March 2004
Environmental Technology
Verification Report
BIOQUELL, Inc.
CLARUS C Hydrogen Peroxide Gas
Generator
Prepared by
Battelle
Battelle
The Business of Innovation
Under a contract with
&EPA U.S. Environmental Protection Agency
ETV ElV ElV

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March 2004
Environmental Technology Verification
Report
ETV Building Decontamination Technology Center
BIOQUELL, Inc.
CLARUS C Hydrogen Peroxide
Gas Generator
by
James V. Rogers
Carol L. Sabourin
Michael L. Taylor
Karen Riggs
Young W. Choi
William R. Richter
Denise C. Rudnicki
Battelle
Columbus, Ohio 43201

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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program
described here. This document has been peer reviewed by the Agency and recommended for
public release. Mention of trade names or commercial products does not constitute
endorsement or recommendation by the EPA for use.
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting
the nation's air, water, and land resources. Under a mandate of national environmental laws,
the Agency strives to formulate and implement actions leading to a compatible balance
between human activities and the ability of natural systems to support and nurture life. To
meet this mandate, the EPA's Office of Research and Development provides data and
science support that can be used to solve environmental problems and to build the scientific
knowledge base needed to manage our ecological resources wisely, to understand how
pollutants affect our health, and to prevent or reduce environmental risks.
The Environmental Technology Verification (ETV) Program has been established by the
EPA to verify the performance characteristics of innovative environmental technologies
across all media and to report this objective information to permitters, buyers, and users of
the technology, thus substantially accelerating the entrance of new environmental
technologies into the marketplace. Verification organizations oversee and report verification
activities based on testing and quality assurance protocols developed with input from major
stakeholders and customer groups associated with the technology area. ETV consists of
seven environmental technology centers. Information about each of these centers can be
found on the Internet at http://www.epa.gov/etv.
Effective verifications of monitoring technologies are needed to assess environmental
quality and to supply cost and performance data to select the most appropriate technology
for that assessment. In 2002, EPA established the Building Decontamination Technology
Center at Battelle. Battelle plans, coordinates, and conducts verification tests of
decontamination technologies and reports the results to the community at large. Information
concerning this specific environmental technology area can be found on the Internet at
http ://www. epa.gov/etv/centers/center9 .html.
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. In particular we would like to
thank John Chang, U.S. Environmental Protection Agency (EPA); Doris Betancourt, EPA;
Shirley Wasson, EPA; Jeff Kempter, EPA; Phil Koga, U.S. Army Research Development
and Engineering Command (RDECOM); Barry Pyle, Montana State University; and Susan
Springthorpe, University of Ottawa, who reviewed the test/quality assurance plan and/or
verification report.
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Contents
Notice	ii
Foreword	iii
Acknowledgments	iv
List of Abbreviations	vii
1.	Background	1
2.	Technology Description	2
3.	Test Design and Procedures	4
3.1	Introduction	4
3.2	Test Design	5
3.3	Agents and Surrogates	5
3.4	Test Sequence	6
3.5	Coupon-Scale Testing	6
3.5.1	Preparation of Test Materials	7
3.5.2	Application of Agents to Test Coupons	7
3.5.3	Confirmation of Surface Applications	8
3.5.4	Decontamination	8
3.5.5	Ob servation of Surface Damage	11
4.	Quality Assurance/Quality Control	12
4.1	Equipment Calibration	12
4.2	Audits 	12
4.2.1	Techni cal Sy stem s Audit	12
4.2.2	Audit of Data Quality	13
4.3	QA/QC Reporting	13
4.4	Data Review	13
5.	Statistical Methods	15
5.1	Efficacy Calculations	15
5.2	Stati sti cal Analy si s	15
6.	Test Results	17
6.1 Efficacy	17
6.1.1	Bacillus anthracis Ames Spores	17
6.1.2	Bacillus subtilis (ATCC 19659) Spores	20
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6.1.3	Geobacillus stearothermophilus (ATCC 12980) Spores	23
6.1.4	Stati sti cal Analy si s	25
6.2	Damage to Coupons	27
6.3	Other F actors	27
6.3.1	Operation of the CLARUS C Unit	27
6.3.2	Operator Bias	29
7.	Performance Summary	30
8.	References	32
Figures
Figure 2-1. BIOQUELL, Inc. CLARUS C	2
Figure 3-1. Test Materials	4
Figure 3-2. Overview of Plas-Labs Compact Glove Box Modifications	9
Figure 3-3. Detailed Views of Plas-Labs Compact Glove Box Modifications (A-C)
and Condensationon on Surfaces Within the Compact Glove Box (D)	10
Figure 6-1. Representative Cycle Parameter Data from a Single Experiment	28
Tables
Table 3-1. Test Sequence and Parameters	6
Table 3-2. Material Characteristics	7
Table 4-1. Summary of Data Recording Process	14
Table 6-1. CLARUS C Decontamination of Bacillus anthracis Ames Spores	18
Table 6-2. Liquid Culture Growth Assessment of Bacillus anthracis Ames Spores	19
Table 6-3. Representative Liquid Culture Growth Assessment of Biological
Indicators/Spores Strips	20
Table 6-4. CLARUS C Decontamination of Bacillus subtilis Spores	21
Table 6-5. Liquid Culture Growth Assessment of Bacillus subtilis Spores	22
Table 6-6. Representative Liquid Culture Growth Assessment of Biological
Indicators/Spores Strips	23
Table 6-7. CLARUS C Decontamination of Geobacillus stearothermophilus Spores	24
Table 6-8. Liquid Culture Growth Assessment of Geobacillus stearothermophilus
Spores	25
Table 6-9. Representative Liquid Culture Growth Assessment of Biological
Indicators/Spores Strips	26
Table 6-10. Statistical Analysis of Mean Efficacy (Log Reduction) for Spores	26
Table 6-11. Representative Data from the CLARUS C Printout	28
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List of Abbreviations
ANOVA	analysis of variance
BDT	Building Decontamination Technology
BWD	Bare wood (pine lumber)
CFU	colony-forming unit
cm	centimeter
DL	decorative laminate
EPA	U.S. Environmental Protection Agency
ETV	Environmental Technology Verification
GM	galvanized metal ductwork
GS	glass
HEPA	high-efficiency particulate air
IC	industrial-grade carpet
in	in
PC	painted (latex, semi-gloss) concrete cinder block
ppm	part per million
PW	painted (latex, flat) wallboard paper
QA	quality assurance
QC	quality control
QMP	Quality Management Plan
SD	standard deviation
TSA	technical systems audit
vii

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viii

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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental tech-
nologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use
of improved and cost-effective technologies. ETV seeks to achieve this goal by providing
high-quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative
technologies by developing test plans that are responsive to the needs of stakeholders, con-
ducting field or laboratory tests (as appropriate), collecting and analyzing data, and
preparing peer-reviewed reports. All evaluations are conducted in accordance with rigorous
quality assurance (QA) protocols to ensure that data of known and adequate quality are
generated and that the results are defensible.
The EPA's National Risk Management Research Laboratory and its verification
organization partner, Battelle, operate the Building Decontamination Technology (BDT)
Center under ETV. The BDT Center recently evaluated the performance of the BIOQUELL,
Inc., CLARUS™ C hydrogen peroxide gas generator for decontaminating buildings.
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Chapter 2
Technology Description
The objective of the ETV BDT Center is to verify the performance characteristics of
technologies that are designed to be used to decontaminate indoor surfaces in buildings
contaminated with either chemical or biological agents as a result of an intentional attack or
accidental release. This verification report provides results for verification testing of the
CLARUS C unit. The following is a description of the CLARUS C unit, based on
information provided by the vendor. The information provided below was not verified in
this test.
The CLARUS C unit is a hydrogen peroxide gas generator (Figure 2-1) that uses a dual
circuit system. The first circuit provides high-efficiency particulate air (HEPA) filtration,
dehumidification, and hydrogen peroxide removal from the air stream via catalytic
conversion. The second circuit delivers high-concentration hydrogen peroxide and water
vapors. During gassing, the CLARUS C unit
recirculates the vapors through the second circuit,
constantly increasing the concentration of hydrogen
peroxide and water vapor within the chamber or
area intended for decontamination. This
recirculation and vapor injection continues until the
chamber reaches saturation, and the process of
microcondensation begins. In microcondensation, a
microscopic film of aqueous hydrogen peroxide
solution is deposited on all surfaces. Once the
gassing phase has been completed, the CLARUS C
unit returns to the first circuit and brings the
chamber to a safe condition by catalytically
converting the hydrogen peroxide to water
(humidity) and oxygen. Excess humidity is removed
via the refrigerant-based dehumidification plant. To
ensure that all essential data are captured, the
CLARUS C unit prints out all critical parameters
recorded throughout the cycle. The CLARUS C unit
has a personal computer connection for more in-
depth cycle analysis.
Figure 2-1. BIOQUELL, Inc.
CLARUS™ C
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The CLARUS C unit was designed to decontaminate enclosures of up to 7,000 cubic feet
(200 cubic meters). It weighs 300 pounds (128 kilograms), and is 26 in (68 cm) wide by 35
in (90 cm) in depth by 45 in (106 cm) in height. The dehumidification system is designed to
run continuously. Because there is no need for dehumidification regeneration down-time,
the CLARUS C unit can operate continuously, if required, from a normal domestic power
supply. The CLARUS C unit is controlled by a Siemens programmable logic controller,
which is complemented by optional sensors (including a microcondensation sensor),
allowing repeatable validated decontamination cycles.
For this verification test, the CLARUS C unit was attached to a Plas-Labs compact glove
box modified according to the vendor's instructions (see Section 3.5.4.1). The CLARUS C
unit and the glove box were connected by flexible supply and delivery gassing hoses that
were HEPA-filtered. A hydrogen peroxide sensor, relative humidity sensor, and pressure
sensing tube also were connected to the inside of the glove box, and data were transmitted
through the glove box wall to the CLARUS C unit.
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Chapter 3
Test Design and Procedures
3.1 Introduction
BWD GS
This verification test was conducted according to procedures specified in the Test/OA Plan
for Verification of Hydrogen Peroxide Vapor Technologies for Decontaminating Indoor
Surfaces Contaminated with Biological or Chemical Agents0' The biological and chemical
agents that pose a threat to buildings include toxic industrial chemicals, chemical warfare
agents, and biological warfare agents
(including biotoxins). The biological
agent selected for this verification test
was Bacillus anthracis (Ames strain). In
addition, two biological surrogates were
used: B. subtilis (ATCC 19659) and
Geobacillus stearothermophilus (ATCC
12980). Seven materials representing
indoor surfaces commonly found in
buildings were used for the verification
testing. The indoor surfaces tested (Figure
3-1) include
¦	Industrial-grade carpet (IC)
¦	Bare wood (pine lumber) (BWD)
¦	Glass (GS)
¦	Decorative laminate (DL)
¦	Galvanized metal ductwork (GM)
¦	Painted (latex, flat) wallboard paper
(PW)
¦	Painted (latex, semi-gloss) concrete
cinder block (PC).
GM
Figure 3-1. Test Materials
The objective of the verification testing was to evaluate the efficacy of the CLARUS C unit
to decontaminate a biological agent/surrogate. Efficacy was tested by applying a biological
agent and surrogates to the surfaces of test coupons and, after using CLARUS C, comparing
the number of viable spores on decontaminated and control (non-decontaminated) samples.
Visual inspection of the physical integrity of the test materials was performed, and
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observations were recorded before and after implementing the CLARUS C unit technology
in an effort to detect any degradation or chemical destruction of the material itself.
3.2	Test Design
Coupons were cut from larger pieces of the representative materials for each of the seven
indoor surfaces (Section 3.1), measuring 3/4 x 3 in (1.9 x 7.5 cm) and having varying
thickness from about 1/32 in (0.79 cm) to 3/8 in (0.95 cm), depending upon the material. In
triplicate, the coupons were placed into a biological agent safety hood, and aliquots of an
aqueous suspension of the biological agent were added to the surface of each coupon. Based
upon the concentration of the spores in the aqueous suspension, the number of spores added
to each coupon was calculated. The coupons were allowed to dry overnight. After drying,
the inoculated coupons intended for decontamination were transferred into a custom-
modified glove box and placed horizontally on a wire rack. Both blank (uncontaminated;
N=2) and control (inoculated with spores, but not decontaminated; N=3) coupons were
prepared, together with the inoculated coupons that were to be decontaminated (N=3).
Efficacy of the decontamination technology was determined by comparing the number of
viable spores on the control coupons (not decontaminated) to the number present on the
decontaminated coupons, expressed as a log reduction. Following extraction of spores from
the test, control, and blank coupons, efficacy was further evaluated for each biological
agent/surrogate by transferring each coupon into liquid growth medium and assessing
bacterial growth after 1 and 7 days.
Physical degradation of the indoor materials used as test surfaces was evaluated informally
in conjunction with the efficacy testing procedure. After decontaminating the test coupons,
the appearance of the decontaminated coupons was observed; and any obvious changes in
the color, reflectivity, and apparent roughness of the coupon surfaces were noted.
3.3	Agents and Surrogates
The following biological agent was used for verification testing:
¦	Bacillus anthracis spores (Ames strain).
To provide correlations with the biological agent results, two biological surrogates also were
used:
¦	Bacillus subtilis spores (ATCC 19659)
¦	Geobacillus stearothermophilus spores (ATCC 12980).
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Biological indicators and spore strips that were used to evaluate decontamination efficacy
included:
¦	Biological indicators, approximately 106 spores each: Bacillus subtilis (ATCC 19659)
and Geobacillus stearothermophilus (ATCC 12980) spores on steel sealed in Tyvek®
pouches
¦	Spore strips: with Bacillus atrophaeus (ATCC 9372) spores, approximately 106 spores
per strip, on a filter paper matrix in sealed glassine envelopes.
3.4 Test Sequence
In Table 3-1, a summary of the verification testing of the the CLARUS C unit is presented.
Verification testing was performed during a 10-week period that commenced in September
2003 and concluded in November 2003.
Table 3-1. Test Sequence and Parameters
Test
Procedure
Parameters l.\ alii a led
Dala Produced
ISiological
Enumerations
Log reduction
Elficacy Test
B. anthracis


B. subtilis


G. stearothermophilus


Liquid culture assessment of coupons
Positive/negative bacterial growth (1 and 7 days)

B. anthracis


B. subtilis


G. stearothermophilus


Biological indicators/spore strips
Positive/negative bacterial growth (1 and 7 days)

B. subtilis


G. stearothermophilus


B. atrophaeus

Coupon
Damage to test coupons
Visual observation of every test coupon in all
Damage

biological efficacy tests before and after


decontamination
3.5 Coupon-Scale Testing
Coupon-scale testing was used to evaluate the decontamination efficacy of the CLARUS C
unit by extracting and measuring the viable biological spores on test coupons.
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3.5.1 Preparation of Test Materials
Coupons used for biological agent decontamination were cut to about 3/4 x 3 in (1.9 x 7.5
cm) and prepared as shown in Table 3.2 by Battelle staff. Test coupons were visually
inspected, and the condition of each coupon was recorded. The length, width, and thickness
of the test coupons were measured and recorded. Chain-of-custody forms were used to
ensure that the test coupons were traceable throughout all phases of testing.
Table 3-2. Material Characteristics
Material
Lot, Batch, or
ASTM No., or
Observation
Manufacturer
/Supplier
Name
Approximate
Coupon Size,
L x W x Thick, in
Material Preparation
Decorative
Laminate
Laminate/ Formica/
White Matte Finish
Solid Surface
Design
3x3/8
Wiped with 70% isopropanol
Galvanized
Metal
Ductwork
Industry HVAC
standard 24 Gauge
Galvanized Steel
Accurate
Fabrication
3 x 3/8x0.0234
Cleaned with Acetone; wiped
with 70% isopropanol
Glass
C1036
Brooks Brothers
3 x 3/8 x 1/8
Cleaned with Acetone; wiped
with 70% isopropanol
Industrial-
grade Carpet
ShawTek,
EcoTek 6
Shaw Industries,
Inc
3x3/8
Wiped with 70% isopropanol
Concrete,
Cinder Block
ASTM C90
Wellnitz
3 x 3/8 x 3/8
Brush and roller painted all
sides. One coat Martin Senour
latex primer (#71-1185) and
one coat Porter Paints latex
semi-gloss finish (#919); wiped
with 70% isopropanol
Wallboard
Paper
05-16-03; Set-E-
493; Roll-3
United States
Gypsum
Company
3x3/8
Roller painted on one side using
Martin Senour Paints. One
primer (#71-1185) and two
finish (flat, #70-1001) coats;
wiped with 70% isopropanol
Wood
Screen Molding
(Pine Wood)
Kingswood
Lumber
3 x 3/8 x 1/4
Wiped with 70% isopropanol
3.5.2 Application of Agents to Test Coupons
Biological agent test coupons were laid flat in a Biological Safety Cabinet (BSC) Class III
and contaminated at challenge levels of approximately 1 x 108 spores per coupon. Working
stock suspensions of the spores at the required concentration were prepared, transferred to
the coupon using a micropipette, placing the suspension over the surface as small droplets.
After contamination with biological agent or surrogate suspension, the test coupons were
allowed to dry overnight, undisturbed. The next day, the inoculated test materials intended
for decontamination (and one blank) were transferred to an isolator glove box that was
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attached to the CLARUS C unit (see Section 3.5.4.1). The control inoculated test materials
(not intended for decontamination) and one blank were left undisturbed in the BSC II.
3.5.3	Confirmation of Surface Applications
To confirm the application density of biological agents and surrogates, the B. anthracis and
surrogate spore suspensions used to contaminate the coupons were re-enumerated on each
day of use. This enumeration was carried out as described in Section 3.5.4.2.
3.5.4	Decontamination
3.5.4.1 Verification Testing Apparatus and Parameters
A Plas-Labs Compact Glove Box (Model 830-ABC, modified according to BIOQUELL's
specifications (Figures 3-2 and 3-3 A-C), was used as the test chamber. The Plas-Labs
Compact Glove Box is 28 in (71 cm) wide by 23 in (59 cm) in depth by 29 in (74 cm) in
height and has a volume of 11.2 cubic feet (317 liters). The BIOQUELL unit was connected
to the test chamber. The cycle parameters specified by BIOQUELL to be used for the
testing were as follows:
•	Cycle pressure: 20 Pascals
•	Conditioning time: 10 minutes
•	Gassing time: 20 minutes
•	Gassing dwell: 20 minutes
•	H2O2 injection rate: 2.0 grams per minute
•	H2O2 dwell rate: 0.5 grams per minute
•	Aeration time: set for 9,999 minutes.
Temperature parameters were not specified, and the CLARUS C unit did not measure
temperature. Time, pressure, relative humidity, and hydrogen peroxide concentrations are
monitored by the CLARUS C unit. Data collected with respect to these parameters can be
printed by the CLARUS C unit. Using the specified cycle parameters, operation of the
CLARUS C unit resulted in condensation on the surfaces inside the compact glove box
(Figure 3-3 D).
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Direct Injection Port
Figure 3-2. Overview of Plas-Labs Compact Glove Box Modifications
3.5.4.2 Decontamination Efficacy
Biological agent/surrogate decontamination efficacy was quantified by measuring the viable
spores on both exposed (test) and unexposed (control) coupons. Each coupon was placed in
a 50-milliliter (mL) test tube containing 10 mL of sterile phosphate-buffered saline to which
0.1% Triton X-100 and -210 micrograms of catalase had been added. The purpose of the
Triton X-100 was to minimize clumping of spores, and the purpose of the catalase was to
neutralize residual hydrogen peroxide. For spore extraction, the tubes were agitated on an
orbital shaker for 15 minutes at room temperature. Each tube was then heat-shocked at 60 -
65°C for one hour to kill vegetative bacteria. Following heat-shock, 1.0 mL of each extract
was removed, and a series of dilutions through 10"7 were prepared in sterile water.
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Direct Injection Port
Figure 3-3. Detailed Views of Plas-Labs Compact Glove Box Modifications (A-C) and
Condensation on Surfaces Within the Compact Glove Box (D)
Spore viability was determined by dilution plating, using both the undiluted extracts and the
successive dilutions of each extract. One hundred microliters of the undiluted extract and of
each serial dilution were plated onto tryptic soy agar plates in triplicate, allowed to dry, and
incubated overnight at 35 to 37°C fori?, anthracis and B, suhli/is and at 55 to 60°C for G.
stearothermophilus. Plates were enumerated the next day, and the colony-forming units
(CFU)/mL were determined by multiplying the average number of colonies per plate by the
reciprocal of the dilution. Data were expressed as a mean ± standard deviation (SD) of the
number of CPUs observed. To calculate the efficacy of the decontamination treatment, the
number of spores remaining on the decontaminated test coupons was compared to the
number of spores on the control coupons. Efficacy for biological agents was expressed in
terms of a log reduction.
10
Microcondens

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An additional qualitative assessment of the CLARUS C unit efficacy was conducted
following spore extraction. After the extraction process described above, each coupon was
transferred to a sterile 50-mL tube containing 20 mL of tryptic soy broth culture medium.
The vials were sealed and incubated on an orbital shaker at the appropriate temperatures
(see above) for each organism. At 1 and 7 days post-decontamination, the tubes were
visually assessed qualitatively for viability as "growth" or "no growth." The biological
indicators and spore strips were also evaluated at 1 and 7 days post-decontamination for
"growth" or "no growth."
3.5.5 Observation of Surface Damage
Following decontamination, each test surface was examined visually to establish whether
decontamination using the CLARUS C unit caused any obvious damage to the surface. The
coupons were observed immediately after completing the decontamination process, but
before post-decontamination sampling. The surface was inspected by comparing the
decontaminated test surface with control coupons of the same test material. Differences in
color, reflectivity, contrast, and roughness were assessed and recorded.
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Chapter 4
Quality Assurance/Quality Control
QA/quality control (QC) procedures were performed in accordance with the Quality
Management Plan (QMP) for the BDT Center'2' and the test/QA plan for this verification
test.(1) QA/QC procedures and results are described below.
4.1	Equipment Calibration
All equipment (e.g., pipettes, incubators, Biosafety cabinets, etc.) used at the time of testing
was verified as being certified, calibrated, or validated.
4.2	Audits
Two types of audit were performed during the verification test: a technical systems audit
(TSA) of the verification test performance and an audit of data quality. Audit procedures are
described below.
4.2.1 Technical Systems Audit
The Battelle Quality Assurance Unit conducted a TSA on September 10, 2003, to ensure
that the verification test was being conducted in accordance with the test/QA plan(1) and the
BDT Center QMP.(2) As part of the TSA, test procedures were compared to those specified
in the test/QA plan, and data acquisition and handling procedures were reviewed.
Observations and findings from the TSA were documented and submitted to the Battelle
Verification Test Coordinator for response. None of the findings of the TSA required
corrective action. TSA records are permanently stored with the Quality Assurance Manager.
The EPA Quality Manager for the BDT Center conducted a TSA on September 10-11,
2003. A final TSA report from the EPA was received by Battelle on October 27, 2003.
Battelle responded to the TSA finding, and submitted a final response to the TSA report on
November 19, 2003. On November 28, 2003, it was noted by the EPA Quality Manager that
Battelle's responses to findings were acceptable and that the audit was complete.
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4.2.2 Audit of Data Quality
At least 10% of the data acquired during the verification test were audited. A Battelle
Quality Assurance Officer traced the data from the initial acquisition, through reduction and
statistical analysis, to final reporting to ensure the integrity of the reported results. All
calculations performed on the data undergoing the audit were checked.
4.3 QA/QC Reporting
Each audit was documented in accordance with Section 3.3.4 of the QMP for the ETV BDT
Center.(2) Once the audit reports were prepared, the Battelle Verification Test Coordinator
ensured that a response was provided for each adverse finding or potential problem and
implemented any necessary follow-up corrective action. A Battelle Quality Assurance
Officer ensured that follow-up corrective action was taken. The results of the TSA were
submitted to the EPA.
4.4 Data Review
Records generated in the verification test received a QC/technical review and a QA review
before they were used to calculate, evaluate, or report verification results. Table 4-1
summarizes the types of data recorded and reviewed. All data were recorded by Battelle.
The person performing the review added his/her initials and the date to a hard copy of the
record being reviewed.
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Table 4-1. Summary of Data Recording Process
Dillil lo lie
\\ here
How Often
Disposition of
Recorded
Keeorded
Recorded
l);il;i
Dates, times of test
Data forms
Start/end of test, and at
Used to organize/check test
events

each change of a test
results; manually incorporated


parameter
into spreadsheets as necessary
Test parameters
Data forms
When set or changed, or
Used to organize/check test
(agent/surrogate

as needed to document the
results; manually incorporated
identities,

sequence of test
in data spreadsheets as
concentrations, test


necessary
surfaces, test conditions,



etc.)



Sampling data
Data forms
At least at start/end of
Used to organize/check test


reference sample, and at
results; manually incorporated


each change of a test
into spreadsheets as necessary


parameter

Biological enumeration
Data forms
Throughout sample
Transferred to spreadsheets
and liquid culture

handling and analysis

assessment, chain of

process

custody, and results



Records and
Printout from the
Throughout
Reviewed and summarized to
observations on
CLARUS C unit;
implementation of the
support data interpretation
CLARUS C unit use
data forms
CLARUS C unit

Surface damage
Data forms
Start/end of test
Used to assess damage of test



materials following use of the



CLARUS C unit
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Chapter 5
Statistical Methods
The statistical methods for evaluating the efficacy of the CLARUS C unit are presented in
this chapter. Qualitative observations also were used to evaluate verification test data.
5.1	Efficacy Calculations
For biological agents and surrogates, decontamination efficacy was calculated as the log
reduction in viable organisms achieved by the CLARUS C unit. The efficacy (E), or log
reduction, for the biological agent, or surrogates was calculated as
E = log (N°IN)
where N° is the mean number of viable organisms applied to the control coupons (i.e., those
not subjected to decontamination), and TV is the number of viable organisms recovered from
each test coupon after decontamination. For decontaminated samples where viable
organisms were not detected, the efficacy was calculated as the log of the mean number of
viable organisms on the control coupons. Using the calculated log reduction for each test
coupon, the mean log reduction (efficacy) ± SD was calculated.
Percent recovery was calculated for each type of test material inoculated with each
biological agent/surrogate. Percent recovery (mean ± SD) was calculated by dividing the
number of biological organisms in the treated sample by the number of biological organisms
in the controls (non-decontaminated).
5.2	Statistical Analysis
For each material and species combination, log reduction was calculated as described above,
resulting in a total of 63 log reduction values. In cases where no viable colonies remained
after decontamination, one colony was assumed to be present for the purpose of this
calculation. A two-way analysis of variance (ANOVA) model with main effects for Bacillus
species and test material and interactions was fitted to the log reduction data. This model
was used to compare each mean to zero, compare each surrogate to B. anthracis (within
material) and compare each surrogate to B. anthracis for porous and non-porous materials.
T-tests or statistical contrasts were used for the comparisons, with no adjustment for
15

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multiple comparisons. The ANOVA model was fitted using the SAS® (version 8.2) GLM
procedure.
16

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Chapter 6
Test Results
The results of the verification test of the CLARUS C unit are presented in this section.
6.1 Efficacy
6.1.1 Bacillus anthracis Ames Spores
Exposure of material test coupons contaminated with B. anthracis Ames spores to the
CLARUS C unit, using the vendor's specified parameters (Section 3.5.4), resulted in decon-
tamination that varied according to the type of the test material (Table 6-1). The mean log
reduction of detectable viable B. anthracis Ames spores ranged from 3.01 to 7.92 across all
seven test materials. Three of these test materials (IC, BWD, PC) can be considered porous
(on the inoculated surface), while the other four test materials (GS, DL, GM, PW) can be
considered non-porous (on the inoculated surface). Based on the results for two of the
porous materials, IC and BWD, decontamination of B. anthracis Ames spores from porous
materials using the unit may be less effective than decontamination of non-porous materials.
The log reduction in viable spores detected on the porous materials was 3.01, 3.70, and 6.36
for IC, BWD, and PC, respectively. The log reduction in viable spores detected on the non-
porous materials was 7.92, 7.85, 7.54, and 6.92 for GS, DL, GM, and PW, respectively.
A liquid culture growth assessment at 1 and 7 days post-decontamination was performed to
determine whether viable B. anthracis Ames spores remained on the test materials following
the extraction step (Table 6-2). The extraction efficiency for spores on all seven test
materials was less than 100%; therefore, it was assumed that viable spores could remain on
the test materials. Each test material was wiped with 70% isopropanol prior to inoculation
(or non-inoculated blanks) with B. anthracis Ames spores; however, this isopropanol wash
does not guarantee sterility, especially with the porous materials. The test materials were not
autoclaved, due to some of the materials being damaged during the autoclaving process.
Therefore, to maintain equivalent treatment/handling of the test materials, a 70%
isopropanol wipe was used. The liquid culture assessment was intended to detect spores that
remained on the test material following the extraction step; however, since the materials
were not sterilized by autoclaving, this type of assessment may not discriminate between the
growth of B. anthracis and/or other bacteria.
17

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Table 6-1. CLARUS C Decontamination of Bacillus anthracis Ames Spores"
Tesl Malerial
Inoculum
Total Spores
V-ii Reco\en
HITicao
Imlusl rial-Grade Carpel (K )




Control
1.15 xlO8
6.87 ± 0.32 x 107
59.7 ±2.79
b
Decontaminated
1.15 xlO8
9.29 ±7.23 x 104
0.081 ±0.063
3.01 ±2.11 (2.62-3.55)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Bare Wood (BWD)




Control
1.07 x 108
9.61 ± 1.38 x 10s
8.98 ± 1.29
-
Decontaminated
1.07 x 108
3.30 ± 2.87 x 103
0.0031 ±0.0027
3.70 ±0.67 (3.20-4.46)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Glass (GS)




Control
1.12 x 108
8.41 ± 2.18 x 107
75.1 ± 19.5
-
Decontaminated
1.12 x 108
0
0
7.92 ± 0 (7.92)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Decorative Laminate (DL)




Control
1.15 xlO8
7.04 ± 1.00 x 107
61.3 ±8.71
-
Decontaminated
1.15 xlO8
0
0
7.85 ±0 (7.85)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Galvanized Metal Ductwork




(GM)
1.12 x 108
3.49 ±0.13 x 107
31.2 ± 1.12
-
Control
1.12 x 108
0
0
7.54 ± 0 (7.54)
Decontaminated
0
0
0
-
Blank (control)
0
0
0
-
Blank (decontaminated)




Painted Wallboard Paper (PW)
Control
1.07 x 108
8.25 ±0.63 x 106
7.71 ±0.59

Decontaminated
1.07 x 108
0
0
6.92 ± 0 (6.92)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Concrete (PC)




Control
1.15 xlO8
3.79 ± 1.68 x 107
32.9 ± 14.6
-
Decontaminated
1.15 xlO8
1.51 ± 2.62 x 103
0.0013 ±0.0023
6.36 ±2.11 (3.92-7.58)
Blank (control)
Blank (decontaminated)
0
0
0
0
0
0
-
aData are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction). The
efficacy range is shown in parentheses.
bNot Applicable
18

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Table 6-2. Liquid Culture Growth Assessment of Bacillus anthracis Ames Spores
Tcsl Malcrial


l);i\ 1


l)a\
1


SI S: S5 151
si s: r.i
Industrial-Grade Carpet (IC)
Control
Decontaminated








Bare Wood (BWD)
Control
Decontaminated
+
+
+
+
+
+
+
+
+
Glass (GS)
Control
Decontaminated
+
+
+

+
+
+
+
Decorative Laminate (DL)
Control
Decontaminated
+
+
+
-
+
+
+
+
+
+
-
Galvanized Metal Ductwork (GM)
Control
Decontaminated
+
+
+

+
+
+

Painted Wallboard Paper (PW)
Control
Decontaminated
+
+
+
+
+
+
+
+
Painted Concrete (PC)
Control
Decontaminated




+
+
+
+
+
-
51	= Sample 1
52	= Sample 2
53	= Sample 3
B1 = Blank (not inoculated with B. anthracis Ames spores)
"+" = growth; = no growth
Following the extraction step, each test coupon was placed into liquid culture to promote
spore germination, thereby enabling the vegetative bacteria to proliferate. Positive growth
was determined if the liquid culture medium turned cloudy, while no growth was
determined when the liquid medium remained clear.
All of the liquid culture samples for IC (both control and decontaminated) were negative for
bacterial growth. The brand of IC used for this test contains a product known as FlorSept®,
which is considered a broad spectrum antimicrobial that is effective against Gram-positive
and Gram-negative bacteria, as well as mold and fungi. It appears that under the conditions
employed for this verification test, the FlorSept® may not be sporicidal since viable
B. anthracis Ames spores were extracted from the IC and cultured on tryptic soy agar plates.
Therefore, it is possible that, in the liquid cultures, FlorSept® may inhibit growth of
vegetative cells derived from germination of the B. anthracis Ames spores. This growth
inhibition was not unique to B. anthracis, as these results were also observed for B. subtilis
and G. stearothermophilus (see Tables 6-5 and 6-8).
19

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For all tests using B. anthracis, the control biological indicators and spore strips exhibited
positive growth in the liquid cultures at both 1 and 7 days. No growth in the liquid cultures
was observed at 1 and 7 days for the biological indicators and spore strips subjected to
hydrogen peroxide exposure using the CLARUS C unit. A representation of the data from a
single test day is shown in Table 6-3.
Table 6-3. Representative Liquid Culture Growth Assessment of Biological
Indicators/Spore Strips
Indicalor (Origin ism)
l)a\ 1
l)a\ ^
si s: si
si s: Si
Biological Indicator (B. subtilis ATCC 19659) Control
Biological Indicator (G. stearothermophilus ATCC 12980) Control
Spore Strip (B. atrophaeus ATCC 9372) Control
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
Biological Indicator (B. subtilis ATCC 19659) Decontaminated
Biological Indicator (G. stearothermophilus ATCC 12980) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated


51	= Sample 1
52	= Sample 2
53	= Sample 3
"+" = growth; = no growth
6.1.2 Bacillus subtilis (ATCC19659) Spores
Exposure of test coupons contaminated with B. subtilis spores to the CLARUS C unit, using
the vendor's specified parameters (Section 3.5.4), resulted in decontamination that varied
according to the type of test material (Table 6-4). The log reduction of detectable viable B.
subtilis spores ranged from approximately 1.63 to 7.66 for all seven test materials. Based on
the results for two of the porous materials, IC and BWD, it appears that decontamination of
B. subtilis (ATCC 19659) spores by the CLARUS C unit was less effective for porous
materials than for non-porous materials. The log reduction in viable spores detected on the
porous materials was 1.63, 2.18, and 6.09 for IC, BWD, and PC, respectively. The log
reduction in viable spores detected on the non-porous materials was 7.57, 7.66, 6.44, and
7.52 for GS, DL, GM, and PW, respectively.
20

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Table 6-4. CLARUS C Decontamination of Bacillus subtilis Spores3
Tesl Malerial
Inoculum
Total Spores
V-ii Recn\er\
HITicao
Industrial-Grade Carpel (IC)




Control
9.26 x 107
4.69 ± 0.19 x 107
50.7 ±2.0
b
Decontaminated
9.26 x 107
1.16 ± 0.42 xlO6
1.25 ±0.45
1.63 ±0.15 (1.46-1.76)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Bare Wood (BWD)




Control
9.26 x 107
8.80 ± 2.24 xlO5
0.95 ±0.24
-
Decontaminated
9.26 x 107
8.06 ±6.11 x 103
0.0087 ± 0.0066
2.18 ±0.50 (1.81-2.75)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Glass (GS)




Control
9.00 x 107
3.71 ± 2.03 xlO7
41.3 ±22.6
-
Decontaminated
9.00 x 107
0
0
7.57 ± 0 (7.57)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Decorative Laminate (DL)




Control
9.00 x 107
4.57 ±0.85 x 107
50.8 ±9.49
-
Decontaminated
9.00 x 107
0
0
7.66 ± 0 (7.66)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Galvanized Metal Ductwork (GM)




Control
8.13 x 107
3.62 ± 0.76 xlO7
44.5 ±9.31
-
Decontaminated
8.13 x 107
3.33 ± 3.35 x 10
<0.0001
6.44 ±0.98 (5.73-7.56)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Wallboard (PW)




Control
8.13 x 107
3.31 ± 2.51 xlO7
40.7 ±30.8
-
Decontaminated
8.13 x 107
0
0
7.52 ± 0 (7.52)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Concrete (PC)




Control
9.26 x 107
1.26 ± 0.16 xlO7
13.6 ± 1.70
-
Decontaminated
9.26 x 107
2.20 ± 1.91 x 10
<0.0001
6.09 ±0.88 (5.58-7.10)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
aData are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction). The
efficacy range is shown in parentheses.
bNot Applicable
A liquid culture growth assessment at 1 and 7 days post-decontamination was performed to
determine whether viable B. subtilis spores remained on the test materials following the
extraction step (Table 6-5). As stated above, each test material (or non-inoculated blank)
was wiped with 70% isopropanol prior to inoculation with B. subtilis spores; however, this
isopropanol wash does not guarantee sterility, especially with the porous materials. There-
fore, positive growth observed in some of the test materials not inoculated with B. subtilis
spores may have resulted from growth of other bacteria not affected by the 70% isopropanol
wash.
21

-------
Table 6-5. Liquid Culture Growth Assessment of Bacillus subtilis Spores
Tcsl Malcrial


l);i\ 1


l)a\
1


SI S: S5 151
si s: S5 r.i
Industrial-Grade Carpet (IC)
Control
Decontaminated








Bare Wood (BWD)
Control
+
+
+
+
+
+
+
+

Decontaminated
+
+
+
-
+
+
+
-
Glass (GS)
Control
Decontaminated
+
+
+

+
+
+

Decorative Laminate (DL)
Control
Decontaminated
+
+
+
+
+
+
+
+
Galvanized Metal Ductwork (GM)
Control
Decontaminated
+
+
+

+
+
+

Painted Wallboard Paper (PW)
Control
Decontaminated
+
+
+
+
+
+
+
+
Painted Concrete (PC)
Control
Decontaminated

+
+


+
+

51	= Sample 1
52	= Sample 2
53	= Sample 3
B1 = Blank (not inoculated with B. subtilis spores)
"+" = growth; = no growth
For all tests using B. subtilis, the biological indicators and spore strips exhibited positive
growth in the liquid cultures at both 1 and 7 days. No growth in the liquid cultures was
observed at 1 and 7 days for the biological indicators and spore strips subject to hydrogen
peroxide exposure using the CLARUS C unit. A representation of the data from a single test
day is shown in Table 6-6.
22

-------
Table 6-6. Representative Liquid Culture Growth Assessment of Biological
Indicators/Spores Strips
Indicalor (Origin ism)

l)a\ 1
l)a\ ^

si s: si
si s: S5
Biological Indicator (B. subtilis ATCC 19b59)
Control
+ + +
+ + +
Spore Strip (B. atrophaeus ATCC 9372)
Control
+ + +
+ + +
Biological Indicator (B. subtilis ATCC 19659)
Decontaminated


Spore Strip (B. atrophaeus ATCC 9372)
Decontaminated


51	= Sample 1
52	= Sample 2
53	= Sample 3
"+" = growth; = no growth
6.1.3 Geobacillus stearothermophilus (ATCC12980) Spores
Exposure of test coupons contaminated with G. stearothermophilus (ATCC 12980) spores
to the CLARUS C unit, using the vendor's specified parameters (Section 3.5.4), resulted in
decontamination, that varied according to the type of test material (Table 6-7). The log
reduction of detectable viable G. stearothermophilus spores (ATCC 12980) ranged from
approximately 0.81 to 5.98 for all seven test materials. The log reduction in viable spores
detected on the porous materials was 0.81, 4.09, and 4.09 for IC, BWD, and PC,
respectively. The log reduction in viable spores detected on the non-porous materials was
4.68, 3.75, 1.97, and 5.98 for GS, DL, GM, and PW, respectively. For B. anthracis Ames
and B. subtilis (ATCC 19659) spores, porosity of the test material appeared to affect the
decontamination efficacy of the CLARUS C unit. For G. stearothermophilus (ATCC
12980), there was no clear trend in decontamination efficacy between the porous and non-
porous materials, as observed with B. anthracis and B. subtilis. Therefore, it is difficult to
determine whether the porosity of the test materials influenced the decontamination efficacy
of the CLARUS C unit. It appears that for one porous and one non-porous surface (IC and
GM, respectively) there was a negative effect on the extent of decontamination of
G. stearothermophilis (ATCC 12980) spores by the CLARUS C unit.
A liquid culture growth assessment at 1 and 7 days post-decontamination was performed to
determine whether viable G. stearothermophilus spores remained on the test materials
following the extraction step (Table 6-8). As stated above, each test material (or non-
inoculated blank) was wiped with 70% isopropanol prior to inoculation with
G. stearothermophilus spores; however, this isopropanol wash does not guarantee sterility,
especially with the porous materials. Therefore, positive growth observed in some of the test
materials not inoculated with G. stearothermophilus spores may have resulted from growth
of other bacteria not affected by the 70% isopropanol wash.
23

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Table 6-7. CLARUS C Decontamination of Geobacillus stearothermophilus Spores3
Tesl Malerial
Inoculum
Total Spores
V-ii Reco\cn
HITicao
Industrial-Grade Carpet (IC)




Control
1.28 x 108
2.69 ±0.051 x 107
21.0 ±0.4
b
Decontaminated
1.28 x 108
4.28 ± 1.08 x 106
3.34 ±0.84
0.81 ±0.10 (0.69-0.89)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Bare wood (BWD)




Control
1.28 x 108
2.76 ±0.081 x 106
2.15 ±0.063
-
Decontaminated
1.28 x 108
3.00 ± 2.02 x 102
<0.001
4.09 ±0.46 (3.80-4.61)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Glass (GS)




Control
1.28 x 108
8.72 ± 0.58 x 106
6.82 ±0.45
-
Decontaminated
1.28 x 108
2.45 ± 2.04 xlO2
<0.001
4.68 ±0.42 (4.27-5.11)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Decorative Laminate (DL)




Control
9.50 x 107
5.89 ± 1.12 x 106
6.20 ± 1.18
-
Decontaminated
9.50 x 107
1.26 ± 2.14 x 104
0.013 ±0.023
3.75 ± 1.37(2.20-4.77)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Galvanized Metal Ductwork (GM)




Control
1.28 x 108
1.50 ± 0.37 x 107
11.7 ±2.89
-
Decontaminated
1.28 x 108
1.64 ± 0.27 x 105
0.13 ±0.021
1.97 ±0.07 (1.90-2.04)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Wallboard Paper (PW)
Control
1.20 x 108
9.73 ±0.81 x 106
8.11 ±0.68

Decontaminated
1.20 x 108
2.20 ± 1.91 x 10
<0.0001
5.98 ±0.88 (5.47-6.99)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Concrete (PC)




Control
1.20 x 108
9.37 ± 1.05 x 10s
7.81 ±0.87
-
Decontaminated
1.20 x 108
2.85 ± 4.11 xlO3
0.0024 ± 0.0034
4.09 ± 1.03 (3.09-5.15)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
aData are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction). The
efficacy range is shown in parentheses.
bNot Applicable
24

-------
Table 6-8. Liquid Culture Growth Assessment of Geobacillus stearothermophilus
Spores
lesl Malerial
l)a\ 1
l)a\ ^
SI s: S3 B1
si s2 S' r.i
luduslrial-Grade Carpel (1C) Cuiiliul
Decontaminated

+
Bare Wood (BWD) Control
Decontaminated
1 1
+ +
+ +
+
i i
+ +
+ +
+ +
Glass (GS) Control
Decontaminated
+ + + +
+ + + +
Decorative Laminate (DL) Control
Decontaminated
+ + + -
+ + + -
Galvanized Metal Ductwork (GM) Control
Decontaminated
1 1
+ +
+ +
+ +
i i
+ +
+ +
+ +
Painted Wallboard Paper (PW) Control
Decontaminated
+ + + +
+ + + +
Painted Concrete (PC) Control
Decontaminated
+ + + -
+ + + -
51	= Sample 1
52	= Sample 2
53	= Sample 3
B1 = Blank (not inoculated with G. stearothermophilus spores)
"+" = growth; = no growth
For all tests using G. stearothermophilus, the biological indicators and spore strips exhibited
positive growth in the liquid cultures at both 1 and 7 days. No growth in the liquid cultures
was observed at 1 and 7 days for the biological indicators and spore strips subject to
hydrogen peroxide exposure using the CLARUS C unit. A representation of the data from a
single test day is shown in Table 6-9.
6.1.4 Statistical A nalysis
Table 6-10 presents the mean log reduction in spores sorted by material type. Significant
differences are also indicated in the table. All means were significantly different from zero
except for the mean log reduction of G. stearothermophilus for carpet, indicating that the
technology decontaminated statistically significant numbers of spores on these materials.
25

-------
Table 6-9. Representative Liquid Culture Growth Assessment of Biological
Indicators/Spores Strips
Indicalor (Origin ism)
l)a\ 1
l)a\ ^
si s: si
si s: Si
liiological Indicator ((#. slcarolhcrmopliilus ATCC 12980) Control
Spore Strip (B. atrophaeus ATCC 9372) Control
+ + +
+ + +
+ + +
+ + +
Biological Indicator (G. stearothermophilus ATCC 12980) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated


51	= Sample 1
52	= Sample 2
53	= Sample 3
"+" = growth; = no growth
Table 6-10. Statistical Analysis of Mean Efficacy (Log Reduction) for Spores
Malcrial
li. anthracis
li. subtilis
< i. slcarolhiTiiiophilus
Porous
Industrial-Grade Carpet
3.01a
1.63 ab
0.81 b
Painted Concrete
6.36a
6.09a
4.09 ab
Bare Wood
3.70a
2.18ab
4.09a
Non-Porous
Glass
7.92a
7.57a
4.68ab
Decorative Laminate
7.85a
7.66a
3.75ab
Painted Wallboard Paper
6.92a
7.52a
5.98a
Galvanized Metal
Ductwork
7.54a
6.44a
1 97ab
aMean significantly different from 0 at the (P<0.05)
bSurrogate significantly different from B. anthracis for specified material (P<0.05).
For both IC and BWD, there appeared to be a negative effect on the degree of
decontamination of B. anthracis and the two surrogates by the CLARUS C unit.
Comparisons within each material indicated that B. subtilis had a significantly lower mean
log reduction in spores from B. anthracis for IC and BWD. G. stearothermophilus had
significantly lower mean log reductions in spores from B. anthracis for IC, PC, GS, DL, and
GM. For two of the three porous materials, both B. subtilis and G. stearothermophilus had
significantly different mean log reductions from B. anthracis. For non-porous materials, G.
stearothermophilus was significantly different than B. anthracis. These overall results are
consistent with the results for each material.
26

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6.2	Damage to Coupons
Subsequent to decontamination, the test coupons were evaluated qualitatively for visible
surface damage. No damage (e.g., change in surface texture, color, etc.) and no visible
changes to any of the test materials were observed during this verification test.
6.3	Other Factors
6.3.1 Operation of the CLARUS C Unit
The CLARUS C unit was operated for approximately 160 hours during this verification test.
By following the user manual, the CLARUS C unit can be set up and programmed for
operation within minutes. The program containing defined test parameters can be stored,
retrieved, and executed by the CLARUS C unit within seconds. The only maintenance
required for the CLARUS C unit during this verification test was the addition of new
hydrogen peroxide at the beginning of each run and disposal of unused hydrogen peroxide
and waste by-product (i.e., water) at the end of each run. The printer paper had to be refilled
once during testing.
Data for the cycle parameters (hydrogen peroxide concentration, relative humidity, and
pressure) are monitored in real-time. The data are stored by a personal computer connected
to the CLARUS C unit, as well as displayed on the control panel. A secondary record of the
data is provided as a printout from the CLARUS C unit. The data on the printout consist of
values from the start of the cycle and the start and completion of each phase. The data
collected with respect to cycle parameters were acquired as the paper printout from the
CLARUS C unit. As a representation of the data collected, Table 6-11 shows values from a
single run. These values include pressure, relative humidity, and hydrogen peroxide
concentration within the Plas-Labs Compact Glove Box, and were acquired as the paper
printout from the CLARUS C unit.
For this verification test, cycle parameter data were not collected in real time by the
personal computer that was provided with the CLARUS C unit. This was due to a non-
functional networking card that is intended to enable communication between the CLARUS
C unit and the personal computer. The networking card was replaced near the end of the
verification test, and sample data were collected for the last two runs. Figure 6-1 is a
graphical representation of the real-time measurements for hydrogen peroxide concentra-
tion, relative humidity, and pressure from 0 to 9 hours of an approximately 16-hour run.
From the real-time data collected, beyond 9 hours, the hydrogen peroxide concentration and
relative humidity were approximately 0 ppm and 0%, respectively, while the pressure was
approximately 20 Pascals inside the Plas-Labs Compact Glove Box.
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Table 6-11. Representative Data from CLARUS C Printout
Phase
Pressure (Pa)
Kelali\e llumidil> ("<")
ll;(); (o neon Million (ppm)
Conditioning



Start
0
25.2
0
Finish
20.4
41.4
0
Pre-Gassing



Start
16.0
NRa
0
Finish
20.8
NR
0
Gassing



Start
20.8
NR
0
Finish
14.5
NR
923.6
Gassing Dwell



Start
14.5
NR
923.6
Finish
25.2
NR
1000.0
Aeration



Start
25.2
NR
1000.0
Finish
8.9
NR
0
aNR = not reported on printout.
1200
1000
800
600
400
200
0.0
Cycle Parameter Data
¦Relative Humidity (%)
Pressure (Pascals)
¦ Hydrogen Peroxide
Concentration (ppm)

1.0 2.0 3.0 4.0 5.0
Time (hours)
6.0
7.0
9.0
Figure 6-1. Representative Cycle Parameter Data from a Single Experiment
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6.3.2 Operator Bias
Due to the automated capabilities of the CLARUS C unit, there is little room for operator
error. The CLARUS C unit provides storage of specific cycle parameters, thereby enabling
the user to turn on the machine, select the pre-programmed cycle parameters, and press start.
The machine runs through the cycle and remains in the aeration phase (set for 9,999
minutes) until the machine is turned off. The aeration phase was allowed to run overnight
and shut off the next morning; therefore, a total run time from start to finish was
approximately 16 to 18 hours. By the end of the aeration phase, the hydrogen peroxide
concentration was 0 ppm. During the run, the hydrogen peroxide concentration, relative
humidity, and pressure was monitored and regulated by the CLARUS C unit, thereby
preventing operator error associated with these parameters.
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Chapter 7
Performance Summary
For this verification test, the CLARUS C unit demonstrated a range of decontamination
efficacy for all three biological organisms on all seven test materials. Based on these results,
different material types influence the efficacy of decontamination differently for various
organisms. IC and BWD had the lowest level of B. anthracis Ames and B. subtilis (ATCC
19659) spore decontamination. However, for G. stearothermophilus (ATCC 12980), IC and
GM resulted in the lowest decontamination efficacy. GM exhibited little or no impact on
decontamination of B. anthracis Ames and B. subtilis (ATCC 19659).
The ETV testing to measure the effectiveness of the CLARUS C unit for inactivating B.
anthracis Ames strain and surrogate spores on seven different indoor surfaces provides a
range of results. A quantitative evaluation of the results indicates that the log reduction
values for detectable viable B. anthracis Ames spores ranged from 3.01 to 7.92 across all
seven test materials. The log reduction values for detectable viable B. subtilis spores ranged
from 1.63 to 7.66 for all seven test materials. The log reduction values for detectable viable
G. stearothermophilus spores (ATCC 12980) ranged from 0.81 to 5.98 for all seven test
materials. In general, significant differences in efficacy between B. anthracis and both
surrogate organisms were observed on porous materials. For non-porous materials,
significant differences in efficacy between B. anthracis and G. stearothermophilus were
observed in most cases.
A qualitative evaluation of the performance of the CLARUS C unit could be based upon the
growth assessment of the biological indicators and spore strips. For all procedures for this
verification test, the control (not exposed to the CLARUS C unit) biological indicators and
spore strips used in this test displayed positive growth in the liquid cultures at both 1 and 7
days. When the biological indicators and spore strips were treated, no growth was observed
in the liquid cultures at 1 and 7 days. Based on these results, the CLARUS C unit
inactivated both the biological indicators (containing B. subtilis and G. stearothermophilus)
and spores strips (containing B. atrophaeus), all of which contain spore loads of
approximately 1 x 106 spores per indicator or spore strip.
The CLARUS C unit can be set up and programmed for operation within minutes. Data for
the cycle parameters are monitored in real-time and stored/displayed via several
mechanisms. During this verification test, cycle parameter data were not collected in real
time by the personal computer that was provided with the CLARUS C unit. Therefore, the
data collected with respect to cycle parameters were derived from the paper printout. Based
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on the data from this printout, the CLARUS C unit appeared to operate within the test
parameters provided by the vendor and programmed into it. The effect of operator skill level
on using the CLARUS C unit, while not verified in this test, should be minimal due to its
automated capabilities, which left little room for operator error.
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Chapter 8
References
1.	Test/QA Plan for Verification of Hydrogen Peroxide Vapor Technologies for
Decontaminating Indoor Surfaces Contaminated with Biological or Chemical Agents,
Battelle, Columbus, Ohio, July, 2003.
2.	Quality Management Plan (QMP) for the Technology Verification of Commercially
Available Methods for Decontamination of Indoor Surfaces Contaminated with
Biological or Chemical Agents, Version 1, prepared by Battelle, Columbus, Ohio,
November 22, 2002.
These references are posted on the ETV web site at:
http: //www .epa.gov/etv/centers/center9 .html
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