  bias in clinical 
trials (see CVM Guidance for Industry (GFI) #85: Good Clinical  Practices: VICH GL9, 
Final Guidance, 05/09/01). 
 
G.  Personnel Training and Experience 
 
As specified in 21 CFR  511.1(b)(7)(i),  all personnel involved  with the investigation 
must have adequate scientific training and experience with the target animal species and 
disease models used in the studies. 
 
H.  Record Keeping 
 
Good record keeping is a critical component in determining  the effectiveness of a drug 
when it is being assessed.  The integrity  and accuracy of the data collected are critical to 
the acceptance of a study as substantial evidence of effectiveness.  CVMs recommended 
standard for record keeping and data management is contained in CVM GFI #85: Good 
Clinical  Practices: VICH GL9, Final Guidance, 05/09/01. 
 
7  Contains Non-Binding Recommendations 
I.  Nutritional Content of Diets 
 
You should feed animals nutritionally-adequate  diets so that observed responses can be 
attributed to the drug, rather than a possible nutritional  effect.  You should formulate 
diets to meet predominant  commercial practices for the species and class of animal being 
fed, and for the geographic region where you conduct the study.  You may use 
agricultural  survey data, e.g., Agrimetrics, Agri-Tech, etc., to support information  on 
predominant  commercial practices. 
 
Nutrient recommendations for different food-producing  animals, published  by the 
National Research Council (NRC), may serve as a reference for formulating  diets.  You 
should specify feed additives,  such as antioxidants,  pellet binders, copper sulfate, etc., in 
the diet formulation  and ensure that any feed additives do not confound the effects of the 
drug.  You should state in the protocol the standard to which the nutrient levels are being 
compared (e.g., NRC or literature). 
 
To ensure that experimental animals receive proper nutrient densities in the diet, you 
should conduct proximate and chemical analyses on a composite sample from the 
uniform basal diet.  You should include a description of the analyses that you intend to 
conduct in your protocol and specify the number of assay replicates.  You should indicate 
whether the analyses are reported on an as-fed or dry-matter basis. 
 
You should divide  each batch equally among all treatment groups. When a batch of feed 
runs out for one or more treatment groups, you should discontinue  feeding that batch of 
feed to all remaining treatment groups and begin feeding the new batch of feed to all 
treatment groups at the same time. Feed from the previous batch should be weighed and 
properly accounted for in the final study report. 
 
J.  Drug Assays 
 
The purpose of conducting drug assays is to verify that the drug mixed in or on feed or 
constituted in water and used in the study is present at the appropriate concentration. 
 
For drugs administered in feed, personnel responsible for mixing  feed should be aware of 
the performance and capabilities  of the feed mixer(s) that will be used to prepare 
experimental diets.  Medicated feeds should be mixed according to 21 CFR Part 225 - 
Current Good Manufacturing Practice for Medicated Feeds, to ensure adequate 
homogeneity.   All standard operating procedures used for feed mixing  should be 
consistent with applicable  GMP regulations for the manufacture of medicated feeds.  The 
protocol should state that the medicated feed will be mixed at a commercial feed mill  and 
shipped to the study site, or that the medicated feed will be mixed using a validated 
mixer.  The final report should include all relevant records. 
   
8  Contains Non-Binding Recommendations 
1.  Assay Limits for Drugs in Feed 
 
Assay limits  for approved drugs in feeds are codified in 21 CFR  558.4.  
Concentrations of approved drugs in experimental feeds should fall within 
these assay limits  regardless of where and when a feed sample is collected. 
 
For investigational  new animal drugs for which assay limits  have not been 
codified,  levels should conform to the investigational  assay limits derived 
through the feed assay method transfer studies.  If the assay limits are not 
established before conducting  studies, the assay method used to determine 
drug concentration(s) should be no more variable than the method that will 
be subjected to the method validation  process. 
 
Note that a permissible analytical  variation (PAV) relates to a single 
application  of the assay method.  Under the proper mixing  conditions 
using the correct quantity of the drug, a single assay of a feed sample 
should fall within  the assay limits.   If a drug assay(s) falls outside of the 
assay limits,  the feed should not be used in the study and the reason for the 
out-of-assay-limit  result(s) should be investigated and discussed in your 
final study report. 
 
2.  Sampling  for Drug Assays 
 
You should propose a feed sampling method for the study drug assay in 
your protocol.  The method used should provide for a representative 
sample for the drug assay. 
 
The most relevant sampling  point is where and when the test article is 
offered to the animals, with the representative composite samples 
collected when the product is presented to the animals.  For feed-based 
drugs, you may collect samples for the assessment of the test article from 
the batch prepared at a feed mill. 
 
In addition,  you should assay every batch of feed (medicated and control 
feed) or water for the presence of the last non-study drug used in the feed 
or water delivery system unless all of the equipment used in the study is 
dedicated and isolated to avoid cross-contamination.   If dedicated 
equipment is used, you should test for the last run drug at the beginning  of 
the study.  Samples for this purpose should be collected from the point that 
the product is provided to the animals. 
   
9  Contains Non-Binding Recommendations 
K.  Combination Approvals 
 
The Animal Drug Availability  Act of 1996 (ADAA) amended the Federal Food, Drug, 
and Cosmetic Act (the act) and changed the requirements for the approval of certain 
combinations  of new animal drugs that have been previously  separately approved.  There 
are important criteria to consider before a sponsor submits a combination  new animal 
drug application.   For example, under section 512(d)(4) of the act, a sponsor seeking 
approval of a combination  of two or more previously  approved new animal drugs may 
not need to conduct additional  effectiveness studies, if each new animal drug in the 
combination  has at least one unique non-overlapping  indication.   However, among other 
things, all of the following  criteria must be met:  the new animal drugs must provide for 
appropriate concurrent use, must be physically  compatible,  and not have disparate dosing 
regimens (21 U.S.C.  360b(d)(4)(C) & (D)).  If you intend to submit a combination 
new animal drug application,  we recommend you contact CVM for specific requirements. 
 
III.  RECOMMENDED STUDY DESIGNS AND ANALYSES 
 
Drugs that are intended to reduce the exposure risk of consumers to pathogenic STECs are 
expected to predominantly  exert their effect on finishing  cattle by reducing the shedding or 
colonization  of the rectal mucosal surface of asymptomatically-affected  cattle by pathogenic 
STECs.  Drug intervention  in the live animal therefore represents a pre-harvest intervention  that 
should be used in concert with other interventions  to reduce the exposure of beef carcasses to 
fecal contamination  by these pathogens. 
 
CVM limits  indications  for pathogenic STEC reduction drugs to the effect of the drug in live 
cattle prior to shipment to slaughter, and also considers the duration of the drugs effect to allow 
for transit time to slaughter.  For this reason, CVM will evaluate study designs to support the 
following  suggested indications  for the reduction in the prevalence or quantity of pathogenic 
STECs in feces, with E. coli O157:H7 as the principal  target: 
 
For the reduction in fecal pen prevalence of Escherichia coli O157:H7 among feedlot 
cattle prior to shipment to slaughter 
 
For the reduction in fecal pen quantity of Escherichia coli O157:H7 among feedlot 
cattle prior to shipment to slaughter 
 
For the reduction in fecal pen prevalence and fecal pen quantity of Escherichia coli 
O157:H7 among feedlot cattle prior to shipment to slaughter 
 
You should clearly state the intended targeted indication(s)  in the protocol. 
   
10  Contains Non-Binding Recommendations 
A.  Variables for Evaluation of Effectiveness 
 
Pathogenic STECs contaminate beef carcasses primarily due to exposure to fecal matter 
during the removal of an animals hide.  The sampling variability  and uncontrollable 
environmental  factors commonly  encountered in a field study precludes the use of hide 
samples as evidence of effectiveness.  Because the ultimate source of hide contamination 
is feces and because significant  fecal cross-contamination  of hides occurs during transit 
of cattle, fecal samples (fecal grab samples from the rectum) should be collected from 
live cattle prior to shipment to evaluate the effectiveness of pathogenic STEC reduction 
drugs. 
 
You should collect fecal samples from all animals at several time points during the study 
and used to calculate results for groups or pens of cattle.  You should collect at least one 
sample prior to treatment with the drug and at least two post-treatment samples for the 
pivotal analysis of the below variables. 
 
Because the contamination  of beef carcasses by strains of E. coli O157:H7 appears to be 
more closely associated with the contamination  of hides during lairage at the slaughter 
facility than a source feedlot, any approvable product should demonstrate effectiveness 
for at least the greater of the pre-slaughter withdrawal period or 24 hours.  An ideal live 
animal intervention  would be one that demonstrates a period of effectiveness for roughly 
9 to 14 days prior to shipment to slaughter in order to provide time for any fecal 
reduction in E. coli O157:H7 to be mirrored by a reduction on the hides of treated cattle. 
 
1.  Fecal Pen Prevalence 
 
You should measure the prevalence of E. coli O157:H7 among all animals 
within a pen using an appropriate and sensitive culture technique at each 
sampling  time point.  You should confirm suspect colonies as E. coli 
O157:H7 using confirmatory genetic or immunologic  methods. 
 
For studies that seek a fecal pen prevalence reduction indication,  these 
data represent the primary response variable.  For studies that seek a fecal 
pen quantity reduction indication,  these data will be used to evaluate 
whether the drug may unintentionally  increase the fecal pen prevalence of 
E. coli O157:H7 despite any apparent reduction in the concentration of the 
pathogen. 
 
2.  Fecal Pen Quantity 
 
You should collect and quantify the fecal concentration of E. coli 
O157:H7 from each animal in the field studies at each sampling  time 
point.  You should confirm suspect colonies as E. coli O157:H7 using 
confirmatory genetic or immunologic  methods in a manner that will  allow 
CVM to assess the concentration of the specific pathogen in feces, 
expressed in colony-forming  units (CFU) per gram of dry feces. 
11  Contains Non-Binding Recommendations 
 
For studies that seek a fecal pen quantity reduction indication,  these data 
represent the primary response variable.  For studies that seek a fecal pen 
prevalence reduction indication,  these data will be used to evaluate 
whether the drug may unintentionally  increase the fecal pen quantity of   
E. coli O157:H7 pathogen in feces despite any apparent reduction in the 
prevalence of the pathogen in feces. 
 
3.  Screen for Induced Resistance 
 
If it is possible that the pathogenic STEC reduction drug may engender 
resistance in E. coli O157:H7 to the drug and thus limit  its effectiveness 
over time, you should assess this potential  within the field study.  CVM 
recommends that you use a selective medium that has a concentration of 
drug that will  allow you to more sensitively  detect a change in 
susceptibility  to the drug among E. coli O157:H7. 
 
B.  Statistical Methods 
 
The final report should contain: 
 
  Raw data in its original  form; 
  Raw data in an electronic format (Excel, SAS or text file); 
  Program code to read raw data and create calculated variables; 
  Statistical programs with documentation;  and 
  All statistical output (e.g., analysis results). 
 
You should also provide a document identifying  the purpose or content of each file and a 
document that describes variable names, abbreviations,  formats, and how variables are 
used in the analysis. 
 
C.  Basis for Study Conclusions 
 
In order to conclude that the drug is effective for a reduction in fecal pen prevalence 
indication,  the study should meet the following  success criteria: 
 
1.  A statistically  significant  (=0.05, two-sided) difference in fecal pen prevalence of  
E. coli O157:H7 between the treated and control group with a lower prevalence in the 
treated group to establish a post-treatment effectiveness period of at least 24 hours in 
duration throughout a minimum  of two sampling  time points within this period; and, 
 
2.  No numerically  higher pen average fecal concentration of E. coli O157:H7 in treated 
group pens compared to control group pens throughout the above-defined 
effectiveness period at each site. 
 
12  Contains Non-Binding Recommendations 
In order to conclude that the drug is effective for a reduction in fecal pen quantity 
indication,  the study should meet the following  success criteria: 
 
1.  A statistically  significant  (=0.05, two-sided) reduction in the fecal pen average 
concentration of E. coli O157:H7 in the treated group compared to the control group 
to establish a post-treatment effectiveness period of at least 24 hours in duration 
throughout  a minimum  of two sampling time points within this period; and, 
 
2.  No numerically  higher fecal pen prevalence of E. coli O157:H7 in treated group pens 
compared to control group pens throughout the above-defined effectiveness period at 
each site. 
 
For both indications,  the effect of the drug treatment should be biologically-meaningful.   
You should propose and provide information  to support the biological  relevance of the 
observed effect, which may include such things as the similarity  to seasonal effects (e.g., 
prevalence of pathogens at winter levels), Hazard Analysis and Critical Control Point 
(HACCP) interventions,  or other similar  relevant comparators. 
 
In addition,  for the evaluation of effectiveness, there should be no scientifically 
significant  increase in resistance to the drug among E. coli O157:H7 isolated at the post-
treatment time points in the treated group compared with those isolated prior to treatment. 
 
Other reasonable scientific approaches will be considered. 
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