EPA/600/R-10/087 | September 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Biological Agent
Decontamination
Technology Testing
TECHNOLOGY EVALUATION REPORT
pH-Amended Bleach
CASCAD™ Surface Decontamination Foam
(Allen-Vanguard)
Decon Green
EasyDECON® 200 (EFT Holdings, Inc.)
Spor-Klenz® RTU (STERIS Corporation)
Peridox® RTU (GET, LEG)
Office of Research and Development
National Homeland Security Research Center
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TECHNOLOGY EVALUATION
pH-Amended Bleach
CASCAD1M Surface Decontamination Foam
(Allen-Vanguard)
Decon Green
EasyDECON® 200 (EFT Holdings, Inc.)
Spor-Klenz" RTU (STERIS Corporation)
RTU (GET, LLC)
M. WORTH CALFEE
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF AND DEVELOPMENT
NATIONAL
CENTER
RESEARCH TRIANGLE PARK, NC 27711
Office of Research and Development
National Homeland Security Research Center
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and directed this technology
evaluation through a Blanket Purchase Agreement under General Services Administration
contract number GS23F0011L-3 with Battelle. This report has been peer and administratively
reviewed and has been approved for publication as an EPA document. Mention of trade names
or commercial products does not constitute endorsement or recommendation for use of a specific
product. The EPA received funding for this project from the Defense Threat Reduction Agency
via Interagency Agreement RW-97-92282401-0.
If you have difficulty accessing this PDF document, please contact Kathy Nickel (Nickel.Kathy(@,
epa.gov) or Amelia McCall (McCall. Amelia(@,epa. govl for assistance.
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Foreword
Following the events of September 11, 2001, addressing the critical needs related to homeland
security became a clear requirement with respect to EPA's mission to protect human health and
the environment. Presidential Directives further emphasized EPA as the primary federal agency
responsible for the country's water supplies and for decontamination following a chemical,
biological, and/or radiological (CBR) attack. To support the EPA mission with respect to response
and recovery from incidents of national significance, the National Homeland Security Research
Center (NHSRC) was established to conduct research and deliver products that improve the
capability of the Agency to carry out its homeland security responsibilities.
One specific goal of NHSRC's research is to provide information on decontamination methods
and technologies that can be used in the response and recovery efforts resulting from a CBR
contamination event. In recovering from an event, it is critical to identify and implement
decontamination technologies that are appropriate for the given situation. In a wide-area attack
scenario, the decontamination approach must be effective; while at the same time must be readily
available, and easily deployed. The current study investigated several currently-available liquid and
foam sporicides technologies for their ability to inactivate spores of Bacillus anthracis on the surface
of common outdoor building materials. Information on the effectiveness of these technogies is
provided to inform both decontaminant selection and implementation.
These results, coupled with additional information in separate NHSRC publications (available at
www.epa. gov/nhsrcX can be used to determine whether a particular decontamination technology
can be effective in a given scenario. With these factors in consideration, the best technology
or combination of technologies can be chosen that meets the clean up, cost and time goals for a
particular decontamination scenario.
NHSRC has made this publication available to assist the response community prepare for and
recover from disasters involving chemical contamination. This research is intended to move EPA
one step closer to achieving its homeland security goals and its overall mission of protecting human
health and the environment while providing sustainable solutions to our environmental problems.
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Contributions of the following individuals and organizations to the development of this document
are gratefully acknowledged.
United States Environmental Protection Agency (EPA)
Joseph Wood
Lcroy Mickelsen
Carlton Kempter
Frank Schaefer
United States Defense Threat Reduction Agency (DTRA)
Ryan Madden
Bruce Hinds
Battelle Memorial Institute
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Notice iii
Foreword iv
Acknowledgments v
Abbreviations/Acronyms xii
Executive Summary xv
1.0 Introduction 1
2.0 Technology Description 3
3.0 Summary of Test Procedures 5
3.1 Preparation of Test Coupons 5
3.2 DecontammantTesting 6
3.3 Decontamination Efficacy 8
3.4 Qualitative Assessment of Residual Spores 8
3.5 Qualitative Assessment of Surface Damage 9
4.0 Quality Assurance/Quality Control 11
4.1 Equipment Calibration 11
4.2 QC Results 11
4.3 Audits 11
4.3.1 Performance Evaluation Audit 11
4.3.2 Technical Systems Audit 11
4.3.3 Data Quality Audit 11
4.4 Test/QA Plan Amendments and Deviations 11
4.5 QA/QC Reporting 11
4.6 Data Review 11
5.0 pH-Amended Bleach Test Results 13
5.1 QC Results 13
5.2 Decontamination Efficacy 13
5.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms 13
5.2.2 Qualitative Assessment of Residual Spores 15
5.3 Damage to Coupons 15
5.4 Other Factors 17
5.4.1 Operator Control 17
5.4.2 Technology Spray Deposition 17
5.4.3 Neutralization Methodology 17
6.0 CASCAD™SD Test Results 19
6.1 QC Results 19
6.2 Decontamination Efficacy 19
6.2.1 Quantitative Assessment of the Log Reduction of
Viable Organisms 19
6.2.2 Qualitative Assessment of Residual Spores 19
6.3 Damage to Coupons 20
6.4 Other Factors 20
6.4.1 Operator Control 20
6.4.2 Technology Spray Deposition 20
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6.4.3 Neutralization Methodology 25
7.0 Decon Green Test Results 27
7.1 QC Results 27
7.2 Decontamination Efficacy 27
7.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms 27
7.2.2 Qualitative Assessment of Residual Spores 27
7.3 Damage to Coupons 30
7.4 Other Factors 30
7.4.1 Operator Control 30
7.4.2 Technology Spray Deposition 30
7.4.3 Neutralization Methodology 32
8.0 EasyDECON® 200 Test Results 33
8.1 QC Results 33
8.2 Decontamination Efficacy 33
8.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms 33
8.2.2 Qualitative Assessment of Residual Spores 35
8.3 Damage to Coupons 36
8.4 Other Factors 36
8.4.1 Operator Control 36
8.4.2 Technology Spray Deposition 37
8.4.3 Neutralization Methodology 38
9.0 Spor-Klenz® RTU Test Results 41
9.1 QC Results 41
9.2 Decontamination Efficacy 41
9.2.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 41
9.2.2 Qualitative Assessment of Residual Spores 43
9.3 Damage to Coupons 45
9.4 Other Factors 45
9.4.1 Operator Control 45
9.4.2 Technology Spray Deposition 45
9.4.3 Neutralization Methodology 46
10.0 Peridox* RTU Test Results 47
10.1 QC Results 47
1.0.2 Decontamination Efficacy 47
10.2.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 47
10.2.2 Qualitative Assessment of Residual Spores 47
10.3 Damage to Coupons 50
10.4 Other Factors 50
10.4.1 Operator Control 50
10.4.2 Technology Spray Deposition 51
10.4.3 Neutralization Methodology 51
11.0 Performance Summary 53
11.1 pH-Amended Bleach Results 53
11.2 CASCAD™ SDF Results 53
11.3 Decon Green Results 53
11.4 EasyDECON8 200 Results 53
11.5 Spor-Klenz® RTU Results 53
11.6 Peridox® RTU Results 53
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Appendices
A Preparation and Application of pH-Amended Bleach 55
B Preparation and Application of CASCAD™ SDF 57
C Preparation and Application of Decon Green 59
D Preparation and Application of EasyDECON® 200 61
E Preparation and Application of Spor-Klenz* RTU 63
F Preparation and Application of Peridox® RTU 65
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Table E-l. Summary of Quantitative Efficacy Results [or Bacillus cmthracis (Ames) by
Dccontaminant and Test Material xviii
Table 2-1. Technology Information 3
Table 3-1. Summary of Materials Used for Decontaminant Testing 5
Table 3-2. Summary of Spore Recover}' Trials on Eight Test Materials 7
Table 5-1. Inactivation of Bacillus anthracis (Ames) Spores—pH-Amended Bleach on
Nonporous Materials 13
Table 5-2. Inactivation of Bacillus anthracis (Ames) Spores—pH-Amended Bleach on
Porous Materials 14
Table 5-3. Summary of Efficacy Values (Log Reduction) Obtained for
pH-Amended Bleach 15
Table 5-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
Bacillus anthracis (Ames) Spores—pH-Amended Bleach 16
Table 5-5. Deposition/Runoff Weight of pH-Amended Bleach on Test Materials 17
Table 5-6. Neutralization Testing with Bacillus anthracis (Ames) Spores for
pH-Amended Bleach 18
Table 6-1. Inactivation of Bacillus anthracis (Ames) Spores—CASCAD™ SDF on
Nonporous Materials 21
Table 6-2. Inactivation of Bacillus anthracis (Ames) Spores—CASCAD™ SDF on
Porous Materials 22
Table 6-3. Summary of Efficacy Values (Log Reduction) Obtained for CASCAD™ SDF 23
Table 6-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
Bacillus anthracis (Ames) Spores—CASCAD™ SDF 24
Table 6-5. Deposition/Runoff Weights of CASCAD™ SDF on Test Materials 25
Table 6-6. Neutralization Testing with Bacillus anthracis (Ames) Spores for
CASCAD™ SDF on Nonporous Test Materials 25
Table 6-7. Neutralization Testing with Bacillus anthracis (Ames) Spores for
CASCAD™ SDF on Porous Test Materials 25
Table 7-1. Inactivation of Bacillus anthracis (Ames) Spores—Decon Green on
Non-Porous Materials 28
Table 7-2. Inactivation of Bacillus anthracis (Ames) Spores—Decon Green on
Porous Materials 29
Table 7-3. Summary of Efficacy Values (Log Reduction) Obtained for Decon Green 30
Table 7-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
Bacillus anthracis (Ames) Spores—Decon Green 31
Table 7-5. Deposition/Runoff Weight of Decon Green on Test Materials 32
Table 7-6. Neutralization Testing with Bacillus anthracis (Ames) Spores for Decon Green on
Nonporous Test Materials 32
Table 7-7. Neutralization Testing with Bacillus anthracis (Ames) Spores for Decon Green on
Porous Test Materials 32
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Table 8-1. Inactivation of Bacillus anthracis (Ames) Spores—EasyDECON® 200
on Nonporous Materials 33
Table 8.2. Inactivation of Bacillus anthracis (Ames) Spores—EasyDECON®
200 on Porous Materials 34
Table 8-3. Summary of Efficacy Values (Log Reduction) Obtained for
EasyDECON® 200 35
Table 8-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
Bacillus anthracis (Ames) Spores-—EasyDECON® 200 36
Table 8-5. Deposition/Runoff Weight of EasyDECON® 200 on Test Materials 38
Table 8-6. Neutralization Testing with Bacillus anthracis (Ames) Spores for
EasyDECON® 200 on Nonporous Test Materials: Glass, Aluminum,
and Porcelain (3 applications) 38
Table 8-7. Neutralization Testing with Bacillus anthracis (Ames) Spores for
EasyDECON® 200 on Nonporous Test Materials: Stainless Steel
and Granite (6 applications) 38
Table 8-8. Neutralization Testing with Bacillus anthracis (Ames) Spores for
EasyDECON® 200 on Porous Test Materials (6 applications) 39
Table 9-1. Inactivation of Bacillus anthracis (Ames) Spores-Spor-Klenz®
RTU on Nonporous Materials (30 minute contact time with one
reapplicalion at 25 minutes) 41
Table 9-2. Inactivation of Bacillus anthracis (Ames) Spores-Spor-Klenz® RTU on
Porous Materials (60 minute contact time with reapplications at 10, 25, 30,
and 50 minutes) 42
Table 9-3. Summary of Efficacy Values (log Reduction) Obtained for Spor-Klenz® RTU 43
Table 9.4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
Bacillus anthracis (Ames) Spores-Spor-Klenz® RTU 44
Table 9-5. Deposition/Runoff Weight of Spor-Klenz® RTU on Test Materials 45
Table 9-6. Neutralization Testing with Bacillus anthracis (Ames) Spores for Spor-Klenz®
RTU on Nonporous Test Materials 46
Table 9-7. Neutralization Testing with Bacillus anthracis (Ames) Spores for Spor-Klenz®
RTU on Porous Test Materials 46
Table 10-1. Inactivation of Bacillus anthracis (Ames) Spores-Peridox® RTU on Nonporous
Materials (30 minute contact time with re-applications at 10 and 25 minutes) 48
Table 10-2. Inactivation of Bacillus anthracis (Ames) Spores-Pcridox® RTU on Porous
Materials (60 minute contact time with re-applications at 10, 20, 30, 40,
and 50 minutes) 49
Table 10-3. Summary of Efficacy Values (Log Reduction) Obtained for Peridox® RTU 50
Table 10-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
Bacillus anthracis (Ames) Spores-Peridox® RTU 50
Table 10-5. Deposition/Runoff Weight of Peridox® RTU on Test Materials 52
Table 10-6. Neutralization Testing with Bacillus anthracis (Ames) Spores for
Peridox® RTU on Nonporous Test Materials 52
Table 10-7. Neutralization Testing with Bacillus anthracis (Ames) Spores for
Peridox® RTU on Porous Test Materials 52
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List of Acronyms and Symbols
ACQ
APHA
B. anthracis
BBRC
BSC
C
CPU
CI
cm
DTRA
EPA
FSP
g
H2°2
hr
L
min
mL
uL
NaOCl
NHSRC
NIST
OC1
OPP
ORD
PBS
ppm
psi
QA
QC
QMP
RH
rpm
RTU
SD
SDF
SE
SFW
STS
alkaline copper quaternary
American Public Health Association
Bacillus anthracis (Ames strain)
Battelle Biomedical Research Center
biosafety cabinet
Celsius
colony-forming unit(s)
confidence interval
Centimeter
U.S. Defense Threat Reduction Agency
U.S. Environmental Protection Agency
Facility Safety Plan
gram
hydrogen peroxide
hour
liter
minute
milliliter
microliter
sodium hypochlorite
National Homeland Security Research Center
National Institute of Standards and Technology
Hypochlorite ion
Office of Pesticide Programs
U.S. EPA Office of Research and Development
phosphate-buffered saline
Parts per million
pounds per square inch
quality assurance
quality control
quality management plan
relative humidity
revolutions per minute
ready-to-use
standard deviation
Surface Decontamination Foam
standard error
sterile filtered water (cell-culture grade)
sodium thiosulfate
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TOPO
TSA
TTEP
wt
Task Order Project Officer
technical systems audit
Technology Testing and Evaluation Program
weight
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Executive Summary
The U.S. Environmental Protection Agency's (EPA)
National Homeland Security Research Center (NHSRC)
helps to protect human health and the environment
from adverse impacts of terrorist acts by carrying out
performance tests on homeland security technologies.
Through NHSRC's Technology Testing and Evaluation
Program (TTEP), the performance of six liquid and
foam decontamination technologies was evaluated
for decontaminating test coupons prepared from the
materials listed below. These materials include building
materials typical of surfaces found outdoors in urban and
residential buildings.
Nonporous materials:
• Stainless steel
• Glass
• Aluminum
• Porcelain (glazed)
• Granite (sealed surface)
Porous materials:
• Concrete
• Brick
• Asphalt paving
• Treated wood
• Butyl rubber
Experimental Procedures. Test coupons were
approximately 1.9 cm by 7.5 cm in size. For testing,
coupons were "contaminated" by spiking with spores
of the biological agent, Bacillus anihracis (Ames). The
technologies evaluated for their ability to inactivate B.
anthracis (Ames) on test coupons of the listed surface
materials were:
• pH-amended bleach (Ultra Clorox® Germicidal
bleach diluted with commercial certified cell-
culture-grade sterile filtered water (SFW) and 5%
acetic acid to obtain pH-amended solution)
• Allen-Vanguard's CASCAD™ Surface
Decontamination Foam (SDF)
• Decon Green
• EFT Holdings'EasyDECON® 200
• STERIS Corporation's Spor-Klenz® RTU (Ready-
to-Use)
• CET, LLC's Peridox® RTU (Ready-to-Use).
With the exception of pH-amended bleach and Spor-
Klenz® RTU, each decontaminant was tested using
application apparatus and conditions specified by
the respective vendor and according to the vendor's
instructions. For pH-amended bleach, no single vendor
exists. That product was tested for decontamination of
outdoor surfaces using a conventional hand-pumped
household garden sprayer to apply the product.
For Spor-Klenz® RTU, in the absence of vendor
specifications, a 500 mL hand-held plastic spray
bottle was used as the applicator, consistent with other
decontaminants tested. Application procedures for all
the decontaminants tested are included as appendices to
this report. Spray distance, humidity, and temperature
were the same for all applications, and all test coupons
were oriented horizontally (i.e., lying flat) for testing.
The following performance characteristics of the
decontamination technologies were evaluated:
• Decontamination efficacy
° Quantitative assessment of the decontamination
efficacy for viable organisms (log reduction)
° Qualitative assessment for residual spores on the
test coupons
• Qualitative assessment of damage to material
surfaces following decontamination.
Results. Results of the technology evaluation are as
follows:
Table E-l summarizes the quantitative efficacy results
(as log reduction in the number of viable spores) for all
six decontaminants on the 10 test materials. Efficacy
results shown with the ">" symbol indicate that complete
inactivation of B. anthracis spores was achieved with the
indicated decontaminant on that material, for all replicate
test coupons. The results in Table E-l show that the
porous materials, especially concrete, asphalt paving,
and treated wood, were more difficult to decontaminate
than the non-porous materials. CASCAD™ SDF foam
was the only one of the six decontaminants tested that
achieved complete inactivation of B. anthracis spores
on all 10 test materials. Porcelain and granite were the
only two test materials on which all six decontaminants
achieved complete inactivation of B. anthracis, although
efficacy was relatively high (i.e., almost always
exceeding 7 log reduction) on all of the nonporous
materials.
pH-amended bleach - This liquid decontaminant was
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applied to the test coupons until they were fully wetted
and then reapplied 15, 30, and 45 minutes after Hie
initial application, with a total contact time before
spore extraction of 60 minutes. This procedure was
sufficient to maintain wetting of the stainless steel,
glass, aluminum, porcelain, and butyl rubber coupons
throughout the contact lime. Granite, concrete, brick,
asphalt, and treated wood coupons did not remain wetted
throughout the entire contact time. Quantitative efficacy
for B. anthracis was > 7.62 log reduction on all five
non-porous materials and > 6.91 log reduction on the
porous materials brick and butyl rubber. On those seven
materials, inactivation of B. anthracis was, complete,
i.e., no viable spores were found on any decontaminated
coupons. Efficacy on concrete, asphalt paving, and
treated wood was 6.27, 3.60, and 1.90 log reduction,
respectively. Only the latter two materials showed
growth from decontaminated test coupons after one and
seven days of incubation, consistent with the quantitative
efficacy results. No visible damage was observed on any
of the test materials after the 60 minute contact time with
pH-amended bleach in the quantitative efficacy testing,
or seven days later after completion of the qualitative
assessment of residual spores.
CASCAD™ SDF - This decontaminant was applied to
the test coupons as a foam, using a two-compartment
spray bottle that mixed separate component solutions
and ejected them through a fine mesh screen to create
the foam. For nonporous materials, a single application
and 30-minute contact time were used; for porous
materials a second application was made 30 minutes
after the first, and the total contact time was 60 minutes.
CASCAD™ SDF foam covered both the non-porous
and porous material coupons throughout the respective
contact times. Quantitative efficacy of CASCAD™ SDF
for B. anthracis was > 6.80 log reduction on all ten
materials. On all materials, inactivation of B. anthracis
was complete; i.e., no viable spores were found on any
decontaminated coupons. Qualitative efficacy results
were consistent with quantitative efficacy results, in that
no growth was seen with decontaminated test coupons
of any materials. No visible damage was observed on
any of the test materials after the 30 or 60 minute contact
times with CASCAD™ SDF in the quantitative efficacy
testing, or seven days later after completion of the
qualitative assessment of residual spores.
Decon Green - This liquid decontaminant was applied
to all test coupons until they were fully wetted, and
then reapplied 30 minutes after the initial application.
The total contact time before spore extraction was 60
minutes. This application procedure was sufficient to
maintain wetting of the stainless steel, glass, aluminum,
porcelain, and butyl rubber coupons throughout
the contact time. Granite, concrete, brick, asphalt,
and treated wood coupons did not remain wetted
throughout the entire contact time. The quantitative
efficacy of Decon Green for B. anthracis was > 7.32
log reduction on all five non-porous materials, and >
7.25 and > 6.94 log reduction on the porous materials
brick and butyl rubber, respectively. No viable spores
were found on any of these seven test materials after
decontamination. Efficacy on concrete, asphalt, and
treated wood was 4.00, 2.97, and 1.91 log reduction,
respectively. Qualitative efficacy results were consistent
with quantitative efficacy results, in that no viable spores
were found on decontaminated coupons of seven of
the ten test materials. The decontaminated coupons of
concrete, asphalt, and treated wood all were positive
for growth at both one and seven days' incubation. No
visible damage was observed on any of the test materials
after the 60 minute contact time with Decon Green in
the quantitative efficacy testing, or seven days later after
completion of the qualitative assessment of residual
spores.
EasyDECON® 200 - This liquid decontaminant
was applied to test coupons of glass, aluminum, and
porcelain until they were fully wetted, and then reapplied
10 and 20 minutes after the initial application, with a
total contact time before spore extraction of 30 minutes.
This decontaminant was applied to test coupons of
stainless steel and granite until they were fully wetted,
and then reapplied 5, 10, 15. 20, and 25 minutes after
the initial application, with a total contact time of 30
minutes. Finally, this decontaminant was applied to
test coupons of the five porous materials until they were
fully wetted, and then reapplied 10, 20, 30, 40. and 50
minutes after the initial application, with a total contact
time of 60 minutes. These application schedules were
sufficient to maintain wetting of the stainless steel, glass,
aluminum, porcelain, granite, and butyl rubber coupons
throughout the contact time. Concrete, brick, asphalt
and treated wood coupons did not always remain wetted
throughout the entire contact time. The quantitative
efficacy of EasyDECON® 200 for B. anthracis was >
7.51 log reduction on all five non-porous materials, and
approximately > 7.14, > 7.28 and > 6.99 log reduction
on the porous materials unpainted concrete, brick, and
butyl rubber, respectively. No viable spores were found
on any of these eight test materials after decontamination
with EasyDECON® 200. Efficacy on asphalt and treated
wood was 1.63 and 0.82 log reduction, respectively.
Qualitative efficacy results were consistent with
quantitative efficacy results, in that no growth was seen
with decontaminated coupons of eight of the ten test
materials. The decontaminated coupons of asphalt and
treated wood all were positive for growth at both one and
seven days' incubation. No visible damage was observed
on any of the test materials after the 30 or 60 minute
contact times with EasyDECON® 200 in the quantitative
efficacy testing, or seven days later after completion of
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the qualitative assessment of residual spores.
Spor-Klenz® RTU - This liquid decontaminant was
applied to test coupons of both nonporous and porous
materials until they were fully wetted, and then reapplied
as necessary to keep the coupons wetted throughout
the contact time. With nonporous materials, the
contact time was 30 minutes and one reapplication
was needed 25 minutes after the first application. With
porous materials, the contact time was 60 minutes;
one planned reapplication was done 30 minutes after
the first application, and additional reapplications
were needed 10, 25, and 50 minutes after the first
application. The quantitative efficacy of Spor-Klenz®
RTU for B. anthracis was > 7.57 log reduction on the
non-porous materials porcelain and granite, and > 7.27
log reduction on the porous materials brick and butyl
rubber. No viable spores were found on any of these
four test materials after decontamination with Spor-
Klenz® RTU. Efficacy was relatively high on stainless
steel, glass, and aluminum (7.28, 7.36, and 7.17 log
reduction, respectively), but a small number of viable
spores were found on one of the replicate test coupons of
each of these materials after decontamination. Efficacy
on concrete, asphalt, and treated wood was 1.02, 2.56,
and 6.06 log reduction, respectively. Qualitative efficacy
results were consistent with quantitative efficacy
results, in that no growth was seen with decontaminated
coupons of porcelain, granite, brick, or butyl rubber.
Decontaminated coupons of other materials were
positive for growth at both one and seven days'
incubation. No visible damage was observed on any
of the test materials after the 30 or 60 minute contact
times with Spor-Klenz® RTU in the quantitative efficacy
testing, or seven days later after completion of the
qualitative assessment of residual spores.
Peridox® RTU - This liquid decontaminant was applied
to test coupons until they were fully wetted, and then
reapplied as necessary to keep the coupons wetted
throughout the contact time. With nonporous materials,
the contact time was 30 minutes and reapplication was
needed 10 and 25 minutes after the first application.
With porous materials, the contact time was 60 minutes,
and reapplication was needed 10, 20, 30, 40, and 50
minutes after the first application. Quantitative efficacy
of Peridox® RTU for B. anthracis was > 6.65 log
reduction on all five non-porous materials and on the
porous materials treated wood and butyl rubber. No
viable spores were found on any of these seven test
materials after decontamination with Peridox® RTU.
Efficacy was relatively high (7.22 log reduction) on
asphalt paving, but a small number of viable spores were
found on one of the replicate asphalt test coupons after
decontamination. Efficacy of Peridox® RTU on concrete
and brick was 1.39 and 3.81 log reduction, respectively.
Qualitative efficacy results were largely consistent with
the quantitative results, in that no growth was seen with
decontaminated test coupons of the five nonporous
materials and one of the porous material (treated
wood). However, three test coupons of butyl rubber
showed positive growth after both one and seven days'
incubation. All decontaminated coupons of unpainted
concrete and brick and two coupons of asphalt paving
were positive for growth at both one and seven days
incubation. No visible damage was observed on any of
the test materials after the 30 or 60 minute contact times
with Peridox® RTU in the quantitative efficacy testing
or seven days later after completion of the qualitative
assessment of residual spores.
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Table E-l. Summary of Quantitative Efficacy Results for Bacillus amthracis (Ames) by Decontaminant and Test Material
Quantitative Efficacy (log reduction)
Test Material
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
pH-Amended
Bleach
>7.73
>7.81
>7.91
>7.80
>7.62
6.27
>6.91
3.60
1.90
> 7.00
CASCAD™
SDF
> 7.67
>7.74
> 7.80
> 7.68
> 7.59
>6.93
>7.40
> 7.58
>6.97
> 6.80
Decon
Green
>7.64
>7.78
>7.80
>7.67
>7.32
4.00
> 7.25
2.97
1.91
>6.94
Easy DECON®
" 200
> 7.61
>7.79
>7.75
>7.78
>7.51
>7.14
>7.28
1.63
0.82
>6.99
Spor-Klenz®
RTU
7.28
7.36
7.17
>7.72
>7.57
1.02
>7.27
2.56
6.06
>7.39
Peridoi®
RTU
> 6.69
> 7.76
>7.82
>7.71
>7.42
1.39
3.81
7.22
>6.99
> 6.65
-------
1.0
Introduction
NHSRC, through its Technology Testing and Evaluation
Program (TTEP) works in partnership with recognized
testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, scientists.
and permitters; and with participation of individual
technology developers in carrying out performance
tests on homeland security technologies. In response
to the needs of stakeholders, NHSRC evaluates
the performance of innovative homeland security
technologies by developing test plans, conducting
evaluations, collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted
in accordance with rigorous quality assurance (QA)
protocols to ensure the generation of high quality
data and defensible results. NHSRC, through its
TTEP, provides unbiased, third-party information
supplementary to vendor-provided information that is
useful to decision makers in purchasing or applying
the evaluated technologies. Stakeholder involvement
ensures that user needs and perspectives are incorporated
into the evaluation design to produce useful performance
information for each evaluated technology.
NHSRC, through its TTEP, evaluated the performance
of six liquid and foam sporicidal decontamination
technologies for inactivating Bacillus anihracis (Ames)
spores on materials representative of outdoor surfaces.
The technologies, which were evaluated on test coupons
often outdoor surface materials, included the following:
• pH-amended bleach (Ultra Clorox® Germicidal
bleach diluted with commercial certified cell-
culture-grade sterile filtered water (SFW) and 5%
acetic acid to obtain pH-amended solution)
• Allen-Vanguard's CASCAD™ Surface
Decontamination Foam (SDF)
• Decon Green
• EFT Holdings' Easy DECON® 200
• STERIS Corporation's Spor-Klenz® RTU (Ready-
to-Use)
• CET, LLC's Peridox® RTU (Ready-to-Use).
Testing was performed using application procedures
specified by each vendor, or developed by EPA and
Battelle (pH-amended bleach and Spor-Klenz® RTU)
based on past experience with similar decontaminants.
The application procedures for all decontaminants
are included as appendices to this report. The
decontaminant test procedures were specified in a
peer-reviewed test/QA plan, as amended to meet the
specific requirements of this evaluation. The following
performance characteristics of the decontamination
technologies were evaluated:
• Decontamination efficacy
° Quantitative assessment of the decontamination
efficacy for viable organisms (log reduction)
° Qualitative assessment for residual spores on the
test coupons
• Qualitative assessment of damage to material
surfaces following decontamination.
-------
-------
2.0
Technology Description
Table 2-1 describes the decontamination
technologies evaluated, based on vendor-provided
information (except in the case of pH-amended
bleach and Spor-Klenz® RTU) and shows the
contact times used. The information provided
Table 2-1. Technology Information
in Table 2-1 on product composition was not
confirmed in this evaluation. The application
procedures used in testing of these products are
included as appendices to this report.
Product
Ultra Clorox®
Germicidal
Bleach
CASCAD™
SDF
Decon Green
EasyDECON®
200
Spor-Klenz®
RTU
Peridox® RTU
Vendor
Clorox®
Professional
Products Co.
Allen-
Vanguard
NA
EFT
Holdings,
Inc.
STERIS
Corp.
CET,
LLC
General
Description/
Active
Ingredients
Sodium
hypochlorite,
hypochlorous
acid
Hypochlorite
Hydrogen
peroxide
Hydrogen
peroxide
Hydrogen
peroxide,
peracetic acid
Hydrogen
peroxide,
peracetic acid
Components
Sodium hypochlorite 5-6% (pH-amended by
Battelle by adding acetic acid 5%)b
Sodium myristyl sulfate 10-30%, sodium
(C14-16) olefin sulfonate 10-30%; ethanol
denatured 3-9%; alcohols (C10-16) 5-10%,
sodium sulfate 3-7%; sodium xylene sulfonate
1-5%; proprietary mixture of sodium and
ammonia salts along with co-solvent >9%;
dichloroisocyanuric acid, sodium salt 48-85%;
sodium tetraborate 3-7%; sodium carbonate
10-15%.
Three separate component solutions:
A: propylene glycol, propylene carbonate,
Triton® X- 100
B: hydrogen peroxide, 35%
C: potassium citrate monohydrate, potassium
bicarbonate, potassium molybdate, propylene
glycol.
Three separate component solutions:
A. benzyl C12-C16 alkyl dimethylammonium
chlorides, 5.5 to 6.5 %, N,N,N,N',N'-
pentamethyl-N' tallow alkyl-
trimethylenediammonium chloride, 1.5 to 2.5%;
B. hydrogen peroxide, < 8.0%;
C. diacetin (i.e., glycerol diacetate) 30 to 60%.
Hydrogen peroxide 1.0%, peracetic acid 0.08%,
acetic acid < 10%.
Hydrogen peroxide 4.4%; peracetic
acid 0.22 %.
EPA
Registration"
67619-8
None
1043-121
74436-1
74436-2
1043-119
81073-3
Contact
Time
(min)
60
30/60C
60
30/60C
30/60C
30/60C
" Registration with EPA's Office of Pesticide Programs (OPP) indicates EPA/OPP has evaluated the pesticide to show it is effective and has no
unreasonable adverse effects on humans, the environment, and non-target species, and has issued a registration or license for use in the United
States. Spor-Klenz* RTU and Peridox* RTU are registered as sporicides but none of the products tested is registered specifically for use against
B. anthracis.
b As recommended by TTEP stakeholders, 5% acetic acid was added to the bleach to obtain a pH-amended bleach solution. Mixing 9.4 parts
SEW, 1 part Ultra Clorox* Germicidal bleach, and 1 part 5% glacial acetic acid yielded a solution having a mean pH of 6.36 and mean total
chlorine content of approximately 6,200 ppm. Ultra Clorox* Germicidal bleach is registered as a disinfectant but pH-amended bleach is not.
0 Total contact times on nonporous/porous materials.
-------
Below are brief physical descriptions of the without dilution, and was applied to test coupons
decontamination technologies (their form, appearance as using a 500 mL hand-held plastic spray bottle.
received) and preparation instructions:
• pH-Amended Bleach - Ultra Clorox® Genmicidal
bleach was purchased in a one-gallon container from
a local retail store. The pH-adjusted decontaminanl
solution was prepared by mixing 9.4 parts SFW, 1
part Ultra Clorox® Germicidal bleach, and 1 part 5%
glacial acetic acid. The final solution was applied
using a hand-pressurized noncorroding portable
garden sprayer.
• CASCAD™ SDF - This decontaminant was prepared
as two separate solutions. One CASCAD"" solution
was prepared by dissolving 31.2 g of GP2100
(decontaminant) in SFW and diluting to 300mL
volume, and the other solution was made by
dissolving 7.2 g of GPB-2100 (buffer) and 18 mL of
GCE2000 (surfactant) in SFW and diluting to 300
mL volume. The application process used a vendor-
supplied dual spray bottle designed to deliver equal
portions of the two solutions through a single spray
nozzle equipped with a diffuser mesh to produce the
foam.
• Dccon Green - This decontaminant is pre-packaged
as three separate solutions, premeasured and ready
to mix. Parts B and C were mixed together and then
that mixture was added to Part A. The final solution
of Decon Green has a pH of about 8 and a density of
approximately 1.1 g/mL. Decon Green was applied
to test coupons using a 500 mL hand-held plastic
spray bottle.
• EasyDECON® 200 - This decontaminant consists
of three components pre-packaged in separate
containers, premeasured and ready to mix. To
prepare EasyDECON® 200. the prepackaged Part
One and Part Two solutions were mixed together
in a clean container, and then the Part 3 solution
was added and all three components were mixed
thoroughly. The final solution of EasyDECON®
200 lias a pH of about 9.6 to 9.9 and a density of
approximately 1.08 g/mL. EasyDECON® 200 was
applied to test coupons using a 500 mL hand-held
plastic spray bottle.
• Spor-Klenz® RTU - Spor-Klenz® RTU is a ready-to-
use clear, colorless, aqueous solution with pH 1.5 to
2.0, density of 1.01 g/mL, and a sharp acidic odor.
The solution was used without dilution and applied
using a 500 mL hand-held plastic spray bottle.
• Peridox® RTU - This decontaminant is an aqueous
solution of 4.4 % hydrogen peroxide (H2O2) and
0.23% peroxyacelic acid. The product is a colorless
liquid with a pH of about 2.2 and a density of
approximately 1.02 g/mL. This solution was used
-------
3.0
Summary of Test Procedures
Test procedures were performed in accordance with the
peer-reviewed test/QA plan and are briefly summarized
here.
3.1 Preparation of Test Coupons
The Bacillus anthracis (Ames) spores used for this
testing were prepared from a qualified stock of the
Ames strain at the Battelle Biomedical Research
Center (BBRC). All spore lots were subject to a
stringent characterization and qualification process,
required by Battelle's standard operating procedure
for spore production. Specifically, all spore lots were
characterized prior to use by observation of colony
morphology, direct microscopic observation of spore
morphology and size, and determination of percent
refractivity and percent encapsulation. In addition the
number of viable spores was determined by colony count
and expressed as colony forming units per milliliter
(CFU/mL). (Theoretically, once plated onto bacterial
growth media, each viable spore germinates and yields
one CPU.) Variations in the expected colony phenotypes
were recorded. Endotoxin concentration of each spore
preparation was determined by the Limulus Ameocyte
Lysate assay. Genomic DNA was extracted from the
spores and DNA fingerprinting was done to confirm the
genotype. The virulence of the spore lot was measured
by challenging guinea pigs intradermally with a dilution
series of spore suspensions, and virulence was expressed
as the intradermal median lethal dose. In addition,
testing was conducted for robustness of the spores
via HC1 resistance. The stock spore suspension was
prepared in SFW at an approximate concentration of 1 x
109 spores/mL and stored by refrigeration at 4 °C.
B. anthracis (Ames) spores were inoculated onto test
coupons in an appropriate level three biosafety cabinet
(BSC) according to established BBRC procedures.
Inoculated coupons were prepared prior to each day of
experimental work. Coupons were placed flat in the
BSC and inoculated at approximately 1 x 10s viable
spores per coupon. This inoculation was accomplished
by dispensing a 100-uL aliquot of the spore stock
suspension (approximately 1 x 109 spores/mL) using
a micropipette as 10 droplets (each of 10 uL volume)
across the surface of the test coupon. This approach
provided more uniform distribution of spores across
the coupon surface than would be obtained through a
single drop of the suspension. After inoculation, the test
coupons remained undisturbed overnight in the BSC to
dry. Test coupons were then decontaminated the next
day, i.e., within 24 hours after inoculation.
The origin and specifications of the materials used for
test coupons are shown in Table 3-1. All materials
were selected as representative types of the respective
building materials, based on consultation with materials
suppliers. With the exception of unpainted concrete
which was poured into coupons by Battelle staff and
asphalt which was salvaged used material, all test
Table 3-1. Summary of Materials Used for Decontaminant
Testing
Materials
Origin
Specifications
NONPOROUS
Stainless Steel
Glass
Aluminum
Porcelain
Granite
POROUS
Concrete
Brick
Alro Steel Inc.
Columbus, OH
Brooks Brothers
Glass and Mirror
Columbus, Ohio
Petersen Aluminum
Elk Grove Village,
IL
AF Supply
Corporation
New York, NY
Lang Stone Co.
Columbus, OH
Stainless, 304,20
gauge, 2B Finish
C1036, 0.32cm
thick
0.81mm thick, 300
Clear Anodized
BRIX Frimmenti
DEF70 Black MOS.
Tile (7 cm x 1.9 cm
x 0.7 cm)
Giallo Ornamental,
Brushed finish,
milled to 1.9 cm
thick
Wysong Concrete
Fairfield, OH
Hamilton Parker
Co.
Columbus, OH
5 parts sand and
2 parts cement, 1
cm thick (Battelle-
made)
Belcrest 560,
common red,
chemical resistant
-------
Asphalt
Paving
Shelly Aggregate
and Asphalt
Columbus, OH
Treated Wood
Lowe's Top Choice
Columbus, OH
Butyl Rubber
Copperstate
Roofing Supply
Phoenix, AZ
Used upper layer
asphalt (fine
aggregate, salvaged
material from urban
parking lot, washed
with water before
cutting coupons)
Alkaline Copper
Quaternary
(ACQ) treated,
5 cm x 10 cm x
2.4 m, 6.4 kg/m3
retention (no water
proofing). Item
#46905, Model #
TC248T225N
GSSI #9897, high
temperature self
adhering double-
sided butyl rubber
sealant tape, 1.9 cm
x 0.48 cm thick
coupons were made from new materials. Test coupons
were approximately 1.9 x 7.5 cm in size. Coupons were
sterilized before use by gamma irradiation (for asphalt,
treated wood, and butyl rubber) or autoclaving (for all
other materials).
Prior to testing of any decontaminants, spore recovery
trials were conducted on eight of the ten test materials
to define suitable spore recovery procedures. This
effort was needed because of the ten materials only two
(glass and concrete) had been used as test substrates in
previous decontaminant tests performed by NHSRC.
Spore recovery trials were conducted by inoculating
three coupons of each material with B. anthracis (Ames)
spores at the same target inoculation (1 x 10s spores/
coupon (±25%)) planned for the decontaminant testing
and allowing the usual overnight drying time. Those
triplicate coupons, along with one blank (uninoculated)
coupon, of each material were then subjected to
spore recovery and enumeration using the procedures
described in Section 3.2. In the spore recovery trials, the
primary approach to spore recovery was agitation of a
coupon in extraction solution for 15 minutes at 200 rpm.
The alternative approach, which was found necessary
for concrete coupons through previous testing, was
sonication of the coupons in extraction solution for 45
minutes. This alternative spore recovery approach was
found in the recovery trials to be necessary for granite,
brick, and asphalt coupons as well.
The results of the spore recovery trials are summarized
in Table 3-2, which shows the spore inoculation
amount, the number of spores recovered from each of
the three coupons of the eight materials, the resulting
recovery values, and the average recovery (± standard
deviation[SD]) on each material. The inoculation and
recovered spore values in Table 3-2 are reported as CPU,
as determined by the enumeration process (Section
3.2). The recovery values are the ratio of recovered to
inoculated CPU, expressed as a percentage. The results
in Table 3-2 arise from using the 45-minute sonication
approach on granite, brick, and asphalt coupons, and
the 15-minute agitation approach on coupons of the
other five materials. Table 3-2 shows that average spore
recovery values ranged from about 59 to 75% on the
four nonporous materials (stainless steel, aluminum,
porcelain, and granite) and were somewhat lower on the
porous materials (brick, asphalt, treated wood, and butyl
rubber), ranging from about 10 to 56%. Treated wood
was the only material which exhibited an average spore
recovery less than 10%. However, all recovery values
are well within the target range of 1 to 150% required by
the test/QA plan, and are fully sufficient for performance
of decontaminant testing.
Spore recoveries were also determined for all ten coupon
materials in each decontaminant test. Those recovery
results are shown in the respective results chapters
(Chapters 5 to 10).
3.2 Decontaminant Testing
On the day following inoculation, test coupons
intended for decontamination (including blanks) were
transferred into a glove box (test chamber) where
the decontamination technology was applied using
the apparatus and application conditions specified in
the appendices of this report. The decontamination
spray distance (30.5 cm), humidity (< 70% relative
humidity), and temperature (22 ± 1°C) were the same
for all applications. For most decontaminants tested, the
amount of decontaminant, contact time, spray pressure,
application and reapplication procedures, etc., were as
specified by the vendor. For pH-amended bleach and
Spor-Klenz® RTU, these parameters were chosen by EPA
with input from Battelle based on previous experience
and reasonable application procedures.
Five replicate test coupons (inoculated with B. anthracis
spores and decontaminated), five replicate positive
control coupons (inoculated and not decontaminated),
one procedural blank (not inoculated, decontaminated),
and one laboratory blank (not inoculated, not
decontaminated) of each coupon material were used
in testing with each decontaminant. In testing of all
six decontaminants, all test coupons were oriented
horizontally (i.e., lying flat). Decontaminant runoff and
decontaminant pooled on top of each test coupon were
captured, neutralized, and subjected to spore extraction
along with the associated test coupon.
-------
Table 3-2. Summary of Spore Recovery Trials on Eight Test Materials
_, ,„ . , Inoculum"
Test Material (CWV)
Stainless Steel 1.02 x 10s
Aluminum 1.02 x 10s
Porcelain 1.02 x 10s
Granite" 8.5 x K)7
Brick" 8.5 x 1CP
Asphalt
Paving" 8.5 x K)7
Treated Wood 8.5 x if)7
Butyl Rubber 1.02 x 108
Recovered
Spores1'
(CFU)
7.07 >
6.47 >
6.13 >
7.77 >
7.77 >
7.57 >
6.63 >
7.13 >
7.77 >
3.87>
6.50 >
4.73 >
3.50 >
1.51 >
2.83 >
4.89 >
4.87>
4.43 >
8.77 >
8.57>
7.57 >
2.67 >
3.47 >
3.17 >
< 107
< 107
'- W
< 107
< 107
< 107
< 107
< io7
< 107
< 107
< 107
'- W
< 107
'- W
< 107
< 107
< 107
'- W
< 106
< 106
< 106
< 107
< 107
'- 107
Recovery'"
(%)
69.3
63.4
60.1
76.2
76.2
74.2
65.0
69.9
76.2
45.5
76.5
55.7
41.2
17.8
33.3
57.5
57.3
52.1
10.3
10.1
8.9
26.2
34.0
31.1
Average Recovery
(°/.)(±SD)
64.3 (±4.7)
75. 5 (±1.1)
70.4 (±5.6)
59.2 (±15. 8)
30.8 (±11. 9)
55. 7 (±3.1)
9.8 (±0.8)
30.4 (±4.0)
B. anthmcis (Allies) spores inoculated onto materials coupons, CFU - colony-forming units.
Results shown for triplicate coupons of each material.
Recover}' is ratio of recovered to inoculated spores; see text.
Spores recovered from these materials by sonication for 45 minutes; for other materials listed spore
recover}' was by agitation at 200 rpm for 15 minutes.
Following decontamination, each coupon (along with
any associated runoff and pooled decontaminant) was
transferred aseptically to a sterile 50mL conical vial
containing 10 mL of sterile phosphate-buffered saline
(PBS) solution with 0.1% Triton® X100 surfactant (i.e.,
99.9% PBS. 0.1% Triton® X-100) and the neutralizer
needed to stop the decontaminant. The required
concentration of neutralizer was determined in trial
runs for each decontaminant tested, according to a
detailed procedure stated in the test/QA plan. In each
of those trial runs, a range of neutralizer concentrations
was tested to determine the concentration that most
effectively stopped the action of the decontaminant (as
indicated by the maximum recovery of viable spores
in simulated coupon extracts). The results of those
trial runs are shown in the respective results chapters
(Chapters 5 to 10). As noted in Section 3.1, most
coupons were then extracted by agitation on an orbital
shaker for 15 minutes at approximately 200 revolutions
per minute (rpm) at room temperature. For granite.
concrete, brick, and asphalt the recover}' of spores used
an alternate procedure in which 45 minutes of sonication
was used, instead of the period of agitation. For all
coupons, following extraction 1 mL of the coupon
extract was removed, and a series of dilutions through
10"7 was prepared in SFW. An aliquot (0.1 mL) of the
undiluted extract and each serial dilution were then
spread-plated onto tryptic soy agar plates (in triplicate)
and incubated overnight at 35 to 37 °C. Resulting
colonies were enumerated within 18 to 24 hours of
plating. The number of CFU/mL was determined by
multiplying the average number of colonies per plate by
the reciprocal of the dilution and accounting for the 0.1
mL volume of the extract or dilution that was plated.
Before further decontamination tests, the test chamber
was cleaned using the vendor-supplied method for
neutralizing the decontamination reagent (see the
appendices to this report). If no instructions for
neutralization were provided, the test chamber was
cleaned following procedures established under the
BBRC Facility Safety Plan.
-------
Laboratory blanks were controlled for sterility and
procedural blanks were controlled for viable spores
inadvertently introduced to test coupons. The procedural
blanks were spiked with an equivalent amount of
0.1 mL of "stock suspension" that did not contain the
biological agent. The target acceptance criterion was
that extracts of laboratory or procedural blanks were to
contain no CPU. The mean percent spore recovery from
each coupon material was calculated using results from
positive control coupons (spiked, not decontaminated
(sprayed with SFW instead of the decontaminant)) by
means of the following equation:
Mean % Recovery = [Mean CFUpc/CFUspike] * 100 (1)
where Mean CPU is the mean number of CPU
PL'
recovered from five replicate positive control coupons
of a single material and CFUspiko is the number of
CPU spiked onto each of those coupons. The value
of CPU .. is known from enumeration of the stock
spike
spore suspension. Spore recovery was calculated for B.
anthracis on each coupon material, and the results are
included in Chapters 5 through 10.
3.3 Efficacy
The performance or efficacy of the decontamination
technology was assessed by determining the number
of viable organisms remaining on each test coupon and
in any decontaminant run-off from the coupon, after
decontamination. Those numbers were compared to the
number of viable organisms extracted from the positive
control coupons, which were sprayed with SFW (the
matrix for the spore suspension inoculation) instead of
with the decontaminant.
The number of viable spores ofB. anthracis in extracts
of test and positive control coupons was determined
to calculate efficacy of the decontaminant. Efficacy is
defined as the extent (as Iog10 reduction) by which viable
spores extracted from test coupons after decontamination
were less numerous than the viable spores extracted
from positive control coupons subjected only to an inert
aqueous spray, at the same temperature and contact time
as the decontaminant application. First, the logarithm
of the CPU abundance from each coupon extract was
determined, and then the mean of those logarithm values
was determined for each set of control and associated
test coupons, respectively. Efficacy of a decontaminant
for a test organism on the /* coupon material was
calculated as the difference between those mean log
values, i.e.:
Efficacy =(log10 C/'T/c,,) - (Iog10 CFUtv)
(2)
and Iog10 CFUt.. refers to they individual logarithm
values obtained from the corresponding test coupons,
and the overbar designates a mean value. In tests
conducted under this plan, there were five control and
five corresponding test coupons (i.e.,7 = 5) for each
coupon material. In the case where no viable spores
were found in any of the five test coupon extracts after
decontamination, a CPU abundance of 1 was assigned,
resulting in a Iog10 CPU of zero for that material. This
situation occurred frequently when a decontaminant was
highly effective and no viable spores were found on the
decontaminated test coupons. In such cases, the final
efficacy on that material was reported as greater than or
equal to (>) the value calculated by Equation 2.
The variances (i.e., the square of the standard deviation)
of the log,, CFUc.. and log,, CFUt.. values were also
G10 // G10 ij
calculated for both the control and test coupons (i.e., S2c..
and S2t.), and were used to calculate the pooled standard
error (SE) for the efficacy value calculated in Equation
2, as follows:
5
S2t..
_ 'L
5
where log]0 CFUc.. refers to they individual logarithm
values obtained from the positive control coupons
where the number 5 again represents the number j of
coupons in both the control and test data sets. Thus each
efficacy result is reported as a log reduction value with
an associated SE value.
The significance of differences in efficacy across
different coupon materials and spore types was assessed
based on the 95% confidence interval of each efficacy
result. The 95% confidence interval (CI) is:
95% CI = Efficacy ± (1.96 x SE) (4)
Differences in efficacy were judged to be significant if
the 95% CIs of the two efficacy results did not overlap.
The efficacy results are presented in a series of tables
in Chapters 5 through 10 for each decontaminant
technology by coupon material.
3.4 of
Spores
Based on previous decontamination studies and the spore
recover}' trials, it was anticipated that spores might not
be completely recovered from coupons by the extraction
process. Therefore, viable spores might remain on the
test coupons following decontamination and extraction.
As in previous decontamination studies, a qualitative
assessment was performed to determine whether viable
spores remained on the test coupons after extraction,
including both the decontaminated test coupons
and the positive control coupons not subjected to
decontamination. This qualitative assessment involved
different conditions and a much longer growth period
than was used in the quantitative assessment of efficacy
-------
and was made to determine whether the decontaminated
coupons with no growth in the quantitative measurement
also showed no growth in the qualitative method.
To conduct the qualitative assessment, the test coupons
from the quantitative assessment, following extraction,
were transferred into tryptic soy broth culture medium
and incubated for seven days at appropriate temperatures
for growth. The culture media were visually inspected
after one and seven days of incubation. A cloudy-
liquid culture after incubation indicated that viable
organisms of some type remained on the coupon after
decontamination and extraction. For liquid cultures
in which cloudiness was observed, a loop of the liquid
sample was streaked onto a tryptic soy agar plate and
incubated under appropriate conditions for growth for B.
anthracis. After incubation, the plates were examined
to determine qualitatively (morphologic comparison
performed visually) if the observed growth was a
pure culture characteristic of the B. anthracis that was
inoculated onto the coupons, a mixture of the inoculated
organism and other endogenous organisms, or a mixture
of organisms, for example molds and bacteria. Thus, the
indication of the presence of viable organisms (cloudy
appearance in growth medium) did not necessarily
indicate the presence of residual viable organisms that
had been spiked onto the test coupon.
3.5 of
Damage
Trial runs were conducted before any testing with each
decontaminant. using coupons that had not been spiked
with spores. In these trial runs the decontaminant
was applied exactly as specified in the test/QA plan
and measurements were made with multiple coupons
of each material type to determine the amount of the
decontaminant that remained on, or ran off from, each
material. This information was used in the calculation
of efficacy for each respective material and in trial runs
to determine the amount of neutralizing agent needed to
stop the action of the decontaminant after the prescribed
contact time. In addition, visual inspection of each
coupon surface by two test personnel took place after the
prescribed decontaminant contact time, through side-by-
side comparison of the decontaminated test surface and
control coupons of the same test material. Differences
in color, reflectivity, and roughness were assessed
qualitatively, and observations were recorded by the test
personnel. The same inspection was conducted after
the conclusion of the 7-day growth period that assessed
qualitative efficacy (Section 3.4).
-------
-------
4.0
Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were
performed in accordance with the test/QA plan for this
evaluation, as amended, except as noted below. QA/QC
procedures are summarized below.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological
safety cabinets) and monitoring devices (e.g.,
temperature, relative humidity) used at the time of
evaluation were verified as being certified, calibrated, or
validated.
4.2 QC
Quality control efforts conducted during decontaminant
testing included positive control coupons (spiked,
not decontaminated), procedural blanks (not spiked,
decontaminated), laboratory blanks (not spiked, not
decontaminated), and spike control samples (analysis of
the stock spore suspension). The results for these QC
samples in each deconlaminanl evaluation are included
in the results chapter for each respective decontaminant
(i.e., see Chapters 5 through 10).
4.3
4.3.1 Performance Evaluation Audit
No performance evaluation audit was performed for
B. anthracis fAmes) organisms because quantitative
standards for these biological materials do not exist.
4.3.2 Technical Systems A
Battcllc QA staff conducted a technical systems audit
(TSA) at the BBRC on July 14 and 17, 2009, during
testing of CASCAD™ SDF, to ensure that the evaluation
was being conducted in accordance with the test/QA
plan and the QMP.(1) 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 Task
Order Leader for response. The only finding of the
TSA concerned the expiration date of CASCAD™ SDF
solutions, as noted in Section 6.1. TSA records were
permanently stored with the Battelle QA Manager.
4.3.3 Data Quality Audit
At least 10% of the data acquired during the evaluation
were audited. A Battelle Q A auditor 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.
4.4 Test/QA Plan
Deviations
The test/QA plan for this evaluation was adapted
by amendment to a peer-reviewed, fully approved
plan established for a previous evaluation under the
TTEP program. Three amendments to the test/QA
plan relevant to this testing were prepared, reviewed,
approved, and distributed to all parties involved in this
evaluation. Those amendments identified the materials
and decontaminant technologies to be used in this
evaluation and indicated the spore extraction procedures
used for various materials.
Five deviations were prepared, approved, and retained
in the test files for this evaluation. Two deviations
were related to acceptance of spore inoculations
slightly outside the target range of 1 x 10s spores/
coupon (±25%). Those inoculations are noted in
Sections 6.1 and 7.1. The third deviation was related
to contamination of three laboratory blank coupons
in testing of Decon Green due to a departure from
usual test procedures. That occurrence is noted in
Section 7.1. The fourth deviation addressed slight
contamination of blank coupons by B. anthracis spores
during testing. That occurrence is noted in Section 5.1.
The fifth deviation addressed slight contamination of
blank material coupons after testing, due to departure
from the prescribed order of handling the coupons.
That occurrence is noted in Section 10.1. None of
those deviations had any significant effect on efficacy
determinations for the respective decontaminants.
4.5 QA/QC Reporting
Each audit was documented, and results of the audits
were submitted to the EPA (i.e., to the NHSRC Quality
Assurance Manager and the Task Order Project Officer
(TOPO)).
4.6
Records and data generated in the evaluation received
a QC/tecnnical review before they were utilized
in calculating or evaluating results and prior to
incorporation in reports. All data were recorded by
Battelle staff. The person performing the QC/technical
review was involved in the experiments and added his/
her initials and the date to a hard copy of the record
being reviewed. This hard copy was returned to the
Battelle staff member who stored the record.
-------
-------
5.0
pH-Amended Bleach Test Results
5.1 QC Results
In testing of pH-amended bleach, all positive control
results were well within the target recovery range of 1
to 150% of the spiked spores. Positive control recovery
values for B. anthracis spores ranged from 8.6 to 93.0%,
with the lowest recovery occurring on brick.
In testing of pH-amended bleach, most procedural
and laboratory blanks met the criterion of no observed
CPU, with the exceptions of two laboratory blanks (not
inoculated, not decontaminated) and two procedural
blanks (not inoculated, decontaminated). Specifically,
laboratory blank coupons for granite and brick, and
procedural blank coupons for asphalt and brick, showed
CPU counts of 36 to 467 CPU per coupon. This
contamination likely occurred during coupon extraction,
but is very slight relative to the spore inoculation of 1 x
10s spores on each test coupon. The blank CPU results
do not enter into the efficacy calculations; nevertheless,
a deviation was prepared regarding the acceptance of
these blank coupon results. Further, decontaminated
test coupons of brick and granite had no recoverable
CPU following treatment, while asphalt test coupons
had on average 3.83 log CPU. Thus, no contamination
was apparent for brick and granite test coupons, and
contamination of asphalt control coupons was on the
order of 10 percent of recovered CPU from test coupons.
Finally, no growth was observed in the qualitative
assessment of residual spores for all procedural and
laboratory blanks, which involves a much longer
incubation period.
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1
x lOVmL (± 25%), leading to a spike of 1 x 10s spores
(± 25%) on each test coupon. The actual spike values
for three days of B. anthracis testing were 8.73 x 10V
coupon, 7.73 x lOVcoupon and 9.63 x lOVcoupon, each
within the required range.
5.2 Decontamination Efficacy
The decontamination efficacy of pH-amended bleach
was evaluated for B. anthracis (Ames) on ten outdoor
material surfaces. The following sections summarize the
results found with this decontaminant.
5.2.1 Quantitative Assessment of the Log
Reduction of Viable Organisms
The decontamination efficacy of pH-amended bleach
for B. anthracis was >7.62 log reduction on all five
nonporous materials, as shown in Table 5-1, and was
> 6.94 log reduction on the porous materials brick and
butyl rubber, as shown in Table 5-2. For all of these
Table 5-1. Inactivation of Bacillus anthracis (Ames) Spores8—pH-Amended Bleach on Nonporous Materials
(60 minute contact time with reapplications at 15, 30, and 45 minutes)
Test Material
Inoculum (CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Decontamination
Efficacy ± CI
Stainless Steel
Positive Controls11
Test Coupons0
Laboratory Blank11
Procedural Blank'
7.73 x 107
7.73 x 107
0
0
7.73 ± 0.04
0
0
0
69.9 ±6.5
0
> 7.73 ±0.03
Glass
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.73 x 107
8.73 x 107
0
0
7.81 ±0.06
0
0
0
73.9 ±9.6
0
> 7.81 ±0.05
-------
Aluminum
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Porcelain
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Granite
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.73 x 107
8.73 x 107
0
0
8.73 x 107
8.73 x 107
0
0
7.73 x 107
7.73 x 107
0
0
7.91 ±0.05
0
0
0
7.80 ±0.06
0
0
0
7.62 ±0.03
0
1.55f
0
93.0 ±10.3
0 > 7.91 ±0.04
-
-
73.2 ±9.6
0 > 7.80 ±0.05
-
-
53. 8 ±3.9
0 > 7.62 ±0.03
-
-
" Data are expressed as the mean (± SD) of the logs of the number of spores (CPU) observed on five individual coupons, the
mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = Inoculated, decontaminated coupons.
11 Laboratory Blank = Not inoculated, not decontaminated coupon.
e Procedural Blank = Not inoculated, decontaminated coupon.
* CPU consistent with B. anthracis morphology observed during spore enumeration.
"-" Not Applicable.
Table 5-2. Inactivation of Bacillus anthracis (Ames) Spores8— pH-Amended Bleach on Porous Materials
(60 minute contact with reapplications at 15, 30, and 45 minutes)
Test Material
Inoculum (CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Decontamination
Efficacy ± CI
Concrete
Positive Controls'1
Test Coupons0
Laboratory Blank11
Procedural Blank'
8.73 x 107
8.73 x 107
0
0
7.47 ±0.33
1.20 ±1.67
0
0
42.1 ±28.1
0
6.27 ±1.49
Brick
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
9.63 xlO7
9.63 xlO7
0
0
6.91 ±0.08
0
1.55f
1.55f
8.6 ±1.6
0
-
_
> 6.91 ±0.07
Asphalt Paving
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
9.63 xlO7
9.63 xlO7
0
0
7.42 ± 0.26
3.82 ±0.47
0
2.67f
30.8 ±16.2
0.010 ±0.009
3.60 ±0.47
-------
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
7.73 x 107
7.73xl07
0
0
7.05 ±0.13
5.15±0.89
0
0
15.1 ±5.0
0.45 ±0.41
1.90 ±0.79
Butyl Rubber
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
7.73 x 107
7.73 x 107
0
0
7.00 ± 0.04
0
0
0
13.1 ±1.2
0 > 7.00 ± 0.03
-
-
" Data are expressed as the mean (• SD) of the logs of the number of spores (CPU) observed on five individual coupons, the
mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
C! - confidence interval (± 1.96 x SE).
b Positive Controls — Inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons — Inoculated, decontaminated coupons.
d Laboratory Blank — Not inoculated, not decontaminated coupon.
' Procedural Blank — Not inoculated, decontaminated coupon.
f CFU consistent with B. anthracis morphology observed during spore enumeration.
"-" Not Applicable.
seven materials the efficacy result is equivalent to
complete inactivation. as no viable spores were found
on any decontaminated coupons. Concrete, asphalt.
and treated wood exhibited lower efficacy values, at
6.27, 3.60 and 1.90 log reduction, respectively. The
quantitative efficacy results arc summarized in Table
5-3.
Table 5-3. Summary of Efficacy Values (Log Reduction)
Obtained for pH-Amended Bleach
Test Material
Nonporous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Efficacy for
B. anthracis (Ames)
> 7.73
>7.81
>7.91
> 7.80
>7.62
6.27
>6.91
3.60
1.90
> 7.00
5.2.2 Qualitative Assessment of
Spores
Results from the liquid culture growth assessment
of the test coupons at one and seven days' post-
decontamination are provided in Table 5-4. In this
assessment, cultures showing positive growth (i.e., a
cloudy growth medium) were subjected to streak plating
and the identity of the growing organism was checked
by colony morphology. The qualitative results in Table
5-4 are consistent with the quantitative efficacy results
shown above for pH-amended bleach on all materials.
Only die asphalt and treated wood test coupons were
positive for growth at both one and seven days of
incubation. Although not a definitive identification,
colony morphology was consistent with all observed
colonies being B. anthracis. The laboratory and
procedural blanks were all negative for growth.
5.3 to Coupons
No visible damage was observed on the test materials
after the 60 min contact time with pH-amended bleach,
or after seven days incubation in the qualitative efficacy
test. The extraction buffer solution showed a yellowish
hue when extracting the treated wood test and control
coupons, most likely from wood treatment chemicals
dial leached out of the material.
-------
Table 5-4. Liquid Culture Assessment of Eitracts from Coupons Inoculated with Bacillus anthrads
(Ames) Spores—pH-Amended Bleach
Test Material
SI
S2
Day 1
S3 S4
S5
B
SI S2
Day?
S3 S4 S5
B
Stainless Steel
Positive Controls
Test Coupons
Glass
Positive Controls
Test Coupons
Aluminum
Positive Controls
Test Coupons
Porcelain
Positive Controls
Test Coupons
Granite
Positive Controls
Test Coupons
Concrete
Positive Controls
Test Coupons
Brick
Positive Controls
[ Coupons
Asphalt Paving
Positive Controls
Test Coupons
Treated Wood
Positive Controls
Test Coupons
Butyl Rubber
Positive Controls
Test Coupons
SI to S5 - Sample 1 to Sample 5.
B — Blank (not inoculated with B. anthrads (Ames) spores); a — laboratory blank, b — procedural blank.
Positive controls — Coupons inoculated with B. anthrads (Ames) spores, but not subjected to decontamination.
Test coupons — Coupons inoculated with B. anthrads (Ames) spores, and subjected to decontamination.
"-:-" - growth; "-" — no growth.
-------
5.4 Other
5.4.1 Operator Control
On each day of testing, pH-amended bleach was
prepared according to the instructions detailed in
Appendix A. To 9.4 parts water (940 mL), 1 part (100
niL) 5% acetic acid was added and mixed, then 1 part
(100 mL) Ultra Clorox® Germicidal bleach was added
and mixed. The pH was verified prior to use for testing
as being 6.6 to 6.8. The pH-amended bleach was then
transferred to a handheld garden sprayer modified with
a pressure gauge to ensure that the spray was applied
using 4 to 6 psi pressure. The bleach solution was then
sprayed onto the test coupons and close observation
of the respective material surfaces was made to ensure
that they were thoroughly wetted (spray duration of
approximately 3 to 5 seconds was needed to produce
wetting across the surfaces of all five replicates and
corresponding blank for each material type).
All tests were conducted under ambient conditions
inside a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature, measured to be 22 °C (± 1 °C).
The RH inside the test chamber was monitored with a
NIST-traceable hygrometer. Whenever Hie RH reached
70%, the dehumidification system attached to the testing
chamber was actuated until the RH dropped below 70%.
Therefore, the testing chamber was always < 70% RH
during the decontamination of test materials with pH-
amended bleach.
5.4.2 Technology Spray Deposition
pH-Amended bleach was applied according to the
procedure included as Appendix A of this report. The
pH-amended bleach was applied from a distance of
30.5 cm to the horizontally-oriented materials until the
materials were fully wetted. Reapplication of Hie pH-
amended bleach was made on all coupon surfaces at
15. 30, and 45 minutes after the initial application, for a
total of four applications. At 60 minutes after the initial
application, each material coupon was placed in the 50
mL vial that also served to collect excess decontaininant
runoff. The test coupons stayed in their horizontal
orientation throughout the 60 minute contact time.
To assess pH-amended bleach deposition, triplicate
coupons of each test material were weighed prior to
application of the pH-amended bleach in the trial runs,
and these values were recorded. Then the triplicate
coupons were sprayed with pH-amended bleach
until fully wetted in their horizontal orientations,
reapplications were made at the 15. 30, and 45 minute
contact times for a total of four applications, and after 60
minutes contact time each coupon was weighed again.
The pre-application weights were then subtracted from
the post-application weights, and that difference was
added to the weight of decontaminant runoff captured
separately from each coupon. The average deposition/
runoff weight of the pH-amended bleach from each
of the test materials is shown in Table 5-5. The total
averaged value (0.75 g) over all ten materials was then
used to estimate the amount of sodium thiosulfate (STS)
needed to effectively neutralize the pH-amended bleach.
Table 5-5. Deposition/Runoff Weight of pH-Amcndcd
Bleach on Test Materials
Test Material
Nonporous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Average
Average Deposition/Runoff
Weight (g)
0.63
0.64
0.42
0.51
0.33
0.82
1.14
0.62
1.82
0.60
0.75
5.4.3
Neutralization of the pH-amended bleach was achieved
with STS. The concentrations of STS tried during
the neutralization trials were 0.5, 1.0, and 1.5% in the
extraction solution. These STS concentrations were
based on unpublished data from previous testing. The
results of the neutralization trials are shown in Table 5-6.
It was determined from these trials that 0.5% STS was
sufficient for neutralization of pH-amended bleach.
-------
Table 5-6. Neutralization Testing with Bacillus anthracis (Ames) Spores for pH-Amended Bleach
Treatment
pH-Amended Bleach + Spores1
pH-Amended Bleach + PBS + Triton'8 X-100 + Spores"-1'
PBS + Triton® X-100 + Spores (Control)1-1
pII-Amended Bleach + PBS + Triton® X-100 + 0.5% STS +
pH-Amended Bleach + PBS + Triton8 X-1 00 + 1 .0% STS +
pH-Amended Bleach + PBS + Triton® X-100 + 1 .5% STS +
Spores3*
Sporesa-b
Spores*
Inoculum
(CPU)
6.70 x 107
6.70 x 107
6.70 xlO7
6.70 xlO7
6.70 x 107
670x10"
Total
Observed
(CFU)
0
0
7.62 x 107
8.09 x 107
7.28 xlO7
8.74 x 107
%of
Control
0
0
100
106.1
95.5
114.7
pll- \mcndcd Bleach volume of 0.75 mL corresponds to mean giavimetric deposition on test materials and density of approximately
1 (I g mL
10 mL Volume of PBS includes 0.1% of Triton* X-100 surfactant and indicated % of STS; total volume for all samples withpH-
amcndcd bleach - 10.75 mL (10 mL PBS/Triton» X-100/STS + 0.75 mL pH-amended bleach).
-------
6.0
CASCAD™ SDF Test Results
6.1 QC Results
In testing of CASCAD™ SDF, all positive control results
were well within the target recovery range of 1 to 150%
of the spiked spores. With the nonporous materials
positive control recovery values ranged from 52.0 to
76.1 %, with the lowest recovery from porcelain and the
highest from stainless steel. With the porous materials,
positive control recovery values ranged from 6.7 to 62.5
%; the lowest recovery was from butyl rubber and the
highest from asphalt paving.
In quantitative efficacy testing of CASCAD™ SDF
with B. anthracis, all procedural and laboratory blanks
met the criterion of no observed CPU. Also no growth
of B. anthracis was observed for any procedural and
laboratory blanks in the qualitative assessment of
residual spores, which involves a much longer nutrient
growth period. (Growth of native organisms, with
colonies morphologically distinct from those of B.
anthracis, was observed from blank coupons of two
materials in the qualitative assessment as described in
Section 6.2.2.)
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1
x lOVmL (± 25%), leading to a spike of 1 x 10s spores
(± 25%) on each test coupon. The actual spike values
for three days of B. anthracis testing were 9.97 x 10V
coupon, 9.40 x lOVcoupon, and 6.17 x lOVcoupon. The
spike value for the third day of testing thus was outside
the target range of 1 x 10s spores (± 25%). However,
spore recovery values were relatively high (i.e., greater
than 62%) for all materials tested on that day, allowing
efficacy up to about 7.6 log reduction to be determined.
As a result, no tests were repeated from that third day
of testing, but a deviation was prepared noting this
acceptance of a spike outside the target range.
In the TSA conducted during testing of CASCAD™
SDF, it was noted that the expiration date of the three-
component solutions provided by the vendor (Allen-
Vanguard) had passed. When this issue was brought
to the vendor's attention, the vendor indicated that the
solutions were still acceptable for use and approved
proceeding with testing.
6.2 Decontamination Efficacy
The decontamination efficacy of CASCAD™ SDF
was evaluated for B. anthracis (Ames) on ten outdoor
material surfaces. The following sections summarize the
results found with this decontaminant.
6.2.1 Quantitative Assessment of the Log
Reduction of Viable Organisms
The decontamination efficacy of CASCAD™ SDF for
B. anthracis was greater than 6.8 log reduction on
all test materials, as shown for the non-porous and
porous materials in Tables 6-1 and 6-2, respectively,
and summarized in Table 6-3. No viable spores were
recovered from any test coupon decontaminated with
CASCAD™ SDF, so all efficacy results are shown as
">" log reduction values. The only efficacy results
lower than 7.0 log reduction were on the porous
materials concrete, treated wood, and butyl rubber, for
which relatively low spore recoveries (i.e., < 10%) limit
the efficacy result.
6.2.2 Qualitative Assessment of Residual
Spores
Results from the liquid culture growth assessment of
test, control, and blank coupons at one and seven days
post-decontamination are provided in Table 6-4. In this
assessment, cultures showing positive growth (i.e., a
cloudy growth medium) were subjected to streak plating
and the identity of the growing organism was checked
by colony morphology. Only B. anthracis colonies
were found in cultures of coupons inoculated with B.
anthracis.
Table 6-4 shows that for all ten materials, the coupons
that were decontaminated with CASCAD™ SDF
showed no growth for B. anthracis, whereas the positive
control (non-decontaminated) coupons were all positive
for growth. These qualitative results are consistent with
the quantitative efficacy results found for CASCAD™
SDF on these materials. Laboratory and procedural
blanks were all negative for growth of B. anthracis. The
concrete laboratory blank coupons showed a cloudy
growth medium at both one and seven days' incubation,
but no growth of any organism was seen when the
cultures were plated. The cloudiness is attributed to
suspended material from the coupons (e.g., concrete
dust) in the growth media. Laboratory blank coupons
of brick and butyl rubber also showed cloudy growth
media, but when plated the morphology of the colonies
was clearly distinct from that of B. anthracis. As a
result, laboratory blanks of concrete, brick, and butyl
-------
rubber are all shown as negative in Table 6-4.
An unusual observation was seen in the qualitative
assessment with the treated wood positive control
coupons. As described in Section 6.3, the extraction
solutions from those coupons were yellowish, apparently
due to leaching of some of the wood treatment from
the coupons. The liquid culture growth assessments
for those extraction solutions were negative at both
Day 1 and Day 7 post-inoculation, even though the
treated wood positive control coupons had not been
decontaminated. On the suspicion that the wood
treatment chemicals may have inhibited growth of
organisms, aliquots of those negative liquid culture
growth assessments were then streaked onto nutrient
agar. The following day all agar plates from the liquid
culture growth assessments of the treated wood positive
controls clearly exhibited colonies morphologically
characteristic of B. anthracis. This observation supports
the possibility that a compound from the treated wood
inhibited the growth of B. anthracis in liquid culture
(where the concentration of the inhibitory compound
was the greatest), but once a small aliquot of this liquid
culture was streaked onto nutrient agar, the organism
flourished. On the basis of this extra confirmation
step, the treated wood positive controls were reported
as positive for growth in Table 6-4. It is unclear why
treated wood positive control coupon were positive for
growth during testing with pH-amended bleach, and
negative in these tests.
6.3 to Coupons
No visible damage was observed on any of the test
materials after either the 30 min or the 60 min contact
time with CASCAD™ SDF. However, the extract
solutions from coupons of treated wood all had a
yellowish color, which is presumed to be from the wood
preservative chemicals leaching out of the material. The
treated wood coupons showed no visible change as a
result of the extraction process. The impact of these
chemicals on the qualitative assessment of residual
spores is noted in Section 6.2.2, above.
6.4 Other
6.4.1 Operator Control
On each day of testing, the two separate component
solutions of Allen-Vanguard's CASCAD™ SDF were
prepared according to the vendor's instructions as
stated in Appendix B. Each half of the dual spray bottle
supplied by Allen-Vanguard was then filled with one
of the two component solutions, and the spray nozzle
was attached to the bottle. Prior to each application,
the CASCAD™ SDF spray nozzle was primed by
repeatedly spraying into an absorbent cloth to clear any
air bubbles that may have formed between applications.
After each application, the spray nozzle was cleaned by
spraying deionized water from a separate bottle. After
the 30 minute contact time for nonporous materials or
60 minute contact time for the porous materials (with
a reapplication at 30 minutes), each material coupon
was placed in the 50 rnL conical vial that also served to
collect pooled CASCAD™ SDF from the coupon surface.
All tests were conducted under ambient conditions
inside a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature, measured to be 22 °C (± 1°C).
The RH inside the test chamber was monitored with a
NIST-traceable hygrometer. Whenever the RH reached
70%, the dehumidification system attached to the testing
chamber was actuated until the RH dropped below 70%.
Therefore, the testing chamber humidity was always
< 70% RH in the decontamination of materials with
CASCAD™ SDF.
6.4.2 Technology Spray Deposition
Allen-Vanguard's CASCAD™ SDF was applied
according to the procedure included as Appendix B
of this report. CASCAD™ SDF was applied from a
distance of 30.5 cm to the horizontally-oriented materials
until the materials were covered with an approximately
3/8-inch layer of foam. The total contact time was 30
minutes for nonporous materials and 60 minutes for
porous materials. Reapplication of the CASCAD™ SDF
was done only on the porous coupon surfaces at 30
minutes after the initial application, for a total of two
applications.
-------
Table 6-1. Inactivation of Bacillus anthracis (Ames) Spores8—CASCAD™ SDF on Nonporous Materials
(30 minute contact time, no reapplication
Test Material
Stainless Steel
Positive Controls11
Test Coupons0
Laboratory Blank11
Procedural Blank'
Glass
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Aluminum
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Porcelain
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Granite
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
6.17xl07
6.17xl07
0
0
9.40 xlO7
9.40 xlO7
0
0
9.40 xlO7
9.40 xlO7
0
0
9.40 xlO7
9.40 xlO7
0
0
6.17xl07
6.17xl07
0
0
Mean of Logs of
Observed CFU
7.67 ±0.06
0
0
0
7.74 ±0.10
0
0
0
7.80 ±0.06
0
0
0
7.68 ±0.08
0
0
0
7.59 ±0.09
0
0
0
Mean % Decontamination
Recovery Efficacy ± CI
76.1 ± 10.9
0 > 7.67 ±0.05
-
-
59.7 ±14. 8
0 > 7.74 ± 0.09
-
-
66.9 ±8. 9
0 > 7.80 ±0.05
-
-
52.0 ±9. 8
0 > 7.68 ±0.07
-
-
64. 5 ±13. 8
0 > 7.59 ±0.08
-
-
" Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual coupons, the
mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = Confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = Inoculated, decontaminated coupons.
11 Laboratory Blank = Not inoculated, not decontaminated coupon.
' Procedural Blank = Not inoculated, decontaminated coupon.
"-" Not Applicable.
-------
Table 6-2. Inactivation of Bacillus anthmcis (Ames) Spores"— CASCAD™ SDF on Porous Materials (60
minute total contact time with reapplication at 30 min)
Test Material
Concrete
Positive Controls11
Test Coupons0
Laboratory Blank11
Procedural Blank'
Brick
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Asphalt Paving
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Butyl Rubber
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.97 xlO7
9.97 xlO7
0
0
9.97 xlO7
9.97 xlO7
0
0
6.17 xlO7
6.17 xlO7
0
0
9.97 xlO7
9.97 xlO7
0
0
9.97 xlO7
9.97 xlO7
0
0
Mean of Logs of Mean %
Observed CFU Recovery
6.93 ±0.18 9.2 ±4.4
0 0
0
0
7.40 ±0.17 26.6 ±9.1
0 0
0
0
7.58 ±0.06 62.5 ±9.1
0 0
0
0
6.97±0.15 9.8±3.2
0 0
0
0
6.80 ±0.20 6.7 ±2.2
0 0
0
0
Decontamination
Efficacy ± CI
-
> 6. 93 ±0.16
-
-
-
> 7.40 ±0.15
-
-
-
> 7.58 ±0.05
-
-
-
>6.97±0.13
-
-
-
> 6. 80 ±0.18
-
-
" Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual coupons, the
mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = Confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
c Test Coupons = Inoculated, decontaminated coupons.
11 Laboratory Blank = Not inoculated, not decontaminated coupon.
e Procedural Blank = Not inoculated, decontaminated coupon.
"-" Not Applicable.
-------
Table 6-3. Summary of Efficacy Values (Log Reduction)
Obtained for CASCAD™ SDF
Test Material
Nonporous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Efficacy for
E. anthracis fAmes)
>7.67
>7.74
> 7.80
> 7.68
>7.59
> 6.93
> 7.40
>7.58
>6.97
>6.80
To assess CASCAD™ SDF deposition, triplicate
coupons of each test material were weighed prior to
application of the CASCAD™ SDF, and these weights
were recorded. Then the triplicate coupons were sprayed
with CASCAD™ SDF until fully wetted in a horizontal
orientation, and then each coupon was weighed again
after its respective contact time. The prc-application
weights were then subtracted from the post-application
weights, and that difference was added to the weight
of decontaminant runoff captured separately from
each coupon. The resulting average deposition/runoff
weight of the CASCAD™ SDF from each of the test
materials is shown in Table 6-5. The average deposition
amounts were 0.28 g on nonporous materials and 0.96
g on porous materials. The former quantity was then
used in trials to estimate the amount of STS needed to
effectively neutralize the CASCAD™ SDF in testing with
nonporous materials. However, an amount of 1.29 g was
erroneously used in the corresponding neutralization trial
for porous materials. The results of the neutralization
trials, including the use of that erroneous CASCAD™
SDF amount to represent deposition on porous materials,
are presented in the next section.
-------
Table 6-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis (Ames) Spores
CASCAD™ SDF
Test Material
SI
Davl
S2 S3 S4 S5 B
SI
S2
Day?
S3 S4
B
Stainless Steel
Positive Controls
Test Coupons
Glass
Positive Controls
Test Coupons
Aluminum
Positive Controls
Test Coupons
Porcelain
Positive Controls
Test Coupons
Granite
Positive Controls
Test Coupons
Concrete
Positive Controls
Test Coupons
Brick
Positive Controls
Test Coupons
Asphalt Paving
Positive Controls
Test Coupons
Treated Wood
Positive Controls"
Test Coupons
Butyl Rubber
Positive Controls
Test Coupons
+ +
+ + + + +
+ + + + +
+ + + + +
SI to S5 = Sample 1 to Sample 5.
B - Blank (not inoculated with B. anthracis (Ames) spores); a - laboratory blank, b - procedural blank.
Positive controls = Coupons inoculated with B. anthracis (Ames) spores, but not subjected to decontamination.
Test coupons — Coupons inoculated with B. anthracis (Ames) spores, and subjected to decontamination.
" I" = growth; "-" = no growth.
c A cloudy extraction solution was observed, but no organisms were detected when solution was plated (see text).
d Positive growth was indicated by a cloudy solution after incubation, but morphology of organisms not consistent with B. anthracis (see
text).
e Positive control coupons of treated wood showed no growth in one-day or seven-day incubation, but showed growth consistent with B.
anthracis morphology when culture was plated (see text).
-------
Table 6-5. Deposition/Runoff Weights of CASCAD™ SDF
on Test Materials
Test Material
lest Material
Average Deposition/
Runoff wdght fe)
Nonporous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Average
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Average
0.11
0.23
0.23
0.56
0.28
0.28
0.75
1.40
0.60
1.59
0.46
0.96
6,4.3 Neutralization Methodology
Neutralization of CASCAD™ SDF was achieved with
STS. For both nonporous and porous materials, the
concentrations of STS tested in the neutralization trials
were 0.5, 1.0, and 1.5% in the PBS/Triton® X-100
extraction solution. This range of STS concentrations
was chosen based on previous experience with
CASCAD™ SDF. The results of the neutralization trials
are shown in Tables 6-6 and 6-7. for the nonporous
and porous materials, respectively. On the basis of
these results 0.5% STS was chosen for neutralization
of CASCAD™ SDF in testing with the nonporous
materials and 1.0% STS was chosen for testing with
the porous materials. The use of an erroneously large
amount of CASCAD™ SDF to represent deposition on
porous materials (i.e.. 1.29 g rather than 0.96 g; see
Section 6.4.2) does not affect the results in Table 6-7.
The 1% STS concentration chosen was sufficient to
neutralize 1.29 g of CASCAD™ SDR and therefore
would certainly neutralize the average actual deposition
of 0.96 g of CASCAD™ SDF on the porous coupons.
On the basis of the results in Table 6-7, the 1% STS
concentration was used for neutralization in the tests
with porous materials.
Table 6-6. Neutralization Testing with Bacillus anthracls
(Ames) Spores for CASCAD™ SDF on Nonporous Test
Materials
Table 6-7. Neutralization Testing with BociUus omthracis
(Ames) Spores for CASCAD™ SDF on Porous Test
Materials
Treatment
Inoculum
(CFU)
r» J°tal A % of
Observed _ ,
(CFU) C°ntro1
CASCAD™ SDF +
Spores3
CASCAD™ SDF +
PBS + Triton® X-100
+ Spores51-1'
PBS + Triton® X-100
+ Spores (Control)b
CASCAD™ SDF
+ PBS + Triton®
X-100+ 0.5% STS +
Spores3-5
CASCAD™ SDF
+ PBS + Triton®
X-100+1.0% STS +
Spores3-5
CASCAD™ SDF
+ PBS + Triton®
X-100+1.5%STS +
Spores1'1'
* CASCAD™ SDF volume of 0.28 ml, corresponds to mean
gravimetric deposition on nonporous materials and liquid density
of approximately 1.0 g/'mL.
b 10 ml, volume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of STS; total volume for all samples with
CASCAD™ SDF - 10.28 ml- (10 ml- PBS/Triton* X-100/STS +
0.28 ml- CASCAD™ SDF).
9.47xl07
9.47x107
9.47 xlO7 1.04xl08
9.47 xlO7 1.06 x 10s
9.47 xlO7 9.24 x 107
9.47xl07 9.46 x 107
100
102.
89.1
91.2
Treatment
Inoculum
(CFU)
Total
Observed
(CFU)
%of
Control
8.67 xlO7
0
0
8.67 xlO7 1.06 x 10s
8.67 xlO7 8.47 x 107
CASCAD™ SDF +
Spores3
CASCAD™ SDF +
PBS + Triton® X-l 00 8.67 x 107
+ Sporesa-b
PBS + Triton® X-l 00
+ Spores (Control)b
CASCAD™ SDF
+ PBS + Triton®
X-100+ 0.5% STS+
Spores3'5
CASCAD™ SDF +
PBS + Tnton®X-100 + 8.67 x 107 9.76 x 107
1.0%STS + Spores"-b
CASCAD™ SDF
+ PBS + Triton®
X-l 00+1.5% STS +
Spores3-5
* CASCAD'" SDF volume of 1.29 mL corresponds to mean
gravimetric deposition on porous materials and liquid density of
approximately 1.0 g/ml- (see text).
' 10 mL Volume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of STS; total volume for all samples with
CASCAD" SDF - 11.29 ml- (10 mL PBS/Triton* X-100/STS -f
1.29 mL CASCAD" SDF).
8.67 xlO7 9.70 xlO7
100
79.6
91.7
-------
-------
7.0
Decon Green Test Results
7.1 QC Results
In testing of Decon Green, all positive control results
were within the target recovery range of 1 to 150% of
the spiked spores. Positive control recovery values
on nonporous materials ranged from about 27 to 90%,
with the lowest recovery on granite and the highest on
aluminum; positive control recovery values on porous
materials ranged from about 7 to 57%, with the lowest
recovery on concrete and the highest on asphalt.
Laboratory blanks showed no indication of B. anthracis,
except for the laboratory blanks of glass, aluminum, and
porcelain from one of the Decon Green test days, which
were found to be contaminated with small numbers of
B. anthracis spores. This contamination apparently
occurred because these blanks (i.e., non-inoculated
material coupons) were placed with the inoculated test
and positive control coupons during the overnight drying
period. In previous testing, the blank coupons were
segregated from the inoculated coupons during drying
and only placed into the test chamber after the inoculated
coupons had been placed there. That approach avoided
transfer of any spores to blank coupons by agitation
or air movement during drying, or by contact with the
inoculated coupons during the transfer to the chamber
for decontaminant testing. However, this procedure
was not followed for the blank coupons on the day of
Decon Green testing that involved these three materials.
Consequently, those blanks showed positive growth
in the qualitative assessment of residual spores. A
report detailing the deviation from the test/QA plan was
prepared noting this departure from procedures. No
viable spores were detected in the quantitative efficacy
testing, and no growth was observed in the qualitative
assessment of residual spores, for any of the procedural
blanks of any materials (i.e., not inoculated with spores
but subjected to Decon Green application).
Spike control samples were taken from the spore
suspension on each day of testing, serially diluted,
nutrient plated, and counted to establish the spore
density used to inoculate the coupons. This process
takes approximately 24 hours, so the spore density is
known after completion of each day's testing. The
target criterion is to maintain a spore suspension density
of 1 x lOVmL (± 25%), leading to an inoculation of 1
x 10s spores (± 25%) on each test coupon. The actual
inoculation values for three days of B. anthracis testing
were 7.13 x lOVcoupon, 7.13 x lOVcoupon and 8.30 x
lOVcoupon. It is unknown why the spore suspension
density was below the target criterion for the first two
testing days. Spore recovery results were good on those
days, allowing determination of quantitative efficacy of
6.67 log reduction or more. Consequently, the tests from
those days were not repeated, but a deviation from the
test/QA plan was prepared noting this acceptance of a
spike outside the target range.
7.2 Decontamination Efficacy
The decontamination efficacy of Decon Green was
evaluated for B. anthracis (Ames) on ten outdoor
material surfaces. The following sections summarize the
results found with this decontaminant.
7.2.1 Quantitative Assessment of the Log
Reduction of Viable Organisms
The results for decontamination efficacy of Decon Green
on nonporous and porous materials are shown in Tables
7-1 and 7-2, respectively, and summarized in Table 7-3.
Decontamination efficacy was > 7.3 log reduction for all
five of the nonporous materials, as shown in Table 7-1.
No viable spores were found on any of the nonporous
materials after decontamination with Decon Green.
The decontamination efficacy of Decon Green was not
as consistent for the porous materials, as is shown in
Table 7-2. Efficacy was > 7.3 and > 6.9 log reduction,
respectively, on brick and butyl rubber, whereas
concrete, asphalt, and treated wood had log reductions of
4.0, 3.0, and 1.9, respectively. Table 7-3 lists all efficacy
results from both nonporous and porous materials.
7.2.2 Qualitative Assessment of Residual
Spores
Results from the liquid culture growth assessment
of coupon extracts at one and seven days post-
decontamination are provided in Table 7-4 for coupons
inoculated with B. anthracis (Ames). In this assessment,
cultures showing positive growth (i.e., a cloudy growth
medium) were subjected to streak plating and the
identity of the growing organism was checked by colony
morphology. Only colonies indicative of B. anthracis
were observed in cultures of coupons inoculated with B.
anthracis.
Table 7-4 shows qualitative efficacy results for all
materials which are consistent with the quantitative
efficacy results reported in Section 7.2.1 (Table
7-3). For all five nonporous materials, and for brick
and butyl rubber, no growth was observed from the
decontaminated test coupons after either one or seven
-------
days' incubation. The decontaminated coupons of
concrete, asphalt, and treated wood all were positive for
growth at both one and seven days' incubation. One of
the concrete test replicates (S5) was negative for growth
after one day of incubation, but was positive for growth
and verified as B. anthracis after seven days as shown in
Table 7-4.
Table 7-4 also shows that the laboratory and procedural
blanks were all negative for growth with the exception of
the laboratory blanks of glass, porcelain, and aluminum.
Those blank coupons were inadvertently left with the
inoculated coupons during the overnight drying period,
as described in Section 7.1, and apparently became
contaminated.
Table 7-1. Inactivation of Bacillus anthracis (Ames) Spores"—Decon Green on Nonporous Materials (60 minute
contact time with reapplication at 30 minutes)
Test Material
Stainless Steel
Positive Controls11
Test Coupons0
Laboratory Blank11
Procedural Blank'
Glass
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Aluminum
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Porcelain
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Granite
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum (CFU)
8.30 xlO7
8.30 xlO7
0
0
7.13 xlO7
7.13 xlO7
0
0
7.13 xlO7
7.13 xlO7
0
0
7.13 xlO7
7.13 xlO7
0
0
8.30 xlO7
8.30 xlO7
0
0
Mean of Logs of ,, „. _.
™ i ^T^TT Mean % Recovery
Observed CFU J
7.64 ±0.04 52. 9 ±4.2
0 0
-
-
7.78 ±0.04 84.6 ±6. 9
0 0
3.47f
0
7.80 ±0.07 90.0 ±13.6
0 0
3.49f
0
7.67 ±0.09 66.2 ±14.4
0 0
3.63f
0
7.32 ±0.19 27.2 ±12.3
0 0
0
0
Decontamination
Efficacy ± CI
-
>7.64 ± 0.03
-
-
-
>7.78 ± 0.03
-
-
-
>7.80±0.06
-
-
-
>7.67 ± 0.08
-
-
-
>7.32±0.17
-
-
" Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual coupons, the mean percent
recovery on those five coupons, and decontamination efficacy (log reduction).
CI = Confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = Inoculated, decontaminated coupons.
11 Laboratory Blank = Not inoculated, not decontaminated coupon.
e Procedural Blank = Not inoculated, decontaminated coupon.
* Contamination of blanks with B. anthracis occurred during drying (see text).
"-" Not Applicable.
-------
Table 7-2. Inactivation of Bacillus anthracis (Ames) Spores8— Decon Green on Porous Materials (60 minute contact
time with reapplication at 30 minutes)
Test Material
Concrete
Positive Controls'1
Test Coupons0
Laboratory Blank11
Procedural Blank'
Brick
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Asphalt Paving
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Butyl Rubber
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum (CFU)
7.13 xlO7
7.13 xlO7
0
0
7.13 xlO7
7.13 xlO7
0
0
8.30 xlO7
8.30xl07
0
0
7.13 xlO7
7.13 xlO7
0
0
7.13 xlO7
7.13 xlO7
0
0
Mean of Logs of
Observed CFU
6.67±0.13
2.67 ±1.90
0
0
7.25 ± 0.04
0
0
0
7.67 ±0.06
4.71 ±0.23
0
0
6. 93 ±0.23
5.02 ±1.69
0
0
6.94 ±0.05
0
0
0
,„ „, _ Decontamination
Mean % Recovery
Efficacy ± CI
6.9 ±2.1
0.020 ±0.032 4.00 ±1.67
-
-
24.7 ±2.1
0 >7.25±0.03
-
-
57.0 ±7.9
0.069 ±0.040 2. 97 ±0.21
-
-
13.1±5.5
0.77 ±0.69 1.91 ±1.50
-
-
12.3 ±1.4
0 >6.94 ± 0.04
-
-
" Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual coupons, the mean percent
recovery on those five coupons, and decontamination efficacy (log reduction).
CI = Confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = Inoculated, decontaminated coupons.
11 Laboratory Blank = Not inoculated, not decontaminated coupon.
e Procedural Blank = Not inoculated, decontaminated coupon.
"-" Not Applicable.
-------
Table 7-3. Summary of Efficacy Values (Log Reduction)
Obtained for Decon Green
Test Material
Nonpo rous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Efficacy for
B. amthracis (Ames)
>7.64
>7.78
>7.80
>7.67
>7.32
4.00
>7.25
2.97
1.91
>6.94
The same observation noted in testing of CASCAD™
SDF (Section 6.2.2) was seen in the qualitative
assessment with Decon Green, with both the test and
positive control coupons of treated wood. The liquid
culture growth assessments for treated wood test and
positive control coupons were negative after both one
and seven days' incubation, even though the positive
control coupons had not been decontaminated and the
test coupons had been minimally decontaminated (i.e.,
Decon Green efficacy on treated wood was only 1.91
log reduction). As noted in Section 7.3, the growth
assessment solutions from the treated wood coupons
had a slight yellow hue. To investigate the possibility of
inhibition from the wood treatment itself, these visibly
negative liquid culture growth assessments from the
Decon Green testing were plated on nutrient agar. By
the following day all agar plates clearly showed colonies
exhibiting B. anthracis morphology. This observation
strongly suggested that an inhibitor}' compound from
the treated wood prevented the growth of B. anthracis in
liquid culture (where the concentration of the inhibitory
compound was the greatest), but the organism flourished
once a small amount of the liquid culture was plated
out onto nutrient agar. Therefore, the positive control
and test coupons of treated wood were indicated as
positive for growth in Table 7-4 because the plating step
established the presence and viability of B. anthracis in
the liquid culture.
7.3 to Coupons
No visible damage was observed on Hie test materials
after the 60 rnin contact time with Decon Green, or after
seven days' incubation in the qualitative efficacy test.
The extract solutions of treated wood coupons had a
noticeable yellowish hue, probably due to leaching of
treatment chemicals from the coupon material.
7.4 Other
7.4.1 Operator Control
On each day of testing, the three components of Decon
Green were prepared according to the vendor's explicit
instructions, as stated in Appendix C. Prior to each
application, the Decon Green spray nozzle was primed
by repeatedly spraying into an absorbent cloth to
clear any air bubbles that may have formed between
applications. After each application, the spray nozzle
was removed from the bottle and any residual Decon
Green was removed by repeated pulls on the trigger of
the spray nozzle. The spray nozzle was then placed into
a reservoir that contained only sterile, cell culture-grade
water so as to completely clean out the spray nozzle
until its next use. All material coupons were oriented
horizontally (i.e., lying flat) and stayed in that orientation
throughout the entire contact time.
All tests were conducted under ambient conditions
inside a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature, measured to be 22 °C (± 1 °C).
The relative humidity (RH) inside the test chamber was
monitored with a NIST-traceablc hygrometer. Whenever
the RH reached 70%. the dehumidification system
attached to the test chamber was actuated until the RH
dropped below 70%. Therefore, the test chamber RH
was always < 70% RH during the decontamination of
test materials with Decon Green.
7.4,2 Technology Spray Deposition
Decon Green was applied according to the procedure
included as Appendix C of this report. Decon Green was
applied from a distance of 30.5 cm to the horizontally-
oriented materials until the materials were fully wetted.
Rcapplication of the Decon Green was made on both the
nonporous and porous coupon surfaces at 30 minutes
after the first application, for a total of two applications.
After the 60 minute contact time, each material coupon
was carefully placed into the respective extraction tube
that also served to collect excess decontaminant runoff.
To assess Decon Green deposition, triplicate coupons
of each test material were weighed prior to application
of the Decon Green in the trial runs, and these values
were recorded. Then the triplicate coupons were sprayed
with Decon Green until fully wetted in a horizontal
orientation, sprayed again 30 minutes later, and then
each coupon was weighed again after the 60 minute
contact time. The pre-application weights were then
subtracted from the post-application weights, and that
difference was added to the weight of decontaminant
runoff captured separately from each coupon. The
average deposition/runoff weight of the Decon Green
from each of the test materials is shown in Table 7-5.
The average deposited amounts of 0.65 g on nonporous
materials and 0.75 g on porous materials were then used
in trials to determine the amount of sodium thiosulfate
(STS) needed to effectively neutralize the Decon Green.
-------
Table 7-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus amthracis (Ames) Spores
—Decon Green
Test Material
S2
Day 1
S3 S4
S5
B
SI
S2
Day?
S3 S4
S5
B
Stainless Steel
Positive Controls
Test Coupons
Glass
Positive Controls
Test Coupons
Aluminum
Positive Controls
Test Coupons
Porcelain
Positive Controls
Test Coupons
Granite
Positive Controls
Test Coupons
Concrete
Positive Controls
Test Coupons
Brick
Positive Controls
Test Coupons
Asphalt Paving
Positive Controls
Test Coupons
Treated Wood
Positive Controls'1
Test Coupons'1
Butyl Rubber
Positive Controls
Test Coupons
+
+ +
+ +
+ + +c
+ + + + + +c
+ + + + + +c
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
SI to S5 = Sample 1 to Sample 5. B = Blank (not inoculated with5. anthracis (Ames)); a = laboratory blank, b = procedural blank.
Positive controls = Coupons inoculated with B. anthracis (Ames) spores, but not subjected to decontamination.
Test coupons = Coupons inoculated with B. anthracis (Ames) spores, and subjected to decontamination.
"•f" = growth; "-" = no growth.
'' These laboratory blanks inadvertently exposed to inoculated coupons during drying (see text).
d Treated wood coupons showed no growth in one-day or seven-day incubation, but showed growth consistent with B. anthracis morphology
when culture was plated (see text).
-------
7,4.3 Neutralization Methodology
Neutralization of Decon Green was achieved with STS.
The concentrations of STS used during the neutralization
trials were 2.0, 2.5, and 3.0% in the PBS/Triton X-100
extraction solution, because initial trials showed that
lower concentrations were inadequate to neutralize the
Decon Green. The results of the neutralization trials
are shown for nonporous test materials in Table 7-6 and
for the porous test materials in Table 7-7. From these
results it was concluded that 2.0% STS and 3.0% STS
concentrations in the extraction solution were sufficient
for neutralization of Decon Green for the nonporous and
porous materials, respectively.
Table 7-5. Deposition/Runoff Weight of Decon Green on
Test Materials
Test Material
Average Deposition/
Runoff Weight (g)
Nonporous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Average
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Average
0.52
0.81
0.62
0.92
0.37
0.65
0.39
0.79
0.51
1.05
1.03
0.75
Table 7-6. Neutralization Testing with Bacillus anthracis
(Ames) Spores for Decon Green on Nonporous Test
Materials
Treatment
Inoculum
(CFU)
Total
Observed
(CFU)
% of
Control
Decon Green +
Spores3
Decon Green + PBS
+ Triton® X-100 +
Spores8*
PBS + Triton8
X-100 +Spores
(Control)5
Decon Green + PBS
+ Triton® X-100
+ 2.0% STS +
Spores8*
8.70 xlO7 0
8.70 xlO7
8.70 xlO7 S.lOxlO7
8.70 xlO7 7.68 xlO7
100
94.8
Decon Green + PBS
+ Triton8 X-100
+ 2.5%STS +
Sporesa-b
Decon Green + PBS
+ Triton® X-100
+ 3.0% STS +
Spores3-1-1
8.70 xlO7 7.12xl07 87.9
8.70 xlO7 7.42 xlO7 91.6
" Decon Green volume of 0.65 ml, corresponds to mean gravimetric
deposition on nonporous materials, and density of approximately
1.0 g/mL.
b 10 ml, Volume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of STS; total volume for all samples with Decon
Green - 10.65 mL (10 ml, PBS/Triton* X-100/STS - 0.65 mL
Decon Green).
Table 7-7. Neutralization Testing with Bacillus anthracis
(Ames) Spores for Decon Green on Porous Test Materials
Treatment
Decon Green +
Spores"
Decon Green + PBS
+ Triton® X- 100 +
Spores3'15
PBS + Triton®
X- 100 + Spores
(Control)b
Inoculum
(CFU)
8.70 x 107
8.70 xlO7
8.70 x 107
Total
Observed
(CFU)
0
0
7.89 x 107
%of
Control
0
0
100
Decon Green + PBS
+ Triton'8 X-100
+ 2.0% STS +
Spores*
Decon Green + PBS
+ Triton® X-100
+ 2.5% STS +
Spores3-5
Decon Green + PBS
+ Triton® X-100
+ 1 0% STS +
Spores1''
Q DLLOU Green volume of 0.75 mL corresponds to mean gravimetric
deposition on porous materials, and density of approximately 1.0
g/mL.
" 10 mL Volume of PBS includes 0.1% of Triton" X-100 surfactant
and indicated % of STS; total volume for all samples with Decon
Green - 10.75 mL (10 mL PBS/Triton« X-100/STS ' 0.75 mL
Decon Green).
8.70 xlO7 7.16xl07
8.70 x 107 7.70 xlO7
8.70 x 107 8.22 xlO7
90.8
97.6
104.1
-------
8.0
EasyDECONf 200 Test Results
8.1 QC
In testing of EasyDECON® 200, all positive control
results were well within the target recovery range of 1
to 150% of the spiked spores. Positive control recover}'
values on the nonporous materials ranged from 40 to
74%, with the lowest recovery occurring on granite and
the highest on glass. Positive control recovery values
on the porous materials ranged from 12 to 39%, with
the lowest recovery occurring on butyl rubber and the
highest on asphalt paving.
In testing of EasyDECON® 200, all procedural and
laboratory blanks met the criterion of no observed CPU
in quantitative efficacy testing with B. anlhracis. No
growth was observed in the qualitative assessment of
residual spores for all procedural and laboratory blanks.
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1
x lOVmL (± 25%), leading to a spike of 1 * 10s spores
(± 25%) on each test coupon. The actual spike values
for three days of B. anthracis testing were all within that
criterion, at 8.40 x lOVcoupon, 8.07 x lOYcoupon and
8.77 x lOVcoupon, respectively.
8.2 Efficacy
The decontamination efficacy of EasyDECON® 200
was evaluated for B. anthracis (Ames) on ten outdoor
material surfaces. The following sections summarize the
results found with this decontaminant.
8.2.1 Quantitative /Issessment of the Log
Reduction of Organisms
The results for decontamination efficacy of
EasyDECON® 200 on nonporous and porous materials
are shown in Tables 8-1 and 8-2, respectively,
and summarized in Table 8-3. A relatively large
deposition amount (approximately 2 g per coupon) was
recommended by the vendor of EasyDECON® 200, but
it was difficult to achieve such a high deposition rate
in practice. Testing of EasyDECON® 200 began with
nonporous materials, using a procedure that called for
three applications of the decontaminant. The resulting
deposited amount of EasyDECON® 200 was lower than
Table 8-1. Inactivation of Bacillus anthracis (Ames) Spores"—EasyDECON® 200 on Nonporous Materials (30
minute contact time with reapplicatlon at 10 and 20 miniites; or 30 miniite contact time with reapplkation at 5,10,
15, 20, and 25 minutes)
Test Material
Inoculum (CFU)
Mean of Logs of
Observed CFU
Mean %
Recover*'
Decontamination
Efflcacv ± CI
Stainless Steel
Positive Controls5
Test Coupons'
Laboratory Blank'1
Procedural Blank0
8.07 xlO7
8.07 xlO7
0
0
7.61 ±0.03
0
0
0
50.4 ±3.9
0
0
0
> 7.61 ±0.03
Glass'
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.40 x 107
8.40 x 107
0
0
7.79 ± 0.05
0
0
0
74.2 ± 8.4
0
0
0
> 7.79 ± 0.04
Aluminum1'
Positive Controls
Test Coupons
Laboratory Blank
8.40 xlO7
8.40 x 107
0
7.75 ± 0.07
0
0
67.2 ±11.7
0
0
> 7.75 ± 0.06
-------
Procedural Blank
Porcelain'
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.40 xlO7
8.40 x 107
0
0
7.78 ± 0.01
0
0
0
71.3 ±2.0
0
0
0
> 7.78 ±0.01
Granite
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.07 x 107
8.07 x 107
0
0
7.51 ±0.05
0
0
0
40.0 ±5.0
0
0
0
>7.51 ±0.05
* Data arc expressed as the mean (± SD) of the logs of the number of spores (CPU) observed on five individual coupons, the mean
percent recovery on those live coupons, and decontamination efficacy (log reduction).
CI - Confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
c Test Coupons = Inoculated, decontaminated coupons.
d Laboratory Blank = Not inoculated, not decontaminated coupon.
* Procedural Blank ~ Not inoculated, decontaminated coupon.
f Tills material tested with three applications of EasyDECON 200®; others tested with six applications.
"-" Not Applicable.
Table 8-2. Inactivatiom of Bacillus anthracis (Ames) Spores"— EasyDECON 200 on Porous Materials (60 minute
contact time with reapplications at 10, 20, 30, 40, and 50 minutes)
Test Material
Inoculum (CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Decontamination
Efficacy ± CI
Concrete
Positive Controls11
Test Coupons'
Laboratory Blank"1
Procedural Blank0
8.77 xlO7
8.77 xlO7
0
0
7.14 ±0.03
0
0
0
15.6 ±1.2
0
0
0
> 7.14 ±0.03
Brick
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.77 xlO7
8.77 xlO7
0
0
7.28 ±0.23
0
0
0
24.6 ±13.8
0
0
0
> 7.28 ± 0.20
Asphalt Paving
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.07 xW
8.07 xlO7
0
0
7.47 ±0.16
5.84 ±0.05
0
0
38.5 ±11.5
0.87 ± 0.096
0
0
1.63 ±0.14
Treated Wood
Positive Controls
8.77 xlO7
6.96 ±0.30
12.9 ±11.3
-------
Test Coupons
Laboratory Blank
Procedural Blank
8.77 xlO7
0
0
6.13 ±0.04
0
0
1.55±0.16
0
0
0.82 ±0.26
Butyl Rubber
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.77 xlO7
8.77 xlO7
0
0
6.99 ±0.12
0
0
0
11.6±3.3
0
0
0
-
> 6.99 ±0.10
-
-
b Data are expressed as the mean (± SD) of the logs of the number of spores (CPU) observed on five individual coupons, the mean
percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = Confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = Inoculated, decontaminated coupons.
11 Laboratory Blank = Not inoculated, not decontaminated coupon.
e Procedural Blank = Not inoculated, decontaminated coupon.
"-" Not Applicable.
Table 8-3. Summary of Efficacy Values (Log Reduction)
Obtained for EasyDECON® 200
Test Material
Efficacy for
B. anthracis (Ames)
Nonporous
Stainless Steel
Glass1
Aluminum1
Porcelain1
Granite
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
>7.61
>7.79
>7.75
>7.78
>7.51
>7.14
>7.28
1.63
0.82
>6.99
" These three materials tested with three applications of
EasyDECON8 200; all others tested with six applications.
expected, but nevertheless for three nonporous materials
(glass, aluminum, and porcelain) complete kill of
B. anthracis spores was achieved. To increase the
deposition rate, a revised procedure of six applications
was then developed and used with the vendor's approval
on all the porous materials and on the two other
nonporous materials (stainless steel and granite). Both
application procedures are described in Appendix D, and
footnotes to Tables 8-1 and 8-3 indicate which materials
were tested with three and which with six applications
of EasyDECON® 200.
The decontamination efficacy of EasyDECON® 200
for B. anthracis was > 7.51 log reduction with all of
the nonporous materials, as shown in Table 8-3. No
viable spores were found on any of the nonporous
materials after decontamination with EasyDECON®
200. However, the decontamination efficacy of
EasyDECON® 200 was not as consistent with the porous
materials, as shown in Table 8-3. Efficacy values of
about 7.0 log reduction were achieved with unpainted
concrete, brick, and butyl rubber, and no viable spores
were found on these materials after decontamination
with EasyDECON® 200. In contrast, efficacy values
of 1.63 and 0.82 log reduction were found with asphalt
paving and treated wood, respectively.
8.2.2 Qualitative Assessment of Residual
Spores
Results from the liquid culture growth assessment of
coupons at one and seven days post-decontamination
are provided in Table 8-4 for coupons spiked with B.
anthracis (Ames). In this assessment, cultures showing
positive growth (i.e., a cloudy growth medium) were
subjected to streak plating and the identity of the
growing organism was checked by colony morphology.
Only colonies consistent with the morphology of B.
anthracis were found in cultures of coupons inoculated
with B. anthracis.
Table 8-4 shows qualitative efficacy results for all
materials which are consistent with the quantitative
efficacy results reported in Section 8.2.1 (Table 8-3).
For all five nonporous materials, and for concrete, brick,
and butyl rubber, no growth was observed from the
decontaminated test coupons after either one or seven
days' incubation. The decontaminated coupons of
asphalt and treated wood all were positive for growth
at both one and seven days' incubation, consistent
with the relatively low quantitative efficacy results for
those materials (Table 8-3). Table 8-4 also shows that
-------
all laboratory and procedural blanks were negative for
growth.
The same observation noted in testing of CASCAD™
SDF (Section 6.2.2) and Decon Green (Section
7.2.2) was seen in the qualitative assessment with
EasyDECON* 200, with both the test and positive
control coupons of treated wood. That is. the liquid
culture growth assessments for treated wood test and
positive control coupons were negative after both one
and seven days' incubation, even though the positive
control coupons had not been decontaminated and the
test coupons had been minimally decontaminated (i.e..
EasyDECON® 200 efficacy on treated wood was only
0.82 log reduction). As noted in Section 8.3, the growth
assessment solutions from the treated wood coupons
had a slight yellow hue. These visibly negative liquid
culture growth assessments from the EasyDECON®
200 testing were plated on nutrient agar, and by the
following day all agar plates clearly showed colonies
exhibiting B. anthracis morphology. This observation
strongly suggested that an inhibitory compound from
the treated wood prevented the growth ofB. anthracis in
liquid culture (where the concentration of the inhibitory
compound was the greatest), but the organism flourished
once a small amount of the liquid culture was plated
out onto nutrient agar. Therefore, the positive control
and test coupons of treated wood were indicated as
positive for growth in Table 8-4 because the plating step
established the presence and viability- of B. anthracis in
the liquid culture.
Table 8-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis
(Ames) Spores—EasyDECON8 200
8.3 to Coupons
No visible damage was observed on the test materials
after the 30 min contact time for non-porous materials
and 60 min contact time for the porous materials with
EasyDECON® 200, with either three or six applications
of the decontaminant. The treated wood extracts had a
noticeable yellowish hue, probably due to leaching of
treatment chemicals from the coupon material.
8.4 Other
8.4.1 Operator Control
On each day of testing, the three components of
EasyDECON® 200 were weighed and mixed according
to the vendor's explicit instructions, as incorporated into
the application procedure in Appendix D. Prior to each
application, the EasyDECON® 200 spray nozzle was
primed by repeatedly spraying into an absorbent cloth
to clear any air bubbles that may have formed between
applications. After each application, the spray nozzle
was removed from the bottle and
any residual EasyDECON® 200 was removed by
repeated pulls on the trigger of the spray nozzle. All
material coupons were oriented horizontally (i.e., lying
flat) and stayed in that orientation throughout the entire
contact time.
Test Material
SI
Dayl
S2 S3 S4 S5
Stainless Steel
Positive Controls
Test Coupons
Glass
Positive Controls
Test Coupons
Aluminum
Positive Controls
Test Coupons
Porcelain
Positive Controls
Test Coupons
Granite
Positive Controls
Test Coupons
Day 7
SI S2 S3 S4
S5 B
-------
Concrete
Positive Controls
Test Coupons
Brick
Positive Controls
Test Coupons
Asphalt Paving
Positive Controls
Test Coupons
Treated Wood
Positive Controls'
Test Coupons0
Butyl Rubber
Positive Controls
Test Coupons
SI to S3 = Sample 1 to Sample 5.
B = Blank (not inoculated with B. anthracis (Amos) spores); a = laboratory blank, b = procedural blank.
Positive controls = Coupons inoculated with B. anthracis (Amos) spores, but not subjected to decontamination.
Test coupons = Coupons inoculated with -B. anthracis (/tales) spores, and subjected to decontamination.
' V = growth; "-" = no growth.
" Treated wood coupons showed no growth in one-day or seven-day incubation, but showed growth consistent with 5. anthracis
morphology when culture was plated (sec text).
All tests were conducted under ambient conditions
inside a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature, measured to be 22 °C (± 1°C).
The RH inside the test chamber was monitored with a
NIST-traceable hygrometer. Whenever the RH reached
70%. the dehumidification system attached to the testing
chamber was actuated until the RH dropped below 70%.
Therefore, the testing chamber RH was always < 70%
during the decontamination of all test materials with
EasyDECON® 200.
8.4.2 Technology Spray Deposition
EasyDECON® 200 was applied according to the
procedures included as Appendix D of this report.
EasyDECON® 200 was applied from a distance of
30.5 cm to the horizontally-oriented material coupons
until the coupons were fully wetted. As described in
Section 8.2.1, reapplication of the EasyDECON® 200
was then made on three of the nonporous materials
(glass, aluminum, porcelain) at 10 and 20 minutes after
the initial application for a total of three applications,
and on the other two nonporous materials (stainless
steel, granite) at 5, 10, 15, 20, and 25 minutes after the
initial application for a total of six applications, within
a 30-minute total contact time. Reapplication of the
EasyDECON® 200 was made on the porous materials at
10, 20, 30, 40, and 50 minutes after the initial application
for a total of six applications within a 60-minute total
contact time. After the 30-minute contact time for the
nonporous materials or the 60-minute contact time for
the porous materials each coupon was placed in the
50 niL conical vial that also served to collect excess
decontaminant that may have pooled on the coupon
surface.
To assess EasyDECON® 200 deposition, triplicate
coupons of each test material were weighed prior to
application of the EasyDECON® 200 in the trial runs,
and these values were recorded. Then the triplicate
coupons were sprayed with EasyDECON® 200 in their
horizontal orientation according to the procedures in
Appendix D, and each coupon was weighed again after
their respective contact times. The pre-application
weights were then subtracted from (lie post-application
weights, and that difference was added to the weight
of decontaminant runoff captured separately from
each coupon. The average deposition/runoff weight of
EasyDECON® 200 from each of the test materials is
shown in Table 8-5, for the application procedure used
with each material in testing. The average deposition
values of 0.12 g for glass, aluminum, and porcelain. 0.32
g for stainless steel and granite, and 0.95 g for porous
materials were used to estimate the amount of sodium
thiosulfate (STS) needed to effectively neutralize the
EasvDECON® 200.
-------
8,4.3 Neutralization Methodology
Neutralization of EasyDECON* 200 was achieved
with STS. The concentrations of STS tested during
the neutralization trial were 0.5, 1.0, and 1.5% in
the PBS/Triton® X-100 extraction solution. The
results of the neutralization trials are shown for the
nonporous materials in Tables 8-6 and 8-7 (three and six
applications, respectively), and for the porous materials
in Table 8-8. On the basis of these results 1.0% STS was
used for neutralization of EasyDECON® 200 in testing
with three applications on nonporous materials, and
1.5% STS was used for neutralization of EasyDECON®
200 in testing with six applications, on both porous and
nonporous materials.
Table 8-5. Deposition/Runoff Weight of EasyDECON® 200
on Test Materials
Average Deposition/
Test Material
Nonporous
Glass1
Aluminum51
Porcelain8
Average
Stainless Steel'1
Granitek
Average
Porous0
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Average
Runoff Weight (g)
0.08
0.14
0.13
0.12
0.27
0.36
0.32
0.80
0.87
0.99
1.27
0.80
0.95
" These materials tested with three applications. 30-minute contact
time.
b These materials tested with six applications, 30-minute contact time.
" All porous materials tested with six applications, 60-minute contact
time.
Table 8-6. Neutralization Testing with Bacillus anthracis
(Ames) Spores for EasyDECON® 200 on Nonporous Test
Materials: Glass, Aluminum, and Porcelain (3 applications)
Treatment
Inoculum
(CFU)
Total
Observed
(CFU)
%of
Control
7.47 xlO7
EasyDECON® 200
Spores"
EasyDECON® 200 +
PBS + Triton® X-l 00 7.47 x 107
+ Sporeslb
0
PBS + Triton® X-100
+ Spores (Control)1"
EasyDECON® 200
+ PBS + Triton®
X-100+ 0.5% STS+
Spores3-1"
EasyDECON® 200
+ PBS + Triton®
X-100 + 1.0%STS +
Spores8-11
EasyDECON® 200
+ PBS + Triton®
X-100 + 1.5% STS +
Sporesa-b
" EasyDECON" 200 volume of 0.12 mL corresponds to mean
gravimetric deposition on glass, aluminum, and porcelain.
assuming density of 1.0 g/mL.
" 10 mL Volume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of SI'S; total volume for all samples with
EasyDECON* 200-10.12 mL (10 mL PBS/Triton* X-100/STS -
0.12 mL EasyDECON* 200).
Table 8-7. Neutralization Testing with Bacillus anthracis
(Ames) Spores for EasyDECON® 200 on Nonporous Test
Materials: Stainless Steel and Granite (6 applications)
7.47 xlO7 7.01 xlO7
7.47 xlO7 7.73 x 107
7.47 xlO7 7.10 xlO7
7.47 x 107 6.60 x 107
100
110.3
101.3
94.1
Treatment
Inoculum
(CFU)
Total
Observed
(CFU)
%of
Control
EasvDECON8 200 +
9.30xl07
Spores3
EasyDECON8 200 +
PBS + Triton® X-100 9.30 x 107
+ Spores3-1-1
PBS + Triton® X-l 00
+ Spores (Control)11
EasyDECON8 200
+ PBS + Triton®
X-100+ 0.5% STS +
Spores3-1"
EasyDECON8 200
+ PBS + Triton®
X-100 + 1.0% STS +
Spores3-'"
EasyDECON® 200
+ PBS + Triton®
X-100 + 1.5%STS +
Spores3-1-1
0
9.30 xlO7 8.49 xlO7
9.30 xlO7 8.32 xlO7
9.30 xlO7 8.44 xlO7
9.30 x 107 8.62 x 107
100
98.1
99.4
101.6
EasyDECON* 200 volume of 0.32 mL corresponds to mean
gravimetric deposition on stainless steel and granite, assuming
density of 1.0 g/mL.
10 ml. Volume of PBS includes 0.1% of Triton* X-l 00 surfactant
and indicated % of STS; total volume for all samples with
EasyDECON* 200 - 10.32 mL (10 ml. PBS/Triton* X-100/STS ~
0.32 mL EasyDECON* 200).
-------
Table 8-8. Neutralization Testing with Bacillus anthracis (Ames) Spores for EasyDECON® 200 on Porous Test
Materials (6 applications)
Treatment
EasyDECON8 200 + Spores"
EasyDECON8 200 + PBS + Triton8 X-100 + Spores1-5
PBS + Triton® X-100 + Spores (Control)'1
EasyDECON® 200 + PBS + Triton® X-100 + 0.5% STS + Spores'-"
EasyDECON® 200 + PBS + Triton® X-100 + 1 .0% STS + Spores'-"
EasyDECON8 200 + PBS + Triton® X-100 + 1 .5% STS + Spores1-5
Inoculum
(CFU)
9.30xl07
9.30xl07
9.30xl07
9.30xl07
9.30xl07
9.30xl07
Total
Observed
(CFU)
0
0
8.85 x 107
8.37 x 107
7.85 x 107
8.41 x 107
%of
Control
0
0
100
94.7
88.8
95.0
" EasyDECON* 200 volume of 0.95 mL corresponds to mean gravimetric deposition on porous materials, assuming density of 1.0
g/mL.
b 10 mL Volume of PBS includes 0.1% of Triton® X-100 surfactant and indicated % of STS; total volume for all samples with
EasyDECON* 200 - 10.95 mL (10 mL PBS/Triton* X- 100/STS -f 0.95 mL EasyDECON* 200).
-------
-------
9.0
Spor-Klenz® RTU Test Results
9.1 QC
In testing of Spor-Klenz® RTU, all positive control
results were well within the target recovery range of 1
to 150% of the spiked spores. Positive control recovery
values on the nonporous materials ranged from 48 to
75%. with the lowest recovery occurring on granite and
the highest on anodized aluminum. Positive control
recovery values on the porous materials ranged from 17
to 29%, with the lowest recover}' occurring on treated
wood and the highest on butyl rubber.
In testing of Spor-Klenz® RTU, all procedural and
laboratory blanks met (lie criterion of no observed CPU
in quantitative efficacy testing with B. anthracis. No
growth was observed in the qualitative assessment of
residual spores for all procedural and laboratory blanks.
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density- is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1
x lOVmL (± 25%), leading to a spike of 1 x 10s spores
(± 25%) on each test coupon. The actual spike values
for three days of B. anthracis testing were all within that
criterion, at 9.30 x lOVcoupon, 8.63 x lOVcoupon, and
7.80 x lOVcoupon. respectively.
9.2 Efficacy
The decontamination efficacy of Spor-Klenz® RTU
was evaluated for B. anthracis (Ames) on ten outdoor
material surfaces. The following sections summarize the
results found with this decontaminant.
9.2.1 Quantitative of tfie Log
Reduction of Viable Organisms
The results for decontamination efficacy of Spor-Klen/®
RTU on nonporous and porous materials are shown in
Tables 9-1 and 9-2, respectively, and summarized in
Table 9-3. The decontamination efficacy of Spor-Klenz®
RTU was greater than 7.5 log reduction on porcelain and
granite, as shown in Table 9-1. No viable spores were
found on any coupons of these two nonporous materials.
Efficacy on the other three nonporous materials ranged
from 7.17 to 7.36 log reduction. With each of these
three materials, viable spores were found on only one of
the five test coupons after decontamination.
Table 9-1. Inactivation of Bacillus anthracis (Ames) Spores"—Spor-Klenz® RTU on Nonporous Materials (30
minute contact time with one rcapplication at 25 minutes)
Test Material
Inoculum
(CFU)
Mean of Logs of
Observed CFU
Mean % Recovery
Decontamination
Efficacy ± CI
Stainless Steel
Positive Controls5
Test Coupons0
Laboratory Blankd
Procedural Blank"
9.30XKF
9.30 x 107
7.74 ± 0.04
0.46 ±1.03
59.9 ±6.0
0.000045 ±
0.000097
0
0
7.28 ±0.91
Glass
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
9.30 xlO7
9.30 xlO7
0
0
7.76 ± 0.06
0.40 ±0.90
0
0
62.4 ±9.1
0.000023 ±
0.000048
0
0
7.36 ± 0.79
Aluminum
Positive Controls
Test Coupons
9.30 xlO7
9.30 xlO7
7.84 ±0.04
0.67 ±1.50
75.5 ±6.4
0.00050 ±0.0011
7.17 ±1.32
-------
Laboratory Blank
Procedural Blank
Porcelain
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Granite
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
0
0
9.30xl07
9.30x107
0
0
7.80x107
7.80 x It)7
0
0
0
0
7.72 ± 0.05
0
0
0
7.57 ± 0.07
0
0
0
0
0
56.4 ± 7.0
0
0
0
47.8 ±7.7
0
0
0
-
-
-
> 7.72 ± 0.05
-
-
-
> 7.57 ±0.06
-
-
" Data are expressed as the mean (± SD) of the logs of the number of spores (CPU) observed on five individual coupons, the
mean percent recover)' on those live coupons, and decontamination efficacy (log reduction).
CI - Confidence interval (± 1.96 x SE).
b Positive Controls ~ Inoculated, not decontaminated coupons (sprayed with SFW).
c Test Coupons ~ Inoculated, decontaminated coupons.
d Laboratory Blank = Not inoculated, not decontaminated coupon.
* Procedural Blank ~ Not inoculated, decontaminated coupon.
"-" Not Applicable.
Table 9-2. Inactivation of Bacillus anthracis (Ames) Spores3— Spor-Kienz'1' RTU on Porous Materials (60
minute contact time with reapplications at 10, 25, 30, and 50 minutes)
Test Material
Inoculum
(CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Decontamination
Efficacy ± CI
Concrete
Positive Controls5
Test Coupons0
Laboratory Blankd
Procedural Blank0
8.63 x 107
8.63 x 107
0
0
7.19 ±0.36
6.17 ±0.21
0
0
23.5 ±18.3
2.35 ±1.41
0
0
1.02 ±0.36
Brick
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
7.80 x 107
7.80 x 107
0
0
7.27 ±0.26
0
0
0
27.3 ±16.1
0
0
0
> 7.27 ±0.22
Asphalt Paving
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
7.80 x 107
7.80x107
0
0
7.27 ±0.14
4.71 ±0.92
0
0
24.8 ±8.8
0.25 ± 0.33
0
0
2.56 ±0.81
Treated Wood
Positive Controls
Test Coupons
8.63 x 107
8.63 x 107
7.05 ± 0.26
0.89 ±1.25
17.1 ±14.7
0.00011 ±0.00021
6.16±1.12
-------
Laboratory Blank
Procedural Blank
Butyl Rubber
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
8.63 x 107
8.63 xlO7
0
0
7.39 ±0.10
0
0
0
29.1 ±7.3
0
0
0
-
> 7.39 ± 0.09
-
-
* Data arc expressed as the mean (± SD) of the logs of the number of spores (CPU) observed on five individual coupons, the
mean percent recovery on those live coupons, and decontamination efficacy (log reduction).
CI - Confidence interval (± 1.96 • SE).
b Positive Controls ~ Inoculated, not decontaminated coupons (sprayed with SFW).
c Test Coupons ~ Inoculated, decontaminated coupons.
d Laboratory Blank = Not inoculated, not decontaminated coupon.
* Procedural Blank = Not inoculated, decontaminated coupon.
"-" Not Applicable.
Table 9-3. Summary of Efficacy Values (Log Reduction)
Obtained for Spor-Klenz® RTU
Test Material
Efficacy for
B. anthracis (Ames)
Nonporous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
7.28
7.36
7.17
>7.72
>7.57
1.02
>7.27
2.56
6.16
>7.39
The decontamination efficacy of Spor-Klenz* RTU
was greater than 7.27 log reduction on brick and butyl
rubber, as shown in Table 9-2. No viable spores were
found on any coupons of these two porous materials.
Efficacy on unpainted concrete, asphalt paving, and
treated wood was approximately 1.02. 2.56, and 6.16 log
reduction, respectively, as shown in Table 9-2.
9.2.2 Qualitative of
Spores
Results from the liquid culture growth assessment of
coupons at one and seven days post-decontamination
are provided in Table 9-4 for coupons spiked with B.
anthracis (Ames). In this assessment, cultures showing
positive growth (i.e., a cloudy growth medium) were
subjected to streak plating and the identity of the
growing organism was checked by colony morphology.
Colonies consistent with the morphology of B. anthracis
were found only in cultures of coupons inoculated with
B. anthracis.
The qualitative efficacy results in Table 9-4 are
consistent with the quantitative efficacy results
summarized in Table 9-3 in that no growth was
observed at either one or seven days incubation from
the four materials that showed complete inactivation
in the quantitative testing (i.e.. porcelain, granite.
brick, and butyl rubber). Also, the other nonporous
materials (stainless steel, glass, and aluminum) each
showed growth for B. anthracis on the same single test
coupon on which viable spores were observed after
decontamination in the quantitative testing. The other
porous materials (concrete, asphalt, and treated wood)
were all strongly positive for growth as shown in Table
9-4, consistent with the relatively low quantitative
efficacy values on these materials (Table 9-3). Table
9-4 also shows that all laboratory and procedural blanks
were negative for growth.
The same observation noted in previous chapters was
made with the treated wood positive control and test
coupons. That is, the liquid culture growth assessments
for all treated wood coupons were negative (i.e., clear)
after both one and seven days' incubation, even though
-------
Table 9-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis (Ames)
Spores—Spor-Klenz® RTU
Test Material
S2
Day 1
S3 S4
SI
Day 7
S2 S3 S4
S5
Stainless Steel
Positive Controls
Test Coupons
Glass
Positive Controls
Test Coupons
Aluminum
Positive Controls
Test Coupons
Porcelain
Positive Controls
Test Coupons
Granite
Positive Controls
Test Coupons
Concrete
Positive Controls
Test Coupons
Brick
Positive Controls
Test Coupons
Asphalt Paving
Positive Controls
Test Coupons
Treated Wood
Positive Controls'
Test Coupons0
Butyl Rubber
Positive Controls
Test Coupons
+
+
+
+
+
+
+
+
SI to S5 - Sample 1 to Sample 5.
B = Blank (not inoculated with B. anthracis (Ames) spores); a = laboratory blank, b = procedural blank.
Positive controls = Coupons inoculated with B. anthracis (Ames) spores, but not subjected to decontamination.
Test coupons = Coupons inoculated with B. anthracis (Ames) spores, and subjected to decontamination.
" I " = growth; "-" = no growth.
0 Treated wood coupons showed no growth in one day or seven day incubation, but showed growth consistent with B. anthracis
morphology when culture was plated (see text).
-------
positive controls had not been decontaminated and the
Spor-Klcnz® RTU efficacy on the test coupons was
incomplete (6.16 log reduction). These negative liquid
culture growth assessments were plated on nutrient
agar, and all such plates clearly exhibited B. anihracis
colonies the following day. This observation suggested
that an inhibitory compound from the treated wood
may have prevented the growth of B. anthracis in
liquid culture (where the concentration of the inhibitory
compound was the greatest), but the organism flourished
once a small amount of this liquid culture was streaked
out on nutrient agar. Therefore, all treated wood
coupons were indicated as positive for growth in Table
9-4 because the plating step established the presence and
viability of B. anthracis in the liquid culture.
9.3 to Coupons
No visible damage was observed on the test materials
after the 30 min contact time for non-porous materials
or the 60 min contact time for the porous materials with
Spor-Klenz® RTU. The treated wood extracts had a
noticeable yellowish hue, probably due to leaching of
treatment chemicals from the coupon material.
9.4 Other
9.4,1 Operator Control
On each day of testing, Spor-Klenz® RTU was
transferred to a new, handheld, plastic spray bottle. Prior
to each application, the Spor-Klenz® RTU spray bottle
was primed by repeatedly spraying into an absorbent
cloth to clear any air bubbles that may have formed
between applications. After each application the spray
nozzle was removed from the bottle and any residual
Spor-Klenz® RTU was removed by repeated pulls on the
trigger of the spray nozzle. All coupons were oriented
horizontally (i.e., lying flat) and stayed in that orientation
throughout the entire contact time.
All tests were conducted at ambient conditions inside
a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature, measured to be 22 °C (± 1 °C).
The RH inside the test chamber was monitored with a
NIST-traceable hygrometer. Whenever the RH reached
70%, the dehumidification system attached to the testing
chamber was actuated until the RH dropped below 70%.
Therefore, the testing chamber was always at < 70% RH
during the decontamination of test materials with Spor-
Klenz® RTU.
9.4.2 Technology Spray Deposition
Spor-Klenz® RTU was applied according to the
procedure included as Appendix E of this report.
Specifics of the application procedure (i.e., the schedule
of reapplications needed to maintain wetting of the
test coupons) were defined in test runs to determine
deposition on the test coupons. Spor-Klenz® RTU was
applied from a distance of 30.5 cm to the horizontally
oriented coupons until the materials were fully wetted.
A single reapplication of the Spor-Klenz® RTU was
needed to maintain wetting of the nonporous materials at
25 minutes after the initial application, for a total of two
applications in the 30 minute total contact time on those
materials. Four reapplications of the Spor-Klenz® RTU
were needed to maintain wetting on the porous materials
at 10, 25, 30, and 50 minutes after the initial application,
for a total of five applications in the 60 minute total
contact time on those materials. The application
procedures thus established were used in the deposition
measurements, and in turn were used in all efficacy
testing of Spor-Klcnz® RTU. After the respective contact
times, each material coupon was placed in the 50 mL
conical vial that also screed to collect excess formulation
that may have pooled on its surface.
To assess Spor-Klenz® RTU deposition, triplicate
coupons of each test material were weighed prior to
application of the Spor-Klenz® RTU in trial runs, and
those values were recorded. Then the triplicate coupons
were sprayed with Spor-Klenz® RTU in their horizontal
orientation according to the procedures established based
on Appendix E, and each coupon was weighed again
after the respective contact time. The pre-application
weights were then subtracted from the post-application
weights, and that difference was added to the weight
of decontaminant runoff captured separately from each
coupon. The average deposition/runoff weight of the
Spor-Klenz® RTU from each of the test materials is
shown in Table 9-5. The average deposition amounts
of 0.16 and 0.48 g for nonporous and porous materials,
respectively, were used to estimate the amount of STS
needed to effectively neutralize the Spor-Klenz® RTU.
Table 9-5. Deposition/Riinoff Weight of Spor-Klenz® RTU
on Test Materials
Test Material
Average Deposition/
Runoff Weight (g)
Nonporous
Glass
Aluminum
Stainless Steel
Granite
Porcelain
Average
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Average
0.12
0.21
0.14
0.12
0.21
0.16
0.22
0.78
0.20
0.81
0.37
0.48
-------
9,4.3 Neutralization Methodology
Neutralization of Spor-Klenz® RTU was achieved
with STS. The concentrations of STS used during the
neutralization trials were 0.5, 1.0, and 1.5% in the PBS/
Triton® X-100 extraction solution. The results of the
neutralization trials are shown in Tables 9-6 and 9-7
for the nonporous and porous materials, respectively.
These tables show that in both the nonporous and
porous material trials, the action of Spor-Klenz® RTU
was inhibited by dilution with PBS/Triton® X-100
extraction solution, substantial recovery of spores was
seen with Spor-Klenz® RTU plus PBS solution in the
absence of STS (second row of Tables 9-6 and 9-7).
This observation implies that partial neutralization of
Spor-Klenz® RTU would occur in coupon extraction
with the PBS/Triton*X-100 solution. However, added
STS was needed to achieve complete neutralization of
Spor-Klenz® RTU. On the basis of these trials 0.5% STS
was used for neutralization of Spor-Klenz® RTU for both
nonporous and porous materials.
Table 9-6. Neutralization Testing with Bacillus anthrads
(Ames) Spores for Spor-Klenz8 RTU on Nonporous Test
Materials
Total
n. Inoculum „, , %of
Treatment ^T^-TV Observed „ ,
(CFU) fr-vwT. Control
v (CFU)
Table 9-7. Neutralization Testing with Bacillus anthrads
(Ames) Spores for Spor-Klenz® RTU on Porous Test
Materials
Treatment
Spor-Klenz® RTU
+ Spores*
Spor-Klenz® RTU
+ PBS + Triton®
X-100 + Sporesa-b
PBS + Triton®
X- 100 + Spores
(Control)1'
Spor-Klenz® RTU
+ PBS + Triton®
X-100 + 0.5%
SI'S + Spores1*
Spor-Klenz® RTU
+ PBS + Triton®
X-100+ 1.0%
STS + Spores3*
Spor-Klenz® RTU
+ PBS + Triton8'
X-100 +1.5%
STS + Sporesa-b
, . Total 0/ „
Inoculum /« ot
,„_,..,. Observed „ . ,
(CFU) /-XT™ Control
(CFU)
7.00xl07c 0 Od
7.00xl07c 1.43xl07 21.7"
9.07 xlO7 8.55 xlO7 100
9.07xl07 9.45 xlO7 110.4
9.07x10' 8.94x10' 104.6
0 07 Y 1 1V 8 Q4 Y 1 O7 1 04 fi
s .\J 1 \ i\J o . J^ A I U 1 UH . U
1 Spor-Klenz1 RTU volume of 0.48 niL corresponds to mean
od
97. ld
100
Spor-Klenz® RTU +
Spores3
Spor-Klenz® RTU
+ PBS + Triton®
X-100 +Spores3-11
PBS + Triton8
X-100 +Spores
(Control)5
Spor-Klenz® RTU
+ PBS + Triton8
X-100+ 0.5% STS
+ Spores3-5
Spor-Klenz® RTU
+ PBS + Triton®
X-100+1.0% STS
+ Spores3-5
Spor-Klenz® RTU
+ PBS + Triton®
X-100+1.5% STS
+ Spoies'b
* Spoi-Klenz* Rl'U A olume of 0.16 rnL corresponds to mean
gijMiuetiic deposition on non-porous materials, assuming
deiiMt\ of 1 0 g/mL.
b 10 mL\olume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of STS; total volume for all samples with Spor-
Klenz* RTU - 10.16 niL (10 ml PBS/Triton* X-100/STS + 0.16 ml,
Spor-Klenz* RTU).
' Inadequate number of dilutions prepared in initial trial with 9.07 x
107 inoculum; these trials redone, resulting in different inoculum.
d Percentage calculated by applying observed spore recovery with
7.00 x 107 inoculum to control inoculum of 9.07 x 107.
7.00x107c
7.00xl07c 6.84 xlO7
9.07x10' 9.13x10'
9.07x10' 9.05x10'
9.07x10' 1.55x108
9.07x10' 9.23 xlO7
gravimetric deposition on porous materials, assuming density
of l.Og/mL.
10 mL Volume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of SI'S; total volume for all samples with Spor-
Klenz* RTU - 10.48 mL (10 ml PBS/Triton* X-100/STS + 0.48 mL
Spor-Klenz* RTU).
Inadequate number of dilutions prepared in initial trial with 9.07 x
107 inoculum; these trials redone, resulting in different inoculum.
Percentage calculated by applying observed spore recovery with
7.00 x 107 inoculum to control inoculum of 9.07 x 107.
170.1
101.1
-------
10.0
Peridox® RTU Test Results
10.1 QC
In testing of Pcridox® RTU, all positive control recovery
results were well within the target range of 1 to 150% of
Hie spiked spores. Positive control recovery values on
the nonporous materials ranged from about 6.4 to 76%,
with the lowest recovery occurring on stainless steel
and the highest on anodized aluminum. The stainless
steel recovery of 6.4% was notably lower than in other
tests, but recoveries from the materials inoculated at the
same lime (i.e., granite, brick, and butyl rubber) were not
unusually low for those materials, so no systematic error
in inoculation is suspected. Positive control recovery
values on the porous materials ranged from about 6.3 to
45%, with the lowest recover}' occurring on butyl rubber
and the highest on asphalt paving.
In quantitative efficacy testing of Peridox* RTU with B.
anthracis, most procedural and laboratory blanks met the
criterion of no observed CPU. However, all procedural
and laboratory blanks of the four materials tested on
the third day of Peridox® RTU testing (i.e., stainless
steel, granite, brick, and butyl rubber) were found to
produce CPU of characteristic B. anthracis morphology
upon streak plating. An investigation of test procedures
disclosed that in that test, a laboratory trainee mistakenly
handled positive control coupons using a laboratory
forceps and then used the same forceps to handle the
blank coupons, thereby contaminating the extraction
solutions into which the blank coupons were placed.
This procedure violated established testing procedures,
which call for handling blank coupons before handling
any inoculated coupons to avoid contamination. The
observed contamination had no impact on the log
reduction determined for Peridox® RTU on these test
materials, as test coupons were handled properly.
However, a test/QA plan deviation was prepared and
placed in the project files to document this departure
from test procedures.
No growth was observed in the qualitative assessment of
residual spores for all procedural and laboratory blanks.
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1
x 10°/mL (± 25%), leading to a spike of 1 * 108 spores
(± 25%) on each test coupon. The actual spike values
for three days of .B. anthracis testing were all within that
criterion, at 8.83 x lOVcoupon, 8.87 x lOVcoupon and
8.20 x lOVcoupon, respectively.
10.2 Decontamination
The decontamination efficacy of Peridox® RTU was
evaluated for B. anthracis (Ames) on 10 outdoor
material surfaces. The following sections summarize the
results found with this decontaminant.
10.2.1 Quantitative Assessment of the Log
Reduction of Viable Organisms
The results for decontamination efficacy of Peridox®
RTU on nonporous and porous materials are shown in
Tables 10-1 and 10-2, respectively, and summarized in
Table 10-3. The contaminated blank coupons of four
materials noted in Section 10.1 are identified in Tables
10-1 and 10-2, and denoted by a footnote in each table.
The decontamination efficacy of Peridox® RTU for
B. anthracis was > 6.69 log reduction on stainless
steel, and greater than 7.42 log reduction on the other
nonporous materials, as shown in Table 10-1. No viable
spores were found on any coupons of the nonporous
materials decontaminated with Peridox® RTU. The
decontamination efficacy of Peridox® RTU was not as
consistent on the porous materials, as shown in Table
10-2. Log reductions on treated wood and butyl rubber
were > 6.99 and > 6.65 logs, respectively, and no viable
spores were found on any coupons of those materials
decontaminated with Peridox® RTU. Efficacy on asphalt
paving coupons was similarly high (7.22 log reduction),
although viable spores were recovered from one
decontaminated asphalt coupon. Unpainted concrete and
brick had log reductions of approximately 1.39 and 3.81,
respectively, and viable spores were recovered from all
decontaminated coupons of those materials.
10.2.2 Qualitative Assessment of Residual
Spores
Results from the liquid culture growth assessment of
coupons at one and seven days post-decontamination
are provided in Table 10-4 for coupons spiked with B.
anthracis (Ames). In this assessment, cultures showing
positive growth (i.e., a cloudy growth medium) were
subjected to streak plating and the identity of the
growing organism was checked by colony morphology.
Only colonies consistent with the morphology of B.
anihracis were found in cultures of coupons inoculated
with B. anthracis.
-------
The qualitative efficacy results in Table 10-4 are
largely consistent with the quantitative efficacy results
summarized in Table 10-3 in that no growth was
observed at either one or seven days incubation from
the five nonporous materials that showed complete
inactivation in the quantitative testing, or from the
porous material treated wood, which also showed
complete inactivation in the quantitative testing. All test
coupons of concrete and brick showed positive growth
at both one and seven days' incubation, consistent with
the relatively low quantitative efficacy found with those
materials. Two test coupons of asphalt paving showed
positive growth, whereas viable spores were recovered
from only one coupon in the quantitative testing.
The exception in terms of consistency relative to the
quantitative results was for butyl rubber, in that three test
coupons of that material showed positive growth at both
one and seven days incubation, although no viable spores
were recovered from that material in the quantitative
Table 10-1. Inactivation of Bacillus anthrack (Ames) Spores"—Peridox'1' RTU on Nonporous Materials
(30 minute contact time with re-applications at 10 and 25 minutes)
Test Material
Stainless Steel
Positive Controls1'
Test Coupons'
Laboratory Blank11
Procedural Blank'
Glass
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Aluminum
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Porcelain
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Granite
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
8.20 x It)7
8.20 x 107
0
0
8.83 x 107
8.83 x 107
0
0
8.83xl07
8.83 x 107
0
0
8.83 x 107
8.83 x 107
0
0
8.20 x 107
8.20 x 107
0
0
Mean of Logs of
Observed CFU
6.69 ±0.17
0
2.01f
3.42f
7.76 ± 0.03
0
0
0
7. 82 ±0.05
0
0
0
7.71 ±0.05
0
0
0
7.42 ±0.12
0
3.54f
2.31f
Mean %
Recovery
6.4 ±2.7
0
0.0001
0.003
65. 3 ±5.1
0
0
0
75. 8 ±9.2
0
0
0
57.8 ±7.5
0
0
0
32.7 ±8.6
0
0.004
0.0003
Decontamination
Efficacy ± CI
-
> 6.69 ±0.1 5
-
-
-
> 7.76 ± 0.03
-
-
-
> 7.82 ± 0.05
-
-
-
> 7.71 ± 0.05
-
-
-
> 7.42 ±0.11
-
-
Data are expiessed as the mean ( I SD) of"Hie log", of'lhe numbet of"spores (CFt ) observed on five individual coupons,
the meanpeuenl ieto\ei\ on those Ine coupons, and decontamination efficacy (log ieduction).
CI - Confidence interval (± 1.96 x SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
c Test Coupons = Inoculated, decontaminated coupons.
d Laboratory Blank = Not inoculated, not decontaminated coupon.
'' Procedural Blank = Not inoculated, decontaminated coupon.
* Blank coupons contaminated after testing due to improper handling procedure; see text.
"-" Not Applicable.
-------
Table 10-2. Inactivation of Bacillus anthmeis (Ames) Spores"— Peridox® RTU on POTOHS Materials (60
minute contact time with re-applications at 10, 20, 30, 40, and 50 minutes)
Test Material
Concrete
Positive Controls1'
Test Coupons1'
Laboratory Blank'1
Procedural Blank'
Brick
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Asphalt Paving
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Butyl Rubber
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
8.87 xlO7
8.87 xlO7
0
0
8.20 xlO7
8.20 x 107
0
0
8.87 xlO7
8.87 xlO7
0
0
8.87 xlO7
8.87 xlO7
0
0
8.20 x 107
8.20 xlO7
0
0
Mean of Logs of
Observed CFU
7.10 ±0.07
5.71 ±0.16
0
0
6.78 ±0.21
2.97±1.11
4.131'
4.17r
7.59 ±0.04
0.37 ±0.83
0
0
6.99 ±0.04
0
0
0
6.65 ± 0.28
0
2.15f
2.55f
Mean %
Recover}'
14.2 ±2.4
0.61 ±0.26
0
0
7.9 ±3. 3
0.0087 ±0.017
0.016
0.018
44. 5 ±4.4
0.000017 ±
0.000035
0
0
11.1 ±1.0
0
0
0
6.3 ±3.4
0
0.0002
0.0004
Decontamination
Efficacy ± CI
-
1.39 ±0.15
-
-
-
3. 81 ±0.99
-
-
-
7.22 ± 0.73
-
-
-
> 6.99 ±0.03
-
-
-
> 6.65 ± 0.25
-
-
a Data are expressed as the mean (;;; SD) of the logs oi'the number of spores (CFU) observed on five individual coupons, the
mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI - Confidence interval (= 1.96 - SE).
b Positive Controls = Inoculated, not decontaminated coupons (sprayed with SFW).
c Test Coupons = Inoculated, decontaminated coupons.
d Laboratory Blank = Not inoculated, not decontaminated coupon.
' Procedural Blank = Not inoculated, decontaminated coupon.
f Blank coupons contaminated after testing due to improper handling procedure; see text.
"-" Not Applicable.
-------
Table 10-3. Summary of Efficacy Values (Log Reduction)
Obtained for Peridox® RTU
Test Material
Efficacy for B. anthracis
(Ames)
Nonporous
Stainless Steel
Glass
Aluminum
Porcelain
Granite
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
>6.69
> 7.76
>7.82
>7.71
>7.42
1.39
3.81
7.22
> 6.99
>6.65
efficacy testing. Growth in the qualitative testing
was also observed with a few coupons of porcelain.
but the morphology of the resulting CPU upon plating
was clearly not consistent with the morphology of
B. anthracis, so all porcelain coupons are shown as
negative for growth in Table 10-4. The laboratory and
procedural blanks were all negative for growth.
10.3 Damage to Coupons
No visible damage was observed on the test materials
after the 30 minute contact time on non-porous materials
and the 60 minute contact time on the porous materials
with Peridox® RTU. The treated wood extracts had a
yellowish hue, probably due to leaching of treatment
chemicals from the coupon material.
10.4 Other Factors
10.4,1 Operator Control
On each day of testing, Peridox® RTU was transferred
to anew, handheld, plastic spray-bottle. Prior to each
application, the Peridox® RTU spray-bottle was primed
by repeatedly spraying into an absorbent cloth to
clear any air bubbles that may have formed between
applications. After each application the spray nozzle
was removed from the bottle and any residual Peridox®
RTU was removed by repeated pulls on the trigger of the
spray nozzle. All coupons were oriented horizontally
(i.e., lying flat) and stayed in that orientation throughout
the entire contact time.
All tests were conducted at ambient conditions inside
a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature, measured to be 22 °C (± 1°C).
The RH inside the test chamber was monitored with a
NIST-traceable hygrometer. Whenever the RH reached
Table 10-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis (Ames)
Spores—Peridox® RTU
Davl
Test Material
SI S2 S3 S4 S5 B
Stainless Steel
Positive Controls + + + + +
Test Coupons - -b
Glass
Positive Controls + + + + +
Test Coupons - -
Aluminum
Positive Controls + + + + +
Test Coupons - -
Porcelain
Positive Controls + + + + +
Test Coupons - -
Granite
Positive Controls + + + + +
Test Coupons ... ...
Concrete
Positive Controls + + + + +
Test Coupons + + + + +
Day 7
SI S2 S3 S4 S5 B
+ + + + +
+ + + + +
+ + + + +
+ + + + +
+ + + + +
+ + + + +
+ + + + +
-------
Brick
Positive Controls + + + + +
Test Coupons + + + + + -
Asphalt Paving
Positive Controls + + + + +
Test Coupons + + - - -
Treated Wood
Positive Controls + + + + + -
Test Coupons - -
Butyl Rubber
Positive Controls + + + + + -
Test Coupons +. + + ..
+ + + + +
+ + + + +
+ + + + +
+ +
+ + + + +
.
+ + + + +
4- - -f -f
SI to S5 = Sample 1 to Sample 5.
B = Blank (not inoculated with B. anthracis (Ames) spores); a = laboratory blank, b = procedural blank.
Positive controls = Coupons inoculated with B. anthracis (Ames) spores, but not subjected to decontamination.
Test coupons = coupons inoculated with B. anthracis (Ames) spores, and subjected to decontamination.
"+" = growth; "-" = no growth.
70%, the dehumidification system attached to the testing
chamber was actuated until the RH dropped below 70%.
Therefore, the testing chamber humidity was always <
70% RH during the decontamination of test materials
withPeridox®RTU.
10.4.2 Technology Spray Deposition
Peridox® RTU was applied according to the procedure
included as Appendix F of this report. Peridox®
RTU was applied from a distance of 30.5 cm to the
horizontally-oriented materials until the materials
were fully wetted. With nonporous materials two
reapplications of Peridox® RTU were needed to maintain
wetting, at 10 and 25 minutes after the initial application,
for a total of three applications in the 30 minute total
contact time on those materials. With porous materials,
five reapplications were needed at 10, 20, 30, 40, and
50 minutes, for a total of six total applications in the
60 minute total contact time on those materials. These
application procedures were used in the deposition
measurements, and in turn were used in all efficacy
testing of Peridox® RTU. After the respective contact
times, each material coupon was placed in the 50 mL
conical vial that also served to collect excess formulation
that may have pooled on the coupon surface.
To assess Peridox® RTU deposition, triplicate coupons
of each test material were weighed prior to application
of the Peridox® RTU in the trial runs, and those values
were recorded. Then the triplicate coupons were sprayed
with Peridox® RTU until fully wetted in their horizontal
orientation according to the procedures established based
on Appendix F, and each coupon was weighed again
after the respective contact time. The pre-application
weights were then subtracted from the post-application
weights, and that difference was added to the weight
of decontaminant runoff captured separately from each
coupon. The average deposition/runoff weight of the
Peridox® RTU from each of the test materials is shown in
Table 10-5. The average deposition amounts of 0.30 and
0.64 g for nonporous and porous materials, respectively,
were used to estimate the amount of STS needed to
effectively neutralize the Peridox® RTU.
10.4.3 Neutralization Methodology
Neutralization of Peridox® RTU was achieved with STS.
The concentrations of STS used during the neutralization
panel were 0.5, 1.0, and 1.5% in the PBS/Triton® X-100
extraction solution. The results of the neutralization
panel are shown in Tables 10-6 and 10-7 for the
nonporous and porous materials, respectively
Table 10-6 shows that in the nonporous material trial the
action of Peridox® RTU was inhibited by dilution with
PBS/Triton® X-100 extraction solution, as substantial
recovery of spores was seen with Peridox® RTU plus
extraction solution in the absence of STS (second
row of Table 10-6). This observation implies that
partial neutralization of Peridox® RTU would occur
in extraction of nonporous material coupons with the
PBS/Triton® X-100 solution. However, added STS
was needed to achieve complete neutralization of
Peridox® RTU with those coupons. This behavior was
not observed in the porous material neutralization trial
(Table 10-7), presumably because of the larger quantity
of Peridox® RTU used with those materials. On the basis
of these trials, 1.0% STS was used for neutralization of
Peridox® RTU for the nonporous materials and 1.5%
STS was used for neutralization with porous materials.
-------
Table 10-5. Deposition/Runoff Weight of Peridox® RTU on
Test Materials
Table 10-7. Neutralization Testing with BaciUus anthracis
(Ames) Spores for Peridox® RTU on Porous Test Materials
Test Material
Average Deposition/
Runoff Weight (g)
Nonporous
Glass
Aluminum
Stainless Steel
Granite
Porcelain
Average
Porous
Concrete
Brick
Asphalt Paving
Treated Wood
Butyl Rubber
Average
0,25
0.34
0.36
0.26
0.27
0.30
0.33
0.96
0.39
1.08
0.46
0.64
Table 10-6. Neutralization Testing with Bacillus anthracis
(Ames) Spores for Peridox8 RTU on NonporoHS Test
Materials
Treatment
Peridox® RTU +
Spores"
Inoculum
(CFU)
7.73 xlO7
Total
Observed
(CFU)
0
%of
Control
0
Peridox® RTU + PBS
+ Triton® X-100+ 7.73 xlO7 3.01 x 107 45.1
Spores"*
PBS + Triton® X-100
+ Spores (Control)"
Peridox® RTU + PBS
+ Triton® X-100 +
0.5% STS + Spores"-"
Peridox® RTU + PBS
+ Triton® X-100 +
1.0% STS + Spores"-"
Peridox® RTU + PBS
+ Triton® X-100 +
1.5% STS + Spores"-1'
7.73x10' 6.67x10' 100
7.73x10' 5.81x10' 87.2
7.73 x 107 5.93 x 10s
7.73x10' 5.88x10' 88.3
Peridox* RTU volume of 0.30 mL corresponds to mean gravimetric
deposition on nonporous materials, assuming density of 1.0 g/'ml,.
10 mL Volume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of STS; total volume for all samples with Peridox®
RTU - 10.30 mL (10 mL PBS/Triton* X-100/STS + 0.30 mL
Peridox* RTU).
Treatment
Peridox® RTU +
Spores"
Inoculum
(CFU)
7.73 x 1 07
Total
Observed
(CFU)
0
%of
Control
0
Peridox® RTU + PBS
+ Triton® X-100+ 7.73 x 107
Spores1*
PBS + Triton® X-100
+ Spores (Control)1'
Peridox® RTU + PBS
+ Triton® X-100 +
0.5% STS + Spores"-"
Peridox® RTU + PBS
+ Triton® X-100 +
1.0% STS + Spores"-"
Peridox® RTU + PBS
+ Triton® X-100 +
1.5% STS + Spores"-"
7.73 xlO7 5.77xl07
7.73 xlO7 8.97xl06
7.73 xlO7 5.71 xlO7
7.73 xlO7 5.87 xlO7
100
15.6
99.0
101.9
Peridox" RTU volume of 0.64 mL corresponds to mean gravimetric
deposition on non-porous materials, assuming density of 1.0 g/'inL.
10 mL Volume of PBS includes 0.1% of Triton* X-100 surfactant
and indicated % of STS; total volume for all samples with Peridox*
RTU - 10.64 mL (10 mLPBS/Triton* X- 100/STS ~ 0.64 mL
Peridox* RTU)-
-------
11.0
Performance Summary
11.1
* The quantitative efficacy of pH-ainended bleach
for B. anthracis was > 7.62 log reduction on all five
nonporous materials and > 6.91 log reduction on the
porous materials, brick and butyl rubber. On those
seven materials inactivation of B. anthracis was
complete; i.e., no viable spores were found on any
decontaminated coupons. Quantitative efficacy was
6.27 log reduction on concrete, 3.60 log reduction
on asphalt paving, and 1.90 log reduction on treated
wood.
* Qualitative efficacy results were consistent with
quantitative efficacy results, in that no growth was
seen with decontaminated test coupons of any
materials except for asphalt paving and treated wood
after one and seven days incubation. Morphological
analysis was consistent with the growth observed
being only B. anthracis.
* No visible damage was observed on any of the test
materials after the 60 minute contact time with
pH-amended bleach in the quantitative efficacy
testing, or seven days later after completion of the
qualitative assessment of residual spores.
11.2 SDF
• The quantitative efficacy of CASCAD™ SDF
for B. anthracis was > 6.80 log reduction on all
ten materials. On all materials inactivation ofB.
anthracis was complete, i.e., no viable spores were
found on any decontaminated coupons.
• Qualitative efficacy results were consistent with
quantitative efficacy results, in that no growth was
seen with decontaminated test coupons of any
materials after one and seven days incubation.
• No visible damage was observed on any of the test
materials after the 30 or 60 minute contact times
with CASCAD™ SDF in the quantitative efficacy
testing, or seven days later after completion of the
qualitative assessment of residual spores.
11.3
* The quantitative efficacy of Decon Green for B.
anthracis was > 7.32 log reduction on all five
nonporous materials, and was > 7.25 and > 6.94
log reduction, respectively, on the porous materials
brick and butyl rubber. No viable spores were
found on any of these seven test materials after
decontamination with Decon Green. Efficacv on
concrete, asphalt, and treated wood was lower, with
4.00, 2.97, and 1.91 log reductions, respectively.
* Qualitative efficacy results were consistent with
quantitative efficacy results, in that no growth
was seen with decontaminated test coupons of
seven of the ten test materials after one and seven
days incubation. The decontaminated coupons of
concrete, asphalt and treated wood all were positive
for growth at both one and seven days incubation.
Morphological analysis was consistent with the
growth observed being only B. anthracis.
* No visible damage was observed on any of the test
materials after the 60 minute contact time with
Decon Green in the quantitative efficacy testing, or
seven days later after completion of the qualitative
assessment of residual spores.
11.4
• The quantitative efficacy of Easy DECON® 200 for
B. anthracis was >7.51 log reduction on all five
nonporous materials, and >6.99 log reduction on the
porous materials concrete, brick, and butyl rubber.
No viable spores were found on decontaminated
coupons of those eight materials. Efficacy on
asphalt paving and treated wood was approximately
1.63 and 0.82 log reduction, respectively.
• Qualitative efficacy results were consistent with
the quantitative results, in that no growth was seen
with decontaminated test coupons of eight of the ten
test materials after one and seven days incubation.
All decontaminated coupons of asphalt and treated
wood were positive for growth at both one and
seven days incubation. Morphological analysis was
consistent with Hie growth observed being only B.
anthracis.
* No visible damage was observed on any of the test
materials after the 30 or 60 minute contact times
with EasyDECON® 200 in the quantitative efficacy
testing, or seven days later after completion of the
qualitative assessment of residual spores.
11.5 RTU
• The quantitative efficacy of Spor-Klenz® RTU
for B. anthracis was > 7.57 log reduction on the
nonporous materials porcelain and granite, and >
7.27 log reduction on the porous materials brick
and butyl rubber. No viable spores were found on
decontaminated coupons of those four materials.
-------
Efficacy was also relatively high on stainless steel, testing, or seven days later after completion of the
glass, and aluminum (approximately 7.28, 7.36, and qualitative assessment of residual spores.
7.17 log reduction, respectively), but small numbers
of viable spores were found on one test coupon
of each of these materials after decontamination.
On concrete, asphalt paving, and treated wood
efficacy was approximately 1.02, 2.56, and 6.16 log
reduction, respectively.
* Qualitative efficacy results were consistent with
the quantitative results, in that no growth was
seen with decontaminated test coupons of four
of the ten test materials. Growth was seen with
one decontaminated test coupon each of stainless
steel, glass, and aluminum. All decontaminated
test coupons of concrete, asphalt, and treated wood
were positive for growth at both one and seven days
incubation. Morphological analysis was consistent
with the growth observed being only B. anthracis.
• No visible damage was observed on any of the test
materials after the 30 or 60 minute contact times
with Spor-Klenz® RTU in the quantitative efficacy
testing, or seven days later after completion of the
qualitative assessment of residual spores.
11.6 RTU
• The quantitative efficacy of Peridox® RTU for
B. anthracis was > 6.65 log reduction on all the
nonporous materials and on the porous materials
treated wood and butyl rubber. No viable spores
were found on decontaminated coupons of any of
these seven materials. Efficacy was also relatively
high (7.22 log reduction) on asphalt paving,
although a small number of viable spores were
found on one test coupon of that material after
decontamination. The efficacy of Peridox RTU on
concrete and brick was relatively low, at 1.39 and
3.81 log reduction, respectively.
* Qualitative efficacy results were largely consistent
with the quantitative results, in that no growth was
seen with decontaminated test coupons of the five
nonporous materials and of the porous material
treated wood. However, three test coupons of butyl
rubber showed positive growth after both one and
seven days incubation, although no viable spores
had been found in the quantitative efficacy testing
with this material. All decontaminated coupons of
unpainted concrete and brick, and two coupons of
asphalt paving, were positive for growth at both one
and seven days' incubation. Morphological analysis
was consistent with the growth observed being B.
anthracis.
• No visible damage was observed on any of the test
materials after the 30 or 60 minute contact times
with Peridox® RTU in the quantitative efficacy
-------
Appendix A
Preparation and Application of pH-Amended
Bleach
General Description
For testing of efficacy against B. anthracis on outdoor
materials, pH-amended bleach consists of a specialized
germicidal bleach formulation (Clorox® Commercial
Solutions Ultra Clorox® Germicidal Bleach), which is
diluted in cell-culture grade sterile filtered water (SFW)
and has its pH adjusted by addition of a small amount
of acetic acid. Specifically, Ultra Clorox® Germicidal
Bleach contains a total of about 6.15% by weight of
sodium hypochlorite (NaOCl) in aqueous solution. The
recipe for preparation of pH-amended bleach for use as a
decontaminant is as follows:
• Prepare 5% acetic acid solution by diluting 50
mL of glacial acetic acid up to 1 L with SFW in a
volumetric flask.
• Mix 9.4 parts SFW, 1 part Ultra Clorox® Germicidal
Bleach, and 1 part 5% acetic acid. The resulting
solution will have a mean pH of about 6.8 and a
mean total chlorine content of about 6,200 ppm.
The active decontaminating agents in this solution
are hypochlorite (OC1") and hypochlorous acid. The
effectiveness of bleach as a biological decontaminant
is widely known, and in particular the vendor indicates
that Ultra Clorox® Germicidal Bleach is the only
product registered with U.S. EPA as effective in killing
Clostridium difficile bacteria.
In previous testing of pH-amended bleach as a
decontaminant, neutralization of the bleach solution
was achieved using sodium thiosulfate (STS). Based on
the chemical composition of the pH-amended bleach,
the amount of that solution (0.325 mL) retained or
run off from a test coupon with a specified 10-second
application period, and the use of 10 mL of an extraction
solution containing phosphate-buffered saline (PBS)
+ 0.1% Triton® X-100, it was determined that an STS
concentration of 0.086% in the extraction solution was
optimal for neutralizing the pH-amended bleach. The
application equipment and procedures used in this
evaluation differed from those used in previous testing,
so the determination of the neutralization procedure
was repeated to establish neutralization conditions
appropriate for this evaluation.
Application Procedure for Testing
Based on previous test results with pH-amended bleach,
and considering the surface materials to be used in this
testing, an application procedure for use in testing was
developed. The intent of this procedure was to employ
conventional and readily available equipment in a
relatively simple application process. Trial runs were
conducted to establish the appropriate concentration of
STS for neutralization of the pH-amended bleach.
The test coupon materials used with pH-amended bleach
included the nonporous materials steel, glass, aluminum,
porcelain, and granite, and the porous materials concrete,
brick, asphalt paving, treated wood, and butyl rubber.
The pH-amended bleach was prepared fresh shortly
before use on each day of testing, as described above.
The pH of the solution was measured and recorded as
part of the test data. A new noncorroding garden pump
sprayer was used to apply the solution of pH-amended
bleach to the test coupon surfaces. An identical sprayer
was used to apply SFW to positive control test coupons.
Each sprayer was fitted with a pressure gauge to indicate
the internal delivery pressure of the sprayer. The internal
pressure of each sprayer was maintained in a normal
range for use (i.e., 4 to 6 psi) throughout all applications.
Based on laboratory tests, such a range of pressures
produces a stable spray suitable for application on the
scale of coupon testing. The step-by-step application
procedure was:
• Apply the pH-amended bleach solution to the test
coupons (or SFW to the positive control coupons)
from a distance of about one foot (30.5 cm) using
the sprayer at a delivery pressure within the
specified range, until the test coupon surfaces are
fully wetted by the solution.
• Reapply the solution three times, i.e., at 15 minutes
after the first application, 30 minutes after the
first application, and 45 minutes after the first
application.
• If necessary, pump up the pressure in the sprayer
before application to maintain pressure within the
specified range.
• When 60 minutes have elapsed since the start of
the first application, place the coupons into the
extraction solution (containing the neutralization
agent) along with any collected runoff of pH-
amended bleach.
-------
-------
Appendix B
Preparation and Application of
CASCAD™SDF
General Description
CASCAD™ Surface Decontamination Foam (SDF)
uses two liquid solutions (A and B) which react to
form a foam as they are mixed upon release from the
application device. These two solutions are made from
three separate reagents, having chemical composition as
follows:
• GPA-2100 (decontaminant) - solid reagent in
powder form consisting of dichloroisocyanuric acid
sodium salt, 70 to 100% by weight;
• GPB-2100 (buffer) - solid reagent in powder form
consisting of sodium tetraborate 10 to 30%, sodium
hydroxide 1 to 5 %, and sodium carbonate 40 to
65% by weight;
• GCE-2000 (surfactant) - liquid reagent consisting of
sodium myristyl sulfate 10 to 30%, sodium (C14 to
C16) olefin sulphonate 10 to 30%, ethanol denatured
3 to 9%, alcohols (C10 to C16) 5-10%, sodium sulfate
3 to 7%, sodium xylene sulfonate 1 to 5%, and a
proprietary mixture of sodium and ammonium salts
along with water and co-solvent >9% by weight.
The A and B solutions are prepared from these reagents
by the following procedure:
1. Make solution A by adding 31.2 grams (four 7.8
gram packets) of GPA-2100 to 250 mL of water in
a graduated cylinder, and then dilute with SFW to
300 mL.
2. Mix with a micro stir bar until dissolved.
3. Make solution B by adding 7.2 grams (four 1.8
gram packets) of GPB-2100 to 250 mL of SFW in a
graduated cylinder.
4. Mix with a micro stir bar until dissolved.
5. Add 18 mL (four 4.5 mL packets) of GCE-2000 to
the solution from Step 4, mix, and then dilute with
SFW up to 300 mL final volume.
For use on the small scale needed for testing, a manual
spray application bottle (the 600 mL Hand Held
Decontamination System) has been developed by Allen-
Vanguard that draws solutions A and B from separate
compartments and delivers them as a foam through a
single spray head. To fill and operate the Hand Held
System, follow these steps:
1. Pull the Locking Lever on the front of the bottle
housing forward and lift to open the housing and
expose the solution bottles, which are labeled "A"
and"B".
2. With the housing opened remove the caps (turn
counter clockwise) and pull out the solution
suction lines from the solution bottles.
3. With the caps and suction lines removed from both
the "A" and "B" solution bottles:
a. Pour solution A into the bottle labeled "A",
and pour solution B into the bottle labeled
"B".
b Assure that both bottles are seated in the
housing with the "B" bottle at the front.
c. Place the suction lines back into the "A" and
"B" bottles and tighten both the "A" and "B"
caps by turning them in a clockwise direction.
4. Hold the suction line up with one hand while
closing the top of the housing with the other
hand. Make certain that the Locking Lever snaps
into its recess when the housing top closes
The suction line may be pinched closed if
this procedure is not followed correctly;
openness of the suction line can be checked by
looking through the housing and checking the
suction line.
5. To use the 600 mL Hand Held Decontamination
System, grasp the neck of the housing with your
dominant hand and place the finger of this hand
on the trigger of the foam nozzle. Aim the tip
of the foam nozzle in the direction of the area to be
decontaminated and pump the trigger. The trigger
may have to be squeezed three or four times
to evacuate the air in the suction line before foam
is discharged.
Application Procedure for Testing
CASCAD™ SDF was applied to test coupons using the
vendor-developed dual spray applicator. In previous
testing, neutralization of the CASCAD™ SDF was
achieved by addition of 0.5% sodium thiosulfate
(STS) to the extraction solution. Trial runs were
conducted before testing to establish the appropriate
STS concentration for neutralization of the applied
CASCAD™ SDF.
-------
The step-by-step application procedure for testing was
as indicated below. Note that the procedure for porous
materials differed from that for nonporous materials. All
test coupons were oriented horizontally (i.e., lying flat)
for testing.
* Follow the instructions provided above for
preparation of the reagent solutions and loading of
the manual spray applicator.
* Squeeze the trigger of the applicator head a few
times while pointing the applicator into a laboratory
sink or other waste container, until any air is
cleared from the applicator and CAS CAD™ SDF is
delivered from the applicator as a foam.
• Apply the CASCAD™ SDF to the test coupons using
the manual applicator from a distance of about one
foot (30.5 cm) while moving the nozzle, until the
test coupons are entirely covered with no less than
one (1) centimeter (3/8") deep foam.
For nonporous coupon materials (glass, steel,
aluminum, porcelain, granite):
• Allow the foam to remain on the coupons for 30
minutes. Do not re-apply.
* When 30 minutes have elapsed since the
application, place each coupon into the extraction
solution (containing the STS neutralization agent)
along with any CASCAD™ SDF accumulated on the
coupon.
For porous coupon materials (concrete, brick, asphalt
paving, treated wood, rubber):
* Allow the foam to remain on the coupons for 30
minutes.
« Reapply more CASCAD™ SDF and allow the
foam to remain on the coupons for an additional 30
minutes.
* When a total of 60 minutes have elapsed since
the first application, place each coupon into
the extraction solution (containing the STS
neutralization agent) along with any CASCAD™
SDF accumulated on the coupon.
After use, empty and clean the manual spray applicator
according to the instructions below.
Cleaning the Hand Decontamination
System
Clean the CASCAD™ SDF system after use by the
following procedure.
1. Dump any remaining decontamination solution
from both the "A" and "B" bottles and dispose of
the solutions following appropriate waste disposal
procedures.
2. Thoroughly rinse both bottles with SFW, and then
4.
fill each bottle with clean water.
Place the filled bottles back into the housing,
insert the suction lines, and close the housing.
Pump the trigger until the suction lines and foam
nozzle are free from the decontamination solution.
5. Flush the interior and the exterior of the housing.
and the caps used while mixing the solution,
thoroughly with SFW.
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c
and of
Description
Decon Green is a hydrogen peroxide-based
decontaminant designed for biological, chemical, and
radiological efficacy. Decon Green consists of a three-
part formula which is mixed just prior to use. "Part A"
contains surfactants and solvents which impart surface
cleaning and penetration ability; "Part B" is aqueous 35
% hydrogen peroxide (H2O2), the active ingredient; "'Part
C" contains activators and buffers. The specific chemical
components of each solution are as follows:
« Part A- propylene carbonate (C4H6O3) + Triton®
X-100 + propvlene glycol (1.2-propanediol.
C3HS02).
* Part B - hydrogen peroxide 35% aqueous solution.
• Part C - aqueous solution of potassium bicarbonate
(potassium hydrogen carbonate, KHCO3) +
potassium citrate monohydrate (CfH5K3O7«H2O) +
potassium molybdate (K,MoO4) + propylene glycol.
These three parts of Decon Green are packaged in three
corresponding separate containers. To mix Decon Green,
the contents of containers B and C are added to the
contents of container A. It is important not to mix the
contents of containers B and C without first adding them
to the contents of container A, as excessive heating may
result. The final solution of Decon Green has a pH of
about 8 and a density of approximately 1.1 g/mL.
Sodium thiosulfate (STS) was used to stop the action of
Decon Green so that efficacy could be determined. Trial
runs were conducted before efficacy testing to establish
the appropriate concentration of STS for neutralization
of Decon Green.
Application Procedure for Testing
An application procedure for use of Decon Green in
testing was developed based on information provided
by the manufacturer of the product. The aim is to use a
relatively simple application process that is likely to be
effective when carried out with conventional and readily
available equipment.
Decon Green was applied to test coupons as a liquid
solution using a hand-held plastic spray bottle. A similar
bottle was used to apply deionized (DI) water to positive
control test coupons. The step-by-step application
procedure was as follows:
* Apply the Decon Green solution to the test coupons
(or SFW to the positive control coupons) from a
distance of 30.5 cm using the handheld spray bottle,
until all test coupon surfaces are fully wetted by the
solution.
When 30 minutes have elapsed since the first
application, re-apply Decon Green to all test
coupons.
When 60 minutes total contact time has elapsed
since the first application of Decon Green, place
each coupon into the extraction solution (containing
the pre-dctemiincd amount of STS neutralization
solution) along with any Decon Green solution
pooled on the test coupon.
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D
and of
200
General Description
EasyDECON® 200 is a liquid decont.amina.nt consisting
of a. three-part formula which is mixed just prior to use.
The specific chemical components of each solution arc
as follows:
* Part One - quaternary ammonium compounds and
benzyl-C12 to -C16 alkyl dimethyl chlorides, 5.5 to
6.5 % aqueous solution;
Part Two -
solution:
hydrogen peroxide < 8% aqueous
* Part Three - diacetin (glycerol diacetate;
1.2,3-propanetriol-l,3-diacetate), 30 to 60%
aqueous solution.
These three parts of EasyDECON® 200 are packaged in
three corresponding separate containers labeled "'Part
One." "Part Two." and "Part Three,", premeasured and
ready to mix. To prepare EasyDECON® 200 in any
amount the required proportions by weight are 49%
of Part One and 49% of Part Two mixed in a clean
container, and then 2% of Part Three is added and all
three components mixed thoroughly. The final solution
of EasyDECON® 200 lias apH of about 9.6 to 9.9 and a
density of approximately 1.08 g/niL.
Sodium thiosulfate (STS) was used to stop the action of
EasyDECON® 200 so that efficacy could be determined.
Trial runs were conducted before efficacy testing to
establish the appropriate concentration of STS for
neutralization of EasyDECON® 200.
Application Procedure for Testing
An application procedure for use of EasyDECON® 200
in testing was developed based on information provided
by the vendor. The aim is to use a relatively simple
application process that is likely to be effective when
earned out with conventional and readily available
equipment.
EasyDECON® 200 was applied to test coupons as a
liquid solution using a hand-held plastic spray bottle. A
similar bottle was used to apply SFW to positive control
test coupons. A target application rate of 0.12 to 0.14
g/cm2 (0.11 to 0.13 mL/cm2) is recommended by the
vendor. This application rate is relatively large, being
equivalent to approximately 1.7 to 2.0 g (1.6 to 1.9 mL)
applied to a 1.9 x 7.5 cm test coupon. The step-by-step
application procedures as indicated below were designed
to achieve this target application rate. Note that the
application procedure for porous materials differed from
that for nonporous materials.
Primary Procedures:
• Apply the EasyDECON® 200 solution to the test
coupons (or SFW to the positive control coupons)
from a distance of 30.5 cm using the handheld spray
bottle, until all test coupon surfaces are fully wetted
by the solution.
For nonporous materials (steel, aluminum, glass,
porcelain, granite):
• Reapply the EasyDECON® 200 10 minutes after
the first application, and again 20 minutes after the
first application. Perform additional applications as
needed if the test coupon surfaces become dry.
« When 30 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pre-determined amount
of STS neutralization solution) along with any
EasyDECON® 200 solution pooled on the test
coupon.
For porous materials (concrete, brick, asphalt paving,
treated wood, butyl rubber):
• Reapply the EasyDECON® 200 20 minutes after
the first application, and again 40 minutes after the
first application. Perform additional applications as
needed if the test coupon surfaces become dry.
« When 60 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pre-determined amount
of STS neutralization solution) along with any
EasyDECON® 200 solution pooled on the test
coupon.
If die actual application rate of EasyDECON® 200 fell
short of the target rate when die primary procedures
above are used, the alternate procedures below
were used. These alternate procedures relied on six
applications of EasyDECON® 200 rather than three.
(NOTE: the alternate procedures were not used if high
efficacy of decontamination was observed even with
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a less-than-target application rate with the primary
procedures.)
Alternative Procedures:
« Apply the EasyDECON® 200 solution to the test
coupons (or SFW to the positive control coupons)
from a distance of 30.5 cm using the handheld spray
bottle, until all test coupon surfaces are fully wetted
by the solution.
For nonporous materials (steel, aluminum, glass,
porcelain, granite):
« Reapply the EasyDECON® 200 5 minutes after
the first application, and again 10, 15. 20, and
25 minutes after the first application. Perform
additional applications as needed if the test coupon
surfaces become dry.
* When 30 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pie-determined amount
of STS neutralization solution) along with any
EasyDECON® 200 solution pooled on the test
coupon.
For porous materials (concrete, brick, asphalt paving,
treated wood, butyl rubber):
• Reapply the EasyDECON® 200 10 minutes after
the first application, and again 20, 30, 40, and
50 minutes after the first application. Perform
additional applications as needed if the test coupon
surfaces become dry.
• When 60 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pie-determined amount
of STS neutralization solution) along with any
EasyDECON® 200 solution pooled on the test
coupon.
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E
and of
RTU
General Description
Spor-Klenz® Ready to Use (RTU) is a liquid
deconlaminanl consisting of 1.00 % hydrogen peroxide
(H2O,), 0.08% peroxyacetic acid, and < 10% acetic acid
in aqueous solution. Spor-Klenz® RTU is designed to be
used directly, without dilution. The product is a clear,
colorless liquid with a pH of 1.5 to 2.0 and a density of
approximately l.Og/mL.
Sodium thiosulfate (STS) was used to stop the action of
Spor-Klenz® RTU so that efficacy could be determined.
Trial runs were conducted before efficacy testing to
establish the appropriate concentration of STS for
neutralization of Spor-Klenz® RTU.
Application Procedure for Testing
An application procedure for use of Spor-Klenz® RTU in
testing was developed based on information provided on
the product label, in particular for its use as a sporicide.
The aim is to use a relatively simple application process
that is likely to be effective when carried out with
conventional and readily available equipment.
Spor-Klenz® RTU was applied to test coupons using a
hand-held plastic spray bottle. A similar bottle was used
to apply SFW to positive control test coupons. The step-
by-step application procedure was as indicated below.
Note that the procedure for porous materials differed
from that for nonporous materials.
* Apply Spor-Klenz® RTU to the test coupons (or
SFW to the positive control coupons) from a
distance of 30.5 cm using the handheld spray bottle,
until all test coupon surfaces are fully wetted by the
solution.
For nonporous materials (steel, aluminum, glass.
porcelain, granite):
• Reapply Spor-Klenz® RTU as needed to keep the
test coupon surfaces wetted throughout the test.
* When 30 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pre-determined amount of
STS neutralization solution) along with any Spor-
Klenz® RTU pooled on the test coupon.
For porous materials (concrete, brick, asphalt paving,
treated wood, butyl rubber):
Reapply the Spor-Klenz® RTU as needed to keep the
test coupon surfaces wetted throughout the test.
Regardless of the wetness of the coupons, reapply
SporKlenz® 30 minutes after the first application.
When 60 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pre-determined amount of
STS neutralization solution) along with any Spor-
Klenz® RTU pooled on the test coupon.
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F
of RTU
General Description of STS neutralization solution) along with any
Peridox® RTU is a liquid decontaminant consisting Peridox® RTU pooled on the test coupon)
of 4.0 to 4.5 % hydrogen peroxide (H2O,) and 0.17 to
0.22% pcracetic acid, in aqueous solution. Peridox®
RTU is intended to be used as is. without further
dilution. The product is a colorless liquid with a pH of
about 2.2 and a density of approximately 1.02 g/mL.
Sodium thiosulfate (STS) was used to stop the action
of Peridox® RTU so that efficacy could be determined.
Trial runs were conducted before efficacy testing to
establish the appropriate concentration of STS for
neutralization of Peridox® RTU.
Application Procedure for Testing
An application procedure for use of Peridox® RTU in
testing was developed based on information provided
by the vendor. The aim is to use a relatively simple
application process that is likely to be effective when
carried out with conventional and readily available
equipment.
Peridox® RTU was applied to test coupons using a hand-
held plastic spray bottle. A similar bottle was used to
apply SFW to positive control test coupons. The step-
by-step application procedure was as indicated below.
Note that the procedure for porous materials differed
from that for non-porous materials.
• Apply Peridox® RTU to the test coupons (or SFW
to the positive control coupons) from a distance of
30.5 cm using the handheld spray bottle, until all
test coupon surfaces are fully wetted by the solution.
For nonporous materials (steel, glass, aluminum,
porcelain, granite):
• Rcapply Peridox® RTU as needed to keep the test
coupon surfaces wetted.
* When 30 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pre-determined amount
of STS neutralization solution) along with any
Peridox® RTU pooled on the test coupon.
For porous materials (concrete, brick, asphalt paving,
treated wood, butyl rubber):
• Reapply the Peridox® RTU as needed to keep the
test coupon surfaces fully wetted.
* When 60 minutes have elapsed since the first
application, place each coupon into the extraction
solution (containing the pre-determined amount
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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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