United States
Environmental Protection
Agency
EPA/600/R-16/149 I July 2016
www.epa.gov/homeland-security-research
Evaluation of Sporicidal Wipes and
Liquid Agents for Decontamination of
Anthrax-Contaminated Surfaces by
Robotic Cleaners
Office of Research and Development
National Homeland Security Research Center

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EPA/600/R-16/149
July 2016
Evaluation of Sporicidal Wipes and
Liquid Agents for Decontamination of
Anthrax-Contaminated Surfaces by
Robotic Cleaners
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711

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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development's
National Homeland Security Research Center, funded and managed this investigation through Contract
No. EP-C-09-027, Work Assignment 72 with ARCADIS U.S., Inc. (ARCADIS), and Contract No. EP-
C-15-008 with Jacobs Technology Inc. (Jacobs). This report will be peer and administratively reviewed
and will be approved for publication as an EPA document. This report does not necessarily reflect the
views of the EPA. No official endorsement should be inferred. This report includes photographs of
commercially available products. The photographs are included for the purposes of illustration only and
are not intended to imply that EPA approves or endorses the products or their manufacturers. EPA does
not endorse the purchase or sale of any commercial products or services.
Questions concerning this document and its application should be addressed to the following individual:
Sang Don Lee, Ph.D.
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
109 T.W. Alexander Dr. (E343-06)
Research Triangle Park, NC 27711
Telephone No.: (919) 541-4531
Fax No.: (919) 541-0496
e-Mail Address: lee.sangdon@epa.gov

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Acknowledgments
The following individuals and organizations are acknowledged for review of this document:
United States Environmental Protection Agency
Leroy Mickelsen
Lukas Oudejans
Doris Betancourt

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Table of Contents
Disclaimer	i
Acknowledgments	ii
Figures	iv
Tables 	iv
Acronyms and Abbreviations	v
Executive Summary	vi
1	Introduction	1
2	Experimental Approach	2
2.1	Project Objectives and Robots Tested	2
2.2	Experimental Methods and Materials	3
2.2.1	Test Coupon Preparation and Sterilization	4
2.2.2	B. atrophaeus Spore Preparation	4
2.2.3	Coupon Inoculation	5
2.2.4	Robotic Cleaner Decontamination Procedures	7
2.2.4.1	R1 Decontamination Procedure	7
2.2.4.2	R2 Decontamination Procedure	7
2.3	Data Reduction	8
3	Sampling Approach	9
3.1	Sampling Site Environmental Conditions	9
3.2	Sampling Media	9
3.3	Wipe Sampling Procedures	9
3.4	Sampling Frequency	10
3.5	Critical Measurements	11
3.6	Prevention of Cross-Contamination of Samples during Sampling	11
3.7	Collection of Representative Samples	12
3.8	Sample Storage and Preservation	12
4	Analytical Procedures for Microbiological Analyses	13
5	Test Results	14
5 1 R1 (Mint Automatic Floor Cleaner 4200) Test Results	14
5.2 R2 (iRobot Scooba® 390) Test Results	16
6	Quality Assurance and Quality Control	20
8.1	Criteria for Critical Measurements/Parameters	20
6.2	Quality Control Checks	21
6.2.1	Integrity of Samples and Supplies	21
6.2.2	NHRSC Biolab Control Checks	21
8.3	QA/QC Sample Acceptance Criteria	21
6.3.1	QA/QC Test Results Validation	23
6.3.2	Sample Holding Time	23
7	Summary	24
8	References	25
Appendix A: Spatial Sampling Data	A-1
Appendix B: Sample Holding Time	B-1
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Figures
Figure 2-1. R1 (Left) and R2 (Right) Robotic Cleaners	2
Figure 2-2. ADA	5
Figure 2-3. MDI and MDI Actuator	5
Figure 2-4. Large Coupon Hot Spot Test Inoculation Area (Section e)	6
Figure 2-5. Large Coupon Broad Area Test Inoculation Areas (Sections a, e, and i)	6
Figure 4-1. Dilution Plate (Left) and Filter Plate (Right) Showing Colonies of B. atrophaeus	13
Figure 5-1. R1 Vinyl Hot Spot Test Results, Replicate 1 of (a) Hype-Wipe™ Cloth and
(b) pAB Cloth	15
Figure 5-2. Highest R2 Hot Spot LR Results for: (a) pAB on Vinyl, Replicate 2; and (b) Spor-
Klenz® on Vinyl, Replicate 1, and (c) pAB on Laminate	17
Figure 5-3. Highest R2 LR Results, Broad Area Inoculation	19
Tables
Table 2-1. Commercially Available Robotic Cleaners Evaluated	2
Table 2-2. Test Matrix for Robotic Cleaners	3
Table 2-3. Robotic Cleaner Decontamination Test Coupon Material Specifications	4
Table 3-1. Sampling Frequency	10
Table 3-2. Critical and Noncritical Measurements	11
Table 5-1. R1 Decontamination Results Summary	14
Table 5-2. R2 Decontamination Results Summary	16
Table 6-1. Critical Measurement Criteria	20
Table 6-2. QA/QC Sample Acceptance Criteria	22
Table 6-3. QA/QC Test Results	23
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Acronyms and Abbreviations
ADA
aerosol deposition apparatus
Anthrax
Bacillus anthracis
ATCC
American Type Culture Collection
B.
Bacillus
Biolab
National Homeland Security Research Center Biocontaminant Laboratory
CFU
colony forming unit(s)
DQI
data quality indicator
DQO
data quality objective
EPA
U.S. Environmental Protection Agency
EtO
ethylene oxide
ft2
square foot/feet
HSPD
Homeland Security Presidential Directive
H2O2
hydrogen peroxide
Lowe's
Lowe's Home Improvement
LR
log reduction
MDI
metered-dose inhaler
mL
milliliters)
MOP
miscellaneous operating procedure
NHSRC
National Homeland Security Research Center
NIST
National Institute of Standards and Technology
PAA
peracetic acid
pAB
pH-adjusted bleach
PBST
phosphate-buffered saline with 0.05% Tween® 20
PRB
polyester rayon blend
QA
quality assurance
QAPP
quality assurance project plan
QC
quality control
RH
relative humidity
STD
standard deviation
STS
sodium thiosulfate
TSA
tryptic soy agar
VHP®
vaporized hydrogen peroxide
V

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Executive Summary
In 2001, U.S. Postal Service system facilities were contaminated through the introduction of a few letters
containing Bacillus anthracis (anthrax) spores. The likelihood of occurrence of another such incident has
prompted the U.S. Environmental Protection Agency (EPA) to develop strategies, guidelines, and plans
for decontaminating persons, equipment, and facilities to mitigate the risks of contamination after a
biological weapons attack. This work is being conducted in accordance with Homeland Security
Presidential Directives (HSPDs)-5, -7, -8, and -10 in a coordinated effort with other federal agencies.
During the response to the U.S. Postal Service incident, fumigation was used primarily in heavily
contaminated facilities. Other cleaning methods were used in (1) secondarily contaminated areas (areas
handling cross-contaminated letters potentially in contact with the letters containing the anthrax spores
and areas where anthrax contamination was tracked from primarily contaminated areas), and (2) primarily
contaminated areas showing a minimal presence of anthrax spores. These other cleaning methods
included combinations of disposal of contaminated items, vacuuming, and the use of liquid sporicides
such as a pH-adjusted (pAB) bleach solution.
The use of commercially available robotic cleaners is a potentially effective decontamination technique for
the complete removal or inactivation of Bacillus (B.) spores. This report discusses a project that
investigated the ability of robotic cleaners to apply sporicidal liquid/wipe to floor surfaces and
inactivate/remove B. atrophaeus spores, formerly known as B. globigii, as a surrogate for anthrax spores.
Specifically, this report addresses the operational aspects of decontamination using robotic cleaners for
large surface areas tested under conditions that mimic their realistic field use.
This project evaluated the robotic cleaners for deactivation of B. atrophaeus on two test material surfaces:
laminate and vinyl. The decontamination procedures were performed using the following two robotic
cleaners:
•	Mint Automatic Floor Cleaner 4200 supplied with a North Star navigation system to track the
area cleaned tested with ready-to-use Hype-Wipe™ cloths or the manufacturer's cloth wetted
with a decontamination solution of pAB;
•	iRobot Scooba® 390 that uses a four-stage, hard-floor cleaning process (preparation,
washing, scrubbing, and using squeegees) tested with pAB or Spor-Klenz® directly sprayed
onto the floor surface and then vacuumed back up.
The Mint Automatic Floor Cleaner 4200 tests using both the Hype-Wipe™ cloths and the pAB-treated
cloths demonstrated a very low decontamination efficacy, with an overall log reduction (LR) in viable
spores of less than 1. Furthermore, a set of experiments was conducted to determine the extent of the
potential spreading of contamination by the Mint Automatic Floor Cleaner 4200 robotic cleaner using both
types of cloths, and results show that not only is the robotic cleaner ineffective at decontaminating a hot-
spot area, the Mint Automatic Floor Cleaner 4200 robotic cleaner also increases the total area
contaminated by spreading the contamination.
The iRobot Scooba® 390 tests showed better decontamination efficacy, with Spor-Klenz® showing better
results than pAB in limiting the spread of contamination outside the inoculation zone. However, the iRobot
Scooba® decontamination efficacy was below a 3 LR in viable spores overall regardless of the type of
liquid decontaminant used. For laboratory based testing, demonstration of a 6 LR is the target for an
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effective sporicide under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).
In summary, neither robotic cleaner successfully showed a decontamination efficacy of a 6 LR, in total
spore count nor significantly reduced the surface contamination amount. The maximum result was a 2.9
LR for the iRobot Scooba® 390 using Spor-Klenz® on vinyl. However, the average efficacy result for all
three replicates was only a 2.29 LR. Spor-Klenz® appears to be more successful at decontamination than
pAB on these surfaces with this application type, and increasing the reservoir volume of the iRobot
Scooba® 390 may increase the LR. Spor-Klenz® did help limit the spread of contamination to new areas,
although some spread was still observed.
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1 Introduction
In 2001, the introduction of a few letters containing Bacillus anthracis (anthrax) spores into the U.S.
Postal Service system resulted in the contamination of several facilities. Although most of the facilities in
which these letters were processed or received were heavily contaminated, the facilities were
successfully remediated using approaches such as fumigation with chlorine dioxide or Vaporous
Hydrogen Peroxide (VHP®). The likelihood of occurrence of another such incident has prompted the U.S.
Environmental Protection Agency (EPA) to develop strategies, guidelines, and plans for decontaminating
persons, equipment, and facilities to mitigate the risks of contamination after a biological weapons attack.
This work is being conducted in accordance with Homeland Security Presidential Directives (HSPDs)-5, -
7, -8, and -10 in a coordinated effort with other federal agencies. It is well agreed that available, effective,
and economical decontamination methods that can be used over wide areas (outdoors and indoors) are
required to increase preparedness to respond to such releases.
During the response to the U.S. Postal Service incident, fumigation was used primarily in heavily
contaminated facilities. Other cleaning methods were used in (1) secondarily contaminated areas (areas
handling cross-contaminated letters potentially in contact with the letters containing the anthrax spores
and areas where anthrax contamination was tracked from primarily contaminated areas) and (2) primarily
contaminated areas showing a minimal presence of anthrax spores. These other cleaning methods
included combinations of disposal of contaminated items, vacuuming, and the use of liquid sporicides
such as a pH-adjusted (pAB) bleach solution.
The use of commercially available robotic cleaners is a potentially effective decontamination technique for
the complete removal or inactivation of Bacillus (B.) spores. This report discusses a project that
investigated the ability of robotic cleaners to apply sporicidal liquid/wipe to floor surfaces and
inactivate/remove B. atrophaeus spores, formerly known as B. globigii, as a surrogate for anthrax spores.
Specifically, this report addresses the operational aspects of decontamination using robotic cleaners for
large surface areas tested under conditions that mimic their realistic field use.
The primary objective of this project is to provide agencies responding to occurrences of toxic biological
contamination with operational criteria that would lead to safe and efficacious decontamination of
bacterial agents on various materials and surfaces. This report specifically addresses the use of two
commercially available robotic cleaners in the removal or inactivation of B. anthracis spores under various
conditions (type of sporicide and type of surface).
Consistent with sporicidal efficacy tests used to register sporicides under FIFRA, the current study utilized
the generally accepted criterion of 6 Log Reduction to consider an approach effective. Tests were
conducted using commonly used surface materials and environmental conditions. Recovery of no viable
spores following treatment was considered highly effective.
Section 2 of this report discusses the experimental approach, Section 3 discusses the sampling
approach, Section 4 discusses analytical procedures for microbial analysis, Section 5 presents the test
results, and Section 6 discusses quality assurance (QA) and quality control (QC) for the project.
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2 Experimental Approach
Testing was conducted at EPA's facility at Research Triangle Park, NC This section describes the project
objectives and robots tested, experimental methods and materials, and the test organism and spore
preparation.
2,1 Project Objectives and Robots Tested
The project objective was to test commercially available, off-the shelf robotic cleaners equipped with
commercially available wipe cloths and decontamination solution for decontaminating indoor flooring
surface materials (laminate and vinyl) contaminated with B. atrophaeus spores (surrogate for anthrax).
The two robots used this study have been previously evaluated to determine their effectiveness for
sampling B, atrophaeus spores [1], These two selected robots use liquid or wet wipes for floor cleaning,
and these approaches are commonly used for surface decontamination. These robots were chosen to
represent two major groups: cloth-wipe type and spray type.
Table 2-1 lists the two types of robotic cleaners evaluated under this project. Figure 2-1 shows each type
of robot tested.
Table 2-1. Commercially Available Robotic Cleaners Evaluated
Robot No.
Model
Manufacturer
Cleaning type
Applicable
Surface
R1
Mint Automatic Floor
Cleaner 4200
Evolution Robotics, Pasadena,
CA
Sweep and
mop
Hard floor
R2
iRobot Scooba® 390
iRobot, Bedford, MA
Wet vacuum
Hard floor
Figure 2-1. R1 (Left) and R2 (Right) Robotic Cleaners
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The R1 robotic cleaner is the Mint Automatic Floor Cleaner 4200 supplied with a North Star navigation
system to track the area cleaned. This robotic cleaner uses dry or wet disposable cleaning cloths or
reusable microfiber cleaning cloths. The North Star navigation system tracks the area cleaned so that the
cleaner will not miss a spot. For this project, the R1 used ready-to-use Hype-Wipe™ cloths or the
manufacturer's cloth wetted with a decontamination solution of pH adjusted bleach (pAB).
The R2 robotic cleaner is the iRobot Scooba® 390, which uses a four-stage, hard-floor cleaning process
(preparation, washing, scrubbing, and using squeegees). For this project, the R2 used pAB orSpor-
Klenz® in the cleaner's reservoir directly sprayed onto the floor surface and then vacuumed back up. The
amount of liquid remaining in the R2 reservoir was measured as an indication of further cleaning capacity.
Table 2-2 summarizes the test matrix. This matrix was tested in triplicate.
Table 2-2. Test Matrix for Robotic Cleaners
Surface Material
Test Area Size
Robot
Decontamination Wipe
or Solution
Inoculated Area
Laminate
42 by 336 inches,
L shape
R1
pAB
Hype-Wipe™
Hot spot test area measuring 12 by 12
inches inoculated at minimum target
concentration of 107 spores per square
foot (ft2)
R2
pAB
Spor-Klenz®
Vinyl
R1
pAB
Hype-Wipe™
R2
pAB
Spor-Klenz®
Vinyl
R1
pAB
Hype-Wipe™
Broad area inoculation test areas include
12- by 12- inch sections inoculated at a
minimum target concentration of 10s
spores/ft2
R2
pAB
Spor-Klenz®
Laminate
R1
pAB
Hype-Wipe™
R2
pAB
Spor-Klenz®
2.2 Experimental Methods and Materials
This section describes the experimental testing and materials, including test coupon preparation and
sterilization, B. atrophaeus spore preparation, coupon inoculation, and the robotic cleaner
decontamination procedures. Digital video was collected during inoculation and decontamination.
Photographs were taken of selected material coupons with any visible change due to the decontamination
procedure.
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2.2.1 Test Coupon Preparation and Sterilization
The representativeness and uniformity of test materials are essential in achieving defensible evaluation
results. Materials are considered representative if they are typical of materials commonly found in facilities
and buildings in terms of quality, surface characteristics, and structural integrity. Material uniformity
means that all test materials of each type are equivalent. Uniformity was maintained by obtaining a
quantity of material sufficient to allow the preparation of multiple test coupons with presumably uniform
characteristics (that is, test coupons were cut from the interior rather than the edge of a large piece of
material).
For testing the robotic cleaners, two coupon materials were used: laminate and vinyl. Materials were
purchased locally. The test coupons were L-shaped and had a surface area of approximately 100 ft2 (42
by 336 inches). The positive and negative control coupons were 42 by 42 inches. The vinyl coupon was
made of one continuous piece of vinyl. Each laminate coupon had two layers of substrate with joints
offset. Joints of laminate were typical of office construction in accordance with the manufacturer's
recommendations. A 6-inch-high border was installed around each coupon to allow the robotic cleaner to
navigate as it would in an indoor area. Table 2-3 lists the coupon specifications.
Table 2-3. Robotic Cleaner Decontamination Test Coupon Material Specifications
Material
Description
Manufacturer or Supplier
Name
Coupon Surface
Size
Laminate
Flooring
7-5/8- by 50-3/4-inch Laminate Wood Locking
Flooring (#103553)
Lowe's store, Mooresville, NC
42 by 336
inches
Vinyl
Flooring
12-foot-wide River Park Staggered Slate
Brown Multi Vinyl Sheet (# G4820)
Armstrong World Industries Inc.,
Lancaster Township, PA
The test coupons were sterilized before inoculation using Dispatch® wipes (Clorox® Company, Oakland
CA). After 5 minutes, each coupon was wiped with a wipe soaked with 3% sodium thiosulfate (STS)
solution to neutralize or remove any remaining hypochlorite and then an alcohol wipe (Catalog No.
21910-110; VWR® Premoistened Clean-Wipes™ with isopropyl alcohol/deionized water, VWR,
Radnor, PA).
2.2.2 B. atrophaeus Spore Preparation
The test organism for this project was a powdered spore preparation consisting of a mixture of B.
atrophaeus (formerly B. globigii) spores (American Type Culture Collection [ATCC] 9372) and silicon
dioxide particles. The preparation was obtained from the U.S. Army Dugway Proving Ground Life Science
Division. The preparation procedure is fully reported in Brown et al. [2] and is summarized as follows.
After 80 to 90% sporulation, the suspension was centrifuged to generate a preparation of approximately
20% solids. The final product was a powdered matrix containing approximately 1 x 1011 viable spores per
gram prepared by dry blending and jet milling the dried spores with fumed silica particles (Degussa,
Frankfurt am Main, Germany).
The powdered preparation that was received was loaded into metered-dose inhalers (MDIs) at the EPA
facility in Research Triangle Park in accordance with a proprietary protocol. Control checks for each MDI
were included in each batch of coupons contaminated using a single MDI. The initial weight of each MDI
was verified using an Ohaus Adventurer Pro balance ARC120 (Ohaus Corporation, Parsippany, NJ).
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Ongoing control checks for each MDI were included in the batches of coupons contaminated using a
single MDi. The ongoing checks were performed using a Mettler-Toledo PL303 balance (Mettler-Toledo,
Inc., Columbus, OH).
2.2.3 Coupon Inoculation
Both the test and positive control coupons were inoculated independently with spores of 6. atrophaeus in
a separate dosing chamber called an aerosol deposition apparatus (ADA) designed to fit one 14- by
14-inch coupon of any thickness [3], The ADA consisted of a stainless-steel hood sized to exactly cover
the square test coupon. In the center at the top of the hood was an opening to attach an MDI and an MDI
actuator. ADAs were sterilized with a 250-part-per-million, four-hour VHP® sterilization cycle, and MDI
actuators were sterilized with ethylene oxide (EtO). Figures 2-2 and 2-3 show the ADA and MDi and MDI
actuator used in this project.
Figure 2-2. ADA	Figure 2-3. MDI and MDI Actuator
The MDI was discharged a single time into the ADA. The MDIs are claimed to provide 200 discharges per
MDI. The number of discharges per MDI was tracked so that use did not exceed this value. Additionally,
the weight of each MDI was determined after completion of the contamination of each coupon. The 14- by
14-inch stainless steel contamination control coupons were inoculated as the first and last coupons within
a single group of coupons inoculated by any one MDI within a single test. Spores were allowed to settle
onto the coupon surfaces for a minimum of 18 hours.
For the robotic cleaner hot spot tests, coupons were divided into eight areas (42- by 42-inch per area,
Areas A through H), each area containing nine 14- by 14-inch sections (sections a through i). Section e of
Area A was inoculated as shown in Figure 2-4.
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Figure 2-4. Large Coupon Hot Spot Test Inoculation Area (Section e)
The minimum target concentration was 107 spores/ft2. The robotic cleaner decontamination test was
started in section c of Area B.
For the robotic cleaner broad area inoculations, coupons were divided into 42- by 42-inch areas (Areas A
through H), each containing nine 14- by 14-inch sections (sections a through i). Sections a, e, and i of
Areas A, B, C, and D were inoculated as shown in Figure 2-5.
a
b
c
d
e
f
g
h
i
42"
Figure 2-5. Large Coupon Broad Area Test Inoculation Areas (Sections a, e, and i)
The minimum target concentration was 106 spores/ft2 for this test. The robotic cleaner decontamination
test was started in section c of Area B with the robot facing Area C. Routinely, the depositions exceeded
107 spores/ft2. In one of the broad area replicate tests, this minimum target concentration was not
reached. However, the results for this test did not differ significantly from results for the other two replicate
tests. Similar sections of all replicate test and positive control coupons were inoculated before moving on
to the next section. All section "a" areas were dosed before the section "e" areas, which were dosed
before the section "i" areas.
A log was maintained for each set of coupons dosed. Each record in this log recorded the unique coupon
identifier, the MDI unique identifier, the date, the operator, the weight of the MDI before dissemination into
the ADA, the weight of the MDI after dissemination, and the difference between these two weights. The
coupon codes were pre-printed on the log sheet before coupon inoculation (dosing) began.
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2.2.4 Robotic Cleaner Decontamination Procedures
The robotic cleaner decontamination procedures were performed using R1 and R2. R1 used pAB and
Hype-Wipe™ cloth wipes for decontamination, and R2 used pAB and Spor-Klenz®in the cleaning
reservoir. The pAB solution consisted of approximately 6,000 parts per million hypochlorite (measured as
free available chlorine) adjusted to a pH of 7 with acetic acid. Ready-to-Use Spor-Klenz®, an off-the-shelf
product, is an aqueous solution of hydrogen peroxide (H2O2) and peracetic acid (PAA). Three R1 units
and three R2 units were tested, one for each replicate test. The decontamination procedures are
discussed below for each robotic cleaner unit.
2.2.4.1	R1 Decontamination Procedure
1.	The Mint Automatic Floor Cleaner 4200 was removed from the box and placed where contamination
was unlikely.
2.	The R1 unit was checked to make certain that its batteries were fully charged. If necessary, the
batteries were charged to full capacity using the supplied cable. According to the manufacturer's
instructions, the batteries should be fully charged before any cleaning cycle begins. Battery installation
and removal procedures are discussed in the user's manual.
3.	The gray mopping cloth (8 by 11 inches) was sterilized over an 18-h cycle using an EtO sterilization
cabinet (EOGas 333, Anderson Products, Haw River, NC) before being secured to the cleaning pad.
Each mopping cloth was placed in a surgical EtO sterilization flat paper bag and then transferred to a
larger EOGas bag along with an EtO cartridge, a Humidichip®, and an EtO dosimeter.
4.	Directly before sampling, 60 milliliters (mL) of decontamination liquid was pipetted uniformly across the
gray mopping cloth.
5.	For Hype-Wipe™ testing, the decontamination wipes were used directly from their individual sachets
(each wipe was folded to fit the Mint cleaner pad [8 by 6 inches]). No additional decontamination liquid
was added.
6.	The R1 unit was deployed in the test area.
2.2.4.2	R2 Decontamination Procedure
1.	The iRobot Scooba® 390 was removed from the box and placed where contamination was unlikely.
2.	The R2 was inspected to make certain all parts were connected and installed in a functional manner
(such as making sure the filter was in its proper position).
3.	The R2 unit was checked to make certain that its batteries were fully charged. If necessary, the
batteries were charged to full capacity using the supplied cable. According to the manufacturer's
instructions, the batteries should be fully charged before any cleaning cycle begins. Battery installation
and removal procedures are discussed in the user's manual.
4.	Directly before sampling, 335 mL of the decontamination solution was loaded into the R2 reservoir using
the procedures summarized below.
a.	The reservoir was opened to remove the R2 tank.
b.	Using a serological pipette, 335 mL of the decontamination liquid was aseptically added to the tank.
c.	The tank was closed and tightly capped.
d.	The tank was inserted into the reservoir by pressing down on the tank. When a click was heard, the
tank was installed correctly.
5.	The R2 unit was deployed in the test area.
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2.3 Data Reduction
Data reduction was performed on measurements of the total spores (CFU) recovered from each replicate
coupon, average recovered CFU, and standard deviation (STD) for each group of coupons. The groups of
coupons included the following for each combination of material type and extracted sample type:
•	Positive control areas (replicate CFU, average recovered CFU, and STD);
•	Test areas (replicate CFU, average recovered CFU, and STD);
•	Procedural blank coupons.
Efficacy is defined as the extent (by LR) to which the agent recovered from the surface of the coupons
after decontamination has been reduced compared to the positive control areas (not decontaminated).
Efficacy was calculated using Equation 2-1, below, for each material within each combination of
decontamination procedure (i) and test material (j).
LR,, = lWCFUt,VN»,-lWCFU»,)iN;
C=1
Where,
LR,
^(cfuj'n,
c—1
2>&(CFUlt)'N
ijk
k=1
ijk
[2-1]
Average LR of spores on a specific material surface
Average of the logarithm of the number of viable spores (determined
by CFU) recovered on the control coupons [C = control, j = coupon
number, and Nc = number of coupons (1,7)]
Average of the logarithm of the number of viable spores (determined
by CFU) remaining on the surface of a decontaminated coupon [S =
decontaminated coupon, k = coupon number, and Nt = number of
coupons tested (1, k)]
When no viable B. atrophaeus spores were detected, the detection limit of the sample was used, and the
efficacy was reported as greater than or equal to the value calculated using Equation 2-1.
The pooled standard error for the LR was calculated using Equation 2-2 as follows:
/ SUn \
\A'yn/
+
- Tr
NTr
[2-2]
Where,
Nun
NTr
=	STD of LR values for untreated carriers (positive controls)
=	STD of LR values for treated carriers (post-decontamination samples)
=	Number of control samples
=	Number of post-decontamination samples
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3 Sampling Approach
This section discusses the sampling approach, including sampling site environmental conditions,
sampling media, wipe sampling procedures, sampling frequency, critical measurements, prevention of
cross-contamination during sampling, collection of representative samples, sample storage and
preservation, and sample holding time. Digital video was collected during sampling. Photographs were
taken of selected material coupons with any visible change due to the sampling procedure.
3.1	Sampling Site Environmental Conditions
Ambient environmental conditions such as temperature, relative humidity (RH), and barometric pressure
can affect the evaporation rate of liquids from surfaces. All tests were conducted at room temperature,
ambient RH, and ambient barometric pressure. RH and temperature were monitored during testing, and
tests were conducted on days when environmental conditions were within 35 to 60% RH and 20 to 25 °C.
All coupons were conditioned at ambient conditions for one week before use.
3.2	Sampling Media
Polyester rayon blend (PRB) 2- by 2-inch wipe (Curity™ All Purpose Sponges, Covidien, Mansfield, MA)
samples were collected from each 42- by 14-inch section (three linear 14- by 14-inch sections labeled a,
b, and c in Figure 2-4) of the positive control and test coupons. The PRB wipe samples were collected
from coupon surfaces after the surfaces had dried following decontamination. Atypical drying time of 30
minutes was used unless some combination of surface and decontamination method was still visually wet
after that time.
The used cloths from R1 and liquid from the reservoirs from R2 were collected, neutralized, and plated as
samples for initial tests. Wipe sampling always began in Area H. The robot decontamination always
started in Area B (section g), but the robot always followed a random path. Stainless-steel coupons (14 by
14 inches) were sampled to provide results indicating the MDI stability during inoculation.
3.3	Wipe Sampling Procedures
Wetted wipe sampling is typically used for small sample areas on nonporous smooth surfaces such as
ceramics, vinyl, metals, painted surfaces, and plastics. The general approach is to use a moistened,
sterile, nonwoven PRB cloth to wipe a specified area to recover bacteria, viruses, and biological toxins.
The protocol used for this project was adapted from that provided by Brown et al [2], The wetted wipe was
used to sample a 14- by 42-inch area. Wipe samples were extracted in 20 mL of Phosphate Buffered
Saline with 0.05% TWEEN®20 (PBST), sonicated, vortexed, and subjected to serial 10-fold dilution and
spread-plating. The sampling sequence started from Area H (sections c, f, and i or strip H3) and ended in
Area A (sections a, d, g or strip A1).
LR was determined using the sum of viable spores collected on wipes from all sections of each coupon
compared to the number of viable spores collected on wipes from positive control coupons not subjected
to decontamination.
9

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3.4 Sampling Frequency
Table 3-1 lists the sampling frequency for all samples collected.
Table 3-1. Sampling Frequency
Sample Type or
Measurement
Quantity
Frequency
Location or
Condition
Purpose
Test Coupon
Samples
Three samples per
coupon section for a
total of 24 wipe
samples (8 sections)
One set per test
Decontaminated
To determine the number of
viable spores after
decontamination
Negative Control
Coupon Samples
One per test coupon
replicate for a total
of 3 samples
One per
decontamination
Not
decontaminated
To determine extent of
cross-contamination and the
sterility of coupons
Positive Control
Coupon Samples
One per test coupon
replicate for a total
of 3 samples
One set per
material
Not
decontaminated
To determine the number of
viable spores recoverable
from the coupons
MDI Control
Coupon (stainless
steel) Samples
At least two per
inoculation event as
the first and the last
actuation of the MDI
One set per
inoculation
Not
decontaminated
To determine the number of
viable spores deposited
onto the coupons, and to
assess the stability of the
MDI
Laboratory Material
Blank Samples
Three per material
One per use of
material
Not applicable
To demonstrate sterility of
extraction and plating
materials
Material Sterility
Check (swab)
Samples
One per sterilized
batch of ADAs,
coupons, and robots
Once before
testing
Not applicable
To demonstrate sterility of
ADAs
Decontamination
Liquid
Test-dependent
Once before and
after each test
Not applicable
To determine the
concentration of active
ingredients in the solution,
and to verify the solution is
within the target
concentration
RH and
Temperature
1
Logged every 10
seconds
Glovebox
To determine environmental
conditions during
decontamination
10

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3.5 Critical Measurements
Table 3-2 lists critical and noncritical measurements for each relevant sample type.
Table 3-2. Critical and Noncritical Measurements
Sample Type
Critical Measurements
Non-critical
Measurements
Test Coupon
Samples
Plated volume, incubation temperature, extracted volume, and colony
forming units (CFU)
Storage time and
temperature
Negative Control
Coupon Samples
Plated volume, incubation temperature, extracted volume, and CFU
Storage time and
temperature
Positive Control
Coupon Samples
Plated volume, incubation temperature, extracted volume, and CFU
Storage time and
temperature
Field Blank
Coupon Samples
Plated volume, incubation temperature, extracted volume, and CFU
Storage time and
temperature
Laboratory Blank
Coupon Samples
Plated volume, incubation temperature, extracted volume, and CFU
Storage time and
temperature
Laboratory Material
Blank Samples
Plated volume, incubation temperature, extracted volume, and CFUs
Storage time and
temperature
pAB
pH, hypochlorite concentration, and storage time
Temperature
Spor-Klenz®
pH, H2O2 concentration, PAA concentration, and storage time
Temperature
3.6 Prevention of Cross-Contamination of Samples during Sampling
Sampling posed a potentially significant opportunity for cross-contamination of samples. To minimize the
possibility of cross-contamination, the management controls summarized below were followed.
•	Lines were drawn with a permanent marker to delimit the sampling sections, and sampling was
contained within these lines.
•	In accordance with aseptic technique, a two-person sampling team was used, consisting of a
"sampler" and a "sample handler."
•	The sample handler was designated as the only person to operate the automated dosing apparatus
(ADA) and handle the test coupons during the sampling event.
•	The sampler handled only the sampling media (wipes and liquids) and performed the surface
sampling of the test coupons.
•	At the completion of each sampling event, each member of the sampling team changed gloves in
preparation for working with the next sample.
As a further precaution to avoid cross-contamination of samples, the order of coupon sampling was from
coupons expected to be the least contaminated with B. atrophaeus to those expected to be most
contaminated as follows: (1) all blank coupons, (2) all decontaminated coupons, and (3) positive control
coupons.
The National Homeland Security Research Center (NHSRC) Biocontaminant Laboratory (biolab) located
at the EPA facility in Research Triangle Park, NC, analyzed the samples with strict adherence to aseptic
laboratory techniques to recover, plate, culture, and analyze samples. The order of analysis was the
same as the order of sampling as follows: (1) all blank coupons, (2) all decontaminated coupons, and (3)
all positive control coupons.
11

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3.7	Collection of Representative Samples
The representativeness and uniformity of test coupon materials was considered essential in achieving
defensible evaluation results. Material representativeness was achieved by using materials typical of
those currently used in buildings and facilities in terms of quality, surface characteristics, and structural
integrity. The materials conformed to industry standards or specifications for indoor use. Material
uniformity was achieved by obtaining a quantity of material sufficient to allow multiple test samples to be
prepared with presumably uniform characteristics.
3.8	Sample Storage and Preservation
After sample collection, sample integrity was accomplished through triple containment of the samples in:
(1) a sample collection container, (2) a sterile bag with the exterior sterilized during the sample packaging
process, and (3) a clean container holding all samples from a test. All individual sample containers
remained sealed in the coupon decontamination laboratory and during transport to the NHSRC
Biocontaminant Laboratory. The sampling person did not handle any samples after they were
relinquished to the sample handler during placement into the primary sample container.
All samples received were stored in a refrigerator at 4 °C ± 2 °C until analysis. All samples were allowed
to stabilize at room temperature for one hour before analysis.
12

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4 Analytical Procedures for Microbiological Analyses
This section discusses analytical procedures for microbiological analyses, including the filtration and
plating of bacteria from the PRB wipe samples used to determine the spores remaining on the coupons.
The NHSRC Biocontaminant Laboratory analyzed all samples for spore presence (sterility check
samples) and to quantify the CFU per sample. A laboratory notebook was used to document the details of
each sampling event (or test).
Spores were extracted from the PRB wipe samples by adding 20 mL PBST to each tube, then agitating
the tubes using a vortex mixer (set to maximum rotation) for wo minutes in ten-second intervals For all
sample types, after extraction, the liquid extracts were serially diluted ten-fold (in PBST). The PBST buffer
used was subjected to a five-stage serial dilution (10 1 to 10 5). Then 0.1 mL of the extract was spread-
plated onto tryptic soy agar (TSA) plates in triplicate and incubated overnight (18 to 24 hours) at 35 °C ± 2
°C. CFU were enumerated visually. Only dilution plates containing between 30 and 300 CFU were used
for recovery estimates. Extracts were diluted and replated if none of the 10-fold dilutions resulted in all
three plates containing CFU counts within the acceptable range.
Total spore recovery was calculated by multiplying the mean CFU counts from triplicate plates by the
inverse of the volume plated, by the dilution factor, and finally by the total volume of the extract. Any
samples with CFU counts below the acceptable range (30 to 300 CFU) on the primary dilution plates
were subsequently filter-plated through 0.2-micrometer-pore-size filters (Nalgene, Rochester, NY), with
the filters placed onto TSA plates, followed by incubation at 35 °C ± 2 °C for 18 to 24 hours. The CFU
counts from these plates were used to calculate recovery in these circumstances. Figure 4-1 shows a
dilution plate and a filter plate with colonies of B. atrophaeus.
Figure 4-1. Dilution Plate (Left) and Filter Plate (Right) Showing Colonies of B. atrophaeus
13

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5 Test Results
This section discusses data reduction and the results of the tests evaluating the R1 and R2 robotic
cleaners for inactivating B. atrophaeus on two test material surfaces (laminate and vinyl). Appendix A to
this report provides a complete set of spatial sampling data for each test condition.
5.1 R1 (Mint Automatic Floor Cleaner 4200) Test Results
The first set of R1 tests were conducted following a hot spot inoculation (14- by 14-inch section, e, of
Area A) on a vinyl coupon (42 by 336 inches) with a Hype-Wipe™ cloth mounted to the robotic cleaner.
Table 5-1 summarizes the results. The positive controls column shows the total number of CFU recovered
from the positive control coupon, and the test coupon column shows the total number of CFU recovered
from the entire 42- by 336-inch test coupon surface for each replicate experiment (3 replicates total for
each test). The LR calculated using Equation 2-1 shows that none of the tests were effective at removing
spores below a single LR, and the average of all three replicates gives a sample LR of 0.67, with a
sample pooled standard error of 0.17. This average is well below the 6 LR set as the target for laboratory
based testing if sporicidal decontaminants. Further, the results suggest strongly that the R1 robotic
cleaner would not be effective overall. It was initially decided to run only a single test using a pAB-treated
cloth to determine if additional testing would be needed. Table 5-1 also summarizes the results of this
test, which showed only a 0.73 LR in spore count. Therefore, it was decided to halt experiments using the
R1 robotic cleaner because it proved ineffective under the best circumstances.
Table 5-1. R1 Decontamination Results Summary
Test Area
Surface
Type
Cloth Type
Average Recovery (CFUs)
LR Average
Positive Controls
Test Samples
Hot Spot
A
Vinyl
Hype-Wipe™
1.30 ±0.31 x 107
3.06 ± 0.18 x107
0.67 ±0.17
pAB
1.30 107
2.40 107
0.73
Figure 5-1 shows the results of the spatial sampling for: (a) replicate 1 of the Hype-Wipe™ on vinyl test,
and (b) the single pAB-treated cloth on vinyl. Areas A through H in the figure represent the 42- by 42-inch
test coupon areas, P represents the full positive control coupon, and M represents the stainless-steel test
coupon used to check the sampling efficiency of the test swabs. The color scale indicates the order of
magnitude of the total number of CFU recovered from each sampling area.
The initial deposition was only in section A2 of the large test coupon. Figure 5-1 shows that a significant
number of spores were recovered far from the inoculation zone, indicating not only that the R1 robotic
cleaner was ineffective at decontaminating a single area. In addition it might also spread the spores.
Since the current test area (100 ft2) is within the manufacturer's recommended wet mopping area (~150
ft2), this spore spread during R1 robotic cleaner operation may be due to the insufficient decontamination
agent deployment. The positive control sampling results should have shown deposition only in the P2
zone. The CFU in the P1 and P3 zones likely are attributable to human error from sampling overlap into
zone P2. The CFU in the P1 and P3 zones were added to the P2 sample data to determine the total
spore inoculation. No broad-area inoculation tests were conducted using the R1 robotic cleaner.
14

-------
E-01
E+00
E+01
E+02
E+03
E+04
E+05
| E+06
E+07
E+08
1.08E+05
	
7.27E+05
2.01E+05
	
4.46E+04
5.42E+04
1.56E+05
5.20E+04
7.93E+05
5.11E+07
4.79E+07
Ml
M2
Positive Control
1.06E+07
Test Coupon
1.55E+06
LR
0.83
1.62E+05
3.40E+04
2.51E+04
4.98E+03
3.90E+03
3.60E+03
2.52E+03
1.88E+03
2.0SE+03
1.32E+03
2.17E+03
2.82E+03
2.75E+03
4.29E+03
3.07E+03
1.92E+03
510
340
B1 B2	B3 CI C2 C3 D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(a)
4.90E+05
1.70E+05
5.93E+05
1.83 E+05
Positive Control
1.30E+07
TestCoupon
2.40E+06
LR
0.73
1.01E+05 I 1.65E+04
3.06E-K54
2.16E+04
1.25E+04
1.13E+04
2.64E+04
1.15E+04
7.93E+03
3.87E+03
670
620
1460
980
4.17E+03
1030
1010
800
1300
980 I 240
B1	B2 B3 CI	C2 C3 D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(b)
Figure 5-1. R1 Vinyl Hot Spot Test Results, Replicate 1 of (a) Hype-Wipe™ Cloth and (b) pAB Cloth
15

-------
5.2 R2 (iRobot Scooba® 390) Test Results
The R2 tests were conducted on both hot spot and broad area inoculations on vinyl and laminate
coupons using both pAB and Spor-Klenz® in the reservoir of the cleaner. Table 5-2 summarizes the
results. The positive controls column shows the total number of CFU recovered from the positive control
coupon, and the test coupon column shows the total number of CFU recovered from the entire 14- by
336-inch test coupon surface for each replicate experiment. The hot spot inoculation tests showed a
maximum LR of 2.91 for Spor-Klenz® on vinyl. The average LR of the Spor-Klenz® hot spot
decontamination experiments was 2.29, with a sample pooled standard error of 0.56. The pAB hot spot
on vinyl test was not as successful as the Spor-Klenz® on vinyl test. Replicate 2 of the pAB hot spot tests
on vinyl showed an LR of 2.71, which was much higher than the other two replicates. The overall average
LR of the pAB on vinyl hot spot tests was 1.17, with a sample pooled standard error of 0.70. Only a single
test was conducted with pAB on laminate, and result showing a 1.50 LR was consistent with the vinyl test.
Therefore, no more hot spot tests were conducted.
Table 5-2. R2 Decontamination Results Summary
Test Area
Surface
Type
Decontaminant
Type
Average Recovery (CFUs)
LR Average
Positive Controls
Test Samples
Hot Spot A
Vinyl
pAB
1.57 x 107 ± 5.73 x 106
3.43 x 107 ± 3.16 x 106
1.17 ±0.70
Vinyl
Spor-Klenz0
1.47 x 107± 1.63 x 106
1.57 x 107 ± 5.73 x 106
2.29 ± 0.56
Laminate
pAB
1.08 x 107 ± 0.00 x 10°
3.46 x 107± 0.00 x106
1.50 ± 0.00
Broad Area
Vinyl
pAB
9.41 x 107±8.14x 107
1.57 x 107 ± 5.73 x 106
0.84 ± 0.48
Vinyl
Spor-Klenz0
1.96 x 108 ± 9.83 x 107
2.05 x 107 ± 2.03 x 107
1.10 ± 0.33
Figure 5-2 shows the spatial distribution of CFU left after decontamination for (a) replicate 2 of pAB on vinyl,
(b) replicate 1 of Spor-Klenz® on vinyl, and (c) the single pAB on laminate test. Here, (a) and (b) represent
the replicates with the highest LR for their respective test conditions. Areas A through H in the figure
represent the 42- by 42-inch test coupon areas, P represents the full positive control coupon, and M
represents the stainless-steel test coupon used to check the sampling efficiency of the test swabs. The color
scale indicates the order of magnitude of the total number of CFU recovered from each sampling area.
Spor-Klenz® was more successful than pAB at decontamination and much more successful at limiting
spread of the inoculum to previously uncontaminated areas (Areas B through H). Figures A-3 and A-4 in
Appendix A show the spatial sampling data for each triplicate of the pAB on vinyl and Spor-Klenz® on vinyl
tests, respectively. These replicates also show the limited spread of inoculum in the Spor-Klenz® tests
compared to the pAB tests. Although the Spor-Klenz® tests showed promise, all of the LR results were well
below the 6 LR target used to define effective sporicides in laboratory based testing.
However, to be thorough, two broad area decontamination tests were conducted for pAB and Spor-Klenz®
on vinyl. These tests showed an LR below 2 in surface spores.
Figure 5-3 shows the best decontamination replicate results from the two broad area inoculation tests for (a)
replicate 2 of pAB on vinyl and (b) replicate 2 of Spor-Klenz® on vinyl. In this case, as opposed to the hot
spot area tests, the total initial inoculation was calculated by sampling the positive control coupon and
multiplying the results by 4 because Areas A through D were inoculated. Spor-Klenz® is, on average,
significantly better than pAB at limiting the spread of inoculum to areas outside the initial contamination zone
(Areas A through D), although significant spread is shown in Figure A-6 (c) in Appendix A.
16

-------
E-01
E+00
E+01
E+02
E+03
E+04
E+05
| E+06
E+07
E+08
810
6.73E+03
1040
52
14
260
A1
A2
A3
5.35E+04
6.73 E+04
PI
P3
Positive Control
2.15E+07
Test Coupon
4.20E+04
LR
2.71
1460
3975
180
153
J	L
94 I 1320
'
H	
4.93E+03
4.83E+03
6.01 E+03
6.60E+03
2.87E+03 I 340
I
	i	
34
146
72
58
31
23
J	I	I	I	L
B1 B2 B3 CI C2 C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(a)
1260
9.27E+03
780
8.20E403 I 120
A1
A2
A3
800
2.58E+04
PI
P3
Positive Control
1.66E+07
Test Coupon
2.05 E+04
LR
2.91
"i	r
27
16
201
51
81
118
221
140
B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
(b)
Figure 5-2. Highest R2 Hot Spot LR Results for: (a) pAB on Vinyl, Replicate 2; and (b) Spor-Klenz® on Vinyl, Replicate 1, and (c) pAB on
Laminate
17

-------
3.70E+04
I-5LE4i5
L56E-KJ5
7.00E403
9.07E+03
6.68E+03
Positive Control
1.08E+07
Test Coupon
3.46E+05
LR
1.50
8.70E+03
287
_L
433
770
530
467
770
247
320
49
J	L
220
143
244
353
340
140
120
207
131
B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
(C)
Figure 5-2. [Continued]
18

-------
E-01
E+00
E+01
E+02
E+03
E+04
E+05
E+06
E+07
E+08
3.09E+05
1.11E+05
Positive Control
1.58E+08
Test Coupon
8.55E+06
LR
1.27
6.24E+05 9.67E+05
2.72 E+05
3.77E+05	5.33E+05
4.25E+05
2.93E+05
4.44E+04
2.69 E+04
4.64E+04
4.65E+04
_L
3.87E+04
3.03E+04
1.81 E+04
3.14E+04
2.36E+04
1.71E+04
2.10E+O4
B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
(a)
1.33E+05
A1
A2
Positive Control
2.84E+08
Test Coupon
4.87E+06
LR
1.77
2.33E+05 I 3.73E+03 I 2.84E+04	I 1.65E+05
6.67E+04 gWilJiUI 2.40E+03
100
i	r
		I
ii

528
B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
(b)
Figure 5-3. Highest R2 LR Results, Broad Area Inoculation
1S

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6 Quality Assurance and Quality Control
This project was performed under the approved Category III quality assurance project plan (QAPP),
"Evaluation of Sporicidal Wipes and Liquid Agents for Decontamination of Anthrax-Contaminated
Surfaces by Hand and Robotic Cleaners," November 2014.
All test activities were documented via narratives in laboratory notebooks and the use of digital
photography. The documentation included, but was not limited to, a record for each decontamination
procedure, any deviations from the QAPP, and physical impacts on materials. All tests were conducted in
accordance with established Decontamination Technologies Research Laboratory (DTRL) and NHSRC
Biocontaminant Laboratory procedures to ensure repeatability and adherence to the data quality
validation criteria set for this project.
6.1 Criteria for Critical Measurements/Parameters
The data quality objectives (DQOs) are used to determine the critical measurements needed to address
the stated objectives and specify tolerable levels of potential errors associated with simulating the
prescribed decontamination environments. The following measurements were deemed critical to
accomplish part or all of the project objectives:
•	Weight of the wipes;
•	Sample volume collected;
•	Plated volume; and
•	Counts of CFU.
The data quality indicators (DQIs) for the critical measurements listed in Table 6-1 were used to
determine if the collected data met the quality assurance objectives. If a measurement method or device
resulted in data that did not meet these goals, the data derived from the critical measurement were
rejected. For instance, if the plated volume of a sample was not known (i.e., was not 100% complete),
then that sample was declared invalid. If a collected sample was lost or did not meet the criteria for other
reasons, then another sample was collected to take its place.
Table 6-1. Critical Measurement Criteria
Critical Measurement
Measurement Device
Accuracy
Precision
Detection Limit
Sample volume
Serological pipette
Subdivision 0.5 mL
+ 0.2 mL
+ 0.1 mL
Plated volume
Pipette
± 2%
± 1%
NA
CFU/plate
Counting
± 10% (between 2 counters)
± 5
1 CFU
Weight
Scale
0.004 g
0.0001 g
NA
NA = not applicable
20

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6.2	Quality Control Checks
Many QA/QC checks were used in this project to ensure that the data collected met all the critical
measurements listed in Table 6-1. The measurement/parameter criteria were set at the most stringent
level that can routinely be achieved. The integrity of the sample during collection and analysis was
evaluated. Control samples and procedural blanks were included along with the test samples so that well-
controlled quantitative values were obtained. Background checks for the presence of bacterial spores
were included as part of the standard protocol. Replicate coupons were included for each set of test
conditions. Specific quality control checks that were performed in this project are described in the
following sections.
6.2.1	Integrity of Samples and Supplies
Samples were carefully maintained and preserved to ensure their integrity. Samples were stored away
from standards or other samples which could possibly cross-contaminate them.
Project personnel carefully checked supplies and consumables prior to use to verify that they met
specified project quality objectives. All pipettes were calibrated yearly by an outside contractor (Calibrate,
Inc.), incubation temperature was monitored using NIST-traceable thermometers, and balances were
calibrated yearly by the EPA Metrology Laboratory.
6.2.2	NHRSC Biolab Control Checks
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, the CFU were
enumerated manually and recorded. If the CFU count for bacterial growth did not fall within the target
range, the sample was either filtered or re-plated. For each set of results (per test), a second count was
performed on 25 percent of the plates within the quantification range (plates with 30 - 300 CFU). All
second counts were found to be within 10 percent of the original count.
6.3	QA/QC Sample Acceptance Criteria
The acceptance criteria for the critical CFU measurements were set at the most stringent level that could
be achieved routinely. Positive controls and procedural blanks were included along with the test samples
in the experiments so that well-controlled quantitative values were obtained. Background checks were
also included as part of the standard protocol. Replicate coupons were included for each set of test
conditions. Further QC samples were collected and analyzed to check the ability of the NHSRC Biolab to
culture the test organism, as well as to demonstrate that materials used in this effort did not themselves
contain spores. The checks included the following:
•	Negative control coupons: sterile coupons that underwent the same sampling process without
spore deposition.
•	Field blank coupons: sterile coupons carried to the decontamination location but not
decontaminated.
•	Laboratory blank coupons: sterile coupons not removed from NHSRC Biolab.
•	Laboratory material coupons: includes all materials, individually, used by the NHSRC Biolab in
sample analysis.
•	Stainless steel positive control coupons: coupons inoculated but not decontaminated.
21

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QA/QC acceptance criteria are shown in Table 6-2. These provide assurances against cross-
contamination and other biases of microbiological samples.
Table 6-2. QA/QC Sample Acceptance Criteria
Sample Type
Purpose
Acceptance Criteria
Corrective Actions
Frequency
Negative control
coupons
Determine extent of
cross-contamination in
test area.
No detectable
spores.
No corrective actions
were taken. Values
on coupons were
considered to have
resulted from cross-
contamination.
One per test
Field blank
coupons
Verify the presence of
coupons does not
introduce contamina-
tion into samples.
No detectable
spores.
Determine source of
contamination and
remove.
One per sample
type per test
Laboratory blank
coupons
Verify the sterility of
coupons using swabs
No detectable
spores.
Determine source of
contamination and
remove.
One per test per
coupon type
Laboratory
material coupons
Verify the sterility of
materials used to
analyze viable spore
count.
No detectable
spores.
Determine source of
contamination and
remove.
Three per material
per test
Blank TSA
sterility control
(plate incubated,
but not
inoculated)
Controls for sterility of
plates.
No observed growth
following incubation.
All plates are
incubated prior to
use, so any
contaminated ones
were discarded.
Each plate
Positive control
coupons
Used to determine the
extent of inoculation
on the coupons.
5 x 10® CFU, ± 0.5
log or 5 x 104 CFU,
± 0.5 log.
Outside target range:
discuss potential
impact on results
with EPA WACOR;
correct loading
procedure for next
test and repeat
depending on
decided impact.
One per test
Inoculation
control coupons
Used to determine
drift in the MDI.
CFU recovered from
the first coupon
must be ±0.5 log of
the last coupon.
Reject results and
repeat test.
Two per inoculation
Replicate plating
of diluted
microbiological
samples
Used to determine
variability in CFU
counts.
Reportable CFU of
triplicate plates
must be within
100%. Reportable
CFU are between
30 and 300 CFU per
plate.
Re-plate sample.
Each sample
22

-------
6.3.1 QA/QC Test Results Validation
The QA/QC test results for the whole sampling campaign are shown in Table 6-3. All field blanks and
inoculum blanks were found to be non-detects. However, some of the negative control blanks (2 out of 17)
were found to have been on the order of 1 x 103 CFU per coupon. Although some of the negative controls
were detects, they do not have a significant bearing on the overall results since decontamination results of
no more than 3 LR were found.
Table 6-3. QA/QC Test Results
Sample
Robot
Material
Inoculation
Type
Decontamination
Type
Average Recovery (CFU per coupon)
Negative
Control
Field Blank
Inoculum
Control
Blank
Repl
cate 1
Mint
Vinyl
Hot-Spot A
Hype-Wipe™
9
ND
1
Repl
cate 2
83
ND
ND
Repl
cate 3
671
ND
ND
Repl
cate 1
pAB
375
ND
ND
Repl
cate 1
Scooba
Broad Area
pAB
2
ND
ND
Repl
cate 2
ND
ND
ND
Repl
cate 3
7
ND
ND
Repl
cate 1
Spor-Klenz0
49
ND
ND
Repl
cate 2
6
ND
ND
Repl
cate 3
26
ND
ND
Repl
cate 1
Hot-Spot A
pAB
33
ND
ND
Repl
cate 2
956
4
ND
Repl
cate 3
22
ND
2
Repl
cate 1
Spor-Klenz0
ND
ND
ND
Repl
cate 2
4
ND
ND
Repl
cate 3
32
ND
ND
Repl
cate 1
Laminate
pAB
4
ND
ND
6.3.2 Sample Holding Time
After sample collection for a single test was completed, all biological samples collected for that test were
immediately transported to the NHSRC biolab along with the appropriate chain of custody form(s). The
QAPP for this project stated that samples were to be stored no longer than five days before beginning
primary analysis. However, given the volume of samples generated over a short period of time, the
samples had to be stored in the refrigerator for longer than five days before primary analysis began.
Therefore, tests were conducted to evaluate the potential reduction of bacterial spores in the wipe sample
containers as a function of time. The results from the sample holding tests showed no reduction of
bacterial spores. Details of this testing and the results are provided in Appendix B.
23

-------
7 Summary
The R1 tests using either Hype-Wipe™ or pAB treated wipe mounted to the robot demonstrated an
overall log reduction of less than 1. The results of a set of experiments conducted to determine the extent
of potential spreading of the contamination, using the R1 with either Hype-Wipe™ or a regular mopping
cloth impregnated with 60 mL of pAB, show that the R1 was not effective (less than 1 log reduction)
enough to decontaminate a hot spot area and R1 operation increases the total area contaminated by
spreading. This may be due to the insufficient liquid decontamination agent available for the entire test
surfaces. The robot is programmed to cover the entire test area (100 ft2) which is within the manufacture
estimated coverage area (150ft2), so it is highly possible that the decontamination agent from the robot
might have run out before completion.
The R2 tests resulted in a better decontamination efficacy, with Spor-Klenz faring better than pAB in
limiting the spread of the contamination and less spreading of the contamination outside the inoculation
zone. However, a sub 3 log reduction overall decontamination efficacy was found for this robot,
independent of the type of liquid decontaminant used. For laboratory based testing, demonstration of a 6
LR is the target for an effective sporicide under the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA). The low efficacy may be due to the lack of contact time between decontamination agent and
surface since the R2 is designed to apply the liquid and retrieve it in a relatively short period of time (less
than 5 seconds). The previous field study used the 10 min contact time of pH-amended bleach on all
surfaces as application procedure and this application method was highly efficacious.4
In summary neither robot successfully performed a 6 Log reduction in total spore count nor significantly
reduced the spores loading on the material surfaces. Spor-Klenz appears to be more successful than
pAB and increasing the reservoir for the Scooba robot may increase the log reduction. Spor-Klenz did
help limit the spread of the contaminant to new areas though some spread of viable spores was still seen.
24

-------
References
1.	Lee, S.D., Calfee, M.W., Mickelsen, L., Clayton, M., and Touati, A. Scenario-based evaluation of
commercially available cleaning robots for collection of Bacillus spores from environmental
surfaces. Remediation, 2014, 24(2):123-133.
2.	Brown, G.S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.S., Boucher, R.M.,
Tezak, M.S., Wilson, M.C., Rudolph, T., Lindquist, H.D., and Martinez, K.F. Evaluation of rayon
swab surface sample collection method for Bacillus spores from nonporous surfaces. Journal of
Applied Microbiology, 2007, 103(4):1074-1080.
3.	Lee, S.D., Ryan, S.P., and Snyder, E.G. Development of an aerosol surface inoculation method for
Bacillus spores. Applied and Environmental Microbiology, 2011, 77(5):1638-1645.
4.	U.S. EPA. Bio-Response Operational Testing and Evaluation (BOTE) Project - Phase 1:
Decontamination Assessment. U.S. Environmental Protection Agency, Washington, DC,
EPA/600/R-13/168, 2013.
25

-------
Appendix A: Spatial Sampling Data
E-01
E+00
E+01
E+02
E+03
E+04
E+05
E+07 E+08


1.08E»05
7.27E+05
2.01Ei05
4.46EI04
5.42EI04
A1
A2
7.93E+05
Positive Control
1.06E+07
Test Coupon
1.55E+06
LR
0.83
F>3
1.56Ei05
1.62E+05
3.40E>04
231E(04 I 4.98EKB
3.D0E103
3.60Ei03
2 52EI03
J	L
1.88Ei03
2.05EI03
1 32E>03
J	L
217E103
2.82Et03
275E(03
4.29Et03
3.07E+03
1.92E+03
J	1	L
510
340
B1
B2
B3
CI
C2
C3
01
02
03
El
11 E3
F1
F2
F3
G1
G2
G3
HI
H2
H3

(a)
7.62Ei04
2.87Et04
A1
Positive Control
1.65E+07
Test Coupon
2.55E+06
4.07E<03
2.67E.03
1.93E+05
LR
0.81
1.81E<04
1.83E»04
I	I	I
1340
787
J	L
440
480
413
84
330
150
i	r
148
284
293
i	r
320
453
427
t	r
469
J	I	I	I	I	I	1	I	I	L
340
B1 B2 B3 CI C2 C3 01 02 03 El C2 £3 F1 F2 F3 G1 G2 G3 HI H2 H3
(b)
Figure A-1. R1 Test Results on Vinyl, Hype-Wipe™ Cloth, Hot Spot Inoculation
A-1

-------
1.23E+05
3.37E+04 I 9.33E+04
A1
A2
A3
2.53E+05
2.53E+05
4.77E+07
1.05E+08
Ml
M2
Positive Control
1.18E+07
Test Coupon
5.07E+06
LR
0.37
7.60E+04
3.70E+04
6.47E+03
1.58E+04
7.07E+04
3.22E+04
7.93 E+04
3.12E+04
3.71E+03
9.3 3E+03
1950
1260
3.40E+03
J	L
1680
2.60E-K53
8.06 E+03
7.73E+04
2.67E+03
6.80E-t03
B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
(C)
Figure A-1 [continued]. R1 Test Results on Vinyl, Hype-Wipe™ Cloth, Hot Spot Inoculation
A-2

-------
4.90E+05
1.70E+05
1.01E+05 I 1.65E+04
3.06E+04
5.93 E+05
1.83E+05
7.80E+07
6.67E+07
Ml
M2
Positive Control
1.30E+07
Test Coupon
2.40E+06
LR
0.73
2.16E+04
1.25E+04 I 1.13E+04
2.64E+04
1.15E404
7.93E+03
3.87E+03
670
620
1460
980
4.17E+03
1030
1010
800
1300
980
240
B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
Figure A-2. R1 Test Results on Vinyl, pAB Cloth, Hot Spot Inoculation
A-3

-------
1.49E+05
A1
5.02E+05
4.26E+05
3.91E+07
3-21E407
Ml
M2
Positive Control
1.55E+07
Test Coupon
6.22E+06
LR
0.40
2.48E+05
A3
2.93E+04 I 3.23E+04 ! 2.49E-t04 I 1.61E-H34
1.35 E+04
8.33E+03 I 4.58E+03 I 1.04E+04 1 7.87E-t03 I 6.67E+03 I 1.19E+04 I 1.19E+04 I 3.91E+03 I 1917 2.28E-t03 I 3.99E+03 I 1720 I 1680 I 720 I 720 I 1200
B1 B2 B3 CI C2 C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(a)
810
6.73E+03
1040
T
A1
A2
A3
5.35E+04
6.73E+04
3.27E+07
4.05E-KJ7
Ml
M2
Positive Control
2.15E+07
Test Coupon
4.20E+04
LR
2.71
52
14
260
1460
Jl
3975
180
153

i
94 I 1320
!
4.93E+03
4.83 E+03
6.01 E+03
6.60E+03
2.87E+03
340
34
146
72
58
I	J	I	L
31
23
B1 B2 B3 CI C2 C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(b)
Figure A-3. R2 Test Results on Vinyl, pAB Solution, Hot Spot inoculation
A-4

-------
1.39E+0S
S.47E+04
3.41E+04
2.95E+04
3.10E+07
4.46E+07
Ml
M2
Positive Control
1.01E+07
Test Coupon
4.03E+06
LR
0.40
4.12E+03 I 1.67E+04
1.31E+04
1940
1660
1320
980
630
1460
1560
1020
680
920
407
600
373
243
207
104
127
192

B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
(C)
Figure A-3 [continued]. R2 Test Results on Vinyl, pAB Solution, Hot Spot Inoculation
A-5

-------

1260

9.27E+03
780
8.20E+03
I
i
120 1
1
i
800
2.5 8 E+04
4.15E+07
3.95E+07
Ml
M2
Positive Control
1.66E+07
Test Coupon
2.05 E+04
LR
2.91
i	r
27
J	I
16
201
51
81
118
221
140
81	B2	B3 CI C2	C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(a)
1.91 E+04
70
600
A1
A2
A3
26
1.19E+04
PI
P3
Positive Control
1.34E+07
Test Coupon
2.18E+04
LR
2.79
i	r
1 I 37 I 137 I 11
330
J	L
224
85
1 I 90

I	!	I
513
320
253
B1 B2 B3 CI C2 C3	D1 D2 D3
El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(b)
Figure A-4. R2 Test Results on Vinyl, Spor-Klenz® Solution, Hot Spot Inoculation
A-6

-------
2.48E+04
5.03E+03
1.87E+U4
1.75E+U5
5.66E+07
530E+07
Ml
M2
Positive Control
1.43E+07
Test Coupon
9.73E+05
LR
1.17
1740
52
24
65

1 I 1
450
B1 B2 B3 CI C2 C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(C)
Figure A-4. [continued] R2 Test Results on Vinyl, Spor-Klenz® Solution, Hot Spot Inoculation
A-7

-------
5.53E+04
1.15E+04
1.73E+04
A1
A2
A3
2.19E+05
1.46E+05
2.40E+05
4.73E+05
Positive Control
2.42E+06
Test Coupon
2.81E+05
M2
LR
0.93
1.37E+05
1.07 E+04
3.90E+03
1.94E-HD4
3.10E+03
2.52 E+03
1.18E+04
1750
1460
1100
440
820
640
1053
887 I 20
115
600
53 I 1
B1 B2 B3 CI C2 C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 63 HI H2 H3
(a)
3.09E+05
6.20E+05
1.50E+06
7.80E+05
1.11E+05
A1
A2
A3
1.35E+07
8.87E+06
1.71E+07
PI
P2
P3
3.13E+07
3.46E+07
Ml
M2
Positive Control
1.58E+08
Test Coupon
8.55E+06
LR
1.27
6.24E405 9.67E-KJ5
2.72E+05

3.77E+05 BlfctlJTiH 5.33E+05
4.25E+05
2.93E+05
4.44E+04
2.69 E+04
4.64E+04
4.65E+04
3.87E+04
3.03E404
1.81 E+04
3.14E+04 I 2.36E+04
i
i
	i	
1.71E+04
2.10E+04
B1 B2 B3 CI C2 C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(b)
Figure A-5. R2 Test Results on Vinyl, pAB Solution, Broad Area Inoculation
A-8

-------
1.40E+06 1.97E+06
5.61E+05
Positive Control
1.22E+08
Test Coupon
5.73E+07
LR
0.33
2.06E+06 2.77E+06 Wl.-Mnil.-1 7.80E+06 6.73E+05
1.88E+05
8.40E+04
3.72 E+04
3.62 E+04
1.77E+04
2.73E+04 I 1.97E-KJ4
2.93E+04
1.77 E+04
5.47E+03
8.27E+03
9.35E+03
1.34E+04
B1	B2	B3	CI	C2	C3	D1	D2	D3	El	E2	E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
(C)
Figure A-5 [continued]. R2 Test Results on Vinyl, pAB Solution, Broad Area Inoculation
A-9

-------

2.86E+06

1.30E+06

5.30E+06
3.26E+06
1.05E+05 3.29E405
B1
B2 B3
1.89E+06
1.33E405
7.33E+OS
2.33E405
I
3.73E+03 2.84E+04
B1
B2 B3
A1
A2
A3
1.77E+07
1.37E+07
2.22E+07
IP1
|P2
IP3
5.05E+07
5.15E+07
Ml
IM2
Positive Control
2.14E+08
Test Coupon
4.35E+07
LR
0.69
1.01E+07 2.18E+06
9.87 E+04
4.18E+04
339
(a)
A1
A2
3.99E+07
1.18E+07
1.94E-KJ7
|P1
|P2
|P3
3.40E+07
3.97E+07
IM1
|M2
Positive Control
2.84E+08
Test Coupon
4.87E+06
LR
1.77
1.65E+05
6.67 E+04
C3
2.40E-KJ3
100
11
528
D3	El
E2
(b)
E3	F1	F2	F3	G1	G2	G3	HI	H2	H3
Figure A-6. R2 Test Results on Vinyl, Spor-Klenz® Solution, Broad Area Inoculation
A-10

-------
1.61E+04
3.46E+05
7.73E+06
PI
P2
P3
3.98E+07
3.72E+07
Ml
IM2
Positive Control
9.01E+07
Test Coupon
1.31E+07
LR
4.44E+04
2.93 E+05
4.84E+0S	1.57E-t04
1.75E+03
3.38E+03
I	I	i	i	i	|
3.88E+03
53
510
12
11
J	!	!	!	!	L
0.84
8 I 6
330
B1 B2 B3 CI C2 C3	D1 D2 D3 El	E2	E3	F1	F2	F3	G1 G2 G3 HI H2 H3
(C)
Figure A-6 [continued]. R2 Test Results on Vinyl, Spor-Klenz® Solution, Broad Area Inoculation
A-11

-------
Appendix B: Sample Holding Time
The QAPP for this project stated that samples were to be stored no longer than five days before
beginning primary analysis. However, given the volume of samples generated over a short period of time,
the samples had to be stored in the refrigerator for longer than five days before primary analysis began.
Therefore, the tests described here were conducted to evaluate the potential reduction of bacterial spores
in the wipe sample containers as a function of time.
In this testing sequence, the following hypochlorite sporicidal wipes were tested on large coupon surfaces
to estimate the occurrence and potential reduction of bacterial spores as a function of remediation
activities:
•	Clorox® Healthcare™ bleach germicidal wipe;
•	Hype-Wipe™ bleach towelette; and
•	pAB wipe prepared in house by soaking Fisherbrand™ dry wipes in 4 mL pAB
The surface tested was glass (to represent nonporous indoor building materials). The coupons measured
3.5 by 3.5 feet (12.25 ft2). The test coupons were first subjected to a specific decontamination wipe. The
coupons were not inoculated before decontamination. Table B-1 summarizes the test matrix.
The testing approach consisted of the following steps:
1.	After an exposure time of 30 minutes, the coupons were wipe sampled as follows:
a)	Sections a, b, and c were sampled together and labeled as the Day 1 sample;
b)	Sections d, e, and f were sampled together and labeled as the Day 4 sample; and
c)	Sections g, h, and i were sampled together and labeled as the Day 7 sample.
2.	Each sampled wipe container was inoculated on Day 1 (1 day after sampling) with 0.1 mL of 2 x 108
CFU/mL microbial suspension (test organism solution) to result in a final suspension containing
approximately 2 x 107 CFU of the microorganism.
3.	The samples were analyzed by adding 20 mL of PBST to each container on the scheduled day (Day 1,
4, or 7), followed immediately by extraction and plating.
Table B-1. Holding Time Testing Sequence Test Matrix
Test ID
Decontaminant
Coupon
Section
Analyzed and
Plated (Day)
No. of Wipe
Samples
73-(C/H/P)-0-G-1-Ta-1
C = Clorox® Healthcare™
bleach germicidal wipe
H = Hype-Wipe™ bleach
towelettes
P = pAB wipe
a
Day 1
C test samples (3)
H test samples (3)
P test samples (3)
73-(C/H/P)-0-G-1 -Tb-1
b
73-(C/H/P)-0-G-1-Tc-1
c
73-(C/H/P)-1 -G-1 -T d-1
d
Day 4
C test samples (3)
H test samples (3)
P test samples (3)
73-(C/H/P)-1 -G-1 -T e-1
e
73-(C/H/P)-1 -G-1 -Tf-1
f
73-(C/H/P)-7-G-1-Tg-1
g
Day 7
C test samples (3)
H test samples (3)
P test samples (3)
73-(C/H/P)-7-G-1 -Th-1
h
73-(C/H/P)-7-G-1 -Ti-1
i
B-1

-------
The positive control and negative control coupons were not subjected to the decontamination wipe. The
negative control coupon and each of the positive control coupon sections were directly wipe-sampled with
a PRB wipe. While the positive coupons were inoculated with spores, the negative control coupons were
not. The procedural blank coupon was decontaminated with the hypochlorite wipe and then sampled but
not inoculated. Table B-2 shows the control sample test matrix for this sample holding time study.
Table B-2. Holding Time Testing Sequence Control Sample Test Matrix
Test ID
Type
Decontaminant
Coupon
Section
Analyzed
and Plated
(Day)
No. of Wipe
Samples
73-(C/H/P)-0-G-1 -Pa-1
Positive
control
C = Clorox®
Healthcare™
bleach
germicidal wipe
H = Hype-
Wipe™ bleach
towelette
P = pAB
a
Day 1
C test samples (3)
H test samples (3)
P test samples (3)
73-(C/H/P)-0-G-1 -Pb-1
b
73-(C/H/P)-0-G-1 -Pc-1
c
73-(C/H/P)-1 -G-1 -Pd-1
d
Day 4
C test samples (3)
H test samples (3)
P test samples (3)
73-(C/H/P)-1 -G-1 -Pe-1
e
73-(C/H/P)-1 -G-1 -Pf-1
f
73-(C/H/P)-7-G-1 -Pg-1
g
Day 7
C test samples (3)
H test samples (3)
P test samples (3)
73-(C/H/P)-7-G-1 -Ph-1
h
73-(C/H/P)-7-G-1 -Pi-1
i
73-( C/H/P)-(0/117 )-G-1 -XT -1
Procedural
blank
NA
0	= Day 1
1	= Day 4
7 = Day 7
1 per
decontamination
wipe per day (9)
73-( C/H/P)-(0/117 )-G-1 -NT-1
Negative
control
NA
1 per
decontamination
wipe per day (9)
The results from the hold tests are summarized in Table B-3 and indicate that the samples were not
affected by the longer holding time before analysis. There was no reduction of bacterial spores in the
sample containers as a function of time.
Table B-3. Sample Holding Test Results for Decontamination Efficacy Tests
Wipe Cloth
Day
Positive Control
Average Recovery
(CFU)
Test Sample Average
Recovery
(CFU)
LR
Average
STD
Average
STD
Average
STD
Clorox®
Healthcare™
bleach germicidal
wipe
1
1.42 x107
9.33 x105
1.34 x107
9.34 x105
0.02
0.03
4
1.40 x107
1.04 x10s
1.35 x107
1.83x10®
0.08
0.06
7
1.26 x107
4.82 x105
1.55 x107
1.08x10®
-0.04
0.03
Hype-Wipe™
bleach towelette
1
1.37 x107
1.48 x10s
1.46 x107
7.57 x105
-0.03
0.02
4
1.44 x107
5.18 x 105
1.40 x107
4.54 x105
-0.01
0.01
7
1.21 x 107
4.73 x105
1.36 x107
3.85 x105
0.00
0.01
pAB wipe
1
1.42 x107
1.34x10®
1.47 x107
1.77x10®
-0.01
0.05
4
1.35 x107
1.03x10®
1.38 x107
7.49 x105
0.01
0.02
7
1.29 x107
9.05 x105
1.27 x107
4.23 x105
0.05
0.01
B-2

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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300

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