Clinical trial NCT03861780
Conditions: Bacteria Microflora Reduction
Sponsor: Professional Disposables International, Inc.
Start date: 2019-04-02; Completion date: 2019-06-06
Enrollment: 152 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Comparative Study of Antimicrobial Effectiveness Evaluation of 26ml Project X, 5.1ml Project X and Prevantics Maxi Swabstick following ASTM E1173 - Standard Test Method for Evaluation of Preoperative, Precatheterization, or Preinjection Skin Preparations, ASTM International, West Conshohocken, Pennsylvania, 2015

Detailed description:

Arms: arm 1: 3.15% w/v CHG (chlorhexidine gluconate) / 70% v/v IPA (isopropyl alcohol) contained within a saturated at use applicator. 26ml volume. Single use. arm 2: 3.15% w/v CHG (chlorhexidine gluconate) / 70% v/v IPA (isopropyl alcohol) contained within a saturated at use applicator. 5.1ml volume. Single use. arm 3: 3.15% w/v CHG (chlorhexidine gluconate) / 70% v/v IPA (isopropyl alcohol). Swabstick. Single use.
Interventions: intervention 1: Project X 26ml intervention 2: Project X 5.1ml intervention 3: Prevantics Maxi Swabstick
Intervention descriptions: intervention 1: Application of antiseptic drug to the inguinal and abdomen areas of the subjects intervention 2: Application of antiseptic drug to the inguinal and abdomen areas of the subjects intervention 3: Application of antiseptic drug to the inguinal and abdomen areas of the subjects

Protocol documents:
Prot_SAP_000.pdf: 
Document Cover Page 
Official Title of Study: 
Pilot Evaluation of Two Test Materials With a Positive Control When Used as a Patient 
Preoperative Skin Preparation 
NCT Number: NCT03861780 
Date of Document: March 5, 2019 


BIOSCIENCE LABORATORIES, INC. PROTOCOL 1810480-103.01 
PILOT EVALUATION OF TWO TEST MATERIALS WITH A POSITIVE CONTROL 
WHEN USED AS A PATIENT PREOPERATIVE SKIN PREPARATION 
1.0 
TITLE PAGE 
Investigational Products: 
Sponsor: 
Study Number: 
Sponsor Representative: 
Principal Investigator: 
Subinvestigators: 
Clinical Site: 
Date: 
26 mL Project X Chlorhexidine Gluconate 3.15% (w/v) solution 
and Isopropyl Alcohol (70% v/v) 
5.1 mL Project X Chlorhexidine Gluconate 3.15% (w/v) solution 
and Isopropyl Alcohol (70% v/v) 
Professional Disposables International, Inc. (PDI) 
400 Chestnut Ridge Road 
Woodcliff Lake, New Jersey 
07677 
1810480-103 
James Clayton 
Collette Duley 
Chelsey Allison, CCRC, Christopher Beausoleil, CCRP, Brett 
Griggs, Eric Olson 
BioScience Laboratories, Inc. (BSLI) 
1755 South 19th Avenue 
Bozeman, Montana 59718 
Telephone: (406) 587-5735 
March 5, 2019 
PROTOCOL# 1810480-103.01 
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BIOSCIENCE LABORATORIES. INC. 
This Protocol has been approved by the GIRB on t4Jfv@ 6-f o/ 
rfL 


Confidentiality Statement 
This document contains the confidential information of Professional Disposables International, 
Inc. (PDI) and BioScience Laboratories, Inc. (BSI::I). It is intended solely for the guidance of the 
clinical investigation. This protocol may not be disclosed to parties not associated with the clinical 
investigation or used for any purpose without the prior written consent of PDI and BSLI. 
Permission to release the Protocol and Study Results to the United States Food and Drug 
Administration (FDA) or other regulatory agency to which this study will be submitted is explicitly 
granted. 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC. 
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2.0 
PROTOCOL SYNOPSIS 
Name of Sponsor: 
Professional Disposables International, Irie. (PDI)' 
Name and Concentration of Test Product Active Ingredients: 
26 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Protocol Number 
1810480-103.01 
5.1 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Title of Study: 
Principal Investigator: 
Sub-Investigators: 
Study Center: 
Publications (References) 
Study Duration: 
Objectives: 
Pilot Evaluation of Two Test Materials with a Positive 
Control When Used as a Patient Preoperative Skin 
Preparation 
Collette Duley 
Chelsey Allison, CCRC, Christopher Beausoleil, CCRP, Brett 
Griggs, Eric Olson 
BioScience Laboratories, Inc. 
ASTM Standard Test Method El 173-15, Evaluation of 
Preoperative, Precatheterization, or Preinjection Skin 
Preparations 
ASTM E1054-08(2013), Standard Test Methods for 
Evaluation of Inactivators of Antimicrobial Agents. 
2017 FDA, 21 CFR Part 310, Safety and Effectiveness of 
Health Care Antiseptics; Topical Antimicrobial Drug 
Products for Over-the-Counter Human Use, Final Rule. 
(FR82: No. 243, December 20, 2017. pp 60474 to 60503) 
A pre-test period of 14 days and a test period of 1 day. 
The purpose of this pilot study is to evaluate the antimicrobial 
efficacy of two test products containing a chlorhexidine 
gluconate and isopropyl alcohol solution (26 mL and 5.1 mL), 
when used as a patient preoperative skin preparation 
compared to a positive control (Prevantics® Maxi Swabstick 
5.1 mL). The evaluation will determine the correct 
application procedures and the sample size to be used for the 
pivotal study. The log,o recoveries of the normal flora will be 
used to determine the antimicrobial effectiveness of the two 
test materials using a multiple linear regression model, 
consistent with the requirements of the 2017 FDA 21 CFR 
PROTOCOL# 18 I 0480-103 0 I 
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BIOSCIENCE LABORATORIES, INC 
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Name of Sponsor: 
Professional Disposables International, Inc. (PDI) 
Name and Concentration of Test Product Active Ingredients: 
26 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Protocol Number 
1810480-103.01 
5.1 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Methodology: 
Number of Subjects: 
Part 310, Safety and Effectiveness of Health Care Antiseptics; 
Topical Antimicrobial Drug Products for Over-the-Counter 
Human Use, Final Rule (December 20, 2017); the difference 
between the two regression lines is the Average Treatment 
Effect (ATE). 
The testing methods are based on the standardized test 
method ASTM El 173-15, Evaluation of Preoperative, 
Precatheterization, or Preinjection Skin Preparations. 
Following a 14-day restriction period, subjects will be 
sampled for baseline, 3O-seconds, 10-minutes, and 6-hours 
microbial reduction evaluations post-product application. 
Subjects will be sequestered for the 6-hour evaluation. Test 
day baseline criteria will be set at: abdomen: 3.0 - 5.5 log10 
CFU/cm2, and inguen: 5.0 - 7.5 log10 CFU/cm2. 
A sufficient number of subjects will be entered into testing to 
complete 50 subjects per each of the 2 test and 1 control 
materials. A minimum of 75 subjects meeting the test day 
baseline criteria will test bi-laterally (150 abdomen and 
inguinal sites; 50 abdomen and inguinal sites per test and 
control materials). 
Main Criteria for Inclusion: 
A sufficient number of overtly healthy subjects at least 18 
years of age will be admitted into the study so that a minimum 
of 75 subjects complete the study. Insofar as possible, the 
groups of subjects selected will be of mixed sex, age, and 
race. The subject recruiters will verbally and/or visually 
verify with subjects that the skin of the abdomen and inguen 
are free from clinically evident dermatoses, injuries, or any 
other disorders that may compromise the subject or the study. 
Duration of treatment: 
For each subject, two of the three test materials will be 
applied bi-laterally to abdomen and inguinal sites, a single 
time. 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC 
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Name of Sponsor: 
Professional Disposables International, Inc. (PDI) 
Name and Concentration of Test Product-Active-Ingredients: · 
26 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Protocol Number 
1810480-103.01 
5.1 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Criteria for Evaluation: 
Efficacy: 
The primary efficacy variables for this study are the 
immediate antimicrobial effects at 3O-seconds and 10-minutes 
post-application and persistent antimicrobial effects at 6-hours 
post-application. The effectiveness criteria are that the log10 
recoveries of the two test products (26 mL and 5.1 mL) are 
non-inferior to the Positive Control (Prevantics® Maxi 
Swabstick 5.1 mL) with a 0.5 margin (log10 scale) per square 
centimeter on the abdominal and inguinal sites within 30 
seconds after drying and within 10 minutes after drying. The 
determination of non-inferiority will be based on a log10 
multiple linear regression model. The Average Treatment 
Effect (ATE) is the mean difference of the test and the 
positive control products used in performing a non-inferiority 
statistic. To demonstrate persistence for the test materials, the 
6-hour post-treatment measurement should be lower than or 
equal to the baseline measurement for 100 percent of the 
subjects on the abdominal and inguinal sites. The efficacy 
criteria will be consistent with the requirements of the 2017 
FDA 21 CFR Part 310, Safety and Effectiveness of Health 
Care Antiseptics; Topical Antimicrobial Drug Products for 
Over-the-Counter Human Use, Final Rule (December 20, 
2017). 
Safety: 
Evaluation for safety of use of the test materials will consist 
of Adverse Event-reporting and assessment for skin reactions 
during testing. 
PROTOCOL# 1810480-103.01 
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BIOSCIENCE LABORATORIES, INC 
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Name of Sponsor: 
Professional Disposables International, Inc. (PDI) 
Name-and Concen-tration-of-Test-Produet-Act-ive Ingredients:· 
26 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Protocol Number 
1810480-103.01 
5.1 mL Chlorhexidine Gluconate 3.15% (w/v) and Isopropyl 
Alcohol 70% (v/v) solution 
Statistical Methods: 
A statistical analysis of the data will be performed to 
determine the non-inferiority of the two test products to the 
Positive Control. Separate analyses will be performed for 
data sets for the abdomen and the inguinal test sites and for 
the 3O-seconds and 10-minutes post-application sample times. 
The statistic to be used is a multiple linear regression with one 
quantitative and one qualitative variable. 
The average treatment effect (ATE) is the mean difference of 
the two products with the 95% confidence intervals in place 
that will determine if the test products will pass the non-
inferiority evaluation. To pass, the upper bounds of the 95% 
confidence interval must be equal to or less than 0.5 log10. 
The number of subjects necessary to pass this requirement 
will be determined. 
Descriptive statistics will be generated, including sample 
sizes, means, standard deviations, and ranges. 
PROTOCOL# 1810480-103.01 
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3.0 
TABLE OF CONTENTS 
SECTION 
PAGE 
1.0 
TITLE PAGE ....................................................................................................................... l 
2.0 
PROTOCOL SYNOPSIS .................................................................................................... 3 
3.0 
TABLE OF CONTENTS .................................................................................................... 7 
4.0 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ...................................... 9 
5.0 
ETHICS ............................................................................................................................. 10 
5.1 
Institutional Review Board ........................................................................................................................... 10 
5.2 
Ethical Conduct of Study .............................................................................................................................. 10 
5.3 
Subject Information and Consent .................................................................................................................. 10 
6.0 
INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE ........................ 10 
6.1 
Monitoring .................................................................................................................................................... 11 
7.0 
INTRODUCTION ............................................................................................................. 12 
8.0 
STUDY OBJECTIVES ..................................................................................................... 12 
9.0 
INVESTIGATIONAL PLAN ............................................................................................ 12 
9.1 
Overall Study Design and Plan ..................................................................................................................... 12 
9.2 
Discussion of Study Design, Including Choice of Sample Size .................................................................... 13 
9 .3 
Selection of Study Population ....................................................................................................................... 13 
9 .3 .1 
Inclusion Criteria ................................................................................................................................... 14 
9.3.2 
Exclusion Criteria .................................................................................................................................. 14 
9.3.3 
Subject Responsibilities ......................................................................................................................... 15 
9.3.4 
Subject Withdrawal ............................................................................................................................... 15 
9.4 
Test Methods ................................................................................................................................................. 16 
9.4.1 
Equipment, Supplies, Test Solutions and Media ................................................................................... 16 
9.4.2 
Identity of Test Materials ...................................................................................................................... I 6 
9.4.3 
Method of Assigning Subjects to Treatment Groups ............................................................................. 17 
9 .4.4 
Pre-Test Conditioning Period ................................................................................................................ 17 
9.4.5 
Test Period ............................................................................................................................................. 18 
9.4.6 
Blinding ................................................................................................................................................. 19 
9.4.7 
Cylinder Sampling Technique ............................................................................................................... 19 
9.4.8 
Plating .................................................................................................................................................... 19 
9.5 
Efficacy and Safety Variables ......................................................................................................... .............. 19 
9.5.1 
Efficacy and Safety Measurements and Flow Chart .............................................................................. 19 
9.5.2 
Appropriateness of Measurements ........................................................................................................ 20 
9.5.2.1 
Neutralization ............................................................................................................................... 20 
9.5.3 
Primary Efficacy Variables ................................................................................................................. .. 21 
9.5.4 
Data Collection and Microbial Recoveries ............................................................................................ 21 
9.6 
Data Quality Assurance ................................................................................................................................ 22 
9.7 
Statistical Methods and Determination of Sample Size ................................................................................ 22 
9. 7. I 
Statistical Methods ................................................................................................................................ 22 
9.7.2 
Determination of Sample Size .................................................................. ............................................. 24 
9.8 
Changes in the Conduct of the Study or Planned Analyses .......................................................................... 24 
10.0 
STUDY SUBJECTS .......................................................................................................... 24 
10.1 
10.2 
11.0 
1 I.I 
11.2 
Disposition of Subjects .............................................................................................................................. 24 
Protocol Deviations ...................................................................................... .............................................. 25 
SAFETY EVALUATION ................................................................................................. 25 
Safety Assessments .................................................................................................. _ ................................ 25 
Evaluation of Test Sites on Completion ........... ......................................................................................... 26 
PROTOCOL II 18 I 0480-103.01 
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11.3 
Adverse Events .......................................................................................................................................... 26 
11.3.1 
Adverse Event/Experience ................................................................................................................ 26 
11.3.2 
Causal Relations of Adverse Event/Experience ................................................................................ 27 
11.3.3 
Serious Adverse Event/Experience - During this Study ................................................................... 27 
11.3 .4 
Unexpected Adverse Event/Experience · ............................................................................................ 27 
11.3.5 
Follow-up ........................................................................................................ .................................. 27 
11.3 .6 
Notification ....................................................................................................................................... 28 
11.3.7 
Anticipated Reactions ....................................................................................................................... 28 
12.0 
EXCEPTIONAL CONDITIONS ...................................................................................... 29 
13.0 
REFERENCES .................................................................................................................. 29 
14.0 
FINAL REPORT ............................................................................................................... 29 
15.0 
DOCUMENTATION AND RECORD KEEPING .......................................................... .29 
15.1 
Study Center File Management ................................................................................................................. 29 
16.0 
LIABILITY AND INDEMNIFICATION ........................................................................ .30 
17 .0 
ACCEPTANCE ................................................................................................................. 31 
APPENDIX 1 ................................................................................................................................. 32 
APPENDIX 2 ................................................................................................................................. 33 
APPENDIX 3 ................................................................................................................................. 36 
APPENDIX 4 ................................................................................................................................. 37 
PROTOCOL # 1810480-103 .0 I 
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4.0 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
AE 
ASTM 
ATCC 
ATE 
BBP++ 
BSLI 
CFR 
CFU 
CHG 
cm 
CRF 
CRO 
FDA 
FR 
GCP 
GLP 
GIRB 
HIPAA 
ICH 
m 
mL 
PBS 
SAE 
SD 
SSF++ 
SOP 
TSA 
TSA+ 
TSB 
Adverse Event 
American Society for Testing and Materials 
American Type Culture Collection 
Average Treatment Effect 
Butterfield's Phosphate Buffer Solution with product neutralizers 
BioScience Laboratories, Inc. 
Code of Federal Regulations 
Colony Forming Units 
Chlorhexidine gluconate 
Centimeter 
Case Report/Record Form 
Contract Research Organization 
Food and Drug Administration 
Federal Register 
Good Clinical Practice 
Good Laboratory Practice 
Gallatin Institutional Review Board 
Health Insurance Portability and Accountability Act 
International Conference on Harmonization 
Inch 
Milliliter 
Phosphate Buffered Saline 
Serious Adverse Event 
Standard Deviation 
Stripping Suspending Fluid with product neutralizers 
Standard Operating Procedure 
Tryptic Soy Agar 
Tryptic Soy Agar with product neutralizers 
Tryptic Soy Broth 
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TFM 
Tentative Final Monograph 
UV 
Ultraviolet 
Cylinder Sampling Procedure 
A procedure used to sample bacteria from the skin 
using a sterile cylinder, sterile rubber policemen and 
a specified volume of sampling fluid. 
Mean log10 reductions 
Positive Control 
Source Documents 
Subjects 
Test Material 
5.0 
ETHICS 
Average of the differences between the baseline 
microbial populations expressed as log10 CFU/cm2 
and the populations in log10 CFU/cm2 recovered 
from the post-application samples. 
A product that is to be tested according to 
procedures in this protocol in order to validate the 
testing procedures and to be used as a control. 
Recorded results of original observations and 
activities of a clinical investigation. 
Healthy human paid participant that has consented 
to test in the study. 
The test products or positive control that are to be 
tested according to procedures in this protocol. 
5.1 
Institutional Review Board 
Informed Consent Forms and any other supportive material relevant to the safety of the 
subjects will be supplied to the Gallatin Institutional Review Board (GIRB) for their 
review and approval. The primary purpose of the GIRB is the protection of the rights and 
welfare of the subjects involved (21 CFR Parts 50, 56, 312, and 314). This study will 
begin only after GIRB approval has been obtained. 
5.2 
Ethical Conduct of Study 
The study will be conducted in compliance with Good Clinical Practice standards (21 
CFR Parts 50, 56, 312, and 314, and ICH E6), the United States Food and Drug 
Administration regulations, Standard Operating Procedures of BioScience Laboratories, 
Inc., the study protocol, and any protocol amendments. 
5.3 
Subject Information and Consent 
The Informed Consent Form and List of Restricted Products will be provided to each 
subject prior to beginning the study. Subjects must also have a current Authorization to 
Use and Disclose Protected Health Information Form on file at the testing facility. 
Trained personnel will explain the study to each subject and will be available to answer 
PROTOCOL# 1810480-103.0 I 
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any questions that may arise. 
6.0 
INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE 
BioScience Laboratories, Inc. 
17 5 5 South 19th A venue. 
Bozeman, Montana 59718 
Principal Investigator: Collette Duley 
Contact: Collette Duley 
Phone: (406) 587-5735 Ext. 110 
Fax: (406) 586-7930 
Email: cduley@biosciencelabs.com 
Subinvestigators: Chelsey Allison, CCRC, Christopher Beausoleil, CCRP, Brett Griggs, 
Eric Olson 
Quality Assurance Monitor: Amy L. Juhnke, RQAP-GLP 
Statistician: Daryl S. Paulson, Ph.D. 
Consulting Medical Experts: David McLaughlin, M.D., Gabor Benda, M.D. 
Sponsor Study Monitor: Clinical Research Works 
Gallatin Institutional Review Board (GIRB) 
168 Timberview Circle 
Bozeman, Montana 59718 
Phone: (406) 581-8559 
DHHS Number: IRB00005939 
6.1 
Monitoring 
Professional Disposables International, Inc. (PDI), as Sponsor of this study, is responsible 
for ensuring the proper conduct of the study with regard to Protocol adherence and 
validity of the data recorded on the study documents. PDI has therefore assigned a study 
monitor to this study. The progress of the study may be monitored by: 
Periodic on-site review 
Telephone communications 
E-mail communications 
• 
Review of sample data sheets and source documents 
The Investigator will give PDI study monitor direct access to source documents that 
support data on the study documents and make available such records to authorized PDI 
personnel, quality assurance, IRB, and regulatory personnel for inspection and/or 
copymg. 
Note: The Federal Privacy rule (HIP AA) 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-
PROTOCOL # 1810480-103.0 I 
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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)(l)(iii)]. 
7.0 
INTRODUCTION 
Prior to surgery or other invasive procedures, skin must be treated with a topical 
antimicrobial product to minimize the risk of nosocomial infection by reducing the 
number of microorganisms on the skin. The 2017 FDA 21 CFR Part 310, Safety and 
Effectiveness of Health Care Antiseptics; Topical Antimicrobial Drug Products for Over-
the-Counter Human Use, Final Rule requires that the immediate and IO-minute log10 
recoveries of the test product is non-inferior to an positive control by a margin of 0.5 
log10, superior to that of a negative control by a margin of 1.2 log10, and that 100% of 
subjects' microbial populations recovered in the 6-hour samples are less than baseline 
populations. The testing methods for performing such evaluations are also described in 
the standardized test method ASTM El 173-15, Evaluation of Preoperative, 
Precatheterization, or Preinjection Skin Preparations. 
8.0 
STUDY OBJECTIVES 
The purpose of this pilot study is to evaluate the antimicrobial efficacy of two test 
products containing a chlorhexidine gluconate and isopropyl alcohol solution (26 mL and 
5.1 mL), when used as a patient preoperative skin preparation. The evaluation will 
determine the correct application procedures and the number of subjects needed to pass 
the pivotal study. The log10 recoveries of the normal flora will be used to determine the 
antimicrobial effectiveness of the two test products consistent with the requirements of 
the 2017 FDA 21 CFR Part 310, Safety and Effectiveness of Health Care Antiseptics; 
Topical Antimicrobial Drug Products for Over-the-Counter Human Use, Final Rule 
(December 20, 2017). 
9.0 
INVESTIGATIONAL PLAN 
9.1 
Overall Study Design and Plan 
This pilot study is designed to determine the antimicrobial effectiveness of two test 
products (Test Products #1 and #2), intended for use as Patient Preoperative Skin 
Preparations. The testing methods are based on the standardized test method ASTM 
E 1173-15, Evaluation of Preoperative, Precatheterization, or Preinjection Skin 
Preparations, and using the requirements specified by the 2017 FDA 21 CFR Part 310, 
Safety and Effectiveness of Health Care Antiseptics; Topical Antimicrobial Drug 
Products for Over-the-Counter Human Use, Final Rule (December 20, 2017). The 
immediate and persistent antimicrobial effects will be measured to evaluate the efficacy. 
PROTOCOL#l810480-l03 01 
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At least 75 subjects will be treated bilaterally with two of the three test materials (Test 
Product #1, Test Product #2, and Positive Control), one per each side of the abdomen and 
inguinal !est sites. Subjects will be required to complete a 14-day pre-test conditioning 
period. Subjects will complete a I-day test period, during which time subjects' sites will 
be treated with the test materials and samples taken following treatment. The Cylinder 
Sampling Technique will be performed for baseline and for sampling 3O-seconds, 
10-minutes, and 6-hours post-test material-application on Test Day. 
The effectiveness criteria are that the log10 recoveries of the two test products (26 mL and 
5.1 mL) are non-inferior to the Positive Control (Prevantics® Maxi Swabstick 5.1 mL) 
with a 0.5 margin (log10 scale) per square centimeter on the abdominal and inguinal sites 
within 30 seconds after drying and within 10 minutes after drying. The determination of 
non-inferiority will be based on the upper bounds of the 95% confidence interval being 
equal to or less than 0.5. Superiority will not be evaluated in this study. To show 
persistence for the test materials, the 6 hours post-treatment measurement should be 
lower than or equal to the baseline measurement for microbial reductions for 100 percent 
of the subjects on the abdominal and inguinal sites. This evaluation will determine the 
correct application procedures and the power for the pivotal study. 
9.2 
Discussion of Study Design, Including Choice of Sample Size 
The sample size of a minimum of 100 subjects treated per test material is required for 
performing this type of evaluation based on the 2017 FDA 21 CFR Part 310, Safety and 
Effectiveness of Health Care Antiseptics; Topical Antimicrobial Drug Products for Over-
the-Counter Human Use, Final Rule (December 20, 2017). For this pilot study, only a 
minimum of 50 subjects treated per test material will be evaluated to determine if a study 
utilizing 100 subjects will meet the requirements in a pivotal study. 
9.3 
Selection of Study Population 
A sufficient number of overtly healthy subjects at least 18 years of age will be admitted 
into the study so that 50 abdomen and 50 inguinal site evaluations are completed per each 
test material at each post-treatment sample time from sites passing Treatment Day 
baseline criteria (3.0 -5.5 log10 CFU/cm2 from the skin of the abdomen and 5.0 -7.5 log10 
CFU/cm2 from the skin of the inguen). Insofar as possible, the group of subjects selected 
will be of mixed gender, age, and race. All subjects will be free from clinically evident 
dermatoses, injuries, wounds, and/or any other disorders that may compromise the 
subject and the study. All subjects will sign the Informed Consent Form and List of 
Restricted Products prior to participating in the study. The above forms are provided as 
separate Informed Consent documents. Subjects will receive a copy of these documents 
once consented to the study. Subjects must also have a current Authorization to Use and 
Disclose Protected Health Information Form on file at the testing facility. The Informed 
Consent Form will include a list of the active ingredients and post study product-removal 
PROTOCOL# 1810480-103.01 
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materials to which subjects may be exposed. The Informed Consent Form provides the 
subject with contact information for the Principal Investigator, and Subinvestigator(s), as 
appropriat~. 
9.3.1 
Inclusion Criteria 
• 
Subjects may be of either sex, at least 18 years of age, and of any race. 
• 
Subjects must be able to read and understand English. 
• 
Subjects must have read and signed an Informed Consent Form and List of Restricted 
Products prior to participating in the study; located in the separate Informed Consent 
documents, as well as have a signed Authorization to Use and Disclose Protected 
Health Information Form on file at the test facility. 
• 
Subjects must be in good general health and have no medical diagnosis of a physical 
condition, such as a current or recent severe illness, medicated or uncontrolled 
diabetes, hepatitis B, hepatitis C, an organ transplant, mitral valve prolapse with heart 
murmur, fibromyalgia, ulcerative colitis, Crohn's disease, an immunocompromised 
condition such as AIDS ( or HIV positive), lupus, or medicated multiple sclerosis. 
• 
Subjects will have test sites on the skin of the abdomen and/or inguinal free of injury 
and in good condition (no active skin rashes, excessive freckling, moles, scratches, 
breaks in the skin, etc.) and have no currently active skin diseases or skin conditions 
(for example, contact dermatitis, psoriasis or eczema) that may compromise subject 
safety or study integrity. 
• 
Subjects must have skin within 6 inches of the test sites that is free of tattoos, 
dermatoses, abrasions, cuts, lesions or other skin disorders. Subjects with tattoos, 
scars, active skin rashes, or breaks in the skin of test sites, skin blemishes, such as dry 
scabs or warts, may be admitted at the discretion of the Principal Investigator, 
Subinvestigators, or Consulting Physicians. 
9.3.2 
Exclusion Criteria 
Potential subjects will be excluded from this study if any of the following criteria apply 
to them: 
• 
Known allergies to vinyl, latex (rubber), alcohols, metals, tapes or adhesives, inks, 
sunscreens, deodorants, laundry detergents, topically applied fragrances, cleansers, or 
to common antibacterial agents found in soaps or lotions, particularly chlorhexidine 
gluconate or ethanol. 
• 
Have experienced hives (raised welts) as a reaction to anything that contacted the skin 
with the exception of items that cause hives as a reaction to the general population 
(e.g. poison oak and poison ivy). 
• 
Use of systemic or topical antibiotic medications during the 14-day pre-test period 
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through completion of testing on the single test day. 
• 
Use of systemic or topical steroids, other than for contraception, hormone therapy, 
post-menopausal indications, during the 14-day pre-test period through completion of 
testing on the single test day. This includes steroid medications used to treat asthma. 
Note: topically applied hormonal steroids used for post-menopausal reasons must not 
get on the test sites. 
• 
Any type of port (or portacath) or Peripherally Inserted Central Catheter (PICC). 
• 
Pregnancy, plans to become pregnant or impregnate a sexual partner within the pre-
test and test period of the study, or nursing a child. Female subjects must have a 
negative urine pregnancy test documented before treatment with test materials. 
• 
Current participation or participation in a clinical study in the past 14 days. 
• 
Any medical condition or use of any medications that, in the opinion of the Principal 
Investigator or Consulting Physicians, would preclude participation. 
9.3.3 
Subject Responsibilities 
During the 14-day pre-test period or through completion of testing on the single test day, 
subjects: 
• 
Must not expose test sites to strong detergents, solvents, other irritants, antimicrobial 
agents, medicated soaps, medicated shampoos, medicated lotions, hot waxes or 
depilatories, including shaving (in the applicable test areas). 
• 
Must not wear fabric softener-treated or antimicrobial-treated clothing (including 
bug-repellent and UV-treated clothing). 
• 
Must not use biocide-treated pools or hot tubs, tanning beds, or sunbathe. 
• 
Must not shower, bath, or swim within the 72-hour period prior to sampling (sponge 
baths may be taken, for example to wash the feet, however, the test areas must be 
avoided). 
• 
May clip hair from the test sites prior to the day product restriction begins if they 
have hair density on the abdomen or inguen as pictured in the bottom two images in 
Appendix 3. 
• 
Must be willing to fulfill the performance requirements of the study. 
9.3.4 
Subject Withdrawal 
After admission to the study, the subject may withdraw at any time for any reason. If 
possible, the reason for withdrawal will be recorded. Any subject not adhering to Protocol 
requirements will be disqualified and withdrawn from the study. 
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9.4 
Test Methods 
9.4.1 
Equipment, Supplies, Test Solutions and Media 
The equipment ancrsupplies used during' this study ·wrn ·be detailed-on tracking forms. 
The Test Solutions and Media used for this study are listed below: 
Sampling Solution 
Stripping Suspending Fluid with product neutralizers (SSF++). Neutralizers in this 
solution are Lecithin, Polysorbate 80, Sodium thiosulfate, and Tamol™ SN. 
Diluting Fluid 
Butterfield's 
Phosphate 
Buffer 
Solution 
with 
product 
neutralizers 
(BBP++). 
Neutralizers in this solution are Lecithin, Polysorbate 80, Sodium thiosulfate, and Tamol™ 
SN. 
Media 
--
Tryptic Soy Agar with product neutralizers (TSA+). Neutralizers in this agar are Lecithin 
and Polysorbate 80 
Tryptic Soy Agar (TSA) 
Tryptic Soy Broth (TSB) 
Phosphate Buffered Saline (PBS) 
9.4.2 
Identity of Test Materials 
The test materials will be provided to the Testing Facility by the Study Sponsor, complete 
with appropriate documentation. The test materials will be received and stored by 
BioScience Laboratories, Inc. (BSLI) in accordance with instructions from the Sponsor 
and retained in secure quarantine when not being used in testing. BSLI will maintain an 
inventory of the test materials and a log of use. Responsibility for determination of the 
identity, strength, purity, composition, stability, and solubility of the test materials, as 
well as responsibility for retention of the test materials rests with the Sponsor. Unused, 
sealed test materials will be stored by BSLI until the Sponsor specifies their disposition. 
In the absence of a disposition request from the Sponsor within 1 year of their planned 
usage, the test materials will be returned to the Sponsor. No test materials will be 
destroyed unless so requested by the Sponsor. 
Test Product #1: 
26 mL Project X Chlorhexidine Gluconate 3.15% (w/v) solution 
and Isopropyl Alcohol (70% v/v) 
Active Ingredients: 
Chlorhexidine Gluconate 3.15% (w/v) solution and Isopropyl 
Alcohol (70% v/v) 
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Lot Number: 
Expiration Date: 
Manufacture Date: 
Test Product #2: 
Active Ingredients: 
Lot Number: 
Expiration Date: 
Manufacture Date: 
Positive Control: 
Active Ingredients: 
Lot Number: 
Expiration Date: 
Manufacture Date: 
FA81 
7/24/2019 
1/24/2019 
5.1 mL Project X Chlorhexidine Gluconate 3.15% (w/v) solution 
and Isopropyl Alcohol (70% v/v) 
Chlorhexidine Gluconate 3.15% (w/v) solution and Isopropyl 
Alcohol (70% v/v) 
FASO 
7/21/2019 
1/21/2019 
Prevantics® Maxi Swabstick 5.lmL 
Chlorhexidine Gluconate 3.15% (w/v) solution and Isopropyl 
Alcohol (70% v/v) 
11801602 
11/2020 
11/2018 
9.4.3 
Method of Assigning Subjects to Treatment Groups 
The three test materials will be assigned randomly to the test sites per a computer-
generated randomization schedule, such that two of the three test materials are used on 
each subject at the abdomen and/or inguinal sites, one test material on one side, and 
another test material on the opposite side of the subject. The randomization schedule will 
be continued until it is determined that the required sample sizes are fulfilled. 
The specific sites for post-application sampling (i.e., 30 seconds, 10 minutes, and 
6 hours after skin preparation on the abdominal and inguinal test sites) will be assigned 
according to a computer-generated randomization scheme. 
9.4.4 
Pre-Test Conditioning Period 
The 14 days prior to the test portion of the study will constitute the pre-test period. 
During this time, subjects will avoid the use of medicated soaps, lotions, deodorants and 
shampoos, as well as skin contact with solvents, detergents, acids and bases, or any other 
products known to affect the normal microbial populations of the skin. Subjects will be 
supplied a personal hygiene kit containing non-medicated soap, shampoo, antiperspirant, 
lotion, and rubber gloves to be worn when contact with antimicrobials, solvents, 
detergents, acids, or bases cannot be avoided. Subjects will be instructed to use the 
contents of this kit exclusively during their participation in the study. Subjects must also 
avoid using UV tanning beds or sunbathing and swimming or bathing in biocide-treated 
pools or hot tubs. 
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At least 3 days (72 hours) prior to the test period, the subjects will be examined 
physically to ensure that there is no evidence of injury, dermatosis, or dermatitis present 
on the test area~. Hair present on test areas may be clipped if needed, to ensure that 
bandages used in testing will remain secure to the test sites. 
9.4.5 
Test Period 
Prior to sampling, the subjects will be questioned regarding adherence to the protocol. 
Females must have a negative urine pregnancy test documented before treatment with test 
materials. Subjects will don a disposable undergarment prior to sampling and will be 
examined physically to ensure no evidence of injury, dermatosis, or dermatitis is present 
at the sampling sites. Subjects will also don disposable exam gloves prior to product 
application to guard against contamination of the test sites during testing. 
On the abdomen (dry-skin sites), a sterile surgical marker will be used to demarcate 
bilaterally two 5" x 5" areas of skin (Appendix 1) that appear to be similar in condition, 
and not including the waistband area of clothing the subject was wearing, to the right and 
left side of the navel. In the inguinal region (moist-skin sites), a sterile surgical marker 
will be used to demarcate bilaterally two 1.5" x 5" areas of skin (Appendix 1) that appear 
to be similar in condition. 
Subjects will be sampled for baseline microbial population at the abdominal and inguinal 
sites on both sides using the Cylinder Sampling Technique (Section 9.4.7). 
The randomly assigned test materials will be applied to the abdominal and inguinal test 
sites per directions presented in Appendix 2. All test materials, including packaging, will 
be weighed before and immediately following application (Appendix 2) and the weights 
will be recorded. 
The abdominal and inguinal test sites will be sampled 30 seconds+ 5 seconds (30 
seconds to 35 seconds) and 10 minutes± 30 seconds post-product application using the 
Cylinder Sampling Technique (Section 9.4.7). At the completion of the IO-minute 
sample, the assigned 6-hour sample site will be covered with a dressing consisting of 
sterile gauze and semi-occlusive bandages to protect the site until the 6-hour sample 
window. All sampling times will be calculated from the completion of the dry time of 
each test material following application. Samples will be taken from the assigned sites at 
6 hours± 30 minutes post-product application using the Cylinder Sampling Technique 
(Section 9.4.7). If the semi-occlusive bandage has lifted from a test site such that the 
gauze sponge is no longer secured, the test site will be sampled and the occurrence noted. 
Each subject who volunteers to participate in this study will be required to remain/be 
sequestered at the test facility through the 6-hours post-treatment sampling. Following 
collection of the last 6-hour ± 30 minutes post-product application sample of a test site, 
the remaining test material will be wiped/cleansed from the test site with a mild soap 
and/or tap water; test sites will be dried with paper towels. 
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9.4.6 
Blinding 
Technicians who participate in test material application or bacterial sample collections, 
will not be blinded to the test material identity due to the physical differences of the test 
materials. 
Technicians who participate in plating samples and/or counting plates from samples will 
not participate in the test product application or sample collection procedures. 
9.4.7 
Cylinder Sampling Technique 
A sterile cylinder with an inside area of 3 .46 cm2 will be held firmly onto the test site to 
be sampled. 3.O-mL of sterile Stripping Suspending Fluid with product neutralizers 
(SSF++) will be placed into the cylinder, and the skin area inside the cylinder will be 
massaged in a circumferential manner for 1 minute with a sterile rubber policeman. The 
SSF++ will be removed with a sterile pipette and transferred to a sterile test tube. A 
second 3.O-mL aliquot of SSF++ will be placed into the cylinder, and the skin area again 
massaged for 1 minute with a sterile rubber policeman. Both aliquots will be pooled 
together for enumeration of microorganisms. 
9.4.8 
Plating 
Aliquots of the microorganism suspension (10° dilution) will be serially diluted in 
Butterfield's Phosphate Buffer Solution with product neutralizers (BBP++), as 
appropriate. Triplicate pour-plates will be prepared from each of these dilutions in 
Tryptic Soy Agar with product neutralizers (TSA+) and incubated at 30 °C ± 2 °C for 
approximately 72 hours, or until sufficient growth is observed. 
9.5 
Efficacy and Safety Variables 
Subject safety will be monitored by irritation evaluations and Adverse Event reporting 
(Section 11.0). 
9.5.1 
Efficacy and Safety Measurements and Flow Chart 
Mean log10 reductions in normal flora will be used to determine the antimicrobial 
effectiveness of the test materials. 
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Flow Chart 
-
.. 
-. . ... -. . -- .. 
--•-
-
... .. 
Day. . 
. . 
•·• . . 
Procedure 
-14 or more 
-14 to 0 
0 
Informed Consent Signed 
X 
Inclusion/Exclusion Criteria Reviewed 
X 
X 
Pre-Test Conditioning Period 
X 
Evaluation of Skin Condition 
X 
Test Material Application Locations and 
X 
Sampling Sites Assigned 
Baseline Sample 
X 
Test Material Application 
X 
30-second Post-Application Sample 
X 
10-minute Post-Application Sample 
X 
6-hour Post-Application Sample 
X 
9.5.2 
Appropriateness of Measurements 
The testing methods are based on the standardized test method ASTM El 173-15, 
Evaluation of Preoperative, Precatheterization, or Pre injection Skin Preparations. The 
effectiveness criteria are that the log10 recoveries of the two test products (26 mL and 5.1 
mL) are non-inferior to the Positive Control (Prevantics® Maxi Swabstick 5.1 mL) by no 
more than the upper margin of the 95% confidence interval of a 0.5 margin (log10 scale) 
per square centimeter on the abdominal and inguinal sites within 30 seconds after drying 
and within 10 minutes after drying. To show persistence of effect for the test materials, 
the 6 hours post-treatment measurement should be lower than or equal to the baseline 
measurement for 100 percent of the subjects on the abdominal and inguinal sites. The 
efficacy criteria will be consistent with the requirements of the 2017 FDA 21 CFR Part 
310, Safety and Effectiveness of Health Care Antiseptics,· Topical Antimicrobial Drug 
Products for Over-the-Counter Human Use, Final Rule (December 20, 2017). 
9.5.2.1 Neutralization 
A neutralization study will be performed to assure that the neutralizers used in the 
recovery medium quench the antimicrobial activity of each test material and are not toxic 
to the challenge species. The study will comprise both an In-vivo component performed 
using human subjects, and an In-vitro component performed based on ASTM El 054-
08(2013), Standard Test Methods for Evaluation of lnactivators of Antimicrobial Agents. 
Escherichia coli (A TCC 11229), Staphylococcus aureus (A TCC 653 8) and methicillin-
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resistant Staphylococcus aureus MRSA (ATCC 33591) will be used as the challenge 
species in both components of the neutralizer validation study. Reference Appendix 4 for 
the procedure for Vc!lidcttion of neutralization effectiveness. 
9.5.3 
Primary Efficacy Variables 
The primary efficacy variables for this study are the immediate antimicrobial effects at 
3O-seconds and 10-minutes post-application and persistent antimicrobial effects at 
6-hours post-application. The effectiveness criteria are that the log1 o recoveries of the 
two test products (26 mL and 5.1 mL) are non-inferior to the Positive Control 
(Prevantics® Maxi Swabstick 5.1 mL) with a 0.5 margin (log10 scale) per square 
centimeter on the abdominal and inguinal sites within 30 seconds after drying and within 
10 minutes after drying. The determination of non-inferiority will be based on the 
regression model used with a 95% confidence interval around the Average Treatment 
Effect (ATE). The upper bound of the 95% confidence interval cannot exceed 0.5. To 
show persistence of effect for the test materials, the 6-hours post-treatment measurement 
should be lower than or equal to the baseline measurement for 100 percent of the subjects 
on the abdominal and inguinal sites. The efficacy criteria will be consistent with the 
requirements of the 2017 FDA 21 CFR Part 310, Safety and Effectiveness of Health Care 
Antiseptics; Topical Antimicrobial Drug Products for Over-the-Counter Human Use, 
Final Rule (December 20, 2017). 
9.5.4 Data Collection and Microbial Recoveries 
Colonies will be manually counted and data recorded on appropriate data collection 
forms. If one of the plates is uncountable, the count on the remaining plate will be used 
for calculations and noted in the source record. 
The estimated log10 number of viable microorganisms recovered from each sample site 
will be designated the "R-value." 
To convert the volumetric measure of the sample into the number of colony-forming 
units per square centimeter ( cm2), the following formula will be employed: 
R = Log10 
A 
where: 
R 
= the average colony-forming unit count in log10 scale per cm2 of sampling 
surface 
F 
= total number of mL of stripping fluid added to the sampling cylinder; in this 
study, F= 6 mL 
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L c; = average of the triplicate colony counts used for each sample collected 
n 
D 
dilution_factor of the plate counts 
A 
= inside area of the cylinder in cm2; in this study, A= 3.46 cm2 
NOTE: 
The reason that a log10 transformation will be performed on the collected data 
is to obtain a linear scale. A linear scale, more appropriately a log10 linear 
scale, is a basic requirement of the statistical models used in this study. 
Average colony counts ( L c i ) of O will be set to 0.5 prior to calculating R-values. Any 
n 
Fe::ci)10-D 
calculated CFU/cm2 values ( 
n 
) ofless than 1 will be set to 1 CFU/cm2, such 
A 
that the log1 o transformation will be zero for the calculated R-Value. 
Data collected from subjects with low or high test-day baseline counts on an anatomical 
test site(< 3.0 log10 CFU/cm2/ > 5.5 log10 CFU/cm2 from the skin of the abdomen, or 
< 5.0 log10 CFU/cm2/> 7.5 log10 CFU/cm2 on the skin on the inguen) will not be used in 
the Efficacy Analyses (Section 9.7.1). This is a common occurrence in clinical-
simulation studies. Hence, more subjects will be enrolled and tested to account for test-
day baseline failures. 
9.6 
Data Quality Assurance 
The Sponsor's designated Quality Assurance Representative may conduct audits at the 
study site. Audits will include, but are not limited to test materials, presence of required 
documents, the informed consent process, and review of source documents. The 
Investigator agrees to participate with audits conducted at a reasonable time in a 
reasonable manner. 
The study will be inspected by Quality Assurance at BioScience Laboratories, Inc., and 
reports will be submitted to the Principal Investigator and Management in accordance 
with BioScience Laboratories, Inc., Standard Operating Procedures. 
9. 7 
Statistical Methods and Determination of Sample Size 
The statistical analysis will be performed by BSLI statistician, Daryl S. Paulson, PhD, to 
determine the correct sample size to use for the abdominal and inguinal sites with a Beta 
error of0.20 (Power of 1 - 0.20 = 0.80), an alpha error of 0.05, and a detectable 
difference of 0.5. The PASS Version 16 statistical package will be used to determine the 
number of subjects needed in the pivotal study in order to pass the evaluation. 
9.7.1 
Statistical Methods 
The Minitab® Version 18 statistical computer package will be used for all statistical 
calculations. 
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Non-brferiorirv Analvsis 
An analysis of the data will be performed to determine the non-inferiority of the two test 
products to the Positive Control. Separate analyses will be performed for data sets for 
both the abdomen and the inguinal test sites and for the 30-seconds and 10-minutes post-
application sample time. Only those data from sites meeting the baseline criteria (3.0 -
5.5 log10 CFU/cm2 from the skin of the abdomen and 5.0 - 7.5 log10 CFU/cm2 from the 
skin of the inguen) will be used in the analysis. 
The statistic to be used is a multiple linear regression with one quantitative and one 
qualitative predictor. It has the form: 
y = B0 + B1x1 + B2x2 + e 
where: 
y = Log10 Recoveries 
Bo = y intercept 
B1 = Regression slope 
B2 = Difference between the two products 
x1 = Log10 Baseline 
x2 = Products 
1, if Test Product 
2, if Positive Control 
e = Error term 
The average treatment effect (ATE) is the mean of the 95% confidence interval. For the 
non-inferiority test, the upper bound of the 95% confidence interval must be equal to or 
less than 0.5 log10. 
For the non-inferiority test: 
Xe - Xr::;; 0.5 
where: 
Xr = mean of the test product 
Xe = mean of the positive control product 
0.5 = non-inferiority margin 
Descriptive statistics will be generated, including sample sizes, means, standard 
deviations, and ranges of the recovery data. 
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Sample Sfae Determination Analysis 
When the study is complete, the PASS 16 statistical package will determine the number 
of subjects used in a pivotal study for-the-abdominal and inguinal-regions;· · · ·· · - · - ·· · 
The formula that will be used is: 
> 2s
2 (za1z +zpf 
n -
0.s2 
where: 
n 2: minimum number of subjects employed in the study 
2 = number of products tested; 3 test materials (Test Product #1, Test Product #2 
and Positive Control) 
s2 = the largest variance of the two products tested at the abdominal or inguinal 
sites 
za12 = 1.96 (z-scale) for 1 - 0.05 = 95% confidence of Type I error 
z~ = 0.842 (z-scale) for 1 - 0.20 = 0.80% confidence of Type II error 
0.5 = Detection level requirement 
9.7.2 
Determination of Sample Size 
The sample size of a minimum of 100 subjects treated per test material is required for 
performing this type of evaluation based on the 2017 FDA 21 CFR Part 310, Safety and 
Effectiveness of Health Care Antiseptics; Topical Antimicrobial Drug Products for Over-
the-Counter Human Use, Final Rule (December 20, 2017). For this pilot study, only a 
minimum of 50 subjects treated per test material will be evaluated to determine if a study 
utilizing 100 subjects will be likely to meet the requirements in a pivotal study. 
9.8 
Changes in the Conduct of the Study or Planned Analyses 
Neither the Investigators nor the Sponsor will modify or alter this protocol without first 
obtaining agreement from the other parties with one exception: when necessary to 
eliminate an apparent immediate hazard to the subject. All protocol modifications 
including, but not limited to, changes in the Principal Investigator, inclusion/exclusion 
criteria, number of subjects to be enrolled, study sites, or procedures must be submitted to 
the GIRB as a written amendment for review and approval prior to implementation. 
10.0 
STUDY SUBJECTS 
10.1 
Disposition of Subjects 
A written consent form will be obtained from each subject and filed by the Investigator 
with the subject's records, in accordance with 21 CFR Parts 50 and 56. 
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The Investigator may discontinue individual subjects from the study at any time. Subjects 
may voluntarily withdraw from the study at any time. The Investigator or designee will 
provide a written report_~n_th~ ~ppropriat~ s_t~~y_cio_c_1:1111~1?-~_cie_~c-~i~i[!g_~h~_reas()n for 
discontinuance and the date of discontinuance. The discontinued or withdrawn subject 
who participated in treatment may not re-enter the study. The Investigator will provide a 
disposition table that specifies the number of subject's enrolled, randomized, treated, and 
discontinued as well as reasons for discontinuations. 
In order to implement a valid revocation of authorization to use and disclose private 
health information, the subject or their representative must make the request in writing to 
BioScience Laboratories, Inc., 1755 South 19th Avenue, Bozeman, Montana 59718. The 
revocation cannot stop the use or disclosure of information that has been collected prior 
to the revocation, is needed to ensure complete and accurate study results, 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 will 
make the subject ineligible to receive the study treatment or care. 
10.2 
Protocol Deviations 
This study will be conducted as described in this Protocol, except for an emergency 
situation in which the protection, safety, and well-being of the subject requires immediate 
interventions, based on the judgment of the Principal Investigator. In the event of a 
significant deviation from the Protocol due to an emergency, accident, or mistake, the 
Investigator or designee will document the details of the situation and any subsequent 
decisions. All deviations from the Protocol or approved amendments shall be 
documented by BSLI. Any deviation to the protocol that may have an effect on the 
safety or rights of the subjects must be reported immediately (within 24 hours) to the 
local GIRB and sponsor representative. Any deviation that may compromise the 
statistical analysis of the study must be reported immediately (within 24 hours) to the 
sponsor representative. 
The Sponsor or the Investigator has the right to discontinue the study at any time for 
medical and/or administrative reasons. This should occur after mutual consultation, 
where possible. 
11.0 
SAFETYEVALUATION 
11.1 
Safety Assessments 
The subject's safety will be monitored by evaluations ofreactions observed on the skin of 
the test sites and any adverse reactions. Adverse reactions will be fully documented, 
reported as an Adverse Event, and followed to resolution. 
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11.2 
Evaluation of Test Sites on Completion 
Skin reactions will be evaluated prior to and post-product application (baseline, 30-
seconds, 10-minutes, and 6-hours) using the following-Skin Irritation Scoring System 
(Draize). 
SKIN IRRITATION SCORING SYSTEM (Draize) 
0 
No reaction 
Erythema 
1 
Mild and/or transient redness limited to sensitive area 
2 
Moderate redness persisting over much of the test material-exposed area 
3 * 
Severe redness extending over most or all of the test material-exposed area 
0 
No reaction 
Edema 
1 
Mild and/or transient swelling limited to sensitive area 
2 
Moderate swelling persisting over much of the test material-exposed area 
3 * 
Severe swelling extending over most or all of the test material-exposed area 
0 
No reaction 
Rash 
1 
Mild and/or transient rash limited to sensitive area 
2 
Moderate rash persisting over much of the test material-exposed area 
3 * 
Severe rash extending over most or all of the test material-exposed area 
0 
No reaction 
Dryness 
1 
Mild and/or transient dryness limited to sensitive area 
2 
Moderate dryness persisting over much of the test material-exposed area 
3 * 
Severe dryness extending over most or all of the test material-exposed area 
* = A score of 3 in one or more of the conditions evaluated represents significant irritation and 
qualifies as an Adverse Event. 
11.3 
Adverse Events 
Adverse events will be captured for all subjects from the time of informed consent to the 
time of subject discharge from the study. Adverse events will be categorized in 
relationship to the test materials that were applied. Medical facilities/personnel are in 
close proximity. 
In the event that either the Principal Investigator or the Sponsor determines that 
continuation of the study poses a hazardous risk of serious injury or death to the subjects, 
the study will be stopped. 
11.3.1 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 materials. All adverse 
event/experiences will be recorded and reported using appropriate Adverse Event 
documentation according to the Standard Operating Procedures of the laboratory. 
All adverse events, regardless of severity or the cause/effect relationship, are to be 
recorded. The severity of the effect will be noted as "Mild, 
11 "Moderate, 
11 or "Severe" 
according to the following definitions: 
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Mild 
Moderate 
Severe 
Awareness of signs or symptom, but easily tolerated. 
Discomfort to a degree as to cause interference with normal daily life 
ac_tivities_ a11:cl /o_r_ r~qt1i~ing ~_e4icat~cm: 
Incapacity with inability to work or do usual daily life activities and 
requiring medical attention/intervention. 
11.3.2 Causal Relations of Adverse Event/Experience 
When determining the causal/effect relationship to a test material, the relationship will be 
described as "None, 
11 "Possible," "Probable, 
11 or "Definite." The following definitions 
will be utilized: 
None 
Possible 
Probable 
Definite 
No association to the test materials. Related to other etiologies such as 
concomitant medications or conditions or subject's known clinical 
state. 
Uncertain association. Other etiologies are also possible. 
Clear-cut association with improvement upon withdrawal of the test 
materials. Not reasonably explained by the subject's known clinical 
state but not an anticipated event. 
An adverse event with a clear-cut temporal association and laboratory 
confirmation if possible. 
11.3.3 Serious Adverse Event/Experience - During this Study 
A Serious Adverse Event/Experience is any adverse experience occurring 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. 
11.3.4 Unexpected Adverse Event/Experience 
An Unexpected Adverse Event/Experience is any adverse event/experience not listed in 
the current labeling for the test materials, the current investigator's brochure, or the 
Anticipated Reactions Section 11.3. 7 of this Protocol. Where test material labeling or 
investigator's brochure is not available, anticipated experiences will be based on the 
known pharmacological/toxicological properties of the test materials or ingredients. 
11.3.5 Follow-up 
If an adverse event/experience occurs, the Sponsor will be monetarily responsible for all 
costs associated with the follow-up for said event including, but not limited to, medical 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on _ O~i'-'-/4_t>~/;,'----'lj___.___..1 __ 


visits and medication prescribed by a medical professional directly related to the adverse 
event along with an administration fee that covers the Principal Investigator's time 
resolving the Adverse Event._ If it is detennined ~y Test Facility _Management that the 
adverse event is due to negligence on the part of the Test Facility, no cost will be passed 
through to the Sponsor. The subject under the direction of the Principal Investigator (or 
designee) may be referred to the nearest acute care facility for treatment. Serious or 
Unexpected Event/Experiences will be followed to resolution. Any adverse event will be 
documented on an Adverse Event Report Form. 
11.3.6 Notification 
The Sponsor and the reviewing IRB will be notified of all adverse events/experiences that 
are related to product usage or require medical treatment within 2 business days. Any 
Serious or Unexpected (in nature or severity) Adverse Event/Experience that occurs 
during the study must be reported immediately by the Principal Investigator to the 
Sponsor and the reviewing IRB, followed by written notification within one business day 
of the information being reported to the investigative study team. Adverse 
events/experiences not linked to test product exposure or not requiring medical treatment 
will be reported to the Sponsor and reviewing IRB approximately weekly or monthly, as 
determined by the Principal Investigator ( or more frequently at the discretion of the 
Principal Investigator). 
The Principal Investigator is required to review all unanticipated problems involving risk 
to volunteers or others, serious adverse events, and all subject deaths associated with the 
protocol and provide an unbiased written report of the event. At a minimum, the 
Principal Investigator must comment on the outcomes of the event or problem, and in the 
case of a serious adverse event or death, comment on the relationship to participation in 
the study. 
11.3. 7 Anticipated Reactions 
Minimal risks to the study subjects are anticipated during the study. The risks associated 
with this study are primarily related to application of the test materials. Reactions to 
adhesives are also possible. Mild skin irritation or mild local irritant reactions such as 
burning and itchiness are anticipated. Although not anticipated, more severe reactions 
might occur, including moderate to severe erythema, swelling, cracking, peeling, or, in 
rare cases, blistering and/or an allergic reaction. Other anticipated reactions that may 
occur may result from testing procedures. Irritation and/or folliculitis may occur after 
clipping. A mild or moderate abrasion may occur from the Cylinder Sampling 
Technique. 
A Chlorhexidine gluconate (CHG) solution will be utilized during testing. CHG can 
cause a rare, but serious allergic reaction that may be life-threatening. Subjects will be 
instructed to seek immediate emergency medical help if they experience any of the 
PROTOCOL# 1810480-103,01 
Page 28 of 43 
BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on 


following symptoms: hives, severe skin rash, wheezing, difficulty breathing, cold sweats, 
feeling light-headed, or swelling of the face/lips/tongue/throat. 
12.0 
EXCEPTIONAL CONDITIONS 
The Sponsor will be notified within 24 hours by telephone, email, and/or letter of any 
exceptions encountered in this study that may affect subject safety or the results of the 
study. The exceptional conditions or occurrences will be detailed in full and formally 
recorded. Exceptional conditions that occur and are not addressed in this Protocol will be 
subject to Out-of-Scope charges (see Proposal/Contract). 
13.0 
REFERENCES 
ASTM E1054-08(2013), Standard Test Methods for Evaluation of Inactivators of 
Antimicrobial Agents. 
ASTM Ell 73-15, 
Standard 
Test 
Methods for 
Evaluation of Preoperative, 
Precatheterization, or Preinjection Skin preparations. 
Code of Federal Regulations Title 21 Parts 50, 54, 56, 58, 310, and 312. 
Food and Drug Administration Tentative Final Monograph (TFM) for Effectiveness 
Testing of a Patient Preoperative Skin (Vol. 59, No. 116, June 17, 1994, pp. 31450-
31452). 
ICH E6 Good Clinical Practice Guidelines. 
14.0 
FINAL REPORT 
A Final Report will be prepared in ICH format describing the methodology and results of 
the study in a clear and concise manner. 
15.0 
DOCUMENTATION AND RECORD KEEPING 
All documentation and records will be compiled and retained by BioScience 
Laboratories, Inc., at its facility in Bozeman, Montana. All raw data for this study, as 
well as the Final Report, will be retained in safe storage by the Testing Facility for a 
period of at least 2 years following the date a marketing application is approved for the 
test products for the indication for which they are being investigated. BioScience 
Laboratories, Inc. will notify the Study Sponsor before any documents or records are 
destroyed. 
15.1 
Study Center File Management 
It will be the responsibility of the Investigator to ensure that the Study File is maintained. 
The Study File for this protocol may contain, but will not be limited to, the information 
listed below: 
PROTOCOL #1810480-103 01 
Page 29 of 43 
BIOSCIENCE LABORATORIES, INC, 
This Protocol has been approved by the GIRB on 


• 
Investigational Brochure (if applicable) or other appropriate test material 
safety information 
• 
GIRB-Approved_ Sig~ed Protocol 
• 
Revised Protocol (if applicable) 
• 
GIRB-Approved Informed Consent Form (blank) 
• 
Financial Disclosure for the Principal Investigator and Subinvestigators (if 
applicable) 
• 
Curriculum Vitae of Principal Investigator and Subinvestigators 
• 
DHHS Number for GIRB, or other documentation of IRB compliance 
with FDA regulation (Included in this Protocol) 
• 
Documentation of GIRB approval of protocol, consent form, any protocol 
amendments and any consent form revisions 
• 
All correspondence between the Principal Investigator, IRB, and Sponsor 
relating to study conduct 
• 
Copies of information related to SAE and the information on Immediately 
Reported Adverse Events 
• 
Copy of the Approval Letter from the GIRB 
• 
Copy of Notification of Completion of Clinical Testing (Completion 
Letter to the GIRB) 
• 
Research Site Signature Log/Delegation of Duties 
To protect privacy and maintain the confidentiality of data, each subject will be assigned 
a unique study number, with all study samples and research records identified using the 
subject's study number. Research records will be kept in a room with access limited to 
study personnel, and electronic databases will be maintained on password-protected 
computers. 
16.0 
LIABILITY AND INDEMNIFICATION 
Test Facility's liability to Sponsor under this Protocol shall be limited to the price of this 
evaluation. Sponsor shall be responsible to Study Participants (when applicable) and to 
other third parties for the fitness of the test material for use as defined in the Study Protocol. 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES. INC. 
This Protocol has been approved by the GIRB on __t23/tJ(p /ttJ 


-····--· -- .... 17.0 
ACCEPTANCE 
PILOT EVALUATION OF TWO TEST MATERIALS WITH A POSITIVE 
CONTROL WHEN USED AS A PATIENT PREOPERATIVE SKIN 
PREPARATION 
ACCEPTED BY: 
BIOSCIENCE LABO RA TORIES, INC. (TESTING FACILITY) 
1755 South 19th Avenue 
Bozeman, Montana 59718 
President \:) ,.. . .}. . 
(_ .J 
& CEO: _ __ 
u.>-'\ 
__ 
" ----'-/ __ 
'\ _______ _ 
Daryl S. Paulson, Ph.D. 
Date 
Investigator: 
C/ ] /0&:,/ /~ 
Principal 
/ 
9 
I 
I 
___ 
C_o.._11-et-te_D_u_le-y-------~ 
Date of Study 1nitiati6n 
REVIEWED BY: 
Director of 
Quality 
,, 
· 
fl 
Assurance: 
t--{l.wJ/(2 I~ 
Amy L. Juhnke, RQAP-G~ 
.. 
ACCEPTED BY: 
PROFESSIONAL DISPOSABLES INTERNATIONAL, INC. (PDI) (SPONSOR) 
400 Chestnut Ridge Road 
Woodcliff Lake, New Jersey 
07677 
Date 
PROTOCOL# 1810480-103.01 
P~gc 31 of43 
BIOSCIENCE LABORATORIES, INC. 
Thi, Pro..,,1 h" b«• •Pl'ro~d by •• GIRB M _ /f}.ai7,/1 _k::_z 


17.0 
ACCEPTANCE 
PILOT EVALUATION OF TWO TEST MATERIALS WITH A POSITIVE 
CONTROL WHEN USED AS A PATIENT PREOPERATIVE SKIN 
PREPARATION 
ACCEPTED BY: 
BIOSCIENCE LABORATORIES, INC. (TESTING FACILITY) 
1755 South 19th Avenue 
Bozeman, Montana 59718 
President \:) ,t . .\.. 
(_ -{ 
&CEO: 
u.M_'-1 
/ 
-----------------
Daryl S. Paulson, Ph.D. 
Principal 
Inv stigator: _______________ _ 
Date 
Collette Duley 
Date of Study Initiation 
REVIEWED BY: 
Director of 
Quality 
,, 
· 
Assurance: ~A<~ tr J{Y~ 
Amy L. Juhnke, RQAP-G . 
ACCEPTED BY: 
PROFESSIONAL DISPOSABLES INTERNATIONAL, INC. (PDI) (SPONSOR) 
400 Chestnut Ridge Road 
Woodcliff Lake, New Jersey 
07677 
Representative 
Title 
PROTOCOL# 1810480-103.01 
Page 31 of43 
Date 
BIOSCIENCE LABORATORIES, INC. 
/M 
This Protocol has been approved by the GIRB on ;14/lft/tJ 6 .. -; It 


APPENDIX 1 
ANATOMICAL DIAGRAM OF SAMPLING SITES 
INGUINAL TEST SITES 
ABDO UNAL TEST SITES 
2 
l 
l 
2 
3 
4 
4 
3 
A 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on ~()~~ h._.b_l:._,b_i ...,..f __ 


APPENDIX2 
PRODUCT APPLICATION INSTRUCTIONS 
Abdominal Te t Site (Sin. x Sin.) 
1) 
Test Product #1 
• 
Peel open package and remove applicator - do not touch swab. 
• 
Hold the applicator with swab down. 
• 
Press down the antiseptic reservoir to release the antiseptic to the swab. 
• 
Wet the swab by repeated blotting against the treatment area until antiseptic is 
visible on the skin. 
• 
Thoroughly wet the treatment area with antiseptic using back-and-forth, left-and-
right strokes repeated for 30 seconds. 
• 
Do not apply swab beyond borders of marked skin areas by more than one swab 
width 
• 
Allow antiseptic to completely dry (minimum of 3 minutes). 
• 
Do not blot or wipe dry. 
2) 
Test Product #2 
• 
Peel open package and remove applicator - do not touch swab. 
• 
Hold the applicator with swab down. 
• 
Press down the antiseptic reservoir to release the antiseptic to the swab. 
• 
Wet the swab by repeated blotting against the treatment area until antiseptic is 
visible on the skin. 
• 
Thoroughly wet the treatment area with antiseptic using back-and-forth, left-and-
right strokes repeated for 30 seconds. 
• 
Do not apply swab beyond borders of marked skin areas by more than one swab 
width. 
• 
Allow antiseptic to completely dry (minimum of 30 seconds). 
• 
Do not blot or wipe dry. 
3) 
Positive Control 
• 
Peel open package and remove swabstick - do not touch swab. 
• 
Place one flat side of the foam tip within the pre-measured treatment area, and 
prep with back-and-forth repeated strokes for 15 seconds. 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC 
This Protocol has been approved by the GIRB 011 


• 
Turn the Maxi-swabstick over and repeat the procedure for 15 seconds. 
• 
Do not apply swab beyond borders of marked skin areas by more than one swab 
width. 
• 
Allow the area to completely air-dry; approximately 30 seconds. 
Inguinal Test Site (I.Sin. x Sin.) 
1) 
Test Product# 1 
• 
Peel open package and remove applicator - do not touch swab. 
• 
Hold the applicator with swab down. 
• 
Press down the antiseptic reservoir to release the antiseptic to the swab. 
• 
Wet the swab by repeated blotting against the treatment area until antiseptic is 
visible on the skin. 
• 
Thoroughly wet the treatment area with antiseptic using back-and-forth, left-and-
right strokes repeated for 2 minutes. 
• 
Do not apply swab beyond borders of marked skin areas by more than one swab 
width. 
• 
Allow antiseptic to completely dry (minimum of 3 minutes). 
• 
Do not blot or wipe dry. 
2) 
Test Product #2 
• 
Peel open package and remove applicator - do not touch swab. 
• 
Hold the applicator with swab down. 
• 
Press down the antiseptic reservoir to release the antiseptic to the swab. 
• 
Wet the swab by repeated blotting against the treatment area until antiseptic is 
visible on the skin. 
• 
Thoroughly wet the treatment area with antiseptic using back-and-forth, left-and-
right strokes repeated for 2 minutes. 
• 
Do not apply swab beyond borders of marked skin areas by more than one swab 
width. 
• 
Allow antiseptic to completely dry (minimum of 90 seconds). 
• 
Do not blot or wipe dry. 
3) 
Positive Control 
• 
Peel open package and remove swabstick - do not touch swab. 
• 
Place one flat side of the foam tip within the pre-measured treatment area, and 
PROTOCOL# 1810480-103.01 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on _P._'3_,_/4'---'--v ...... U-'--l'l..i..__ 


prep with back-and-forth repeated strokes for 60 seconds. 
• 
Turn the Maxi-swabstick over and repeat the procedure for 60 seconds 
• 
Do riot apply swab beyond boraers of marked skin areas by more than one swab 
width. 
• 
Allow the area to completely air-dry; approximately 90 seconds. 
• 
Do not blot or wipe dry. 
PROTOCOL # 1810480-103 .0 I 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on ~~-3 ..... fa_~__,_/4"-'/'f,__ 


APPENDIX3 
HAIR DENSITY IMAGE 
Four Typica l Abdominal Hair Patterns 
S<i ntlf C dcwiptlon ...., lh Q'\'-Oryd y ngllsh 
. 
dtStript:1Qn 
I ·1 
0 
Hotizo,ntal 
(Jlone) 
cu.mi11,ate 
~t~pilicd or \I) 
Sa•gi.ttaf 
(lln ortra I) 
Disperse 
(;ug) 
https://en.wikipedia.org/wiki/ Abdominal_ hair 
PROTOCOL #1810480-103.01 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on ~t)~J+
1h_0_6-+-f-/lJ~-


APPENDIX4 
VALIDATION OF NEUTRALIZER EFFECTIVENESS 
1.0 
PURPOSE OF THE NEUTRALIZER EFFECTIVENESS STUDY 
The purpose of this neutralization study is to assure that the neutralizers used in the 
recovery medium quench the antimicrobial activity of the test materials, and are not toxic 
to the bacteria. The study will comprise both an In-Vivo component performed using 
human subjects, and an In-Vitro component performed based on ASTM E1054-08(2013), 
Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents. 
Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 6538) and methicillin-
resistant Staphylococcus aureus MRSA (ATCC 33591) will be used as the challenge 
species in both components of the neutralizer validation study. 
2.0 
SCOPE 
An effective nontoxic method of neutralization must be employed to eliminate the 
antimicrobial activity of a test material quickly. Sufficient supporting data are required to 
show that the neutralizing method employed is effective. A known population of 
microorganism must be exposed to the antimicrobial test materials, diluent/recovery 
media, the neutralizing solution, and the neutralizing solution plus antimicrobial test 
material in order to determine whether microbial inhibition is present. 
Neutralizing methods include chemical inactivation, dilution of antimicrobial test 
material to a sub-inhibitory concentration, and membrane filtration. The procedures 
detailed here deal with chemical inactivation and dilution of antimicrobial test material, 
as well as recovery from human subjects. 
The In-Vivo component of the neutralization study will evaluate the three test materials. 
At least 18 human subjects will be tested using product applications on the skin of the 
abdomen, to obtain a minimum of four samples per test material challenged with 
Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 6538) and methicillin-
resistant Staphylococcus aureus MRSA (ATCC 33591). 
3.0 
TEST MATERIALS 
Test Materials are as described in the Study Protocol, Section 9.4.2. 
4.0 
EQUIPMENT AND SUPPLIES 
The equipment and supplies used during this neutralization will be detailed on tracking 
form(s). 
5.0 
TEST SOLUTIONS AND MEDIA 
Sampling olution 
Stripping Suspending Fluid with product neutralizers (SSF++). Neutralizers in this 
solution are Lecithin, Polysorbate 80, Sodium thiosulfate, and Tamol™ SN. 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on ~{) 
............ ~+,h~~~,+-,h'--'f t/,___ 


Diluting Fluid 
Butterfield's Phosphate Buffer Solution with product neutralizers (BBP++). 
Neutralizers in this so~ution are Lecithin,-Pel-yserbate-89-,-Sediumthiosulfate, and 
Tamol™ SN. 
Media 
Tryptic Soy Agar with product neutralizers (TSA+). Neutralizers in this agar are Lecithin 
and Polysorbate 80 
Tryptic Soy Agar (TSA) 
Tryptic Soy Broth (TSB) 
Phosphate Buffered Saline (PBS) 
6.0 
SUBJECT SELECTION 
6.1 
A sufficient number of overtly healthy subjects at least 18 years of age will be 
admitted into the study to ensure collection of at least four samples for each of the 
three test materials (18 subjects). 
6.2 
Subjects must meet the inclusion and exclusion criteria in Sections 9 .3 .1 and 9 .3 .2 
of the Protocol to which this neutralizer validation is attached, except for the 72-
hour exclusion from showering/bathing criteria, and the length of the washout 
period. Neutralization subjects only need to avoid topical and systemic 
antimicrobials for 7 days (not 14 days) prior to Test Day. One additional criterion 
for subjects is that they must not have hair on the test sites of the abdomen; this to 
minimize normal flora in the samples. Subjects will be asked to provide 
information on demographics and inclusion/exclusion criteria and sign the 
Informed Consent and List of Restricted Products before beginning the 7-day 
washout period (subjects will be provided with a product restriction kit). When 
subjects return to begin their participation in the study they will again be asked to 
provide information relative to inclusion/exclusion criteria. If they meet all 
inclusion/exclusion criteria, they may be enrolled. These subjects will be 
identified by the letter "N" for neutralization and a subject number starting with 
001. 
6.3 
Each subject will receive two of the three test materials, randomly assigned to a 
side of the abdomen, which will be applied to two separate 1.5" x 5" test sites on 
the abdomen. See Figure 1 below. 
PROTOCOL #1810480-103 .01 
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BIOSCI ENCE LABORATORIES, INC. 
This Prolocol has been approved by the GIRB 011 


Figure 1. Neutralization Test Sites Diagram 
7.0 
PROCEDURES 
Compliance with Good Clinjcal l ractices and Regulatory Requirement 
7 .1 
The study will be conducted in compliance with the Good Clinical Practice 
regulations in Section 5.0 of the Protocol to which this neutralizer validation is 
attached. 
7.2 
Subjects will be questioned prior to and during the study to ensure compliance 
with study requirements. 
Product Restriction Period 
7.3 
The 7-day period prior to the neutralization will be designated the "product-
restriction" period. During this time, subjects will avoid the use of medicated 
soaps, lotions, shampoos, deodorants (with exception of deodorant provided in the 
product-restriction kit), etc., as well as skin contact with solvents, acids, and 
bases. Subjects will also avoid sunbathing, using UV tanning beds or bathing in 
biocide-treated (e.g., chlorinated) pools and/or hot tubs. Subjects will be provided 
a kit containing products that they will be instructed to use exclusively for 
personal hygiene during the course of the study. 
7.4 
Subjects must not remove hair from the anatomical sites to be treated from the 
beginning of the product restriction period until after the neutralization has been 
completed. 
Randomization 
7 .5 
The test materials will be assigned randomly to test sites per a computer-
generated randomization schedule, such that each subject receives two of the three 
test materials at the abdominal sites. 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on 


Test lnoculum Pr paration 
7 .6 
Prior to beginning the neutralization assay, Escherichia coli (A TCC 11229), 
Staphylococcus .aureus (ATCC 6538)_and methicillin-resistant Staphylococcus 
aureus MRSA (ATCC 33591) from a stock culture slant, lyophilized vial, or 
cryogenic stock culture will be transferred into separate tubes containing Tryptic 
Soy Broth (TSB). The tube will be incubated for 24 hours± 4 hours at 35 °C ± 
2 °C. 
7.7 
One day prior to beginning the neutralization assay, loopfuls of each broth culture 
will be streaked onto Tryptic Soy Agar (TSA) plates, and the plates will be 
incubated for 24 hours± 4 hours at 35 °C ± 2 °C. 
7.8 
Immediately prior to initiating the neutralization assay, an inoculum suspension 
will be prepared in Phosphate Buffer Saline (PBS) solution from each culture on 
an agar plate, and the concentrations adjusted to approximately 3.0 x 108 to 1.0 x 
109 CFU/mL. The suspensions will then be serially diluted in PBS to achieve an 
inoculum titer of approximately 3.0 x 103 to 1.0 x 104 CFU/mL, and used as test 
inoculum for each species. 
lnoculu:m Assay .(Initial Population) - Test 
7.9 
Test Inoculum will be assayed by adding a 0.1 mL aliquot of the inoculum to 
5.0 mL of PBS solution (IP), vortexing for at least 
3 seconds, and immediately (within 1 minute) pour-plating, in triplicate, 1 mL 
aliquots of the test material with TSA. This assay will be performed three 
additional times for a total of four replicates. 
7 .10 
The diluted test inoculum suspensions will be allowed to stand for at least 
30 minutes, following which, 1 mL aliquots will be pour-plated, in triplicate, 
with TSA. 
Product Efli acy Evaluatj n (ln Vitro) - Test D 
7.11 
This phase of the neutralization assay determines whether the antimicrobial test 
material is able to reduce the population of the challenge microorganism. This 
assay will be performed in four replicates. 
7.12 
To each of four test tubes containing 5.0 mL of each test material, a 0.1 mL 
aliquot of the test inoculum will be added. The suspensions will be vortexed for 
at least 3 seconds and, immediately (within 1 minute), 1 mL aliquots of each 
replicate will be pour-plated, in triplicate, with TSA. 
7.13 
The tubes will be allowed to stand for at least 30 minutes, following which 1 mL 
aliquots of each replicate will be pour-plated, in triplicate, with TSA. 
Neutralizing/Re overy M dium fnhi bition Evaluations (in Vitro)- Tesl B 
7.14 
This phase of the neutralization assay assures that the sterile Stripping Suspending 
Fluid with product neutralizers (SSF++) the sampling solution employed in the 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORI ES, INC. 
This Protocol has been approved by the GIRB on _lJ~3-A_tl~'--+/4~1 .... 'f _ _ 


evaluation, is not inherently toxic to the microorganism. This assay will be 
performed in four replicates. 
7 .15 
To each of four test tubes containing 5 . .0 mL of one of the sampling solutions, a 
0.1 mL aliquot of the test inoculum will be added. The suspensions will be 
vortexed for at least 3 seconds and, immediately (within 1 minute), 1 mL aliquots 
of each replicate will be pour-plated, in triplicate, with Tryptic Soy Agar with 
product neutralizers (TSA + ). 
7 .16 
The tubes will be allowed to stand for at least 30 minutes, following which 1 mL 
aliquots of each replicate will be pour-plated, in triplicate, with TSA+. 
Diluent Broth Inhibition Evaluation Un Vitro)-Test B 
7 .17 
This phase of the neutralization assay assures that the Butterfield's Phosphate 
Buffer Solution with product neutralizers (BBP++) employed in the evaluation is 
not inherently toxic to the microorganism. This assay will be performed in four 
replicates. 
7.18 
Four test tubes containing 5.0 mL of diluent broth to be used in the test will be 
prepared, and a 0.1 mL aliquot of the test inoculum will be transferred to each 
tube. The suspension will be vortexed for at least 3 seconds, and immediately 
(within 1 minute), 1 mL aliquots of each replicate will be pour-plated, in 
triplicate, with TSA+. 
7 .19 
The tubes will be allowed to stand for at least 30 minutes, following which, 1 mL 
aliquots of each replicate will be pour-plated, in triplicate, with TSA+. 
Neutralizer Efficacy Evaluati n (In Vivo)-Test A 
7 .20 
This phase of the evaluation determines whether the neutralizing method chosen 
effectively eliminates the antimicrobial activity of the test materials. 
7.21 
Prior to sampling, the subjects will be questioned regarding adherence to the 
protocol. Subjects will also be examined physically to ensure no evidence of 
injury, dermatosis, or dermatitis is present at the sampling sites. Female subjects 
will be required to provide a urine sample for a pregnancy test. Only those female 
subjects with a negative test will be allowed to proceed into testing. 
7 .22 
A 1.5" x 5" test area will be demarcated on each side of the abdomen. After the 
test areas are marked, each area will be processed using three 70% isopropyl 
alcohol swabs for a total of~ 90 seconds ( ~ 30 seconds each), followed by an air-
dry for at least 1 minute. This step will prepare the skin for the neutralization test. 
7.23 
A test material (reference Section 9.4.2 of the Study Protocol) will be applied to a 
1.5" x 5" site on the abdomen of the subjects following the instructions for 
inguinal application in Appendix 2, with the randomly assigned test material. 
7.24 
The site will then be sampled using the Cylinder Sampling Technique 
30 seconds+ 5 seconds (30 seconds to 35 seconds) post product-application 
completion. 
PROTOCOL# 1810480-103.0 I 
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BIOSCIENCE LABORATORIES, INC. 
This Protocol has been approved by the GIRB on 


7.25 
The Cylinder Sampling Technique will be performed as described in Section 9.4.7 
of the Study Protocol (using SSF++). 
7.26 
The volume of sample will be adjusted to 5.0 mL, inoculated with 0.1 mL of the 
test inoculum, and vortexed for at least 3 seconds. Immediately (within 
approximately 1 minute), 1 mL aliquots will be pour-plated, in triplicate, with 
TSA+. 
7.27 
The tube containing the sample and inocula will be allowed to stand for at least 
30 minutes. Following the exposure, 1 mL aliquots of each sample will be pour-
plated, in triplicate, with TSA+. 
7.28 
The process will be repeated on the remaining abdominal site with the randomly 
assigned test material. A total of at least 18 subjects will be treated with two of 
the three test materials with samples from at least 6 subjects being used for each 
challenge microorganism. 
7.29 
Following all sampling, each test site will be cleaned using a towelette saturated 
with tap water and/or mild soap to remove the test material from the skin. 
Incubation 
7.30 
The inoculated plates will be incubated at 30 °C ± 2 °C for approximately 72 
hours, or until sufficient growth is visible and then enumerated. 
8.0 
CALCULATIONS 
8 .1 
The following formula will be used to calculate the number of viable bacteria 
recovered: 
{A+ B + C} 
LoglO 
3 
Where: A, B, and C = Individual Plate Counts 
8.2 
If colonies on one of the plates are uncountable, the count(s) from the remaining 
plate(s) will be used. 
9.0 
STATISTICS 
9.1 
After calculating the log10 populations recovered from Tests D, B, and A, these 
will be statistically compared to the Initial Population Test C (Control) using a 
One-Way Analysis of Variance (ANOVA) with Dunnett Multiple Comparisons 
with the Control. All statistical calculations will be performed using the 0.05 
level of significance for Type I (a) error. 
9.2 
Prior to comparing Phases to the Initial Population (Control), the 30-minute 
evaluation of the Initial Population should be shown to be statistically equivalent 
to the "time-zero" Initial Population of the microorganism. Hypotheses are: 
Ho: Initial Population= Test Phase, or 
HA: Initial Population -:t:- Test Phase. 
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9.3 
If p 
0.05 for each comparison to the control, Ho will be rejected and the Test 
Phase will be considered to be significantly different from the Initial Population. 
There is potential for low variance of the data, which would result in rejecting Ho. 
The difference between the Initial Population and the Test Phase will also be used 
to confirm significant differences. If the difference is greater than or equal to 
0.20, the two tests will be determined to be significantly different. Differences 
less than 0.20 between the two tests will be determined to be not significantly 
different. 
9 .4 
The Product Efficacy evaluation is effective if the antimicrobial test product 
produces a significant reduction in the population of the microorganism. 
9.5 
The Neutralizing/Recovery Medium Inhibition Evaluations are considered non-
inhibitory if all recovered populations are not statistically different from the Initial 
Population of the microorganism. 
9.6 
The Diluent Broth Inhibition Evaluation is considered non-inhibitory if all 
recovered populations are not statistically different from the Initial Population of 
the microorganism. 
9. 7 
Neutralization is considered adequate if all recovery populations are not 
statistically different from the Initial Population. 
10.0 
REFERENCES 
ASTM E 1054-08(2013), Standard Test Methods for Evaluation of Inactivators of 
Antimicrobial Agents, ASTM International, 100 Barr Harbor Drive, P.O. Box C700, West 
Conshohocken, PA 19428-2959, United States. 
Beausoleil, Christopher M. 2003. A Guide for Validation of Neutralizer Systems Used 
in Topical Antimicrobial Evaluations. In: Handbook of Topical Antimicrobials, D.S. 
Paulson, Ed. Marcel Dekker, New York. 452 pp. 
BSLI SOP CT-1006. Adequacy of Neutralization Assay: Clinical Trials Evaluations. 
Sutton, Scott V. W. 1996. Neutralizer Evaluations as Control Experiments for 
Antimicrobial Efficacy Tests. In: Handbook of Disinfectants and Antiseptics, J. 
M. Ascenzi, Ed. Marcel Dekker, New York. 300 pp. 
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