Clinical trial NCT02831998
Conditions: Surgical Skin Preparation
Sponsor: Zurex Pharma, Inc.
Start date: 2016-07-27; Completion date: 2017-04-09
Enrollment: 440 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The objective of the study is to demonstrate the antimicrobial efficacy of the ZP Preoperative Prep on skin flora of the abdomen and inguinal regions of human subjects.

Detailed description:
The purpose of this study is to evaluate the antimicrobial properties of one finished test product (ZP) with a positive control (CP) and a negative control (ZP™ Vehicle without IPA) when used as a patient preoperative skin preparation.

Arms: arm 1: Isopropyl alcohol (IPA) 70% arm 2: Chlorhexidine gluconate (CHG) 2% / Isopropyl alcohol (IPA) 70% arm 3: ZP without IPA
Interventions: intervention 1: ZuraPrep intervention 2: ChloraPrep intervention 3: ZP Vehicle
Intervention descriptions: intervention 1: Apply topically. intervention 2: Apply topically. intervention 3: Apply topically.

Protocol documents:
Prot_SAP_000.pdf: 


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TITLE PAGE (continued) 
 
 
Zurex Pharma, Inc.  
Sponsor Representative: 
 
 
 
 
 
 
 
 
 
Study Monitor 
 
 
 
 
 
 
 
Qualified Physician: 
 
 
 
 
 
Statistical Consultant: 
 
 
 
 
 
 
 
 
 
 
 
 




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Table of Contents 
 
 
 
1. 
Background Information ...................................................................................................... 5 
2. 
Study Objectives And Purpose ........................................................................................... 6 
3. 
Study Design ...................................................................................................................... 7 
4. 
Subject Selection ...............................................................................................................16 
5. 
Study Treatment ................................................................................................................19 
6. 
Assessment Of Efficacy .....................................................................................................26 
7. 
Assessment Of Safety .......................................................................................................27 
8. 
Statistics ............................................................................................................................30 
9. 
Monitoring ..........................................................................................................................32 
10. Quality Control And Quality Assurance ..............................................................................33 
11. Ethics .................................................................................................................................33 
12. Data Handling And Record Keeping ..................................................................................33 
13. References ....................................................................................................................... 35 
14. Appendices ........................................................................................................................35 
 
 
 
 
 
 
 
 
 


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1. Background Information 
 
1.1 Name, Description and Intended Use of the Test and Control Articles 
(Treatments) 
 
The single investigational test article, ZuraPrep™ solution 
 
 is being evaluated for efficacy as a preoperative skin preparation solution 
by demonstrating its immediate and persistent antimicrobial properties.  Testing will 
be performed based upon procedures outlined in the FDA Tentative Final 
Monograph (TFM) for Health-Care Antiseptic Drug Products (vol. 59, No. 116, 
June17, 1994, pp. 31450-31452) and ASTM E1173 – 15 Evaluation of Preoperative, 
Precatheterization, or Preinjection Skin Preparations. 
 
ZuraPrep™ Vehicle 
 will be 
evaluated as a negative control.  
 
ChloraPrep® 
 (Scrub Teal® Tint) will be evaluated as a reference 
control. 
 
1.2 Risk/Benefit Summary 
 
There are minimal anticipated adverse health risks for the participants of this study.  
Considerable safety and efficacy data are available for the active ingredient in the 
test and reference formulations. 
  
1.3 Treatment Application Configurations 
 
The treatments will be applied topically to intact skin of the abdomen and groin 
regions of each subject. The treatments will be applied per the Treatment Application 
Instructions (Appendix 14.7). 
 
1.4 Good Clinical Practice and Regulatory Requirements 
 
This study will be conducted in compliance with this protocol, Good Clinical Practice 
(GCP) including 45 CFR 160 and 164 (Authorization for Use/Disclosure of Protected 
Health Information (PHI), 21 CFR 50 (Protection of Human Subjects), 56 
(Institutional Review Boards), 330 (Over-The-Counter  Human Drugs which are 
generally recognized as safe and effective and not misbranded) and Tentative Final 
Monograph for Health Care Antiseptic Drug Products (TFM), the Standard Operating 
Procedures of MicroBioTest, the study protocol and any protocol amendments. 
 
 
 




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3. 
Study Design 
 
3.1 Study Type 
 
This is a randomized, paired-comparisons design where each subject receives two of 
the planned treatments. 
 
 
Table 3.1:  Treatments, Anatomical Sites of Evaluation, Application and Dry Times and 
Coverage Areas (See Appendix 14.7 for detailed application instructions) 
Treatment 
(Quantity/Volume) 
Body Site 
Application 
Time 
Dry Time 
Area of 
Coverage 
ZuraPrep™ 
 
 
Abdomen 
(sebaceous poor) 
30 seconds 
3 minutes 
5” x 5” 
Groin  
(sebaceous rich) 
2 minutes 
3 minutes 
1.5” x 5” 
ChloraPrep® 
 
  
Scrub Teal® Tint,  
(positive control)   
Abdomen 
(sebaceous poor) 
30 seconds 
3 minutes 
5” x 5” 
Groin  
(sebaceous rich) 
2 minutes 
3 minutes 
1.5” x 5” 
ZuraPrep™ Vehicle, 
 
(negative control) 
Abdomen 
(sebaceous poor) 
30seconds 
3 minutes 
5” x 5” 
Groin  
(sebaceous rich) 
2 minutes 
3 minutes 
1.5” x 5” 
 
 
3.2 Primary Endpoint/Analysis 
 
The study will be analyzed per the standards of the 1994 Tentative Final Monograph 
(TFM) for Effectiveness Testing of a Patient Preoperative Skin Preparation (FR 
59:116, 17 June 94, pp. 31450-31452), as updated by recent recommendations by 
the FDA on responder rates.  The primary efficacy criteria will be assessed based on 
log10 CFU/cm2 reductions at the 10-minute sampling time. The TFM indicates log10 
CFU/cm2 reductions as the primary efficacy measure while the Proposed 
Amendment of the Tentative Final Monograph (FR 80:84, 01 May 2015) indicates 
responder rates as the primary efficacy requirement. This study will use responder 
rate at 10 minutes post-prep as the primary measure of efficacy but will also 
calculate and show log10 CFU/cm2 reductions from baseline. 
 


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The primary measure of antimicrobial efficacy is the reduction of skin flora on the 
abdominal and groin sites at 10 minutes following application of the study treatments 
relative to the Treatment Day baseline counts.  
The reduction will be calculated by a percent successes approach (i.e. percent of 
subjects meeting required reductions = responder rate).  A site is considered a 
responder for the treatment at the indicated time point if the criteria in the following 
table are met:  
Table 3.2:  Minimum and Maximum Treatment Day Baseline and Expected Mean Log10 
Reduction per Anatomical Site 
Anatomical Site 
Minimum and Maximum  
Treatment Day Baseline* 
Expected Mean Log10 
Reduction 
Abdomen 
1.0 x 103 – 3.2 x 105 CFU/cm2
  
(3.0 Log10 – 5.5 Log10) 
2.0 log10 @ 10-minutes 
Groin 
1.0 x 105 – 3.2 x 107 CFU/cm2
  
(5.0 Log10 – 7.5 Log10) 
3.0 log10 @ 10 minutes 
*Note: For the Screening Day baseline, the maximum upper limit does not apply.  Only the 
minimum criteria must be met. 
  
Responder status will be calculated separately for the abdomen and groin, for each 
test substance, for each post-treatment sample time, and for each subject. The 
individual responses will then be grouped to generate an overall responder rate for 
each anatomical area, for each test substance, and for each post-treatment time. 
The primary efficacy goal for active products is to have the lower bound of the 95% 
confidence interval for the responder rates at 10 minutes to be greater than or equal 
to 70%.  
 
Secondary Endpoints/Analysis 
 
Responder rates at 30 seconds will be calculated identically to the 10 minute rates. 
At 6 hours a site is considered a responder if it is below baseline; otherwise, 6-hour 
responder rates will be calculated identically to the 10-minute responder rates. The 
secondary efficacy goals are to have the 95% confidence intervals for the responder 
rates to be greater than or equal to 70%. Log10 CFU reductions from baseline will 
also be calculated for all post-prep samples.  
 
Study Validity 
 
For a valid study, the active control must make the same objectives that the test 
article does and both the test article and the active control should be superior to the 
inactive control for the primary endpoint. The responder rates for both active and 
inactive treatments at 6 hours are near 100%. Therefore, comparison of responder 
rates at 6 hours is unlikely to show any difference. Differences in log10 CFU/cm2 
reductions from baseline will be used at 6 hours in order to show that the active 
treatments are more effective than the inactive control. 


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3.3 Randomization and Blinding 
 
Subjects will be randomized before treatment, after screening eligibility is 
determined.  Subjects will be randomized to treatment using the following block 
design: 
 
Treatment Balance 
Each subject will receive two different treatments, one on the right side of the 
body and one on the left.   
 
The treatment assignments will be balanced such that the number of readings 
per anatomical site matches the calculated requirements.  
 
Left/Right Balance 
The application will be randomized so that each treatment is used on an equal 
number of left and right sides of the body. 
 
Site and Sample Time Balance 
Within each anatomical site, sampling order will be randomized for baseline, 30 
second, 10 minute, and 6 hour samples based on the statistical consultant’s 
randomization plan. 
 
The randomization plan has been updated
 
  
 
. 
 
The Investigator is responsible for ensuring that the randomization is followed. A 
basic outline of a randomization schedule for the abdominal and groin sites is 
provided in Appendix 14.4. The final randomization schedule will be prepared before 
the initial treatment.  The test and control articles will be labeled with the appropriate 
codes as designated by the study randomization.   
 
Subjects will be identified by their initials, a screening ID number, and a subject 
number corresponding to each body region in the study for which they qualify.  
Subject numbers will not be assigned until a subject has passed the screening 
criteria, including baseline bacterial counts (at least 1.0 x 105 CFU/cm2 in the groin 
and at least 1.0 x 103 CFU/cm2 on the abdomen). Subjects whose abdominal and 
groin regions qualify will be assigned the subject number. Therefore, for each of the  
participating subject will be assigned two identification numbers. 
 
 
 


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• 
Screening subjects will be assigned numbers ranging from 9001 to 9999. 
 
• 
Subjects to be treated (including treatment day baseline collection) will be 
assigned sequential numbers ranging from 0001 up to the total number of 
test subjects treated.  
 
The study materials will not be blinded from the Investigator or other study staff 
performing the study material application or bacterial sample collections. The staff 
member(s) performing bacterial enumeration will be blinded from the identification of 
treatment assignment.  The study staff performing the bacterial enumeration will not 
be involved in the study material application or the collection of samples. The Raw 
Data Sheet sections of the case report form will be maintained separately (from the 
pages within the case report form which include study treatment identifications) 
during the conduct phase of the study. The study staff performing the bacterial 
enumeration will record counts directly onto the Raw Data Sheet pages of the case 
report form without accessing the subject study documentation folder containing the 
other case report form pages. The Raw Data Sheets will be compiled with the entire 
case report form after all data recording has been completed. The CRF will serve as 
the source document. 
 
 






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• 
Timers or stopwatches 
• 
Pipette Aid or similar apparatus 
• 
Vortex mixer 
• 
Surgical Clipper & clipper blades 
• 
Water bath (45 ± 2C) 
• 
Incubator (30 ± 2C) 
• 
Disposable underwear for subjects 
• 
Tryptic Soy Broth (for the Neutralization Validation Procedure only – See 
Appendix 14.10) (TSB) 
• 
 Urine Strip Test (for Pregnancy) or equivalent.  
 
3.5 Study Duration 
 
The expected duration of this study for each subject is up to 3-4 weeks.  Subjects will 
undergo at least a 14-day washout period followed by a qualification screening 
baseline visit. Subjects whose screening baseline samples meet the minimum values 
described in the Inclusion Criteria (Section 4.1) will be notified and invited to 
participate in the treatment phase of the study. The treatment phase will be 
scheduled no sooner than 72 hours from the screening baseline collection.    
 
3.6 Study Termination/Subject Discontinuation or Withdrawal/Subject Revocation 
of Authorization 
 
3.6.1 Study Termination 
 
Zurex Pharma, Inc. or the Investigator has the right to discontinue the study 
at any time for medical and/or administrative reasons.  As far as possible, this 
should occur after mutual consultation. 
 
3.6.2 Subject Discontinuation and Withdrawal 
 
The Investigator may discontinue individual subjects from the study at any 
time.  Subjects may voluntarily withdraw from the study at any time without 
reason or consequence. The subject will be asked to report the reason for 
withdrawal. The Investigator will provide a written report on the appropriate 
case report form (CRF) including the date and reason for discontinuance.  
Subjects who qualify on Screening Day and begin the treatment phase may 
not be re-entered into the study, regardless of whether or not they completed 
the study. 
 
 
 
 


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Any enrolled subject will be discontinued for the following reasons:  
 
1. A skin irritation rating of 3 for any individual skin condition at any 
evaluation following the application of study treatment. (A skin 
irritation rating of 2 for any individual skin condition at any evaluation 
following the application of study material may also be the cause for 
subject discontinuation at the discretion of the Investigator.) 
2. Experiencing a serious protocol deviation that compromises the data 
results, for example, using a topical antibiotic at a test site during the 
study. 
 
See section 8.5 for handling of withdrawn subject data.  
 
3.6.3 Subject Revocation of Authorization to Use and Disclose Protected 
Health Information 
 
In order to implement a valid revocation of authorization, the subject or their 
representative must make the request in writing to MicroBioTest, 
 
 The revocation cannot stop the use or 
disclosure of information that has been collected prior to the revocation, or is 
needed to ensure complete and accurate study results, and/or is required by 
law or government regulation (e.g., reporting adverse events, etc.).  
Revocation of an authorization may not be used to withhold normal medical 
care from the subject, but may [or will] make the subject ineligible to receive 
the study treatment or care. 
  
3.7 Treatment Material Accountability 
 
Zurex Pharma, Inc. requires Investigators to maintain accountability and adequate 
inventory security of the study material at all times. The Investigator or designee will: 
 
• 
complete the Confirmation of Release and Receipt of Study Materials form 
(Appendix 14.8) upon receipt of the shipment and maintain and account for 
inventory on the Study Material Disposition form (Appendix 14.9). 
• 
keep study materials in a secure storage area, accessible only to authorized 
individuals. 
• 
dispense study material only to subjects properly enrolled into the study. 
• 
return all unused study materials to Zurex Pharma, Inc. at the end of the study or 
dispose of unused study materials as agreed upon. 
 
 
 


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3.8 Source Data 
 
The CRF will serve as the source document. Data will be recorded directly onto the 
CRF and will not be transcribed.  
 
Includes any original documents, data, and records where any data are first recorded 
(e.g., questionnaires, consent forms, and laboratory notes). If data are recorded for 
the first time directly onto the CRF, then the CRF is considered the source document 
for these data. 
 
3.9 Protocol Modifications 
 
3.9.1 Protocol Amendments 
 
The party initiating an amendment must confirm it clearly in writing using the 
Amendment/Administrative Revision form. It must be signed and dated by 
Zurex Pharma, Inc. and, in the case of a significant amendment, the 
Investigator. A significant amendment means one that affects the safety, 
rights or welfare of subjects, the scope of the investigation or the scientific 
quality of the study. 
 
Zurex Pharma, Inc. will submit significant protocol amendments to the 
Investigator for submission to the IRB.  Zurex Pharma, Inc. will also notify the 
Investigator when a protocol amendment may be implemented. 
 
 
3.9.2 Protocol Deviations 
 
A deviation is a departure from the protocol that will likely affect the safety, 
rights or welfare of subjects, the scope of the investigation or the scientific 
quality of the study. Protocol deviations are documented on a Protocol 
Deviation Form and appropriate CRF, if applicable. 
 
Sponsor Notification 
Deviations that potentially affect 1) subject safety, rights or welfare,  
2) data integrity or 3) compromise the statistical analysis of the study require 
immediate communication to Zurex Pharma, Inc. The Investigator must 
contact the Zurex Pharma, Inc. study monitor within 24 hours of occurrence 
at 
the 
following 
phone 
number: 
 
 
 A Protocol 
Deviation Form must be completed by the Investigator and include a 
description of the circumstances surrounding and the reason for the 
deviation, any actions taken, and whether or not the subject was allowed to 


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continue in the study. A copy must be sent to the Zurex Pharma, Inc. study 
monitor within 24 hours of identifying the occurrence. 
 
IRB Notification 
Deviations which are made to protect the life or physical well-being of a 
subject in an emergency must be reported by the Investigator to the IRB as 
soon as possible, or no later than 5 working days after the investigative site 
learns of the occurrence. 
 
3.10 Computerized System  
 
• 
 
 
4. 
Subject Selection 
 
Healthy volunteers with no dermatological conditions or known history of sensitivity to 
natural rubber latex, adhesive skin products (e.g., Band-Aids, medical tapes), isopropyl 
alcohol, citric acid, methylene blue, methylparaben, propylparaben, or chlorhexidine 
gluconate will be enrolled into this study. A sufficient number of volunteers will be enrolled 
in the screening phase such that the total number of abdominal regions and the total 
number of groin regions meets or exceeds the number determined for the study (320 
abdominal regions and 320 groin regions for the test article and reference article; and 64 
abdominal regions and 64 groin regions for the negative control). Subjects must satisfy all 
Screening Day and Treatment Day Inclusion/Exclusion Criteria prior to screening sample 
collection and Treatment Day procedures. Volunteers will be recruited and treated in 
blocks until the count of results in the mITT population meets or exceeds the required test 
material/region counts. 
 
A subject must qualify for both the abdominal portion and the groin portion of the study. 
The right and left sides of the abdomen and groin must meet the minimum baseline values 
stated in the Inclusion Criteria.  
 
Subjects who are treated but discovered to have Treatment Day baseline counts below the 
minimum values or above the maximum values stated in Table 3.2 will not be included in 
the primary efficacy analysis based on the body site(s) that did not qualify due to not 
meeting the treatment day baseline inclusion criteria - see section 4.1. 
 
Subjects will be identified at screening by their initials and a screening ID number. Subject 
numbers will not be assigned until a subject has passed the screening criteria, including 
baseline bacterial counts. 
 
All volunteers will be given verbal and written information about the study procedures, and 
Subject Instructions (Appendix 14.6) will be provided to each subject for the pretreatment 


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phase of the study. The following Inclusion/Exclusion Criteria will be reviewed on 
Screening Day and on Treatment Day to establish eligibility for participation: 
 
4.1 Subject Inclusion Criteria 
 
Subjects to whom all of these conditions apply will be eligible for enrollment in this 
study: 
 
• 
Healthy male and female volunteers, 18 years of age or older. 
• 
Are in good general health. 
• 
Have skin within 6 inches of the test sites that is free of tattoos, dermatoses, 
abrasions, cuts, lesions or other skin disorders.  
• 
Cooperative and willing to follow Subject Instructions (Appendix 14.6). 
• 
Cooperative and willing to sign Consent Form and HIPAA Authorization Form. 
• 
Have Screening Day baseline counts of at least 1.0 x 103 CFU/cm2 per 
abdominal site (left and right) and at least 1.0 x 105 CFU/cm2 per groin site (left 
and right).  
 
4.2 Subject Exclusion Criteria 
 
Subjects to whom any of these conditions apply will be excluded from this study: 
 
• 
Topical or systemic antimicrobial exposure within 14 days prior to Screening Day.  
Restrictions 
include, 
but 
are 
not 
limited 
to 
antimicrobial 
soaps, 
antiperspirants/deodorants, 
shampoos, 
lotions, 
perfumes, 
after 
shaves, 
colognes, and topical or systemic antibiotics. 
• 
Swimming in chemically treated pools or bathing in hot tubs, spas and whirlpools 
within 14 days prior to Screening Day. 
• 
Use of tanning beds, hot waxes, or depilatories, including shaving (in the 
applicable test areas) within 14 days prior to Screening Day. 
• 
Contact with solvents, acids, bases, fabric softener-treated clothing or other 
household chemicals in the applicable test areas within 14 days of the Screening 
Day.  
• 
Subjects who have a history of sensitivity to natural rubber latex, adhesive skin 
products (e.g., Band-Aids, medical tapes), isopropyl alcohol, citric acid, 
methylene blue, methylparaben, propylparaben, or chlorhexidine gluconate 
chlorhexidine gluconate products. 
• 
Subjects who have a history of skin allergies. 
• 
Subjects who have a history of skin cancer within 6 inches of the applicable test 
areas. 


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• 
Subjects who are pregnant, attempting pregnancy or nursing. For all females of 
child-bearing potential (<60 years of age), the pregnancy test will be performed 
before treatment on treatment day. 
• 
Subjects who have showered or bathed within 72 hours of the Screening Day or 
Treatment Day (sponge baths may be taken, however, the lower abdomen and 
upper thigh region must be avoided). 
• 
Subjects who receive an irritation score of 1 for any individual skin condition prior 
to the Screening Day baseline or Treatment Day baseline sample collection.  
• 
Participation in another clinical trial in the 30 days prior to Test Day of this study 
(treatment with test materials in this study), or be currently enrolled in another 
clinical trial, or has previously participated in this study.   
 
4.3 Subject Consent 
 
The Investigator or designated sub-investigators trained by the Investigator must 
ensure that written informed consent to participate in the investigation is obtained 
before including any individual as a subject in the investigation. The Investigator or 
designated sub-investigators trained by the Investigator must provide the prospective 
subject or the representative sufficient opportunity to consider whether or not to 
participate, and minimize the possibility of coercion or undue influence. The process 
is designed to 1) give the subject all the information needed, 2) ensure that the 
subject understands the information, and 3) give the subject a chance to consider 
study participation. The process should permit the subject to ask questions and 
exchange information freely. 
 
Specifically, the Investigator or designated sub-investigators trained by the 
Investigator is to explain to each subject all elements of informed consent as specified 
in 21 CFR 50.25 (Appendix 14.5). After the explanation, subjects or their 
representative will voluntarily sign and date the consent form if they wish to participate 
in the study.  A copy of the consent form must be provided to the subject. A signed 
and dated consent form must be maintained in the Investigator study documentation 
file at all times. 
 
4.4 Subject Authorization for Use and Disclosure of Protected Health Information  
 
The Investigator or designated sub-investigators trained by the Investigator must 
ensure that written authorization for use and disclosure of Protected Health 
Information (PHI) is obtained before including any individual as a subject in the 
investigation. 
 
Specifically, the Investigator or designated sub-investigators trained by the 
Investigator is to explain to each subject all elements of authorization as specified in 
45 CFR 164.508. After the explanation, subjects or their representative must 


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voluntarily sign and date the authorization form if they wish to participate in the study.  
A copy of the authorization form must be provided to the subject. A signed and dated 
authorization form must be maintained in the Investigator study documentation file at 
all times. 
 
An authorization form may be combined with a consent form (i.e., compound 
authorization) if required by the IRB. All required elements for both informed consent 
and authorization must be included in a compound authorization. 
 
5. 
Study Treatment  
 
These study procedures are based on the American Society for Testing and Materials 
(ASTM) “Standard Test Method for Evaluation of Preoperative, Precatheterization, or 
Preinjection Skin Preparations” (E 1173-011).   
 
5.1 Study Procedures 
Procedures will be performed by the Investigator or designated personnel trained by 
the Investigator.  
 
5.1.1 Screening Phase Study Procedures 
 
5.1.1.1 Washout Period 
 
The Inclusion/Exclusion Criteria will be reviewed with each subject 
to ensure eligibility for the study.  If these criteria are satisfied, 
subjects will sign the consent form and HIPAA Authorization Form 
before screening phase study procedures begin. Prior to the 
scheduled Screening Day, subjects will undergo a minimum 14-
day washout period. The subjects will be instructed to avoid 
contact with any topical or systemic antimicrobial products for the 
duration of their involvement in the study as written in the Subject 
Instructions (Appendix 14.6). If it becomes necessary to take 
systemic antibiotics or to apply topical medications to the test 
areas within this pretreatment period, the subject must contact the 
Investigator as soon as reasonably possible so that another 
volunteer may be recruited. 
 
Restrictions include, but are not limited to: 
• 
Use of antimicrobial soaps, shampoos, lotions, perfumes, after 
shaves, colognes, antiperspirants, deodorants 
• 
Contact with materials such as acids, bases, solvents 
• 
Swimming in chemically treated pools and bathing in hot tubs, 
spas and/or whirlpools 


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• 
Use of tanning beds, hot waxes or depilatories (including 
shaving) 
 
Subjects will be provided a kit with non-antimicrobial personal care 
products for exclusive use during the study.  Subjects will also be 
provided with written instructions regarding the use of these 
products (Appendix 14.6). 
 
A visual skin assessment of the test areas will be performed. If 
subjects require hair removal to facilitate sample collection, the 
subject will be asked to return to the test facility at least 48 hours 
before the Screening Day. 
 
Subjects will be required to refrain from bathing or showering for 
72 hours prior to both the Screening Day and Treatment Day. 
 
Sponge bathing will be allowed, however, the subject must avoid 
the lower abdomen and upper thigh region. 
 
5.1.1.2 Screening Day (Qualification Baseline Sampling) 
 
Subjects will be required to refrain from bathing or showering 72 
hours prior to Treatment Day and hair will be clipped at least 48 
hours prior to Screening Day. 
 
After the washout period and at least 3-days before Treatment 
Day, the Investigator or a designated assistant will complete the 
Screening Day Inclusion/Exclusion Criteria page in each subject’s 
CRF.   
 
Prior to performing the Screening Day baseline sample collection, 
a skin irritation assessment will be performed  
 
If an irritation score of 1 for any individual skin condition at the 
Screening Day baseline is assigned, the subject will be excluded 
from the study.   
 
A baseline screening sample will be collected from each test area 
using the Williamson-Kligman scrub cup technique3. Baseline 
samples will be taken from the center of each contra lateral test 
area within each anatomical region. Samples from both the left 
and right sides of a body region must meet the minimum value 
indicated in the Inclusion Criteria for the subject to be enrolled into 
the treatment phase of the study for that region. Subjects must 


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qualify for both the abdominal portion and the groin portion of the 
study.  
 
Subjects who qualify for the study will be notified and will continue 
to follow the subject instructions until completion of the scheduled 
Treatment Day. 
 
Subjects will again be required to refrain from bathing or 
showering 72 hours prior to Treatment Day and hair will be clipped 
at least 48 hours prior to Treatment Day. 
 
5.1.2 Treatment Phase Study Procedures 
 
A sufficient number of subjects who meet the entrance criteria will be enrolled 
into the treatment phase of the study for each region, such that the total 
number of abdominal regions and the total number of groin regions meets or 
exceeds the number required (320 abdominal regions and 320 groin regions 
for the test article and reference article; and 64 abdominal regions and 64 
groin regions for the negative control). The randomization schedule will 
designate the treatment to each side of the abdomen and groin. 
 
The Treatment Day Inclusion/Exclusion Criteria CRF will be completed. If 
these criteria are satisfied, a visual skin assessment will be performed to 
evaluate the condition of each test area. 
 
5.1.2.1 Preparation of Test Areas on Treatment Day  
 
A Test Site Diagram for the abdominal and groin test areas is 
shown in Appendix 14.3. Note: skin irritation will be assessed for 
all assigned test areas prior to treatment of any test area.   
 
 
5.1.2.1.1 Preparation of Abdominal Test Area 
 
The test site within the abdominal region (abdominal 
test area) is defined as the area below the umbilicus 
and above the groin. Using a 5” x 5” sterile template, 
the corners of each abdominal test area will be marked 
directly on the skin using a non-toxic skin marker. Four 
sampling sites will be numbered within each abdominal 
test area, on each side of the abdominal region. The 
positioning and numbering of the abdominal sampling 
sites are standard for all subjects. Sampling sites on 
the contra-lateral side of the abdomen will be 
numbered in a mirror-image orientation. The four 


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sampling sites within each abdominal test area 
represent one baseline (pre-prep) site, and one post-
prep sampling site for each of three sampling times (30 
seconds, 10 minutes, and 6 hours).   
 
Prior to performing the Treatment Day baseline sample 
collection, a skin irritation assessment will be 
performed. 
 
If an irritation score of 1 for any individual skin 
condition at the Treatment Day baseline is assigned, 
the subject will be excluded from the treatment phase 
of the study. After abdominal test areas are marked 
and sample sites numbered, baseline samples will be 
collected 
from 
the 
appropriate 
site 
per 
the 
randomization schedule (Appendix 14.4) in each test 
area using the scrub cup technique. 
 
5.1.2.1.2 Preparation of Groin Test Area 
 
The test site within the groin region (groin test area) is 
defined as the inner aspect of the upper thigh within 
and parallel to the inguinal crease below the groin.  
Using a 1.5” x 5” sterile template, the corners of each 
groin test area will be marked directly on the skin using 
a non-toxic skin marker. Four sampling sites will be 
numbered within each groin test area, on each side of 
the groin region. The positioning and numbering of the 
groin sampling sites are standard for all subjects.  
Sampling sites on the contra-lateral side of the groin 
will be numbered in a mirror-image orientation. The 
four sampling sites within each groin test area 
represent one baseline (pre-prep) site, and one post-
prep sampling site for each of three sampling times (30 
seconds, 10 minutes, and 6 hours).   
 
Prior to performing the Treatment Day baseline sample 
collection, a skin irritation assessment will be 
performed. 
 
If an irritation score of 1 for any individual skin 
condition at the Treatment Day baseline is assigned, 
the subject will be excluded from the treatment phase 
of the study. After groin test areas are marked and 




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from the subject’s skin using mild soap and/or tap water with a 
paper towel. 
 
5.2 Microbiological Methods 
 
5.2.1 Microbial Sample Collection / Scrub Cup Technique 
 
Quantitative cultures (screening baselines, treatment baselines and post 
treatment application) will be obtained by a modification of the cylinder 
sampling technique of Williamson-Kligman scrub cup technique.  To collect 
the samples, a sterile scrub cup 
 will be placed on the site and 
held firmly to the skin. Sampling solution (SS) (
) will be pipetted into 
the cup and the skin will be scrubbed in a circular motion with moderate 
pressure for 1 minute using a sterile rubber policeman. Using a sterile 
transfer pipette, the SS will be removed and placed in a sterile test tube.  An 
additional 
 of fresh sampling solution will be pipetted into the cup and 
the scrub procedure will be repeated. This solution will be pooled with the first 
solution collected.   
 
5.2.2 Sampling Solution (SS) 
 
The SS consists of sterile [75mM phosphate buffer (
 
 containing 
 lecithin, 
 Tween® 
80, and 
 Tamol,
]2 (SS). 
 
5.2.3 Bacterial Enumeration Methods 
 
Following sample collection, 10-fold serial dilutions (1 mL sample + 9 mL 
PBW) will be prepared using Butterfield’s phosphate buffered water with 
neutralizers (PBW). One mL aliquots of appropriate dilutions will be pour-
plated in duplicate using trypticase soy agar containing neutralizers (TSA+N).  
Samples must be plated within 30 minutes of collection. After 72 ± 4 hours of 
aerobic incubation at 30 ± 2C, colonies will be counted and viable cells in the 
original sample will be calculated according to Standard Operating 
Procedures. After incubation, plates may be refrigerated up to 48 hours prior 
to counting.   
 
Raw colony counts from each dilution will be recorded on the appropriate 
CRFs for each subject. The average number of microorganisms recovered 
(CFU/cm2) of skin for the screening and treatment day baseline samples will 
be calculated using the following formula to convert the plate CFU values into 
CFU/cm2 of skin: 
 
 


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R = log10 

















Σ
A
D
n
c
F
i
 
 
Where: 
 
 
R = the average CFU count in log10 scale per cm2 of skin. 
 
F = total mL of stripping fluid added to the sampling cylinder (
);  
 
Σci/n = average of the duplicate colony counts for each sample collected 
 
D = Dilution of the plates counted (100, 101, 102, 103, 104, or 105) 
 
A = Inside area of the sampling cylinder (
 
) 
Dilution may be reported as dilution factor, which is the base 10 logarithm of the 
dilution. In that case, the factor of ‘D’ in the equation above will be replaced with 
10D. 
 
5.2.4 Growth Promotion Control 
 
For each batch of plating medium, TSA+N, fewer than 100 CFU of 
 will be inoculated in a single plate 
pour plate. A 
 aged culture of 
 will be 
serially diluted in dilution fluid. The CFU added will be confirmed using 
duplicate trypticase soy agar (TSA) spread plates. The plates will be 
incubated for 72 ± 4 hours of aerobic incubation at 30 ± 2C. 
 
5.2.5 Neutralizer Validation 
 
The effectiveness of the neutralizer system must be validated prior to the 
study start date to demonstrate that the antimicrobial is effectively neutralized 
and there is no effect on the growth of microorganisms. A procedure that will 
include in-vivo sampling will be combined with an in-vitro evaluation using 
procedures in accordance with ASTM E1054-084. The procedure is attached 
as Appendix 14.10. These data must be provided in the final report.  
 
5.3 Medication(s)/Treatment(s) Not Permitted 
 
Topical or systemic antimicrobial exposure is not permitted within 14 days prior to the 
day of Screening Day, and during the study.  Restrictions include, but are not limited 
to antimicrobial soaps, antiperspirants/deodorants, shampoos, lotions, perfumes, 
after shaves, colognes, and topical or systemic antibiotics. 
 


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5.4   Subject Compliance 
 
Answers to the inclusion/exclusion questions (Inclusion/Exclusion CRFs) asked at 
the beginning of the screening and treatment phases will determine compliance to 
the Subject Instructions (Appendix 14.6) provided to each subject upon study 
participation. Documentation of the Inclusion/Exclusion criteria shall serve as 
confirmation of subject compliance with the Subject Instructions. 
 
6. 
Assessment of Efficacy 
 
6.1 Efficacy Parameters 
 
The primary measure of antimicrobial efficacy is the reduction of skin flora on the 
abdominal and groin sites 10 minutes following application of the study treatments 
relative to the Treatment Day baseline counts. 
 
The reduction will first be calculated as log10 CFU/cm2 changes from baseline, then 
the percentage of successes will be calculated from the log10 CFU/cm2 reductions 
(i.e. percent of subjects meeting required reductions = responder rate). A site is 
considered a responder for the treatment at 10 minutes if it achieves ≥ 2.0 log10 
CFU/cm2 reduction on the abdomen or ≥ 3.0 log10 CFU/cm2 reduction on the groin.  
 
Responder status will be calculated separately for the abdomen and groin, for each 
test substance, for each post-treatment sample time, and for each subject. The 
individual responses will then be grouped to generate an overall responder rate for 
each anatomical area, for each test substance, and for each post-treatment sample 
time. The efficacy goal for active products is to have the lower bound of the 95% 
confidence interval for the responder rate to be greater than or equal to 70%. 
 
Secondary Endpoints/Analysis 
 
Responder rates at 30 seconds will be calculated identically to the 10 minute rates. 
At 6 hours a site is considered a responder if it is below baseline; otherwise, 6-hour 
responder rates will be calculated identically to the 10-minute responder rates. The 
secondary efficacy goals are to have the 95% confidence intervals for the responder 
rates to be greater than or equal to 70%. Log10 CFU reductions from baseline will 
also be calculated for all post-prep samples. 
 
Study Validity 
 
For a valid study, the active control must make the same objectives that the test 
article does and both the test article and the active control should be superior to the 
inactive control at the primary endpoint. The responder rates for both active and 
inactive treatments at 6 hours are near 100%. Therefore, comparison of responder 
rates at 6 hours is unlikely to show any difference. Differences in log10 CFU/cm2 
reductions from baseline will be calculated for 6 hours in order to show that the active 
treatments are more effective than the inactive control. 


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6.2 Assessment Methods  
 
Efficacy will be assessed by sampling the skin using the cup scrub method described 
in Section 5.2.  See Section 8.0 for analysis methods. 
 
7. 
Assessment of Safety 
 
7.1 Safety Parameters 
 
The principal measures of safety will be the recording of skin irritation scores and the 
incidence of adverse events reported during the study. 
 
7.2 Assessment Methods for Skin Irritation 
 
After the washout period, the Investigator or designated sub-investigators trained by 
the Investigator will assess the subject’s skin condition and assign a skin irritation 
rating score (see Appendix 14.11).  A skin irritation score will be recorded at both the 
screening and treatment phases prior to baseline sample collections and prior to 
each post treatment application sample collection (30 seconds, 10 minutes, and 6 
hours).  
   
A corresponding rating score for each individual skin condition, for each site will be 
recorded in the subject’s CRF. (See Appendix 14.11, which includes the following 
four independent evaluation categories:  Erythema, Edema, Rash, and Dryness).  
If an irritation score of 1 or greater for any individual skin condition prior to the 
baseline sample collection (at either the screening or treatment day phases) is 
assigned, the subject will be excluded from the study (no study treatment will be 
applied). 
 
If an irritation score of 3 for any individual skin condition at any observation period is 
assigned, the subject will be discontinued from the study and an adverse event will 
be recorded. See Section 7.3 (Adverse Events). 
 
7.3 Adverse Events 
 
 
The Investigator is responsible for identifying adverse events that occur to each 
subject throughout the study and follow-up period. An adverse event can occur at 
any time during the conduct of the study. Adverse events will be captured for all 
subjects from the time of screening baselines are taken until the time of discharge 
from the study. An adverse event can be identified by the Investigator or reported by 
the subject.  
 


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Note: The Federal Privacy Rule (HIPAA) specifically permits the use and disclosure 
of protected health information “without written authorization of the individual” when 
used for public health activities such as reporting adverse events, tracking FDA-
related products, enabling recalls, repairs, replacements, lookbacks, or conducting 
post-market surveillance [45 CFR 164.512]. This use and disclosure is subject to the 
minimum necessary standard, i.e. “the minimum necessary to accomplish the 
intended use, disclosure, or request” [45 CFR 164.502(b)(1)]. 
 
Definitions: 
 
Adverse Event/Experience 
 
An Adverse Event/Experience is any unexpected or undesirable experience 
occurring to a subject during a study, which may or may not be related to the test 
article. All adverse event/experiences will be recorded and reported according to the 
Standard Operating Procedures of the laboratory. 
All adverse events, regardless of severity or the causal/effect relationship, will be 
recorded. The severity of the effect will be noted as “Mild,” “Moderate,” or “Severe” 
according the following definitions: 
Mild 
Awareness of signs or symptom, but easily tolerated. 
Moderate 
Discomfort to a degree as to cause interference with normal daily 
life activities and /or requiring medication. 
Severe 
Incapacity with inability to work or do usual daily life activities and 
requiring medical attention/intervention. 
Causal Relations of Adverse Event/Experience 
When determining the causal/effect relationship to the test article, the relationship 
will be described as “None,”  “Possible,” “Probable,” or “Definite.” The following 
definitions will be utilized: 
 
 None 
No association to the test article. Related to other etiologies such as 
concomitant medications or conditions or subject’s known clinical 
state. 
 
Possible 
Uncertain association. Other etiologies are also possible.  
 
Probable 
Clear-cut association with improvement upon withdrawal of the test 
article. Not reasonably explained by the subject’s known clinical 
state  
Definite 
An adverse event with a clear-cut temporal association with 
exposure to study materials and cannot reasonably be explained by 
the subject’s known clinical state.  Association with study material is 
confirmed by laboratory testing if possible.  


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Serious Adverse Event/Experience 
 
A Serious Adverse Event/Experience is any adverse experience occurring at any 
dose that results in any of the following outcomes: 
• 
Death; 
• 
A life-threatening adverse drug experience; 
• 
Inpatient hospitalization or prolongation of existing hospitalization; 
• 
A persistent or significant disability/incapacity; 
• 
Congenital anomaly/birth defect; 
• 
An important medical event that may require medical or surgical 
 
intervention to prevent one of the previously listed outcomes. 
Unexpected Adverse Event/Experience 
An Unexpected Adverse Event/Experience is any adverse drug event/experience 
not listed in the current labeling for the test article or the current Investigator’s 
brochure. Where test article labeling or Investigator’s brochure is not available, 
anticipated experiences will be based on the known pharmacological/toxicological 
properties of the test article or ingredients. 
 
Recording and Reporting 
 
The Investigator or designee records all adverse events on an Adverse Event 
Record in the subject’s CRF. Documentation includes the AE description, severity, 
seriousness, date of onset and resolution, relationship to the test article, action taken 
and outcome. 
The Investigator must promptly report all treatment related adverse events to the 
Sponsor . All serious adverse events must be reported to the Sponsor and to the IRB 
within 24 hours of the Investigator awareness/notification of the event. 
  
If a subject has no adverse event during the study, the absence of such must be 
recorded on the CRF. 
 
7.4 Follow-up 
 
If an adverse event/experience occurs, the subject under the direction of the     
Investigator (or designee) may be referred to the nearest acute care facility for 
treatment. Serious or Unexpected Drug Event/Experience will be followed to 
resolution. Any adverse event will be documented on an Adverse Event Report.  
 
 
 
 
 


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8. 
Statistics 
 
8.1 Data Sets Analyzed 
 
The Intent-to-Treat (ITT) Population will consist of all subjects who pass the pre-test 
period prior to baseline screening and are assigned a subject number for treatment. 
The ITT population (all randomized subjects) will be used for the safety analysis. 
 
 
a Modified Intent-to-Treat (mITT) 
Population will be used for analysis and consist of all subjects who have at least one 
site (left or right for abdominal or inguinal) that passed the treatment day baseline 
and has CFU results for any other sample time for that site. Body sites will be 
included in the mITT population if and only if they meet the treatment day baseline 
criteria. The mITT data set will be evaluated for efficacy. 
 
8.2 Statistical Methods 
 
8.2.1 Efficacy Analyses 
 
CFU values will be calculated as per the Bacterial Enumeration methods above. 
Individual plate CFU counts that are zero will be treated as 0.5 for further 
calculations. Changes from baseline will be calculated separately for each subject 
and for each of the three non-baseline sites by taking the baseline log10 CFU/cm2 
values and then subtracting the log10 CFU/cm2 values for the samples taken after the 
baseline. Therefore, a subject who completes all treatments and passes all baseline 
requirements will have 4 (sites) * 3 (post-application samples per site) = 12 changes 
from baseline reported. Responder status will be calculated for each reported log10 
CFU/cm2 reduction. The sites will be considered responders based on the sample 
time and body area: 
 
For the groin at 30 seconds or 10 minutes, a log10 CFU/cm2 reduction ≥ 3.0 is 
considered a responder. 
 
For the abdomen at 30 seconds or 10 minutes, a log10 CFU/cm2 reduction ≥ 
2.0 is considered a responder. 
 
For either the groin or the abdomen at 6 hours, a log10 CFU/cm2 value less 
than baseline (i.e., a log10 CFU/cm2 reduction > 0) is considered a responder. 
 
Responder statuses will be grouped by body area, sample time, and test substance. 
Exact confidence intervals will be calculated for responder rates. The primary 
efficacy goal is to have the 95% confidence intervals for the responder rate at 10 


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minutes to be ≥ 70%. The secondary efficacy goals are to have the 30 second and 6 
hour 95% confidence intervals for the responder rates to be ≥ 70%. 
 
Differences in responder rates between treatments and their confidence intervals will 
be calculated using asymptotic (approximate) methods. If differences in responder 
rates are not reliable based on approximate methods, which happens when rates are 
near either 100% or 0%, log10 CFU/cm2 confidence intervals for the differences will 
be used for comparison instead.  
 
Two-sided confidence intervals for log10 CFU/cm2 changes from baseline will also be 
calculated. These calculations will use an ANOVA model with subject as a random 
variable and test substance as a fixed variable. Body area (abdomen or groin) will be 
a fixed variable. Sample times (30 seconds, 10 minutes, or 6 hours) will be 
calculated separately using identical models. Differences in log10 CFU/cm2 
reductions will be calculated based on the same model. 
 
8.2.2 Safety Analysis 
 
All treated subjects will be considered evaluable for safety. Skin irritation 
scores assessed in accordance with Appendix 14.11 will be reported for any 
subject who is scored with a 1 or more at any observation [baseline 
(screening day and treatment day), post-application/prior to 30 second, 10 
minute, or 6 hour sampling procedures], in any category for any site.    
 
Adverse events (including post treatment skin irritation scores of 3 in 
accordance with Appendix 14.11, will also be summarized. Summary tables 
will present incidence rates of adverse events by treatment group for all 
subjects who enter the treatment period. Listings of adverse events will be 
provided. 
 
The statistical significance of differences in skin irritation between the three 
treatments will be evaluated using Fisher’s exact test on skin irritation data 
summarized as follows: any reaction above zero (no reaction) on the skin 
irritation rating scale for any category (erythema, edema, rash, and dryness) 
will be considered a positive signal for that substance. If Fisher’s exact test 
shows statistically significant skin irritation between the three treatments, a 
secondary analysis will be conducted to determine how the reactions differ. 
 
8.3 Sample Size Justification  
 
The number of subjects for the active treatments (ZuraPrepTM and the active 
comparator) is based off of the following assumptions: the primary efficacy 
parameters are as defined above; the power is 80%; and the minimum responder 
rate for active substances is 77.8%. Using 
 for a single 


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proportion method, this requires 267 subjects per active treatment. The number 320 
was used to make randomization blocking easier and to be conservative. 
 
The numbers required for the inactive/negative control group were based on results 
from 
. The sample size is designed to be sufficient to show there is 
a significant difference in responder rates between the active substances and 
inactive control at 30 seconds and 10 minutes and a significant difference in log10 
CFU/cm2 reductions from baseline at 6 hours. Based on these assumptions, the 
sample size should be at least 40. A sample size of 64 was chosen for easier 
balance of factors during randomization.  
 
  
 
8.4 Subject Discontinuation Criteria 
 
The Investigator may discontinue individual subjects from the study at any time.  
Subjects may voluntarily withdraw from the study at any time. The Investigator will 
provide a written report on the appropriate CRF including the date and reason for 
discontinuation.    
 
8.5 Procedures for Accounting for Missing Data and Protocol Deviations 
 
Missing microbiological data at 30 seconds, 10 minutes, or 6 hours will be reported 
as missing; if the entire sample is missing the site will be treated as a nonresponder 
for that sample time. These subjects will be included in the mITT data set based on 
the criteria defined above. Details of any other missing data handling will be specified 
in the statistical analysis. 
 
8.6   Deviations to Statistical Plan 
 
Any deviation(s) from the original statistical plan will be described and justified in the 
final report. 
 
9. 
Monitoring 
 
Zurex Pharma, Inc., as sponsor of this study along with the Investigator, is responsible for 
ensuring the proper conduct of the study with regard to protocol adherence and validity of 
the data recorded on the CRFs. Zurex Pharma, Inc. has, therefore, assigned a study 
monitor to this study. The progress of the study will be monitored by: 
 
• 
Periodic on-site review 
• 
Telephone communications and e-mail 
• 
Review of CRFs and source documents (CRF serves as source document) 
 


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The Investigator will give the Zurex Pharma, Inc. study monitor direct access to source 
documents that support data on the CRFs and make available such records to authorized 
Zurex Pharma, Inc., quality assurance, IRB, and regulatory personnel for inspection and/or 
copying. 
 
Note: The Federal Privacy rule (HIPAA) specifically permits the use and disclosure of 
protected health information “to a person subject to the jurisdiction of the Food and Drug 
Administration (FDA) [e.g., study sponsor] with respect to an FDA-related product or 
activity for which that person has responsibility, for the purpose of activities related to the 
quality, safety, or effectiveness of such FDA-regulated product or activity” [45 CFR 
164.512(b)(1)(iii)]. 
 
10. Quality Control and Quality Assurance 
 
Zurex Pharma, Inc. and the Investigator are responsible for implementing and maintaining 
quality assurance and quality control systems through written standard operating 
procedures (SOPs) to ensure that this study is conducted and data are generated, 
documented and reported in compliance with the protocol, GCP and regulations cited in 
Section 1.4 of this protocol. Study monitoring may be carried out to accomplish this. 
 
11. Ethics 
 
This study will be conducted in accordance with the principles that have their origin in the 
Declaration of Helsinki, 21 CFR 50 (Informed Consent) and 56 (IRBs). Laboratory 
operations will be conducted in accordance with 21 CFR 58 (Good Laboratory Practice for 
Nonclinical Laboratory Studies). 
 
The study will start only after approval of the protocol and consent form by the IRB. The 
approval letter or notice must contain the IRB name and identification number, meeting 
date, and sufficient information to identify the protocol and informed consent by name and 
number that were reviewed. Zurex Pharma, Inc., prior to study initiation, must receive a 
copy of the IRB approval letter. 
 
12. Data Handling and Record Keeping 
 
12.1 Study Personnel 
 
Prior to study initiation, the Investigator must provide Zurex Pharma, Inc. with a 
signed Investigator agreement as documentation of the Investigator’s commitment to 
conduct the study according to the protocol and all applicable state and federal 
regulations.  
 
 


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12.2 Pre-Study Documentation Requirements 
 
Prior to study initiation, the Investigator must provide Zurex Pharma, Inc. with the 
following documents: 
12.2.1 
Signed protocol including any amendments in place prior to study initiation 
12.2.2 
Curriculum vitae for the Investigator and any co-Investigators 
12.2.3 
IRB-approved informed consent form containing subject authorization for 
use and disclosure of Protected Health Information statement.  
12.2.4 
IRB study approval letter (including approval of the protocol, consent form, 
Investigator and study facility) 
12.2.5 
IRB name, location and board membership listing  
12.2.6 
Signed Investigator agreement 
12.2.7 
Signed Study Agreement and Confidentiality agreement 
 
12.3 Completion of Case Report Forms 
 
The Investigator will review all CRF entries for completeness and accuracy. If a 
correction is required, a single line must be drawn through the error. The person 
making the correction will initial, date, and provide a reason for the error (if not self-
evident). 
 
The Investigator must review and sign each CRF in a timely fashion following 
completion and make them available to the Zurex Pharma, Inc. study monitor for 
inspection. Before acceptance, the monitor will review the CRFs to ensure accuracy 
and completeness. The copies of CRFs will be submitted to Zurex Pharma, Inc. and 
the original will be maintained at the study site. In addition, any data queries 
prepared after the original CRFs have been completed must be answered promptly. 
 
12.4 Final Report 
 
The Investigator will prepare a final study report. The final report will be a record of 
the total study conduct and will be subject to review by Zurex Pharma, Inc. The final 
report will assess and summarize all data collected and include: test material 
identification, type of assay, randomization schedule, dates of study initiation and 
completion, CRFs, and data demonstrating neutralization effectiveness. A review of 
the final report by Quality Assurance, including compliance statements will also be 
included with the final report. A copy of the final report will be sent to the study 
sponsor. 
 
 


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12.5 Records, Reports and Retention Requirements 
 
The Investigator will maintain study records for a minimum of 2 years following the 
date a marketing application is approved for the indication for which is being 
investigated, or, if no application is to be filed or if the application is not approved for 
such indication, until 2 years after the investigation is discontinued and FDA is 
notified. Records that must be maintained by the Investigator include, but are not 
restricted to: 
 
● Signed study protocol, amendments, deviations 
● IRB approval of protocol, consent form, authorization form, waiver of consent 
and/or authorization and amendments to any of these documents 
● Applications to the IRB 
● Signed consent and authorization forms 
● Case report forms 
● Adverse event reports 
● Records of receipt, use or disposition of the study material 
● Correspondence relating to the study 
● Investigator Final Report 
● Sponsor Final Report (if provided) 
 
13. References 
 
1. Annual Book of ASTM Standards, Vol 11.05.  E 1173-01 (Reapproved 2009) Standard 
Test Method for Evaluation of Preoperative, Precatheterization, or Preinjection Skin 
Preparations. 
2. Butterfield, C.T.  The Selection of a Dilution Water for Bacteriological Examinations.  J. 
Bacteriol. 23: 355-368, 1931. 
3. Williamson, P., Kligman, A.M.  A New Method for the Quantitative Investigation of 
Cutaneous Bacteria.  J. Invest. Dermatol.  45:498-503, 1965.  
4. ASTM International. ASTM E1054-08, standard test methods for evaluation of 
inactivators of antimicrobial agents.  West Conshohocken [PA]: ASTM Int’l; 2013. 
 
14. Appendices 
 
14.1 Key Study Personnel, Titles, Responsibilities 
 
Zurex Pharma, Inc. Personnel: 
  
 
 
 
 
 


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MicroBioTest Personnel: 
M. Hamid Bashir, MD, CCRP, Laboratory Manager              Principal Investigator 
 
                   
 
 
                    
  
  
 
 
 
 
 
 
Statistical Consultant: 
  
             
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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14.2 Study Summary 
 
Pre-Study 
Screening Phase 
Treatment Phase 
Preparation 
14 Day Washout 
Period 
Screening Day 
Abdominal Region 
Groin Region 
 
Staff reviews 
study protocol 
 
Initiation of 
consenting 
process 
 
 
Complete 
Screening 
Inclusion/ 
Exclusion Criteria 
form 
 
Complete Treatment  
Inclusion/ Exclusion Criteria form 
Prepare 
consent form 
Review study 
Consent Form 
and Inclusion/ 
Exclusion Criteria 
Visual skin 
assessment 
 
Visual skin assessment 
 
Obtain IRB 
approval 
Review subject 
instructions 
Collect screening 
baseline samples 
from abdominal 
and groin regions 
Mark test areas, 
Collect baseline samples 
Recruit 
volunteers for 
screening 
phase 
(14 day washout / 
screening day) 
Subject signs 
consent form 
Count screening 
plates, determine 
which volunteers 
qualify for study 
 
Apply test articles 
 
Prepare 
subject kits 
Visual skin 
assessment  
(abdominal and 
groin regions) 
Contact and enroll 
eligible subjects, 
schedule 
Treatment Day 
Visual skin 
assessment,  
30 seconds (± 5 
sec.), 10 minute (± 
30 sec.), 6 hours (± 
30 min.) post-prep 
sample 
  
 
Visual skin 
assessment,  
30 seconds (± 5 
sec.), 10 minute (± 
30 sec.), 6 hours (± 
30 min.) post-prep 
sample 
  
 
 
Dispense subject 
kits 
Schedule for 
clipping, if needed, 
48 hrs. prior to 
Treatment Day 
visit 
 
Schedule for 
clipping, if 
needed, 48 hrs. 
prior to screening 
visit 
No bathing / 
showering 72 hrs. 
prior to Treatment 
Day visit 
Count Treatment 
Day Baseline plates, 
determine 
qualification and 
enroll additional 
subjects as required 
Count Treatment Day 
Baseline plates, 
determine 
qualification and 
enroll additional 
subjects as required 
 
No bathing / 
showering 72 hrs. 
prior to screening 
visit 
 
 
 
 
 
 


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14.3 Abdomen and Groin Diagram 
 
Follow the randomization scheme for each subject for the exact placement of study materials.  
 
 
 
 
 
Groin (anterior) – 1.5”’ x 5” area                                         Abdomen (anterior) – 5” x 5” area 
 
 
 


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14.4 Randomization Scheme 
 
Abdominal and Groin Sites Bilateral Sampling Procedure Configurations  
GROUP 1 
                  
Key: 
A = ZuraPrep™ 
 
B = ChloraPrep® 
ator (Scrub Teal® Tint) 
C = ZuraPrep Vehicle (negative control) 
Subject 
Treatment 
Site 1 
Site 2 
Site 3 
Site 4 
0001 
Subjects will be numbered sequentially from 0001 to the number of subjects treated 
during the study. 
Randomization will be performed by group issued prior to study initiation. 
0002 
0003 
0004 
0005 
0006 
0007 
0008 
0009 
0010 
0011 
0012 
0013 
0014 
0015 
0016 
0017 
0018 
0019 
0020 
0021 
0022 
0023 
0024 
… 
 


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14.5 Required Elements of Informed Consent 
 
These elements of consent should be included as applicable to the study being 
conducted. 
 
1. Statement that the study involves research. 
2. Purpose(s) of the research. 
3. Expected duration of subject’s participation. 
4. Procedures to be followed and identification of any procedures that are 
experimental. 
5. A description of any reasonable foreseeable risks or discomforts to the subject. 
a) 
Risks/discomforts from study procedures. 
b) 
Foreseeable risks, which include adverse experiences listed in the 
Investigator’s Brochure or package insert. 
6. A description of any benefits to the subject or to others which may reasonably 
be expected from the research. 
7. A disclosure of appropriate alternative procedures or courses of treatment, if 
any, that might be advantageous to the subject. 
8. Extent to which confidentiality of records identifying subject will be maintained. 
a) 
Possibility that representatives of Zurex Pharma, Inc. and the FDA may 
inspect and make copies of the records. 
b) 
Suggested text: “Information on the Confidential Follow-up form (where 
used) will be held and treated with strict confidentiality and will be used 
only in the event that long-term follow-up is needed. 
c) 
Suggested text: “I understand that, at any time, an agent of Zurex 
Pharma, Inc. may also review any hospital, physician, or insurance billing 
or any other costs which relate to therapy incurred as a direct result of my 
participating in this study. 
9. An explanation as to whether any compensation or medical treatments are 
available if injury occurs for research involving more than minimal risk.  The 
explanation should involve a description of the compensation or treatment 
available or a statement describing where further information may be obtained. 
10. Whom to contact for answers to pertinent questions about research and 
research subject’s rights. 
11. Whom to contact in the event of research-related injury to the subject. 
12. Participation is voluntary: 
a) 
Refusal to participate will involve no penalty or loss of benefits to which 
subject is otherwise entitled. 
b) 
Subject may discontinue participation at any time without penalty or loss 
of benefit to which subject is otherwise entitled. 
 
 
 
 


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ADDITIONAL ELEMENTS OF CONSENT 
 
When appropriate, one or more of the following elements of information shall also 
be provided to each subject. 
 
13. A statement that the particular treatment or procedure may involve risks to the 
subject (or embryo or fetus, if subject became pregnant) which are currently 
unforeseeable. 
14. Anticipated circumstances under which subject’s participation may be 
terminated by the Investigator without regard to subject’s consent. 
15. Any additional costs to the subject that may result from participation in the 
research. 
16. A statement explaining the consequences of subject’s decision to withdraw 
during the course of the research which may relate to subject’s willingness to 
continue participation will be provided to the subject. 
17. A statement that significant new findings developed during the course of the 
research which may relate to subject’s willingness to continue participation will 
be provided to the subject. 
18. Approximate number of subjects involved in the study. 
 
Nothing in these regulations is intended to limit the authority of a physician to 
provide emergency medical care to the extent the physician is permitted to do so 
under applicable federal, state, or local laws. 
 
Informed consent allows the subject to fully understand his/her participation and 
serves to protect the Investigator and Sponsor from potential negligence claims. A 
fully informed subject is the best protection against such claims. 
 
The informed consent requirements in these regulations are not intended to 
preempt any applicable federal, state, or local laws that require additional 
information be disclosed for informed consent to be legally effective.  Some states, 
such as California and Oregon, require further action on the Investigator’s part 
concerning subject consent. 
 
 
 
 
 
 
 
 
 
 
 




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14.7 Treatment Application Instructions 
 
ZuraPrep™ 
 
 
 
 
 
 
 
 
 
 
Treatment Site Application Instructions 
 
Abdominal Test Site  
 
1. Using repeated back-and-forth strokes of the sponge for thirty (30 ) seconds, 
completely wet the treatment area with test material.  
2. Allow the area to air-dry for three (3) minutes. Do not blot or wipe away.  
 
Inguinal Test Site  
 
1. Using repeated back-and-forth strokes of the sponge for two (2) minutes, 
completely wet the treatment area with test material.  
2. Allow the area to air-dry for three (3) minutes. Do not blot or wipe away.  
 
 
ChloraPrep® 
 (Scrub Teal® Tint) (positive control) 
 
 
 
 
 
 
 
 
 
 


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Treatment Site Application Instructions 
 
Abdominal Test Site  
 
1. Using repeated back-and-forth strokes of the sponge for thirty (30) seconds, 
completely wet the treatment area with test material.  
2. Allow the area to air-dry for three (3) minutes. Do not blot or wipe away.  
 
Inguinal Test Site  
 
1. Using repeated back-and-forth strokes of the sponge for two (2) minutes, 
completely wet the treatment area with test material.  
2. Allow the area to air-dry for three (3) minutes. Do not blot or wipe away. 
 
 
  ZuraPrep Vehicle (negative control) 
 
 
 
  
 
 
 
 
 
Treatment Site Application Instructions 
 
Abdominal Test Site  
 
1. 
Using repeated back-and-forth strokes of the sponge for thirty (30 ) seconds, 
completely wet the treatment area with test material.  
2. 
Allow the area to air-dry for three (3) minutes. Do not blot or wipe away.  
 
Inguinal Test Site  
 
1. 
Using repeated back-and-forth strokes of the sponge for two (2) minutes. 
Completely wet the treatment area with test material.  
2. 
Allow the area to air-dry for three (3) minutes. Do not blot or wipe away.  
 


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14.8 Confirmation of Release and Receipt of Study Materials 
 
 
 
CONFIRMATION OF RELEASE and RECEIPT OF STUDY MATERIALS 
 
Investigator:  M. Hamid Bashir, MD, CCRP 
Study No: MBT 865-105 
Test Site: MicroBioTest 
 
.  
 
Quantity 
(Units) 
Description 
ID/Lot 
Number 
Exp. Date 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
Supplies Released to Site by:     
_________________________________ 
 
 
 
 
 
 
Sponsor Signature 
 
 
 
Supplies Sent to Site (Date):  
 
___________________________________ 
 
 
 
Supplies Checked and Verified by:   
____________________________________________ 
 
 
 
 
 
 
Signature 
 
 
                           Date 
 
 
 
 
 
 
 
_____________________________________________ 
 
 
 
 
 
 
Print Name, Title 
 
Once the supplies have been verified and this form is signed / dated, a signature copy will be sent to the 
sponsor representative: 
  
 
 


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14.9 Study Material Disposition Form 
 
Use one form for each study material. 
Study Number: MBT 865-105 
Investigator: M. Hamid Bashir, MD, CCRP 
Investigator Site: MicroBioTest 
 
Study 
 Material ID: 
Date Received: 
Quantity 
Received: 
Lot Number/ Serial 
Number: 
Date Returned 
to Sponsor: 
 
 
 
 
 
 
Date Dispensed/Distributed 
Subject 
Number 
Quantity Dispensed 
Quantity Remaining 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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14.10 
Procedure for Neutralization Validation 
 
1. 
Background 
 
The sampling solution, SS, sterile [75mM phosphate buffer (
 
) containing 
 lecithin, 
 Tween® 80, and 
 Tamol, 
]2 is a buffered detergent solution that is commonly used in 
studies where microbial sampling of skin is conducted. Neutralizers have been added 
to inactivate the antimicrobial, isopropyl alcohol and chlorhexidine gluconate, present 
in test treatments. The effectiveness and toxicity of this neutralizer system must be 
assessed to demonstrate that there is no effect on the growth of microorganisms and 
that the active ingredient is inactivated. 
 
The density of normal human skin flora generally ranges from 102 to 105 CFU/cm2 
depending on the body site.  However, since significant neutralizer or toxic effects are 
more easily detected at a lower cell density, the efficacy and toxicity of this neutralizer 
system will be assessed against a lower bacterial concentration. 
 
This is a test where the study materials are applied to the abdomen and the treated 
areas will be sampled. After sampling, 
, a selected 
representative of normal skin flora will be added into a portion of each sample. Each 
sample will then be processed using procedures in accordance with ASTM E1054-08.  
 
 
2. 
Objective 
 
This control assay will determine the ability of the SS to completely neutralize the 
active ingredients in the test treatments when applied to the abdomen by recovering 
and quantifying microorganism populations on agar media and is appropriate for 
antimicrobial agents that can be chemically inactivated or diluted to sub-inhibitory 
levels.   
 
3. 
Subject Entry Criteria 
 
subjects will be used for the neutralization validation required for this study.  Each 
subject must meet the inclusion and exclusion criteria described in Sections 4.1 and 
4.2 except for the baseline bacterial count, the 72-hour exclusion from 
showering/bathing and the length of the washout period. No minimum bacterial count 
is required and the washout period is only necessary for 7 days (not 14 days). The 
subjects will be asked to provide information on demographics and inclusion/exclusion 
criteria and sign the Consent and Authorization Forms before beginning the 7-day 
washout period. When the subjects return to begin their participation in the study they 
will again be asked to provide information relative to inclusion/exclusion criteria.  If they 


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meet all inclusion/exclusion criteria, they may be enrolled. The subjects will be 
identified by the letter “N” for neutralization and a subject number of 
. 
 
The test, vehicle and reference control articles will be applied to the abdomen regions 
using bilateral applications so that 
 applications are performed for each treatment 
using bilateral application (a total of 
 subjects). One area will be located on one 
side of the body and the remaining area on the other side. 
 
The treatments per subject will be randomized. The randomization scheme will be 
generated before the test.  
 
4. 
Test Organisms 
 
The test organisms for this study are: 
 
a. 
  
b. 
  
  
5. 
Treatment Materials 
 
a. 
ZuraPrep™ 
 (containing 70% v/v Isopropyl Alcohol) 
b. 
ChloraPrep® 
 (Scrub Teal® Tint) (containing 2% chlorhexidine 
gluconate) 
c. 
ZuraPrep Vehicle (negative control) 
 
 
6. 
Materials, Supplies and Equipment 
 
See section 3.4.3 of the MBT 865-105 Protocol. In addition, 70% isopropyl alcohol 
swabs. 
 
7. 
In-vivo Test Procedures (collection of samples) 
 
Preparation of Test Area and Post-Prep Sampling: Neutralization samples will be 
taken from the abdomen. The subject number, location of the prep application, 
location of the sites sampled within the prep area, and the time of sample collection 
will be documented on the CRF. The subject will be treated with the study materials 
based on the following. 
 
• 
For each side of the body, mark the abdominal test areas using a sterile 2” x 
5” template. The 2” x 5” areas will be delineated with each containing one 1” x 
1” sampling site. 
• 
After the test areas are marked, each area will be processed using three 70% 
isopropyl alcohol swabs for a total of one minute to prepare the site; the areas 


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will be allowed to dry. This step is to prepare the skin for the neutralization 
test.  
• 
Prep the test areas with the appropriate treatment according to the instructions 
provided in Appendix 14.7, MBT 865-105 Protocol for groin. 
• 
Using the scrub cup technique at approximately 30 seconds post-prep, begin 
collecting samples from each site using SS. This technique is described in 
Section 5.2.1 of the MBT 865-105 Protocol.  
 
8. 
In-vitro Test Procedures (performed using the collected samples in accordance with 
ASTM E1054-08) 
 
8.1. Inoculum Preparation:  For each test organism, the organism will be prepared 
from and overnight broth culture (24 ± 4 hours) grown in TSB at 35 ± 2C to yield 
a concentration of approximately 
  
CFU/mL.  The culture will be diluted 
using PBW in a manner such that 
 CFU/mL will be delivered into the 
neutralizer tube (a 
 aliquot of the collected SS sample will be used).   
 
8.2 Test: (note – the reference to test article applies to test article, the vehicle and the reference 
control article; all procedures outlined, where applicable will employ all articles.) 
 
 
8.2.1   Neutralizer effectiveness (Test 1): 
 
a. 
Out of the 
 aliquot of sampling solution taken from the volunteer, 
 
will be transferred to a new sterile tube and inoculated with the challenge 
microorganism so that the final concentration will equal 
 colony-
forming units (CFU) / mL of the challenge microorganism (the prepared 
inoculum will be diluted using PBW to achieve the desired concentration, a 
 aliquot from the 
 dilution will be used). 
b. 
Within one min after the addition of the challenge microorganism, the 
microorganisms will be enumerated by standard microbiological methods 
extant in the laboratory.  
c. 
Duplicate 
 aliquots will be removed and plated using TSA+N pour 
plates. 
d. 
After 30 minutes, the microorganisms will be enumerated a second time 
using the same procedures.  
e. 
Duplicate 
 aliquots will be removed and plated using TSA+N pour 
plates. 
8.2.2  Neutralizer toxicity (Test 2): 
 
a. A 
 aliquot of sampling solution will be inoculated with the challenge 
microorganism so that the final concentration will equal 
 colony-
forming units (CFU) / mL of the challenge microorganism in the same manner 


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as Test 1. 
 
b. Within one min after the addition of the challenge microorganism, the 
microorganisms will be enumerated by standard microbiological methods 
extant in the laboratory.  
c. Duplicate 
 aliquots will be removed and plated using TSA+N   pour 
plates. 
d. After 30 minutes, the microorganisms will be enumerated a second time 
using the same procedures.  
e. Duplicate 
 aliquots will be removed and plated using TSA+N pour 
plates. 
f. This procedure will be repeated two times for a total of three replicates. 
 
8.2.3 Test microorganism viability control (Test 3): 
 
a. A 
 aliquot of PBW will be inoculated with a volume of the challenge 
microorganism so that the resulting suspension contains 
 CFU/mL in 
the same manner as Test 1. 
b. Within one min the microorganisms will be enumerated (in triplicate) by 
standard microbiological methods extant in the laboratory. 
c. Duplicate 
 aliquots will be removed and plated using TSA pour plates. 
d. After 30 minutes, the microorganisms will be enumerated a second time 
using the same procedures.  
e. Duplicate 
 aliquots will be removed and plated using TSA pour plates. 
f. This procedure will be repeated two times for a total of three replicates. 
 
8.2.4 Test article control (Test 4): 
 
a. A 
 aliquot of the test or control article will be inoculated with a volume 
of the challenge microorganism so that the resulting suspension contains 
 
CFU/mL in the same manner as Test 1. 
b. Within one min the microorganisms will be enumerated (in triplicate) by 
standard microbiological methods extant in the laboratory. 
c. Duplicate 
 aliquots   will be removed and plated using TSA pour plates. 
d. After 30 minutes, the microorganisms will be enumerated a second time 
using the same procedures.  
e. Duplicate 
 aliquots   will be removed and plated using TSA pour plates. 
f. This procedure will be repeated two times for a total of three replicates. 
 
 
 


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8.3.  Incubation: 
 
 All plates for Tests 1, 2, 3 and 4 will be incubated for 48±2 hours at 35°±2°C. 
 
8.4. Interpretation of data: 
 
a. The number of surviving challenge microorganisms for each replicate from each 
test will be average count of the three plates. 
 
 
 
b. The number of survivor values will be transformed to log10. 
c. The number of survivors (log10) from each test (1, 2, and 4) will be compared to 
the test microorganism viability population (test 3). 
d.  Neutralization aspects of the sampling solution will be considered adequate if the 
mean log10 CFU/mL of Test 1 is not more than 0.20 log10 less than the mean 
log10 CFU/mL of Test 3 (Mean log10 CFU/mL from Test 3 – Mean log10 CFU/mL 
from Test 1 using corresponding time points). 
e. The sampling solution will be considered non-toxic if the mean log10 CFU/mL of 
Test 2 is not more than 0.20 log10 less than the mean log10 CFU/mL of Test 3 
(Mean log10 CFU/mL from Test 3 − Mean log10 CFU/mL from Test 2 using 
corresponding time points). 
f. The mean log10 CFU/mL from Test 4 must be at least 0.20 log10 less than the 
mean log10 CFU/mL of Test 3. 
g. The amount of CFU added for each aspect must be confirmed to yield a  final 
suspension containing 
/mL (validated in test 3). 
h. The sterility controls must be negative for growth. 
  
 
8.5. Controls: 
 
8.5.1 Sterility control: 
 
Triplicate plates of TSA and TSA+N used will be incubated with the test. In 
addition, triplicate 
 aliquots of sampling solution and PBW will be plated 
using TSA pour plates used for a particular test date. All plates will be incubated 
with the test.  
 
8.5.2   Challenge microorganism confirmation: 
 
In order to confirm growth consistent with the challenge microorganism, Gram 
stains will be performed from a representative colony from a test plate. The 
colony morphology will be noted as well. 
 
 
 


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14.11 Skin Irritation Rating Scale 
 
 
a = A rating of 3 on the skin irritation scale in any category will be recorded as Adverse Event 
and will require subject’s removal from the study.  
 
 
 
 
 
 
 
 
Skin Irritation Rating Scale 
Reactive area(s) within the treatment site only 
Condition 
Rating Description 
Erythema 
0 
No reaction 
1 
Mild and/or transient redness   
2 
Moderate redness  
3a 
Severe redness  
Edema 
0 
No reaction 
1 
Mild and/or transient swelling   
2 
Moderate swelling   
3a 
Severe swelling   
Rash 
0 
No reaction 
1 
Mild and/or transient rash   
2 
Moderate rash   
3a 
Severe rash   
Dryness 
0 
No reaction 
1 
Mild and/or transient dryness  
2 
Moderate dryness   
3a 
Severe dryness   


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14.12 Estimation of Sample Size Report 
 
 
 
See attached report. 
 
 
 
14.13     Informed Consent Forms 
 
 
 
 See attached forms