United States Office of Research and EPA 600-R-06-146
Environmental Protection Development September 2006
Agency Washington, D.C. 20460 www.epa.gov/nhsrc
v-xEPA
Technology Evaluation Report
Evaluation of Spray-Applied Sporicidal
Decontamination Technologies
-------
EPA600/R-06/146
September 2006
Technology Evaluation Report
Evaluation of Spray-Applied Sporicidal
Decontamination Technologies
By
James V. Rogers, William R. Richter, Young W. Choi,
Emily J. Fleming, Adrienne M. Shesky, Jing Cui,
Michael L. Taylor, Karen B. Riggs, Zachary J. Willenberg, and Harry J. Stone
Battelle
505 King Avenue
Columbus, Ohio 43201
Blanket Purchase Agreement under
General Services Administration Contract No.
GS23F0011L-3
Task Order Project Officer
Joseph P. Wood
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development's
National Homeland Security Research Center (NHSRC), funded and directed this technology evaluation
through a Blanket Purchase Agreement (BPA) under General Services Administration Contract No.
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.
Abstract
The Technology Testing and Evaluation Program (TTEP) is an effort to provide reliable information
regarding the performance of commercially available technologies that may have application for
homeland security. This effort is an outgrowth of EPA's successful and internationally recognized
Environmental Technology Verification (ETV) Program.
As part of EPA's Office of Research and Development, the National Homeland Security Research
(NHSRC) rigorously tests technologies against a wide range of performance characteristics,
requirements, and specifications. The technology categories of interest include detection, monitoring,
treatment, decontamination, and computer modeling. Stakeholder involvement is important to the
success of the program. Stakeholders are engaged in identifying and selecting technologies for testing
and in developing test plans.
This report presents both quantitative and qualitative results for the spray-applied sporicidal
technologies that were evaluated for their effectiveness in decontamination of surfaces. Test coupons
that are typical of surfaces found in an office or transportation terminal were selected for the study. The
technologies evaluated were:
pH-Amended Bleach (Clorox®)
CASCAD™ Surface Decontamination Foam (Allen-Vanguard)
DeconGreen (Edgewood Chemical Biological Center)
DioxiGuard (Frontier Pharmaceutical)
EasyDecon 200 (Envirofoam Technologies)
Exterm-6 (ClorDiSys Solutions)
Hi-Clean 605 (Howard Industries)
HM-4100 (Biosafe)
KlearWater (Disinfection Technology)
Peridox (Clean Earth Technologies)
Selectrocide (BioProcess Associates)
The decontamination efficacy results varied by technology, bacterial spore specie, and coupon material.
Following testing, the technology vendors were given the opportunity to review and comment on the
draft results.
in
-------
Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the nation's
air, water, and land resources. Under a mandate of national environmental laws, the Agency strives to
formulate and implement actions leading to a compatible balance between human activities and the
ability of natural systems to support and nurture life. To meet this mandate, the EPA's Office of
Research and Development (ORD) provides data and scientific support that can be used to solve
environmental problems and to build the scientific knowledge base needed to manage our ecological
resources wisely, to understand how pollutants affect our health, and to prevent or reduce environmental
risks.
In September 2002, EPA announced the formation of the National Homeland Security Research Center
(NHSRC). The NHSRC is part of the Office of Research and Development; it manages, coordinates, and
supports a variety of research and technical assistance efforts. These efforts are designed to provide
appropriate, affordable, effective, and validated technologies and methods for addressing risks posed by
chemical, biological, and radiological agents. Research focuses on enhancing our ability to detect,
contain, and decontaminate materials in the event of such attacks.
NHSRC's team of scientists and engineers is dedicated to understanding the threat scenarios,
communicating the risks, and mitigating the results of attacks. Guided by the roadmap set forth in EPA's
Strategic Plan for Homeland Security, NHSRC ensures rapid production and distribution of security
related products.
The NHSRC's Technology Testing and Evaluation Program (TTEP) is an effort to provide reliable
information regarding the performance of homeland security related technologies. TTEP provides
independent, quality assured performance data that is useful to decision makers in purchasing or
applying the tested technologies. It provides potential users with unbiased, third-party information that
can supplement vendor-provided information and data. Stakeholder involvement ensures that user needs
and perspectives are incorporated into the test design so that useful performance information is produced
for each of the tested technologies. The technology categories of interest include detection and
monitoring, water treatment, air purification, decontamination, and computer modeling tools for use by
those responsible for protecting buildings, drinking water supplies and infrastructure, and for
decontaminating structures and the outdoor environment.
The evaluation reported herein was conducted by Battelle as part of TTEP. Information on NHSRC and
TTEP can be found at http://www.epa.gov/ordnhsrc/index.htm.
IV
-------
Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the evaluation,
analyze the data, and prepare this report. We also would like to thank Dr. Lloyd Larsen, U.S. Army
Dugway Proving Ground as well as U.S. Environmental Protection Agency reviewers.
-------
Contents
Notice iii
Abstract iii
Foreword iv
Acknowledgments v
Abbreviations/Acronyms xi
Executive Summary xii
1.0 Introduction 1
2.0 Technology Description 3
3.0 Quality Assurance/Quality Control 5
3.1 Equipment Calibration 5
3.2 Audits 5
3.2.1 Performance Evaluation Audit 5
3.2.2 Technical Systems Audit 5
3.2.3 Data Quality Audit 5
3.3 QA/QC Reporting 6
3.4 Data Review 6
4.0 Test Results 7
4.1 pH-Amended Bleach 7
4.1.1 Decontamination Efficacy 7
4.1.1.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 7
4.1.1.2 Qualitative Assessment of Residual Spores 14
4.1.2 Damage to Coupons 19
4.1.3 Other Factors 19
4.1.3.1 Operator Control 19
4.1.3.2 Technology Spray Deposition 20
4.1.3.3 Neutralization Methodology 20
4.2 Ten Technologies Evaluated by Screening Test 21
4.2.1 Decontamination Efficacy 22
4.2.1.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 22
4.2.2 Damage to Coupons 25
4.2.3 Other Factors 25
4.2.3.1 Operator Control 25
4.2.3.2 Technology Spray Deposition 25
4.2.3.3 Neutralization Methodology 26
4.3 CASCAD Surface Decontamination Foam 29
4.3.1 Decontamination Efficacy 31
4.3.1.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 31
4.3.1.2 Qualitative Assessment of Residual Spores 33
vi
-------
4.3.2 Damage to Coupons 35
4.3.3 Other Factors 35
4.3.3.1 Operator Control 35
4.3.3.2 Technology Spray Deposition 35
4.3.3.3 Neutralization Methodology 35
4.4 HI-Clean605 35
4.4.1 Decontamination Efficacy 36
4.4.1.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 36
4.4.1.2 Qualitative Assessment of Residual Spores 38
4.4.2 Damage to Coupons 40
4.4.3 Other Factors 40
4.4.3.1 Operator Control 40
4.4.3.2 Technology Spray Deposition 40
4.4.3.3 Neutralization Methodology 41
4.5 KlearWater 41
4.5.1 Decontamination Efficacy 41
4.5.1.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 41
4.5.1.2 Qualitative Assessment of Residual Spores 43
4.5.2 Damage to Coupons 45
4.5.3 Other Factors 46
4.5.3.1 Operator Control 46
4.5.3.2 Technology Spray Deposition 46
4.5.3.3 Neutralization Methodology 46
4.6 Peridox 46
4.6.1 Decontamination Efficacy 46
4.6.1.1 Quantitative Assessment of the Log Reduction of Viable
Organisms 46
4.6.1.2 Qualitative Assessment of Residual Spores 49
4.6.2 Damage to Coupons 51
4.6.3 Other Factors 51
4.6.3.1 Operator Control 51
4.6.3.2 Technology Spray Deposition 51
4.6.3.3 Neutralization Methodology 51
5.0 Performance Summary 52
5.1 pH-Amended Bleach Results 52
5.2 Ten Technologies Evaluated by Screening Test Results 53
5.3 CASCAD SDF Results 53
5.4 Hi-Clean 605 Results 54
5.5 KlearWater Results 55
5.6 Peridox Results 56
5.7 Comparison of pH-Amended Bleach with Down-Selected Technologies 56
6.0 References 58
vn
-------
Tables
Table 2-1. Technology Information (Vendor Supplied) 3
Table 4-1. Inactivation of Bacillus anthracis Ames Spores (pH-Amended Bleach; Ten Minute Contact
Time) 10
Table 4-2. Inactivation of Bacillus anthracis Sterne Spores (pH-Amended Bleach) 11
Table 4-3. Inactivation of Bacillus subtilis Spores (pH-Amended Bleach) 12
Table 4-4. Inactivation of Geobacillus stearothermophilus Spores (pH-Amended Bleach) 13
Table 4-5. Summary of Efficacy Values (Log Reduction) Obtained for pH-Amended Bleach 14
Table 4-6. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames Spores
following Extraction (pH-Amended Bleach) 15
Table 4-7. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Sterne Spores
following Extraction (pH-Amended Bleach) 16
Table 4-8. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores following
Extraction (pH-Amended Bleach) 17
Table 4-9. Liquid Culture Assessment of Coupons Inoculated with Geobacillus stearothermophilus
Spores following Extraction (pH-Amended Bleach) 18
Table 4-10. Summary of Results Obtained from the Quantitative and Qualitative Assessments when
Comparing Decontaminated Coupons (pH-Amended Bleach) 19
Table 4-11. Mean± (SD) Weight of pH-Am ended Bleach Deposited on Test Coupons (g) 21
Table 4-12. Neutralization Testing for pH-Amended Bleach 21
Table 4-13. Inactivation of Bacillus anthracis Ames Spores on Glass (Ten Sporicidal
Technologies) 24
Table 4-14. Spray Deposition of Water and Individual Technologies 26
Table 4-15. Neutralizer for Each of Ten Commercially Available Technologies 27
Table 4-16. Neutralization Testing for CASCAD SDF 27
Table 4-17. Neutralization Testing for DeconGreen 27
Table 4-18. Neutralization Testing for DioxiGuard 28
Vlll
-------
Table 4-19. Neutralization Testing for EasyDecon 200 28
Table 4-20. Neutralization Testing for Exterm-6 28
Table 4-21. Neutralization Testing for Hi-Clean 605 29
Table 4-22. Neutralization Testing for HM-4100 29
Table 4-23. Neutralization Testing for KlearWater 29
Table 4-24. Neutralization Testing for Peridox 30
Table 4-25. Neutralization Testing for Selectrocide 30
Table 4-26. Inactivation of Bacillus anthracis Ames Spores (CASCAD SDF) 31
Table 4-27. Inactivation of Bacillus subtilis Spores (CASCAD SDF) 32
Table 4-28. Inactivation of Geobacillus stearothermophilus Spores (CASCAD SDF) 32
Table 4-29. Summary of Efficacy Values Obtained for CASCAD SDF 33
Table 4-30. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames
Spores following Extraction (CASCAD SDF) 33
Table 4-31. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (CASCAD SDF) 34
Table 4-32. Liquid Culture Assessment of Coupons Inoculated with Geobacillus stearothermophilus
Spores following Extraction (CASCAD SDF) 34
Table 4-33. Summary of Results Obtained from the Quantitative and Qualitative Assessments when
Comparing Decontaminated Coupons (CASCAD SDF) 35
Table 4-34. Inactivation of Bacillus anthracis Ames Spores (Hi-Clean 605) 36
Table 4-35. Inactivation of Bacillus subtilis Spores (Hi-Clean 605) 37
Table 4-36. Inactivation of Geobacillus stearothermophilus Spores (Hi-Clean 605) 37
Table 4-37. Summary of Efficacy Values Obtained for Hi-Clean 605 38
Table 4-38. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames Spores
following Extraction (Hi-Clean 605) 38
Table 4-39. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (Hi-Clean 605) 39
ix
-------
Table 4-40. Liquid Culture Assessment of Coupons Inoculated with Geobacillus stearothermophilus
Spores following Extraction (Hi-Clean 605) 39
Table 4-41. Summary of Results Obtained from the Quantitative and Qualitative Assessments
when Comparing Decontaminated Coupons (Hi-Clean 605) 40
Table 4-42. Inactivation of Bacillus anthracis Ames Spores (KlearWater) 42
Table 4-43. Inactivation of Bacillus subtilis Spores (KlearWater) 42
Table 4-44. Inactivation of Geobacillus stearothermophilus Spores (KlearWater) 43
Table 4-45. Summary of Efficacy Values Obtained for KlearWater 43
Table 4-46. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames Spores
following Extraction (KlearWater) 44
Table 4-47. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (KlearWater) 44
Table 4-48. Liquid Culture Assessment of Coupons Inoculated with Geobacillus stearothermophilus
Spores following Extraction (KlearWater) 45
Table 4-49. Summary of Results Obtained from the Quantitative and Qualitative Assessments when
Comparing Decontaminated Coupons (KlearWater) 45
Table 4-50. Inactivation of Bacillus anthracis Ames Spores (Peridox) 47
Table 4-51. Inactivation of Bacillus subtilis Spores (Peridox) 48
Table 4-52. Inactivation of Geobacillus stearothermophilus Spores (Peridox) 48
Table 4-53. Summary of Efficacy Values Obtained for Peridox 49
Table 4-54. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames Spores
following Extraction (Peridox) 49
Table 4-55. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (Peridox) 50
Table 4-56. Liquid Culture Assessment of Coupons Inoculated with Geobacillus stearothermophilus
Spores following Extraction (Peridox) 50
Table 4-57. Summary of Results Obtained from the Quantitative and Qualitative Assessments when
Comparing Decontaminated Coupons (Peridox) 51
-------
Abbreviations/Acronyms
ATCC
C
CPU
C102
cm
CT
ECBC
EPA
g
hr
L
min
mL
NHSRC
NIST
OPP
ORD
PBS
ppm
psi
QA
QC
QMP
RH
SD
SDF
STS
TOPO
ISA
TTEP
American Type Culture Collection
Celsius
colony-forming unit
chlorine dioxide
centimeter
concentration x time
Edgewood Chemical Biological Center
U.S. Environmental Protection Agency
gram
hour
liter
minute
milliliter
National Homeland Security Research Center
National Institute of Standards and Technology
EPA Office of Pesticide Programs
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
standard deviation
Surface Decontamination Foam
sodium thiosulfate
Task Order Project Officer
technical systems audit
Technology Testing and Evaluation Program
XI
-------
Executive Summary
The U.S. Environmental Protection Agency's (EPA) National Homeland Security Research Center
(NHSRC) Technology Testing and Evaluation Program (TTEP) helps to protect human health and the
environment from adverse impacts of terrorist acts by carrying out performance tests on homeland
security technologies. Under TTEP, Battelle evaluated the performance of spray-applied technologies to
decontaminate test coupons (1.9 cm by 7.5 cm) prepared from the following materials (typical of
surfaces found in an office or transportation terminal):
For testing, coupons were 'contaminated' by spiking with a biological warfare agent - Bacillus anthracis
Ames - or one of the following, B. anthracis Sterne, B. subtilis (ATCC 19659), and Geobacillus
stearothermophilus (ATCC 12980). The spray-applied technologies evaluated and the scope of testing
were:
• pH-amended bleach (Clorox® bleach, with water and 5% acetic acid added to obtain pH-amended
solution) to inactivate B. anthracis Ames, B. anthracis Sterne, B. subtilis., and G. stearothermophilus
on test coupons of seven indoor surface materials:
o Industrial-grade carpet
o Bare wood (pine lumber)
o Glass
o Decorative laminate
o Galvanized metal ductwork
o Painted (latex, flat) wallboard paper
o Painted (latex, semi-gloss) concrete cinder block.
• Ten sporicidal technologies (including four aqueous chlorine dioxide technologies, two hydrogen
peroxide technologies, one hydrogen peroxide/peracetic acid technology, two hypochlorous acid
technologies and one quaternary ammonium technology) to inactivate B. anthracis Ames on glass;
the results of the evaluation served as a screening test to down-select four technologies for further
evaluation. These four technologies represent four different types of sporicidal chemical
formulations that are available
• Four technologies (down-selected from results obtained in the screening tests on glass) to inactivate
B. anthracis Ames, B. subtilis, and G. stearothermophilus on test coupons of three indoor surface
materials - galvanized metal, carpet, and bare wood.
Testing was performed using a spray application test apparatus developed by Battelle under TTEP. The
spray application test apparatus allows for precise control of parameters that could affect the efficacy of
spray-applied decontamination technologies, such as mass of spray-applied technology.
The following performance characteristics of sprayed-applied 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 material surface damage following decontamination.
Results obtained in these tests indicated the sprayed pH-amended bleach inactivated extractable, viable
spores from the test coupons. The decontamination efficacy of amended bleach was relatively high (i.e.,
xii
-------
7.2-7.9 log reduction) for hard, nonporous surfaces (glass, decorative laminate, and galvanized metal
ductwork) and low (0.28-2.0 log reduction) for the porous surfaces (industrial grade carpet, bare wood,
and painted concrete) for B. anthracis Ames. For B. anthracis Sterne and B. subtilis, the results were
similar; however, for G. stearothermophilus, the log reductions were much lower for hard, nonporous
surfaces (0.75-5.90), as well as for porous surfaces (0.02-1.40). Statistically significant differences in the
decontamination efficacy were observed when comparing B. anthracis to each of the other organisms.
G. stearothermophilus appeared to be the most resistant to the sprayed, pH-amended bleach.
The results from the qualitative tests using the amended bleach are generally consistent with the results
from the quantitative tests. Whereas in the quantitative tests, the amended bleach achieved high
decontamination efficiency on hard, non-porous surfaces against B. anthracis Ames, B. anthracis
Sterne, and B. subtilis, similar results were seen in the qualitative tests (i.e., few or no positive samples
found). Also, whereas the quantitative tests show notably less decontamination efficacy for amended
bleach when used against G. stearothermophilus on hard surfaces, the qualitative tests showed all
positive cultures for this spore type on hard surfaces. No visible damage was observed for any of the test
coupons subjected to the sprayed pH-amended bleach.
For the ten technologies, a screening test was used to down-select the four most efficacious technologies
that were subjected to more in-depth decontamination efficacy testing. In addition, these four
technologies were selected for additional testing because they represent four different types of sporicidal
chemical formulations that are available. The screening test evaluated the decontamination efficacy for B.
anthracis Ames spores on glass coupons. Results obtained in the screening test showed varying
decontamination efficacies for the ten technologies ranging from 0.37 to >7.8 log reductions. Based on these
results, the four down-selected technologies included CASCAD™ Surface Decontamination Foam
(SDF), Hi-Clean 605, KlearWater, and Peridox. Results for in-depth testing of the down-selected
technologies showed that the degree of inactivation varied with respect to the porosity of the test
material where greater decontamination efficacy was predominantly observed for hard, non-porous
surfaces compared to more porous surfaces. Statistically significant differences in the decontamination
efficacy were observed when comparing B. anthracis Ames to each of the other organisms. Qualitative
assessment of positive liquid cultures resulting from residual viable microorganisms (inoculated spores
or endogenous microorganisms) on the test coupons revealed bacterial growth of only the inoculated
organism on the streak plates. This reflects improved procedures for sterilizing the coupons (i.e., gamma
irradiation) prior to testing that were implemented following the pH-amended bleach decontamination
evaluation. There was no physical damage observed for any of the test coupons subjected to the sprayed
technologies.
In general, treatment of inoculated coupons with sprayed pH-amended bleach and the four down-
selected technologies yielded higher log reductions on non-porous compared to porous materials.
However, one notable exception to this is that sprayed Peridox promoted higher log reductions of G.
stearothermophilus on the porous materials (carpet and wood) compared to the non-porous galvanized
metal. The spray-applied CASCAD SDF, Hi-Clean 605, KlearWater, and Peridox consistently yielded
higher log reductions in B. anthracis Ames, B. subtilis, or G. stearothermophilus spores on industrial
carpet coupons compared to pH-amended bleach with the exception of KlearWater for B. anthracis
Ames. Amended bleach performed the best on galvanized metal, for all spores, with the exception of
CASCAD SDF against G. stearothermophilus. Moreover, log reductions in B. anthracis Ames, B.
subtilis, or G. stearothermophilus spores on bare wood coupons sprayed with Peridox were greater than
those sprayed with pH-amended bleach or the other technologies.
xiii
-------
Following testing, the technology vendors were given the opportunity to review and comment on the
draft results. Three of the four chlorine dioxide based technology vendors expressed concern that the
spray system used in testing may not have been operated optimally for their product. An air pressure of
40 psi was used to atomize the liquid, producing a fine mist (10 - 50 micron diameter droplet size). One
vendor indicated that this high pressure spray would create relatively small size droplets, leading to
increased mass transfer of chlorine dioxide from the liquid to gas phase, thus potentially decreasing the
chlorine dioxide concentration in the liquid and rendering it less effective. The other two vendors had
made similar comments. Although this phenomenon has not been verified, the reader is thus cautioned
about the screening test results reported herein for the aqueous chlorine dioxide based technologies, and
that testing of this type of technology at more optimal conditions may be warranted.
xiv
-------
1.0 Introduction
NHSRC's 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, TTEP 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. 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.
Under TTEP, Battelle recently evaluated the performance of spray-applied sporicidal decontamination
technologies. The primary objective of testing spray-applied sporicidal decontamination technologies
was to evaluate their ability to inactivate Bacillus anthracis Ames spores and spores of one or more of
the following: Bacillus anthracis Sterne, Bacillus subtilis (ATCC 19659), and Geobacillus
stearothermophilus (ATCC 12980), on representative indoor surface materials. The spray-applied
technologies (note that each technology was applied as liquid droplets, foaming action was not apparent
during application) were evaluated as indicated below:
• pH-amended bleach (Clorox® bleach and 5% acetic acid to obtain pH-amended solution) to
inactivate B. anthracis Ames, B. anthracis Sterne, B. subtilis, and G. stearothermophilus on test
coupons of seven indoor surface materials
• Ten sporicidal technologies (including four aqueous chlorine dioxide technologies, two hydrogen
peroxide technologies, one hydrogen peroxide/peracetic acid technology, two hypochlorous acid
technologies and one quaternary ammonium technology) to inactivate B. anthracis Ames on glass;
the results of the evaluation served as a screening test to down-select four technologies for further
evaluation. The four technologies were selected for additional testing also because they represent
four different types of sporicidal chemical formulations that are available
• Four technologies (down-selected from results obtained in the screening tests on glass) to inactivate
B. anthracis Ames, B. subtilis., and G. stearothermophilus on test coupons of three indoor surface
materials - galvanized metal, carpet, and bare wood.
Testing was performed using a spray system developed by Battelle and specifically used for the present
study under TTEP. The spray system allowed for precise-control of parameters that could affect the
efficacy of spray-applied decontamination technologies. This spray test system and approach is currently
not a standardized method.
-------
These evaluations were conducted according to a peer-reviewed test/QA plan(1) that was developed
according to the requirements of the quality management plan (QMP) for the TTEP program.(2) The
following performance characteristics of spray-applied technologies were evaluated:
• Decontamination efficacy
—Quantitative assessment of the decontamination efficacy for viable organisms
—Qualitative assessment for residual spores
• Qualitative assessment of material surface damage following decontamination.
-------
2.0 Technology Description
The table below is a description of the spray-applied sporicidal decontamination technologies evaluated
and contact times used based on information provided by the vendor. The information provided in Table
2-1 was not confirmed in this evaluation. Spray-application of the technologies was performed using a
spray system developed by Battelle under TTEP and in accordance with the test/QA plan(1).
Table 2-1. Technology Information (Vendor Supplied)
Product
Bleach
CASCAD
SDF
DeconGreen
DioxiGuard
EasyDecon
200
Exterm-6
Hi-Clean 605
HM-4100
KlearWater
Peridox
Selectrocide
Vendor
Clorox®
Allen-Vanguard
Edgewood
Chemical &
Biological Center
Frontier
Pharmaceutical
Envirofoam
Technologies
ClorDiSys
Solutions
Howard Industries
Biosafe
Disinfection
Technology
Clean Earth
Technologies
BioProcess
Associates
General
Description/
Formula
Type
Sodium
hypochlorite
Hypochlorite
Hydrogen
peroxide
Chlorine
dioxide
Hydrogen
peroxide
Chlorine
dioxide
Hypochlorous
acid
Quaternary
ammonia
Chlorine
dioxide
Hydrogen
peroxide
Chlorine
dioxide
Components
Sodium hypochlorite 5-6% (pH-amended by
Battelle by adding acetic acid 5% and water )
Sodium myristyl sulfate 10-30%, sodium (C14-
16) olefin sulphonate 10-30%; ethanol denatured
3-9%; alcohols (C10-16) 5-10%, sodium sulfate
3-7%; sodium xylene sulphonate 1-5%;
proprietary mixture of sodium and ammonia salt
along with co-solvent >9%; dichloroisocyanuric
acid, sodium salt 48-85%; sodium tetraborate 3-
7%; sodium carbonate 10-15%.
Potassium molybdate; potassium carbonate;
propylene carbonate 25%; H2O2 35%, Triton X-
100; polyethylene glycol 4-(tert-octyl)phenyl
25%
Inerts
Hydrogen Peroxide <8%; quaternary ammonium
compounds, benzyl-C12-C16 alkyl di-methyl
chlorides 5.5-6.5%; diacetin 30-60%
Inorganic acid 25-35%; sodium chlorite 15-30%;
inorganic salt 35-45%; activator 5-10%
Sodium dichlorisocyanurate 1 1%; trichloro-s-
triazinetrione 3%
Octadecylaminodimethyltrimethoxysilylpropyl
ammonium chloride 84%;
chloropropyltrimethoxysilane 15%; dimethyl
octadecylamine 1%
<0.30% C1O2 suspended in de-ionized water
H2O2 23-25%; peroxyacetic acid 1-1.4%; acetic
acid 1-1.4%; inert ingredients 1-2%
Sodium chlorite 15-40%; activator 55-85%; inert
ingredients <2%
EPA
Registration*
5813-1
None
None
None
74436-1 and
74436-2
70060-19
None
None
None
81073-1
74986-4
Contact
Time (min)
10
30
30
10
60
60
90
30
30
10
10
Registered with the EPA Office of Pesticide Programs (OPP). Registration indicates EPA/OPP has evaluated the pesticide to ensure that it
will not have unreasonable adverse effects on humans, the environment and non-target species and has issued a registration or license for
use in the United States. Note: No product is registered for use against B. anthracis.
" Using procedure recommended by stakeholders, water and 5% acetic acid was added to the household bleach to obtain a pH-amended
bleach solution. The solution was prepared using 9.4 parts water, 1 part bleach, and 1 part 5% glacial acetic acid to yield a solution having
a mean pH of 6.81 ±0.15 andamean total chlorine content of 6,215 ±212 ppm. This "pH-amended bleach" was evaluated for sporicidal
activity.
-------
Below are brief, physical descriptions of the spray-applied technologies (their form, appearance as
received from the vendor) and preparation instructions (as supplied by the vendor). The concentrations
of active ingredients in the prepared solutions, as reported by the vendor, were not confirmed.
• Bleach - Clorox® bleach purchased in a one gallon container from a local retail store
• CASCAD SDF - The CASCAD™ vehicle/equipment laboratory decontaminant packets are
prepared by mixing 9.6 g of GP2100 (decontaminant) and 4.5 mL of GCE2000 (surfactant) in water
to yield a final volume of 150 mL
• DeconGreen - Received in a 6.5 gallon pail containing pre-measured component A and two smaller
containers, containing pre-measured components B & C. Components B & C are added to
component A yielding 5 gallons of activated DeconGreen solution
• DioxiGuard - Two component product that was mixed in equal volumes prior to use. Component A
was a chlorine dioxide solution and component B was an inert solution
• EasyDecon 200 - Received in a 6.5 gallon pail kit containing one pre-measured liquid bladder of
Penetrator (Part 1), one pre-measured liquid bladder of Fortifier (Part 2), and a pre-measured plastic
bottle of Fortifier Booster, (Part 3). The combination of all three pre-measured components yields
five gallons of EasyDECON 200 Decontamination Solution finished blend
• Exterm-6 - This chlorine dioxide generating system is a quarter sized tablet that is dissolved into one
half gallon of water, yielding a 200 ppm chlorine dioxide solution.
• Hi-Clean 605 - Comes as a powder, when mixed with water yields a hypochlorous acid solution. For
this testing, a 4% solution was used.
• HM-4100 - Biosafe is an antimicrobial coating (organosilane) that can be applied to metal surfaces,
or in the case of plastics and textiles the entire substrate can be treated. The product was used neat.
• KlearWater - Contains 0.15% chlorine dioxide in de-ionized water; the product was used neat.
• Peridox - Comes as a concentrate (24%) which is diluted 1:5 to yield the working solution (4%).
The pH of the final solution is 2.7 ± 0.5
• Selectrocide - This product is designed to generate a chlorine dioxide solution in a self contained
pouch by adding 2 L of water. Filling the pouch with water initiates the generation of chlorine
dioxide from an inner sachet, resulting in a 500 ppm solution (0.05%) of chlorine dioxide dissolved
in the water.
-------
3.0 Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the program
QMP(2) and the test/QA plan(1) for this evaluation except as noted below. QA/QC procedures are
summarized below.
3.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety cabinets) used at the time of evaluation was
verified as being certified, calibrated, or validated.
3.2 Audits
3.2.1 Performance Evaluation Audit
No performance evaluation audit was performed for biological agents and organisms because
quantitative standards for these biological materials do not exist.
3.2.2 Technical Systems A udit
Battelle QA staff conducted a technical systems audit (TSA) on December 12, 2005 to ensure that the
evaluation was being conducted in accordance with the test/QA plan(1)and the QMP.(2) As part of the
TSA, test procedures were compared to those specified in the test/QA plan; and data acquisition and
handling procedures were reviewed. Observations and findings from the TSA were documented and
submitted to the Battelle Task Order Leader for response. In response to the findings of the TSA, a
deviation was prepared that accounted for cases where serial dilutions of coupon extracts down to 10"5
(not 10"7 as stated in the test/QA plan) were performed. The latter approach was used in cases where
coupons were treated with a technology that had appreciable (determined during neutralization studies)
efficacy and thus dilution plating below the 10"5 was not necessary. This approach eliminated
unnecessary analyses and conserved resources. TSA records were permanently stored with the TTEP
QA Manager.
3.2.3 Data Quality Audit
At least 10% of the data acquired during the evaluation were audited. A Battelle QA 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.
-------
3.3 QA/QC Reporting
Each audit was documented in accordance with the QMP.(2) The results of the ISA were submitted to
the EPA (the NHSRC Quality Assurance Manager and the TOPO).
3.4 Data Review
Records and data generated in the evaluation received a QC/technical 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.
-------
4.0 Test Results
4.1 pH-Amended Bleach
The decontamination efficacy of pH-amended bleach was evaluated for a biological warfare agent and
three additional organisms on seven indoor surfaces. The evaluation followed the EPA-approved
Technology Testing and Evaluation Program Test/QA Plan for Evaluating Liquid and Foam Sporicidal
Spray Decontaminants (Version 1).(1) Various structural, decorative, and functional surfaces typically
found inside an office building or a mass transit station were used to evaluate the sporicidal
decontamination technology. The test surfaces (coupons measuring 1.9 cm x 7.5 cm) are listed below:
• Industrial-grade carpet
• Bare wood (pine lumber)
• Glass
• Decorative laminate (Formica®, white matte finish)
• Galvanized metal ductwork
• Painted (latex, flat) wallboard paper
• Painted (latex, semi-gloss) concrete cinder block.
The decontamination technologies were tested against the biological agent, B. anthracis Ames spores.
To provide comparative data with the B. anthracis Ames, other organisms frequently used in
decontamination testing, B. anthracis Sterne, B. subtilis, and G. stearothermophilus spores, were tested
in parallel. The following sections summarize the results of these evaluations.
4.1.1 Decontamination Efficacy
4.1.1.1 Quantitative Assessment of the Log Reduction of Viable Organisms
Decontamination efficacy (E) was calculated as the mean log reduction in viable organisms
achieved by the decontamination technology. The spraying system treats two coupons at a time;
therefore, for the controls and decontaminated samples (for each test material/organism), three
separate spray replicates (trials) were employed. This accounted for the six controls and six
decontaminated samples (e.g., two coupons x three replicates = six control/decontaminated
coupons). The blanks (sprayed with water) were treated separately. The log reduction in viable
spores for each individual coupon (E,) was calculated for each of the six replicates of each
material type and biological agent or organism as:
Et = lo§io ~
-------
where TV was the mean number of viable organisms recovered from the six positive control
coupons of a given material type and X . was the number of viable organisms of a given type
recovered from a replicate test coupon (/') after decontamination. (Positive controls are spiked
with biological agent and run at the same test chamber temperature and RH and analyzed at the
same time points as test coupons, but without exposure to the decontaminant technology.) If no
viable organisms were recovered from a test coupon after decontamination, the value 1 was
substituted for X . . Since the value 1 is greater than the observed value of zero, the estimate with
this substitution becomes a lower bound for the true log reduction. Next, the mean log reduction
for a given material type was calculated as decontamination efficacy (£) for a given biological
agent or organism as:
n
where the sum of the log reductions (Et values) was divided by the number of replicates (n). The
mean log reduction calculated as described above is the decontamination efficacy (£).
The decontamination efficacy of amended bleach was high (i.e., 7.2-7.9 log reduction) for hard,
nonporous surfaces (glass, decorative laminate, and galvanized metal ductwork) and low (0.28-
2.0 log reduction) for the porous surfaces (industrial grade carpet, bare wood, and painted
concrete) for B. anthracis Ames (Table 4-1). For B. anthracis Sterne and B. subtilis, the results
were similar; however, for G. stearothermophilus, the log reductions were much lower for hard,
nonporous surfaces (0.75-5.90), as well as for porous surfaces (0.02-1.40). These results are
presented in Tables 4-2, 4-3, and 4-4. No viable organisms were detected by the quantitative
method in any of the blank samples. The decontamination efficacy results for B. anthracis Ames,
B. anthracis Sterne, B. subtilis, and G. stearothermophilus spores are summarized in Table 4-5.
The decontamination efficacy varied according to the type and porosity of the test material with
a greater decontamination efficacy observed for hard, non-porous surfaces compared to more
porous surfaces. G. stearothermophilus appeared to be the most resistant to the pH-amended
bleach.
It should be noted that, in some cases, there were average percent recoveries of <25% of
organisms spiked onto control coupons as shown in Table 4-1; however, these values were
within the acceptable limits as defined in the test/QA plan. These recoveries of inoculated
organisms may be attributed to interactions (adherence or sorption) to the material comprising
each test coupon. The recoveries reported herein are similar to the recoveries achieved in
previous testing. (3"6) Note also that in the present evaluation as well as previous studies,(3"6)
recoveries obtained for B. anthracis Ames are generally not the same as the recoveries obtained
for the organisms, B. subtilis and G. stearothermophilus.
The null hypothesis(1) was tested that there were no differences in the efficacy results for B.
anthracis Ames and the other organisms. Statistically significant differences were observed for
the results obtained for five of the seven test materials (Table 4-5). For B. anthracis Ames and
Sterne as well as B. subtilis, no viable spores were detected in extracts from glass and galvanized
metal test coupons that were decontaminated with pH-amended bleach. Also, no viable B.
anthracis Ames and Sterne spores were detected in extracts from decontaminated decorative
-------
laminate coupons following decontamination with pH-amended bleach. For industrial carpet,
painted wallboard paper, and painted concrete, the decontamination efficacy for pH-amended
bleach inactivation of B. anthracis Sterne spores was statistically greater than that of B. anthracis
Ames. When compared to B. anthracis Ames, statistically lower decontamination efficacies were
observed in the case of inactivation of G. stearothermophilus spores on decorative laminate,
galvanized metal, and painted wallboard paper. Statistically lower decontamination efficacy
values were obtained for B. subtilis spores compared to B. anthracis Ames spores on painted
wallboard paper. However, statistically greater decontamination efficacy values were obtained
for B. subtilis spores compared to B. anthracis Ames spores on painted concrete. The log
reduction values for G. stearothermophilus shown in Table 4-5 generally indicate that these
spores were more resistant to pH-amended bleach than other spores tested. This trend is
especially noticeable upon comparison of the log reduction values obtained for decorative
laminate for spores from the four test organisms. The 0.75 log reduction value is not, when one
examines the log reduction range (rounded numbers) for the six replicates of this data point
(0.84, 0.74, 1.09, 0.50, 0.79, and 0.52) an outlier. G. stearothermophilus often, as seen in
previous testing (References 3-6), behaves differently.
-------
Table 4-1. Inactivation of Bacillus anthracis Ames Spores3 (pH-Amended Bleach; Ten Minute
Contact Time)
Test Material
Industrial-Grade Carpet
Positive Control13
Decontaminated0
Laboratory Blankd
Procedural Blank6
Bare Wood
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Glass
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Galvanized Metal Ductwork
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Wallboard Paper
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Concrete
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
8.47 x 107
8.47 x 107
0
0
9.03 x 107
9.03 x 107
0
0
9.20 x 107
9.20 x 107
0
0
9.03 x 107
9.03 x 107
0
0
9.20 x 107
9.20 x 107
0
0
8.17 xlO7
8.17 xlO7
0
0
8.47 x 107
8.47 x 107
0
0
Total Observed CFU % Recovery
4.10 ± 0.68 xlO7 48.4 ±8.04
2.14 ± 0.09 xlO7 25.3 ±1.02
0 0
0 0
4.66 ± 0.80 xlO6 5. 17 ±0.89
1.27 ± 0.47 xlO6 1.41 ±0.52
0 0
0 0
7.31 ± 0.93 xlO7 79.5 ±10.1
0 0
0 0
0 0
1.52 ± 1.22 xlO7 16.9 ±13. 5
0 0
0 0
0 0
5.29±1.18xl07 57.5 ±12.8
0 0
0 0
0 0
4.12±1.10xl07 50.4 ±13.4
4.09 ± 0.97 xlO5 0.50 ±0.12
0 0
0 0
4.42 ± 0.71 xlO7 52.1 ±8.38
4.26±1.36xl06 5.03 ±1.60
0 0
0 0
Decontamination
Efficacy
.
0.28 ±0.02
-
-
-
0.59 ±0.15
-
-
-
>7.9
-
-
.
>7.2
-
-
.
>7.7
-
-
.
2.0±0.11
-
-
-
1.0 ±0.12
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated coupon
"-" Not Applicable
10
-------
Table 4-2. Inactivation of Bacillus anthracis Sterne Spores3 (pH-Amended Bleach)
Test Material
Industrial-Grade Carpet
Positive Control13
Decontaminated0
Laboratory Blankd
Procedural Blank6
Bare Wood
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Glass
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Galvanized Metal Ductwork
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Wallboard Paper
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Concrete
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.24 xlO8
1.24 xlO8
0
0
1.24 xlO8
1.24 xlO8
0
0
4.73 x 107
4.73 x 107
0
0
4.73 x 107
4.73 x 107
0
0
4.73 x 107
4.73 x 107
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
Total Observed CFU
6.09 ± 0.56 xlO7
9.53±5.59xl06
0
0
1.46 ± 0.64 xlO7
2.35 ± 1.46 xlO5
0
0
5.96±3.36xl06
0
0
0
4.03 ± 2.00 xlO6
0
0
0
2.27±1.53xl07
0
0
0
3.11±0.39xl07
3.38 ± 1.00 xlO4
0
0
7.07 ± 2.07 x 107
4.23 ± 2.69 xlO5
0
0
% Recovery
49.2 ±4.50
7.68 ±4.50
0
0
11.8±5.16
0.19±0.12
0
0
12.6 ±7.11
0
0
0
8.51 ±4.23
0
0
0
47.9 ±32.4
0
0
0
26.9 ±3.37
0.03 ±0.01
0
0
61.0 ±17.9
0.36 ±0.23
0
0
Decontamination
Efficacy
.
0.88 ±0.30
-
-
.
1.9±0.31
-
-
.
>6.8
-
-
.
>6.6
-
-
-
>7.4
-
-
-
3.0 ±0.12
-
-
.
2.3 ±0.35
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated coupon
"-" Not Applicable
11
-------
Table 4-3. Inactivation of Bacillus subtilis Spores3 (pH-Amended Bleach)
Test Material
Industrial-Grade Carpet
Positive Control13
Decontaminated0
Laboratory Blankd
Procedural Blank6
Bare Wood
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Glass
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Galvanized Metal Ductwork
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Wallboard Paper
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Concrete
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.20 xlO8
1.20 xlO8
0
0
1.20 xlO8
1.20 xlO8
0
0
1.15xl08
1.15xl08
0
0
1.15xl08
1.15xl08
0
0
1.15xl08
1.15xl08
0
0
LlOxlO8
LlOxlO8
0
0
LlOxlO8
LlOxlO8
0
0
Total Observed CFU
2.99 ± 0.20 xlO7
1.98 ± 0.56 xlO7
0
0
1.03 ± 0.19 xlO7
3.81 ± 2.19 xlO6
0
0
5.39 ± 0.49 xlO7
0
0
0
1.76 ± 0.19 xlO7
3.43 ± 8.41 xlO1
0
0
6.24 ± 0.85 xlO7
0
0
0
1.83 ± 0.84 xlO7
9.60 ± 4.87 xlO6
0
0
3.61±1.38xl07
2.85 ± 3.75 xlO5
0
0
% Recovery
25.0 ±1.66
16.5 ±4.63
0
0
8.57 ±1.56
3. 18 ±1.83
0
0
46.9 ±4.25
0
0
0
15.3 ±1.65
0
0
0
54.2 ±7.40
0
0
0
16.6 ±7.59
8.72 ±4.43
0
0
32.8 ±12.5
0.26 ±0.34
0
0
Decontamination
Efficacy
0.19±0.11
.
0.49 ±0.27
-
-
-
>7.7
-
-
.
6.90 ±0.94
-
-
-
>7.8
-
-
-
0.33 ±0.22
-
-
.
2.40 ±0.50
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated coupon
"-" Not Applicable
12
-------
Table 4-4. Inactivation of Geobacillus stearothermophilus Spores3 (pH-Amended Bleach)
Test Material
Industrial-Grade Carpet
Positive Control13
Decontaminated0
Laboratory Blankd
Procedural Blank6
Bare Wood
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Glass
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Galvanized Metal Ductwork
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Wallboard Paper
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Painted Concrete
Positive Control
Decontaminated
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
8.23 x
8.23 x
0
0
9.07 x
9.07 x
0
0
9.20 x
9.20 x
0
0
9.07 x
9.07 x
0
0
9.20 x
9.20 x
0
0
8.23 x
8.23 x
0
0
9.20 x
9.20 x
0
0
107
107
107
107
107
107
107
107
107
107
107
107
107
107
Total Observed CFU
1.
9.
3.
1.
5.
1.
2.
4.
4.
0.
2.
5.
2.
8.
03 ±
92 ±
66 ±
83 ±
0.20 x
1.78 x
0
0
2.07 x
1.65 x
0
0
94 ± 0.65 x
81 ±
2.80 x
0
0
10 ± 0.84 x
16±
90 ±
87 ±
56 ±
78 ±
51 ±
.54 ±
1.94 x
0
0
0.56 x
1.38 x
0
0
0.71 x
3.20x
0
0
1.03 x
5.30 x
0
0
107
106
106
105
107
105
107
106
107
107
107
106
107
106
% Recovery
12.5
12.1
4.03
0.20
64.6
0.20
23.2
4.58
53.3
9.46
31.1
7.02
27.2
9.29
±
±
0
0
±
±
0
0
±
±
0
0
±
±
0
0
±
±
0
0
±
±
0
0
±
±
0
0
2.
2.
2.
0.
7.
0.
9.
2.
6.
36
16
28
18
04
30
21
14
06
15.0
8.
3.
59
88
Decontamination
Efficacy
-
0.02 ±0.08
-
-
.
1.40±0.39
-
-
.
5.90 ±3.0
-
-
.
0.75 ±0.22
-
-
.
2.60 ±2.8
-
-
-
0.74 ±0.37
-
-
11.2
5.
77
0.54 ±0.27
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated coupon
"-" Not Applicable
13
-------
Table 4-5. Summary of Efficacy Values (Log Reduction) Obtained for pH-Amended Bleach3
Material
Industrial-Grade Carpet
Bare Wood
Glass
Decorative Laminate
Galvanized Metal Ductwork
Painted Wallboard Paper
Painted Concrete
B. anthracis
Ames
0.28
0.59
>7.9
>7.2
>7.7
2.0
1.0
B. anthracis
Sterne
0.88
1.9
>6.8
>6.6
>7.4
3.0
2.3
B. subtilis
0.19
0.49
>7.7
6.9
>7.8
0.33
2.4
G. stearothermophilus
0.02
1.4
5.9
0.75
2.6
0.74
0.54
a Numbers in bold are statistically different (p < 0.05) from B. anthracis Ames
4.1.1.2 Qualitative Assessment of Residual Spores.
Based on previous decontamination studies,(3"6) it was anticipated that spores would 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 decontaminated and extracted test coupons; an assessment was also made
to determine whether the decontaminated coupons with zero growth (zero growth indicated in
Table 4-5 by ">" values) 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. 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.
After incubation the plates were examined to determine qualitatively (morphologic comparison
performed visually) if the observed growth was a pure culture of the organism 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. Because test coupons
were not sterilized (only the coupon surface was wiped with 70% isopropanol) prior to
inoculation, the presence of endogenous organisms was likely. 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 were spiked onto the test coupon. The percent of
streak plates displaying only growth from the inoculated organism was 40, 58, 56, and 100% for
B. anthracis Ames, B. anthracis Sterne, B. subtilis, and G. stearothermophilus spores,
respectively.
Results from the liquid culture growth assessment of coupons at one and seven days post-
decontamination are provided in Tables 4-6, 4-7, 4-8, and 4-9 for coupons spiked with B.
anthracis Ames, B. anthracis Sterne, B. subtilis, and G. stearothermophilus spores, respectively.
It should be noted that in several cases, growth was observed in blanks (for example see Table 4-
14
-------
6, results for bare wood, Sample Bl for Day 1 and Day 7). This is due to growth of endogenous
organisms.
Table 4-6. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames Spores
following Extraction (pH-Amended Bleach)
Test Material Day 1 Day 7
SI S2 S3 S4 S5 S6 Bl SI S2 S3 S4 S5 S6 Bl
Industrial-Grade Carpet
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated + + + + + + - + + + + + +
Bare Wood
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated + + + + + + - + + + + + +
Glass
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated . ..
Decorative Laminate
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated . ..
Galvanized Metal Ductwork
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated . ..
Painted Wallboard Paper
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated + + + + + + + + + + + + + +
Painted Concrete
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated + + + + + + - + + + + + +
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated withB. anthracis Ames spores)
"+" = growth; "-" = no growth
15
-------
Table 4-7. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Sterne
Spores following Extraction (pH-Amended Bleach)
Test Material Day 1 Day 7
SI S2 S3 S4 S5 S6 Bl SI S2 S3 S4 S5 S6 Bl
Industrial-Grade Carpet
Inoculated, Not Decontaminated ..............
Inoculated, Decontaminated ..............
Bare Wood
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated + + + + + + - + + + + + + -
Glass
Inoculated, Not Decontaminated + + + + + + - + + + + + + -
Inoculated, Decontaminated ..............
Decorative Laminate
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated ..............
Galvanized Metal Ductwork
Inoculated, Not Decontaminated + + + + + + - + + + + + + -
Inoculated, Decontaminated ..............
Painted Wallboard Paper
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated + + + + + + + + + + + + + +
Painted Concrete
Inoculated, Not Decontaminated + + + + + + - + + + + + + -
Inoculated, Decontaminated + + + + + + - + + + + + + -
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated withB. anthracis Sterne spores)
"+" = growth; "-" = no growth
16
-------
Table 4-8. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (pH-Amended Bleach)
Test Material Day 1 Day 7
SI S2 S3 S4 S5 S6 Bl SI S2 S3 S4 S5 S6 Bl
Industrial-Grade Carpet
Inoculated, Not Decontaminated -- + + - + - - . + + . + .
Inoculated, Decontaminated ..............
Bare Wood
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated + + + + + + + + + + + + + +
Glass
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated ..............
Decorative Laminate
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated . + + ..... + + ....
Galvanized Metal Ductwork
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated ..............
Painted Wallboard Paper
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated + + + + + + + + + + + + + +
Painted Concrete
Inoculated, Not Decontaminated + + + + + + + + + + + + + +
Inoculated, Decontaminated + + + + + + - + + + + + +
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated withB. subtilis spores)
"+" = growth; "-" = no growth
17
-------
Table 4-9. Liquid Culture Assessment of Coupons Inoculated with Geobacillus stearothermophilus
Spores following Extraction (pH-Amended Bleach)
Test Material Day 1 Day 7
SI S2 S3 S4 S5 S6 Bl SI S2 S3 S4 S5 S6 Bl
Industrial-Grade Carpet
Inoculated, Not Decontaminated - ... + ..
Bare Wood
Inoculated, Not Decontaminated + + + + + +NR + + + + + +NR
Inoculated, Decontaminated + + + + + + - + + + + + +
Glass
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated + + . + .+ . + + . + .+
Decorative Laminate
Inoculated, Not Decontaminated + + + + + +NR + + + + + +NR
Inoculated, Decontaminated + + + + + + - + + + + + +
Galvanized Metal Ductwork
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated .... + +. .... + +.
Painted Wallboard Paper
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated + + + + + + - + + + + + +
Painted Concrete
Inoculated, Not Decontaminated + + + + + + - + + + + + +
Inoculated, Decontaminated + + + + + + - + + + + + +
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with G. stearothermophilus spores)
NR = Not Recorded (data inadvertently not recorded)
"+" = growth; "-" = no growth
These results from the qualitative tests are generally consistent with the results from the quantitative
tests (Table 4-10). Whereas in the quantitative tests, the amended bleach achieved high decontamination
efficiency on hard, non-porous surfaces against B. anthracis Ames, B. anthracis Sterne, and B. subtilis,
similar results were seen in the qualitative tests (i.e., few or no positive samples found). Also, whereas
the quantitative tests shows notably less decontamination efficacy for amended bleach when used
against G. stearothermophilus on hard surfaces, the qualitative tests showed all positive cultures for this
spore type on hard surfaces. Finally, except for industrial grade carpet (due to its antimicrobial activity),
amended bleach was not effective on porous surfaces for either the quantitative or qualitative tests for
any spore type.
18
-------
Table 4-10. Summary of Results Obtained from the Quantitative and Qualitative Assessments
when Comparing Decontaminated Coupons (pH-Amended Bleach)
Material
Industrial-Grade Carpet
Bare Wood
Glass
Decorative Laminate
Galvanized Metal Ductwork
Painted Wallboard Paper
Painted Concrete
B. anthracis Ames
A B
+ +
-
-
-
+ +
B. anthracis Sterne
A B
+ I
-
-
-
+ +
B. subtilis
A B
+ +
-
c
-
+ +
G. stearothermophilus
A B
? - J
+ c
+ +
+ c
+ +
A = Quantitative Assessment
B = Qualitative Assessment at seven days
"+" = observed CFU or growth; "-" = no observed CFU or no growth; "c" = combination of growth and no growth
* A small amount of growth was observed on only two of the seven replicates
Shading denotes inconsistent results
In the case of industrial-grade carpet, results were inconsistent. This is likely due to the susceptibility of
vegetative growth to a broad-spectrum antibacterial compound (FlorSept®) in the carpet that leaches
into the medium. It has been observed in previous testing (ref. 3-6) that FlorSept® (zinc omadine; also
known as zinc pyrithione) appears to be both bactericidal and bacteriostatic in qualitative assessments.
Apparently, the FlorSept® is not sporicidal, as it did not inactivate spores that were extracted and
subsequently plated onto tryptic soy agar in this evaluation's quantitative assay. However, in the
qualitative assessment, the carpet samples are incubated in the liquid broth for seven days. It is likely
that during the seven day incubation the FlorSept® in the carpet leaches into the liquid broth, thereby
asserting its bactericidal/bacteriostatic properties. In the past, we have demonstrated that if a sample of
these "negative" liquid cultures is plated onto tryptic soy agar and incubated overnight that growth of the
inoculated organism is observed. These results support the bactericidal/bacteriostatic properties of
FlorSept®.
4.1.2 Damage to Coupons
Before and after decontamination of the test coupons, the decontaminated coupons were visually
inspected; and any obvious changes in the color, reflectivity, and apparent roughness of the coupon
surfaces were recorded. No damage (e.g., change in surface texture, color) or visible change was
observed during this evaluation to any of the test coupons.
4.1.3 Other Factors
4.1.3.1 Operator Control
On each day of testing, the pH-amended bleach was prepared fresh by mixing household
Clorox® bleach (5-6% sodium hypochlorite), 5% acetic acid, and distilled water. Titrations
determined that in the pH-amended bleach solutions prepared, the mean total chlorine content
was 6,215 ± 212 ppm and the pH was 6.81 ± 0.15. A NIST-traceable thermometer/hygrometer
indicated that the temperature and RH were maintained in the test chamber within the specified
range of 22 to 35°C and <70% RH.
19
-------
4.1.3.2 Technology Spray Deposition
The pH-amended bleach was applied to glass and bare wood test coupons using a ten-second
spray duration. The liquid was gravity-fed into an ultrasonic nozzle where it was mixed in an air
stream at 40 psi to convert the liquid to droplets (fine mist, 10-50 microns in diameter for water).
Gravimetric analysis was used to measure total spray deposition (as described in the test/QA
plan(1)), which included the liquid adhering to the coupon surface as well as any collected run-
off. This total mass was used to determine the total amount of spray deposition, or total mass of
liquid contacting the surface of the coupon. For this assessment, separate pre-weighed coupons
were sprayed simultaneously as two replicates of two coupons (N=4 total coupons). Following a
ten-second spray, each coupon (plus collected runoff) was individually weighed and the mass
determined. During each spray replicate, there was no observable difference in spray deposition
(based on gravimetric results) between the two coupons. Total deposition was recorded at 0.32 ±
0.02 grams and 0.33 ± 0.02 grams of the pH-amended bleach onto glass and bare pine wood test
coupons, respectively. Other spray times were used and the reproducibility of the spray
application process is indicated by the data shown in Table 4-11. The high level of
reproducibility ensures that the differences in efficacy are not likely due to differences in
deposition of the decontamination technology.
4.1.3.3 Neutralization Methodology
Methods demonstration was performed to determine the neutralization efficiency against the pH-
amended bleach using sodium thiosulfate. Neutralization of residual pH-amended bleach was
necessary in order to obtain accurate decontamination efficacy data for the ten minute contact
time. The neutralization results are shown in Table 4-12. The ten-second spray time resulted in
approximately 0.325 mL of pH-amended bleach deposited on the coupon + collected runoff in a
50 mL conical tube, to which 10 mL of extraction buffer [phosphate-buffered saline (PBS) +
0.1% Triton X-100] containing sodium thiosulfate (STS) was added. It is known that the molar
ratio for neutralization of hypochlorite with STS is 2:1; therefore, based upon the 0.325 mL of
pH-amended bleach and 10 mL of extraction buffer, there were multiple variables that were
calculated or measured so that neutralization efficiency could be determined. These variables
included total chlorine concentration in the pH-amended bleach, total amount of pH-amended
bleach deposited on the coupon + collected runoff for a ten second spray duration, molarity of
pH-am ended bleach, molarity of STS in the 10 mL of extraction buffer, and molar ratio of STS
to hypochlorite. The results shown in Table 4-12 were based on a starting pH-amended bleach
concentration of 6200 ppm. The target STS concentration for this 10 mL solution was calculated
to be 0.086%. Higher (0.17%) and lower (0.043%) concentrations of STS were also tested to
help demonstrate the most effective neutralization. STS can inhibit bacterial growth; therefore,
the higher STS concentration was used to demonstrate the potential for any unreacted STS
remaining in the extraction buffer to reduce neutralization efficiency by inactivating spores. The
lower STS concentration was used to demonstrate the potential for any remaining non-
neutralized bleach in the extraction buffer to potentially continue inactivating spores, thereby
reducing neutralization efficiency. When compared to the controls (extraction buffer + spores
with no STS), the calculated target STS concentration of 0.086% was the optimal STS
concentration for neutralizing the bleach.
20
-------
Table 4-11. Mean ± (SD) Weight of pH-Amended Bleach Deposited on Test Coupons (g)
Material3
Glass
Bare Wood
1
0.04 ± 0.002
0.04 ± 0.004
Spray Time (seconds)1"
5 10 15
0.17 ±0.01
0.16 ±0.01
0.32 ±0.02
0.33 ±0.02
0.60 ± 0.02
0.56 ±0.08
20
0.81 ±0.08
0.76 ±0.10
Correlation
Coefficient (R2)
0.9873
0.9946
aN = 4 coupons per time point
b Spray distance of 12 inches; spray pressure of 40 psi
Table 4-12. Neutralization Testing for pH-Amended Bleach
Treatment
NaOCl + Spores3
NaOCl + PBS + Triton X-100 + Sporesab
PBS + Triton X-100 + Spores (Control)13
NaOCl + PBS + Triton X-100 + 0.043% STS + Sporesab
NaOCl + PBS + Triton X-100 + 0.086% STS + Sporesab
NaOCl + PBS + Triton X-100 + 0. 17% STS + Sporesab
Inoculum
(CFU)
9.80 x 107
9.80 x 107
9.80 x 107
9.80 x 107
9.80 x 107
9.80 x 107
Total Observed
CFU
0
0
8.59 xlO7
4.30 xlO7
8.45 x 107
5.62 x 107
%of
Control
0
0
50.1
98.3
65.4
aNaOCl volume corresponds to gravimetric deposition for ten-sec, spray duration
b Total volume is 10 mL
"-" Not Applicable
4.2 Ten Technologies Evaluated by Screening Test
Ten decontamination technologies were evaluated for decontamination efficacy against a biological
warfare agent on one indoor surface. The technologies evaluated in the screening test are listed in Table
2-1. The evaluation followed the Technology Testing and Evaluation Program Test/QA Plan for
Evaluating Liquid and Foam Sporicidal Spray Decontaminants (Version 1).(1)
Testing was undertaken as described in this technology evaluation report in order to rapidly screen
sporicidal, spray-applied technologies for efficacy for inactivating spores on indoor materials. Prior to
the screening test, a Vendor Agreement (including a Quick-Screen Protocol) was reviewed and signed
by each vendor. The Quick-Screen Protocol specified that an automated spray apparatus would be used
to apply the decontaminant and that spray application would be performed as specified by the vendor.
Decontaminants were prepared daily in clean mixing chambers per vender instructions. Pre-established
parameters - 12 inch spray distance, ten-second spray time, and 40 psi spray pressure would be used if
vendors did not specify parameters. Some vendors expressed preference that their own spray equipment
would be used and two vendors expressed concern that 40 psi might not be optimal for their
technologies; however, none of the vendors recommended an alternate spray pressure. All of the ten
technologies tested were sprayed using the same automated sprayer and the same spray-application
conditions (12 inch spray distance, ten second spray time, and 40 psi spray pressure) in order to obtain
comparative performance data. Also a contact time (contact time is the time that the spray-applied liquid
was allowed to be in or on the test coupon prior to extraction and neutralization) following spraying was
specified by the vendor (in the absence of specified spray time a ten minute contact time was used). The
testing reported herein was performed in the Quick-Screen mode for the purpose of obtaining a
preliminary assessment of the efficacy of different spray-applied technologies. Following this Quick-
Screen, more testing was anticipated and performed for different types of technologies and for those that
exhibited the greatest log reduction values.
21
-------
Following the testing, the technology vendors were given the opportunity to review and comment on the
draft results. Three of the four chlorine dioxide based technology vendors (ClorDiSys, Frontier
Pharmaceutical, and BioProcess Associates) expressed concern that the spray system used in testing may
not have been operated optimally for their product, and in particular, expressed concern with the high
operating pressure. One vendor indicated that an air pressure of 40 psi would produce a fine mist (10 -
50 micron diameter droplet size). These relatively small size droplets, along with the air flow used to
atomize the liquid, could lead to increased mass transfer of chlorine dioxide from the liquid to gas phase,
thus potentially decreasing the chlorine dioxide concentration in the liquid and rendering it less
effective. This phenomenon has not been verified.
Since the same test conditions were used for each technology, it is probable that the conditions for
application would not be ideal for every technology. The data herein suggest that the method of
application may impact efficacy. Therefore, based upon the results of this report, certain technologies
(e.g., DioxiGuard, Exterm-6, and Selectrocide) should be further tested to evaluate various spray
conditions as modification of these conditions may affect decontamination efficacy. We do note
however that these technologies achieved complete inactivation of approximately 108 spores in the
neutralization tests conducted (solution of spore inoculum + liquid decontamination technology); see
Tables 4-18, 4-20, and 4-25.
The data also indicate that efficacy of spray applied technologies varies, depending upon the type of
spores and the material on which or in which the spores reside. In addition, increasing the concentration
of the decontaminant(s) (active ingredients) in a spray applied technology might lead to a marked
improvement in the efficacy of the technology. In summary, the reader should view the data and results
contained herein in the context of results of "Quick-Screening". More definitive testing may be needed
in order to obtain data that fully characterize the sporicidal efficacy of each spray-applied technology.
Glass, a surface typically found inside an office building or a mass transit station, was used for this
screening test. The test coupon surface measured 1.9 cm x 7.5 cm.
The biological agent used to evaluate the sporicidal decontamination technologies was B. anthracis
Ames spores.
Four of the ten technologies that exhibited the greatest decontamination efficacy (Table 4-13) were
selected for further evaluation. These four technologies were also selected because they represent four
different types of sporicidal formulations that are available. Sections 4.3, 4.4, 4.5, and 4.6 detail the
results of the further evaluation. The following sections summarize the results of the initial screening
evaluations.
4.2.1 Decontamination Efficacy
4.2.1.1 Quantitative Assessment of the Log Reduction of Viable Organisms
Decontamination efficacy was calculated as the mean log reduction in viable organisms achieved
by the decontamination technology. Decontamination efficacy was calculated as described in
Section 4.1.1.1.
22
-------
The decontamination efficacy of the ten sporicidal technologies for inactivating extractable,
viable spores from the test materials ranged from 0.37 to > 7.8 for B. anthracis Ames spores on
glass coupons (Table 4-13) and varied statistically between technologies. Each of the ten
technologies did reduce the number of viable spores that could be extracted from the glass
coupons. No viable organisms were detected in any of the blank samples.
23
-------
Table 4-13. Inactivation of Bacillus anthracis Ames Spores3 on Glass (Ten Sporicidal
Technologies)
Technology (Contact Time)
CASCAD SDF (30 minutes)
Sprayed H2O
Sprayed CASCAD SDF
Laboratory Blankb
Procedural Blank0
DeconGreen (30 minutes)
Sprayed H2O
Sprayed DeconGreen
Laboratory Blank
Procedural Blank
DioxiGuard (10 minutes)*1
Sprayed H2O
Sprayed DioxiGuard
Laboratory Blank
Procedural Blank
EasyDecon 200 (60 minutes)
Sprayed H2O
Sprayed EasyDecon 200
Laboratory Blank
Procedural Blank
Exterm-6 (60 minutes)*1
Sprayed H2O
Sprayed Exterm-6
Laboratory Blank
Procedural Blank
ffl-Clean 605 (90 minutes)
Sprayed H2O
Sprayed Hi-Clean 605
Laboratory Blank
Procedural Blank
HM-4100 (30 minutes)
Sprayed H2O
Sprayed HM-4100
Laboratory Blank
Procedural Blank
KlearWater (30 minutes)
Sprayed H2O
Sprayed KlearWater
Laboratory Blank
Procedural Blank
Peridox (10 minutes)
Sprayed H2O
Sprayed Peridox
Laboratory Blank
Procedural Blank
Selectrocide (10 minutes)*1
Sprayed H2O
Sprayed Selectrocide
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.67 xlO7
9.67 xlO7
0
0
9.67 xlO7
9.67 xlO7
0
0
9.40 xlO7
9.40 xlO7
0
0
9.77 xlO7
9.77 xlO7
0
0
9.77 xlO7
9.77 xlO7
0
0
9.60 xlO7
9.60 xlO7
0
0
9.50 xlO7
9.50xl07
0
0
9.50 xlO7
9.50xl07
0
0
9.40 xlO7
9.40 xlO7
0
0
9.40 xlO7
9.40 xlO7
0
0
Total Observed CFU
5.83 ± 0.14 xlO7
2.78 ± 3.32 x 102
0
0
5.83 ± 0.14 xlO7
2.44±1.49xl04
0
0
6.87 ± 0.73 xlO7
4.04±1.35xl04
0
0
5.96±0.89xl07
7.54 ± 1.75 xlO6
0
0
5.96±0.89xl07
4.65 ± 2.47 x 106
0
0
5.81 ± 0.91 xlO7
0
0
0
5.93 ± 0.74 xlO7
2.76±1.07xl07
0
0
5.93 ± 0.74 xlO7
0
0
0
6.87 ± 0.73 xlO7
0
0
0
6.87 ± 0.73 xlO7
3.87 ± 0.71 xlO5
0
0
% Recovery
60.3 ±1.5
0
0
0
60.3 ±1.5
0.025 ±0.015
0
0
73.1 ±7.7
0.04 ±0.01
0
0
61.0±9.1
7.72 ±1.80
0
0
61.0±9.1
4.76 ±2.5
0
0
60. 5 ±9. 5
0
0
0
62.4 ±7.8
29.0 ±11.2
0
0
62.4 ±7.8
0
0
0
73.1 ±7.7
0
0
0
73.1 ±7.7
0.41 ±0.08
0
0
Decontamination
Efficacy
-
6.4 ±1.6
-
-
-
3.4 ±0.29
-
_
-
3.2±0.13
-
-
-
0.91 ±0.10
-
_
-
1.1 ±0.20
-
-
-
>7.8
-
-
-
0.37 ±0.22
-
-
-
>7.8
-
-
-
>7.8
-
_
-
2.3 ±0.08
-
-
Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
Laboratory Blank = not inoculated, not decontaminated coupon
Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
Vendor expressed concern that the 40 psi spray pressure utilized for testing could potentially be too high for optimal performance of
their product
-" Not Applicable
24
-------
4.2.2 Damage to Coupons
Before and after decontamination of the test coupons, the decontaminated coupons were visually
inspected; and any obvious changes in the color, reflectivity, and apparent roughness of the coupon
surfaces were recorded. No damage (e.g., change in surface texture, color) or visible change was
observed during this evaluation to any of the test coupons.
4.2.3 Other Factors
4.2.3.1 Operator Control
On each day of testing, each of the ten sporicidal technologies was prepared fresh according to
the vendor's instructions. Preparation procedures required simple procedures (dilution, stirring).
The KlearWater technology did not require any preparation and was used as received.
A NIST-traceable thermometer/hygrometer indicated that the temperature and RH were
maintained in the test chamber within the specified range of 22 to 35°C and <70% RH.
4.2.3.2 Technology Spray Deposition
Each of the ten technologies was applied to the test coupons using a ten-second spray duration.
Gravimetric analysis was performed on glass and bare pine wood coupons as described above in
Section 4.1.3.2. The results of the gravimetric analysis for total spray deposition of each of the
ten sporicidal technologies are shown in Table 4-14. For the four down-selected technologies, a
daily verification of spray deposition was performed using a ten second spray duration and glass
test coupons. This daily verification was performed to demonstrate consistency of the spray
system with each decontaminant technology. This daily verification was performed to
demonstrate consistency of the spray system with each decontaminant technology (p values
calculated using Mest were all greater than 0.05, indicating essentially no difference in the values
being compared).
25
-------
Table 4-14. Spray Deposition of Water and Individual Technologies311
Technology/Material0 Spray Deposition (mass in grams)
Water
Glass 0.52 ± 0.03
Bare Pine Wood 0.52 ±0.01
CASCAD SDF
Glass 0.44 ±0.01
Bare Pine Wood 0.43 ± 0.02
DeconGreen
Glass 0.25 ±0.01
Bare Pine Wood 0.26 ±0.01
DioxiGuard
Glass 0.42 ±0.01
Bare Pine Wood 0.46 ± 0.03
EasyDecon 200
Glass 0.24 ± 0.03
Bare Pine Wood 0.25 ±0.01
Exterm-6
Glass 0.44 ± 0.04
Bare Pine Wood 0.47 ± 0.02
Hi-Clean 605
Glass 0.48 ± 0.04
Bare Pine Wood 0.47 ± 0.04
HM-4100
Glass 0.49 ± 0.06
Bare Pine Wood 0.44 ± 0.05
KlearWater
Glass 0.45 ± 0.04
Bare Pine Wood 0.47 ± 0.02
Peridox
Glass 0.43 ± 0.02
Bare Pine Wood 0.42 ± 0.02
Selectrocide
Glass 0.51 ±0.03
Bare Pine Wood 0.53 ±0.02
aData are expressed as mean (± SD)
b Spray distance of 12 inches; spray pressure of 40 psi; ten-second spray time
°N=4 coupons per time point
4.2.3.3 Neutralization Methodology
Methods demonstration was performed to determine the appropriate concentration of neutralizer
for each technology. Neutralization was necessary in order to terminate the spore inactivating
effect of the technology and obtain accurate efficacy data according to the vendor-specified
contact time. The overall method used for testing neutralization efficiency for each technology is
the same as used for pH-amended bleach (Section 4.1.3.3) and is described in the test/QA plan.(1)
Details of the vendor-recommended neutralizer and selected concentration are shown in Table 4-
15.
The neutralization results, using the neutralizer recommended by the vendors, are shown in
Tables 4-16 to 4-25. A target concentration of neutralizer was calculated based upon vendor-
26
-------
estimated concentration of decontaminant and spray-deposited mass of liquid following
spraying. From this collected information, a range of neutralizer concentrations was evaluated
for each technology. Six treatments (duration of each treatment was 15 minutes while shaking at
200 rpm) shown in Tables 4-16 to 4-25 were used to gather the data necessary to select a
neutralization approach for each technology that was used in subsequent testing.
Table 4-15. Neutralizer for Each of Ten Commercially Available Technologies
Technology
Neutralizer (Vendor Recommended)
Final Concentration of Neutralizera
CASCAD SDF Sodium thiosulfate
DeconGreen Sodium thiosulfate
DioxiGuard Sodium thiosulfate
EasyDecon 200 Sodium thiosulfate
Exterm-6 Sodium thiosulfate
Hi-Clean 605 Sodium thiosulfate
HM-4100 Sodium dodecylbenzene sulfonate
KlearWater Sodium thiosulfate
Peridox Dey/Engley broth + catalase
Selectrocide Sodium thiosulfate
0.5%
0.60%
0.002%
0.5%
0.004%
0.08%
0.467 mL of 0.17%
0.015%
0.13 mL of Dey/Engley broth + 0.336 mL catalase
0.01%
a See Tables 4-16 through 4-25 for additional details
Table 4-16. Neutralization Testing for CASCAD SDF
Treatment
CASCAD SDF + Spores3
CASCAD SDF + PBS + Triton X-100 + Sporesab
PBS + Triton X-100 + Spores (Control)13
CASCAD SDF + PBS + Triton X-100 + 1.0% STS + Sporesab
CASCAD SDF + PBS + Triton X-100 + 0.5% STS + Sporesab
CASCAD SDF + PBS + Triton X-100 + 0.25% STS + Sporesab
Inoculum
(CFU)
4.80 x 107
4.80 x 107
4.80 x 107
4.80 x 107
4.80 x 107
4.80 x 107
Total Observed
CFU
0
0
4.68 x 107
4.07 x 107
5.03 x 107
0
%of
Control
0
0
87.0
108
0
a CASCAD volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g= 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
Table 4-17. Neutralization Testing for DeconGreen
Treatment
DeconGreen + Spores3
DeconGreen + PBS + Triton X-100 + Spores*
PBS + Triton X-100 + Spores (Control)13
DeconGreen + PBS + Triton X-100 + 0.30% STS + Spores*
DeconGreen + PBS + Triton X-100 + 0.60% STS + Spores*
DeconGreen + PBS + Triton X-100 + 1.20% STS + Spores*
noculum
(CFU)
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
.08 x 108
Total Observed
CFU
0
TNTC
9.88 xlO7
1.02 xlO8
9.46 x 107
1.03 x 108
%of
Control
0
103
95.7
104
a DeconGreen volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g= 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
TNTC = Too Numerous to Count
"-" Not Applicable
27
-------
Table 4-18. Neutralization Testing for DioxiGuard
Treatment
DioxiGuard + Spores3
DioxiGuard + PBS + Triton X-100 + Sporesab
PBS + Triton X-100 + Spores (Control)b
DioxiGuard + PBS + Triton X-100 + 0.02% STS+Sporesab
DioxiGuard + PBS + Triton X-100 + 0.002% STS+Sporesab
DioxiGuard + PBS + Triton X-100 + 0.0002% STS+Sporesab
noculum
(CFU)
.02 x 108
.02 x 108
.02 x 108
.02 x 108
.02 x 108
.02 x 108
Total Observed
CFU
0
0
9.54 xlO7
9.00 x 107
9.00 x 107
3.48 xlO4
%of
Control
0
0
94.3
94.3
0.0004
a DioxiGuard volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g = 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
Table 4-19. Neutralization Testing for EasyDecon 200
Treatment
EasyDecon 200 + Spores3
EasyDecon 200 + PBS + Triton X-100 + Spores*
PBS + Triton X-100 + Spores (Control)b
EasyDecon 200 + PBS + Triton X-100 + 0.23% STS+ Sporesab
EasyDecon 200 + PBS + Triton X-100 + 0.50% STS + Spores*
EasyDecon 200 + PBS + Triton X-100 + 1.0% STS + Sporesab
Inoculum
(CFU)
7.93 x 107
7.93 x 107
7.93 x 107
7.93 x 107
7.93 x 107
7.93 x 107
Total Observed
CFU
0
4.64 x 106
7.36 xlO7
6.91 x 107
6.91 x 107
7.06 x 107
%of
Control
0
6.30
93.9
93.9
95.9
aEasyDecon 200volume aEasyDecon 200 volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g = 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
Table 4-20. Neutralization Testing for Exterm-6
Treatment
Exterm-6 + Spores3
Exterm-6 + PBS + Triton X-100 + Spores*
PBS + Triton X-100 + Spores (Control)13
Exterm-6 + PBS + Triton X-100 + 0.04% STS + Spores*
Exterm-6 + PBS + Triton X-100 + 0.004% STS + Spores*
Exterm-6 + PBS + Triton X-100 + 0.0004% STS + Spores*
Inoculum
(CFU)
8.43 x 107
8.43 x 107
8.43 x 107
8.43 x 107
8.43 x 107
8.43 x 107
Total Observed
CFU
0
0
6.96 x 107
7.24 x 107
0
0
%of
Control
0
0
-
104
0
0
aExterm-6 volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g = 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
28
-------
Table 4-21. Neutralization Testing for Hi-Clean 605
Treatment
Hi-Clean 605 + Spores3
Hi-Clean 605 + PBS + Triton X-100 + Spores*
PBS + Triton X-100 + Spores (Control)13
Hi-Clean 605 + PBS + Triton X-100 + 0.06% STS +
Hi-Clean 605 + PBS + Triton X-100 + 0.08% STS +
Hi-Clean 605 + PBS + Triton X-100 + 0. 10% STS +
Sporesab
Sporesab
Sporesab
Inoculum
(CFU)
4.77 x 108
4.77 x 108
4.77 x 108
4.77 x 108
4.77 x 108
4.77 x 108
Total Observed
CFU
0
0
S.lOxlO8
2.68 x 108
2.96 x 108
1.67 xlO8
%of
Control
0
0
86.5
95.5
53.8
aHI-Clean 605 volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g= 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
Table 4-22. Neutralization Testing for HM-4100
Treatment
HM-4 100 + Spores3
HM-4100 + PBS + Triton X-100 + Sporesab
PBS + Triton X-100 + Spores (Control)13
HM-4100 + PBS + Triton X-100 + 1.0% SDS + Spores*
HM-4100 + PBS + Triton X-100 + 0.047% SDS + Spores*
HM-4100 + PBS + Triton X-100 + 0.023% SDS + Spores*
noculum
(CFU)
.00 x 108
.00 x 108
.00 x 108
.00 x 108
.00 x 108
.00 x 108
Total Observed
CFU
1.07 xlO7
8.87 x 107
1.02 xlO8
8.19 xlO7
9.84 x 107
7.82 x 107
%of
Control
10.5
84.7
80.3
96.5
76.7
a HM-4100 volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g= 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
Table 4-23. Neutralization Testing for KlearWater
Treatment
KlearWater + Spores
KlearWater + PBS +
PBS + Triton X-100
KlearWater + PBS +
KlearWater + PBS +
KlearWater + PBS +
Inoculum
(CFU)
a
Triton X-100 + Spores*
+ Spores (Control)13
Triton X-100 + 0.005% STS + Spores*
Triton X-100 + 0.015% STS + Spores*
Triton X-100 + 0.03% STS + Spores*
.05
.05
.05
.05
.05
.05
x
x
X
X
X
X
108
108
108
108
108
108
Total Observed
CFU
0
0
9.99 x
9.12 x
9.69 x
9.59 x
107
107
107
107
%of
Control
0
0
91
97
95
3
2
9
a KlearWater volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g = 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
29
-------
Table 4-24. Neutralization Testing for Peridox
Treatment
Peridox + Spores3
Peridox + PBS + Triton X-100 + Sporesab
PBS + Triton X-100 + Spores (Control)13
Peridox + PBS + Triton X-100 + 0.3mL DE/CAT+Sporesab
Peridox + PBS + Triton X-100 +0.426mL DE/CAT+Sporesab
Peridox + PBS + Triton X-100 + 0.5mL DE/CAT+Sporesab
noculum
(CFU)
.01 x 108
.01 x 108
.01 x 108
.01 x 108
.01 x 108
.01 x 108
Total Observed
CFU
0
7.64 x 107
9.22 x 107
8.44 x 107
1.01 xlO8
8.10 xlO7
%of
Control
0
82.9
91.5
110
87.8
a Peridox volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is lOmL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g= 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
Table 4-25. Neutralization Testing for Selectrocide
Treatment
Selectrocide + Spores3
Selectrocide + PBS + Triton X-100 + Sporesab
PBS + Triton X-100 + Spores (Control)13
Selectrocide + PBS + Triton X-100 + 0.02% STS + Sporesab
Selectrocide + PBS + Triton X-100 + 0.01% STS + Sporesab
Selectrocide + PBS + Triton X-100 + 0.005% STS +Sporesab
noculum
(CFU)
.00 x 108
.00 x 108
.00 x 108
.00 x 108
.00 x 108
.00 x 108
Total Observed
CFU
0
0
1.02 xlO8
9.90 x 107
1.03 x 108
9.88 xlO7
%of
Control
0
0
-
96.9
101
96.8
a Selectrocide volume corresponds to spray deposited mass shown in Table 4-14
b Total volume is 10 mL (PBS + Triton X-100) plus volume (for mass to volume conversion, 1 g= 1 mL) of corresponding spray deposited
mass shown in Table 4-14)
"-" Not Applicable
4.3 CASCAD Surface Decontamination Foam
CASCAD Surface Decontamination Foam (SDF), one of the four down-selected technologies, was
evaluated for decontamination efficacy against a biological warfare agent and two additional organisms
on three indoor surfaces. Various structural, decorative, and functional surfaces typically found inside an
office building or a mass transit station were used to evaluate the sporicidal decontamination
technology. In the case of this further testing, three of the seven coupon types were employed. The test
surfaces (coupons measuring 1.9 cm x 7.5 cm) are listed below:
• Industrial-grade carpet
• Bare wood (pine lumber)
• Galvanized metal ductwork.
The biological agent used to evaluate the sporicidal decontamination technology was B. anthracis Ames
spores. To provide comparisons with the B. anthracis Ames results, the organisms, B. subtilis (ATCC
19659) and G. stearothermophilus (ATCC 12980) were used. The following sections summarize the
results of these evaluations.
30
-------
4.3.1 Decontamination Efficacy
4.3.1.1 Quantitative Assessment of the Log Reduction of Viable Organisms
Decontamination efficacy was calculated as the mean log reduction in viable organisms achieved
by the decontamination technology. Decontamination efficacy was calculated as defined in
Section 4.1.1.1.
The decontamination efficacy of CASCAD SDF for inactivating extractable, viable spores from
the test materials ranged from 0.58 to 5.52 for B. anthracis Ames spores for all three test
materials (Table 4-26). The decontamination efficacy for B. subtilis and G. stearothermophilus
spores ranged from 0.78 to 4.49 and 0.83 to 4.61, respectively (Tables 4-27 and 4-28). No viable
organisms were detected in any of the blank samples. The decontamination efficacy results for B.
anthracis Ames, B. subtilis, and G. stearothermophilus are summarized in Table 4-29. The
decontamination efficacy varied with respect to both the agent or organism and the test material.
Statistically significant differences in efficacy for B. subtilis and G. stearothermophilus
compared to B. anthracis Ames were observed for two of the three test materials (Table 4-29).
For bare wood, the decontamination efficacy for B. subtilis and G. stearothermophilus spores
were statistically greater than that of B. anthracis Ames. However, a statistically lower
decontamination efficacy for B. subtilis and G. stearothermophilus spore compared to B.
anthracis Ames spores was observed on galvanized metal.
Table 4-26. Inactivation of Bacillus anthracis Ames Spores" (CASCAD SDF)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed CASCAD SDFC
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed CASCAD SDF
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed CASCAD SDF
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.60 x 107
9.60 x 107
0
0
9.60 x 107
9.60 x 107
0
0
9.60 x 107
9.60 x 107
0
0
Total Observed CFU
4.20 ± 0.82 xlO7
3.98 ± 0.96 xlO6
0
0
6.95 ± 1.24 xlO6
1.83 ± 0.13 xlO6
0
0
5.91 ± 0.53 xlO7
2.61 ± 3.01 xlO2
0
0
% Recovery
43. 8 ±8.50
4.15 ±1.00
0
0
7.24 ±1.30
1.91 ±0.14
0
0
61.6 ±5.56
<0.001
0
0
Decontamination
Efficacy
-
1.03 ±0.10
-
-
-
0.58 ±0.03
-
-
-
5.52 ±0.37
-
-
aData are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction),"-" Not Applicable
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
-------
Table 4-27. Inactivation of Bacillus subtilis Spores" (CASCAD SDF)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed CASCAD SDFC
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed CASCAD SDF
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed CASCAD SDF
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.06 xlO8
1.06 xlO8
0
0
1.06 xlO8
1.06 xlO8
1.06 xlO8
1.06 xlO8
0
0
Total Observed CFU
4.27 ± 0.54 xlO7
4.87 ± 0.84 xlO6
0
0
4.97 ± 0.66 xlO6
8.24±1.19xl05
0
0
6.87 ± 0.82 xlO7
2.34 ± 0.84 xlO3
0
0
% Recovery
40.3 ±5. 13
4.60 ±0.79
0
0
4.69 ±0.63
0.78 ±0.11
0
0
64.8 ±7.77
0.01
0
0
Decontamination
Efficacy
-
0.95 ±0.08
-
-
-
0.78 ±0.06
-
-
-
4.49 ±0.16
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction),"-" Not Applicable
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
Table 4-28. Inactivation of Geobacillus stearothermophilus Spores3 (CASCAD SDF)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed CASCAD SDFC
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed CASCAD SDF
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed CASCAD SDF
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.02
1.02
1.02
1.02
1.02
1.02
X
X
0
0
X
X
0
0
X
X
0
0
108
108
108
108
108
108
Total Observed CFU
4.
4.
3.
4.
7.
2.
71
.98
.26
.98
.84
.09
±0.36x
±1.42x
0
0
±0.21x
±1.45x
0
0
±0.38x
±0.84x
0
0
107
106
106
105
107
103
% Recovery
46.2
4.88
3.20
0.49
76.9
±
±
0
0
±
±
0
0
±
3.57
1.40
0.20
0.14
3.73
0.01
0
0
Decontamination
Efficacy
-
0.99 ±0.14
-
-
-
0.83 ±0.14
-
-
-
4.61 ±0.19
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
32
-------
Table 4-29. Summary of Efficacy Values Obtained for CASCAD SDFa
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
1.03
0.58
5.52
B. subtilis
0.95
0.78
4.49
G. stearothermophilus
0.99
0.83
4.61
a Numbers in bold are statistically different (p < 0.05) from B. anthracis Ames
4.3.1.2 Qualitative Assessment of Residual Spores
Based on previous decontamination studies,(3"6) it was anticipated that spores would not be
completely recovered from coupons by the extraction process. Therefore, viable spores might
remain on the test coupons. As in previous decontamination studies, a qualitative assessment was
performed, as detailed in Section 4.1.1.2, to determine whether viable spores remained on the
decontaminated and extracted test coupons.
Results from the liquid culture growth assessment of coupons at one and seven days post-
decontamination are provided in Tables 4-30, 4-31, and 4-32 for B. anthracis Ames, B. subtilis,
and G. stearothermophilus, respectively. In all cases viable spores remained on the coupons.
Streak plates displayed only growth from the inoculated organism. This growth in which only the
inoculated organism was observed on the streak plates reflects the improved procedure for
sterilizing the coupons [i.e., gamma irradiation (40 KiloGray) of coupons] prior to testing.
Table 4-30. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames
Spores following Extraction (CASCAD SDF)
Organism/Test Material gl S2 S3 ffi1S5 S6 m gl S2 S3 ^\5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF +- - + + +ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
33
-------
Table 4-31. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (CASCAD SDF)
Organism/Test Material ^ ^ ^ *g\5 S6 Bl SI S2 S3 "T? S5 S6 Bl
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF - + + + --ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
Table 4-32. Liquid Culture Assessment of Coupons Inoculated with Geobacillus
stearothermophilus Spores following Extraction (CASCAD SDF)
Organism/Test Material ^ ^ S3 D^ * S5 S6 m S1 S2 S3 *g\5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF + + + + + +ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed CASCAD SDF + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
The results of the decontaminated coupons from the qualitative assessment were consistent with the
results from the quantitative assessment and are summarized in Table 4-33. In all cases where growth of
spores was observed in the quantitative method, there was a corresponding observation of growth in the
liquid culture medium after seven days incubation. In all cases, viable spores were shown to be present
in the positive controls using both the quantitative and qualitative methods.
34
-------
Table 4-33. Summary of Results Obtained from the Quantitative and Qualitative Assessments
when Comparing Decontaminated Coupons (CASCAD SDF)
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
A B
+ +
+ +
+ +
B. subtilis
A B
+ +
+ +
+ +
G. stearothermophilus
A B
+ +
+ +
+ +
A = Quantitative Assessment
B = Qualitative Assessment at seven days
"+" = observed CFU or growth; "-" = no observed CFU or no growth
4.3.2 Damage to Coupons
Before and after decontamination of the test coupons, the decontaminated coupons were visually
inspected; and any obvious changes in the color, reflectivity, and apparent roughness of the coupon
surfaces were recorded. No damage (e.g., change in surface texture, color) or visible change was
observed during this evaluation to any of the test coupons.
4.3.3 Other Factors
4.3.3.1 Operator Control
On each day of testing, the sporicidal technology was prepared fresh according to the vendor's
instructions. A NIST-traceable thermometer/hygrometer indicated that the temperature and RH
were maintained within the specified range of 22 to 35°C and <70% RH.
4.3.3.2 Technology Spray Deposition
Gravimetric analysis for total deposition (coupon + run-off) for the sporicidal technology and
daily verification of spray performance were performed. The results of the initial CASCAD SDF
spray deposition are shown in Table 4-14 for glass and bare pine wood. Daily verification of the
amount of spray deposition based on three separate days (N=4 glass coupons per day) resulted in
a mean (± SD) mass of 0.45 ± 0.01 grams, which was not statistically different (p = 0.559) than
the initial spray deposition on glass coupons of 0.44 ± 0.01 grams.
4.3.3.3 Neutralization Methodology
The approach used for testing neutralization efficiency for CASCAD SDF is described in Section
4.2.3.3 and is also described in the test/QA plan.(1) Details of the vendor-recommended
neutralizer and selected concentration are shown in Table 4-15.
4.4 HI-Clean605
Hi-Clean 605 was evaluated for decontamination efficacy as described in Section 4.3. The following
sections summarize the results of these evaluations.
35
-------
4.4.1 Decontamination Efficacy
4.4.1.1 Quantitative Assessment of the Log Reduction of Viable Organisms
Decontamination efficacy was calculated as the mean log reduction in viable organisms achieved
by the decontamination technology. Decontamination efficacy was calculated as defined in
Section 4.1.1.1.
The decontamination efficacy of Hi-Clean 605 for inactivating extractable, viable spores from
the test materials ranged from 0.92 to 4.27 for B. anthracis Ames spores for all three test
materials (Table 4-34). The decontamination efficacy for B. subtilis and G. stearothermophilus
spores ranged from 0.02 to 2.46 and 0.47 to 1.31, respectively (Tables 4-35 and 4-36). No viable
organisms were detected in any of the blank samples. The summary of decontamination efficacy
results for B. anthracis Ames, B. subtilis., and G. stearothermophilus are summarized in Table 4-
37. The decontamination efficacy varied with respect to both the agent or organism and the test
material.
Statistically significant differences in efficacy compared to B. anthracis were observed for two
of the three test materials (Table 4-37). For bare wood and galvanized metal, the
decontamination efficacy for B. subtilis and G. stearothermophilus spores were statistically
lower than that of B. anthracis Ames.
Table 4-34. Inactivation of Bacillus anthracis Ames Spores3 (Hi-Clean 605)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Spray ed Hi-Clean 605C
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Spray edHI-Clean 605
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Spray edHI-Clean 605
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.17xl07
9.17xl07
0
0
9.17xl07
9.17xl07
0
0
9.17xl07
9.17xl07
0
0
Total Observed CFU
5.65 ± 0.70 xlO7
1.40 ± 1.23 xlO6
0
0
5.91 ± 0.92 xlO6
7.21 ± 1.67 xlO5
0
0
6.81 ± 0.93 xlO7
3.73 ± 0.96 xlO3
0
0
% Recovery
61.7 ±7.59
1.53 ±1.34
0
0
6.45 ±1.01
0.79 ±0.18
0
0
74.3 ± 10.2
0.0041 ±0.001
0
0
Decontamination
Efficacy
-
1.70 ±0.28
-
-
-
0.92 ±0.11
-
-
-
4.27 ±0.11
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
36
-------
Table 4-35. Inactivation of Bacillus subtilis Spores3 (Hi-Clean 605)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Spray ed Hi-Clean 605C
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Spray edHI-Clean 605
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Spray edHI-Clean 605
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.03 x 108
1.03 x 108
0
0
1.03 x 108
1.03 x 108
0
0
1.03 x 108
1.03 x 108
0
0
Total Observed CFU
2.17±1.18xl07
1.05 ± 0.93 xlO6
0
0
3.76 ± 1.33 xlO6
3.59 ± 0.55 xlO6
0
0
7.13 ± 1.04 xlO7
2.87 ± 1.47 xlO5
0
0
% Recovery
21.1 ±11.5
1.02 ±0.90
0
0
3.65 ±1.29
3.48 ±0.53
0
0
69.2 ±10.1
0.28 ±0.14
0
0
Decontamination
Efficacy
1.42 ±0.30
0.02 ± 0.07
-
2.46 ±0.28
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
Table 4-36. Inactivation of Geobacillus stearothermophilus Spores" (Hi-Clean 605)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Spray ed Hi-Clean 605C
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed Hi-Clean 605
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed Hi-Clean 605
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.09 xlO8
1.09 xlO8
0
0
1.09 xlO8
1.09 xlO8
0
0
1.09 xlO8
1.09 xlO8
0
0
Total Observed CFU
1.76 ± 1.53 xlO7
1.14±1.06xl06
0
0
1.54±1.14xl07
6.17 ± 2.90 xlO6
0
0
9.04 ± 1.08 xlO7
6.95 ± 3.42 xlO6
0
0
% Recovery
16.2 ±14.0
1.04 ±0.97
0
0
14.1 ±10.5
5.66 ±2.66
0
0
82.9 ±9.87
6.38 ±3. 14
0
0
Decontamination
Efficacy
-
1.31 ±0.33
-
-
-
0.47 ±0.32
-
-
-
1.15 ±0.20
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
37
-------
Table 4-37. Summary of Efficacy Values Obtained for Hi-Clean 605a
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
1.70
0.92
4.27
B. subtilis
1.42
0.02
2.46
G. stearothermophilus
1.31
0.47
1.15
a Numbers in bold are statistically different (p < 0.05) from B. anthracis Ames
4.4.1.2 Qualitative Assessment of Residual Spores
Based on previous decontamination studies,(3"6) it was anticipated that spores would not be
completely recovered from coupons by the extraction process. Therefore, viable spores might
remain on the test coupons. As in previous decontamination studies, a qualitative assessment was
performed, as detailed in Section 4.1.1.2, to determine whether viable spores remained on the
decontaminated and extracted test coupons.
Results from the liquid culture growth assessment of coupons as one and seven days post-
decontamination are provided in Tables 4-38, 4-39, and 4-40 for B. anthracis Ames, B. subtilis,
and G. stearothermophilus, respectively. In all cases viable spores remained on the coupons.
Streak plates displayed only growth from the inoculated organism. This growth in which only the
inoculated organism was observed on the streak plates reflects the improved procedures for
sterilizing the coupons prior to testing.
Table 4-38. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames
Spores following Extraction (Hi-Clean 605)
Organism/Test Material gl S2 S3 ffi1S5 S6 m gl S2 S3 ^\5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + + ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
38
-------
Table 4-39. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (Hi-Clean 605)
Organism/Test Material ^ ^ ^ *g\5 S6 Bl SI S2 S3 "T? S5 S6 Bl
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
Table 4-40. Liquid Culture Assessment of Coupons Inoculated with Geobacillus
stearothermophilus Spores following Extraction (Hi-Clean 605)
Organism/Test Material ^ ^ S3 D^ * S5 S6 m S1 S2 S3 *g\5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed Hi-Clean 605 + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
39
-------
The results of the decontaminated coupons from the qualitative assessment were consistent with
the results from the quantitative assessment and are summarized in Table 4-41. In all cases where
growth of spores was observed in the quantitative method, there was a corresponding observation
of growth in the liquid culture medium after seven days incubation. In all cases, viable spores
were shown to be present in the positive controls using both the quantitative and qualitative
methods.
Table 4-41. Summary of Results Obtained from the Quantitative and Qualitative Assessments
when Comparing Decontaminated Coupons (Hi-Clean 605)
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
A B
+ +
+ +
+ +
B. subtilis
A B
+ +
+ +
+ +
G. stearothermophilus
A B
+ +
+ +
+ +
A = Quantitative Assessment
B = Qualitative Assessment at seven days
"+" = observed CPU or growth; "-" = no observed CPU or no growth
4.4.2 Damage to Coupons
Before and after decontamination of the test coupons, the decontaminated coupons were visually
inspected; and any obvious changes in the color, reflectivity, and apparent roughness of the coupon
surfaces were recorded. No damage (e.g., change in surface texture, color) or visible change was
observed during this evaluation to any of the test coupons.
4.4.3 Other Factors
4.4.3.1 Operator Control
On each day of testing, the sporicidal technology was prepared fresh according to the vendor's
instructions.
A NIST-traceable thermometer/hygrometer indicated that the temperature and RH were
maintained within the specified range of 22 to 35°C and <70% RH.
4.4.3.2 Technology Spray Deposition
Gravimetric analysis for total deposition (coupon + run-off) for the sporicidal technology and
daily verification of deposition were performed and are detailed in Section 4.2.3.2. The results of
the initial Hi-Clean 605 spray deposition are shown in Table 4-14 for glass and bare pine wood.
Daily verification of the amount of spray deposition based on three separate days (N=4 glass
coupons per day) resulted in a mean (± SD) mass of 0.47 ± 0.02 grams, which was not
statistically different (p = 0.809) than the initial spray deposition on glass coupons of 0.48 ± 0.04
grams.
40
-------
4.4.3.3 Neutralization Methodology
The approach used for testing neutralization efficiency for Hi-Clean 605 is described in Section
4.2.3.3 and is also described in the test/QA plan.(1) Details of the vendor-recommended
neutralizer and selected concentration are shown in Table 4-15.
4.5 KlearWater
KlearWater was evaluated for decontamination efficacy as described in Section 4.3. The following
sections summarize the results of these evaluations.
4.5.1 Decontamination Efficacy
4.5.1.1 Quantitative Assessment of the Log Reduction of Viable Organisms
Decontamination efficacy was calculated as the log reduction in viable organisms achieved by
the decontamination technology. Efficacy was calculated as defined in Section 4.1.1.1.
The decontamination efficacy of KlearWater for inactivating extractable, viable spores from the
test materials ranged from 0.05 to 0.92 for B. anthracis Ames spores for all three test materials
(Table 4-42). The decontamination efficacy for B. subtilis and G. stearothermophilus spores
ranged from 0.12 to 0.30 and 0.72 to 0.98, respectively (Tables 4-43 and 4-44). No viable
organisms were detected in any of the blank samples. The summary of decontamination efficacy
results for B. anthracis Ames, B. subtilis., and G. stearothermophilus are summarized in Table 4-
45. The decontamination efficacy varied with respect to both the agent or organism and the test
material.
Statistically significant differences in efficacy compared to B. anthracis Ames were observed for
all three test materials (Table 4-45). For industrial carpet, the decontamination efficacy for B.
subtilis and G. stearothermophilus spores was statistically greater than that of B. anthracis
Ames. For bare wood, the decontamination efficacy for G. stearothermophilus spores was
statistically greater than that of B. anthracis Ames. However, statistically lower decontamination
efficacy for B. subtilis spores was observed on galvanized metal when compared to B. anthracis
Ames.
41
-------
Table 4-42. Inactivation of Bacillus anthracis Ames Spores3 (KlearWater)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed KlearWater'
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed KlearWater
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed KlearWater
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.53 x 107
9.53 x 107
0
0
9.53 x 107
9.53 x 107
0
0
9.53 x 107
9.53 x 107
0
0
Total Observed CFU
5.29 ± 0.82 xlO7
4.78 ± 1.00 xlO7
0
0
7.76 ± 1.01 xlO6
4.61 ± 1.43 x 106
0
0
5.02 ± 1.50 xlO7
6.08 ± 1.07 xlO6
0
0
% Recovery
55.5 ±8.58
50.1 ±10.5
0
0
8.14 ±1.06
4.84 ±1.50
0
0
52.7 ±15.7
6.38 ±1.12
0
0
Decontamination
Efficacy
0.05 ±0.09
0.24 ±0.13
-
0.92 ±0.07
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
Table 4-43. Inactivation of Bacillus subtilis Spores" (KlearWater)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed KlearWater'
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed KlearWater
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed KlearWater
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.08 xlO8
1.08 xlO8
0
0
1.08 xlO8
1.08 xlO8
0
0
1.08 xlO8
1.08 xlO8
0
0
Total Observed CFU
2.67 ± 0.79 xlO7
1.35 ± 0.16 xlO7
0
0
3.62 ± 0.85 xlO6
2.82 ± 0.58 xlO6
0
0
4.60 ± 0.60 x 107
3.25 ± 0.22 xlO7
0
0
% Recovery
24.8 ±7.31
12.5 ±1.48
0
0
3.35 ±0.79
2.61 ±0.53
0
0
42.6 ±5.56
30.1 ±2.06
0
0
Decontamination
Efficacy
-
0.30 ±0.05
-
-
-
0.12 ±0.10
-
-
-
0.15 ±0.03
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
42
-------
Table 4-44. Inactivation of Geobacillus stearothermophilus Spores3 (KlearWater)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed KlearWater'
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed KlearWater
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed KlearWater
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.05 x 108
1.05 x 108
0
0
1.05 x 108
1.05 x 108
0
0
1.05 x 108
1.05 x 108
0
0
Total Observed CFU
2.47 ± 1.70 xlO7
4.89 ± 1.45 xlO6
0
0
3.46 ± 0.45 xlO6
3.69 ± 0.69 xlO5
0
0
7.84 ± 0.48 xlO7
8.25 ± 0.66 xlO6
0
0
% Recovery
23.5 ±16.2
4.66 ±1.38
0
0
3.29 ±0.43
0.35 ±0.07
0
0
74.7 ±4.61
7.85 ±0.63
0
0
Decontamination
Efficacy
-
0.72 ±0.13
-
-
-
0.98 ±0.08
-
-
-
0.98 ±0.04
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
Table 4-45. Summary of Efficacy Values Obtained for KlearWater"
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
0.05
0.24
0.92
B. subtilis
0.30
0.12
0.15
G. stearothermophilus
0.72
0.98
0.98
a Numbers in bold are statistically different (p < 0.05) from B. anthracis Ames
4.5.1.2 Qualitative Assessment of Residual Spores
Based on previous decontamination studies,(3"6) it was anticipated that spores would not be
completely recovered from coupons by the extraction process. Therefore, viable spores might
remain on the test coupons. As in previous decontamination studies, a qualitative assessment was
performed, as detailed in Section 4.1.1.2, to determine whether viable spores remained on the
decontaminated and extracted test coupons
Results from the liquid culture growth assessment of coupons at one and seven days post-
decontamination are provided in Tables 4-46, 4-47, and 4-48 for B. anthracis Ames, B. subtilis,
and G. stearothermophilus, respectively. Streak plates displayed only growth from the inoculated
organism. This growth in which only the inoculated organism was observed on the streak plates
reflects the improved procedures for sterilizing the coupons prior to testing.
43
-------
Table 4-46. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames
Spores following Extraction (KlearWater)
Organism/Test Material
Industrial-Grade Carpet
Sprayed H2O
Sprayed KlearWater
Bare Wood
Sprayed H2O
Sprayed KlearWater
Galvanized Ductwork
Sprayed H2O
Sprayed KlearWater
SI S2 S3
+ + +
+ - +
+ + +
+ + +
+ + +
+ + +
Day
+
+
+
+
+
+
1
S5
+
+
+
+
+
+
S6
+
+
+
+
+
+
Bl
-
ND
-
ND
-
ND
SI S2 S3
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
^4
+
+
+
+
+
+
•7
S5
+
+
+
+
+
+
S6
+
+
+
+
+
+
Bl
-
ND
-
ND
-
ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
Table 4-47. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (KlearWater)
Organism/Test Material ^ ^ S3 D^ * S5 S6 m S1 S2 S3 *g\5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed KlearWater + + + + + +ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed KlearWater + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed KlearWater + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
44
-------
Table 4-48. Liquid Culture Assessment of Coupons Inoculated with Geobacillus
stearothermophilus Spores following Extraction (KlearWater)
Organism/Test Material ^ ^ S3 1 S5 S6 m ^ S2 S3 ^ ? S5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed KlearWater + + + + + +ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed KlearWater + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed KlearWater _ + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
The results of the decontaminated coupons from the qualitative assessment were consistent with the
results from the quantitative assessment and are summarized in Table 4-49. In all cases where growth of
spores was observed in the quantitative method, there was a corresponding observation of growth in the
liquid culture medium after seven days incubation. In all cases, viable spores were shown to be present
in the positive controls using both the quantitative and qualitative methods.
Table 4-49. Summary of Results Obtained from the Quantitative and Qualitative Assessments
when Comparing Decontaminated Coupons (KlearWater)
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
A B
+ +
+ +
+ +
B. subtilis
A B
+ +
+ +
+ +
G. stearothermophilus
A B
+ +
+ +
+ +
A = Quantitative Assessment
B = Qualitative Assessment at seven days
"+" = observed CFU or growth; "-" = no observed CFU or no growth
4.5.2 Damage to Coupons
Before and after decontamination of the test coupons, the decontaminated coupons were visually
inspected; and any obvious changes in the color, reflectivity, and apparent roughness of the coupon
surfaces were recorded. No damage (e.g., change in surface texture, color) or visible change was
observed during this evaluation to any of the test coupons.
45
-------
4.5.3 Other Factors
4.5.3.1 Operator Control
The KlearWater technology did not require any preparation and was used as received.
A NIST-traceable thermometer/hygrometer indicated that the temperature and RH were
maintained within the specified range of 22 to 35°C and <70% RH.
4.5.3.2 Technology Spray Deposition
Gravimetric analysis for total deposition (coupon + run-off) for the sporicidal technology and
daily verification of deposition were performed and are detailed in Section 4.2.3.2. The results of
the initial KlearWater spray deposition are shown in Table 4-14 for glass and bare pine wood.
Daily verification of the amount of spray deposition based on three separate days (N=4 glass
coupons per day) resulted in a mean (± SD) mass of 0.46 ± 0.01 grams, which was not
statistically different (p = 0.526) than the initial spray deposition on glass coupons of 0.45 ± 0.04
grams.
4.5.3.3 Neutralization Methodology
The approach used for testing neutralization efficiency for KlearWater is described in Section
4.2.3.3 and is also described in the test/QA plan.(1) Details of the vendor-recommended
neutralizer and selected concentration are shown in Table 4-15.
4.6 Peridox
Peridox was evaluated for decontamination efficacy as described in Section 4.3. The following sections
summarize the results of these evaluations.
4.6.1 Decontamination Efficacy
4.6.1.1 Quantitative Assessment of the Log Reduction of Viable Organisms
Decontamination efficacy was calculated as the mean log reduction in viable organisms achieved
by the decontamination technology. Decontamination efficacy was calculated as defined in
Section 4.1.1.1.
The decontamination efficacy of Peridox for inactivating extractable, viable spores from the test
materials ranged from 0.87 to 1.05 for B. anthracis Ames spores for all three test materials
(Table 4-50). The decontamination efficacy for B. subtilis and G. stearothermophilus spores
ranged from 0.72 to 2.08 and 1.93 to 4.38, respectively (Tables 4-51 and 4-52). No viable
organisms were detected in any of the blank samples. The summary of decontamination efficacy
results for B. anthracis Ames, B. subtilis., and G. stearothermophilus are summarized in Table 4-
53. The decontamination efficacy varied with respect to both the agent or organism and the test
material.
46
-------
Statistically significant differences in efficacy compared to B. anthracis Ames were observed for
all three test materials (Table 4-53). For industrial carpet and galvanized metal, the
decontamination efficacy for B. subtilis spores was statistically greater than that of B. anthracis
Ames. Also, the decontamination efficacy was statistically greater for G. stearothermophilus
spores when compared to B. anthracis Ames spores for all three test materials - industrial carpet,
bare wood, and galvanized metal.
Table 4-50. Inactivation of Bacillus anthracis Ames Spores3 (Peridox)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed Peridox0
Laboratory Blankd
Procedural Blank"
Bare Wood
Sprayed H2O
Sprayed Peridox
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed Peridox
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9
9
9
9
9
9
.83 x
.83 x
0
0
.83 x
.83 x
0
0
.83 x
.83 x
0
0
107
107
107
107
107
107
Total Observed CFU
4.
6.
5.
5.
6.
6.
45 ± 0.70 x
03 ± 0.66 x
0
0
90 ± 1.06 x
46 ± 1.82 x
0
0
18 ± 0.67 x
07 ± 1.42 x
0
0
107
106
106
105
107
106
% Recovery
45.2
6.13
6.00
0.56
62.9
6.17
±
±
0
0
±
±
0
0
±
±
0
0
7.
0.
1.
0.
6.
1.
15
67
08
18
77
45
Decontamination
Efficacy
-
0.87 ±0.05
-
-
-
1.05 ±0.15
-
-
-
1.02 ±0.10
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
47
-------
Table 4-51. Inactivation of Bacillus subtilis Spores3 (Peridox)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed Peridox0
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed Peridox
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed Peridox
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.05
1.05
1.05
1.05
1.05
1.05
x
x
0
0
x
x
0
0
x
x
0
0
108
108
108
108
108
108
Total Observed CFU %
4.22
6.19
3.95
7.97
5.06
6.48
±0.33x
±2.45x
0
0
± 0.74 x
±2.97x
0
0
± 0.62 x
±3.98x
0
0
107
105
106
105
107
105
Recovery
40.2
0.
3.
0.
.59
.76
.76
48.2
0.
.62
±3
±0
0
0
±0
±0
0
0
±5
±0
0
0
.17
.23
.70
.28
.91
.38
Decontamination
Efficacy
-
1.86 ±0.15
-
-
-
0.72 ±0.17
-
-
-
2.08 ±0.58
-
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
Table 4-52. Inactivation of Geobacillus stearothermophilus Spores3 (Peridox)
Test Material
Industrial-Grade Carpet
Sprayed H2Ob
Sprayed Peridox0
Laboratory Blankd
Procedural Blank6
Bare Wood
Sprayed H2O
Sprayed Peridox
Laboratory Blank
Procedural Blank
Galvanized Ductwork
Sprayed H2O
Sprayed Peridox
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.03 x 108
1.03 x 108
0
0
1.03 x 108
1.03 x 108
0
0
1.03 x 108
1.03 x 108
0
o
Total Observed CFU
5.88 ± 1.09 xlO7
3.30±2.03xl03
0
0
3.46 ± 0.51 xlO6
9.03 ± 7.86 xlO2
0
0
7.62 ± 0.32 xlO7
9.71 ± 4.23 xlO5
0
o
% Recovery
57.1 ±10.6
<0.01
0
0
3. 36 ±0.50
<0.01
0
0
73. 9 ±3.09
0.94 ±0.41
0
o
Decontamination
Efficacy
-
4.38 ±0.45
-
-
-
3.72 ±0.37
-
-
-
1.93 ±0.20
-
a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log
reduction)
b Inoculated, not decontaminated coupon
0 Inoculated, decontaminated coupon
d Laboratory Blank = not inoculated, not decontaminated coupon
e Procedural Blank = not inoculated, decontaminated (H2O-sprayed) coupon
"-" Not Applicable
48
-------
Table 4-53. Summary of Efficacy Values Obtained for Peridoxa
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
0.87
1.05
1.02
B. subtilis
1.86
0.72
2.08
G. stearothermophilus
4.38
3.72
1.93
a Numbers in bold are statistically different (p < 0.05) from B. anthracis Ames
4.6.1.2 Qualitative Assessment of Residual Spores
Based on previous decontamination studies, (3"6) it was anticipated that spores would not be
completely recovered from coupons by the extraction process. Therefore, viable spores might
remain on the test coupons. As in previous decontamination studies, a qualitative assessment was
performed, as detailed in Section 4.1.1.2, to determine whether viable spores remained on the
decontaminated and extracted test coupons.
Results from the liquid culture growth assessment of coupons at one and seven days post-
decontamination are provided in Tables 4-54, 4-55, and 4-56 for B. anthracis Ames, B. subtilis,
and G. stearothermophilus, respectively. In all cases viable G. stearothermophilus spores
remained on the coupons after seven days. Streak plates displayed only growth from the
inoculated organism. This growth in which only the inoculated organism was observed on the
streak plates reflects the improved procedures for sterilizing the coupons prior to testing.
Table 4-54. Liquid Culture Assessment of Coupons Inoculated with Bacillus anthracis Ames
Spores following Extraction (Peridox)
Organism/Test Material gl S2 S3 ffi1S5 S6 m gl S2 S3 ^\5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox . + + + .+ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
49
-------
Table 4-55. Liquid Culture Assessment of Coupons Inoculated with Bacillus subtilis Spores
following Extraction (Peridox)
Organism/Test Material "g1 Da/7
5>1 5>z 5>J 5>4 5>J 5>o til 5>1 5>z 5>J 5>4 5>J 5>o JD!
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox - - + - + +ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
Table 4-56. Liquid Culture Assessment of Coupons Inoculated with Geobacillus
stearothermophilus Spores following Extraction (Peridox)
Organism/Test Material ^ ^ S3 D^ * S5 S6 m S1 S2 S3 ^? S5 S6 m
Industrial-Grade Carpet
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox - - + - - -ND+ + + + + +ND
Bare Wood
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox + + + + + +ND+ + + + + +ND
Galvanized Ductwork
Sprayed H2O + + + + + + - + + + + + +
Sprayed Peridox + + + + + +ND+ + + + + +ND
SI = Sample 1
S2 = Sample 2
S3 = Sample 3
S4 = Sample 4
S5 = Sample 5
S6 = Sample 6
Bl = Blank (not inoculated with spores)
ND = Not determined
"+" = growth; "-" = no growth
The results of the decontaminated coupons from the qualitative assessment were consistent with
the results from the quantitative assessment and are summarized in Table 4-57. In all cases where
growth of spores was observed in the quantitative method, there was a corresponding observation
of growth in the liquid culture medium after seven days incubation. In all cases, viable spores
were shown to be present in the positive controls using both the quantitative and qualitative
methods.
50
-------
Table 4-57. Summary of Results Obtained from the Quantitative and Qualitative Assessments
when Comparing Decontaminated Coupons (Peridox)
Material
Industrial-Grade Carpet
Bare Wood
Galvanized Metal Ductwork
B. anthracis Ames
A B
+ +
+ +
+ +
B. subtilis
A B
+ +
+ +
+ +
G. stearothermophilus
A B
+ +
+ +
+ +
A = Quantitative Assessment
B = Qualitative Assessment at seven days
"+" = observed CPU or growth; "-" = no observed CPU or no growth
4.6.2 Damage to Coupons
Before and after decontamination of the test coupons, the decontaminated coupons were visually
inspected; and any obvious changes in the color, reflectivity, and apparent roughness of the coupon
surfaces were recorded. No damage (e.g., change in surface texture, color) or visible change was
observed during this evaluation to any of the test coupons.
4.6.3 Other Factors
4.6.3.1 Operator Control
On each day of testing, the sporicidal technology was prepared fresh according to the vendor's
instructions. A NIST-traceable thermometer/hygrometer indicated that the temperature and RH
were maintained within the specified range of 22 to 35°C and <70% RH.
4.6.3.2 Technology Spray Deposition
Gravimetric analysis for total deposition (coupon + run-off) for the sporicidal technology and
daily verification of spray performance were performed and are detailed in Section 4.2.3.2. The
results of the initial Peridox spray deposition are shown in Table 4-14 for glass and bare pine
wood. Daily verification of the amount of spray deposition based on three separate days (N=4
glass coupons per day) resulted in a mean (± SD) mass of 0.46 ± 0.01 grams, which was not
statistically different (p = 0.073) than the initial spray deposition on glass coupons of 0.43 ± 0.02
grams.
4.6.3.3 Neutralization Methodology
The approach used for testing neutralization efficiency for Peridox is described in Section 4.2.3.3
and is also described in the test/QA plan.(1) Details of the vendor-recommended neutralizer and
selected concentration are shown in Table 4-15.
51
-------
5.0 Performance Summary
5.1 pH-Amended Bleach Results
• The results of the decontamination tests of the pH-amended bleach varied according to the
contaminating biological agent or organism -B. anthracis Ames, B. anthracis Sterne, B. subtilis, or
G. stearothermophilus - and the porosity of the test coupons. Of the seven test coupons, the four
relatively non-porous test materials (glass, galvanized metal ductwork, decorative laminate, and
painted wallboard paper) yielded higher log reductions than the test materials considered relatively
porous (industrial carpet, bare wood, and painted concrete). In fact, no viable spores could be
extracted from glass, galvanized metal ductwork, and decorative laminate following
decontamination by pH-amended bleach.
• The decontamination efficacy of amended bleach was high (i.e., 7.2-7.9 log reduction) for hard,
nonporous surfaces (glass, decorative laminate and galvanized metal ductwork) and low (0.28-2.0
log reduction) for the porous surfaces (industrial grade carpet, bare wood, and painted concrete) for
B. anthracis Ames. For B. anthracis Sterne and B. subtilis, the results were similar; however, for G.
stearothermophilus, the log reductions were generally lower for hard, nonporous surfaces (0.75-
5.90), as well as for porous surfaces (0.02-1.40).
• Statistical analyses comparing the mean spore log reductions of B. anthracis with each of the
corresponding values for the other organisms revealed statistically significant differences for five of
the seven test materials. For industrial carpet, painted wallboard paper, and painted concrete, the log
reduction in B. anthracis Sterne spores was statistically higher than that of B. anthracis Ames. When
compared to B. anthracis Ames, statistically lower mean log reductions in G. stearothermophilus
spores were observed on decorative laminate, galvanized metal, and painted wallboard paper. A
statistically lower log reduction in B. subtilis spores compared to B. anthracis Ames spores was
observed on painted wallboard paper. However, a statistically higher log reduction in B. subtilis
spores compared to B. anthracis Ames spores was observed on painted concrete.
• To assess whether or not viable spores remained in or on the coupons following decontamination
and subsequent extraction (to quantitate extractable, viable spores), both extracted control and
extracted decontaminated coupons were placed in tryptic soy broth and incubated for seven days.
The contents of the tubes were examined at one and seven days for cloudiness as an indicator of
growth. Where no growth was observed using the quantitative extraction method, the qualitative
method showed corresponding no growth of residual spores on the extracted coupon.
• In the cases where the liquid cultures exhibited positive growth, a sample of the culture was further
analyzed by plating on tryptic soy agar and incubating the plates overnight. For B. anthracis Ames,
B. anthracis Sterne, and B. subtilis, the non-sterilized bare wood and painted wallboard coupons
yielded mixed microorganism growth in greater than 70% of the streak plates. The percentage of
streak plates displaying only growth from the inoculated organism was 40%, 58%, 56%, and 100%
52
-------
for B. anthracis Ames, B. anthracis Sterne, B. subtilis, and G. stearothermophilus, respectively for
all coupon types.
• The growth on the non-inoculated decontaminated blanks may have been due to ineffective
disinfection (the 70% isopropanol wipe did not sterilize the internal portions of the coupons) prior to
inoculating the coupons.
• No visual damage was observed for any of the test coupons subjected to the pH-amended bleach.
5.2 Ten Technologies Evaluated by Screening Test Results
• The results of the decontamination screening tests of the ten sporicidal technologies varied according
to the technology employed. All tests were conducted on a glass test coupon contaminated with B.
anthracis Ames spores.
• Decontamination of the glass test coupons spiked with B. anthracis Ames spores using the ten
sporicidal technologies resulted in mean log reduction values ranging from 0.37 to >7.8.
• After reviewing the draft results, three of the four chlorine dioxide based technology vendors
expressed concern that the spray system used in testing may not have been operated optimally for
their product. An air pressure of 40 psi was used to atomize the liquid, producing a fine mist (10 -
50 micron diameter droplet size). One vendor suggested that these relatively small size droplets
could lead to increased mass transfer of chlorine dioxide from the liquid to gas phase, thus
potentially decreasing the chlorine dioxide concentration in the liquid and rendering it less effective.
The other two vendors made similar comments. We agree that this phenomenon may be a
possibility, although it has not been verified. We do note, however, that these three technologies
achieved complete inactivation of approximately 108 spores in the neutralization tests conducted
(solution of spore inoculum + liquid decontamination technology); see Tables 4-18, 4-20, and 4-25.
• The four technologies that exhibited the highest log reductions (see Table 4-13) were selected by the
TOPO for further evaluation. The four technologies selected were CASCAD SDF, Hi-Clean 605,
KlearWater, and Peridox. Results from the further evaluation are outlined in Sections 5.3 through
5.6. These four technologies were also selected because they represent four different types of
sporicidal formulations that are available.
• No visual damage was observed for any of the test coupons subjected to any of the ten sporicidal
technologies.
5.3 CASCAD SDF Results
• The results of the decontamination tests of CASCAD SDF varied according to the contaminating
biological agent or organism - B. anthracis Ames, B. subtilis, or G. stearothermophilus - and the
porosity of the test coupons. In general, the relatively non-porous galvanized metal ductwork yielded
a much higher log reduction than the two test materials classified as relatively porous (industrial
carpet and bare wood).
53
-------
• Decontamination of the three types of test coupons spiked with B. anthracis Ames spores using
CASCAD SDF resulted in mean log reduction values ranging from 0.58 to 5.52. Mean log reduction
values on all three test materials for B. subtilis and G. stearothermophilus spores ranged from 0.78 to
4.49 and 0.83 to 4.61, respectively.
• Statistical analyses comparing the mean spore log reductions of B. anthracis with each of the
corresponding values for the other organisms revealed statistically significant differences for two of
the three test materials. For bare wood, the log reduction in B. subtilis and G. stearothermophilus
spores was statistically higher than that of B. anthracis Ames. However, for galvanized metal, the
log reduction in B. subtilis and G. stearothermophilus spores was statistically lower than the log
reduction in B. anthracis spores.
• To assess whether or not viable spores remained in or on the coupons following decontamination
and subsequent extraction (to quantitate extractable, viable spores), both extracted control and
extracted decontaminated coupons were placed in tryptic soy broth and incubated for seven days.
The contents of the tubes were examined at one and seven days for cloudiness as an indicator of
growth. In all cases qualitative analysis showed that viable residual organisms were present on the
decontaminated coupons (see Tables 4-26, 4-27, and 4-28).
• In the cases where the liquid cultures exhibited positive growth, a sample of the culture was further
analyzed by plating on tryptic soy agar and incubating the plates overnight. The percentage of streak
plates displaying only growth from the inoculated organism was 100% for B. anthracis Ames, B.
subtilis., and G. stearothermophilus. This growth in which only the inoculated organism was
observed on the tryptic soy agar plates reflects the improved procedures for sterilizing (i.e., gamma
irradiation) the coupons prior to testing.
• No visual damage was observed for any of the test coupons subjected to CASCAD SDF.
5.4 Hi-Clean 605 Results
• The results of the decontamination tests of Hi-Clean 605 varied according to the contaminating
biological agent or organism - B. anthracis Ames, B. subtilis, or G. stearothermophilus - and the
porosity of the test coupons. Of the three test coupons, the relatively non-porous galvanized metal
ductwork generally yielded a much higher log reduction than the two test materials considered
relatively porous (industrial carpet and bare wood).
• Decontamination of the three types of test coupons spiked with B. anthracis Ames spores using Ffl-
Clean 605 resulted in mean log reduction values ranging from 0.92 to 4.27. Mean log reduction
values on all three test materials for B. subtilis and G. stearothermophilus spores ranged from 0.02 to
2.46 and 0.47 to 1.31, respectively.
• Statistical analyses comparing the mean spore log reductions of B. anthracis Ames with each of the
corresponding values for the other organisms revealed statistically significant differences for two of
the three test materials. For bare wood and galvanized metal ductwork, the log reduction in B.
subtilis and G. stearothermophilus spores was statistically lower than that of B. anthracis Ames.
54
-------
• To assess whether or not viable spores remained in or on the coupons following decontamination
and subsequent extraction (to quantitate extractable, viable spores), both extracted control and
extracted decontaminated coupons were placed in tryptic soy broth and incubated for seven days.
The contents of the tubes were examined at one and seven days for cloudiness as an indicator of
growth. In all cases qualitative analysis showed that viable residual organisms were present on the
decontaminated coupons (see Tables 4-34, 4-35, and 4-36).
• In the cases where the liquid cultures exhibited positive growth, a sample of the culture was further
analyzed by plating on tryptic soy agar and incubating the plates overnight. The percentage of streak
plates displaying only growth from the inoculated organism was 100% for B. anthracis Ames, B.
subtilis, and G. stearothermophilus. This growth in which only the inoculated organism was
observed on the tryptic soy agar plates reflects the improved procedures for sterilizing the coupons
(i.e., gamma irradiation) prior to testing.
• No visual damage was observed for any of the test coupons subjected to Hi-Clean 605.
5.5 KlearWater Results
• The results of the decontamination tests of KlearWater varied according to the contaminating
biological agent or organism - B. anthracis Ames, B. subtilis., or G. stear other mophilus - and the
porosity of the test coupons. In general, the relatively non-porous galvanized metal ductwork yielded
higher log reduction than the two test materials considered relatively porous (industrial carpet and
bare wood).
• Decontamination of the three types of test coupons spiked with B. anthracis Ames spores using
KlearWater resulted in mean log reduction values ranging from 0.05 to 0.92. Mean log reduction
values on all three test materials for B. subtilis and G. stearothermophilus spores ranged from 0.12 to
0.30 and 0.72 to 0.98, respectively.
• Statistical analyses comparing the mean spore log reductions of B. anthracis with each of the
corresponding values for the other organisms revealed statistically significant differences for all
three of the test materials. For industrial carpet, the log reduction in B. subtilis and G.
stearothermophilus spores was statistically higher than that of B. anthracis Ames. For bare wood,
the log reduction in G. stearothermophilus spores was also statistically higher than the log reduction
in B. anthracis spores. However, for galvanized metal, the log reduction in B. subtilis spores was
statistically lower than the log reduction in B. anthracis spores.
• To assess whether or not viable spores remained in or on the coupons following decontamination
and subsequent extraction (to quantitate extractable, viable spores), both extracted control and
extracted decontaminated coupons were placed in tryptic soy broth and incubated for seven days.
The contents of the tubes were examined at one and seven days for cloudiness as an indicator of
growth. In all cases qualitative analysis showed that viable residual organisms were present on the
decontaminated coupons (see Tables 4-42, 4-43, and 4-44).
• In the cases where the liquid cultures exhibited positive growth, a sample of the culture was further
analyzed by plating on tryptic soy agar and incubating the plates overnight. The percentage of streak
plates displaying growth from only the inoculated organism was 100% for B. anthracis Ames, B.
55
-------
subtilis, and G. stearothermophilus. This growth in which only the inoculated organism was
observed on the tryptic soy agar plates reflects the improved procedures for sterilizing (i.e., gamma
irradiation) the coupons prior to testing.
• No visual damage was observed for any of the test coupons subjected to KlearWater.
5.6 Peridox Results
• The results of the decontamination tests of Peridox varied according to the contaminating biological
agent or organism - B. anthracis Ames, B. subtilis., or G. stearothermophilus - and the porosity of
the test coupons. In the case of B. anthracis Ames, the decontamination efficacy values for the
relatively non-porous galvanized metal ductwork and wood were essentially the same; whereas,
industrial-grade carpet yielded values slightly lower than the values obtained for wood and metal.
• Decontamination of the three types of test coupons spiked with B. anthracis Ames spores using
Peridox resulted in mean log reduction values ranging from 0.87 to 1.05. Mean log reduction values
on all three test materials for B. subtilis and G. stearothermophilus spores ranged from 0.72 to 2.08
and 1.93 to 4.38, respectively.
• Statistical analyses comparing the mean spore log reductions of B. anthracis with each of the
corresponding values for the other organisms revealed statistically significant differences for all
three of the test materials. For industrial carpet and galvanized metal, the log reduction in B. subtilis
and G. stearothermophilus spores was statistically higher than that of B. anthracis Ames. For bare
wood, the log reduction in G. stearothermophilus spores was also statistically higher than the log
reduction in B. anthracis spores.
• To assess whether or not viable spores remained in or on the coupons following decontamination
and subsequent extraction (to quantitate extractable, viable spores), both extracted control and
extracted decontaminated coupons were placed in tryptic soy broth and incubated for seven days.
The contents of the tubes were examined at one and seven days for cloudiness as an indicator of
growth. In all cases qualitative analysis showed that viable residual organisms were present on the
decontaminated coupons (see Tables 4-50, 4-51, and 4-52).
• In the cases where the liquid cultures exhibited positive growth, a sample of the culture was further
analyzed by plating on tryptic soy agar and incubating the plates overnight. The percentage of streak
plates displaying only growth from the inoculated organism was 100% for B. anthracis Ames, B.
subtilis, and G. stearothermophilus. This growth in which only the inoculated organism was
observed on the tryptic soy agar plates reflects the improved procedures for sterilizing (i.e., gamma
irradiation) the coupons prior to testing.
• No visual damage was observed for any of the test coupons subjected to Peridox.
5.7 Comparison of pH-Amended Bleach with Down-Selected Technologies
In general, treatment of inoculated coupons with sprayed pH-amended bleach and the four down-
selected technologies yielded higher log reductions on non-porous compared to porous materials.
However, one notable exception to this is that sprayed Peridox promoted higher log reductions of G.
56
-------
stearothermophilus on the porous materials (carpet and wood) compared to the non-porous galvanized
metal. The spray-applied CASCAD SDF, Hi-Clean 605, KlearWater, and Peridox consistently yielded
higher log reductions in B. anthracis Ames, B. subtilis, or G. stearothermophilus spores on industrial
carpet coupons compared to pH-amended bleach with the exception of KlearWater for B. anthracis
Ames. Amended bleach performed the best on galvanized metal, for all spores, with the exception of
CASCAD SDF against G. stearothermophilus. Moreover, log reductions in B. anthracis Ames, B.
subtilis, or G. stearothermophilus spores on bare wood coupons sprayed with Peridox were greater than
those sprayed with pH-amended bleach or the other technologies.
57
-------
6.0 References
1. Technology Testing and Evaluation Program Test/QA Plan for Evaluating Liquid and Foam
Sporicidal Spray Decontaminants, Version 1, Battelle, Columbus, Ohio, March 2006.
2. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP),
Version 1, Battelle, Columbus, Ohio, January 2005.
3. Rogers JV, Sabourin CL, Taylor ML, Riggs K, Choi YW, Richter WR, Rudnicki DC.
BIOQUELL, Inc. Clarus™ Hydrogen Peroxide Gas Generator. Report submitted to EPA
Environmental Technology Verification, ETV Building Decontamination Technology Center, March
2004. (http://www.epa.gov/etv/verifications/vcenterl 1-1 .html).
4. Rogers JV, Sabourin CL, Taylor ML, Riggs K, Choi YW, Richter WR, Rudnicki DC.
CERTEK® Inc. 1414RH Formaldehyde Generator/Neutralizer. Report submitted to EPA
Environmental Technology Verification, ETV Building Decontamination Technology Center,
August 2004, (http://www.epa.gov/etv/verifications/vcenterll-2.html).
5. Rogers JV, Sabourin CL, Taylor ML, Riggs K, Choi YW, Richter WR, Rudnicki DC.
CDG TECHNOLOGY, Inc. Bench-Scale Chlorine Dioxide Generator: Biological agent
Decontamination. Report submitted to EPA Environmental Technology Verification, ETV Building
Decontamination Technology Center, September 2004,
(http://www.epa.gov/etv/verifications/vcenterll-3.html).
6. Rogers JV, Sabourin CL, Richter WR, Choi YW, Waugh JD, Taylor ML, Riggs KB, Stone HJ,
Willenberg ZJ, Krile RT. Evaluation of Sporicidal Decontamination Technology: Sabre Technical
Services Chlorine Dioxide Gas Generator. Report submitted to EPA Technology Testing and
Evaluation Program, April 2006, (http://www.epa.gov/nhsrc/news/news060506.html).
58
------- |