Clinical trial NCT03817580
Conditions: Bacterial Microflora Reduction
Sponsor: Professional Disposables International, Inc.
Start date: 2019-02-18; Completion date: 2019-06-04
Enrollment: 170 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Comparative Study of Antimicrobial Effectiveness Evaluation of 26 ml 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, PA, 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 swabstick. 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 swabstick. 5.1ml volume. Single use. arm 3: 3.15 % w/v CHG (chlorhexidine gluconate) / 70% v/v IPA (isopropyl alcohol). Swabstick. 5.1ml volume. 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: 
Comparative Study of Antimicrobial Effectiveness 
NCT Number: NCT03817580 
Date of Document: January 14, 2019 


 
 
CLINICAL STUDY PROTOCOL 
 
Study Number:
ER 18/282 
Study Title:
Comparative Study of Antimicrobial Effectiveness Evaluation of 26 ml 
Project X, 5.1 ml Project X and Prevantics® Maxi Swabstick following 
ASTM E1173 
Investigational 
Products:
26 ml Project X (A) 
5.1 ml Project X (B) 
Positive Control:
Prevantics® Maxi Swabstick 5.1 ml (C) 
Principal Investigator:
Rozalia Olsavszky M.D. 
Dermatologist 
Registered Number (Romanian Ministry of Health): 461524 (specialist 
in dermato-venerology doctor, doctor in medical science) 
Sub-Investigator:
Elena Chitoiu M.D. 
Resident Dermatologist 
Research Facility:
EUROFINS EVIC PRODUCT TESTING ROMANIA S.R.L. 
64-66, Marasesti Boulevard 
Bucharest 040256 Romania 
Telephone: +40 21 335 70 90 
IRB:
Advarra, Inc 
6940 Columbia Gateway Drive, Suite 110 
Columbia, Maryland, 21046, USA 
Sponsor:
Professional Disposables International Inc. 
400 Chestnut Ridge Road 
Woodcliff Lake, NJ 
07677 
Sponsor 
Representative:
James Clayton 
Professional Disposables International Inc. 
Email: James.Clayton@pdipdi.com  
Date
14-JANUARY-2019 
 
This document contains confidential information of Professional Disposables International Inc. and may 
not be disclosed to parties not associated with the clinical investigation or used for any purpose without 
the prior written consent of Professional Disposables International Inc. 
 




 
 
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Synopsis 
Study Number: 
ER 18/282 
Protocol Title: 
Comparative Study of Antimicrobial Effectiveness Evaluation of 26 ml Project X, 5.1ml 
Project X and Prevantics® Maxi Swabstick following ASTM E1173 
Planned Sample Size:
This evaluation will be performed with an estimated of a minimum of 75 evaluable 
subjects corresponding to 50 evaluable sites per product with qualifying treatment 
day microbial baseline counts per body area (abdomen and groin). 
Study Design:  
This is a randomized, single-center, single blind study in healthy subjects. The staff 
member(s) performing bacterial enumeration will be blinded from the identification 
of treatment assignment. 
Study Methodology: 
 
ASTM Standard Test Method E1173-15 
Study Population: 
Healthy subjects at least 18 years of age and of any race. 
Screening Day Baseline microbial count requirements are at least 3.11 log10 CFU/cm2 
bilaterally on the abdomen, and at least 5.00 log10 CFU/cm2 bilaterally on the groin 
except for subjects enrolled for Neutralization study. 
Treatment Day Baseline microbial count requirements are in the range of 3.11 to 
5.50 log10 CFU/cm2, inclusive, on the abdomen, and 5.00 to 7.50 log10 CFU/cm2, 
inclusive, on the groin except for subjects enrolled for Neutralization study. 
Objectives: 
Efficacy objectives 
The objective of this study is to demonstrate immediate (30 seconds and 10 
minutes) and persistent (6 hours) antimicrobial efficacy for patient preoperative 
skin preparation of product A and product B compared with product C. 
To demonstrate the immediate antimicrobial efficacy at 10 minutes post-application 
the following criteria should be met on both body area (abdomen and groin): 
- 
non-inferiority of the ATE of the product A and product B compared to 
product C (the upper two-sided 95% confidence bound of the post-application 
microbial counts corrected for Baseline of the A-C or B-C should be less than 0.5 
log10). 
To demonstrate the persistent efficacy for patient preoperative skin preparation, 
the microbial counts (log10 CFU/cm2) at 6 hours post-application should be lower 
than or equal to the baseline for 100 percent of the subjects in each body area. 
 
Safety objectives 
Safety will also be evaluated based on the incidence of adverse events reported 
during the study and assessment of skin irritation ratings. 
Study Endpoints: 
- 
Log10 CFU/cm2 of skin flora at the abdominal and groin sites before and after 
each treatment at 30 seconds, 10 minutes and 6 hours. 
- 
Skin irritation scores (Erythema, Edema, Rash, and Dryness) at each time 
interval and the incidence of AEs reported during the study. 


 
 
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Study Products: 
 
Treatment 
Description 
Body Site 
Application 
Time 
Drying 
Time 
Treatment 
Area 
A – 26 ml 
Project X 
3.15 % w/v 
chlorhexidine 
gluconate / 
70% v/v 
isopropyl 
alcohol, applied 
back and forth 
Abdomen 
30 seconds 
min. 180 
seconds 
5.0” x 5.0”  
(12.7 cm x 
12.7 cm) 
Groin 
120 seconds 
min. 180 
seconds 
1.5” x 5.0” 
(3.81 cm x 
12.7 cm) 
B – 5.1 ml 
Project X 
3.15 % w/v 
chlorhexidine 
gluconate / 
70% v/v 
isopropyl 
alcohol, applied 
back and forth 
Abdomen 
30 seconds 
30 seconds 
5.0” x 5.0” 
(12.7 cm x 
12.7 cm) 
Groin 
120 seconds 
90 seconds 
1.5” x 5.0” 
(3.81 cm x 
12.7 cm) 
C – 5.1ml 
Prevantics® 
Maxi 
Swabstick 
3.15 % w/v 
chlorhexidine 
gluconate / 
70% v/v 
isopropyl 
alcohol, applied 
back and forth 
Abdomen 
30 seconds 
30 seconds 
5.0” x 5.0” 
(12.7 cm x 
12.7 cm) 
Groin 
120 seconds 
90 seconds 
1.5” x 5.0” 
(3.81 cm x 
12.7 cm) 
 
Statistical Methods: 
Subjects with treatment day baseline bacterial count in the range of 3.11 to 5.50 
log10/cm2, inclusive, on the abdomen and 5.00 to 7.50 log10/cm2, inclusive, on the 
groin will be included in the analysis. 
An alpha level of 5% will be used for all analyses. 
The following descriptive statistics for log10 CFU/cm2 and for log10 CFU/cm2 reductions 
from Baseline will be computed for each treatment, grouped by body area and each 
post application sampling time point (30 seconds, 10 minutes and 6 hours): mean, 
median, standard deviation, minimum, maximum, and count. 
Efficacy Analysis 
A linear regression model for each body area (abdomen and groin) and each post-
treatment sampling time point of immediate efficacy (30 seconds and 10 minutes 
post-application) will be used. In the model, the dependent variable used will be the 
post-treatment bacterial count (log10 CFU/cm2) and predictors will be the treatment 
as a fixed effect and the Baseline as a covariate. The ATE corrected for Baseline will 
be estimated from the model and compared to non-inferiority criteria. 
To evaluate the persistent efficacy (6 hours post-application) of the product A and B, 
microbial counts (log10 CFU/cm2) at 6 hours post-application for each product 
application site will be converted to a binary (success/failure), “success” defined as 
having microbial counts less than or equal to the baseline counts. Responder rates 


 
 
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at 6 hours post product application will be summarized descriptively for each product 
on each body area. 
Safety Analysis 
All treated subjects will be considered evaluable for safety. Skin irritation scores will 
be reported for any subject who is scored with a 1 or more at any observation 
(baseline screening day, baseline treatment day, post-application/prior to 30 
seconds, 10 minutes, and 6 hours sampling procedures), in any category for any site.
Adverse events (including post treatment skin irritation scores of 3), will also be 
summarized. Summary tables will present incidence rates of adverse events by 
treatment group for all subjects who enter the treatment period. Listings of adverse 
events will be provided. 
 
 


 
 
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Contents 
Contents  .............................................................................................................................. 6 
Abbreviations ........................................................................................................................... 9 
1 
Introduction ........................................................................................................... 10 
2 
Study Objectives .................................................................................................... 10 
2.1 
Efficacy objectives .................................................................................................. 10 
2.2 
Safety objectives .................................................................................................... 10 
3 
Study Design ......................................................................................................... 11 
3.1 
Study Type ............................................................................................................ 11 
3.2 
Endpoints .............................................................................................................. 11 
3.3 
Randomization and Blinding ..................................................................................... 11 
3.4 
Study Products ....................................................................................................... 12 
 
Study Products Labeling ................................................................................... 12 
 
Study Supplies Provided by Sponsor ................................................................... 12 
 
Study Supplies Provided by Research facility ......................................................... 12 
3.5 
Study Duration ....................................................................................................... 13 
3.6 
Study Termination/Subject Discontinuation or Withdrawal .......................................... 13 
 
Study Termination............................................................................................ 13 
 
Subject Discontinuation and Withdrawal ................................................................ 14 
3.7 
Study Product Accountability .................................................................................... 14 
3.8 
Source Data ........................................................................................................... 14 
3.9 
Protocol Modifications .............................................................................................. 14 
 
Protocol Amendments ....................................................................................... 14 
 
Protocol Deviations .......................................................................................... 15 
4 
Study Population .................................................................................................... 15 
4.1 
Subject Inclusion Criteria ......................................................................................... 16 
4.2 
Subject Exclusion Criteria ......................................................................................... 16 
4.3 
Subject Consent ..................................................................................................... 17 
5 
Study Treatment .................................................................................................... 17 
5.1 
Study Procedures .................................................................................................... 17 
 
Washout Period ............................................................................................... 17 
 
Screening Day ................................................................................................. 18 
 
Treatment Day ................................................................................................ 19 
5.2 
Microbiological Methods ........................................................................................... 21 
 
Microbial Sample Collection / Scrub Cup Technique .............................................. 21 
 
Bacterial Enumeration Methods .......................................................................... 21 
 
Neutralization Validation ................................................................................... 22 
6 
Assessment of Efficacy ............................................................................................ 22 


 
 
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6.1 
Efficacy Parameters ................................................................................................. 22 
6.2 
Assessment Methods ............................................................................................... 22 
7 
Risk / Benefit Assessment ........................................................................................ 23 
7.1 
Potential Risks ........................................................................................................ 23 
7.2 
Potential Benefits.................................................................................................... 23 
8 
Assessment of Safety .............................................................................................. 23 
8.1 
Safety Parameters ................................................................................................... 23 
8.2 
Assessment Methods for Skin Irritation ...................................................................... 24 
8.3 
Adverse Events ....................................................................................................... 24 
9 
Statistics ................................................................................................................ 26 
9.1 
Data sets analyzed ................................................................................................. 26 
9.2 
Sample Size ........................................................................................................... 26 
9.3 
Efficacy Analyses .................................................................................................... 26 
 
Efficacy Analysis .............................................................................................. 26 
9.4 
Safety Analysis ....................................................................................................... 26 
9.5 
Procedures for Accounting for Missing, Unused, and Spurious Data .............................. 27 
10 
PI Responsibilities ................................................................................................... 27 
11 
Monitoring ............................................................................................................. 27 
12 
Quality Control and Quality Assurance ....................................................................... 27 
13 
Audits and Inspections ............................................................................................ 27 
14 
Ethical and Regulatory Standards ............................................................................. 28 
15 
Data Handling and Record Keeping ........................................................................... 28 
15.1 
Completion of Case Report Forms ............................................................................. 28 
15.2 
Final Clinical Study Report ....................................................................................... 29 
15.3 
Records, Reports and Retention Requirements ........................................................... 29 
16 
References ............................................................................................................ 29 
17 
Appendices ............................................................................................................ 30 
17.1 
Key Study Personnel, Titles, Responsibilities .............................................................. 30 
17.2 
Study Summary ...................................................................................................... 31 
 
Main study ...................................................................................................... 31 
 
Neutralization study ......................................................................................... 32 
17.3 
Abdomen and Groin Diagram ................................................................................... 33 
17.4 
Randomization Scheme Example .............................................................................. 34 
17.5 
Subject Instructions ................................................................................................ 35 
 
Subject Instructions for Main Study ................................................................... 35 
 
Subject Instructions for Neutralization Study....................................................... 36 
17.6 
Treatment Application Instructions ........................................................................... 37 
17.7 
Procedure for Neutralization Validation ...................................................................... 39 
 
Background .................................................................................................... 39 
 
Objective ........................................................................................................ 39 
 
Subject Entry Criteria ....................................................................................... 39 


 
 
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Test Organism ................................................................................................. 39 
 
Study Products ................................................................................................ 40 
 
Materials, Supplies and Equipment ..................................................................... 40 
 
In-vivo Test Procedures.................................................................................... 40 
 
In vitro Test Procedures .................................................................................... 40 
 
Incubation ...................................................................................................... 42 
 
Interpretation of data ................................................................................... 42 
 
Controls ...................................................................................................... 42 
17.8 
Skin Irritation Rating Scale ...................................................................................... 43 
17.9 
Confirmation of Release and Receipt of Study Materials ............................................... 44 
17.10 
Study Material Disposition Form ............................................................................ 45 
 
 


 
 
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Abbreviations 
AE 
Adverse event 
AIDS 
Acquired immune deficiency syndrome 
ASTM 
American Society for Testing Materials 
ATCC 
American Type Culture Collection 
ATE 
Average treatment effects 
CFR 
Code of Federal Regulations 
CFU 
Colony Forming Units 
CHG 
Chlorhexidine gluconate 
cm2 
Square centimeter 
CRF 
Case Report/Record Form 
ER 
EUROFINS EVIC PRODUCT TESTING ROMANIA S.R.L. 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIV 
Human immunodeficiency virus 
IB 
Investigator’s brochure 
ICF 
Informed consent form 
ICH 
International Conference on Harmonization 
IEC 
Institutional Ethics Committee 
IPA 
Isopropyl Alcohol 
IRB 
Institutional or Independent Review Board 
MRSE 
methicillin-resistant Staphylococcus epidermidis 
MSSE 
methicillin-sensitive Staphylococcus epidermidis 
PBW 
Phosphate Buffered Water 
PDI 
Professional Disposables International Inc. 
PI 
Principal Investigator 
SAE 
Serious adverse event 
SOP 
Standard Operating Procedure 
SSF++ 
Stripping Suspending Fluid with product neutralizers 
TSA 
Trypticase Soy Agar 
TSA+N 
Trypticase Soy Agar containing neutralizers 
TSB 
Tryptic Soy Broth 
US 
United States of America 
 
 


 
 
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1 
Introduction 
Topical antimicrobial products are used prior to surgery in order to reduce the risk of nosocomial 
infection by reducing the number of microorganisms on the skin. Chlorhexidine gluconate (CHG) was 
first introduced in the United States in the 1970s as a hand washing agent for healthcare workers. 
Today, CHG is widely used as an effective antimicrobial for hand washing and patient skin antisepsis. 
The use of CHG for patient skin antisepsis was studied clinically by Maki et al. in the early 1990s. Maki 
compared the use of 2% aqueous CHG, 70% alcohol, and 10% povidone-iodine (a common iodophor) 
for patient skin preparation prior to placement of a central-line catheter. Aqueous 2% CHG formulation 
was found to significantly reduce the incidence of catheter-related infections, as compared to either 
70% alcohol or 10% povidone-iodine. Moreover, Mimoza et al. showed CHG was more efficacious 
than povidone-iodine skin preparation in reducing contamination of blood cultures. In 2000, the first 
CHG and 70% isopropyl alcohol (IPA), was approved by the US FDA for patient pre-operative skin 
preparation. Since that time, numerous clinical studies support the use of the alcoholic CHG 
formulations, driving its wide adoption by clinicians worldwide. 
The immediate and persistent antimicrobial efficacy of the investigational products will be evaluated 
in this study according to US FDA analysis criteria. 
 
2 
Study Objectives 
2.1 
Efficacy objectives 
The objective of this study is to demonstrate immediate (30 seconds and 10 minutes) and persistent 
(6 hours) antimicrobial efficacy for patient preoperative skin preparation of product A and product B 
compared with product C. 
To demonstrate the immediate antimicrobial efficacy at 30 seconds and 10 minutes post-application 
the following criteria should be met on both body area (abdomen and groin): 
 
- 
non-inferiority of the ATE of the product A and product B compared to product C (the upper 
two-sided 95% confidence bound of the post-application microbial counts corrected for 
Baseline of the A-C or B-C should be less than 0.5 log10). 
To demonstrate the persistent efficacy for patient preoperative skin preparation, the microbial counts 
(log10 CFU/cm2) at 6 hours post-application should be lower than or equal to the baseline for 100 
percent of the subjects in each body area. 
2.2 
Safety objectives 
Safety will also be evaluated based on the incidence of adverse events reported during the study and 
assessment of skin irritation ratings from Appendix 17.8. 
 
 


 
 
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3 
Study Design 
3.1 
Study Type 
This is a randomized, single-center, single blind study in healthy subjects. The staff member(s) 
performing bacterial enumeration will be blinded from the identification of treatment assignment. 
3.2 
Endpoints 
- 
Log10 CFU/cm2 of skin flora at the abdominal and groin sites before and after each treatment 
at 30 seconds, 10 minutes and 6 hours. 
- 
Skin irritation scores (Erythema, Edema, Rash, and Dryness) at each time interval and the 
incidence of AEs reported during the study. 
3.3 
Randomization and Blinding 
The randomization scheme for the study will be provided by the Research facility. 
Each subject will receive two treatments per body area. Assignment of products to the left or right side 
for each body area will be per a computer-generated randomization schedule. 
Specific sites of sampling for baseline and post application sampling time points will be randomized on 
each body site (abdomen/groin and left/right). 
Randomization will continue until required number of subjects with required treatment day baseline 
counts is enrolled. 
The PI is responsible for ensuring that the randomization is followed. The randomization schedule for 
the abdominal and groin sites will be provided in a separate document. The study products will be 
labeled with the appropriate codes as designated by the study randomization. 
 
Each subject will be identified by their initials and a subject number for which they qualify. Inclusion 
subject numbers will not be assigned until a subject has passed the screening criteria, including 
screening bacterial counts.  
- 
Screening subjects will be assigned numbers starting with 001 and the letter “P”: P001 
- 
Treatment subjects will be assigned numbers with 001 and the letter “I”: I001 
The study products will not be blinded from the PI or other study staff performing the study material 
application or bacterial sample collections. The staff member(s) performing bacterial enumeration will 
be blinded from the identification of treatment assignment. The study staff performing the bacterial 
enumeration will not be involved in the study products application or the collection of samples. The 
Raw Data Sheet sections of the CRF will be maintained separately (from the pages within the CRF 
which include study treatment identifications) during the conduct phase of the study. The study staff 
performing the bacterial enumeration will record counts directly onto the Raw Data Sheet pages of the 
CRF without accessing the subject study documentation folder containing the other CRF pages. The 
Raw Data Sheets will be compiled with the entire CRF after all data recording have been completed. 
 
 


 
 
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3.4 
Study Products 
The products identified in Table 1Table 1 will be used in the study. Specific product identification codes 
and lot numbers will be included with the clinical supplies when they are shipped to the Research 
facility. 
PDI is responsible for analytically testing (content and purity) the study products to ensure they comply 
with their specifications and both released as per approved specifications of the marketed control 
product. 
Table 1: Study Products 
Treatment 
Denomination 
Volume 
Description 
A 
26 ml Project X  
26 ml 
3.15 % w/v chlorhexidine gluconate / 70% v/v isopropyl 
alcohol 
B 
5.1 ml Project X  
5.1 ml 
3.15 % w/v chlorhexidine gluconate / 70% v/v isopropyl 
alcohol 
C 
Prevantics® Maxi Swabstick 
5.1 ml 
3.15 % w/v chlorhexidine gluconate / 70% v/v isopropyl 
alcohol 
 
 Study Products Labeling 
PDI will label, package, and ship the study products to the research facility. ER personnel will document 
receipt and storage of the study materials.  
 
 Study Supplies Provided by Sponsor 
 
- 
Study Products  
 
 Study Supplies Provided by Research facility 
- 
Product kits (toiletry items to be used by subjects during study) 
- 
Stripping Suspending Fluid with product neutralizers (SSF++) containing 1.01% Na2HPO4, 
0.04% KH2PO4, 0.1% Triton X-100, 1.167% Lecithin, 10% Polysorbate 80, 0.5% 
Na2S2O3•5H2O, and 1% Tamol™. 
- 
Butterfield’s sterile phosphate-buffered water (PBW), (312 µM KH2PO4, pH 7.2 ± 0.4) 
- 
High-purity deionized water 
- 
Trypticase Soy Agar (TSA) (for the Neutralization Validation Procedure only – See Appendix 
17.7) 
- 
Trypticase soy agar containing 0.5% Tween 80 and 0.07% lecithin (TSA+N) 
- 
Serological pipettes (10 ml), sterile 
- 
Disposable Pasteur Pipettes, sterile 
- 
Tubes with sealable caps, polypropylene or glass, sterile 
- 
Petri dishes, 90 mm, sterile 
- 
Gloves, sterile 


 
 
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- 
Gauze, sterile 
 
- 
Non-toxic marking pen: Chemo Skin Marker- Regular™ 
- 
Rubber policemen, sterile 
- 
Scrub cups (2.20 cm I.D., 3.80 cm 2), sterile 
- 
Timers 
- 
Pipette Aid or similar apparatus 
- 
Vortex mixer 
- 
Surgical Clipper & clipper blades: 3M Surgical Clipper 
- 
Culture Media Preparator 
- 
Peristaltic Pump 
- 
Incubator (30 ± 20C) 
- 
Disposable underwear for subjects 
- 
Sterile, Non-Occlusive dressing (to cover treated test sites for 6 hours post treatment): 
3M TegadermTM 
- 
urine pregnancy test (UPT) 
- 
Tryptic Soy Broth (for the Neutralization Validation Procedure only – See Appendix 17.7) 
(TSB) 
- 
Staphylococcus epidermidis MRSE, ATCC 51625 (for the Neutralization Validation Procedure 
only – See Appendix 17.7) 
- 
Staphylococcus epidermidis MSSE, ATCC 12228 (for the Neutralization Validation Procedure 
only – See Appendix 17.7) 
- 
Biological safety cabinet 
- 
Manual colony counter  
- 
pH-meter 
- 
70% IPA swabs (for the Neutralization Validation Procedure only – See Appendix 17.7) 
 
 
3.5 
Study Duration 
The expected duration of this study for each subject is up to 3 weeks. Subjects will undergo at least a 
14-day washout period followed by a qualification screening baseline visit. Subjects whose screening 
baseline samples meet the minimum values described in the Inclusion Criteria (Section 4.1) will be 
notified and invited to participate in the treatment phase of the study. The treatment phase will be 
scheduled no sooner than 72 hours from the screening baseline collection. Each subject who chooses 
to participate in this study will be required to stay in the test facility for one scheduled Treatment Day 
to include a 6 hours post-prep sampling. 
3.6 
Study Termination/Subject Discontinuation or Withdrawal 
 Study Termination 
PDI or the PI has the right to discontinue the study at any time for medical and/or administrative 
reasons. As much as possible, this should occur after mutual consultation. 
 
 


 
 
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 Subject Discontinuation and Withdrawal 
The PI may discontinue individual subjects from the study at any time. Subjects may voluntarily 
withdraw from the study at any time without reason or consequence. A reason will be reported, if 
provided. The PI will provide a written report on the appropriate CRF including the date and reason for 
discontinuance. Subjects who qualify on Screening Day and begin the treatment phase may not be re-
entered into the study, regardless of whether or not they completed the study.  
Additional subjects will be recruited and screened, as necessary, to meet the required number of 
evaluable treatment sites per body area, per treatment, per collection time. 
3.7 
Study Product Accountability 
PDI requires PI to maintain accountability and adequate inventory security of the study material at all 
times. The PI or designated sub-investigator trained by the PI will: 
- 
complete the Confirmation of Receipt form upon receipt of the shipment and maintain and 
account for inventory on the Study Material Disposition form. 
- 
keep study materials in a secure storage area, accessible only to authorized individuals. 
- 
dispense study material only to subjects properly enrolled into the study. 
- 
return or destroy all unused study materials to PDI at the end of technical execution of the 
study upon Sponsor agreement. 
Before starting the study, investigational products and positive control product will be supplied and 
shipped to the site by PDI and stored at controlled room temperature between 20-25 °C (68-77°F). 
Excursions permitted to 15-30°C (59-86°F) in the product storage area and a retain sample for each 
product kept in the sample storage area of the Research facility for at least 3 years after the end of 
the study, then destroyed, according to the corresponding procedure of the Research facility. 
 
3.8 
Source Data 
Source data includes any original documents, data and records where any data are first recorded (e.g., 
ICFs, laboratory notes, etc) used for study traceability. If data are recorded for the first time directly 
onto the CRF, then the CRF is considered the source document for these data. 
3.9 
Protocol Modifications 
 Protocol Amendments 
Protocol Amendments will not be implemented without prior agreement between the PI and PDI and 
prior submission and approval from the IRB/EC, except when necessary to eliminate an immediate 
hazard to the subject and to protect the safety and well-being of the subject. 
Will constitute Protocol Amendments for administrative inputs and changes: correction of typing 
mistakes, changes in study personnel (other than the PI) or contact information. Protocol Amendments 
for administrative inputs and changes has to be agreed between the PI and PDI and will be submitted 
to the IRB/EC but implementation may proceed without prior IRB/EC approval, unless so required by 
the IRB/EC or site SOPs.  
 
 


 
 
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 Protocol Deviations 
A deviation is a departure from the protocol that will likely affect the safety, rights or welfare of subjects, 
the scope of the investigation, the integrity of the data or the scientific quality of the study. Protocol 
deviations are documented on a Protocol Deviation Form. 
Deviations that potentially affect 1) subject safety, rights or welfare, 2) data integrity or 3) compromise 
the statistical analysis of the study require communication to PDI at the earliest convenience.  
A Protocol Deviation Form must be completed by the PI or designated sub-investigator trained by the 
PI and checked and approved by PI and include a description of the circumstances surrounding and 
the reason for the deviation, any actions taken, and whether or not the subject was allowed to continue 
in the study. 
If during monitoring visits a deviation is identified, the Study Monitor will report it to the PI and PDI  
4 
Study Population 
The trial will utilize ER’s subject database and would not require the recruitment of subjects through 
study-specific advertisement. 
Subjects must satisfy all Screening Day and Treatment Day Inclusion/Exclusion Criteria prior to 
screening sample collection and Treatment Day procedures. 
Screening Day Baseline microbial count requirements are at least 3.11 log10 CFU/cm2 bilaterally on the 
abdomen, and at least 5.00 log10 CFU/cm2 bilaterally on the groin except for subjects enrolled for 
Neutralization study. 
Treatment Day Baseline microbial count requirements are in the range of 3.11 to 5.50 log10 CFU/cm2, 
inclusive, on the abdomen, and 5.00 to 7.50 log10 CFU/cm2, inclusive, on the groin except for subjects 
enrolled for Neutralization study. 
Subjects must qualify on both body area (abdomen and/or groin) to be admitted into testing, except 
for Neutralization study. 
Both the right and left sides of the abdomen and groin must meet the minimum Screening Day values 
stated in the Inclusion Criteria to qualify, except for Neutralization study and except when one 
anatomical area (abdomen or groin) has completed the valid cases for all the products, then additional 
subjects will be screened only for the leftover area.  
Additional subjects will be recruited and screened, as necessary, to meet the required number of 
evaluable treatment sites per body area, per collection time.  
All subjects will be given verbal and written information about the study procedures - Subject 
Instructions for Main Study (Appendix 17.5.1) will be provided to each subject for the Washout phase 
of the study and Subject Instructions for Neutralization (Appendix 17.5.2) will be provided to each 
subject for the Neutralization Study. 
 
 


 
 
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4.1 
Subject Inclusion Criteria 
Subjects will be included in this study if they meet the following requirements: 
1. 
Male or female at least 18 years of age and of any race 
2. 
In good general health 
3. 
Read, understand and sign the ICF 
4. 
If female of child-bearing potential, are willing to use an acceptable method of contraception 
to prevent pregnancy (i.e. oral contraceptive, intra-uterine device, diaphragm, condom, abstinence, 
bilateral tubal ligation, or are in a monogamous relationship with a partner who has had a vasectomy) 
5. 
Female subjects of child-bearing potential, must have a negative Urine Pregnancy Test (UPT) 
on Treatment Day prior to any applications of the study products 
6. 
Screening Day Baseline microbial counts are at least 3.11 CFU/cm2 bilaterally from the skin of 
the abdomen, and 5.00 log10 CFU/cm2 bilaterally from the skin of the groin (not applicable for 
Neutralization study) 
4.2 
Subject Exclusion Criteria 
Potential subjects will be excluded from participation if the following criteria apply to them. 
1. 
Exposure of test sites to antimicrobial agents, medicated soaps, medicated shampoos, or 
medicated lotions, use of biocide-treated pools or hot tubs, use of tanning beds, or sunbathing during 
the 14-day washout conditioning period and during the test period (7-day for Neutralization study) 
2. 
Exposure of the test sites to strong detergents, solvents, or other irritants during the 14-day 
washout conditioning period and during the test period (7-day for Neutralization study) 
3. 
Wear fabric softener, bug repellent or UV-treated clothing during the 14-day washout 
conditioning period and during the test period (7-day for Neutralization study) 
4. 
Receive an irritation score of 1 (any redness, swelling, rash, or dryness present at any 
treatment area) for any individual skin condition prior to the Screening Day baseline or Treatment Day 
baseline sample collection or Neutralization study 
5. 
Use of systemic or topical antibiotic medications, steroid medications (other than for hormonal 
contraception or post-menopausal reasons), or any other product known to affect the normal microbial 
flora of the skin during the 14-day washout conditioning period and during the test period (7-day for 
Neutralization study) 
6. 
Known allergies or sensitivities to vinyl, latex (rubber), alcohols, metals, inks, or tape 
adhesives, or to common antibacterial agents found in soaps, lotions, or ointments, particularly 
isopropyl alcohol or chlorhexidine gluconate  
7. 
A medical diagnosis of a physical condition, such as a current or recent severe illness, asthma, 
diabetes, hepatitis, an organ transplant, mitral valve prolapses, congenital heart disease, internal 
prostheses, or any immunocompromised conditions such as AIDS (or HIV positive) 
8. 
Pregnancy, plans to become pregnant within the washout and test periods of the study, or 
nursing a child 
9. 
Any tattoos or scars within 2” (5.08 cm) of the test sites 
10. 
Dermatoses, cuts, lesions, active skin rashes, or breaks or other skin disorders within 6” on 
or around the test sites 
11. 
A currently active skin disease or inflammatory skin condition (for example, contact dermatitis) 
anywhere on the body that, in the opinion of the Consulting Physician or PI, would compromise subject 
safety or study integrity 
12. 
Showering, bathing, or swimming within the 72-hour period prior to sampling for baseline 
screening, Treatment Day, and throughout the test period (not applicable for Neutralization study) 
 
 


 
 
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13. 
Participation in another clinical study in the past 30 days or current participation in another 
clinical study at time of signing informed consent 
14. 
Any medical condition or use of any medications that, in the opinion of the PI, should preclude 
participation 
15. 
Unwillingness to fulfill the performance requirements of the study. 
4.3 
Subject Consent 
The PI or designated sub-investigator trained by the PI must ensure that written informed consent to 
participate in the investigation is obtained before including any individual as a subject in the 
investigation. The PI or designated sub-investigator trained by the PI must provide the prospective 
subject sufficient opportunity to consider whether or not to participate and minimize the possibility of 
coercion or undue influence.  
The process is designed to: 
1) give the subject all the information needed, 
2) ensure that the subject understands the information, 
3) give the subject a chance to consider study participation.  
The process should permit the subject to ask questions and exchange information freely. 
Specifically, the PI or designated sub-investigator trained by the PI is to explain to each subject all 
elements of ICF as specified in 21 CFR 50.25. After the explanation, subjects will voluntarily sign and 
date the ICF if they wish to participate in the study. A copy of the ICF must be provided to the subject. 
A signed and dated ICF must be maintained in the PI study documentation file at all times. 
 
5 
Study Treatment 
These study procedures are based on the American Society for Testing and Materials (ASTM) “Standard 
Test Method for Evaluation of Preoperative, Precatheterization, or Preinjection Skin Preparations” 
(E1173-15). 
5.1 
Study Procedures 
 Washout Period 
The Inclusion/Exclusion Criteria will be reviewed with each subject to ensure eligibility for the study 
after subjects has signed the ICF.  
A skin irritation assessment using the scale from Appendix 17.8 will be performed by the PI or 
designated sub-investigator trained by the PI. If an irritation score of 1 for any individual skin condition 
is observed, the subject will be excluded from the study. 
Prior to the scheduled Screening Day, subjects will undergo a minimum 14- day restriction period (7-
day for Neutralization study). The subjects will be instructed to avoid contact with any topical or 
systemic antimicrobial products or any other product know to affect the normal microbial flora of the 
skin for the duration of their involvement in the study as written in the Subject Instructions for Main 
Study (Appendix 17.5.1) or Subject Instructions for Neutralization (Appendix 17.5.2).  
If it becomes necessary to take systemic antibiotics or to apply topical medications to the test areas 
within this Washout period, the subject must contact the PI or designated sub-investigator whom will 
inform the PI as soon as reasonably possible so that another volunteer may be recruited. 
 
 


 
 
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Restrictions include, but are not limited to: 
- 
Use of antimicrobial soaps, shampoos, lotions, perfumes, after shaves, colognes, 
antiperspirants, deodorants 
- 
Contact with materials such as acids, bases, solvents 
- 
Swimming in chemically treated pools and bathing in hot tubs, spas and/or whirlpools 
- 
Use of tanning beds, hot waxes or depilatories (including shaving) 
Subjects will be provided a kit with non-antimicrobial personal care products for exclusive use during 
the study.  
If subjects require hair removal to facilitate sample collection, the subject will be asked to return to the 
test facility at approximately 48-96 hours before the Screening Day or Neutralization Day for clipping/re-
clipping. 
Prior to performing the clipping/re-clipping before the Screening Day or Neutralization Day, a skin 
irritation assessment using the scale from Appendix 17.8 will be performed by the PI or designated 
sub-investigator trained by the PI. If an irritation score of 1 for any individual skin condition is observed, 
the subject will be excluded from the study. 
Subjects will be required to refrain from bathing or showering for 72 hours prior to Screening Day. 
 
 Screening Day 
After the restriction period and at least 3-days before Treatment Day, the PI or a designated sub-
investigator will complete the Screening Day Inclusion/Exclusion Criteria page in each subject’s CRF. 
Prior to performing the Screening Day baseline sample collection, a skin irritation assessment using the 
scale from Appendix 17.8 will be performed by the PI or designated sub-investigator trained by the PI. 
If an irritation score of 1 for any individual skin condition at the Screening Day baseline is observed, 
the subject will be excluded from the study. 
A baseline screening sample will be collected from each test area using the Williamson-Kligman scrub 
cup technique. The baseline screening samples will be taken from the center of each contra-lateral 
test area within each body area (abdomen and groin). Samples from both body areas and from both 
left and right sides must meet the minimum value indicated in the Inclusion Criteria for the subject to 
be enrolled into the treatment phase of the study.  
Subjects who qualify for the study will be notified and will continue to follow the subject instructions 
for the duration of the study. 
Subjects will again be required to refrain from bathing or showering 72 hours prior to Treatment Day. 
If subjects require hair removal to facilitate sample collection, the subject will be asked to return to the 
test facility at approximately 48-96 hours prior to Treatment Day for clipping/re-clipping. 
Prior to performing the clipping/re-clipping before the Treatment Day, a skin irritation assessment using 
the scale from Appendix 17.8 will be performed by the PI or designated sub-investigator trained by the 
PI. If an irritation score of 1 for any individual skin condition is observed, the subject will be excluded 
from the study. 
 
 


 
 
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 Treatment Day 
The PI or a designated sub-investigator will complete the Treatment Day Inclusion/Exclusion Criteria 
CRF. If these criteria are satisfied, a skin irritation assessment using the scale from Appendix 17.8 will 
be performed prior to performing the Treatment Day baseline sample collection, by the PI or designated 
sub-investigator trained by the PI. If an irritation score of 1 for any individual skin condition at the 
Treatment Day baseline is observed, the subject will be excluded from the study. 
5.1.3.1 Preparation of Test Areas on Treatment Day 
A Test Site Diagram for the abdominal and groin test areas is shown in Appendix 17.3. 
5.1.3.1.1 
Preparation of Abdomen Test Area 
The test site within the abdominal region (abdominal test area) is defined as the area to the right and 
left sides of the abdomen, adjacent to the umbilicus that appear to be similar in condition and above 
the groin. The corners of each abdominal test area will be marked directly on the skin using a non-
toxic skin marker. Four sampling sites will be numbered within each abdominal test area, on each side 
of the abdominal region. The positioning and numbering of the abdominal sampling sites are standard 
for all subjects. Sampling sites on the contra-lateral side of the abdomen will be numbered in a mirror-
image orientation.  
 
The four sampling sites within each abdominal test area represent one baseline (pre-prep) site and 
three post-prep sample sites (30 seconds, 10 minutes and 6 hours). 
Test sites for study products and active control application on the abdomen will be 5’’ x 5’’ (12.7 cm x 
12.7 cm) in area, occur on areas of skin to the right and left side of the navel, and should appear to 
be similar in condition. 
After abdominal test areas are marked and sample sites numbered, baseline samples will be collected 
from the appropriate site per the randomization schedule in each test area using the scrub cup 
technique (section 5.2.1). 
 
5.1.3.1.2 
Preparation of Groin Test Area 
The test site within the groin region (groin test area) is defined as the inner aspect of the uppermost 
thigh within and parallel to the inguinal crease. The corners of each groin test area will be marked 
directly on the skin using a non-toxic skin marker. Four sampling sites will be numbered within each 
groin test area, on each side of the groin region. The positioning and numbering of the groin sampling 
sites are standard for all subjects. Sampling sites on the contra-lateral side of the groin will be 
numbered in a mirror-image orientation. The four sampling sites within each abdominal test area 
represent one baseline (pre-prep) site and three post-prep sample sites (30 seconds, 10 minutes and 
6 hours). 
Test sites for the investigational product and active control applicator on the groin will be 1.5’’ x 5’’ 
(3.81 cm x 12.7 cm) in area, occur at the uppermost inner aspects of both thighs, and appear to be 
similar in condition. 
After groin test areas are marked and sample sites numbered, baseline samples will be collected from 
the appropriate site per the randomization schedule in each test area using the scrub cup technique 
(section 5.2.1). 
 
 


 
 
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5.1.3.2 Study Products Application 
The randomization schedule will designate the treatment to each side of the abdomen and groin. 
Following baseline sample collection, randomly assigned contra-lateral test areas will be treated with 
the applicable study products. The post-application sampling times will be randomized among the 
sampling sites within a test area. 
The study products will be applied and the sampling configurations will be performed per the 
randomization scheme and the Treatment Application Instructions (Appendix 17.6). The duration of 
each application procedure will be recorded on the appropriate CRF. The study products, body sites, 
application and dry times and treatment area are summarized in Table 2Table 2. 
 
Table 2: Study products, body sites, application and dry times and treatment area 
Products 
Body Site 
Application Time 
Drying Time 
Treatment  
Area 
A – 26 ml Project X 
Abdomen 
30 seconds 
min. 180 seconds 
5.0” x 5.0”  
(12.7 cm x 12.7 cm) 
Groin 
120 seconds 
min. 180 seconds 
1.5” x 5.0” 
(3.81 cm x 12.7 cm) 
B – 5.1 ml Project X 
Abdomen 
30 seconds 
30 seconds 
5.0” x 5.0” 
(12.7 cm x 12.7 cm) 
Groin 
120 seconds 
90 seconds 
1.5” x 5.0” 
(3.81 cm x 12.7 cm) 
C – 5.1ml Prevantics® Maxi 
Swabstick 
Abdomen 
30 seconds 
30 seconds 
5.0” x 5.0” 
(12.7 cm x 12.7 cm) 
Groin 
120 seconds 
90 seconds 
1.5” x 5.0” 
(3.81 cm x 12.7 cm) 
 
5.1.3.3 Timing of Post Application Sample Collection 
Microbial samples will be collected at 30 seconds (+5 seconds), 10 minutes (± 30 seconds), and 6 
hours (± 30 minutes) post treatment application for both body areas, the abdomen and the groin. Post 
application timing begins upon completion of the study product application, including drying time. 
Microbial samples will be collected using the scrub cup technique (section 5.2.1). 
After the 30 second and 10 minute samples have been collected, a piece of sterile gauze and a non-
occlusive dressing will be secured over the remaining sample site to allow subjects unrestricted mobility 
and to protect the sites from contamination between sampling times. The subjects will stay in the 
clinical test facility, for the final post-application sample collection. 
 
 


 
 
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A skin irritation assessment using the scale from Appendix 17.8 will be performed by the PI or 
designated sub-investigator trained by the PI prior to collection of each post treatment microbial sample 
collection (30 seconds, 10 minutes and 6 hours) and a corresponding rating score for each individual 
skin condition will be recorded in the subject’s CRF.  
If an irritation score of 3 for any individual skin condition at any post treatment observation is assigned, 
the subject will be discontinued from the study and an adverse event will be recorded. See Section 8.3 
(Adverse Events). 
Following final sample collection, the remaining study material will be removed from the subjects’ skin 
with water and soap as necessary and dried with paper towels. 
 
5.2 
Microbiological Methods 
 Microbial Sample Collection / Scrub Cup Technique 
Quantitative cultures (screening baselines, treatment day baselines and post treatment samples) will 
be obtained by a modification of the cylinder sampling technique of Williamson-Kligman scrub cup 
technique following ASTM E1874-14 “Standard Test Method for Recovery of Microorganisms from Skin 
using the Cup Scrub Technique”. To collect the samples, a sterile scrub cup (2.20 cm I.D.) will be 
placed on the site and held firmly to the skin. 3.0 ml of SSF++ will be pipetted into the cup and the 
skin will be scrubbed in a circular motion with moderate pressure for 1 minute using a sterile rubber 
policeman. Using a sterile transfer pipette, the SSF++ will be removed and placed in a sterile test tube. 
An additional 3.0 ml of fresh SSF++ will be pipetted into the cup and the scrub procedure will be 
repeated. This solution will be pooled with the first solution collected. 
 Bacterial Enumeration Methods 
Following sample collection, vortex the sample for 15 seconds and 10-fold serial dilutions will be 
prepared using PBW. One ml aliquots of appropriate dilutions will be pour-plated in TSA+N. Samples 
must be plated within 30 minutes of collection.  
Serial dilutions of collected baseline screening samples as follows: 
- 
for groin sites: 1 serial dilution (10-3) 
- 
for abdomen sites: 1 serial dilution (10-1) 
Serial dilutions of collected treatment day (D0) samples as follows: 
for groin sites: 
- 
Baseline: 3 serial dilutions (10-3, 10-4 and 10-5) 
- 
30 seconds: 4 serial dilutions (100, 10-1, 10-2 and 10-3) 
- 
10 minutes: 4 serial dilutions (100, 10-1, 10-2 and 10-3) 
- 
6 hours: 4 serial dilutions (100, 10-1, 10-2 and 10-3) 
 
for abdomen sites: 
- 
Baseline: 3 serial dilutions (10-1, 10-2 and 10-3) 
- 
30 seconds: 3 serial dilutions (100, 10-1 and 10-2) 
- 
10 minutes: 3 serial dilutions (100, 10-1 and 10-2) 
- 
6 hours: 3 serial dilutions (100, 10-1 and 10-2) 
 
 


 
 
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After 72 ± 4 hours of aerobic incubation at 30 ± 2˚C, colonies will be manually counted and viable 
cells in the original sample will be calculated according to SOPs. After incubation, plates may be 
refrigerated up to 48 hours prior to counting. 
 
Bacterial colonies enumerated manually within the countable range 25-250 from each dilution will be 
recorded on the appropriate CRFs for each subject. The average number of microorganisms recovered 
(CFU/cm2) from the skin during screening and treatment day will be calculated using the formula to 
convert the volume of sample collected into log10CFU/cm2 of skin: 

∑






 
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  
∑



 

 = average of the triplicate colony counts used for each sample collected 
D = dilution factor of the plate counts 
A = inside area of the cylinder in cm2; in this study, A = 3.80 cm2 for the 2.2-cm diameter cylinder. 
 Neutralization Validation 
A Neutralization study will be conducted to demonstrate that the neutralizers used in the sampling 
solution and media are effective in inhibiting the antimicrobial properties of the test products without 
impairing the recovery of challenge microorganisms. Staphylococcus epidermidis, ATCC 12228 (MSSE), 
Staphylococcus epidermidis, ATCC 51625 (MRSE) will be used as challenge microorganisms.  
Neutralization procedures will follow guidelines provided by ASTM E1054-08 (reapproved 2013), 
“Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents”. The full procedure is 
attached as Appendix 17.7. 
6 
Assessment of Efficacy 
6.1 
Efficacy Parameters 
The measure of antimicrobial efficacy will be the log10 CFU/cm2 of skin flora at the abdominal and groin 
sites 30 seconds, 10 minutes and 6 hours following study products application relative to the Treatment 
Day baseline log10 counts. 
6.2 
Assessment Methods 
Efficacy will be assessed by sampling the skin using the cup scrub method and analysis methods 
described in Section 5.2. 
 
 


 
 
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7
Risk / Benefit Assessment 
7.1Potential Risks 
Subjects participating in this study could experience side effects to the skin such as: 
•
Redness 
•
Swelling 
•
Burning, stinging and itching 
•
Cracking 
•
Peeling 
•
Small blisters or sores (in uncommon cases) 
•
Rash or other allergic reaction including in rare cases anaphylaxis 
•
Irritation 
•
Pain 
•
Localized rash or inflammation (dermatitis) 
•
Impetiginized eczema (infected eczema or infected dry skin and recurring skin rashes) 
•
Application site hypopigmentation (lightening of skin) or hyperpigmentation (darkening of 
skin). 
Subjects may also experience folliculitis from clipping. 
There may be risks from participating in this study that are unknown. 
 
7.2Potential Benefits 
There are no direct benefits to the subject for participation in this study. 
8
Assessment of Safety 
8.1
Safety Parameters 
The principal measures of safety will be skin irritation scores and the incidence of adverse events 
reported during the study. 
Adverse events will be captured from the time screening baseline samples are taken to the time of 
subject discharge from the study. Adverse events will be categorized in relationship to the product that 
was applied to the specific skin site. All local and systemic adverse events observed or reported to the 
investigator will be evaluated.  
The severity, duration, causal relationship to the study product and study procedure, action taken 
(study related and subject related), study impact, outcome of the event; will be described for all adverse 
events. 
 
 


 
 
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8.2 
Assessment Methods for Skin Irritation 
All randomized subjects will be considered evaluable for safety. Skin irritation scores based on the scale 
from Appendix 17.8 will be reported for any subject who is scored with a 1 or more at any observation 
[baseline (screening day and treatment day), clipping/re-clipping (prior to screening day, treatment 
day or neutralization day), prior/post-application to 30 seconds, 10 minutes, and 6 hours sampling 
procedures], in any category for any site. 
Adverse events (including post treatment skin irritation scores of 3), will also be summarized.  Summary 
tables will present incidence rates of adverse events by treatment group for all subjects who enter the 
treatment period. Listings of adverse events will be provided. 
A corresponding rating score for each individual skin condition, for each site will be recorded in the 
subject’s CRF. (See Appendix 17.8, which includes the following four independent evaluation 
categories: Erythema, Edema, Rash, and Dryness). 
If an irritation score of 1 or greater for any individual skin condition prior to the baseline sample 
collection (at either the screening or treatment day phases) is assigned, the subject will be excluded 
from the study (no study treatment will be applied). 
If an irritation score of 3 for any individual skin condition at any observation period is assigned, the 
subject will be discontinued from the study and an adverse event will be recorded. See Section 8.3 
(Adverse Events). 
8.3 
Adverse Events 
The PI is responsible for identifying adverse events that occur to each subject throughout the study 
and follow-up period. An adverse event can occur at any time during the conduct of the study, in any 
phase of the study or after the study is completed. An adverse event can be identified by the PI or 
reported by the subject. 
 
Definitions: 
Adverse Event/Experience 
An Adverse Event/Experience is any unexpected or undesirable experience occurring to a subject during 
a study, which may or may not be related to the test product.  
All adverse events, regardless of severity or the causal/effect relationship, will be recorded. The severity 
of the effect will be noted as Mild, Moderate, or Severe according the following definitions: 
Mild 
 
Awareness of signs or symptom, but easily tolerated. 
Moderate 
Discomfort to a degree as to cause interference with normal daily life activities and 
/or requiring medication. 
Severe 
Incapacity with inability to work or do usual daily life activities and requiring medical 
attention/intervention. 
Causal Relations of Adverse Event/Experience 
When determining the causal/effect relationship to the test product, the relationship will be described 
as Not related, Unlikely related, Possibly related or Related. 
 
 


 
 
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The following definitions will be utilized: 
Not related 
No association to the test product. Related to other etiologies such as 
concomitant medications or conditions or subject’s known clinical state. 
Unlikely related  
Uncertain association. Other etiologies are also possible. 
Possibly related 
Clear-cut association with improvement upon withdrawal of the test 
product. Not reasonably explained by the subject’s known clinical state. 
Related 
An adverse event with a clear-cut temporal association with exposure to 
study materials and cannot reasonably be explained by the subject’s known 
clinical state. Association with study material is confirmed by laboratory 
testing if possible. 
Serious Adverse Event/Experience 
A Serious Adverse Event/Experience is any adverse experience occurring at any dose that results in 
any of the following outcomes: 
- 
Death; 
- 
A life-threatening adverse drug experience; 
- 
Inpatient hospitalization or prolongation of existing hospitalization; 
- 
A persistent or significant disability/incapacity; 
- 
Congenital anomaly/birth defect; 
- 
An important medical event that may require medical or surgical intervention to prevent one 
of the previously listed outcomes. 
Unexpected Adverse Event/Experience 
An Unexpected Adverse Event/Experience is any adverse drug event/experience not listed in the current 
labeling for the test product or the current IB. Where test product labeling or IB is not available, 
anticipated experiences will be based on the known pharmacological/toxicological properties of the test 
product or ingredients. 
Follow-up: 
If an adverse event/experience occurs, the subject under the direction of the PI or designated sub-
investigator trained by the PI may be referred to the nearest acute care facility for treatment. Serious 
or Unexpected Drug Event/Experience will be followed to resolution. Any adverse event will be 
documented on an Adverse Event Report Form. 
Recording and Reporting 
The PI or designee records all adverse events on an Adverse Event Form in the subject’s CRF.  
The PI must promptly report all treatment related adverse events to the PDI study monitor.  
All SAE must be reported to the: 
- 
Sponsor within 24 hours of the PI awareness/notification of the event. 
- 
IRB/IEC within 10 working days of the PI awareness/notification of the event. 
If a subject has no adverse event during the study, the absence of such must be recorded on the CRF. 
 
 


 
 
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9 
Statistics 
9.1 
Data sets analyzed 
Subjects with treatment day baseline bacterial count in the range of 3.11 to 5.50 log10/cm2, inclusive, 
on the abdomen and 5.00 to 7.50 log10/cm2, inclusive, on the groin will be included in the analysis. 
9.2 
Sample Size 
This evaluation will be performed with an estimated of a minimum of 75 evaluable subjects 
corresponding to 50 evaluable sites per product with qualifying treatment day microbial baseline counts 
per body area (abdomen and groin). No sample size calculation was performed to provide statistically-
powered evidence of efficacy as part of this study. 
9.3 
Efficacy Analyses 
An alpha level of 5% will be used for all analyses. 
The following descriptive statistics for log10 CFU/cm2 and for log10 CFU/cm2 reductions from Baseline 
will be computed for each treatment, grouped by body area and each post application sampling time 
point (30 seconds, 10 minutes and 6 hours): mean, median, standard deviation, minimum, maximum, 
and count. 
 Efficacy Analysis 
A linear regression model for each body area (abdomen and groin) and each post treatment sampling 
time point of immediate efficacy (30 seconds and 10 minutes post-application) will be used. In the 
model, the dependent variable used will be the post treatment bacterial count (log10 CFU/cm2) and 
predictors will be the treatment as a fixed effect and the Baseline as a covariate. The ATE corrected 
for Baseline will be estimated from the model and compared to non-inferiority criteria. 
To evaluate the persistent efficacy (6 hours post-application) of the product A and B, microbial counts 
(log10 CFU/cm2) at 6 hours post-application for each product application site will be converted to a 
binary (success/failure), “success” defined as having microbial counts less than or equal to the baseline 
counts. Responder rates at 6 hours post product application will be summarized descriptively for each 
product on each body area. 
9.4 
Safety Analysis 
All treated subjects will be considered evaluable for safety. Skin irritation scores will be reported for 
any subject who is scored with a 1 or more at any observation (baseline screening day, baseline 
treatment day, post-application/prior to 30 seconds, 10 minutes, and 6 hours sampling procedures), in 
any category for any site. 
Adverse events (including post treatment skin irritation scores of 3), will also be summarized. Summary 
tables will present incidence rates of adverse events by treatment group for all subjects who enter the 
treatment period. Listings of adverse events will be provided. 
 
 


 
 
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9.5 
Procedures for Accounting for Missing, Unused, and Spurious Data 
Missing microbiological data at 30 seconds, 10 minutes, or 6 hours, such as due to laboratory error or 
subject lost to follow-up, will be reported as missing and will not be imputed. Inclusion of these subjects 
in the per-protocol data set will be based on the criteria defined above. Details of any missing data and 
rationale for inclusion/exclusion in the per-protocol data set will be described in the study report. 
10 
PI Responsibilities 
The PI is responsible for ensuring that this clinical investigation is conducted according to this protocol; 
protecting the rights, safety, and welfare of subjects; and controlling the study products under 
investigation. The PI has overall responsibility for the technical conduct of the study, as well as for the 
interpretation, analysis, documentation and reporting of results, and represents the single point of 
study control. 
11 
Monitoring 
PDI, as sponsor of this study along with the PI, is responsible for ensuring the proper conduct of the 
study with regard to protocol adherence and validity of the data recorded on the CRFs. PDI has, 
therefore, assigned a study monitor to this study. The progress of the study will be monitored by: 
- 
Periodic on-site review 
- 
Telephone communications and e-mail 
- 
Review of CRFs and source documents 
The PI will give the PDI study monitor direct access to source documents that support data on the 
CRFs and make available such records to authorized PDI personnel, quality assurance, IRB/IEC, and 
regulatory personnel for inspection and/or copying. 
 
12 
Quality Control and Quality Assurance 
PDI, and the PI are responsible for implementing and maintaining Quality Assurance and Quality Control 
systems through written SOPs to ensure that this study is conducted and data are generated, 
documented and reported in compliance with the protocol, GCP and regulations cited in Section 14 of 
this protocol. Study monitoring may be carried out to accomplish this. 
13 
Audits and Inspections 
If the study is selected for audit by the Sponsor or if there is an inspection by the appropriate Health 
Authorities, then the PI and her team will make themselves available during the visit.  The PI must 
agree to the inspection of all study related records and give the auditor/inspector direct access to 
source documents for verification of data on CRFs.  The subject’s anonymity must be safeguarded and 
data checked during the audit remain confidential. 
As soon as the PI is aware of an upcoming inspection/audit by the Health Authorities, she will promptly 
inform PDI.  As agreed with the PI, PDI personnel might be present at the site during the inspection. 
 
 


 
 
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14 
Ethical and Regulatory Standards 
The study will be conducted in compliance with this protocol, the regulatory guidelines of the US FDA 
regulations, 21 CFR 50 (Protection of Human Subjects), 56 (IRB), the ethical principles of the 
Declaration of Helsinki, the ICH-GCP Guidelines as currently amended, and all applicable SOPs of ER 
The study will start only after approval of the protocol and ICF including Subjects instruction and 
Subjects calendar (Main study and Neutralization study) by the IRB/IEC. The approval letter or notice 
must contain the IRB name and identification number, meeting date, and sufficient information to 
identify the protocol and ICF form by name and number that were reviewed. PDI prior to study 
initiation, must receive a copy of the IRB/IEC approval letter. 
 
The IRB used for this study will be: 
Name: 
Advarra, Inc 
Address: 6940 Columbia Gateway Drive, Suite 110, Columbia, MD 21046, USA 
The IEC used for this study will be: 
Name: 
Institutional ethics committee 
Address: 64-66, Marasesti Boulevard, 040256 - Bucharest, Romania 
Subject confidentiality is strictly held in trust by the PI, the study staff, and PDI This confidentiality is 
extended to cover testing of biological samples in addition to the clinical information relating to 
participating subjects.  Subject confidentiality and anonymity will be maintained at all times by removal 
of all identifiers from any data, clinical samples or documentation submitted for this study. 
Any data collected meeting the definition of protected health information will be collected and 
maintained using the designated authorizations and following all privacy procedures as specified in the 
applicable health authority regulations. 
PDI will maintain the security and confidentiality of all clinical study data received. 
PDI clinical study databases will not be shared with any third party without the express written consent 
of the PI and/or Research facility. 
15 
Data Handling and Record Keeping 
15.1 
Completion of Case Report Forms 
The PI or sub-investigator will review all CRF entries for completeness and accuracy. If a correction is 
required, a single line must be drawn through the error. The person making the correction will initial, 
date, and provide a reason for the error (if not self-evident). 
The PI or sub-investigator must review and sign each CRF in a timely fashion following completion and 
make them available to the PDI study monitor for monitoring. Before acceptance, the monitor will 
review the CRFs to ensure accuracy and completeness. The original CRFs will be kept at the Research 
facility and electronic/scanned copies will be sent to PDI In addition, any data queries prepared after 
the original CRFs have been completed must be answered promptly. 
 
 


 
 
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15.2 
Final Clinical Study Report 
Final Clinical Study report will be produced by Research facility in collaboration with PDI team. 
15.3 
Records, Reports and Retention Requirements 
The PI will maintain study records for a minimum of 10 years following completion of the study. Sponsor 
will be notified in written regarding the due date of the record retention time.  
Records that must be maintained by the PI include, but are not restricted to: 
- 
Signed study protocol, amendments, deviations 
- 
IRB/IEC approval of protocol, ICF including Subjects instruction and Subjects calendar (Main 
study and Neutralization study) 
- 
Applications to the IRB/IEC 
- 
Signed ICFs 
- 
CRFs 
- 
Adverse event reports 
- 
Records of receipt, use or disposition of the study material 
- 
Correspondence relating to the study 
- 
Investigator Final Report 
- 
Sponsor Final Report (if provided) 
No formal presentation or publication of data from this study can be initiated without the sponsor’s 
explicit written agreement and in direct collaboration with the sponsor. 
 
16 
References 
1. ASTM E 1054-08, Standard Test Methods for Evaluation of Inactivators of Antimicrobial 
Agents, ASTM International, West Conshohocken, PA. 2013,  www.astm.org 
2. ASTM E 1874-14, Standard Test Method for Recovery of Microorganisms from Skin using 
the 
Cup 
Scrub 
Technique, 
ASTM 
International, 
West 
Conshohocken, 
PA. 
2014,  www.astm.org 
3. ASTM E1173-15, Standard Test Method for Evaluation of Preoperative, Precatheterization, 
or Preinjection Skin Preparations, ASTM International, West Conshohocken, PA, 
2015,  www.astm.org 
4. Butterfield, C.T. The Selection of a Dilution Water for Bacteriological Examinations. J. 
Bacteriol. 23: 355-368, 1931. 
5. Food and Drug Administration (FDA). Safety and Effectiveness of Health Care Antiseptics; 
Topical Antimicrobial Drug Products for Over-the-Counter Human Use; Final Rule. December 
2017. Accessed at https://www.gpo.gov/fdsys/pkg/FR-2017-12-20/pdf/2017-27317.pdf 
6. Maki DG, Ringer M, Alvarado CJ. Prospective randomized trial of Povidone-Iodine, alcohol, 
and Chlorhexidine for prevention of infection associated with central venous and arterial 
catheters. The Lancet 1991; Vol. 338: 339-343. 
7. Mimoz O, Karim A, et al. Chlorhexidine compared with Povidone-Iodine as skin preparation 
before blood culture. Annals of Internal Medicine 1999; Vol.131; No. 11; 834-837. 
8. Williamson, P., Kligman, A.M. A New Method for the Quantitative Investigation of 
Cutaneous Bacteria. J. Invest. Dermatol. 45:498-503, 1965. 
 
 


 
 
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17 
Appendices 
17.1 
Key Study Personnel, Titles, Responsibilities 
 
PROFESSIONAL DISPOSABLES INTERNATIONAL INC.: 
James Clayton 
Sponsor Representative 
EUROFINS EVIC PRODUCT TESTING ROMANIA S.R.L.: 
Rozalia Olsavszky MD 
Dermatologist 
Principal Investigator 
EUROFINS EVIC PRODUCT TESTING ROMANIA S.R.L.: 
Elena Chitoiu MD 
Resident Dermatologist 
Sub-Investigator 
EUROFINS EVIC PRODUCT TESTING ROMANIA S.R.L.: 
Getuta Dopcea 
Microbiologist 
Microbiology Laboratory Manager 
 
 


 
 
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17.2 
Study Summary  
  Main study 
Pre-Study 
Preparation 
Screening Phase 
Treatment Phase 
At least 14 Day 
Restriction 
Period 
Screening Day 
Abdominal Region 
Groin Region 
Staff reviews 
study protocol 
Initiation of 
consenting process
Complete Screening 
Inclusion/ Exclusion 
Criteria form 
Complete Treatment Inclusion/ Exclusion 
Criteria form 
Prepare ICF, 
Subjects 
instruction and 
Subjects 
calendar (Main 
study and 
Neutralization) 
Review study ICF 
and Inclusion/ 
Exclusion Criteria 
Visual skin 
assessment 
(abdominal and 
groin regions) 
Visual skin assessment 
(abdominal and groin regions) 
Obtain IRB/ IEC 
approval 
Review subject 
instructions 
Collect screening 
baseline samples 
from abdominal and 
groin regions 
Mark test areas, Collect baseline samples 
Recruit subjects 
Subject signs ICF
Count screening 
plates, determine 
which subjects qualify 
for study 
Apply test products 
Prepare subject kits
Visual skin 
assessment 
(abdominal and 
groin regions) and 
Inclusion/ 
Exclusion Criteria 
Contact and enroll 
eligible subjects, 
schedule Treatment 
Day 
Visual skin 
assessment, within 
30 seconds post-prep 
sample 
Visual skin 
assessment, within 30
seconds post-prep 
sample 
Prepare CRFs and 
appendices to CRFs
(Main study and 
Neutralization) 
Dispense subject 
kits 
Schedule for clipping/
re-clipping, if needed, 
approximately 48-96 
hours prior to 
Treatment Day visit 
Visual skin 
assessment, 10 
minutes post-prep 
sample 
Visual skin 
assessment, 10 
minutes post-prep 
sample 
 
Schedule for 
clipping/re-
clipping, if needed,
approximately 48-
96 hours prior to 
No bathing / 
showering 72 hrs. 
prior to Treatment 
Day visit 
Visual skin 
assessment, 6 hours
post-prep sample 
Visual skin 
assessment, 6 hours 
post-prep sample 
No bathing / 
showering 72 hrs. 
prior to screening 
visit 
 
Count Treatment Day 
Baseline plates, 
determine 
qualification  
Count Treatment Day 
Baseline plates, 
determine qualification 
 


 
 
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 Neutralization study 
Pre-Study Preparation 
Neutralization day 
At least 7 Day Restriction 
Period 
Neutralization day 
 Abdominal Region 
Recruit subjects 
Initiation of consenting process 
Complete Treatment Inclusion/ 
Exclusion Criteria form 
Prepare subject kits 
Review study ICF and Inclusion/ 
Exclusion Criteria 
Visual skin assessment 
(abdominal region) 
 
Review subject instructions 
Mark test areas 
Subject signs ICF 
Apply test products 
Visual skin assessment (abdominal
and groin regions) and Inclusion/ 
Exclusion Criteria 
Visual skin assessment, within 30 
seconds post-prep sample 
Dispense subject kits 
Count Neutralization Day plates 
Schedule for clipping/re-clipping, if 
needed, approximately 48-96 
hours prior to neutralization day 
 
 
 
 


 
 
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17.3 
Abdomen and Groin Diagram 
 
 
 
 


 
 
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17.4 
Randomization Scheme Example 
 
The randomization will be provided in a separate document. 
 
Randomization 
number 
Body Area 
Product 
Left Side 
Product 
Right Side 
Site 1 
Site 2 
Site 3 
Site 4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
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17.5 
Subject Instructions  
 Subject Instructions for Main Study 
The following instructions are to be followed until the completion of the study 
o 
Use only the soap provided for all bathing, sponge bathing and hand washing. 
o 
Use only the shampoo provided when washing your hair. 
o 
Do not use antiperspirants or deodorants (other than those provided to you in the kit), lotions, 
colognes, perfumes, after shaves or powders. 
o 
Do not come in contact with solvents, acids, bases, fabric softener-treated clothing or other household 
chemicals in the abdominal and upper thigh body areas. 
o 
Do not swim in chemically treated pools or bathe in hot tubs, whirlpools or spas. 
o 
Do not use tanning beds. 
o 
Do not shave, use depilatories or hot waxes on the abdomen or upper thigh areas.  If hair is present, 
allow study staff to clip hair at a designated time. 
o 
Do not apply any medicated creams or ointments to any area of your skin, nor should you take any 
antibiotics. If antibiotics are necessary due to illness, please report this to Chitoiu Elena, Dermatologist 
at Phone: +40213357090 (work), or Mobile: +40 721322073 (home) immediately. 
 
Additional Instructions for Screening Day Visit dd/mm/yyyy _ _ / _ _ / _ _ _ _ 
o 
Do not bathe or shower in the 72-hour period before your scheduled appointment. A sponge bath 
may be taken, but avoid the areas of the lower abdomen, and/or upper thigh. 
o 
You may be required to return to the Research facility approximately 48-96 hours before your 
Screening day Visit for hair clipping. 
o 
On the day of the Screening Day Visit, you will be required to return to the Research facility for the 
screening baseline sampling procedures. 
o 
You will be contacted by telephone as to whether you have met the established criteria and therefore 
will be returning for a Treatment Day Visit (if selected). Remember to continue to use your kit products 
until you have been eliminated or have completed the study. 
 
Additional Instructions for Treatment Day Visit (if you are selected)dd/mm/yyyy_ _ / _ _ / _ _  
o 
Do not bathe or shower in the 72-hour period before your scheduled appointment and for the rest of 
the study. A sponge bath may be taken, but avoid the areas of the lower abdomen, and/or upper 
thigh. 
o 
You may be required to return to the Research facility at least approximately 48-96 hours before your 
Treatment Day Visit for hair clipping. 
o 
On the Treatment Day Visit, you will return to the Research facility for treatment and the initial 
sampling. 
o 
You will be required to stay in the test facility for the 6-hours ± 30 minutes post-prep skin sampling 
(as scheduled). 
 
If you have questions about this study or in case of emergency, contact any time 
during business hours or/and after business hours: 
 
CHITOIU ELENA, Resident Dermatologist 
Phone: +40213357090 (work) 
Mobile: +40729066737 (home) 
 
 


 
 
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 Subject Instructions for Neutralization Study 
The following instructions are to be followed until the completion of the study 
o 
Use only the soap provided for all bathing, sponge bathing and hand washing. 
o 
Use only the shampoo provided when washing your hair. 
o 
Do not use antiperspirants or deodorants (other than those provided to you in the kit), lotions, 
colognes, perfumes, after shaves or powders. 
o 
Do not come in contact with solvents, acids, bases, fabric softener-treated clothing or other household 
chemicals in the abdominal and upper thigh body areas. 
o 
Do not swim in chemically treated pools or bathe in hot tubs, whirlpools or spas. 
o 
Do not use tanning beds. 
o 
Do not shave, use depilatories or hot waxes on the abdomen or upper thigh areas. If hair is present, 
allow study staff to clip hair at a designated time. 
o 
Do not apply any medicated creams or ointments to any area of your skin, nor should you take any 
antibiotics. If antibiotics are necessary due to illness, please report this to Chitoiu Elena, Dermatologist 
at Phone: +40213357090 (work), or Mobile: +40 721322073 (home) immediately. 
 
Additional Instructions for Neutralization Study Visit (if you are selected)_ _ / _ _ / _ _ _  
o 
You may be required to return to the Research facility at least approximately 48-96 hours before your 
Treatment Visit for hair clipping. 
o 
On the Treatment Day Visit, you will return to the Research facility for treatment and sampling. 
 
 
If you have questions about this study or in case of emergency, contact any time 
during business hours or/and after business hours: 
 
CHITOIU ELENA, Resident Dermatologist 
 
Phone: +40213357090 (work) 
Mobile: +40729066737 (home) 
 
 


 
 
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17.6 
Treatment Application Instructions 
Investigational Product: Treatment A (Project X 26 ml) 
Treatment Site Application Instructions – Abdomen 
1. Peel open package and remove swabstick – do not touch swab. 
2. Hold the swabstick with swab down. 
3. Press down the antiseptic reservoir to release the antiseptic to the swab. 
4. Wet the swab by repeated blotting against the treatment area until antiseptic is visible on the 
skin. 
5. Thoroughly wet the treatment area with antiseptic using gentle back-and-forth strokes. 
6. Repeat steps 4 & 5 for 30 seconds  
7. Do not apply swab beyond borders of marked skin areas by more than one swab width.  
8. Allow antiseptic to completely dry (minimum of 3 minutes).  
9. Do not blot or wipe dry 
Treatment Site Application Instructions – Groin  
1. Peel open package and remove swabstick – do not touch swab. 
2. Hold the swabstick with swab down. 
3. Press down the antiseptic reservoir to release the antiseptic to the swab. 
4. Wet the swab by repeated blotting against the treatment area until antiseptic is visible on the 
skin. 
5. Thoroughly wet the treatment area with antiseptic using gentle back-and-forth strokes. 
6. Repeat steps 4 & 5 for 2 minutes  
7. Do not apply swab beyond borders of marked skin areas by more than one swab width.  
8. Allow antiseptic to completely dry (minimum of 3 minutes).  
9. Do not blot or wipe dry. 
Investigational Product: Treatment B (Project X 5.1 ml) 
Treatment Site Application Instructions – Abdomen 
1. Peel open package and remove swabstick – do not touch swab. 
2. Hold the swabstick with swab down. 
3. Press down the antiseptic reservoir to release the antiseptic to the swab. 
4. Wet the swab by repeated blotting against the treatment area until antiseptic is visible on the 
skin. 
5. Thoroughly wet the treatment area with antiseptic using gentle back-and-forth strokes. 
6. Repeat steps 4 & 5 for 30 seconds  
7. Do not apply swab beyond borders of marked skin areas by more than one swab width.  
8. Allow antiseptic to completely dry (30 seconds).  


 
 
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9. Do not blot or wipe dry. 
Treatment Site Application Instructions – Groin  
1. Peel open package and remove swabstick – do not touch swab. 
2. Hold the swabstick with swab down. 
3. Press down the antiseptic reservoir to release the antiseptic to the swab. 
4. Wet the swab by repeated blotting against the treatment area until antiseptic is visible on the 
skin. 
5. Thoroughly wet the treatment area with antiseptic using gentle back-and-forth strokes. 
6. Repeat steps 4 & 5 for 2 minutes  
7. Do not apply swab beyond borders of marked skin areas by more than one swab width.  
8. Allow antiseptic to completely dry (90 seconds).  
9. Do not blot or wipe dry. 
Positive Control: Treatment C (Prevantics® Maxi Swabstick) 
Treatment Site Application Instructions – Abdomen 
1. Peel open package and remove swabstick – do not touch swab. 
2. Place one flat side of the foam tip within the pre-measured treatment area, and prep with 
vback-and-forth repeated strokes for 15 seconds. 
3. Turn the Maxi-swabstick over and repeat the procedure for 15 seconds. 
4. Do not apply swab beyond borders of marked skin areas by more than one swab width.  
5. Allow the area to completely air-dry; approximately 30 seconds. 
6. Do not blot or wipe dry. 
Treatment Site Application Instructions – Groin  
1. Peel open package and remove swabstick – do not touch swab. 
2. Place one flat side of the foam tip within the pre-measured treatment area, and prep with 
back-and-forth repeated strokes for 60 seconds. 
3. Turn the Maxi-swabstick over and repeat the procedure for 60 seconds 
4. Do not apply swab beyond borders of marked skin areas by more than one swab width.  
5. Allow the area to completely air-dry; approximately 90 seconds. 
6. Do not blot or wipe dry. 
 
 


 
 
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17.7 
Procedure for Neutralization Validation 
 Background 
The sampling solution, SSF++ is a buffered detergent solution that is commonly used in studies 
where microbial sampling of skin is conducted. Neutralizers have been added to inactivate the 
antimicrobial, CHG, present in test treatments. The effectiveness and toxicity of this neutralizer 
system must be assessed to demonstrate that there is no effect on the growth of microorganisms 
and that the active ingredient is inactivated. 
The density of normal human skin flora generally ranges from 102 to 105 CFU/cm2 depending on the 
body site. However, since significant neutralizer or toxic effects are more easily detected at a lower cell 
density, the efficacy and toxicity of this neutralizer system will be assessed against a lower bacterial 
concentration. 
This is a test where the study materials are applied to the abdomen and the treated areas will be 
sampled. After sampling, Staphylococcus epidermidis, a selected representative of normal skin flora 
will be added into a portion of each sample. Each sample will then be processed using procedures in 
accordance with ASTM E1054-08(2013). Two samples will be taken from each treatment area whereas 
one sample will be processed using a methicillin-sensitive Staphylococcus epidermidis (MSSE) (ATCC 
12228) strain and the remaining sample will be processed using methicillin-resistant Staphylococcus 
epidermidis (MRSE) (ATCC 51625) strain of the test organism.  
 Objective 
This control assay will determine the ability of the SSF++ to completely neutralize the active 
ingredients in the test treatments by recovering and quantifying microorganism populations on agar 
media and is appropriate for antimicrobial agents that can be chemically inactivated or diluted 
to sub-inhibitory levels. Each test will be performed in triplicate. 
 Subject Entry Criteria  
Six subjects will be used for the neutralization validation required for this study. Each subject must 
meet the inclusion and exclusion criteria described in Sections 4.1 and 4.2. 
No minimum bacterial count is required and the washout period is only necessary for 7 days (not 14 
days). The subjects will be asked to provide information on demographics and inclusion/exclusion 
criteria and sign the ICF before beginning the 7-day washout period. When the subjects return for 
clipping/re-clipping or 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. 
- 
At washout subjects will be assigned numbers starting with 01 to 06 
- 
At Neutralization day subjects will be assigned numbers with 01 and the letter “N”: N01 
The test article and active control will be applied to the abdomen regions so that three applications are 
performed for each treatment using bilateral application. The treatments per subject will be 
randomized. One treatment area will be located on one side of the body and the remaining treatment 
area on the other side. Each treatment site will have two sampling sites. 
 Test Organism 
The test organisms for this study are: 
- 
Methicillin-resistant Staphylococcus epidermidis (MRSE), ATCC 51625 
- 
Methicillin-sensitive Staphylococcus epidermidis (MSSE), ATTC 12228 
 
 


 
 
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 Study Products 
See section 3.4 of the protocol. 
 Materials, Supplies and Equipment 
See section 3.4.3 of the Protocol. In addition, 70% IPA swabs.  
 In-vivo Test Procedures  
Preparation of Test Area and Post-Prep Sampling: Neutralization samples will be taken from the 
abdomen. The subject number, location of the prep application, location of the sites sampled within 
the prep area, and the time of sample collection will be documented on the CRF. The subject will be 
treated with the study product based on the following: 
- 
For each side of the body, mark the abdominal test areas using the corresponding sterile 
template per study material. The 1.5” x 5.0” (3.81 cm x 12.7 cm) area will be delineated with 
each containing two 1” x 1” (2.54 cm x 2.54 cm) sampling sites (one each will be used for each 
test organism). 
- 
After the test areas are marked, each area will be processed using three 70% IPA swabs for a 
total of one minute to prepare the site; the areas will be allowed to dry. This step is to prepare 
the skin for the neutralization test. 
- 
Subjects participating in the neutralization portion of the study will be randomized using a 
randomization schedule which will be provided in a separate document. 
- 
Prep the test areas with the appropriate treatment according to the randomization schedule 
and the instructions provided in Appendix 17.6, (using groin treatment application procedures).  
- 
Using the scrub cup technique at approximately 30 seconds post-prep, begin collecting samples 
from each site using SSF++. This technique is described in Section 5.2.1. For the two sites 
within each area; the samples will be collected simultaneously by two technicians. 
 In vitro Test Procedures 
1 7 . 7 . 8 . 1  Inoculum Preparation 
One day prior to beginning the neutralization assay, the test organisms from a working stock culture 
slant or plate will be inoculated onto TSA plates. The plates will be incubated for 24 ± 4 hours at 35° 
± 2°C. Immediately prior to initiating the neutralization assay, an inoculum suspension for each test 
organism will be prepared in PBW solution from the overnight culture on the TSA plate, and the 
concentration adjusted to approximately 108 – 109 CFU/ml. Bacterial suspension will be further diluted 
with PBW to achieve an appropriate concentration (approximately 104 CFU/ml) for inoculation of the 
test samples.  The density of the test inocula will be verified by direct plating at the beginning and 
end of the study. 
17.7.8.2 
Test 
(Note – the reference to test product applies to both the investigational products and the active control; 
all procedures outlined, where applicable will employ both products. In addition, using two samples 
taken within each treatment area; all procedures, as applicable will be performed using one sample for 
each of the two test organisms.) 
 
 


 
 
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17.7.8.2.1 
Neutralizer effectiveness (Test 1) 
a) Out of the 6.0 mL aliquot of sampling solution taken from the volunteer, 5.0 mL will be 
transferred to a new sterile tube and inoculated with the challenge microorganism so that the 
final concentration will equal 30 – 100 CFU/ml of the challenge microorganism (the prepared 
inoculum will be diluted using PBW to achieve the desired concentration, a 0.1 mL aliquot 
from the 10-4 dilution will be used). 
b) Within one min after the addition of the challenge microorganism, the microorganisms will 
be enumerated by standard microbiological methods extant in the laboratory. 
c) Triplicate 0.75 mL aliquots will be removed and plated using TSA+N pour plates. 
d) After 30 minutes, the microorganisms will be enumerated a second time using the same 
procedures. 
e) This procedure will be repeated two times for a total of three replicates per test product per time 
point. 
17.7.8.2.2 
Sampling fluid (SSF++) toxicity (Test 2) 
a) A 5.0 mL aliquot of sampling solution will be inoculated with the challenge microorganism so 
that the final concentration will equal 30 – 100 CFU/ml of the challenge microorganism in the 
same manner as Test 1.  
b) Within one min after the addition of the challenge microorganism, the microorganisms will 
be enumerated by standard microbiological methods extant in the laboratory. 
c) Triplicate 0.75 mL aliquots will be removed and plated using TSA+N pour plates. 
d) After 30 minutes, the microorganisms will be enumerated a second time using the same 
procedures. 
e) This procedure will be repeated two times for a total of three replicates per test product per time 
point. 
17.7.8.2.3 
Test microorganism viability control (Test 3) 
a) A 5. 0  mL aliquot of PBW will be inoculated with a volume of the challenge microorganism 
so that the resulting suspension contains 30 – 100 CFU/ml in the same manner as Test 1.   
b) Within one min the microorganisms will be enumerated by standard microbiological 
methods extant in the laboratory. 
c) Triplicate 0.75 mL aliquots will be removed and plated using TSA pour plates. 
d) After 30 minutes, the microorganisms will be enumerated a second time using the 
same procedures. 
e) This procedure will be repeated two times for a total of three replicates per test product per 
time point. 
 
 


 
 
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17.7.8.2.4 
Test product control (Test 4) 
a) A 5.0 mL aliquot of the investigational product or active control will be inoculated with a 
volume of the challenge microorganism so that the resulting suspension contains 30 – 100 
CFU/ml in the same manner as Test 1.  
b) Within one minute the microorganisms will be enumerated by standard microbiological 
methods extant in the laboratory. 
c) Triplicate 0.75 mL aliquots will be removed and plated using TSA pour plates. 
d) After 30 minutes, the microorganisms will be enumerated a second time using the sample 
procedures. 
e) This procedure will be repeated two times for a total of three replicates per test product. 
 Incubation 
All plates for Tests 1, 2, 3, and 4 will be incubated for 48±2 hours at 35±2 °C. 
 
Interpretation of data 
a) The number of surviving challenge microorganisms for each replicate from each test will 
be average count of the three plates. 
b) The number of survivor values will be transformed to log10. 
c) The number of survivors (log10) from each test (1, 2, and 4) will be compared to the test 
microorganism viability population (Test 3). 
d) Neutralization aspects of the SSF++ will be considered adequate if the mean log10 CFU/ml 
of Test 1 is not more than 0.20 log10 less than the mean log10 CFU/ml of Test 3 (Mean 
log10 CFU/ml from Test 4 – Mean log10 CFU/ml from Test 1 using corresponding time 
points). 
e) The SSF++ will be considered non-toxic if the mean log10 CFU/ml of Test 2 is not more 
than 0.20 log10 less than the mean log10 CFU/ml of Test 3 (Mean log10 CFU/ml from Test 
3 − Mean log10 CFU/ml from Test 2 using corresponding time points). 
f) 
The mean log10 CFU/ml from Test 4 must be at least 0.20 log10 less than the mean log10 
CFU/ml of Test 3. 
g) The amount of CFU added for each aspect must be confirmed to yield a final suspension 
containing 30-100 CFU/ml (validated in test 3). 
h) The absence of growth controls must be negative for growth. 
 
Controls 
Absence of growth control: 
Triplicate plates of TSA and TSA+N used will be incubated with the test. In addition, triplicate 
1.0 ml aliquots of SSF++ and PBW will be plated using TSA. All plates will be incubated with the 
test. 
 
 


 
 
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17.8 
Skin Irritation Rating Scale 
 
Skin Irritation Scoring System 
Condition 
Rating 
Description 
Erythema 
0 
No reaction 
1 
Mild and/or transient redness limited to sensitive area 
2 
Moderate redness persisting over much of the product-exposed area 
3* 
Severe redness extending over most or all of the product-exposed area 
Edema 
0 
No reaction 
1 
Mild and/or transient swelling limited to sensitive area 
2 
Moderate swelling persisting over much of the product-exposed area 
3* 
Severe swelling extending over most or all of the product-exposed area 
Rash 
0 
No reaction 
1 
Mild and/or transient rash limited to sensitive area 
2 
Moderate rash persisting over much of the product-exposed area 
3* 
Severe rash extending over most or all of the product-exposed area 
Dryness 
0 
No reaction 
1 
Mild and/or transient dryness limited to sensitive area 
2 
Moderate dryness persisting over much of the product-exposed area 
3* 
Severe dryness extending over most of all of the product-exposed area 
* A score of 3 in one or more of the conditions evaluated represents significant irritation and qualifies as 
an Adverse Event. 
 
 


 
 
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Page 44 of 45 
17.9 
Confirmation of Release and Receipt of Study Materials 
 
CONFIRMATION OF RELEASE and RECEIPT OF STUDY MATERIALS 
 
Investigator: Rozalia Olsavszky M.D. 
Study No: ER 18/282 
Research facility: EUROFINS EVIC PRODUCT TESTING ROMANIA S.R.L. 
Quantity 
(Units) 
Description 
ID/Lot Number
Exp. Date 
 
Supplies Released to Research facility by:  
_________________________________ 
Sponsor Signature 
 
Supplies Sent to Research facility (Date):  
_________________________________ 
 
 
Supplies Checked and Verified by:  
_________________________________ 
Signature                             Date 
 
________________________________________________________________ 
Print Name, Title 
 
 
Once the supplies have been verified and this form is signed / dated, a signature copy will be sent 
to the sponsor representative: James.Clayton@pdipdi.com. 
 
 


 
 
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Page 45 of 45 
17.10 Study Material Disposition Form 
Use one form for each study material. 
Study Number: ER 18/282 
Principal Investigator:  
Rozalia Olsavszky M.D. 
Research facility: 
EUROFINS EVIC PRODUCT TESTING ROMANIA S.R.L. 
 
Study Material ID 
Date Received Quantity 
Received 
Lot Number/ 
Serial Number 
Date 
Returned 
to Sponsor 
 
 
 
 
 
 
Date Dispensed/ 
Distributed 
Subject 
Number 
Quantity Dispensed 
Quantity Remaining