Conditions: Surgical Skin Preparation Surgical Procedure, Unspecified
Keywords: surgical skin prep CHG IPA
Allocation: RANDOMIZED | Intervention model: SINGLE_GROUP
Enrollment: 28
Healthy volunteers: yes

Brief summary:
This study evaluates reduction of resident flora produced by the test product within a defined prepped area of skin.

Detailed description:
Three different sample sites within a defined prepped area of skin were collected to determine whether reductions in resident flora produced by the test product are consistent.

Arms:
arm 1: Chlorhexidine gluconate (CHG) 2% / Isopropyl alcohol (IPA) 70% solution applied topically to intact skin; sample collected from center test site on skin in application area. arm 2: Chlorhexidine gluconate (CHG) 2% / Isopropyl alcohol (IPA) 70% solution applied topically to intact skin; sample collected from mid-peripheral test site on skin in application area. arm 3: Chlorhexidine gluconate (CHG) 2% / Isopropyl alcohol (IPA) 70% solution applied topically to intact skin; sample collected from peripheral test site on skin in application area.

Interventions: intervention 1: 3M CHG/IPA Prep
Intervention descriptions: intervention 1: Apply topically to intact skin

Protocol document:
Prot_SAP_000.pdf: 
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Clinical Study Protocol
Study Number and Protocol Title
EM-05-012926
Evaluation of Microbial Population 
Reductions Within a Defined Product 
Coverage Area
Principal Investigator
 
 
 
Sub-Investigators:
 
 
 
 
Research Facility
Eurofins Evic Romania/ S.C. BIO HIGH 
TECH S.R.L.
Bucharest 040256 Romania
IRB/EC
Chesapeake Institutional Review Board 
Address: 6940 Columbia Gateway Drive, 
Suite 110, Columbia, Maryland, 21046, USA


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Sponsor
3M Health Care
3M Center
St. Paul, MN  55144-1000
3M Study Monitor
3M Medical Monitor
 
  


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Table of Contents
1.
BACKGROUND INFORMATION.................................................................................6
1.1 Name, Description and Intended Use of the Investigational Product ....................................6
1.2 Summary of Relevant Studies..............................................................................................6
1.3 Risk/Benefit Summary ........................................................................................................6
1.4 Investigational Material Application....................................................................................7
1.5 Good Clinical Practices and Regulatory Requirements ........................................................7
1.6 Study Population .................................................................................................................7
2.
STUDY OBJECTIVES ....................................................................................................8
3.
STUDY DESIGN..............................................................................................................8
3.1 Study Type and Methodology..............................................................................................8
3.2 Primary and Secondary Endpoints .....................................................................................10
3.3 Randomization ..................................................................................................................10
3.4 Study Products and Labeling .............................................................................................10
3.4.1
Study Products.......................................................................................................10
3.4.2
Investigational Products Labeling and Accountability............................................10
3.4.3
Additional Study Supplies......................................................................................11
3.5 Study Duration ..................................................................................................................13
3.6 Study Termination/Subject Discontinuation or Withdrawal ...............................................13
3.6.1
Study Termination .................................................................................................13
3.6.2
Subject Discontinuation or Withdrawal..................................................................13
3.7 Source Data.......................................................................................................................13
3.8 Computerized Systems ......................................................................................................14
3.9 Protocol Modifications ......................................................................................................14
3.9.1
Protocol Amendments............................................................................................14
3.9.2
Protocol Deviations................................................................................................14
4.
SUBJECT SELECTION ................................................................................................14
4.1 Subject Inclusion Criteria ..................................................................................................15
4.2 Subject Exclusion Criteria .................................................................................................15
4.3 Subject Informed Consent .................................................................................................16
5.
TREATMENT OF SUBJECTS .....................................................................................16
5.1 Medication(s)/Treatment(s) Not Permitted.........................................................................16
5.2 Subject Compliance...........................................................................................................17
5.3 Study Procedures...............................................................................................................17
5.3.1
Wash Out (Pre-inclusion) Phase.............................................................................17
5.3.2
Screening Phase.....................................................................................................17
5.3.3
Treatment Phase ....................................................................................................18
6.
ASSESSMENT OF EFFICACY ....................................................................................19
6.1 Efficacy Parameters...........................................................................................................19


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6.2 Assessment Methods .........................................................................................................19
6.2.1
Microbiological Methods.......................................................................................19
6.3 Subject Discontinuation.....................................................................................................20
7.
ASSESSMENT OF SAFETY.........................................................................................20
7.1 Safety Parameters..............................................................................................................20
7.2 Assessment Methods .........................................................................................................20
7.3 Adverse Events..................................................................................................................20
8.
STATISTICS ..................................................................................................................21
8.1 Data Sets Analyzed............................................................................................................21
8.2 Statistical Methods ............................................................................................................22
8.2.1
Efficacy Analyses ..................................................................................................22
8.2.2
Safety Analyses .....................................................................................................22
8.3 Sample Size Justification...................................................................................................22
8.4 Criteria for Termination of the Study.................................................................................22
8.5 Procedures for Accounting for Missing, Unused, and Spurious Data..................................22
8.6 Deviations to Statistical Plan .............................................................................................22
9.
MONITORING ..............................................................................................................22
10.
QUALITY CONTROL AND QUALITY ASSURANCE..............................................23
11.
ETHICS ..........................................................................................................................23
12.
DATA HANDLING AND RECORD KEEPING ..........................................................23
12.1 Study Personnel.................................................................................................................23
12.2 Pre-Study Documentation Requirements ...........................................................................23
12.3 Completion and Return of Case Report Forms...................................................................24
12.4 Final Report.......................................................................................................................24
12.5 Records, Reports and Retention Requirements...................................................................24
13.
REFERENCES...............................................................................................................25
14.
APPENDICES ................................................................................................................26
14.1 Study Flow Diagrams ........................................................................................................26
14.2 Study Product Application Instructions..............................................................................29
14.3 Confirmation of Release & Receipt of Clinical Supplies....................................................30
14.4 Study Product Disposition Form........................................................................................31
14.5 Subject Instructions ...........................................................................................................32
14.6 Elements of Informed Consent...........................................................................................33
14.7 Test Site Diagrams ............................................................................................................35
14.8 Skin Irritation Rating Scale................................................................................................36
14.9 Protocol for Neutralization Validation ...............................................................................37
14.9.1 Background ...........................................................................................................37
14.9.2 Objective ...............................................................................................................37
14.9.3 Subject Entry Criteria ............................................................................................37


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14.9.4 Test Organism .......................................................................................................38
14.9.5 Study Products.......................................................................................................38
14.9.6 Materials, Supplies and Equipment ........................................................................39
14.9.7 Study Procedures ...................................................................................................40
14.9.8 Control of Bias.......................................................................................................41
14.9.9 Data Evaluation .....................................................................................................41


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1.
BACKGROUND INFORMATION
1.1
Name, Description and Intended Use of the Investigational Product
 Surgical Solution
 is an investigational 
antimicrobial skin preparation containing 2% w/v chlorhexidine gluconate (CHG) and 70% v/v 
isopropyl alcohol (IPA) 
 for reducing the bacterial 
flora on patients’ skin prior to surgery.
 
 
he product will be available in the following configurations: 
10.5-mL tint and colorless, and 26-mL tint. The 26-mL tint configuration will be evaluated in the 
current study.
The purpose of this study is to evaluate the ability of a single preoperative preparation product to 
achieve similar reductions of resident flora at three different sample sites within a defined test 
area, 
on the skin of the posterior torso (back region). One sample site will be in 
the center of the application site, a second sample will be taken halfway between (equidistant) 
the center and the periphery of the application site, and a third sample will be taken on the 
periphery of the application site.
1.2
Summary of Relevant Studies
 
 two studies were conducted to evaluate the coverage area
 on human 
subjects. In study 
 the coverage area for the 26-mL applicator was evaluated and 
in study 
 the coverage area for the 10.5-mL applicator was evaluated. The 
coverage area determined in study 
 is being used as the treatment coverage area 
for this study.  No safety concerns were identified in these studies.  
Refer to the Investigator Brochure 
 for a summary of 
studies. 
1.3
Risk/Benefit Summary
There are minimal anticipated adverse health risks for the participants of this study. Considerable 
safety and efficacy data are available for the investigational product and preclinical safety 
studies, 
, have been completed.
 
 
 
There are no direct health benefits to subjects who participate in this study.


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1.4
Investigational Material Application 
This study will be conducted using the tint formulation 
 in the 26-mL applicator 
configuration. The study product will be applied topically to intact skin of the subject’s posterior
torso (back region) in a defined
 area.    
The study product will be applied as intended per instructions for use (Appendix 14.2).
 
   
1.5
Good Clinical Practices and Regulatory Requirements
 
 
This study will be conducted in compliance with the protocol, Good Clinical Practice (GCP), 
Declaration of Helsinki and applicable state, federal and regional regulations including 21 CFR 
312 (Investigational New Drug Application), 50 (Protection of Human Subjects), 56 
(Institutional Review Boards), 54 (Financial Disclosure by Clinical Investigators). And in 
accordance with the European Directive 95/46/EC on the protection of individuals with regard to 
the processing of personal data and on the free movement of such data and with the Romanian 
Order No 677/2001 for the protection of persons concerning the processing of personal data and 
free circulation of such data. And in spirit with Directive of the European Parliament and 
Council 2001/20/EC, and Romanian Order No. 904/25.07.2006.
1.6
Study Population
Healthy male subjects, 18 years of age or older, who meet the study inclusion/exclusion criteria 
are eligible for enrollment into this study.
A sufficient number of subjects will be enrolled in the screening phase to provide 18 treatment 
sites that meet the baseline requirements on treatment day. Subjects must satisfy all Screening 
Day and Treatment Day inclusion/exclusion criteria prior to collection of screening baselines and 
Treatment Day procedures. If the required numbers of subjects do not qualify from the initial 
screening group, additional subjects will be recruited, as needed. Treatment day baseline 
requirement is greater than or equal to 3.0 log10 CFU/cm2 on each of the 3 sampling sites in the 
treatment site.
Each subject will be identified by a screening identification number, and a treatment number.  
Subject treatment numbers will not be assigned until a subject has passed the screening criteria, 
including screening baseline bacterial counts.
All three sampling sites (center, mid-peripheral and peripheral) on the treatment site must meet 
the screening baseline requirements stated in the inclusion criteria to qualify for treatment.


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2.
STUDY OBJECTIVES
The primary objective of this study is to determine whether reductions in resident flora produced 
by the test product are consistent within a prepped area of skin. Sufficient number of subjects 
will be included for testing in order to finish with 18 evaluable subjects.  
 
  
Safety will also be evaluated based on the incidence of adverse events reported during the study 
and assessment of skin irritation ratings.
3.
STUDY DESIGN
3.1
Study Type and Methodology
This is a prospective, randomized, uncontrolled single-center study in healthy subjects.  
 
 This study will be conducted using a modification of ASTM 
E1173-15 Standard Test Method for Evaluation of Preoperative, Precatheterization, or 
Preinjection Skin Preparations.2 The 2-log10 reduction criteria (dry site) will be used to evaluate 
efficacy. Time points for sample collection are baseline (pre-prep) and 10 minutes or 13 minutes
(post-prep).        
Table 3.1A provides a summary of the study procedures. A schematic diagram of the study is 
included in Appendix 14.1.


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Table 3.1A Study Summary
14-day Wash Out        
(Pre-inclusion*) Phase
Screening Phase
Treatment Phase
Perform the Informed 
Consent process.
Complete Inclusion/Exclusion
form; check study constrains.
Perform visual skin 
assessment.
Assign sequential subject 
number.
Complete Inclusion/Exclusion
form; check study constrains.  
Perform visual skin assessment.  
Record information on 
demographics.
Assign subject screening 
number.
Using a
 sterile 
template mark back test area. 
Within the test area mark the 
3 baseline sampling sites
(center, mid-peripheral and 
peripheral).
Using a 
 sterile 
template mark back test area. 
Within the test area mark the 3 
baseline sampling sites (center,
mid-peripheral and peripheral) 
and the 3 post-prep sampling 
sites (center, mid-peripheral 
and peripheral).
Complete Inclusion/
Exclusion Criteria form.
Collect screening baseline 
samples (center, mid-
peripheral and peripheral) 
from test area.
Perform skin irritation rating.  
*Prior to the scheduled 
Screening Phase, subjects 
will undergo a minimum 
14-day Wash Out (Pre-
inclusion) Phase.   
Count screening plates to
determine which subjects 
qualify for study.
Collect baseline samples
(center, mid-peripheral and 
peripheral) from test area.
Review subject 
instructions for wash out 
and dispense subject 
personal hygiene kit
Contact eligible subjects to
schedule Treatment Day 
(schedule for clipping 72 
hours prior to treatment, if 
needed).
Weigh product applicator pre-
application.
Apply study product.
Weigh product applicator post-
application.
Schedule for clipping 72 
hours prior to screening, 
if needed.
10-minutes
post-
prep perform skin irritation 
rating.
Per randomization, collect the
post-prep samples [10-minutes
or 13-minutes
 


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3.2
Primary and Secondary Endpoints
Primary Endpoint:
The primary endpoint, coverage area with efficacy, is measured by log10 reductions calculated by 
subtracting the log10 number of microorganisms recovered at a specified sampling time following 
product application from the log10 number of microorganisms recovered from the baseline 
sampling. Baseline samples must satisfy the inclusion criteria to be used in data analysis, > 3.0 
log10 CFU/cm2 in all three sampling sites on the treatment site. The sampling time for efficacy 
evaluation will be specified per the randomization schedule as 10 minutes or 13 minutes post-
product application.       
 
 
 
   
The principal measure of safety will be the incidence of adverse events reported during the study 
and a summary of the skin irritation rating scores.
 
3.3
Randomization
The three post-product application sampling sites will be randomized so that 2 of the 3 sites will 
be sampled at one of the post-product application sample times (10 minutes or 13 minutes) and 
the third sampling is collected at the remaining sample time.    
The randomization schedule will be provided to the study site in a separate document.
3.4
Study Products and Labeling
3.4.1
Study Products
Investigational prep:
 Surgical Solution, 2% w/v
CHG/70% v/v IPA, tint formulation with 26 mL foam tipped applicator
  
 will be applied topically to intact skin of the treatment site (back region) of the 
subject. The prep will be applied with repeated back-and-forth strokes for 30 seconds.
Detailed instructions for application and removal of the study products are provided in the 
Investigational Product Application Instructions (Appendix 14.2).
3.4.2
Investigational Products Labeling and Accountability
3M will label, package and ship the study products to the research facility. The
applicators will be provided in individual
 packages, and labeled as below.


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Sample Investigational Product Label 
CAUTION: New Drug-Limited by Federal Law to Investigational Use
For use in study EM 05-012926
3M Health Care, St. Paul, MN 55144-1000
 Surgical Solution, Tint
(26-mL)   
Product ID:
Subject Number:
Lot: TBD
Directions: Use as directed in protocol
Warning:  Contents are flammable until dry.
3M requires Investigators to maintain accountability and adequate inventory security of the study
product at all times. The Investigator or designee will: 
●
Document receipt of study product on the Confirmation of Release & Receipt of Clinical 
Supplies form (Appendix 14.3) and return the completed form to 3M, 
●
Store study product in a secure facility accessible only to authorized individuals,
●
Dispense study product only to subjects properly enrolled into the study,
●
Account for inventory and disposition using the Study Product Disposition Form (Appendix 
14.4), and
●
Dispose of used and unused study product as agreed upon. Once accounted for the study 
product may be disposed. Documentation shall be maintained concerning the disposal and 
will contain: the quantity of the study product disposed, the date and manner of disposal, the 
staff member who conducted the disposal. NOTE: Record the subject number on the package 
label and retain the label for inventory purposes. The used applicator can be discarded.
In addition, copies of all completed Study Product Disposition Forms and disposal records must 
be retained in the Investigator Study Documentation File. The originals of all Study Product
Disposition Forms and disposal records must be sent to the Sponsor’s Clinical Monitor.
3.4.3
Additional Study Supplies
Sponsor will provide the following:
●
Study Material Disposition forms
●
Randomization schedule
●
Data Collection Sheets (DCSs)
●
Investigational product


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Study site will provide the following:
●
Subject Informed Consent forms, IRB/EC-approved
●
 
 
 
 
 
)


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3.5
Study Duration
The expected duration of this study for each subject is up to 3 weeks. Each subject’s 
participation will involve at least a 14-day Wash Out (Pre-inclusion) Phase, a 1-day Screening 
Phase, and a 1-day Treatment Phase. Following the Wash Out (Pre-inclusion) Phase, each 
subject will be required to visit the test facility on an arranged date for collection of screening 
baseline samples from the back region. Subjects will remain on washout until notified that they 
do or do not qualify for the study. Subjects whose screening baseline samples meet the 
requirements 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 collections. Each subject who chooses to 
participate in this study will be required to return to the test facility for treatment and sample 
collection. Table 3.1A indicates the sequence of events throughout the study and a schematic 
diagram of the study is included in Appendix 14.1.
3.6
Study Termination/Subject Discontinuation or Withdrawal
3.6.1
Study Termination
Conditions that may warrant the termination of the study by 3M include, but are not limited to 
the following:
●
The discovery of an unexpected, serious, or unreasonable risk to study subjects,
●
Insufficient adherence to protocol requirements,
●
Failure of the Investigator to enroll subjects into the study at an acceptable rate,
●
Failure of the Investigator to comply with pertinent state, federal or regional regulations or 
applicable privacy regulations,
●
Submission of knowingly false information from the Investigator to 3M,
●
Termination of IND or IRB/EC approval, or withdrawal of the IRB/EC approval, 
●
A decision on the part of 3M to suspend or discontinue evaluation of the investigational 
product.
3.6.2
Subject Discontinuation or Withdrawal
The Investigator may discontinue individual subjects from the study at any time. Subjects 
entering the study may withdraw at any time without penalty. The right of each test subject to 
withdraw from the study and the right of each test subject to privacy, confidential treatment of 
personal data, and protection from view by others not conducting the study will be respected at 
all times. The Investigator will provide a written report on the appropriate case report form 
including the date and reason for discontinuance.
3.7
Source Data
Electronic data capture (eDC) will be used for this study. If not entered directly into eDC, data 
will first be recorded onto a DCS, followed by entry into the appropriate electronic case report 
form (eCRF). Wherever the data are entered first is considered the source document.


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3.8
Computerized Systems
All study data will be loaded into the
data management system.
The current 3M Health Care document storage system, 
 will be used to store the study 
documents.  
 may be used 
for study documentation and data analysis.
3.9
Protocol Modifications
3.9.1
Protocol Amendments
A protocol amendment is defined as a change in procedure that is identified and documented 
before initiation. A significant amendment means one that affects the safety, rights or welfare of 
subjects, the scope of the investigation or the scientific quality of the study.
All protocol amendments must be documented and approved by the Investigator and study
monitor prior to implementation. All significant protocol amendments must also be pre-approved 
by the reviewing IRB/EC prior to implementation.
3M will submit significant protocol amendments to the Investigator for submission to the 
IRB/EC. 3M will also notify the Investigator when a protocol amendment may be implemented.
3.9.2
Protocol Deviations
A protocol deviation is defined as a change in procedure that has not been documented before 
initiation and therefore has not been reviewed and approved with the study sponsor and/or 
reviewing IRB/EC prior to initiation. All deviations from the study protocol are to be 
documented and reported to the investigator/study sponsor and reviewing IRB/EC.
A deviation is a departure from the protocol that will likely affect the safety, rights or welfare of 
subjects, the scope of the investigation or the scientific quality of the study.
A protocol deviation is only for an individual subject. Deviations that potentially affect 1) 
subject safety, rights or welfare, 2) data integrity or 3) compromise the statistical analysis of the 
study require immediate communication to 3M. The Investigator or designee must contact the 
3M study monitor 
 A Protocol Deviation Form must be completed 
by the Investigator and include the type of deviation and a description of the circumstances 
surrounding the deviation. A copy is sent to the 3M study monitor 
 of identifying 
the occurrence.
Deviations, which are made to protect the life or physical well-being of a subject in an 
emergency, must be reported by the Investigator to the IRB/EC within 5 working days of 
occurrence.
4.
SUBJECT SELECTION
Healthy male subjects will be recruited. A suitable number of subjects will be enrolled in the 
wash out (pre-inclusion), screening and treatment phase, such that a total of 18 subjects are 
evaluable for efficacy at completion of the study.  


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Subjects must follow all Subject Instructions (Appendix 14.5) and satisfy inclusion/exclusion 
criteria prior to enrollment into the treatment phase of the study.  
All subjects will be provided verbal and written information about the study procedures, and 
subject instructions (Appendix 14.5). The following inclusion/exclusion criteria will be reviewed 
on wash out (pre-inclusion), screening and treatment day to establish eligibility for participation:
4.1
Subject Inclusion Criteria
Subjects to whom all of these conditions apply will be eligible for enrollment in this study:
1. Male subjects of any race who are at least 18 years of age,
2. Subjects must have a 
area on their back region to accommodate the treatment 
coverage area,
3. Subjects must be in good general health,
4. Subjects who satisfy all inclusion/exclusion criteria and will voluntarily read and sign the 
Informed Consent Form,
5. Subjects who have good skin condition on the test sites,
6. Subjects who are willing to report to the study facility approximately 72 hours prior to 
Screening or Treatment Day for clipping, if needed,
7. Subjects who are willing to avoid showering and tub-bathing within 72 hours prior to 
Screening and Treatment Days, and
8. Subjects who have Screening Day baseline counts of > 3.00 log10 per cm2 in each of the 3 
sample sites (center, mid-peripheral and peripheral) in the treatment area (back region).
4.2
Subject Exclusion Criteria
Subjects to whom any of these conditions apply will be excluded from this study:
1. Participation in another clinical study in the past 30 days, current participation in another 
clinical study, or previous participation in this study,
2. Has taken antihistamines in the 48 hours prior to Treatment Day, 
3. Any tattoos, scars, breaks in the skin, or any form of dermatitis, or other skin disorders 
(including acne) on the applicable test areas,
4. A history of skin allergies,
5. A history of skin cancer within 6 inches of the test areas,
6. Known sensitivity to acrylate-, chlorhexidine gluconate-, or alcohol-containing products, or 
to medical tape, metals, natural rubber latex, vinyl, or skin-marking inks, 
7. A medical diagnosis with physical condition that may put the subject at risk, such as a 
current or recent severe illness, hepatitis, organ transplant, congestive heart disease, or any 
immunocompromised conditions, such as AIDS (or HIV positive),
8. Any medical condition or use of any medications that, in the opinion of the Investigator, 
should preclude participation,


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9. Topical antimicrobial exposure within 14 days prior to Screening Day and throughout the 
study. Restrictions include, but are not limited to antimicrobial soaps, medicated shampoos, 
medicated lotions, antiperspirants/deodorants, perfumes, after shaves, and colognes,
10. Use of systemic or topical antibiotic medications, steroid medications, or any other product 
known to affect the normal microbial flora of the skin within 14 days prior to Screening Day 
and throughout the study,
11. Exposure of the test areas to solvents, acids, bases, strong detergents, fabric softener-treated 
clothing, or other household chemicals within 14 days prior to Screening Day and throughout 
the study,
12. Swimming in chemically treated pools or bathing in hot tubs, spas, or whirlpools within 14 
days prior to Screening Day and throughout the study,
13. Use of tanning beds, hot waxes, or depilatories (in the applicable test areas) within 14 days 
prior to Screening Day and throughout the study
14. Bathing and showering within 72 hours prior to Screening Day and throughout the study, or
15. Subject has used moisturizers or any topical treatment (e.g. lotion, sunscreen or shaving 
cream) on the test sites in the 24-hour period prior to Screening Day and Treatment Day 
participation in the study.
4.3
Subject Informed Consent
The Investigator or designee must ensure that written informed consent to participate in the 
investigation is obtained before including any individual as a subject in the investigation. The 
Investigator or designee must provide the prospective subject or the representative sufficient 
opportunity to consider whether or not to participate, and minimize the possibility of coercion or 
undue influence. The process is designed to 1) give the subject all the information that they need, 
2) ensure that the subject understands the information and 3) give the subject a chance to consider 
study participation. The process should permit the subject to ask questions and exchange 
information freely.
Specifically, the Investigator or designee is to explain to each subject all elements of informed 
consent as specified in 21 CFR 50.25 (Appendix 14.6). After the explanation, the subject or 
representative will voluntarily sign and date the consent form if they wish to participate in the 
study. A copy of the consent form must be provided to the subject. A signed and dated copy of the 
consent form must be maintained in the Investigator study documentation file at all times. 
Consent and study participation, with the date, must be documented in the subject’s record.
5.
TREATMENT OF SUBJECTS
5.1
Medication(s)/Treatment(s) Not Permitted
Exposure of the test areas to antimicrobial agents is not permitted within 14 days prior to 
Screening Day and for the duration of the study. Restrictions include, but are not limited to 
antimicrobial soaps, antiperspirants/deodorants, shampoos, lotions, perfumes, after shaves, 
colognes, steroid medications and topical or systemic antibiotics.


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5.2
Subject Compliance
Answers to the inclusion/exclusion criteria questions asked at the beginning of the Screening and 
Treatment Phases will determine compliance to the requirements of the study.
5.3
Study Procedures
These study procedures are based on ASTM E1173-15, Standard Test Method for Evaluation of 
Preoperative, Precatheterization, or Preinjection Skin Preparations.2
5.3.1
Wash Out (Pre-inclusion) Phase
Perform the Informed Consent process.  After the subject has signed the informed consent form
assign the subject a screening number and complete the Inclusion/Exclusion form.  
If the inclusion/exclusion criteria are satisfied, prior to the scheduled Screening Day, subjects 
will undergo a minimum 14-day wash out (pre-inclusion) phase to allow for the removal of any 
antimicrobial agents from the subjects’ skin. Subjects are to refrain from the use of products 
containing antibacterial agents as written in the Subject Instructions (Appendix 14.5). Subjects 
will be given personal hygiene kits containing non-antimicrobial products for use during this 
period. Subjects will be instructed to use these products through completion of the Treatment 
Phase or until notified by the Investigator or designee.
In addition, subjects must avoid systemic or topical antibiotics or medications and contact with 
chemically treated swimming pools or hot tubs, hot waxes, and depilatories on the test area for 
14-days before the scheduled Screening Day. If it becomes necessary to take systemic antibiotics 
or to apply topical medications to the test area within this wash out (pre-inclusion) period, the 
subject must contact the Investigator or designee as soon as reasonably possible so that another 
subject may be recruited.
If subjects require hair removal to facilitate sample collection, the subject will be asked to return 
to the test facility approximately 72 hours before the Screening Day. Subjects will not be allowed 
to shower or bathe for 72 hours prior to their scheduled screening appointment.  
5.3.2
Screening Phase
Prior to collection of the screening baseline samples, the Investigator or a designee will complete 
the screening Inclusion/Exclusion form. A visual skin assessment of the test area will be 
performed. The test area within the back region is defined as the uppermost aspect up to the top 
of the scapula extending down to just above the sacrum as shown in Appendix 14.7. Using a
 
sterile template, the corners of each back test area will be marked directly on the 
skin using a nontoxic skin marker. Three sampling sites will be numbered within the test area.
The 3 sampling sites within each back test area represent the 3 screening baseline sites (center, 
mid-peripheral, and peripheral). The positioning and numbering of the sampling sites are 
standard for all subjects (Appendix 14.7). The screening baseline samples will be collected using 
the cup scrub technique described in Section 6.2.1.1.  
All three sampling sites (center, mid-peripheral and peripheral) on the test area (back region) 
must meet the screening baseline requirements stated in the inclusion criteria to be enrolled into 
the Treatment Phase of the study. Those subjects who qualify will be notified and will continue 


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to follow the instructions in Appendix 14.5 until completion of the scheduled Treatment Day. If 
subjects require hair removal to facilitate sample collection, the subject will be asked to return to 
the test facility approximately 72 hours before Treatment Day. Subjects will not be allowed to 
shower or bathe for 72 hours prior to Treatment Day.  
5.3.3
Treatment Phase
A sufficient number of subjects who meet the inclusion criteria will be enrolled into the 
Treatment Phase of the study, such that at least 18 treatment sites (back region) are evaluable at 
completion of the study.
The Investigator or a designee will complete the treatment Inclusion/Exclusion form. If these 
criteria are satisfied, complete information on demographics and perform a visual skin assessment 
to evaluate the condition of the test area.
5.3.3.1
Preparation of Test Areas
A Test Site Diagram for the test area (back region) is shown in Appendix 14.7.
The test area within the back region is defined as the uppermost aspect up to the top of the 
scapula extending down to just above the sacrum as shown in Appendix 14.7. Using a
 
sterile template, the corners of each back test area will be marked directly on the skin
using a nontoxic skin marker. Six sampling sites will be numbered within the test area. The 
positioning and numbering of the sampling sites are standard for all subjects (Appendix 14.7).
The 6 sampling sites within each back test area represent the 3 baseline (pre-prep) sites (center, 
mid-peripheral, and peripheral) and the 3 post-prep sample sites (center, mid-peripheral, and 
peripheral). The sampling sites are designated as Site 1 baseline (pre-prep) center, Site 2 baseline 
(pre-prep) mid-peripheral, Site 3 baseline (pre-prep) peripheral, Site 4 (post-prep) center, Site 5 
(post-prep) mid-peripheral and Site 6 (post-prep) peripheral. The three post-prep sampling sites, 
sites 4, 5 and 6, will be randomized as the 10-minute and 13-minute post-prep sample sites.
After the back region test area is marked and sampling sites are numbered, the 3 baseline (pre-
prep) samples will be collected using the cup scrub technique described in Section 6.2.1.1.
5.3.3.2
Study Product Application
Following baseline sample collection, the test area will each be prepped with the study product.
 
  
The study product will be applied to the entire test area per the Investigational Product
Application Instructions (Appendix 14.2). The duration of the prep application procedure is 30-
seconds.
 
  
5.3.3.3
Evaluation of Skin Irritation
Prior to collection of the baseline and the
 post-prep samples, the skin in each test area 
will be evaluated for indications of skin irritation, based on the Skin Irritation Rating Scale 


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(Appendix 14.8). Skin irritation ratings for each area will be recorded on the Skin Irritation 
Rating form. 
 
Any area with a rating of 3 
should not be sampled.
5.3.3.4
Timing of Post-Prep Sample Collection
The 3 post-product application sample sites will be randomized so that 2 of the 3 sites will be 
sampled at one of the post-product application sample times (10 minutes or 13 minutes) and the 
third within the sample time remaining. The 3 sample locations within the prepped area will be 
demarcated such that each is taken from the same location on each of the subjects.  
Microbial samples will be collected at 10 minutes 
or 13 minutes 
 
post-prep from the sampling sites within the test area. Post-prep timing begins upon completing
the application of the study products, including the 3-minute drying time. Microbial samples will 
be collected using the cup scrub technique described in Section 6.2.1.1.
Following the final sample collection, residual study products will be removed from the subject’s 
skin 
6.
ASSESSMENT OF EFFICACY
6.1
Efficacy Parameters
The primary measure of coverage area with efficacy
 is measured as > 2 log10
CFU/cm2 reductions calculated by subtracting the log10 number of microorganisms recovered at 
a specified sampling time following product application from the log10 number of 
microorganisms recovered from baseline sampling. Baseline samples must satisfy the inclusion 
criteria, > 3.0 log10 CFU/cm2 in each of the 3 sampling sites, to be used in data analysis.    
6.2
Assessment Methods
6.2.1
Microbiological Methods
6.2.1.1
Sample Collection and Processing
Quantitative cultures will be obtained from sampling sites using a modification of the cup scrub 
method of Williamson and Kligman.3
 
A sterile scrub cup will be placed and held firmly on each site. Scrub 1:
 
SSS will be aseptically pipetted into the scrub cup. The skin will be scrubbed in a 
circular motion with moderate pressure for 1 minute using a sterile policeman. Using a sterile 
transfer pipette, the scrub solution will be transferred to a sterile collection tube. Scrub 2:
 
fresh SSS will be pipetted into the scrub cup and the scrub procedure will be 


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repeated. This solution will be pooled with the solution from Scrub 1 for a total sample volume of 
approximately 6 mL per site.
Following collection, each sample tube will be capped tightly 
 
 followed by 
 
 30-second vortex. A 1.0 mL aliquot of each sample 
will be diluted into sterile tubes containing 9.0 mL PBW. Serial 10-fold dilutions will be 
performed in PBW, as appropriate. One-milliliter (1.0-mL) aliquots of selected dilutions will be 
pour-plated in duplicate using TSA+N.  Samples must be plated within 30 minutes of 
collection.  
After 72 
 hours of aerobic incubation at 
30 ± 2°C, colonies will be counted and viable cells in the original sample will be calculated using 
standard methods. 
 
6.2.1.2
Collection of Raw Data
Raw colony counts from each of the dilutions will be recorded on the appropriate data collection 
sheet (DCS) for each subject.  
6.2.1.3
Neutralization of Investigational Products
Prior to Treatment Day the effectiveness of the neutralizers contained in the microbial sampling 
solution will be tested based on ASTM E1054-08 (2013), Standard Test Methods for Evaluation 
of Inactivators of Antimicrobial Agents.4 The protocol for neutralization validation is provided in 
Appendix 14.9.
6.3
Subject Discontinuation
The Investigator or designee may discontinue individual subjects from the study at any time. 
Subjects may voluntarily withdraw from the study at any time. The Investigator or designee will 
provide a written report on the appropriate form including the date and reason for 
discontinuation. Subjects who have been previously screened may not be re-entered into the 
study.
7.
ASSESSMENT OF SAFETY
7.1
Safety Parameters
The principal measure of safety will be the incidence of adverse events reported during the study 
and a summary of the skin irritation rating scores.
 
7.2
Assessment Methods
The Investigator or designee will assess the subject’s skin condition prior to collection of the 
baseline and post-prep (10-minutes or 13-minutes) samples using the Skin Irritation Rating Scale 
(Appendix 14.8).
7.3
Adverse Events 
The Investigator is responsible for identifying adverse events that occur to each subject. An 
adverse event can be identified by the Investigator or reported by the subject.


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Definitions:
●
Adverse event (AE) means any undesirable clinical occurrence in a subject whether or not it 
is considered to be drug related.
●
Drug-related adverse event (ie, adverse drug experience) is an adverse event that is 
considered by the Investigator to have a reasonable likelihood of being associated with the 
investigational drug.
●
Life-threatening adverse drug experience is any adverse drug experience that places the 
subject, in the view of the Investigator, at immediate risk of death from the reaction as it 
occurred (does not include a reaction that, had it occurred in a more severe form, might have 
caused death).
●
Serious adverse drug experience is an adverse drug experience that is fatal or life-threatening, 
is permanently or significantly disabling/incapacitating, requires inpatient hospitalization or 
prolongation of existing hospitalization, or results in a congenital anomaly/birth defect.
●
Unexpected adverse drug experience is one not previously identified in nature, severity or 
frequency of incidence in the current Investigator brochure, in the clinical plan, or elsewhere 
in the current IND application and amendments.
Recording and Reporting:
The Investigator records each AE occurring in the study, including those not thought to be 
associated with the drug, on the Adverse Event Record. Documentation includes the AE 
description, severity at onset, seriousness, date of onset and resolution, relationship to the 
investigational drug, action taken, and outcomes.
The Investigator must promptly report all adverse drug experiences to the 3M study monitor. If 
the adverse drug experience is also considered by the Investigator to be serious and/or 
unexpected the Investigator must report it to the IRB/EC as soon as possible.
3M reports (by telephone or fax) a death or life-threatening adverse drug experience to the FDA 
within 7 days of notification, and submits a follow-up report within 15 days.
A serious and unexpected adverse drug experience involving a non-3M commercialized product 
is reported to the 3M study monitor and IRB/EC.
If a subject does not experience an AE during the study, the absence of such must be recorded on 
the form.
8.
STATISTICS
8.1
Data Sets Analyzed
Subjects with an average treatment day baseline of >3.0 log10 CFU/cm2 at each of the sampling 
sites (center, mid-peripheral and peripheral) within the test site (back region) will be included in 
the analysis.  


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8.2
Statistical Methods

CFU/ml will be converted to CFU/cm2.  

Summary tables will be produced for all responses. Summary tables for log10 recovery, log10
baseline and log10 reduction from baseline will include n, mean, standard deviation, median, 
minimum and maximum.
8.2.1
Efficacy Analyses
A mean log10 reduction of 2.0 in all 3 sampling locations is needed to support the proposed 
coverage area. 
8.2.2
Safety Analyses
Adverse events, 
 will be summarized.  
Skin irritation tables will be produced at baseline collection and post-prep sample collection.
8.3
Sample Size Justification
 
 
 The rationale for the number of 
subjects is based on EN test methods EN1499, EN1500 and EN12791 which require between 12 
and 18 subjects.
8.4
Criteria for Termination of the Study
3M reserves the right to terminate the study at any time for business reasons.
8.5
Procedures for Accounting for Missing, Unused, and Spurious Data
Any non-use of data will be discussed and justified in the blinded review of data at the data lock 
meeting and documented in the meeting minutes.
8.6
Deviations to Statistical Plan
Any deviation from the statistical plan discussed above will be documented in the study report.
9.
MONITORING
3M, as sponsor of this study, is responsible for ensuring the proper conduct of the study with 
regard to protocol adherence and validity of the data recorded. 3M has therefore assigned a study 
monitor to this study. The progress of the study will be monitored by:
●
Periodic on-site review
●
Ongoing review of electronic documentation 
●
Telephone and e-mail communications 
●
Review of source documents


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The Investigator or designee will give the 3M study monitor direct access to source documents 
that support data on the eCRFs and make available such records to authorized 3M, quality 
assurance, IRB/EC, and regulatory personnel for inspection and/or copying.
10. QUALITY CONTROL AND QUALITY ASSURANCE
3M is responsible for implementing and maintaining quality assurance and quality control 
systems through written standard operating procedures (SOPs) to ensure that this study is 
conducted and data are generated, documented and reported in compliance with the protocol, 
GCP and regulations cited in Section 1.5 of this protocol. Study monitoring is carried out to 
accomplish this.
11. ETHICS
This study will be conducted in accordance with the principles that have their origin in the 
Declaration of Helsinki, 21 CFR 50 (Informed Consent) and 56 (IRBs). The study will start only 
after approval of the protocol and consent form by the IRB/EC. The approval letter or notice 
must contain the IRB/EC name, meeting date, and sufficient information to identify the protocol 
and informed consent by name and number that were reviewed. 3M, prior to study initiation, 
must receive a copy of the IRB/EC approval letter.
12. DATA HANDLING AND RECORD KEEPING
12.1 Study Personnel
Prior to study initiation, the Investigator must provide 3M with a signed Form FDA 1572.
This form identifies all sub-investigators who will be assisting the Investigator in the 
conduct of the study. The Investigator will provide a log of signatures/initials and 
delegation of responsibility log prior to study initiation.
12.2 Pre-Study Documentation Requirements
Prior to study initiation, the Investigator must provide 3M with the following documents:

Signed protocol including any amendments in place prior to study initiation

Curriculum vitae for the Investigator and any sub-investigators

IRB/EC approved consent form

IRB/EC study approval letter

IRB/EC name, location and membership list

IRB/EC certification

Signed Form FDA 1572

Financial Disclosure  

Signed research agreement


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12.3 Completion and Return of Case Report Forms
Electronic data capture (eDC) software will be used for this study. Data may be recorded onto 
data collections sheets prior to data entry or may be entered directly into the eDC system. 
Microbiological count data will be recorded onto DCSs prior to entry into the eDC system. 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). Once the 
forms are completed, the monitor will review the case report form (CRF) to assure accuracy and 
completeness.  
12.4 Final Report
 
 
 
 The Investigator will submit the Final Report to 
3M and the IRB/EC. 
12.5 Records, Reports and Retention Requirements
Study records must be kept until 3M provides notification that the documents can be destroyed.  
This may extend beyond the 2 years required by law. In order to comply with regulatory 
requirements, the Investigator must arrange for the retention of all study records for 2 years
following the date a marketing application is approved for the drug and indication investigated, 
or if no application is filed, until 2 years after completion/termination of the study [see 21 CFR 
312.62 (c) and 21 CFR 812.140(d)].  
Records to be retained in the Investigator study file include, but are not restricted to:

Signed study protocol, amendments, deviations

Applications to the IRB/EC

IRB/EC approval of protocol, consent form, authorization form and amendments to any of 
these documents

Signed consent forms 

Case report forms and/or data collection forms

Adverse event reports

Records of receipt, use, or disposition of the investigational drug

Correspondence relating to the study

Investigator Brochure 

Statement of Investigator

Financial disclosure documents

Final Report


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13. REFERENCES
1
Butterfield, CT.  The selection of a dilution water for bacteriological examinations.  J 
Bacteriol 1932; 23: 355-68.
2
ASTM International. ASTM E1173, standard test method for evaluation of preoperative, 
precatheterization, or preinjection skin preparations. West Conshohocken [PA]: ASTM Int’l; 
2015.
3
Williamson P, Kligman PM.  A new method for the quantitative investigation of cutaneous 
bacteria.  J Invest Dermatol 1965; 45:498-503.
4
ASTM International. ASTM E1054, standard test methods for evaluation of inactivators of 
antimicrobial agents.  West Conshohocken [PA]: ASTM Int’l; 2013.


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14. APPENDICES
14.1 Study Flow Diagrams
                         Informed Consent/Wash out and Screening Day


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                                            Treatment Day


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14.2 Study Product Application Instructions
 
 
 
 
  
 
 
d.


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14.3 Confirmation of Release & Receipt of Clinical Supplies
   
 


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14.4 Study Product Disposition Form
 
 
 
 


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14.5 Subject Instructions
 
 
 
 
 
 
 
 
 
 
 


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


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


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14.7 Test Site Diagrams
 
 
 
 
                       


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14.8 Skin Irritation Rating Scale


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14.9 Protocol for Neutralization Validation
14.9.1 Background
In order to accurately assess the efficacy of an antimicrobial product, it is necessary to 
completely inactivate the antimicrobial agent at the time point being evaluated. Inadequate 
neutralization will allow killing or inhibition of the microorganisms to continue beyond the 
specified contact time, resulting in an overestimation of antimicrobial activity.
Standard sampling solution (SSS) 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 the active study products. 
 
 
he effectiveness and nontoxicity of these neutralizer systems must be assessed to 
demonstrate that there is no effect on the growth of microorganisms and that the antimicrobial 
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 these neutralizers will be assessed against a lower 
bacterial concentration. 
This is an in vivo test where the active study products are applied to the skin using the same 
application instructions used for the study (Appendix 14.2). Scrub cup samples are collected and 
inoculated with low levels of
 
 Two contact times after inoculation 
will be evaluated: immediately (<1 minute) and
 minutes (to test for residual antimicrobial 
activity between sample collection and the time of plating).
14.9.2 Objective
The objective of this assay is to determine the ability of the sampling solutions to completely 
neutralize the active ingredients contained in
 when applied to the abdomen of test 
subjects without exhibiting toxicity to the test organisms.
14.9.3 Subject Entry Criteria
Six subjects will participate in this study. Subjects must meet the inclusion and exclusion criteria 
in Sections 4.1 and 4.2 of the protocol to which this neutralizer validation is attached, except for 
the baseline bacterial count, the 72-hour exclusion from showering/bathing, and the length of the 
washout period. The neutralization subjects do not require a minimum baseline count and they 
only need to avoid topical and systemic antimicrobials for 7 days (not 14 days) prior to 
Treatment Day. Subjects will be asked to sign the Informed Consent Form and provide 
information on demographics and inclusion/exclusion criteria before beginning the 7-day 
washout period. When subjects return to begin their participation in the study they will again be 
asked to provide information relative to inclusion/exclusion criteria. If they meet all 


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inclusion/exclusion criteria, they may be enrolled
 
.
Each subject will receive the active study product,
, which will be applied to the test 
area on the abdomen. See diagram below.
Neutralization Test Area Diagram
14.9.4 Test Organism
The test organisms for this study are
 
14.9.5 Study Products
 Surgical Solution, Tint, 26-mL applicator.


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14.9.6 Materials, Supplies and Equipment

Subject Informed Consent forms, IRB/EC-approved
 
 
 
 


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14.9.7 Study Procedures
14.9.7.1 Preparation of Inoculum
One day prior to beginning the neutralization assay, the test organisms from a stock culture slant 
or from a lyophilized vial will be inoculated on TSA. The plates will be incubated for 24 
 
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.
Dilute the inoculum suspension with PBW to achieve an appropriate concentration 
(approximately 104 CFU/mL) for inoculation of the test samples. The density of the test inocula 
must be verified by direct plating at the beginning and end of the study.
14.9.7.2 Preparation of Test Area and Post-Prep Sampling
Neutralization samples will be evaluated on the abdomen. Each subject will be treated with the
study product in the abdominal test area.
The abdominal test area will be marked using a sterile 1.5” x 5” template
 
A 1.5” x 5” area will be 
delineated on one side of the body containing two 1” x 1” sampling sites, yielding 2 samples per 
subject. The tube from one sampling area will be inoculated with 
 and the other will be 
inoculated with 
After the test area is marked, the test area will be prepped with three 70% isopropyl alcohol 
swabs for a total of 1 minute and allowed to dry for 3 minutes.
The test area will be treated with the study product, according to the instructions provided in 
Appendix 14.2 of the protocol to which this neutralizer validation is attached.
Samples will be collected, using the scrub cup technique, at 10 minutes 
 post-prep.  
Post-prep timing begins upon completion of study product application, including the 3-minute 
drying time.  The sampling technique is described in Section 6.2.1.1 of the protocol to which this 
neutralizer validation is attached.
Residual product should be removed from test area after sample collection is complete 
 
Each pooled sample (~6 mL) will be capped tightly
 
. 
 followed by vortex mixing for 30 seconds. Five mL 
will be transferred to a clean tube and immediately inoculated with 100 μL of the appropriate 


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diluted inoculum (Section 14.9.7.1 of the neutralization validation). This will yield an organism 
concentration of ~20 – 200 CFU/mL.
Immediately (<1 minute) and 
minutes 
 post-inoculation,
aliquots of 
the inoculated samples will be pour-plated in triplicate using TSA+N. Plates will be incubated 
inverted at 35 + 2oC for 48 
 hours.
14.9.7.3 Numbers Control and Toxicity Control
This testing will be performed in triplicate.
Numbers Control (NC):
Add 100 µL of the appropriate diluted inoculum to a tube containing 5 mL PBW to yield final 
inoculum concentrations of ~20-200 CFU/mL. 
 
Pour-plate triplicate
aliquots immediately (<1 minute) and 
 minutes 
 
 post-inoculation in the same manner as 14.9.7.2, except using TSA without 
neutralizers.
Toxicity Control (TC-MSS):
Add 
 5.0 mL SSS
 
 to a clean tube. Add 100 µL of the appropriate 
diluted inoculum to the 5-mL tube to yield final inoculum concentrations of ~20-200 CFU/mL.  
Pour-plate triplicate 
aliquots immediately (<1 minute) and 
minutes 
 
post-inoculation in the same manner as 14.9.7.2.
These controls will provide assurance that the test organisms are not adversely affected by the 
treatments or the sampling procedures.
14.9.7.4 Enumeration
 Enumerate plates and calculate 
CFU/mL for each sample. Convert data to log10 CFU/mL.
14.9.8 Control of Bias
Bias is controlled by internal controls and triplicate plating.
14.9.9 Data Evaluation
Recovery for each of the samples will be expressed as log10 CFU/mL.


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Neutralizer Effectiveness: 
If the mean log10 CFU/mL of the sample is not more than 0.3 log10 less than the mean log10
CFU/mL of the NC, the neutralization will be considered effective.

Mean log10 CFU/mL from NC − Mean log10 CFU/mL from sample (use corresponding time 
points)
Neutralizer Toxicity:
If the mean log10 CFU/mL of the TC is not more than 0.3 log10 less than the mean log10 CFU/mL 
of the NC, the sampling solutions will be considered non-toxic.

Mean log10 CFU/mL from NC − Mean log10 CFU/mL from TC (use corresponding time 
points).