United States
Environmental Protection
Agency
EPA 600/R-11/076 | August 2011 | www.epa.gov/ord
Systematic Investigation
of Liquid and Fumigant
Decontamination Efficacy
against Biological Agents
Deposited on Test Coupons of
Common Indoor Materials
TECHNOLOGY EVALUATION REPORT
Office of Research and Development
National Homeland Security Research Center
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EPA 600-R-11-076
August 2011
Investigation and Technology
Evaluation Report
Systematic Investigation of
Liquid and Fumigant
Decontamination Efficacy
against Biological Agents
Deposited on Test Coupons of
Common Indoor Materials
United States Environmental Protection Agency
Research Triangle Park, North Carolina 27711
in
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Disclaimer
The U.S. Environmental Protection Agency, through its Office of Research and
Development, funded and managed this investigation through a Blanket Purchase
Agreement under General Services Administration contract number GS23F0011L-3 with
Battelle. This document has been subjected to the Agency's review and has been
approved for publication. Note that approval does not signify that the contents necessarily
reflect the views of the Agency.
Mention of trade names or commercial products in this document or in the methods
referenced in this document does not constitute endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Shawn P. Ryan
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-0699
IV
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Foreword
Following the events of September 11, 2001, addressing the critical needs related to
homeland security became a clear requirement with respect to EPA's mission to protect
human health and the environment. Presidential Directives further emphasized EPA as
the primary federal agency responsible for the country's water supplies and for
decontamination following a chemical, biological, and/or radiological (CBR) attack. To
support EPA's mission to assist in and lead response and recovery activities associated
with CBR incidents of national significance, the National Homeland Security Research
Center (NHSRC) was established to conduct research and deliver products that improve
the capability of the Agency and other federal, state and local agencies to carry out their
homeland security responsibilities.
One goal of NHSRC's research is to provide information on decontamination methods
and technologies that can be used in the response and recovery efforts resulting from a
CBR release over a wide area. The complexity and heterogeneity of the wide-area
decontamination challenge necessitates the understanding of the effectiveness of a range
of decontamination options. In addition to effective fumigation approaches, rapidly
deployable or readily available surface decontamination approaches have also been
recognized as a tool to enhance the capabilities to respond to and recover from such an
intentional CBR dispersion.
Through working with ORD's program office partners (EPA's Office of Emergency
Management and Office of Chemical Safety and Pollution Prevention) and Regional on-
scene coordinators, NHSRC is attempting to understand and develop useful
decontamination procedures for wide-area remediation. This report documents the
results of a laboratory study designed to better understand the effectiveness of several
fumigation and liquid decontamination methods for materials contaminated with
biological agents. The primary focus of the work is relevant to Bacillus anthracis spores;
some data is alos presented related to smallpox virus and ricin toxin.
These results, coupled with additional information in separate NHSRC publications
(available at www.epa.gov/nhsrc) can be used to determine whether a particular
decontamination technology can be effective in a given scenario. NHSRC has made this
publication available to the response community to prepare for and recover from disasters
involving chemical and/or biological contamination. This research is intended to move
EPA one step closer to achieving its homeland security goals and its overall mission of
protecting human health and the environment while providing sustainable solutions to our
environmental problems.
Jonathan Herrmann, Director
National Homeland Security Research Center
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Acknowledgments
Contributions of the following individuals and organization to the development of this
document are acknowledged.
United States Environmental Protection Agency (EPA)
Timothy Dean
Richard Rupert
Frank Schaefer
Battelle
VI
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Executive Summary
The U.S. Environmental Protection Agency (EPA), Office of Research and Development is
striving to protect human health and the environment from adverse impacts resulting from
acts of terror by investigating the effectiveness and applicability of technologies for
homeland security (HS) related applications. The purpose of this investigation is to
determine the efficacy of various fumigation and liquid decontamination technologies for
inactivating biological threat agents (spore, virus, and biotoxin) as a function of building
material surface and decontamination parameters (concentration, contact time,
temperature, and percent relative humidity [RH]). The objective of these studies is to
provide an understanding of the performance of technologies or decontamination
methods to guide scenario-specific selection and implementation of technologies of HS
applications. In the assessment of options for decontamination of indoor surfaces
following intentional release of biological agents, it is important to know whether and to
what extent such factors can impact the decontamination efficacy. This investigation
focused on decontamination of indoor surfaces typical of those found in public buildings
or transportation facilities with the ultimate goal of restoring the buildings or
transportation facilities to a usable state using appropriate decontamination technologies.
Bacillus anthracis spores, ricin toxin, and vaccinia virus were selected for this
investigation based upon a review of available information and other ongoing research
and assessment efforts; the selection represents a range of biological agents with an
expected range of ease of decontamination. B. anthracis spores are the causative agent
for anthrax and are the most difficult to kill of the selected agents. Ricin is an extremely
toxic protein extracted from castor beans (Ricinus communis). Vaccinia is the virus used
for vaccination against smallpox.
The building materials included in the investigation were: glass (small), painted concrete
block, decorative laminate, blown cellulose insulation, particle board, industrial carpet,
plate glass, painted I-beam steel, unpainted pine wood, and ceiling tile. Not all materials
were used with every biological agent or decontamination method. Temperatures selected
for use in testing ranged from 20 °C to 37 °C.
Decontamination technologies investigated included three fumigant technologies
(chlorine dioxide [C1O2], methyl bromide [MeBr], and hydrogen peroxide [HP]) and four
liquid decontamination technologies (pH-amended bleach, C1O2 solution, and two
different HP/peracetic acid [HP-PA] solutions).
Summary of Fumigation Results
The C1O2 fumigation results showed efficacy against B. anthracis spores, ricin toxin, and
vaccinia virus. The efficacy was dependent on the type of materials onto which the spores
were inoculated and the strain of B. anthracis. C1O2 fumigation at a concentration x time
(CT) of 1000 ppmv-hour (hr) resulted in no B. anthracis colony-forming units (CPU)
vii
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being recovered from most types of coupons (>7 log reduction). The mass of ricin
remaining on coupons after C1O2 fumigation at a CT of 500 ppmv-hr of C1O2 was
reduced by >99.1% (geometric mean) from all materials tested except cellulose insulation
which exhibited a 92.7% reduction in recovered mass of ricin compared to controls. No
viable vaccinia virus was recovered from any coupon of any of the seven materials tested
after fumigation at a target CT of 125 ppmv-hr of C1O2.
MeBr fumigation demonstrated up to complete kill of B. anthracis spores (>6 log
reduction in CPU, no CPU recovered) dependent on the building material, fumigant
concentration, and contact time. B. subtilis is a surrogate for the virulent Ames strain of
B. anthracis that was included in the MeBr fumigation tests. Little or no efficacy of MeBr
against B. subtilis was observed in the CT range or 1260-1899 mg/L-hr. In contrast, a
high level of MeBr efficacy was observed against B. anthracis Ames spores at CTs of
1575 mg/L-hr or greater; no viable B. anthracis spores were recovered at CTs of 1890
mg/L-hr (105 mg/L for 18 hr) or 1899 mg/L-hr (211 mg/L for 9 hr).
Decontamination at alternative RH was investigated for C1O2 and MeBr. Both C1O2 and
MeBr showed efficacy to be impacted by the RH of the test chamber. Specifically,
fumigation at higher humidity (80%-84% for C1O2 and 75% for MeBr) exhibited higher
efficacy against B. anthracis Ames spores than fumigation at lower humidity (71%-77%
for C1O2 and 40% for MeBr).
HP fumigation demonstrated up to >6 log reduction in B. anthracis Ames spores (no
CPU recovered) dependent on the building material and contact time. The HP
concentration was 500 ppmv for all fumigations. The CT required for a 6-log reduction in
recoverable B. anthracis Ames spores appears to depend on the type of building material.
Summary of Liquid Decontamination Results
All liquids were applied by immersing the coupons directly into the decontaminant. This
approach may be in contrast to actual field application and other results may suggest
difference due to application procedures (e.g., spray application, which was also
evaluated by EPA). ^
Bleach adjusted to pH 7 provided complete kill of B. anthracis Ames spores (0 CPU
recovered, >6 log reduction) dependent on the building material and the contact time. The
efficacy of pH-amended bleach against three strains of B. anthracis spores (virulent
Ames and avirulent Vollum and NNR1 Al) and B. subtilis was evaluated using the
quantitative method. The log reduction in recovery of various types of viable Bacillus
spores on coupons exposed to pH-amended bleach or phosphate buffer solution for
specified time periods was evaluated. In addition, the extracted coupons were placed
individually into tryptic soy broth (TSB) and incubated for seven days to look for
cloudiness, indicating the presence of residual viable spores on the test coupon. In all
cases where no spores were observed using the quantitative methodology, no residual
viable spores were detected using the qualitative method. While strong similarities in
efficacy were observed across spore types, differences were also observed. For example,
viable B. anthracis Vollum and B. subtilis spores were present on painted concrete after
viii
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exposure to pH-amended bleach for five minutes (min); the other two strains of B.
anthracis, Ames and NNR1 Al, did not have viable spores present after exposure to pH-
amended bleach for 5 min.
Exterm (brand) C1O2 solution provided >6 log reduction in recovered viable B. anthracis
Ames spores dependent on the building material and the contact time. In general, B.
subtilis spores appear to be able to survive a longer exposure to C1O2 solution than B.
anthracis Ames spores.
Spor-Klenz® hydrogen peroxide-peracetic acid (HP-PA) solution exhibited a range of
efficacies, up to >6 log reduction in B. anthracis Ames spores (no viable spores
recovered), dependent on the building material and contact time. No viable B. anthracis
Ames or B. subtilis spores were recovered from any glass coupons after a 20-30 min
contact time. A complete kill of spores on decorative laminate, i.e., no viable spores
recovered or detected, occurred at a 30-min contact time with Spor-Klenz® HP-PA for B.
anthracis Ames spores. In contrast, less than a 2-log reduction in viable spores was
observed after a 30-min contact time with Spor-Klenz® HP-PA for B. anthracis Ames
spores on galvanized metal.
Oxonia Active HP-PA solution exhibited a range of efficacies, up to >6 log reduction in
B. anthracis Ames spores (no viable spores recovered), dependent on the building
material and contact time. Oxonia Active HP-PA against B. anthracis Ames and B.
subtilis spores, reported as log reduction, showed similar efficacies. For both B. anthracis
Ames and B. subtilis on glass, a >6 log reduction was observed after a 10-min contact
time (except that only a 5.77 log reduction was observed for B. subtilis after 60 min).
Small numbers of viable spores were recovered from one or more replicate coupons of
some material for both B. anthracis Ames and B. subtilis at each contact time (10, 30, and
60 min).
IX
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Table of Contents
Disclaimer iv
Foreword v
Acknowledgements vi
Executive Summary vii
Abbreviations/Acronyms xvii
1.0 Introduction 1
2.0 Investigation Procedures 3
2.1 Experimental Design 3
2.2 Test and Apparatus 3
2.2.1 Fumigation Test and Control Chambers 4
2.2.2 Apparatus for Testing Liquid Decontamination Technologies 7
2.3 Test Surfaces 8
2.4 Biological Agents and Surrogates 9
2.5 Application of Biological Agents to Test Coupons 11
2.6 Extracting and Quantifying Biological Agent and Surrogates
(Quantitative Method) 13
2.6.1 B. anthracis Spores 13
2.6.2 Ricin 14
2.6.3 Vaccinia Virus 17
2.7 Broth Culture Assay for Viable Spores (Qualitative Method) 17
2.8 Calculations and Statistics 18
2.8.1 Percent Recovery 18
2.8.2 Decontamination Efficacy 18
2.9 C1O2Fumigation 20
2.9.1 Description of C1O2 Technology 20
2.10 MeBr Fumigation 30
2.10.1 Description of MeBr Technology 30
2.10.2Test Matrix for MeBr Fumigation 31
2.11 HP Fumigation 36
2.12 Soak in pH-Amended Bleach 39
2.13 Soak in Liquid C1O2 Technology 42
2.14 Soak in Spor-Klenz® Hydrogen Peroxide-Peractic Acid (HP-PA)
Solution 44
/B\
2.15 Soak in Oxonia Active Solution 44
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3.0 Quality Assurance/Quality Control 48
3.1 Performance Evaluation (PE) Audits 48
3.2 Technical Systems Audit 51
3.3 Data Quality Audit 51
3.4 QA/QC Reporting 51
3.5 Deviations from the Test QA Plans 52
3.5.1 General Deviation 52
3.5.2 Chlorine Dioxide 52
3.5.3 Liquid Testing 52
3.5.4 Methyl Bromide 53
4.0 Fumigation Results 55
4.1 C1O2 Fumigation Results 55
4.1.1 Trial 4g: C1O2 Fumigation Cycle Fractionation Results 55
4.1.2 Trials 4a and 4b: Fumigation Cycle Fractionati on Results for
Two Concentrations of C1O2 against.8. anthracis 56
4.1.3 B. anthracis on Painted Concrete: Quantitative CT
Investigation 58
4.1.4 B. anthracis on Eight Materials: Quantitative CT Investigation 60
4.1.5 Investigation of Effect of Temperature on C1O2 Efficacy 64
4.1.6 Investigation of Effect of Organic Burden, Spore Types, and
RH on C1O2 Efficacy 65
4.1.7 C1O2 Fumigation of Ricin at Two CTs 69
4.1.8 C1O2 Fumigation of Vaccinia Virus at Two CTs 72
4.1.9 Summary of Findings from the C1O2 Fumigation Investigation 72
4.2 MeBr Fumigation Results 76
4.2.1 Results for MeBr Fumigation of B. anthracis on Glass and
Ceiling Tile at Various CT Values (36 °C) 76
4.2.2 Results for MeBr Fumigation of B. anthracis on Various
Materials at Various CT Values (36 °C, 75% RH) 80
4.2.3 Results for MeBr Fumigation of B. anthracis on Various
Materials at 2 CT Values (25 °C and 36 °C, 75% RH) 83
4.2.4 Results for MeBr Fumigation of B. subtilis on Glass at Various
CT Values 84
4.2.5 Results for MeBr Fumigation of B. anthracis on Glass and
Ceiling Tile at Alternate RH Conditions 86
4.2.6 Results for MeBr Fumigation of B. subtilis on Suture Loops at
Various CT Values 87
4.2.7 Results for MeBr Fumigation of B. atrophaeus on Stainless
Steel in Tyvek Packaging at Various CT Values 88
4.2.8 Summary of Findings from the MeBr Investigation 88
4.3 HP Fumigation 89
4.3.1 Results of Varying HP CT at 22 °C 89
4.3.2 Summary of Findings from the HP Investigation 96
XI
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5.0 Liquid Decontamination Technologies Test Results 97
5.1 pH-Amended Bleach 97
5.1.1 Qualitative Results for pH-Amended Bleach Decontamination
of Bacillus Species 97
5.1.2 Quantitative Results for pH-amended Bleach Decontamination
of Bacillus Species 98
5.1.3 Quantitative Results for pH-Amended Bleach Efficacy
Decontamination of Vaccinia Virus 105
5.1.4 Summary of Findings from the pH-Amended Bleach
Investigation 106
5.2 Liquid C1O2 106
5.2.1 Results for Liquid C1O2 Decontamination of Bacillus Species 106
5.2.2 Results for Li quid C1O2 Decontamination of Vaccinia Virus 110
5.2.3 Summary of Findings from the Liquid C1O2 Investigation Ill
5.3 Spor-Klenz® Ready-to-Use HP-PA Solution 112
5.3.1 Qualitative Results for Spor-Klenz HP-PA Decontamination
of Bacillus Species 112
5.3.2 Quantitative Results for Spor-Klenz® HP-PA Decontamination
of Bacillus Species 113
5.3.3 Summary of Findings from the Spor-Klenz® HP-PA
Investigation 116
5.4 Oxonia Active® Solution 116
5.4.1 Quantitative Results for Oxonia Active® Decontamination of
Bacillus Species 116
5.4.2 Summary of Findings from the Oxonia Active® Investigation 117
6.0 Summary 121
6.1 Summary of C1O2 Fumigation Results 121
6.2 Summary of MeBr Fumigation Results 125
6.3 Summary of HP Fumigation Results 126
6.4 Summary of Results from Decontamination of B. anthracis with Liquid
pH-Amended Bleach (10% adjusted to approximately pH 7) 127
6.5 Summary of Results from Decontamination of B. anthracis with Liquid
ExtermClO2 Solution 129
6.6 Summary of Results from Decontamination of B. anthracis with Liquid
Spor-Klenz® HP-PA Solution 131
6.7 Summary of Results from Decontamination of B. anthracis with Liquid
Oxonia Active 131
7.0 References 133
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Figures
Figure 2-1. Glove box used for test and control chambers for C1O2 fumigation testing.... 5
Figure 2-2. Schematic of MeBr decontamination testing 5
Figure 2-3. Ultrasonic fogging system 7
Figure 2-4. Custom apparatus for immersing coupon surface in liquid 8
Figure 2-5. Multichannel micropipette applying biological agent to a coupon 12
Figure 2-6. B. anthracis Ames colonies on TSA 14
Figure 2-7. Visual demonstration of MTT assay on a microplate 16
Figure 2-8. Example of ricin cytotoxic profile with corresponding absorbance measured
using microplate reader 16
Figure 2-9. STERIS VHP® Biodecontamination Cycle.[14] 37
Figure 4-1. Mean B. anthracis log reduction from glass and ceiling tile vs. CT at
various concentrations of MeBr 80
Figure 4-2. Mean B. anthracis (B. subtilis on glass) log reduction from various materials
vs. CT 81
Figure 4-3. Mean B. anthracis log reduction from various materials (-1900 mg/L-hr). 83
Figure 4-4. Mean B. anthracis log reduction from various materials vs. CT (25 °C,
75% RH, [MeBr] = 212 mg/L, contact times = 9 and 24 hr) 84
Figure 4-5. Mean log reduction of B. anthracis on various materials and B. subtilis
spores on glass at various CTs 86
Figure 4-6. Mean B. anthracis spores log reduction from various materials vs. hydrogen
peroxide CT (500 ppmv concentration) at 22 °C 93
Figure 4-7. Mean B. anthracis log reduction from various materials vs. FTP CT (225
ppmv concentration) at 22 °C 96
Figure 5-1. Decontamination efficacy of pH-amended bleach against B. anthracis
Ames spores at 22 °C 102
Figure 5-2. Decontamination efficacy of pH-amended bleach against B. anthracis
spores and surrogates with 5-min contact time 103
Figure 5-3. Decontamination efficacy of Exterm C1O2 solution against B. anthracis Ames
spores at 22 °C 108
Figure 5-4. Decontamination efficacy of Exterm C1O2 solution against B. subtilis
spores 109
Figure 5-5. Decontamination efficacy of Spor-Klenz®FIP-PA solution against B.
anthracis Ames on various materials and B. subtilis spores on glass 116
Figure 5-6. Decontamination efficacy of Oxonia Active® decontamination solution
against.8. anthracis Ames on various materials and.8. subtilis spores on glass.... 120
Xlll
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Tables
Table 2-1. Material Characteristics 10
Table 2-2. Dilution Factors for Various Coupon Materials 15
Table 2-3. Test Matrix for C1O2 Decontamination Investigation 23
Table 2-4. Test Matrix for Generation of MeBr Decontamination CT Curves against
B. anthracis Ames Spores and Surrogates 33
Table 2-5. Formula, Model Parameters, and Relevant Model Results 35
Table 2-6. Test Matrix for Generation of HP Decontamination CT Curves against
B. anthracis Ames Spores 39
Table 2-7. Test Matrix for pH-Amended Bleach Investigation 41
Table 2-8. Test Matrix for Liquid C1O2 Decontamination Investigation 43
Table 2-9. Test Matrix for the Spor-Klenz® HP-PA Decontamination Investigation 46
Table 2-10. Test Matrix for the Oxonia Active® Decontamination Investigation 47
Table 3-1. C1O2PE Audit Results 49
Table 3-2. Liquid PE Audit Results 50
Table 3-3. MeBr PE Audit Results 51
Table 4-1. Results from Qualitative Evaluation of C1O2 Fumigation of B. anthracis
Ames Spores and Surrogates 56
Table 4-2. Results from Qualitative Evaluation of C1O2 Fumigation of B. anthracis
Ames Spores 57
Table 4-3. B. anthracis Ames Spores CFU after C1O2 Fumigation at Various CTs 59
Table 4-4. Mean Log Reduction of B. anthracis Ames Spores after C1O2 Fumigation
of Painted Concrete at Various CTs 60
Table 4-5. B. anthracis Ames Spores Mean CFU after C1O2 Fumigation at Various
CTs, Temperatures 61
Table 4-6. Mean Log Reduction of B. anthracis Ames Spores after C1O2 Fumigation
at Various Temperatures and Contact Times 63
Table 4-7. Statistical Comparison of C1O2 Efficacy against B. anthracis Ames Spores
on Cellulose Insulation at Two Temperatures 65
Table 4-8. Mean C1O2 Fumigation of B. anthracis Ames and NNR1 Al Spores CFU
with and without Added Organic Burden 66
Table 4-9. Mean Log Reduction of C1O2 Fumigation of B. anthracis Ames and
NNR1A1 Spores with and without Added Organic Burden 67
Table 4-10. Statistical Comparison of C1O2 Fumigation of B. anthracis Ames and
NNR1A1 Spores with and without Added Organic Burden 69
Table 4-11. Ricin Cytotoxicity with and without C1O2 Fumigation at Various CTs 71
Table 4-12. Geometric Mean Percent Reduction of Ricin Cytotoxicity with C1O2
Fumigation at Various CTs 72
Table 4-13. Vaccinia PFU with and without C1O2 Fumigation at Various CTs 74
Table 4-14. Mean Log Reduction of Vaccinia PFU with C1O2 Fumigation at Various
CTs 75
Table 4-15. Results of Varying MeBr CT (36 °C and 75% RH) 77
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Table 4-16. Analysis of Results of Varying MeBr CT (36 °C and 75% RH) for Mean
Log Reduction of B. anthracis Ames 79
Table 4- 17. Results of Contact Time Series of MeBr Fumigation (36 °C and 75% RH)
of B. anthracis Spores on Various Building Materials 82
Table 4-18. Results of 9 hr and 24 hr Contact Time of MeBr Fumigation (75% RH)
of B. anthracis on Various Building Materials at a Target of 320 mg/L 84
Table 4-19. Difference in Log Reduction of B. anthracis Ames and B. subtilis on Glass
atVariousCTs 85
Table 4-20. Significance of Differences in Log Reduction Arising from Percent RH at
212 mg/L MeBr and 7-Hr Contact Time at 37 °C 87
Table 4- 21. Qualitative Spore Viability Test 87
Table 4-22. Comparison of Results for Log Reduction and Qualitative Spore Viability
Test at Various CT Conditions 88
Table 4-23. STERIS VHP® Fumigation Results for B. anthracis (500 ppmv) 91
Table 4-24. STERIS VHP® Fumigation Results for B. anthracis Ames Spores
(225 ppmv) 94
Table 5-1. Results from Qualitative Evaluation of pH-Amended Bleach
Decontamination of B. anthracis Ames 97
Table 5-2. B. anthracis Ames Spores CFU after Various pH-Amended Bleach Contact
Times 99
Table 5-3. B. anthracis Vollum Spores CFU after Various pH-Amended Bleach Contact
Times 100
Table 5-4. B. anthracis NNR1 Al Spores CFU after pH-Amended Bleach Contact
Times 100
Table 5-5. B. subtilis CFU after Various pH-Amended Bleach Contact Times 101
Table 5-6. Summary of pH-Amended Bleach Decontamination of Various Bacillus
Species and Strains 104
Table 5-7. Vaccinia PFU after 5 Min pH-amended Bleach Contact Time 105
Table 5-8. B. anthracis Ames spores CFU after Various Liquid C1O2 Contact Times .. 108
Table 5-9. B. subtilis CFU after Various Contact Times with Liquid C1O2 109
Table 5-10. Log Reduction in Bacillus Species after Various Contact Times with
Liquid C1O2 110
Table 5-11. Vaccinia PFU after 10-Min Contact with Li quid C1O2 Ill
Table 5-12. Results from Qualitative Evaluation of Spor-Klenz HP-PA
Decontamination of B. anthracis Ames 112
Table 5-13.5. anthracis Ames Spores CFU after Various Contact Times with Spor-
Klenz®HP-PA 114
Table 5-14. B. anthracis Ames and B. subtilis Spores CFU after Various Contact
Times with Spor-Klenz® HP-PA 115
Table 5-15. Log Reduction in Bacillus Species after Various Contact Times with Spor-
Klenz®HP-PA 115
Table 5-16. B. anthracis Ames Spores CFU after Various Oxonia Active Contact
Times 118
Table 5-17. B. anthracis Ames and B. subtilis Spores CFU after Various Oxonia
Active® Contact Times* 119
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Table 5-18. Log Reduction in Bacillus Species after Various Contact Times with
Oxonia Active®* 119
Table 6-1. Summary of Minimum C1O2 Fumigation CT Resulting in 0 CPU of B.
anthracis Ames Spores, <10% Bioactivity of Ricin Applied, or 0 PFU
of Vaccinia Being Recovered 122
Table 6-2. Bacillus anthracis Ames Spores Fumigated with C1O2 123
Table 6-3. Ricin Fumigated with C1O2 124
Table 6-4. Vaccinia Virus Fumigated with C1O2 124
xvi
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Abbreviations/Acronyms
AOAC
ASTM
ATCC
BBRC
BI
BSCII
BSC III
°C
CDC
CFR
CPU
C102
cm
C02
CT
d
ECBC
EPA
H
HP
HP-PA
hr
HS
HVAC
1
L
mL
^g
ML
min
mm
mg
Association of Official Analytical Chemists (now AOAC
International)
American Society for Testing and Materials
American Type Culture Collection
Battelle Biomedical Research Center
biological indicator
Class II biological safety cabinet
Class III biological safety cabinet
degrees Celsius
Centers for Disease Control and Prevention
Code of Federal Regulations
colony-forming unit(s)
chlorine dioxide
centimeter
carbon dioxide
concentration x contact time
depth
U. S. Army Edgewood Chemical Biological Center
U.S. Environmental Protection Agency
height
hydrogen peroxide
hydrogen peroxide - peracetic acid
hour
homeland security
heating, ventilation, and air conditioning
length
liter
milliliter
microgram
microliter
minute
millimeter
milligram
xvn
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MTT 3-(4,5-dimethylthiazol-2-yl)-2, 5,-diphenyl tetrazolium bromide
nm nanometer
NIST National Institute of Standards and Technology
ORD Office of Research and Development
PBS phosphate-buffered saline
PE performance evaluation
PFU plaque-forming unit(s)
ppm parts per million
ppmv parts per million volume
psi pounds per square inch
PSR perfectly stirred reactor
QA quality assurance
QC quality control
RH relative humidity
rpm revolutions per minute
SD standard deviation
TSA trypitic soy agar
TSB tryptic soy broth
TO time zero
Hg Microgram
vs. versus
w width
xvin
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1.0 Introduction
The U.S. Environmental Protection
Agency, through its Office of Research
and Development (ORD), is helping to
protect human health and the
environment from adverse impacts
resulting from intentional acts of terror.
With an emphasis on decontamination
and consequence management, water
infrastructure protection, and threat and
consequence assessment, EPA is
working to develop tools and
information that will help detect the
intentional introduction of chemical,
radiological, or biological contaminants
in buildings, subways, water systems, or
outdoor environments; contain these
contaminants; decontaminate buildings,
subways, water systems, and/or outdoor
environments; and facilitate the disposal
of material resulting from cleanups.
As part of the above effort, EPA
investigates the effectiveness and
applicability of technologies for homeland
security (HS) related applications by
developing test plans that are responsive
to the needs of stakeholders, conducting
tests, collecting and analyzing data, and
preparing peer-reviewed reports. All
evaluations are conducted in accordance
with rigorous QA protocols to ensure
that data of known and high quality are
generated and that the results are
defensible. EPA provides high-quality
information that is useful to decision
makers in purchasing or applying the
tested technologies. EPA provides
potential users with unbiased, third-party
information that can supplement vendor-
provided information. 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 purpose of this investigation was to
develop an understanding of the
effectiveness of decontamination
approaches for HS applications as a
function of a range of materials,
environmental conditions, and operating
conditions typical of field use. This
investigation focused on
decontamination of indoor surfaces
typical of those found in a public
building and public transportation that
could be contaminated by biological
agent following an intentional release.
Residual amount of biological agent on
indoor surfaces following
decontamination after an intentional
release could present a potential health
risk for personnel re-entering the
building. This is a report of the impact of
various factors on the efficacy of
fumigant and liquid decontamination
technologies against biological warfare
agents and surrogates. (Subsequent use
of the term "biological agents" should be
understood to include surrogates.)
The investigation generated data that are
indicative of the performance or efficacy
of the fumigation and liquid
decontamination technologies under a
variety of parametric conditions,
including non-ideal conditions. This
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bench scale investigation can be
summarized as follows. Biological
agents were applied to a variety of test
coupons representative of porous and
non-porous indoor building surfaces. A
variety of fumigation and liquid
decontamination technologies were
applied under conditions that were
expected to be highly effective as well as
conditions that might be partially
effective. The factors that were varied
included: duration of contact with the
decontamination technology (contact
time), concentrations of the fumigant
decontamination technologies,
temperature, and RH (for fumigation
technologies). Residual biological agent
was extracted from the coupons and a
quantitative measurement was made of
the viable organisms or level of
cytotoxicity. Efficacy of the
decontamination technology under a
given set of conditions was evaluated by
comparing the residual biological agent
after the decontamination treatment to a
measurement of biological agent
extracted from positive control coupons
incubated under environmental
conditions specified for a test and for the
same time period. (Various factors were
varied for different technologies.)
All testing and evaluation were
conducted under the direction of the
EPA. In performing each test, peer-
reviewed test/QA plans and amendments
were developed, approved by EPA, and
followed in execution of the testing.
Chapter 2 summarizes the methods used
in this investigation. Chapter 3 provides
information on the quality assurance and
quality control methods that were
employed and a summary of quality
audits and deviations. Chapter 4
provides the results of the fumigation
testing. Chapter 5 provides the results of
liquid decontamination testing.
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2.0 Investigation Procedures
This section provides an overview of the
investigation procedures that were used.
For all tests, specific test plans detailing
the test methodology and data quality
assurance provisions were developed
prior to testing. Important aspects of
those test plans are provided in this
section (test plan details) and Section 3.0
(quality assurance documentation).
2.1 Experimental Design
This report provides results for the
bench-scale investigation of
technologies to decontaminate biological
agents from indoor surfaces. The general
approach and methods, biological
agents, and types of indoor surfaces used
are summarized in this section. A
pretest-posttest control group design was
used. The independent test parameters
include the type of biological agent, type
of building material, type of
decontamination technology, contact
time, concentration of the
decontamination technology,
temperature, and RH. The dependent
variable is the number of viable
organisms or amount of bioactive toxin
extracted from a test or positive control
coupon with or without exposure to the
decontamination technology.
Key measurements in this investigation
include:
• Quantitative measurement of
viable organisms or level of
bioactivity
• Environmental conditions
including temperature and RH
• Operating conditions including
contact time with the
decontamination technology and,
for fumigants, concentration of
the decontamination technology.
The key outputs of this investigation are
concentration x contact time (the product
henceforth referred to as CT) versus
(vs.) log reduction curves, showing the
efficacy:
• Of the various technologies
• Against various biological
warfare agents
• On various indoor building
surfaces
• Under various environmental
conditions.
Statistical comparisons were conducted
to determine whether the mean log
reduction in viable biological agent or
bioactive toxin exceeded the control
mean log reduction by an amount that
was statistically significant at a 95%
confidence level.
2.2 Test and Apparatus
Decontamination was performed by
placing coupons into customized test
chambers and applying the fumigation
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technology, or by immersing the
coupons in liquid decontamination
technologies either in a custom-built
trough system or capped vials. For
positive controls, corresponding control
chambers, troughs, or capped vials were
used to replicate the conditions in the
test chambers and troughs, but
precluding contact with the
decontamination technology.
2.2.1 Fumigation Test and Control
Chambers
The impact of the critical parameters on
decontamination efficacy of various
fumigation technologies was evaluated
through bench-scale testing in the
laboratory. The fumigation test
chambers were a 317 L glove box for
C1O2, a 23 L glass chamber for MeBr,
and a 1275 L Class III biological safety
cabinet (BSC) for HP fumigation. The
glass chamber was needed for MeBr
because the gas exhibits high penetration
through many materials.
The glove box was a Compact Glove
Box Model 830-ABC (Plas Labs, Inc.,
Lansing, MI), shown in Figure 2-1, with
internal dimensions of 71 cm width (w)
x 59 cm diameter (d) x 74 cm height (h)
and external dimensions of 110 cm (w) x
61 cm (d) x 79 cm (h), having a total
volume of 317 L. The glove box also has
a top opening of 43 cm x 58 cm and an
attached transfer chamber that was 30
cm long and an inner diameter of 28 cm.
For C1O2 investigation, the glove box is
coated with black latex paint to shield
the interior from light. Glove ports
enabled working in the glove box.
For positive control (spiked with
biological agent) and laboratory blank
coupons (not spiked with biological
agent) that were not exposed to C1O2
fumigation, an identical Compact Glove
Box Model 830-ABC (not painted black)
was used.
F91
As was done in previous studies,1 J
multiple coupons of each indoor material
were spiked with the biological agent
and placed horizontally on a wire rack
the test chamber. For C1O2 investigation,
the glove box was coated with black
latex paint to shield the interior from
light. Two 93 mm computer fans in the
glove box provided air flow in the test
chamber to promote uniform exposure to
the fumigant throughout the chamber;
identical fans were included in the
control chamber. The short-term stability
of the C1O2 measurement in the
circulating air suggests that mixing was
sufficient to provide uniform exposure.
Blank (i.e., uncontaminated) and
positive control (i.e., contaminated but
not decontaminated) coupons were also
prepared for each test material, and were
utilized along with data from the test
contaminated and decontaminated (test)
coupons to determine decontamination
efficacy. This approach provides a
highly controlled, reproducible approach
to assess sensitivity of the fumigation
decontamination efficacy to CT,
temperature, and RH.
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Figure 2-1. Glove box used for test and control chambers for ClOi fumigation
testing.
Figure 2-2. Schematic of MeBr decontamination testing.
Figure 2-2 shows a schematic drawing of
the MeBr test chamber and containment
system. The primary test chamber was
glass with a 23 L volume (approximately
29 cm x 29 cm x 29 cm). A second
chamber of the same size made of
polycarbonate plastic was used as the
control chamber. The chambers were
insulated to prevent condensation on the
inside chamber walls. The high toxicity
and penetrability of MeBr required a
primary and secondary containment
chamber for protection of laboratory
personnel. A Class III biological safety
cabinet (BSC III) (SG603, Baker,
Sanford, ME) provided secondary
containment.
For evaluation of the HP fumigation, a
BSC III was used as the fumigation test
chamber having a total volume of
approximately 1275 L. Positive control
coupons were in sealed vials within the
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fumigation test chamber during the
fumigation cycle.
The experimental temperature and RH in
test chambers and the control chambers
were monitored and, where specified in
the test plan, controlled during a given
fumigation event ("trial"). Temperature
and RH were manually recorded at
defined intervals using a calibrated,
National Institute of Standards and
Technology (NIST)-traceable
thermometer/hygrometer (accuracy of
±1 °C and ±5% RH; 14-648-53, Fisher
Scientific, Pittsburgh, PA).
As testing proceeded, decontamination
efficacy was greater at high RH than at
low RH; however, moisture was
observed on surfaces at high RH.
Because moisture may absorb the C1O2
from the atmosphere, the fumigation at
high RH may become a liquid
decontamination technology. To address
this concern, the RH requirements were
revised and tolerances were tightened
(75% ± 5%). In the early C1O2
fumigation testing, where broad RH
levels were specified (<40% RH, >75%
RH), low RH was maintained in the
chambers using calcium sulfate
desiccants (W.A. Hammond Drierite
Co., Xenia, OH). The W.A. Hammond
Drierite Co. web site
(www.Drierite.com) reports that the
National Bureau of Standards has
verified that the moisture remaining in
gases dried with Drierite at 25 °C-30 °C
is 0.005 mg/L. High RH in the test
chamber during the C1O2 fumigation was
generated using a nebulizer approach
previously used to maintain high
humidity during testing. High RH in the
control chamber was achieved by
enclosing a wet paper towel.
Based on knowledge gained during
testing, the RH tolerances were tightened
(e.g., 75% ± 5%) and methods for
adjusting humidity were modified to
prevent possible generation and
deposition of moisture (liquid water) in
the test and control system. To meet
these tighter requirements, a custom-
designed ultrasonic fogging system with
a water trap, shown connected to the
MeBr chamber in Figure 2-3, was
developed and used to humidify the test
chamber. The ultrasonic fogger was
inside a polyvinylchloride pipe that was
surrounded by heating tape and covered
with insulation. A water sight gauge
allowed the water level to be monitored.
Humidified air from the fogging system,
at the specified temperature for the trial,
was pumped through a water trap to
remove liquid water and flushed through
the test chamber until the specified RH
was reached. The system can be
operated in a single-pass mode or the
test chamber air can be recirculated
through the fogging system. During
method development, Water Contact
Indicator Tape (3M, St. Paul, MN) was
used to show that the humidified air
introduced into the test chamber did not
condense into liquid water. The RH was
lowered by introduction of dry air. This
approach provided tightly controlled RH
when the chamber was static and
allowed adjustment of RH when the
chamber atmosphere was dynamic, e.g.,
during introduction of the fumigant.
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Temperature and RH
Monitoring System
Air Pump
Figure 2-3. Ultrasonic fogging system.
Temperatures of all chambers above
ambient, except the MeBr control
chamber, were regulated using a
thermostatically-controlled heating pad.
The MeBr control chamber was placed
in an incubator to maintain the
temperature. When the RH tolerances
were tightened, the MeBr test chamber
was insulated to help maintain a constant
temperature and to prevent any potential
condensation. The MeBr control
chamber was in an incubator; insulation
was not necessary.
2.2.2 Apparatus for Testing Liquid
Decontamination Technologies
The liquid decontamination testing was
performed by immersing the coupon
surface spiked with biological agent in
the liquid decontamination technology.
The immersion of the contaminated
surface in the liquid decontamination
technology provides a well-controlled,
conservative efficacy test. Spraying of
the liquid decontamination technology
onto contaminated materials may yield
different results. For the pH-amended
bleach and Exterm liquid C1O2 testing,
the test coupons were placed in custom-
built trays (shown in Figure 2-4). The
custom tray in Figure 2-4 consists of six
wells separated from each other by
walls. Note: In Figure 2-4, the spiked
surfaces of the numbered wooden
coupons are facing downward. In this
position the spiked surface was in
contact with the decontamination fluid.
The wells were deep enough to hold one
coupon and enough decontamination
liquid to cover the bottom surface of the
coupon without overflowing into
adjoining wells. The specific amount of
decontamination liquid required in the
wells to ensure continuous contact with
each material type during
-------
decontamination was determined.
During the testing, the spiked surface
remained in contact with the liquid
decontaminant for the decontamination
period and at the temperature specified
in the test/QA plan.
For the Spor-Klenz® and Oxonia Active®
testing, test coupons and procedural
blanks were placed into 50 mL conical
vials (21008-714, VWR, West Chester,
PA) holding enough decontamination
liquid to cover the surface of the coupon
for the specified decontamination period.
The spiked surface remained in contact
with the decontamination technology
liquid for the decontamination period
and at the temperature specified in the
test/QA plan.
IS*.
Figure 2-4. Custom apparatus for immersing coupon surface in liquid.
2.3 Test Surfaces
The indoor building material test
coupons used in the evaluations are
described in Table 2-1. Generally, the
indoor building material test coupons
were cut from the interior of a large
piece of test material to a standard size
of approximately 1.9 cm (w) x 7.5 cm
length (1). Edges and damaged areas
were avoided in cutting test coupons.
The thickness of the coupons varied as
shown in Table 2-1. The test coupons
were visually inspected prior to being
spiked with the biological agents.
Coupons were visually inspected for
irregularities and those with anomalies
were rejected.
Some types of coupons varied from the
standard size. The small glass coupons
were 5 mm x 5 mm and were of the size
and type specified in the EPA's modified
Three Step Method for evaluating
decontamination efficacy.^ The small
glass coupons both represent glass that
was found in buildings and provided a
linkage to the Association of Official
Analytical Chemists (AOAC) 2008-05[3]
method for evaluating decontamination
efficacy. The data enable comparison of
results from this investigation with
results using the AOAC 2008-05
method, controlling for coupon material
and size that could result in differences
in efficacy results attributable to the
specific test method used. Such
comparisons are useful to inform
-------
decisions by the EPA Office of Pesticide
Programs.
Coupons of compressed blown cellulose
insulation were round with a 13 mm
diameter. Unpainted wood and painted I-
beam steel coupons were 1.3 cm x 1.3
cm and were provided by the U. S. Army
Edgewood Chemical Biological Center
(ECBC). The latter materials represent
materials found in buildings and provide
a linkage between the decontamination
testing reported here and
decontamination testing performed by
EPA/ECBC. These linkages enable
evaluation of whether comparable
efficacy results are obtained by the two
laboratories. Knowing of potential
interlaboratory differences is important
for interpreting results, identifying
additional key variables, and
standardizing test methods.
On each day of testing, each large
coupon was assigned and marked with a
unique identifier code for traceability.
Small coupons were also assigned a
number and placed into containers
marked with the unique identifier code.
To prevent contamination of test
surfaces, sterile technique, following
developed policies and guidelines was
exercised during all phases of handling
the coupons.
2.4 Biological Agents and
Surrogates
The biological agents used in the testing
include:
• Bacillus anthracis Ames
(produced from BAA365 seed
stock received from the U.S.
Army Medical Research Institute
for Infectious Diseases)
• Bacillus anthracis Vollum strain
received from a confidential
source and maintained in culture
by Battelle (strain verified by
genotyping by independent
laboratory)
• Bacillus anthracis NNKlAl with
0.5% bovine serum albumin (as
prepared and provided by the
U.S. Army ECBC)
• Bacillus subtilis on stainless disk
in Tyvek® pouches (Apex
Laboratories, Apex, NC)
biological indicators
• Bacillus atrophaeus spores on
filter paper strips (spore strip) in
glassine envelopes (Raven
Biological Laboratories, Omaha,
NE) biological indicators
• Ricin toxin (Vector Laboratories,
Burlingame, CA, L-1090 from
Vector Laboratories, product
specification: Ricin communis
agglutinin II, 5 mg/mL protein
concentration)
• Vaccinia virus (American Type
Culture Collection test VR119).
The biological agents were selected
based on an evaluation of potential
threats to buildings and discussions with
and approval by EPA. Each biological
agent was used according to the Centers
for Disease Control and Prevention
(CDC) Select Agents Program (42 CFR
Part 73) and the Biological Defense
Research Program (32 CFR 626 and
627) in adherence with the Battelle
Biomedical Research Center (BBRC)
Facility Safety Plan safety plans.
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Table 2-1. Material Characteristics
Material
Glass
Painted
Concrete Block
Galvanized
Metal
Ductwork
Decorative
Laminate
Blown
Cellulose
Insulation
(compressed)
Particle Board
Industrial-
Grade Carpet
Plate Glass
Painted I-beam
Steel
Unpainted Pine
Wood
Ceiling tile
Lot, Batch, or
ASTM No., or
Observation
Same as in
AOAC 2008.05
ASTM C90
Industry HVAC
standard 24
Gauge
Galvanized Steel
Laminate/
Formica/ White
Matte Finish
Cocoon Attic
Blow-in
Insulation
Batch 06 10-29-
04 15-23
Appearance is
pressed medium
density
fiberboard
ShawTek,
EcoTek 6
Color: mottled
gray/dark
brown/and black
(or equivalent)
C1036, 1/8"
thick
ECBC
ECBC
Armstrong 954,
Classic Fine
Textured (or
equivalent)
Manufacturer/
Supplier Name
Erie Scientific
Company,
Portsmouth, NH
Wellnitz,
Columbus, OH
Accurate
Fabrication,
Columbus, OH
Solid Surface
Design,
Columbus, OH
U.S. GreenFiber,
LLC
Charlotte, NC
Weyerhaeuser
Shaw Industries,
Inc.
(or equivalent)
Brooks Brothers
ECBC
ECBC
Armstrong
(or equivalent)
Approximate
Coupon Size,
w x 1 (thickness)
5 mmx5 mm
(1 mm)
1.9 cmx7.5 cm
(5 mm)
1.9 cmx7.5 cm
(1 mm)
1.9 cmx7.5 cm
(1 mm)
13 mm diameter
containing 100
mg of material
(1 mm)
1.9 cmx7.5 cm
(1.3 cm)
1.9 cmx7.5 cm
(7mm)
1.9 cmx7.5 cm
(3 mm)
1.3 cmx.1.3 cm
1.3 cmx.1.3 cm
3.5 x 1.5 cm
Material Preparation
Autoclaved
Brush and roller painted
all sides. One coat Martin
Senour latex primer (#71-
1 185) and one coat Porter
Paints latex semi-gloss
finish (#919); autoclaved
Cleaned with acetone;
autoclaved or gamma
irradiated
Gamma irradiated
Compressed at 2000 psi in
a mold; gamma irradiated
Gamma irradiated
Gamma irradiated
Autoclaved
Autoclaved
Autoclaved
Gamma irradiated
The B. anthracis Ames and Vollum
spores were prepared according to
established BBRC procedures. Details of
the method are published in the Journal
of Applied Microbiology.[2] Preparations
have >95% refractile spores with <5%
cellular debris. No final additives were
included. Stock spore suspensions
(approximately 1.0 x 109 CFU/mL) are
stored at 2 °C to 8 °C until use. No
additives were included in the final spore
preparations.
10
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The B. anthracisNNKlAl spores were
used in the stock suspension as received.
The biological indicators (Bis), B.
subtilis spores on stainless steel and B.
atrophaeus spores on filter paper strips,
were used as received or with the
packaging removed. Biological
indicators are typically used without
removal from their packaging. Efficacy
testing was performed both using the
biological indicators enclosed in the
glassine or Tyvek® packaging and with
the packaging removed to allow a
comparison of the impact of the
packaging on the surrogate efficacy
results.
Ricin was used in the stock solution as
received.
Vaccinia virus from Battelle stock was
propagated in Vero cells following
Battelle's internal methods (BBRC
Method No. 116-03/Microbiology,
Monkeypox and Vaccinia Plaque Assay
and BBRC Method No. 107-
04/Microbiology, Method for the
Routine Maintenance of Multiple
Adherent Cell Lines). A monolayer of
Vero cells was inoculated with vaccinia
virus at a multiplicity of infection
ranging from 0.01 to 1.0%. Cultures
were maintained in an incubator at 37 °C
± 2 °C under a 95% air, 5% CO2 mixture
until 90%-100% cytopathic effects were
observed. Infected cells were harvested
and subjected to a rapid freeze/thaw
cycle. Cellular debris was removed by
centrifugation at 800 to 1,000 x g. The
resulting supernatant (containing virus)
was harvested and stored in 1.0 mL
o
aliquots, approximately 0.7 x 10 -1.0 x
108 PFU/mL, at < -70 °C until use. The
resulting supernatant, vaccinia in
complete cell culture medium containing
5%-10% fetal bovine serum, was not
filter sterilized since the virus was
propagated in sterile tissue culture. All
manipulations were performed with
sterile technique. The PFU/mL was
determined after the virus was aliquoted
by randomly selecting and assaying vials
of the stock samples. The samples were
assayed using a standard plaque assay
(see Section 2.6.3) to determine the viral
titer of the entire lot.
2.5 Application of Biological Agents
to Test Coupons
Application of B. anthracis (Ames,
Vollum, or NNR1A1) spores was
performed in a BSC III. The large test
coupons (1.9 cm x 7.5 cm) were placed
lying flat in the cabinet and
contaminated at challenge levels of
approximately 1 x 108 CPU of spores per
coupon. A 100 jiL aliquot of a stock
suspension (approximately 1 x 109
CFU/mL) of organisms was dispensed as
shown in Figure 2-5 using a multi-
channel micropipette applied as two
rows of five 10 jiL droplets across the
surface of all the test coupons except for
the small glass, blown cellulose
insulation, and ECBC coupons. The
small glass and blown cellulose
insulation coupons were spiked by a
single 10 jiL drop containing about 107
spores of biological agent or surrogate.
The unpainted pine and painted I-beam
coupons (1.3 cm x 1.3 cm) from ECBC
were spiked with seven-7.1 |iL aliquots
(49.7 |iL total) of a stock suspension of
known concentration of B. anthracis
(Ames orNNRlAl) spores (1.0 x 109
CFU/mL). The 50 |iL spike volume
matched the spike volume used by
ECBC in their tests. (Because results
from the testing at Battelle were being
11
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compared to the testing by EPA/ECBC
to look for potential causes of
differences in test results, potential
sources of variation between the
laboratories, such as volume applied to
coupons, were controlled.) The
application of multiple droplets is
consistent with other testing reported
here, except that only seven droplets,
rather than 10 droplets, were used
because of the small coupon surface.
The coupons spiked with spores were
dried overnight in a BSC III cabinet at
22 °C ± 2 °C and 40% ± 10% RH. The
temperature and RH at the beginning and
end of the drying period were
documented.
The ricin solution was applied onto the
test coupons, lying flat, at approximately
25 jig per test coupon. A 5 jiL aliquot of
a stock suspension (5 mg/mL of ricin)
was dispensed using a micropipette as a
streak across the surface of the test
coupon. The ricin application was
performed in a Class II BSC. The ricin
coupons were left undisturbed in an
appropriate BSC cabinet or container at
ambient laboratory temperature and RH
(22 °C ± 2 °C and 40% ± 10% RH) for 1
hr before use in technology evaluations.
Application of vaccinia virus to test
coupons was performed in a Class II
BSC. Test coupons were placed lying
flat in the cabinet and contaminated at
challenge levels of approximately 1 x
107 PFU of vaccinia virus per coupon. A
100 jiL aliquot of a stock suspension
(approximately 1 x 108 PFU/mL) of
organisms was dispensed using a multi-
channel micropipette applied as two
rows of five 10 jiL droplets across the
surface of all the test coupons except
glass. The small glass coupons were
spiked by a single 10 jiL drop containing
a specified total amount of biological
agent or surrogate. The vaccinia coupons
were immediately transferred into the
test chamber for fumigation.
Except as specified in a given trial, no
additional organisms or organic
materials (organic burden) were added to
the suspensions and stock solutions of
agents and surrogates before application
to the coupons.
Figure 2-5. Multichannel micropipette applying biological agent to a coupon.
12
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2.6 Extracting and Quantifying
Biological Agent and Surrogates
(Quantitative Method)
Aeration followed by dilution in an
extraction buffer was used to terminate
the fumigations. With liquid
decontamination technologies,
neutralization of the technology occurred
at the end of the contact time, at the
same time as the extraction of the
coupon. Neutralization of liquid
decontamination technologies is
described in the section reporting
specific liquid decontamination
technologies. To extract the biological
agent, the coupons were placed
individually into sterile 50 mL conical
vials to which 10.0 mL of sterile
extraction buffer was added. Phosphate-
buffered saline was the extraction buffer
for ricin and vaccinia virus. Phosphate-
buffered saline with 0.1% Triton X-100
(Sigma) was the extraction buffer for
spores. The tubes were agitated on an
orbital shaker for 15 min at
approximately 200 rpm at room
temperature.
2.6.1 B. anthracis Spores
The number of residual viable B.
anthracis spores on test and positive
control coupons was determined using a
dilution plating approach on tryptic soy
agar (TSA). B. anthracis grows well on
ordinary laboratory media.[1] Following
extraction, the extract was removed and
a series of dilutions through a maximum
of 10"7 were prepared in sterile water. An
aliquot (0.1 mL) of the undiluted extract
and each serial dilution were plated onto
TSA plates, shown in Figure 2-6, in
triplicate. The cultures were incubated
for 18-24 hr at 37 °C ± 2 °C. Colonies
(CFU) were counted manually. The
number of CFU/mL was determined by
multiplying the average number of
colonies per plate by the reciprocal of
the dilution. Data were expressed as
mean CFU/mL ± SD based on the
numbers of CFU observed.
Potential confounding organisms were
excluded or controlled by sterilization of
the coupons (by autoclaving or gamma
irradiation), use of sterile technique, and
a pure initial culture. Negative control
coupons were run in parallel with the
inoculated coupons (both test and
positive control coupons). Except as
noted in Section 3.5.3, no CFU were
observed on any blanks so the CFU
observed from inoculated coupons were
assumed to indicate viable B. anthracis
spores and were confirmed by
observation of colony morphology.
13
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Figure 2-6. B. anthracis Ames colonies on TSA.
2.6.2 Ricin
The amount of cytotoxic ricin persisting
on test coupons over time was
determined using a bioassay. The
bioassay used in this investigation for
determining the cytotoxicity of bioactive
ricin was based on the MTT (3-(4,5-
dimethylthiazol-2-yl)-2, 5,-
diphenyltetrazolium bromide) assay
developed by Mosmann (1983).^ The
mechanism of action by which ricin
exerts its toxic effect is through
inhibition of protein synthesis within
cells. Such inhibition of protein
production leads to cell death. Therefore,
an in vitro cytotoxicity assay can be used
to evaluate the cytotoxicity of bioactive
ricin in the applied volume of solution.
Cytotoxicity was reported as remaining
mass of bioactive ricin relative to initial
cytotoxic protein mass applied.
Vero (African Green Monkey kidney)
cells were seeded in wells of a 96-well
microplate at a density of approximately
2 x 104 cells/well. Cells were cultured to
confluence (approximately 16-30 hr) at
37 °C ± 2 °C under 95% air and 5% CO2
and exposed to the coupon extracts.
Following extraction of coupons, 1.0 mL
of the PBS extracts of the ricin test and
control coupons were removed and an 8-
point series of two-fold dilutions was
prepared in complete cell culture
medium. An aliquot (100 |iL) of the
undiluted extract and each serial dilution
were plated onto confluent Vero cells in
the 96-well plate. For quantitation, a
parallel standard curve was prepared
using 2- or 3-fold serial dilutions of 10
ng/mL ricin and plated onto confluent
Vero cells in the 96-well plate.
Following exposure to coupon extracts
for 72 hr at 37 °C ± 2 °C in a
humidified 95% air 5% CC>2 atmosphere,
the cells were incubated in the presence
of MTT (Promega, Madison, WI) for 4
hr, where mitochondrial enzymes within
cells convert the yellow MTT to a purple
formazan salt. The reaction was
terminated by adding 100 jiL of a
Solubilization/Stop Solution (Promega,
Madison, WI) and incubation at 37 °C ±
2 °C in a humidified atmosphere for 1-
24 hr. A SPECTRAmax microplate
reader (Molecular Devices, Silicon
Valley, CA) was used to measure the
absorbance at 570 nm wavelength using
a reference wavelength of 630-750 nm.
For each standard and test sample,
absorbance values of the reference
wavelength (630-750 nm) were
14
-------
subtracted from the absorbance values at
570 nm for each well. Absorbance
values were directly proportional to the
number of viable cells present in the
sample well. For each standard, the
mean absorbance values (Y-axis) were
plotted against the concentration in
ng/mL, and a four-parameter logistic
curve was generated by the software
included in the SPECTRAmax
microplate reader using Equation 1
7 = min+
(max- min)
\ + (XIC)B
Equation 1
where:
X
max
min
B
C
= concentration of ricin, ng/mL
= Y-value of the asymptote at the low values of X, optical density
= Y-value of the asymptote at the high values of X, optical density
= value related to the slope of the curve between the asymptotes
= X-value of the midpoint between max and min, ng/mL.
The absorbance of the purple reaction
product, read at 570 nm using a
microplate reader, was directly
proportional to the number of living
cells. The amount of purple formazan
produced was inversely proportional to
the cytotoxic potential of ricin (Figures
2-7 and 2-8).
To determine the concentration of ricin
toxin from each test sample (i.e., the
mass of ricin toxin extracted in a
specified volume of extraction fluid), the
ricin toxin stock solution (as received
from Vector Laboratories) was assayed
in parallel and used to prepare a standard
curve of absorbance vs. mass of ricin
protein. The absorbance of sample
extracts was plotted onto the standard
curve to determine the mass of cytotoxic
ricin in the sample. Data were expressed
as the concentration of bioactive ricin.
The percent cytotoxic ricin recovery was
calculated.
In the method development, the inherent
cytotoxicity of extracts of the various
types of coupons was determined. The
coupons with background levels of
cytotoxicity were diluted sufficiently
that the cytotoxicity of the extract
(without ricin) killed less than 10% of
the cells in the MTT assay. The dilution
scheme effectively "zeros out" the
cytotoxicity of the test coupons (see
Tables 2-2).
Table 2- 2. Dilution Factors for Various Coupon Materials
Material
Dilution Factors Required to
"Zero Out" Coupon Cytotoxicity*
Glass
Painted Concrete
Galvanized Metal
Decorative Larninate
Cellulose insulation
Particle Board
Industrial Carpet
1:4
1:4
1:4
1:4
1:256
1:32
1:64
* Coupon cytotoxicity is considered to be "zero-ed out" if the optical density readings after cell exposure to
coupon extracts are at least 90% of media alone.
15
-------
Purple = cells alive;
little to no toxin
Increasing ricin
concentration
Yellow = cells dead;
abundant toxin
Figure 2-7. Visual demonstration of MTT assay on a microplate.
Standard Curve
1.4-
1-
0.6-
0.4-
= jalive cells; i
little to no toxin!-f -f
YeUow) 4= jdead cells;
abundant toxin
0.01
0.1
10
Concentration (ngfrnL)
Figure 2-8. Example of ricin cytotoxic profile with corresponding absorbance
measured using microplate reader.
16
-------
2.6.3 Vaccinia Virus
Both vaccinia and variola (smallpox)
viruses are species in the genus
Orthopoxvirus. There is a 96% identity
at the nucleotide level between vaccinia
and variola.^ Given the high levels of
similarity between these viruses,
decontamination of variola is expected
to be similar to that observed for
vaccinia.^ The investigation of the
decontamination efficacy against
vaccinia virus measured the amount of
residual PFU extracted from test and
control coupons. The number of vaccinia
viruses on the coupons was determined
using a dilution plating approach.
Following extraction, 1.0 mL of the PBS
extract was removed and a series of
dilutions through 10~7 was prepared in
sterile water or PBS. An aliquot (100
jiL) of the undiluted extract and each
serial dilution were plated onto
uninfected Vero cells. Following
inoculation, the tissue culture plates
were rocked for approximately 1 hr at 37
°C ± 2 °C to allow adsorption of
vaccinia virus to the Vero cells. The
cultures were then overlaid with
Minimum Essential Medium containing
2% fetal bovine serum, 0.5%
methylcellulose, and antibiotics. The
cultures were incubated for 1-2 days at
37 °C ± 2 °C in 95% air and 5% CO2.
Following incubation, crystal violet dye
was added to the monolayers for 15 min,
removed and the cells rinsed with PBS.
Plaques were visualized as clearings in
the purple monolayer of Vero cells and
the PFU were counted manually. The
number of PFU/mL was determined by
multiplying the average number of
plaques per well by the reciprocal of the
dilution. Data were expressed as mean
PFU/mL ± SD based on the numbers of
PFU observed.
Potential confounding organisms were
excluded or controlled by sterilization of
the coupons, use of sterile technique,
negative control coupons and a pure
initial culture. Negative control coupons
were run in parallel with the inoculated
coupons. No PFU were observed on any
blank control coupons. Therefore, the
PFU observed from inoculated coupons
were assumed to indicate viable vaccinia
virus.
2.7 Broth Culture Assay for Viable
Spores (Qualitative Method)
B. anthracis grows in ordinary
laboratory media, ^ including nutrient
broth (see, for example CDC, American
Society for Microbiology, and
Association of Public Health
Laboratories^), at 37 °C. A qualitative
broth culture assay was used to detect
the presence of viable spores on the Bis
or residual on coupons after extraction.
The Bis or coupons were aseptically
transferred into individual tubes
containing 30 mL of tryptic soy broth
(TSB) culture medium (as specified in
the respective test/QA plans) and
capped. The tubes were cultured for
seven days at 37 °C ± 2 °C to encourage
viable spore germination and subsequent
proliferation of vegetative bacteria.
At one and seven days post-
decontamination, the tubes were visually
assessed for cloudiness. A cloudy culture
medium may indicate "growth" of viable
spores. Clear culture medium indicated
"no growth" and was consistent with a
complete kill of all spores on the BI or
complete kill or extraction of all viable
17
-------
spores on/from the coupon. Data were
expressed as "growth" ("+") or "no
growth" ("-").
In an approach, referred to herein as
qualitative cycle fractionation, coupons
are removed from test conditions at
various contact times during the
decontamination cycle, placed into TSB,
and treated as described in the preceding
paragraphs. Data are reported as
"growth" or "no growth" after a given
contact time. This approach was used
selectively as a screening procedure to
refine or determine test conditions.
Percent recovery (% R) is calculated as:
2.8 Calculations and Statistics
2.5.7 Percent Recovery
Percent recovery for each biological
agent on each test material was
determined prior to investigating
decontamination efficacy. Percent
recovery (mean ± SD) was calculated for
each type of test material inoculated with
each biological agent by dividing the
viable biological agent (spores or
vaccinia virus) or mass of cytotoxic ricin
extracted from control coupons by the
number of biological organisms or ricin
mass applied to each coupon.
%R= - xlOO
[A
Equation 2
where:
x = CFU/mL, PFU/mL or mass of cytotoxic ricin recovered from the control
coupons
A= CFU/mL, PFU/mL or mass of cytotoxic ricin applied to the control
coupons
The quantity applied was determined
during the confirmation of initial titer
using analytical methods described in
Section 2.6. Mean percent recovery was
calculated by summing the replicate
percent recovery values and dividing by
the number of replicates (five).
2.8.2 Decontamination Efficacy
Decontamination efficacy was calculated
as the log reduction (mean ± SD) in
viable biological agent (CFU or PFU) or
percent reduction in mass of cytotoxic
ricin after a given treatment. The higher
the decontamination efficacy (log
reduction or percent reduction) value,
the less biological agent remains on the
test coupon after a given treatment.
The first step in the calculation of
overall decontamination efficacy for
viable biological agents (spores or
viruses) was to determine the efficacy
for each individual coupon in a given set
of replicates. The efficacy was defined
as the extent (expressed as log reduction)
to which the specific agent extracted
from the specific type of coupon after
the treatment with the decontamination
technology at a specific concentration
for a specific contact time (at a given
temperature and RH) was less than what
was extracted from coupons exposed to
the contact time, temperature, and RH
portions of the treatment without
exposure to fumigation. Efficacy of a
given treatment against a specific viable
biological agent on an individual coupon
was calculated in the general form:
18
-------
Efficacy k =
Equation 3
Where:
x = cytotoxicity (|ig), CPU or PFU values of an individual control coupon of a
given type and condition
t = measured cytotoxicity (jig), CPU, or PFU value of an individual test coupon
of a given type and treatment
k = particular coupon type and test condition
/' = number of control coupons
j = number of test coupons
A t-test on the efficacy data was used to
statistically analyze whether a
decontamination treatment resulted in
significant efficacy. The t-test was
performed using PROC TTEST in SAS
v 9.1. It was assumed that the two
underlying populations to be compared
were normally distributed. A folded F-
test was performed to assess whether
they have equivalent variances. If they
had equivalent variances, a pooled
variance estimate was used. Otherwise,
the separate sample variances were used
and the Satterthwaite approximation for
degrees of freedom was employed. The
PROC TTEST generated an estimated
mean log reduction, 95% confidence
interval, and a p-value for statistical
significance. Where the p-value was less
than or equal to 0.05, and assuming a
positive estimate, it was concluded that
the reduction in log recovery due to the
treatment was superior to that of the
controls alone with 95% or greater
confidence. Note that cytotoxicity was
transformed back into the original units
(micrograms) and is reported as a
geometric mean with a 95% confidence
interval. The geometric mean has an
asymmetric 95% confidence interval.
Model diagnostics were examined to
assess whether there were any
difficulties with outliers or the model
assumptions of constant variance and
normality of the residuals. For data that
were not adequate for the model,
appropriate transformations or more
general statistical models (e.g.,
nonparametric) were used. No outlier
was identified and excluded from the
final analysis.
To make comparisons between different
experimental groups (e.g., CTs, Bacillus
strains, humidity levels), the approach
above was extended to a general linear
model with PROC GLM in SAS v 9.1.
If test coupons had zero recovered CFU
or PFU, the base 10 logarithm was
undefined. In these cases, the value of
zero was replaced by 1 CFU or PFU.
The arithmetic average log reduction
was calculated as above, but the reported
value was a conservative reduction, and
was identified as such by "> x" log
reduction in the results. The t-test
assumptions of normality are not valid in
these cases, and the statistical
comparison cannot be done with a t-test.
Instead, a nonparametric approach was
taken. The Kolmogorov-Smirnov test
19
-------
was performed through PROC
NPAR1 WAY in SAS v 9.1. If a p-value
of 0.05 or less was found, the overall
distribution of the treatment was
concluded to be different than the
controls.
If both the control and decontamination
coupons have occurrences of zero
recovered CPU or PFU, the log
reduction becomes indeterminate as
defined above. A statistical test of
differences using the Kolmogorov-
Smirnov procedure was performed with
the interpretation as above.
There were separate statistical analyses
for B. anthracis, B. subtilis, vaccinia
virus, and ricin. The specific factors
included in the analyses vary among
technologies and trials.
The efficacy data for biological
organisms are reported as log reductions.
Efficacy data for ricin toxin are reported
as percent reductions. The primary
decontamination efficacy results from
the coupon testing are shown in a matrix
table in which each entry shows the
"geometric mean log reduction" in
viable organisms or "mean percent
reduction" in ricin cytotoxicity for each
combination of biological agents,
surface materials, concentration, contact
time, temperature, and RH.
Statistical analysis in this section
compares whether the efficacy of the
decontamination treatment at a particular
concentration, contact time, temperature,
RH, and test material was significantly
(p < 0.05) different from zero (null
hypothesis). Additional comparisons
were made of mean efficacy between
materials, contact time, temperature, and
RH. Both means and corresponding p-
values were produced for each
comparison.
2.9 C1O2 Fumigation
2.9.1 Description of CIO2 Technology
The Sabre decontamination technology
in this investigation uses C1O2 as the
active ingredient for decontamination.
C1O2 is not stable as a compressed gas
and, therefore, C1O2 gas must be
produced on-site. For this evaluation,
Sabre Technical Services, LLC,
provided chemicals and a custom-built
bench-scale system for on-site
generation, delivery, removal, and
neutralization of C1O2. The
decontamination technology was
operated as specified an the standard
operating procedure developed with the
vendor and summarized below.
The Sabre equipment includes a 20 cm
base onto which was mounted a 15 cm
square, 91 cm high sparging column. An
aqueous C1O2 solution consisting of
about 3 g/L of C1O2 plus 1000 ppm of
chlorite for a 3000 ppm C1O2 solution
was prepared on-site; however, these
values varied slightly from batch to
batch. Because the C1O2 is sparged into
the test chamber atmosphere, the exact
concentration of the aqueous solution is
not critical. C1O2 solution was pumped
(using a peristaltic pump) into the
sparging column and air from the test
chamber was pumped into and through
the column to sparge the C1O2 from the
liquid into the air stream. The air stream
re-entered the test chamber to establish
the desired gaseous C1O2 concentration.
Liquid introduction from the reservoir of
ClO2/chlorite solution to the sparging
column was initially at the rate of 60 mL
per min; when the desired C1O2
20
-------
concentration in the test chamber was
achieved, the liquid introduction into the
sparging column was turned off. As the
C1O2 concentration dropped, additional
gas was added to the chamber by
manually turning on the flow of air
through the sparging column and back
into the chamber to achieve the target
concentration. The spent liquid exiting
the sparging column was collected in a
reservoir. The air from the test chamber
was recirculated into and out of the
sparging column.
2.9.2 CIO2 Test Matrix
The testing performed in this
investigation is shown in Table 2-3.
Critical parameters include C1O2
concentration, decontamination contact
time, temperature, RH, and the viability
or cytotoxicity of the biological agents.
Efficacy of C1O2 is generally assumed to
be higher at higher concentrations, with
longer contact times, at an RH of 75% or
higher, and at an elevated temperature.
These assumptions were tested. An
adaptive management approach was
used to incorporate new knowledge into
the testing. Based on new knowledge,
EPA revised, added or eliminated trials
and experimental conditions. These
changes were documented in
amendments to the original test/QA plan.
The trial numbers correspond to trials
specified in the test/QA plan and
amendments. The trial numbers are
included for convenience in referencing
back to the test/QA plan and
amendments for details. Numbering gaps
represent deletions by EPA of planned
trials. Numbers with a letter (e.g., 4a)
represent trials that were added by
amendment of the test/QA plan. Results
from all testing under the test/QA plan,
including testing done under
amendments, are included in this report.
Five replicate test coupons (plus one
procedural blank) and five replicate
positive control coupons (plus one
laboratory blank) were included at each
set of conditions and time points except
in Trials 4a, 4b, and 4g. Trials 4a, 4b,
and 4g were run with three replicate test
coupons at each time point, three
replicate positive control coupons, a
procedural blank, and a laboratory
control blank. A smaller number of
replicates were used in the qualitative
screening tests (three) than in the
quantitative tests (five) because no
statistical inferences would be drawn.
Negative controls (procedural blanks and
laboratory blanks) are coupons to which
corresponding diluent, but no biological
agent, was applied.
Decontamination was halted by dropping
the atmospheric concentration of C1O2 to
which the test and procedural blank
coupons were exposed to near zero by
removal of the coupons from the test
chamber into the air inside the BSC III
and then into the extraction medium. No
quenching agents were added to stop the
decontamination reaction because
residual C1O2 was assumed to be rapidly
removed from interaction with the
biological agent through convection,
diffusion, and dilution.
Trials 1-3. The experimental design
tested whether there was a difference
between the decontamination efficacy
using the treatment compared to the
control. For any particular material, the
comparisons included efficacy under
given environmental conditions
(temperature and humidity) at a
particular CT (concentration of fumigant
21
-------
x contact time) inactivating a particular
biological agent. The dependent variable
was extracted residual viable spores
measured as CPU, compared to controls.
Tests were performed with five replicate
test coupons and five replicate positive
controls for each set of conditions and
time points.
Trials 1 through 3, shown in Table 2-3,
used B. anthracis (Ames) spores on
painted concrete to screen for the
impacts of temperature, concentration,
and decontamination contact time on log
reduction in viable spores. The target
C1O2 concentrations ([C1O2]) were 3,000
ppmv, 1,500 ppmv, or 750 ppmv,
respectively. The target RH was >75%.
TO, for all trials, was the time that the
C1O2 reached the target concentration.
The target temperature was 24 °C.
Trial 4, shown in Table 2-3, was run at
the shortest contact time from Trial 1
with 100% kill (no CPU recovered from
any coupon type). The target 3,000
ppmv C1O2 fumigation was used to
decontaminate B. anthracis (Ames)
spores from eight materials. This
concentration was selected for
application against a broad range of
materials because 3,000 ppmv C1O2 is
the typical application in the field. The
target temperature was 24 °C.
22
-------
Table 2-3. Test Matrix for ClOi Decontamination Investigation
Trial
Agent
Material
CT,
ppmv-Hr
Contact Target
Time, [C1O2]
Hr ppmv
Target
Temperature
Target
RH
Number of Coupons
per Material Type and Condition
Test
Lab
Procedural RJ Positive
Coupons Blank Control* Blank
1 B.anthracis Painted 3000 u±2^ >?5%
Ames concrete
1 B.anthracis Painted 3000 ^±2^ >?5%
Ames concrete
1 B.anthmcis Painted 90Q() 3 3QQQ ^±^ >?5%
Ames concrete
2 A75%
Ames concrete
2 A75%
Ames concrete
2 B.anthmcis Painted 90Q() g ^ ^±^ >?5%
Ames concrete
3 B.anthmcis Painted 5() 3 ?5() ^±^ >?5%
Ames concrete
3 B.anthracis Painted g 24±20C >?5%
Ames concrete
3 B.anthmcis Painted 9QQQ u JSQ ^±^ >?5%
Ames concrete
4 A^Arac« Eig.ht 9000 3 3000 24±2oC >75o/o
Ames matenals*
5
5
5
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
5
5
5
5
5
5
5
5
5
5
1
1
1
1
1
1
1
1
1
1
* Eight test materials are small glass, painted concrete, galvanized metal, decorative laminate, cellulose insulation, particle board, industrial carpet, large glass.
f Seven test materials are small glass, painted concrete, galvanized metal ductwork, decorative laminate, cellulose insulation, particle board, and industrial carpet.
*Five positive control coupons extracted at time 0 (the time at which decontamination treatment begins) and five positive control coupons extracted at the end of the treatment time.
Bold: factor varied in trial series
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
23
-------
Table 2-3. Continued
Trial
Agent
Material
„„ Contact Target
' Time, [C1O2]
ppmv-Hr __ ' l J
rr Hr ppmv
Target
Temperature
Number of Coupons
per Material Type and Condition
RTT
Test Procedural RT Positive Lab
Coupons Blank "" Control Blank
5
6
6
B.
anthracis
Ames
B.
anthracis
Ames
B.
anthracis
Ames
materials!
Seven
materials!
except
cellulose
insulation
Cellulose
insulation
9000 3 3000
1000 0.33 3000
12,000 4 3000
30 °C ± 2 °C
24 °C ± 2 °C
24 °C ± 2 °C
>75%
>75%
>75%
5 1151
5 1
1
5
1
5 1151
9 Ricin ^lgh^ „ 500 0.33 1500 24°C±2°C >75%
matenals*
10 Ricin ^lgh^ „ 100 0.5 200 24 °C ± 2 °C >75%
matenals*
13 Vaccinia ^lgh^ * 500 0.33 1500 24°C±2°C >75%
matenals*
14 Vaccinia ^lgh^ * 125 0.5 250 24 °C ± 2 °C >75%
matenals*
5
5
5
5
1
1
1
1
1
1
1
1
10*
10*
10*
10*
1
1
1
1
* Eight test materials are small glass, painted concrete, galvanized metal, decorative laminate, cellulose insulation, particle board, industrial carpet, large glass.
f Seven test materials are small glass, painted concrete, galvanized metal ductwork, decorative laminate, cellulose insulation, particle board, and industrial carpet.
*Five positive control coupons extracted at time 0 (the time at which decontamination treatment begins) and five positive control coupons extracted at the end of the treatment time.
24
-------
Table 2-3. Continued
Trial
Agent
Material
CT,
ppmv-
Hr
Contact
Time, Hr
Target
[C102]
ppmv
Number of Coupons
per Material Type and Condition
Target
Temperature
Target
RH
Test
Coupons
Procedural
Blank
Positive Lab
Control* Blank
4c
4d
4e
B. anthracis
Ames
&
B. atrophaeus
Bis
B. anthracis
Ames with 0.5%
bovine serum
albumin &
B. atrophaeus
Bis
B. anthracis
Ames with 0.5%
bovine serum
albumin (ceiling
tilie only),
B. anthracis
NNR1A1
with 0.5%
bovine serum
albumin,
&
B. atrophaeus
Bis
ECBC3.lx3.lcm
coupons of ceiling
tile, unpainted pine 3000 1 3000 24±2°C >75% 5 1
wood, and painted I-
beam steel
ECBC3.lx3.lcm
coupons of ceiling >75%
tile, unpainted pine 3000 1 3000 24±2°C (83%
wood, and painted I- actual)
beam steel
ECBC3.lx3.lcm
coupons of ceiling
tile, unpainted pine 3000 l 3QQQ 24±20C 75%
wood, and painted I-
beam steel
5
5
1
1
10
10
10
1
1
1
* Eight test materials are small glass, painted concrete, galvanized metal, decorative laminate, cellulose insulation, particle board, industrial carpet, large glass.
f Seven test materials are small glass, painted concrete, galvanized metal ductwork, decorative laminate, cellulose insulation, particle board, and industrial carpet.
*Five positive control coupons extracted at time 0 (the time at which decontamination treatment begins) and five positive control coupons extracted at the end of the treatment time.
25
-------
Table 2-2. Continued
Trial
4a
Agent Material
Painted concrete,
galvanized metal
„ ,, . . ductwork, decorative
B. anthracis Ames , . , ,, ,
laminate cellulose
insulation, particle board,
and plate glass.
Target
[C102]
ppmv
750
Target
Temperature
24 ± 2 °C
Target
RH
>75%
Decontamination Contact Times and Number
of Coupons of Each Material
Three replicates of each material at 0, 10, 20,
30, 40, 50, 60 min are placed into TSB to test
for viable spores. (Qualitative test)
4b
4g
4g
4g
B. anthracis Ames
B. anthracis Ames
B. subtilis
B. subtilis
B. atrophaeus
B. atrophaeus
Painted concrete,
galvanized metal
ductwork, decorative
laminate, cellulose
insulation, and plate glass.
Plate glass
Spores on stainless disk
in Tyvek" envelope
Tyvek* envelope, spores
on stainless disk removed
from Tyvek* envelope)
Spore on filter paper
strips in glassine
envelope
Spore on filter paper
strips removed from
glassine envelope
3000
3000
3000
3000
3000
3000
24 ± 2 °C
24 °C ± 2 °C
24 °C ± 2 °C
24 °C ± 2 °C
24 °C ± 2 °C
24 °C ± 2 °C
Three replicates of each material at 0, 20, 40,
>75% 60, 80, 100, 120 min are placed into TSB to test
for viable spores. (Qualitative test)
Three replicates of each material/ spore
combination at 0, 30, 60, 90, and 120 min are
70%-75% placed into TSB to test for viable spores.
(Qualitative test)
70%-75%
70%-75%
70%-75%
70%-75%
Three replicates of each material/ spore
combination at 0, 30, 60, 90, and 120 min are
placed into TSB to test for viable spores.
(Qualitative test)
Three replicates of each material/ spore
combination at 0, 30, 60, 90, and 120 min are
placed into TSB to test for viable spores.
(Qualitative test)
Three replicates of each material/ spore
combination at 0, 30, 60, 90, and 120 min are
placed into TSB to test for viable spores.
(Qualitative test)
Three replicates of each material/ spore
combination at 0, 30, 60, 90, and 120 min are
placed into TSB to test for viable spores.
(Qualitative test)
* Eight test materials are small glass, painted concrete, galvanized metal, decorative laminate, cellulose insulation, particle board, industrial carpet, large glass.
f Seven test materials are small glass, painted concrete, galvanized metal ductwork, decorative laminate, cellulose insulation, particle board, and industrial carpet.
*Five positive control coupons extracted at time 0 (the time at which decontamination treatment begins) and five positive control coupons extracted at the end of the treatment time.
26
-------
Trials 4a-4b. In order to determine the
CT at which zero growth was observed
in a qualitative spore viability test, cycle
fractionation tests (Table 2-3) were
performed. In a cycle fractionation test,
the maximum fumigation contact time
(decontamination cycle) was divided
into a series of shorter contact times by
administering a given concentration of
fumigant and terminating the exposure
time at various intervals up to the full
CT being evaluated. The presence of
viable spores was evaluated by
qualitative analysis (described in Section
2.7).
Indoor building material coupons were
spiked with approximately 1 x 108 B.
anthracis Ames spores. The spiked
coupons were exposed to a known
concentration of C1O2 (750 ppmv or
3000 ppmv) at a target RH of >75% and
a target temperature of 24 °C ± 2 °C. At
specified time intervals within the
decontamination cycle, three spiked
coupons of each material type were
removed from the test conditions and
placed into nutrient broth and incubated
at 37 °C up to 7 days to perform a
qualitative assay for viable spores
(described in Section 2.7). The time
interval for removal of spiked coupons
was 10 or 20 min over a total period of
60 or 120 min for Trials 4a and 4b,
respectively.
Trials 4c-4e. The EPA was investigating
the effectiveness of C1O2 gas for the
inactivation of Bacillus species spores in
conjunction with ECBC and at Battelle.
The preliminary results from the two
organizations were shared and appeared
to be inconsistent. These trials were
added to the original test matrix so that
Battelle could replicate the ECBC
testing to determine the causes of
differences between C1O2 efficacy
results obtained at Battelle and those
obtained at ECBC.
Three types of indoor building material
coupons previously used in testing at
ECBC were provided to Battelle by
ECBC for use in trials 4c, 4d and 4e.
The coupons, measuring 3.1 cm x 3.1
cm, include ceiling tile, unpainted pine
wood, and painted I-beam steel.
In Trial 4c, B. anthracis Ames spores
(preparation used at Battelle without
added organic burden) were spiked onto
material coupons used in the EPA/ECBC
decontamination testing. In Trial 4c, 15
coupons of each material type were
spiked with seven 7. l-|iL aliquots (49.7
|lL total) of a stock suspension of known
concentration of B. anthracis Ames
(CFU/mL), prepared as described in the
test/QA plan. The spiked coupons were
dried overnight and then placed into the
glove box, described in Section 2.2.1.
The coupons were exposed to a known
concentration of C1O2 (3000 ppmv) for a
specified contact time (1 hr or 3 hr).
After specified contact time intervals,
five spiked coupons of each material
type were removed from the test
conditions, extracted, and enumerated as
described in Section 2.6.1. One blank
control and five positive control coupons
were run at TO and in parallel with the
test coupons.
In the tests at ECBC, organic burden was
added to the spore suspension (0.5%
bovine serum albumin); Battelle testing
does not typically add organic burden to
the spore suspension. Battelle performed
Trial 4d to evaluate the impact on
efficacy of adding organic burden when
compared to Trial 4c without organic
burden. In Trial 4d, organic burden
27
-------
(0.5% bovine serum albumin) was added
to the B. anthracis Ames spores
(preparation used at Battelle), and the
spores were spiked onto EPA/ECBC
material coupons for use in
decontamination testing. This procedure
provided a comparable level of organic
burden to the EPA/ECBC spore
preparations and the spores were applied
to EPA/ECBC material coupons.
In Trial 4d, fifteen coupons of each
material type were spiked with seven
7.1-|oL aliquots (49.7-|iL total) of a
stock suspension of known concentration
of B. anthracis Ames, prepared as
described in the test/QA plan but with
0.5% bovine serum albumin as added
organic burden. The spiked coupons
were dried overnight and then placed
into the glove box, described in Section
2.2.1. The coupons were exposed to a
target concentration of C1O2 (3000
ppmv) at an RH >75% and a temperature
of 24 °C ± 2 °C for 1 hr. After the 1-hr
contact time, five spiked coupons of
each material type were removed from
the test conditions, extracted, and
enumerated as described in the test/QA
plan. One blank control and five positive
control coupons were run at TO and in
parallel with the test coupons.
EPA/ECBC uses B. anthracis NNR1 Al
spores with organic burden for some
efficacy testing; testing is performed at a
75% RH. In Trial 4e, Trial 4d was
replicated, but using the B. anthracis
NNR1A1 with 0.5% bovine serum
albumin provided by ECBC and B.
anthracis Ames with 0.5% bovine serum
albumin. Further, the RH was controlled
at about 75%, a lower RH than used in
previous testing (Trials l-4b) in the
studies conducted at Battelle, but
consistent with testing performed at
ECBC. The target RH was selected
based on the results of Trial 4d. The
actual RH in Trial 4d was 83% and
condensation was visible on the test
chamber. The lower RH in Trial 4e was
selected to avoid visible condensation.
Trials 4d and 4e compared the "Battelle
conditions" of B. anthracis Ames at high
RH with visible condensation to the
"ECBC conditions" of B. anthracis
NNR1A1 with 0.5% bovine serum
albumin at 75% RH (no visible
condensation). In addition, 5 Bis (B.
atrophaeus on stainless steel in a
Tyvek® pouch (Apex Laboratories))
were included at TO and at the one-hr
time point.
Trial 4g. In Trial 4g, qualitative testing
determined the CT at which no viable
spores were detected on various
materials when the RH was controlled in
a target range of 70%-75%, a lower RH
than was used in previous trials (>75%).
At this lower RH, no liquid water was
present in the test chamber. At higher
RH conditions of previous trials,
moisture and condensation were
sometimes observed in the test chamber.
The decontamination cycle fractionation
exposed spores to a given concentration
of fumigant and for exposure time at
various intervals up to the full CT being
evaluated. The presence of viable spores
on coupons after a given CT exposure
was determined by incubating the
coupons in nutrient broth and checking
for cloudiness that would indicate
growth from viable spores.
Plate glass coupons (three per time
point) were each spiked with a 100 |iL
aliquot of a stock suspension of known
concentration of B. anthracis Ames. The
spiked coupons were placed into the test
chamber along with the following Bis:
28
-------
• Bacillus atrophaeus spores on
filter paper strips in glassine
envelopes (Raven Biological
Laboratories)
• Bacillus atrophaeus spore strips
on filter paper strips removed
from glassine (Raven Biological
Laboratories)
• Bacillus subtilis on stainless disk
in Tyvek® pouches (Apex
Laboratories)
• Bacillus subtilis on stainless disk
removed from Tyvek® pouches
(Apex
Laboratories).
The coupons were exposed to 3000
ppmv C1O2 in Trial 4g. The target
temperature was
24 °C ± 2 °C, and the RH was controlled
between 70% and 75% for the total
decontamination cycle. After reaching
the target RH but prior to introducing the
test coupons into the test chamber, strips
of 3M Water Contact Indicator Tape
(3M, St. Paul, MN) were placed into the
test chamber for 15 min. The indicator
tape was removed and examined to
determine whether moisture (liquid
water) was present. The results of the
indicator tape were documented.
B. anthracis exposures were
accomplished by having the spiked
coupons in closed vials in the test
chamber and using the gloves in the
glove box to open them in sequence so
that an appropriate contact time for
exposure to C1O2 was achieved. Contact
time was the time from opening the vial
containing the spiked coupon in the
fumigation test chamber until the coupon
was removed from the test chamber.
Time zero coupons were in sealed vials
in the test chamber for the full
fumigation cycle and removed from the
chamber without opening the vials; there
was no exposure of the TO coupons to
the fumigant.
Trial 5. The experimental design tested
decontamination efficacy by determining
whether there was a difference between
the log reduction in viable B. anthracis
Ames spores after fumigation compared
to positive controls. Efficacy was tested
for spores on a variety of types of indoor
building materials. These trials also
assessed whether there was any
difference in efficacy at a target
temperature of 30 °C ± 2 °C rather than
24 °C ± 2 °C (used in Trial 4) during
decontamination. The target RH was
75%-85%. The fumigation was 3000
ppmv for 3-hr (CT of 9000 ppmv-hr).
Trial 6. The experimental design tested
decontamination efficacy at alternative
contact times. Efficacy was tested for
spores on a variety of types of indoor
building materials. These trials also
assessed efficacy at a target temperature
of 24 °C ± 2 °C during decontamination,
target RH was >75% (desirable range of
75% -85%). The fumigation was 3000
ppmv for 20 min (0.33-hr, CT of 1000
ppmv-hr) for all materials except
cellulose insulation. Cellulose insulation
was fumigated for 4 hr.
Trials 9 and 10. The experimental design
tested whether there was a difference
between the decontamination efficacy
against ricin toxin using the treatment
compared to the positive controls.
Efficacy was tested for ricin on a variety
of types of indoor building materials.
The temperature was 24 °C ± 2 °C and
the RH was controlled at 80% ± 5%.
These trials were run at two CTs: 500
ppmv-hr (Trial 9) and 100 ppmv-hr
(Trial 10).
29
-------
Trials 13 and 14. The experimental
design tested whether there was a
difference between the decontamination
efficacy against vaccinia virus using the
treatment compared to the positive
controls for each coupon material.
Testing was performed using coupons of
a variety of types of indoor building
materials. The temperature was 24 °C ±
2 °C and the RH was controlled at 80%
± 5%. These trials were run at two CTs:
500 ppmv-hr (Trial 13) and 125 ppmv-hr
(Trial 14).
2.10 MeBr Fumigation
2.10.1 Description of MeBr Technology
MeBr has been registered by EPA for
soil fumigation (injected into the soil
before a crop was planted to effectively
sterilize the soil), commodity treatment
(used for post-harvest pest control),
structural pest control (used to fumigate
buildings for termites, and warehouses
and food processing facilities for insects
and rodents), and quarantine uses (used
to treat imported commodities). Target
MeBr fumigant concentrations ([MeBr])
and contact times vary with the
commodity or structure being treated,
the target pest, and temperature. MeBr is
an effective pesticide because it acts as a
methylating agent that disrupts an
organism's internal chemical reactions.
However, the use of MeBr has been
phased out by EPA under the Clean Air
Act due to its being recognized as an
ozone depleting substance. Use, now,
requires an exemption by the EPA under
appropriate provisions in the Clean Air
Act (http://www.epa.gov/ozone/mbr/).
Research on the use of MeBr to
inactivate Bacillus species includes:
• Kolb and Schneiter (1950)[8] used
spores of six virulent cultures of
B. anthmcis, which were
identified only as being strains of
canine, bovine, human, and ovine
origin. Tests were conducted at
room temperature with MeBr
concentrations of 3,400 to 3,900
mg/L for 1 to 72 hr; all moist
spores (filter paper inoculated
with spore suspensions
immediately before exposure)
were inactivated after 24 hr-72 hr
exposures, whereas some dry
spores (filter paper inoculated
with spores and dried at 65 °C-75
°C for 3 to 4 hr prior to exposure)
survived. The study did not
specify the strains of B. anthracis
used, specify RH conditions, use
building materials used in this
investigation, evaluate the effects
of temperature, or calculate
decontamination efficacy by log
reduction.
• Schade and King (1977)[9]
conducted decontamination
testing with MeBr and spores of
B. megaterium B-938 and B.
subtilis var. niger (recently
reclassified as B. atrophaeus^)
applied to filter paper. Only the
highest MeBr concentration
tested (64 mg/L) at the highest
temperature tested (35 °C)
showed appreciable activity
against the spores (the exposure
period was 18 hr and the RH was
32%). The test results indicated
that high temperature (e.g., 35
°C) and moderately low RH
(e.g., 20% RH) were favorable
for the sporicidal activity of
MeBr. The study results were
provided as number of surviving
spores on membrane filters
30
-------
(decontamination efficacy was
not presented), testing was not
conducted with B. anthracis., and
the test did not use building
materials used in this
investigation.
• Weinberg et al. (2004)[11]
reported that a MeBr minimum
effective dose of 80 mg/L was
lethal to 107 spores of B.
anthracis (specifically, nine
different strains: ATCC 10,
ATCC 937, ATCC 4728, ATCC
11966, AMES-RIID, ANR-1,
Sterne, ATCC 14187, AMES-1-
RIID) on glass slides after a 48
hr exposure at 37 °C. Similar
testing with B. atrophaeus and B.
thuringiemis showed that these
bacteria were more resistant to
MeBr with minimum effective
doses of >112 mg/L. Additional
fumigation testing with a MeBr
concentration of 120 mg/L at 27
°C resulted in complete mortality
of two strains and reductions of
seven others. The efficacy of
MeBr was reduced at the lower
temperature, even when spores
were exposed to a 50% higher
MeBr concentration. Although
RH was monitored during the
test, the RH level during
decontamination was not
reported.
• Weinberg and Scheffrahn
(2004)[12]'conducted a field trial
within a 30,000 cubic foot
structure. Filter paper coupons
containing 106 spores of one of
three species, Geobacillus
stearothermophilus, B.
atrophaeus and B. thuringiensis,
and stainless steel coupons with
106 spores of B. atrophaeus were
placed in 50 locations within the
structure. After fumigation with
312 mg/L of MeBr for 48 hr at
35.5 °C (the overall mean RH
was 76%), only one location (a
sealed refrigerator) contained
viable spores of B. atrophaeus on
a single coupon.
2.10.2 Test Matrix for MeBr Fumigation
The research conducted by Weinberg et
al. (2004)[11] and Weinberg and
Scheffrahn (2004)[12] provides the most
pertinent information for the
investigations described in this report,
which applied B. anthracis Ames to new
materials (the Weinberg studies only
used glass, filter paper, and stainless
steel coupons); used a range of MeBr
concentrations above and below the
minimum effective dose of 80 rng/L^11^
and further investigated the potential
influence of RH. Critical parameters
include MeBr concentration,
decontamination contact time,
temperature, RH, and the viability of B.
anthracis Ames and surrogate spores.
The MeBr test matrix is shown in Table
2-4. The preliminary temperature (36
°C) and RH (75%) selected for the test
matrix were based on those
demonstrated to be effective by
Weinberg and Scheffrahn'12^
(temperature 35.5 °C and 76% RH).
Lower temperatures (25 °C) and RH
(40%) were also included in the test
matrix.
The theoretical MeBr concentrations
selected for this investigation were 80,
160, and 320 mg/L, consistent with the
effective levels applied in previous
research summarized above, and at
levels that may be sustained within a
building. The theoretical mass sufficient
to generate MeBr at these concentrations
-------
in the test chamber was introduced to the
test chamber and the concentration was
measured. The measured concentrations
were lower than the theoretical
concentrations. Thus, introduction of a
mass of MeBr theoretically sufficient to
generate a concentration of 80 mg/L
theoretical mass yielded a measured
concentration of about 53 mg/L when
measured; 160 mg/L theoretical was
about 105 mg/L when measured; and
360 mg/L theoretical was about 212
mg/L when measured. The target
measured MeBr concentrations were 53
mg/L, 105 mg/L and 212 mg/L. The
results were reported based on measured
MeBr concentrations.
32
-------
Table 2-4. Test Matrix for Generation of MeBr Decontamination CT Curves against B. anthracis Ames Spores and Surrogates
Trial
Material
Target
„„ ,, „ Contact „„ „ , Target
CT, mg/L-Hr „. , [MeBr] _, 6 ,
w Time, hr . Temperature
Target
RH
Number of Coupons
per Material Type and Condition
Test Procedural RT Positive Lab
Coupons Blank Control Blank
1 Glass, ceiling tile 2520 24 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
2,13 Glass, ceiling tile 126° 12 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
3 Glass, ceiling tile 315 3 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
4 Glass, ceiling tile 63° 6 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
5 Glass, ceiling tile 945 9 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
6 Glass, ceiling tile 1272 6 2 12 ±10% 36± 1 °C 75% ± 5% (full scale)
7 Glass, ceiling tile 1908 9 212 ±10% 36± 1 °C 75% ± 5% (full scale)
8 Glass, ceiling tile 1484 7 2 12 ±10% 36± 1 °C 75% ± 5% (full scale)
9 Glass, ceiling tile 1484 7 2 12 ±10% 36± 1 °C 40% ± 5% (full scale)
10 Glass, ceiling tile 795 15 53 ± 10% 36± 1 °C 75% ± 5% (full scale)
11 Glass, ceiling tile 954 18 53 ± 10% 36± 1 °C 75% ± 5% (full scale)
12 Glass, ceiling tile 1272 24 53 ± 10% 36± 1 °C 75% ± 5% (full scale)
5
5
5
5
5
5
5
5
5
5
5
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
5
5
5
5
5
5
5
5
5
5
5
5
1
1
1
1
1
1
1
1
1
1
1
1
33
-------
Table 2-4. Continued
Trial
Material
CT, Contact
mg/L-Hr Time, Hr
Target
[MeBr]
mg/L
Target
Temperature
Target
RH
Number of Coupons
per Material Type and Condition
Test
Lab
Procedural RT Positive
Coupons Blank Control Blank
14 Seven U6Q u 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
materials
15 Seven 19Q8 g 212 ±10% 36± 1 °C 75% ± 5% (full scale)
matenals
16 Seven Ig9() lg 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
matenals
17 Seven 15?5 15 105 ± 10% 36± 1 °C 75% ± 5% (full scale)
matenals
18 Elght. . 1908 9 212 ±10% 25 ± 2 °C 75% ± 5% (full scale)
matenals
19 Elght, . 5088 24 2 12 ±10% 25 ± 2 °C 75% ± 5% (full scale)
matenals
5
5
5
5
5
5
1
1
1
1
1
1
1
1
1
1
1
1
5
5
5
5
5
5
1
1
1
1
1
1
'Glass, ceiling tile, cellulose insulation, painted concrete, industrial carpet, galvanized metal ductwork, and decorative laminate. Five silk suture loop carriers prepared per
AOAC 966.04 (1 x 104 to 1 x 105) were included in the test chamber during the decontamination; these were evaluated using the qualitative method to test for the presence
of viable spores. In addition, B. subtilis (prepared per AOAC 966.04 Method 2) on three glass coupons (5 mm x 5 mm) was included in the test chamber during the
decontamination; these coupons were evaluated using the quantitative method to enumerate viable spores.
' Glass, ceiling tile, cellulose insulation, painted concrete, industrial carpet, galvanized metal ductwork, decorative laminate, pine wood. Five Bis that are B. atrophaeus on
steel in Tyvek packaging (Raven).
Note [MeBr] indicates the MeBr concentration in the chamber air, in the units indicated.
34
-------
The fumigant technology investigated
was 99.5% pure MeBr with
approximately 0.5% chloropicrin added
as a warning odorant. Results were
reported as log reduction in viable spores
recovered from coupons after a specified
contact time and temperature. Five
replicate test coupons (plus one
procedural blank) and five replicate
positive control coupons (plus one
laboratory blank) were included at each
set of conditions and time points.
Positive control coupons and laboratory
blanks were run under the same
conditions (temperature and RH) and
extracted at the same time points as the
test coupons. Negative controls
(procedural blanks and laboratory
blanks) were coupons to which
corresponding diluent, but no biological
agent, was applied. Because coupons
could not be removed from the test
chamber while it was charged with
MeBr, each contact time constituted a
separate fumigation cycle.
The initial concentration of MeBr was
established by specifying the
concentration, flow rate and time for the
injection of MeBr into the test chamber.
Theoretical concentrations (based on
perfectly stirred reactor [PSR] modeling
performed by EPA) are shown Table 2-
5. For the MeBr investigation,
theoretical MeBr concentrations of 80,
160, and 320 mg/L were selected for the
fumigations. The PSR results were used
to establish the charging times required
to yield these theoretical MeBr
concentrations.
Table 2-5. Formula, Model Parameters, and Relevant Model Results
V(dC/dt) = Q(Cin - C)
Q= 1 L/min = 0.0167L/sec
V = 23L
Cm = 3711.8 mg/L
t [sec]
30
31
60
61
124
125
C [mg/L]
79.8
82.5
157.9
160.5
319.0
321.4
A Fumi scope MeBr meter (Key
Chemical & Equipment, Clearwater,
FL), of the type used in the field to
monitor MeBr concentrations, was used
in this investigation to take reading of
the MeBr concentration in the test
chamber. Fumiscope meter readings
were taken after the appropriate charging
time for each of the selected theoretical
concentrations (80, 160, and 320 mg/L).
The charging and Fumiscope meter
measurements of MeBr concentration
were repeated twice. The means of the
initial concentrations, ±10%, were used
as the target levels for fumigation in this
investigation. For example: Based on the
35
-------
PSR model, the desired MeBr
concentration of 160 mg/L is achieved
by introducing 1 standard liter per min
of 99.5% MeBr for 61 seconds into a
23 L chamber. At the end of the 61
second injection, the concentration in the
chamber was measured using the
Fumiscope meter. The initial meter
reading was about 105 mg/L. Therefore,
the concentration that was maintained in
the subsequent investigation, based on
the Fumiscope meter reading at 105
mg/L ± 10%, was a Fumiscope meter
range of 95 - 116 mg/L for the specified
contact time. The test matrix, shown in
Table 2-4, shows the target MeBr
concentration range based on target
Fumiscope meter readings. The target
concentrations of MeBr used were 53
mg/L ± 10%, 105 mg/L ± 10%, and 212
mg/L± 10%.
A comparison, repeated twice, was made
between measurements using a total
hydrocarbon analyzer (Model M-20,
VIG Industries, Anaheim, CA) and the
Fumiscope meter. The Fumiscope meter
has a positive bias (compared to the total
hydrocarbon analyzer) of 10%-20% over
the range of interest. Thus, a mean
Fumiscope measurement of 53 mg/L
corresponded to a mean measurement of
49 mg/L using the total carbon analyzer;
a 104.5 mg/L Fumiscope measurement
corresponded to a mean measurement of
90.9 mg/L using the total carbon
analyzer; and a mean Fumiscope
measurement of 211.5 mg/L
corresponded to a mean measurement of
179 mg/L using the total carbon
analyzer. The MeBr measurements
shown in Table 2-4 and in the remainder
of this document reflect target or actual
MeBr measurements made using the
Fumiscope meter during the
investigation. Use of Fumiscope-
generated data maximizes the utility of
the results for field applications in which
a Fumiscope meter would likely be used.
The experimental treatments in Trials 1
through 13, shown in Table 2-4,
generated data to prepare CT efficacy
curves for B. anthracis Ames spores on
two types of materials at three different
concentrations of MeBr. The coupons
used were glass and ceiling tile. Except
for Trial 9, in Trials 1 through 13, RH
was 75% ± 5% (full scale) and
temperature was 36 °C ± 1 °C. In Trial 9
the target RH was 40% ± 5% (full scale).
Time zero for all trials, was the time that
the MeBr reached the target
concentration.
The experimental treatments in Trials 14
through 17, also shown in Table 2-3,
generated data necessary to prepare CT
efficacy curves for MeBr fumigation of
B. anthracis Ames spores on seven
material types. The RH was 75% ± 5%
(full scale) and temperature was 36 °C ±
1 °C. Two MeBr concentrations and
three contact times were included in the
investigation. The seven types of coupon
materials used were: glass, painted
concrete, galvanized metal, decorative
laminate, cellulose insulation, ceiling
tile, and industrial carpet. In addition,
silk suture loops of the type utilized for
efficacy testing in the AOAC 966.04
method'13^ were included in qualitative
testing.
2.11 HP Fumigation
The STERIS VHP® Generator Series
1000ED (STERIS Corporation, Mentor,
OH) was used to introduce hydrogen
peroxide (HP) vapor and control the
fumigation cycle parameters. Because
HP vapor is not stable as a compressed
36
-------
gas, HP must be produced on site by
vaporization of concentrated aqueous
solutions of HP. Thus, this technology
includes the equipment and chemicals
for on-site generation, dispersion, and
neutralization of the HP vapor.
The HP fumigation technology operates
at ambient temperature and atmospheric
pressure in a closed loop configuration.
As depicted in Figure 2-9 (from STERIS
literature), the testing chamber was
subjected to four phases:
dehumidification, condition,
decontamination, and aeration. During
dehumidification the RH was reduced by
the HP fumigation technology by re-
circulating the air through a reusable or
disposable desiccant cartridge. Once the
desired RH was reached, the automated
STERIS VHP® system injected HP
vapor at the rate programmed into the
system to achieve the desired
concentration of HP vapor inside the
chamber. The STERIS VHP® system
then maintained the set concentration for
the desired contact period for
decontamination of the biological agent.
Once the decontamination phase was
completed, the STERIS VHP® system
automatically re-circulated the test
chamber air through the STERIS VHP®
system to reduce the HP vapor
concentration to the desired level
necessary to safely open the test
chamber.
Typical Biodecontamination Cycle
More
A
c
c
3
CL
L
Less
, / \
\ !-jf ' N.
\ f N
y •\:::--
'"•-
^" 100%
[Condensation
Point]
£
£
15
I
-- Relatve Humidity ^^^^^^H Condition
^^^^^^=- Condensaton Point ^^^^^^H Sterilization
Figure 2-9. STERIS VHP® Biodecontamination Cycle.
[14]
The experimental design tested whether
there was a difference between the
number of viable B. anthracis Ames
spores recovered from coupons after
exposure to the STERIS VHP®
fumigation treatment (test coupons)
compared to the number of viable spores
recovered from positive control coupons
that were not exposed to the fumigation
treatment. Results are reported as log
reduction in viable spores recovered
from coupons after a specified contact
37
-------
time and temperature. Five replicate test
coupons (plus one procedural blank) and
five replicate positive control coupons
(plus one laboratory blank) were
included at each set of conditions and
time points. Positive control coupons
and laboratory blanks were run under the
same conditions (temperature and RH)
and extracted at the same time points as
the test coupons. Negative controls
(procedural blanks and laboratory
blanks) were coupons to which
corresponding diluent, but no biological
agent, was applied.
During the test cycle, the spiked coupons
were in closed vials in the test chamber.
Using the gloves in the glove box the
vials were opened in sequence and the
coupon removed from each vial so that
an appropriate contact time for exposure
to HP was achieved. Contact time was
the time from opening the vial
containing the spiked coupon in the
fumigation test chamber until the end of
the sterilization cycle. One set of spiked
test coupons, along with procedural
blank coupons, was not in vials, but was
in the test chamber exposed to the full
fumigation cycle. Time zero positive
control coupons were in sealed vials in
the test chamber for the full fumigation
cycle and removed from the chamber
without opening the vials; there was no
exposure of the TO coupons to the
fumigant.
The experimental treatments, shown in
Table 2-6, generated data to prepare CT
efficacy curves for B. anthracis Ames
spores on twelve types of materials at
four different CTs and a full 4-hr
fumigation cycle. The twelve types of
coupon materials used were: finished
aluminum, computer keyboard keys,
industrial grade carpet, painted joint
tape, decorative laminate, galvanized
metal ductwork, latex-painted concrete,
pine wood, plate glass, ceiling tile,
particle board, and compressed cellulose
insulation. The CTs (concentration x
time) that were used for the HP
fumigation testing were: 250 ppmv-hr
(500 ppmv x 0.5 hr), 500 ppmv-hr (500
ppmv x 1 hr), 1,000 ppmv-hr (500 ppmv
x 2 hr), and 2,000 ppmv-hr (500 ppmv x
4hr).
38
-------
Table 2-6. Test Matrix for Generation of HP Decontamination CT Curves against B.
anthracis Ames Spores
Target Type and Number of Coupons or Bis per Contact
Trial
1
2a
2b
3a
3b
D2
Materials
Finished aluminum,
computer keyboard
keys, industrial
carpet, and
painted joint tape
Decorative laminate,
galvanized metal
ductwork,
industrial grade
carpet,
painted concrete
block
Industrial grade
carpet,
decorative laminate,
galvanized metal
ductwork,
painted concrete
block
Pine wood, plate
glass, and
ceiling tile
Pine wood, plate
glass, and
ceiling tile
Particle board,
Cellulose insulation
STERIS
VHP®
Cycle, Time
[HP], 0
ppmv
500 C=3
BL=1
500 C=3
BL=1
200- 250 C=3
BL=1
500 C=3
BL=1
200- 250
BL=1
200-250 c 3
BL=1
Time and Material
Time
1
30
min
N=5
BI=5
30
min
N=5
BI=5
30
min
N=5
BI=5
30
min
N=5
BI=5
30
min
N=5
BI=5
30
mm
N=5
BI=5
Time
2
Ihr
N=5
BI=5
Ihr
N=5
BI=5
Ihr
N=5
BI=5
Ihr
N=5
BI=5
Ihr
N=5
BI=5
Ihr
N=5
gj— 5
Time
3
2hr
N=5
BI=5
2hr
N=5
BI=5
2hr
N=5
BI=5
2hr
N=5
BI=5
2hr
N=5
BI=5
2hr
N=5
gj— 5
Time
4
4hr
N=5
C=2
BI=5
4hr
N=5
C=2
BI=5
4hr
N=5
C=2
BI=5
4hr
N=5
C=2
BI=5
4hr
N=5
C=2
BI=5
4hr
N-5
C=2
BI=5
Time
5
Gassing
+ 4hr
N=5
BI=5
Gassing
+ 4hr
N=5
BI=5
Gassing
+ 4hr
N=5
BI=5
Gassing
+ 4hr
N=5
BI=5
Gassing
+ 4hr
N=5
BI=5
Gassing
+ 4 hr
N=5
BI=5
C = positive control coupon, BL = laboratory blank, N = test coupon, BI = biological indicator
Note [HP] indicates the HP vapor concentration in the chamber air, in the units indicated.
2.12 Soak in pH-Amended Bleach
The decontamination efficacy of "pH-
amended bleach" was investigated. pH-
Amended bleach solution was prepared
as described in the U.S. EPA crisis
exemption requirements for use against
B. anthracis spores.'15^ The solution was
prepared using 9.4 parts water, 1 part
Clorox® bleach (sodium hypochlorite 5-
6%), and 1 part 5% acetic acid to yield a
solution having a mean pH close to, but
not above, neutral (actual pH = 6.51-
6.93) and a mean total chlorine content
of 6,000-6,700 ppm.
To stop the decontamination action of
the pH-amended bleach solution and
39
-------
extract the biological agent from
coupons, the coupons were removed
from the decontaminant and placed
individually into sterile 50 mL conical
vials to which 10.0 mL of sterile
extraction buffer with neutralizer was
added. Neutralization approaches were
tested in an earlier EPA evaluation.'16^
Phosphate-buffered saline with 1%
sodium thiosulfate was the extraction
buffer for vaccinia virus. Phosphate-
buffered saline with 1% sodium
thiosulfate and 0.1% Triton X-100
(Sigma) was the extraction buffer for
spores. The tubes were agitated on an
orbital shaker for 15 min at
approximately 200 rpm at room
temperature.
Qualitative and quantitative test methods
were included in the liquid
decontamination test matrix outlined in
Table 2-7. The qualitative cycle
fractionation method was used to screen
for decontamination efficacy. The
presence of viable spores on coupons
after contact with the pH-amended
bleach for various contact times were
determined by transferring the coupons
into TSB. As described in Section 2.7,
the tubes were cultured for seven days at
37 °C ± 2 °C. Results are reported as
"growth" of viable spores (cloudy
medium) or "no growth" (clear medium)
after one day and seven days of
incubation.
Counting CPU, described in Section
2.6.1, or PFU, described in Section
2.6.2, provided quantitative
measurement of viable spores and
viruses, respectively, after contact with
pH-amended bleach for various contact
times. Both the number of viable
organisms recovered and the log
reduction in viable organisms was
reported.
Ricin was proposed for inclusion in the
test matrix. However, ricin was rapidly
removed from the coupons by PBS in
control conditions, so the efficacy of a
liquid decontaminant could not be
determined. Therefore, ricin was
removed from the liquid
decontamination test matrices.
40
-------
Table 2-7. Test Matrix for pH-Amended Bleach Investigation
Biological
Agent/
Measurement
B . anthracis
(Ames)
(Qualitative
Cycle
Fraction)
B. anthracis
(Ames)
(Quantitative)
B. anthracis
(Vollum)
(Quantitative)
B. anthracis
(Ames)
(Quantitative)
Vaccinia
Virus
(Quantitative)
Materials and
Temperatures
Glass, Painted
Concrete Block,
Galvanized
Metal,
Decorative
Laminate @ 22
°C ± 2 °C
Glass, Painted
Concrete Block,
Carpet,
Galvanized
Metal, Particle
Board,
Decorative
Laminate @ 22
°C ± 2 °C
Glass, Painted
Concrete Block,
Carpet,
Galvanized
Metal, Particle
Board,
Decorative
Laminate @ 22
°C ± 2 °C
Glass, Painted
Concrete Block,
Carpet,
Galvanized
Metal, Particle
Board,
Decorative
Laminate @ 22
°C ± 2 °C
Glass, Painted
Concrete Block,
Carpet,
Galvanized
Metal, Particle
Board,
Decorative
Laminate @ 22
°C ± 2 °C
Test Coupon*
Contact times
(min): 10,20,40,
60 min are
neutralized and
placed into TSB
to test for viable
spores
Five replicate
coupons per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min):
5,10,30
Five replicate
coupons per
contact time
Contact time
(min):
0,5
Five replicate
coupons per
contact time
Positive
Control
Coupons1^
Contact times
(min): 10,20,40,
60 min are
neutralized and
placed into TSB
to test for viable
spores
Five replicate
coupons per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min):
5,10,30
Five replicate
coupons per
contact time
Contact time
(min):
0,5
Five replicate
coupons per
contact time
Laboratory
Blank*
Contact times
(min): 10,20,40,
60 min are
neutralized and
placed into TSB
to test for viable
spores
One coupon per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min):
5,10,30
Five replicate
coupons per
contact time
Contact time
(min):
0,5
Five replicate
coupons per
contact time
Procedural
Blank'
Contact times
(min): 10,20,40,
60 min are
neutralized and
placed into TSB to
test for viable
spores
One coupon per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min): 5
Five replicate
coupons per
contact time
Contact time
(min):
5,10,30
Five replicate
coupons per
contact time
Contact time
(min):
0,5
Five replicate
coupons per
contact time
* Spiked, Decontaminated; in contact with decontamination technology for specified time.
^Spiked, placed in PBS, Not Decontaminated; in contact with PBS for the specified time'
*Not Spiked, Not Decontaminated; not in contact with liquid, but analyzed along with test and control coupons at
specified time.
'Not Spiked, Decontaminated; in contact with the decontamination technology for the specified time.
41
-------
2.13 Soak in Liquid C1O2 Technology
A 1000 ppm liquid C1O2 solution was
prepared following the manufacturer's
instructions by dissolving one Exterm-6
disinfectant tablet (ClorDiSys Solutions,
Inc., Lebanon, NJ) in 500 mL of water.
Exterm-6 disinfectant tablets contain an
inorganic acid (25%-35%), sodium
chlorite (15%-30%), an inorganic salt
(35%-45%), and an activator (5%-10%)
that generates C1O2 when dissolved in
water.
To stop the decontamination action of
the liquid C1O2 and extract the biological
agent from coupons, the coupons were
removed from the decontaminant and
placed individually into sterile 50 mL
conical vials to which 10.0 mL of sterile
extraction buffer with neutralizer was
added. Neutralization approaches were
tested in an earlier EPA evaluation.[16]
Phosphate-buffered saline with 1%
sodium thiosulfate was the extraction
buffer for vaccinia virus. Phosphate-
buffered saline with 1% sodium
thiosulfate and 0.1% Triton X-100
(Sigma) was the extraction buffer for
spores. The tubes were agitated on an
orbital shaker for 15 min at
approximately 200 rpm at room
temperature.
The liquid C1O2 decontamination test
matrix, shown in Table 2.8, used
quantitative methodologies. Counting
CPU, described in Section 2.6.1, or PFU,
described in Section 2.6.2, provided
quantitative measurement of viable
spores and viruses, respectively, after
contact with liquid C1O2 for various
contact times. Both the number of viable
organisms recovered and the log
reduction in viable organisms was
reported.
42
-------
Table 2-8. Test Matrix for Liquid ClOi Decontamination Investigation
Biological
Agent/
Measurement
B. anthracis
(Ames)
(Quantitative)
B. anthracis
(Vollum)
(Quantitative)
B. sub tills
(Quantitative)
Vaccinia Virus
(Quantitative)
Materials and
Temperatures
Glass, Carpet,
Particle Board,
Decorative
Laminate @ 22
°C ± 2 °C
Glass, Carpet,
Particle Board,
Decorative
Laminate @ 22
°C ± 2 °C
Glass, Carpet,
Particle Board,
Decorative
Laminate @ 22
°C ± 2 °C
Glass, Carpet,
Particle Board,
Decorative
Laminate @ 22
°C ± 2 °C
Test Coupon*
Contact time (min):
10, 30, 120
Five replicate
coupons per contact
time
Contact time (min):
5
Five replicate
coupons per contact
time
Contact time (min):
10, 30, 120
Five replicate
coupons per contact
time
Contact time (min):
0,5
Five replicate
coupons per contact
time
Positive Control
Coupons1^
Contact time (min): 10,
30, 120
Five replicate coupons
per contact time
Contact time (min): 5
Five replicate coupons
per contact time
Contact time (min): 10,
30, 120
Five replicate coupons
per contact time
Contact time (min):
0,5
Five replicate coupons
per contact time
Laboratory Blank*
Contact time (min):
10, 30, 120
Five replicate
coupons per contact
time
Contact time (min):
5
Five replicate
coupons per contact
time
Contact time (min):
10, 30, 120
Five replicate
coupons per contact
time
Contact time (min):
0,5
Five replicate
coupons per contact
time
Procedural Blank
Contact time (min): 10,
30, 120
Five replicate coupons
per contact time
Contact time (min): 5
Five replicate coupons
per contact time
Contact time (min): 10,
30, 120
Five replicate coupons
per contact time
Contact time (min):
0,5
Five replicate coupons
per contact time
* Spiked, Decontaminated; in contact with decontamination technology for specified time.
^Spiked, placed in PBS, Not Decontaminated; in contact with PBS for the specified time.
*Not Spiked, Not Decontaminated; not in contact with liquid, but analyzed along with test and control coupons at specified time.
'Not Spiked, Decontaminated; in contact with the decontamination technology for the specified time.
43
-------
(S)
2.14 Soak in Spor-Klenz Hydrogen
Peroxide-Peractic Acid (HP-PA)
Solution
The following description of Spor-
Klenz® Ready-to-Use (STERIS
Corporation, Mentor, OH) is from the
STERIS website
(http://www.steris.com/products/view.cf
m?id=253) and was not verified in this
testing:
A fast acting, liquid cold
sterilant/disinfectant,
filtered to 0.22 micron
and specifically
formulated for use in the
sterilization and
disinfection of hard
surfaces. This product is a
stabilized blend of
peracetic acid, hydrogen
peroxide, and acetic
[acid] that provides fast,
effective microbial
control, including spores.
It offers a low toxicity
profile and requires no
mixing or activation.
According to the Material Safety
Data Sheet for Spor-Klenz®
Ready to Use solution, the
composition includes peracetic
acid (0.8%), hydrogen peroxide
(1.0%), and acetic acid (<10%).
The Spor-Klenz® Ready to Use solution
(referenced hereinafter as Spor-Klenz®
HP-PA) was used as received without
dilution or other preparation against
Bacillus spores. The decontamination
process was terminated by removing
coupons from the Spor-Klenz HP-PA
and placing coupons individually into
sterile 50 mL conical vials containing
10.0 mL of sterile 1% catalase in Dey-
Engley (neutralizing) broth. Dey-Engley
broth is a standard medium used to
neutralize disinfectant components in
order to test for residual bacterial
activity.* Neutralization approaches
were tested in an earlier EPA
evaluation.'16^ The tubes were agitated
on an orbital shaker for 15 min at
approximately 200 rpm at room
temperature to extract the spores.
The Spor-Klenz® HP-PA
decontamination test matrix, shown in
Table 2-8, used a preliminary qualitative
test to identify a contact time with Spor-
Klenz® HP-PA at which no viable spores
were detected on any test coupons that
had been inoculated with approximately
108 CPU of B. anthracis spores/coupon.
The quantitative methodology, described
in Section 2.6.1, measured viable,
colony-forming spores recovered from
coupons after contact with Spor-Klenz
HP-PA for various contact times.
Both the number of viable organisms
recovered and the log reduction in viable
organisms was reported as CPU.
2.15 Soak in Oxonia Active® Solution
The following description Ecolab
Oxonia Active® solution is from the
product label and from the Material
Safety Data Sheet; this information was
not verified in this testing:
Oxonia Active® acid
sanitizer is recommended
for use on pre-cleaned
surfaces such as
* For a further description, see:
www.sigmaaldrich.com/etc/medialib/docs/Fluka/
Datasheet/d3435dat.Par.0001.File.tmp/d3435dat.
pdf.
44
-------
equipment, tanks, vats,
filters, evaporators,
pasteurizers and aseptic
equipment in dairies,
breweries, wineries,
beverage and food
processing plants. This
product is effective as a
sanitizer when solution is
prepared in water of up to
500 ppm hardness
CaCO3.
The label instructions specify use
of a 5% solution of Oxonia
Active® for sterilization. At a
temperature of 20°C, a 6-hr
contact time is required. Per
recommendations from the
vendor, Oxonia Active at a 7%
concentration (rather than label
strength) was used against
Bacillus spores in this
investigation. EPA chose to
follow the vendor
recommendation as to the
concentration (7%) that was used
in this investigation. Contact
times were selected not to exceed
60 min, the maximum time
considered by EPA practitioners
to be practical for field
application of liquid
decontamination technologies.
According to the Material Safety
Data Sheet for Oxonia Active®,
the composition includes
peracetic acid (5% - 10%),
hydrogen peroxide (15% - 40%),
and acetic acid (7% - 13%).
The Oxonia Active decontamination
process was terminated by removing
coupons from the Oxonia Active HP-
PA solution and placing coupons
individually into sterile 50 mL conical
vials containing 10.0 mL of sterile 1%
catalase in Dey-Engley broth. Catalase
was added to the standard Dey-Engley
neutralizing broth to rapidly decompose
the HP. Neutralization approaches were
tested in an earlier EPA evaluation.[16]
The tubes were agitated on an orbital
shaker for 15 min at approximately 200
rpm at room temperature to extract
spores.
The Oxonia Active® test matrix is shown
in Table 2-10. Test coupons were
inoculated with approximately 108 CPU
of B. anthracis or B. subtilis
spores/coupon. The quantitative
methodology, described in Section 2.6.1,
provided quantitative measurement of
viable spores after contact with Oxonia
Active® HP-PA for various contact
times. The decontamination process was
terminated using 1% catalase in Dey-
Engley broth. Both the number of viable
organisms recovered and the log
reduction in viable organisms was
reported.
45
-------
Table 2-9. Test Matrix for the Spor-KlenzR HP-PA Decontamination Investigation
Biological
Agent/
Measurement
B. anthracis
(Ames)
(Qualitative)
B cinthrcicis
(Ames)
(Quantitative)
B. subtilis
(Quantitative)
Materials and
Temperatures
Glass, Painted
Concrete,
Galvanized
Metal,
Decorative
Laminate
@ 20 °C ± 2 °C
Glass
Painted
Concrete
Carpet
Galvanized
Metal
Particle Board
Decorative
Laminate
® 20 °C ± 2 °C
Glass
@ 20 °C ± 2 °C
Test Coupon*
Contact time (min):
5 10 20 30
Three replicate
coupons per contact
time
Contact time (min):
10, 20, 30
Five replicate
coupons per contact
time
Contact time (min):
10, 20, 30
Five replicate
coupons per contact
time
Positive Control Laboratory
Coupons1^ Blank*
None None
Non-contact time
(min):
10 (three replicates) „
~n , , r . / One coupon
20 (one replicate)
30 (one replicate)
Non-contact time
(min):
10 (three replicates) „
~n ,, ,. .^ One coupon
20 (one replicate) r
30 (one replicate)
Procedural
Blank'
None
Non-contact time
(min):
30 (one coupon)
Non-contact time
(min):
30 (one coupon)
* Spiked, decontaminated.
^ Spiked, placed in PBS, not decontaminated.
*Not spiked, not decontaminated.
'Not spiked, decontaminated.
46
-------
si-
Table 2-10. Test Matrix for the Oxonia Active Decontamination Investigation
Biological
Agent/
Measurement
B. anthracis
(Ames)
(Quantitative)
Materials and
Temperatures
Glass
Painted
Concrete
Carpet
Galvanized
Metal
Particle Board
Decorative
Laminate
@ 20 °C ± 2 °C
Test Coupon*
Contact time (min):
10, 30, 60
Five replicate
coupons per contact
time
Positive Control Laboratory
Coupons1^ Blank*
Non-contact time
(min):
10 (three replicates) „
~n ,, r . / One coupon
20 (one replicate)
30 (one replicate)
Procedural Blank'
Non-contact time
(min):
30 (one coupon)
B. sub tills
(Quantitative)
Glass
(a) 20 °C ± 2 °C
Contact time (min):
10, 30, 60
Five replicate
coupons per contact
time
Non-contact time
(min):
10 (three replicates)
20 (one replicate)
30 (one replicate)
One coupon
Non-contact time
(min):
30(one coupon)
* Spiked, decontaminated.
^Spiked, placed in PBS, not decontaminated.
*Not spiked, not decontaminated.
'Not spiked, decontaminated.
47
-------
3.0 Quality Assurance/Quality Control
QA/quality control (QC) procedures
were performed in accordance with the
test/QA plans and associated
amendments developed for the
investigations. QA/QC procedures are
summarized below.
3.1 Performance Evaluation (PE)
Audits
PE audits were conducted to assess the
quality of the results obtained during
these experiments. No PE audits were
performed for biological agents and
surrogates because quantitative
standards for these biological materials
do not exist. The confirmation
procedure, controls, blanks, and method
validation efforts support the biological
evaluation results. For the chlorine
dioxide, all of the PE audits performed
were within the target tolerances
specified in the test/QA plan. For liquid
testing, all of the PE audits performed
were within the target tolerances
specified in the test/QA plan. For methyl
bromide testing, all of the PE audits
performed were within the target
tolerances specified in the test/QA plan.
The results of these analyses are given in
Tables 3-1 through 3-3. All PE audit
results were within the respective target
tolerances.
48
-------
Table 3-1. C1O2 PE Audit Results
Parameter
[C102]
Time
Temperature
RH
Microplate Reader
Date of Audit
1/15/07
1/11/07
1/11/07
1/11/07
1/18/07
Value of Standard
1000 mg/L Sodium Chlorite
10:43:00*
10:53:07*
11:03:10*
11:13:02*
20 °C*
23.5%*
Optical density at 590 nm
0.269
0.539
1.122
1.629
Optical density at 635 nm
0.274
0.533
1.088
1.577
Measured Result
1012 mg/L Sodium Chlorite
10:43:00*
10:53:07*
11:03:10*
11:13:02*
19.8 °C*
17.25 %*
Optical density at 590 nm
0.270'
0.539
1.121
1.628
Optical density at 635 nm
0.273
0.531
1.086
1.574
Difference
1.2%
0 seconds
0 seconds
0 seconds
0 seconds
-0.2 °C
-6%
0.37%
0.00%
-0.09%
-0.06%
-0.36%
-0.38%
-0.18%
-0.19%
Expected Tolerance
±10%
±1 min/30 days
±1°C
±10%
±1.0%
* Average of four measurements.
' All measured results for the microplate reader were an average of 24 measurements.
* Time in hr: min: seconds.
49
-------
Table 3-2. Liquid PE Audit Results
Parameter
Temperature - unit I1
- unit 21
-units1
Time
Microplate reader
Date of Audit
11/16-12/13/06
12/11-13/06
12/11-13/06
11/16/06
11/21/06
12/11/06
12/11/06
12/11/06
12/13/06
12/13/06
12/13/06
1/18/07
1/18/07
Value of Standard
22.2 °C *
21.2 °C*
21.0 °C*
08: 08: 051
09: 30: 02*
14: 50: 36*
14:53:48*
14: 55: 02*
15:08: 17*
15:09:32*
15: 10: 24*
Optical density at 590 nm
0.269
0.539
1.122
1.629
Optical density at 635 nm
0.274
0.533
1.088
1.577
Measured Result
21.7 °C*
22.0 °C*
21.9 °C*
08: 08: 05*
09: 30: 02*
14: 50: 36*
14:53:48*
14:55:02*
15:08: 17*
15:09:32*
15: 10:24*
Optical density at 590 nm
0.270'
0.539
1.121
1.628
Optical density at 635 nm
0.273
0.531
1.086
1.575
Difference
0.5 °C
0.8 °C
0.9 °C
0 seconds
0 seconds
0 seconds
0 seconds
0 seconds
0 seconds
0 seconds
0 seconds
0.37%
0.00%
-0.09%
-0.06%
-0.36%
-0.38%
-0.18%
-0.19%
Expected Tolerance
±1°C
±1 min/30 days
±1.0%
* Average of four measurements for unit 1, average of two measurements for units 2 and 3.
' All measured results for the microplate reader were an average of 24 measurements.
* Time in hr: min: second.
^Units 1-3 refers to the three sensors that were used to measure temperature during the testing.
50
-------
In the audit of the manual plate count,
only one repeat plate was read instead of
five as required by the test/QA plan.
This deviation was noted, but had no
impact on the results.
3.2 Technical Systems Audit
The Battelle QA Manager conducted
technical systems audits to ensure that
the tests were being performed in
accordance with the appropriate test/QA
plans. As part of the audit, the Battelle
QA Manager reviewed the reference
sampling and analysis methods used,
compared actual test procedures with
those specified in the test/QA plan, and
reviewed data acquisition and handling
procedures. No significant findings were
noted in these audits that might impact
the quality of the evaluation results. The
records concerning the technical systems
audits were permanently stored with the
Battelle QA Manager.
Table 3-3. MeBr PE Audit Results
Parameter
[MeBr] *
Time
Temperature
RH
Manual Plate
Count'
Date of
Audit
2/15/08
5/11/07
5/11/07
5/11/07
8/22/07
Value of
Standard
49mg/L
}
19.7 °C
48%
9.47 xlO8 CPU
Measured Result
53 mg/L
}
21.0 °C
48%
9.50 x 108 CPU
Difference
8%
0 seconds/
min
1.3 °C
0.00%
0.32%
Expected
Tolerance
±10%
±1 min/30 days
±2°C
±5% of Ml scale
±10% agreement in
repeated plate counts
* Average of two measurements. Prior to use, the Fumiscope was returned to the manufacturer for
calibration. The Fumiscope measurements were compared to measurements with a calibrated VIG M-20
total hydrocarbon analyzer. Differences between measurements made with the total hydrocarbon analyzer
and the Fumiscope meter varied up to about 20% depending on concentration of MeBr. Fumiscope readings
were reported as specified in the test/QA plan to reflect readings that would be expected in field use.
'Only one plate was read instead of five required by test/QA plan - Noted below under deviations.
* 0 second/min was observed for 44 of 44 comparisons.
3.3 Data Quality Audit
At least 10% of the data acquired during
the evaluation were audited. Battelle's
Q A Manager traced the data from the
initial acquisition through reduction to
final reporting, to ensure the integrity of
the reported results. All calculations
performed on the data undergoing the
audit were checked.
QA/QC Reporting
Each assessment and audit was
documented in accordance with the
appropriate test/QA plan and associated
amendments. For these evaluations, no
significant findings were noted in any
assessment or audit, and no follow-up
corrective action was necessary. Copies
of the TSA and assessment reports were
distributed to the EPA QA Manager and
Battelle staff.
51
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3.4 Deviations from the Test QA
Plans
3.4.1 General Deviation
One general deviation was documented
and reported to the EPA. The test/QA
plans specified that dilutions through 10"
7 would be analyzed. In some cases,
dilutions outside the useful range, based
on expected concentrations of biological
agent, were not run. This judgment was
based on historical data and experience.
For example, for positive controls only
three dilutions, i.e., 10~3, 10~4, and 10~5
would be plated. For decontamination
tests where no viable organisms were
expected, only the undiluted solution and
10"1, 10~2, and 10~3 dilutions might be
plated. In some cases with high
confidence of no organisms surviving,
only the undiluted extract was plated.
The reduced plating saved time and
money without reducing useful data.
There was no impact on results.
3.4.2 Chlorine Dioxide
One deviation was documented. The
test/QA plan specified that test coupons
would be removed from the test chamber
after given periods of exposure to the
fumigant. During the cycle
fractionation, 3000 ppmv C1O2, 75% RH
trials for B. anthracis, this removal was
done by having the spiked coupons in
closed vials in the test chamber and
using the gloves in the glove box to open
them in sequence and remove them from
the vials so that an appropriate contact
time for exposure to C1O2 was achieved.
Contact time was the time from opening
the vial containing the spiked coupon in
the fumigation test chamber until the
coupon was removed from the test
chamber. Time zero (TO), positive
control coupons were in sealed vials in
the test chamber for the full 180 min of
fumigation and removed from the
chamber without opening the vials; there
was no exposure of the TO coupons to
the fumigant. There was no impact on
the results.
3.4.3 Liquid Testing
A number of deviations were
documented and described below.
One procedural blank, rather than two
specified in the test/QA plan, was used
at each time point. The results of the
procedural blanks do not enter into the
calculation of efficacy and therefore did
not impact the results.
Five-min positive controls were used as
TO positive controls for spores. Spores
are known to be highly persistent on the
building materials used under ambient
laboratory conditions so that a difference
of five min in extracting the positive
controls would have no impact on
recoveries and, therefore, no impact on
the results.
Small glass coupons were spiked with a
single 10 jiL drop instead often IjiL
drops as specified in the test/QA plan.
The test/QA plan was intended to require
use of a single 10 jiL drop instead often
1 jiL drops for the small glass coupons to
be consistent with other EPA testing.
The deviation is actually a correction of
the test/QA plan and provides the
desired results.
The cultures of Bacillus on TSA with no
CFU observed after 18-24 hr were not
returned to incubation for up to 72-hr
total and re-examined per Section
B.4.2.1 of the test/QA plan. The practice
52
-------
of incubating for additional time has
been dropped from subsequent test/QA
plans. The additional incubation time
was found not to be useful. There was no
impact on the results.
The test/QA plan specifies that in the
event of contamination of the laboratory
or procedural blank, the test will be
rejected. In the testing of Bacillus
subtilis on glass decontaminated with
Oxonia (6/11/09) the procedural blank
extract contained a low level of
contamination. In this report the data are
flagged and the contamination is noted.
Including the data, rather than rejecting
the data, is a deviation from the test/QA
plan. However, conclusions do not
appear to be compromised and the data
were believed to be useful and therefore
were included in the report with caveats,
rather than rejecting the data. The results
show comparable and high
decontamination efficacy, >5.78 log
reduction, for both B .anthracis and B.
subtilis. If cross contamination of the
test coupons occurred in the B. subtilis
test, B. subtilis decontamination may
have been even higher than reported.
In the cycle fractionation, the target
application range was 7.5 x 107 - 1.25 x
o
10 ; the actual application used was 6.8
x 107. The qualitative cycle fractionation
test was performed to guide the selection
of decontamination parameters and was
effective for that purpose. The results are
easily and correctly interpreted based on
the known number of spores applied.
There was no impact on the results.
During the Spor-Klenz® and Oxonia
Active® efficacy testing the target
application range was 7.5 x 10 - 1.25 x
o
10 . The actual range exceeded the target
range; the actual application range was
1.2 x 10 - 6.9 x 108. The results are
valid, but the potential dynamic range is
0.08 - 0.74 log greater with the higher
applications. The comparison of the
surrogate B. subtilis to B. anthracis is
not impacted. Comparisons across
technologies, not an objective of this
report, would need to consider the
dynamic range of the respective tests.
There was no impact on the conclusions
of this report.
During the Spor-Klenz® and Oxonia
Active® efficacy testing, there were three
cases in which the spore recoveries were
below the acceptance range of 20% -
120%:
• Spor-Klenz®/Glass: 6% recovery
• Oxonia Active®/Laminate: 2%
recovery
• Oxonia Active® /Metal: 5%.
The test/QA plan specifies that in the
event of low recoveries from the positive
control coupons, the test will be rejected.
The tests were not rejected but the data
are flagged in the results. Because the
initial spore load was higher than
specified in the test/QA plan (noted
above), the dynamic range, even with
lower than target recoveries, was
sufficient to observe a 6-log reduction
and observed intended results. There was
minimal or no impact on the results.
3.4.4 Methyl Bromide
Two deviations were documented. The
first deviation was a spore application
outside of the target range. The test/QA
plan stated that the number of B.
anthracis CPU in the stock spore
suspension to be spiked onto coupons
will be acceptable if the application
controls are within ±25% of the target
53
-------
spike level. The stock suspension in
Trial 8 exceeded this range. There was
no impact on the results.
The second deviation covered two
separate items. For the first item, the
test/QA plan specified that temperature
and RH in the test chamber would be
monitored every 20 min during working
hours using a NIST-traceable system,
and monitored continuously during
decontamination with the continuous
monitoring system checked daily with a
NIST-traceable
thermometer/hygrometer. Instead, a
NIST-traceable thermometer/hygrometer
was used to take measurements roughly
every hr during decontamination. The
temperature and RH were stable so there
was no impact on the results.
For the second item, five replicates were
to be plated, counted, and compared with
an expected agreement of ±10%. Only
one replicate plate was prepared and
counted during the PE audit with an
agreement of ±0.32% - well below the
tolerance. The personnel performing the
plate counts are highly experienced and
competent and are expected to have
repeatable and accurate counts. There
was no impact on the results.
54
-------
4.0 Fumigation Results
4.1 C1O2 Fumigation Results
Adaptive management, i.e., making
changes to the test/QA plan based on
knowledge gained during the
investigation, resulted in trials being
added to and removed from the test
matrix. Results are presented in a
sequence intended to provide a logical
flow. However, in some cases this
differs from the sequence in which the
testing was performed (resulting, for
example, in Trial 4g results being
presented ahead of Trial 1). Trial
numbers are included for ease of
reference to the test matrix in Table 2-2.
C1O2 fumigation trials with the same
number were performed in the same
fumigation cycle.
4.1.1 Trial 4g: CIO 2 Fumigation Cycle
Fractionation Results
The results of the qualitative cycle
fractionation test using C1O2 fumigation
of B. anthracis Ames on plate glass and
B. atrophaeus and B. subtilis Bis are
shown in Table 4-1. At specified C1O2
fumigation contact times, the coupons
and Bis were removed from the test
chamber and placed into TSB as
described in Section 2.7. The Bis
included: B. atrophaeus spores on filter
paper strips in glassine (Raven Labs
Division, Lakewood, CO); B.
atrophaeus spores on filter paper strips
(Raven) removed from the glassine prior
to fumigation; B. subtilis spores on
stainless steel disk in Tyvek® envelope
(Apex Laboratories, Apex, NC); and B.
subtilis spores on stainless steel disk
(Apex) removed from the Tyvek
envelope prior to fumigation.
®
Contact for 30 min at 3000 ppmv C1O2
killed all B. anthracis Ames, B.
atrophaeus., and B. subtilis spores,
except the B. atrophaeus spores on filter
paper enclosed in a glassine envelope.
The glassine-enclosed B. atrophaeus
spores were killed on one of three spore
strips at a 90-min contact time and were
killed on all three spore strips at a 120-
min contact time. Glassine paper has the
characteristic of being almost
impervious to water vapor (e.g., see
http://www.papertecinc.eom/specPaper.c
fm) whereas Tyvek® is permeable to
water vapor (e.g., see
http ://www2 . dupont. com/
Tvvek Weatherizati on/en US/products/r
esidential/resi_homewrap.html).
Humidity is a key parameter related to
Bacillus species spore inactivation with
C1O2 gas (discussed later in this section).
55
-------
Table 4-1. Results from Qualitative Evaluation of ClOi Fumigation of B. anthracis
Ames Spores and Surrogates
Bacterium and [C1O2],
Material (Trial 4g) ppmv
B. anthracis Ames, Plate Glass 3000
B. atrophaeus spores on filter
paper strips in glassine (Raven)
B. atrophaeus spore on filter
paper strips not in glassine 3000
(Raven)
_ ... Decontaminated,
„ , Positive „ , , „.
Temperature, „ , Contact Time
RH Control 3Q fi() 9Q UQ
Coupon . .
1 mm mm mm mm
25
71
25
71
25
71
- 26 °C
-74%
-26°C +/-
- 74% (2 +, 1 -)
- 26 °C
-74%
B. subtilis spores on stainless
disk in Tyvek envelope (Apex)
~B. subtilis spores on stainless
disk not in Tyvek envelope 3000
(Apex)
25 - 26 °C
71-74%
25 - 26 °C
71-74%
"+" indicates that all replicates (n = 3) are positive for growth after incubation at 37 °C for seven days; B. anthracis
confirmed morphologically by plating onto tryptic soy monoplates.
"-" indicates that all replicates (n = 3) are negative for growth after incubation at 37 °C for seven days; absence ofB.
anthracis confirmed morphologically by plating onto tryptic soy monoplates.
+/-" indicates that two replicates were positive and one replicate was negative for growth after incubation at 37 °C for
seven days; B. anthracis confirmed morphologically by plating onto tryptic soy monoplates.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
4.1.2 Trials 4a and 4b: Fumigation
Cycle Fractionation Results for
Two Concentrations of CIO2
against B. anthracis
The results of the qualitative cycle
fractionation test using C1O2 fumigation
of B. anthracis Ames spores are shown
in Table 4-2. The qualitative cycle
fractionation test was used to indicate
the contact time required at two target
concentrations of C1O2(750 ppmv and
3000 ppmv) for complete kill (no
observed CPU) of B. anthracis Ames
spores on coupons of various building
materials. Three test coupons of each
material type, spiked with B. anthracis
spores, were exposed to C1O2 for each
specified contact time.
The TO positive control and test coupons
were placed in glass tubes, sealed, and
placed into the decontamination chamber
during the fumigation cycle. Test
coupons were opened and the coupons
removed from the glass tube and
exposed to fumigant in the test chamber
at appropriate intervals, e.g., the tube for
a 40-min contact time exposure would
be opened and the coupon exposed to the
fumigant 40 min prior to aeration.
After the fumigation cycle was
completed, the TO coupons, still in
sealed glass tubes, were removed from
the test chamber, removed from the glass
tube, and cultured in TSB to detect
viable spores. All TO coupons, except
particle board, exhibited growth in TSB.
The particle board coupons did not
exhibit growth in TSB. However,
subculture of an aliquot of the TSB
containing the particle board coupons
onto tryptic soy agar plates resulted in
growth of B. anthracis Ames. (Colony
morphology was consistent with B.
56
-------
anthracis Ames and no colonies were
recovered from procedural blank
coupons.) The particle board appears to
have biostatic properties that inhibited
the growth of B. anthracis Ames in the
TSB. As a result of this interference, no
conclusions can be reached on the
effectiveness of C1O2 fumigation against
B. anthracis Ames on particle board
using the qualitative cycle fractionation
test. Therefore the particle board was not
included in the testing at 3000 ppmv.
Table 4-2. Results from Qualitative Evaluation of ClOi Fumigation of B. anthracis
Ames Spores
Material
B. anthracis
Ames
Glass
Painted
Concrete
Galvanized
Metal
Decorative
Laminate
Cellulose
Insulation
Particle Board
B. anthracis
Ames
Glass
Painted
Concrete
Galvanized
Metal
Decorative
Laminate
Cellulose
Insulation
[C102],
ppmv
750
750
750
750
750
750
3000
3000
3000
3000
3000
,. _ Decontaminated, Contact Time
Mean '
Temperature, 0 20 40 60 80 100
Mean RH min* min min min min min
24'7°C
82.9%
24.7 °C
82.9%
24.7 °C
82.9%
24'7°C
82.9%
24'7°C
82.9%
24.7 °C M
82.9% ~(+) -
0 10 20 30 40 50
min* min min min min min
24.6 °C
78.9%
24.6 °C
78.9%
24.6 °C
78.9%
24.6 °C
78.9%
24'6°C
78.9%
120
min
-
-
+
-
+
-
60
min
-
-
-
-
+
"+" indicates that all replicates (n = 3) are positive for growth after incubation at 37 °C for seven days; B.
anthracis confirmed morphologically by plating onto tryptic soy monoplates.
"-" indicates that all replicates (n = 3) are negative for growth after incubation at 37 °C for seven days; absence of
B. anthracis confirmed morphologically by plating onto tryptic soy monoplates.
"- (+)" indicates that all replicates (n = 3) are negative for growth after incubation at 37 °C for seven days,
however, B. anthracis colonies grew, and were confirmed morphologically, when plated onto tryptic soy
monoplates.
* These inoculated coupons were in sealed tubes in the decontamination chamber during fumigation.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
57
-------
The results of the qualitative testing
showed that the C1O2 concentration and
contact time necessary for complete kill
of B. anthracis Ames spores (negative
for growth) depends on the type of
material to which the spores were
applied. At 750 ppmv C1O2, complete
kill of B. anthracis Ames spores
(negative for growth) was observed in as
little as 60 min for painted concrete
coupons, and 80 min for glass and
decorative laminate. In contrast, viable
B. anthracis Ames spores were still
observed on galvanized metal and
compressed cellulose insulation after a
120-min contact with 750 ppmv C1O2.
At 3000 ppmv C1O2, complete kill of B.
anthracis Ames spores (negative for
growth) was observed in 10 min for all
coupon types except compressed
cellulose insulation. Viable B. anthracis
Ames spores were still observed on
cellulose insulation after 60-min contact
with 3000 ppmv C1O2.
4.1.3 B. anthracis on Painted
Concrete: Quantitative CT
Investigation
Trials 1-3 investigated the efficacy of
C1O2 against B. anthracis Ames spores
on painted concrete using the
quantitative method (Section 2.6) at
three CT values at high RH (>80%). The
three CT values, 2250 ppmv-hr, 4500
ppmv-hr, and 9000 ppmv-hr, were
repeated at three different C1O2
concentrations: 3000 ppmv, 1500 ppmv
and 750 ppmv. The quantitative recovery
results for B. anthracis Ames spores on
painted concrete exposed to C1O2 at
specified concentrations and contact
times are shown in Tables 4-3 and 4-4.
Complete kill (>7 log reduction) was
observed at all CT values (2250 ppmv-hr
and higher) and at all C1O2
concentrations tested (750 ppmv and
higher).
58
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Table 4-3. B. anthracis Ames Spores CFU after ClOi Fumigation at Various CTs
Biological
Trial Agent,
Material
B. anthracis
1 Ames,
Painted Concrete
B. anthracis
2 Ames,
Painted Concrete
B. anthracis
3 Ames,
Painted Concrete
[C102],
Temperature,
RH
3000 ppmv
24 - 25 °C
89-91%
1500 ppmv
24 - 25 °C
81-87%
750 ppmv
24 - 25 °C
72 - 82%
Positive Controls Decontaminated Coupons*
T0t „. „. „. CT, CT, Contact CT,
Mean CFTJ Time, Time, Time, „ , ' . ' . „ , ' .
lYieau ^r u » » » Contact time, time, Contact time,
(SD) Mean CFU Mean CFU Mean CFU ,_ ™TT ,_ ' TT ,_ «^TT
^^ ^ ,„_, ,„_, ,cm Mean CFU Mean CFU Mean CFU
(oUj (oilj (oilj
0.75hr, 1.5hr, 3 hr, 2250 ppmv-hr 4500 ppmv -hr, 4500ppmv-hr,
6.57 xlO7 5.99 x 107 5.67 x 107 7.18 x 107 0.75 hr, 1.5 hr, 3 hr,
(7.39 xlO6) (1.15xl07) (1.36 xlO7) (1.64 x 107) 000
1.5hr, 3 hr, 6 hr, 2250 ppmv-hr 4500 ppmv-hr, 4500 ppmv-hr,
6.21 xlO7 7.86 xlO7 6.96 x 107 5.58 x 107 1.5 hr, 3 hr, 6 hr,
(2.21 xlO7) (6.25 xlO6) (1.09 x 107) (1.02 x 107) 000
3 hr, 6 hr, 12 hr, 2250 ppmv-hr 4500 ppmv-hr, 4500 ppmv-hr,
7.50 xlO7 6.93 xlO7 7.21 x 107 6.34 x 107 3 hr, 6 hr, 12 hr,
(1.38 xlO7) 1 (6.06 xlO6) (1.25 x 107) (9.98 x 106) 000
*SD not calculated when no spores are recovered from any test coupon
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
59
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Table 4-4. Mean Log Reduction of B. anthracis Ames Spores after ClOi Fumigation of Painted
Concrete at Various CTs
rr
Trial C ',
ppmv-hr
2,250
1 4,500
9,000
2,250
2 4,500
9,000
2,250
3 4,500
9,000
[C1O2], Temperature, RH,
ppmv °C %
3000 24'25 89'91
150o 24-25 81-87
750 24 - 25 72 - 82
Time,
hr
0.75
1.5
3
1.5
3
6
3
6
12
Mean Log Reduction
and p-value*' '
>7.77 (5/5) p=0.0135
>7.75 (5/5) p=0.0135
>7.85 (5/5) p=0.0135
>7.89 (5/5) p=0.0135
>7.84 (5/5) p=0.0135
>7.74 (5/5) p=0.0135
>7.84 (5/5) p=0.0135
>7.85 (5/5) p=0.0135
>7.80 (5/5) p=0.0135
Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons. Confidence intervals and p-values are
from two sample t-tests comparing logs of control and treated coupons. Bolded results are statistically significant at 0.05 level.
^One or more of treated coupons had no recovered agent (the exact number is shown in parentheses as test coupons with no
growth/total test coupons). The reported ">x" value is the mean log reduction with one CPU substituted for all zero recovery coupon
values to permit calculation of the log. For these trials, the test of statistical significance is a nonparametric Kolmogorov-Smimov test
where a p-value less than 0.05 indicates statistically significantly greater reduction in the treated group than in the controls.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
4.1.4 B. anthracis on Eight Materials:
Quantitative CT Investigation
In Trials 4, 5, and 6, the efficacy of C1O2
fumigation was evaluated against B. anthracis
Ames spores on coupons of eight materials
using the quantitative method (Section 2.6).
All test and positive control coupons (except
the small glass coupons) were inoculated with
Ix 108 spores; the small glass coupons were
inoculated with a single droplet of 107 spores.
The results, shown in Tables 4-5 and 4-6,
include C1O2 fumigations at two CT values
and at two temperatures.
At 3000 ppmv for 3 hr (9000 ppmv-hr) (24 -
25 °C and 85 - 95% RH), no viable spores
were observed on any material coupons except
for one of the five painted concrete coupons
and all of the coupons of compressed cellulose
insulation. The results correspond to a >6 log
reduction in viable spores for all fumigated
materials except cellulose insulation.
Repeating the 9000 ppmv-hr test at a higher
temperature and essentially the same RH (30 -
32 °C and 74 - 89% RH) resulted in no CPU
being observed on any fumigated material,
including painted concrete and cellulose
insulation. These results correspond to a >6
log reduction in viable spores for all fumigated
materials.
60
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Table 4-5. B. anthracis Ames Spores Mean CFU after ClOi Fumigation at Various CTs,
Temperatures
Trial
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
B. anthracis Ames
Glass (small),
Ix 107 spores
applied
Painted Concrete,
Ix 108 spores
applied
Galvanized Metal,
Ix 108 spores
applied
Decorative
Laminate,
Ix 108 spores
applied
Cellulose Insulation,
Ix 108 spores
applied
Particle Board,
Ix 108 spores
applied
Industrial Carpet,
Ix 108 spores
applied
Plate Glass,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Painted Concrete,
Ix 108 spores
applied
Galvanized Metal,
Ix 108 spores
applied
Decorative
Laminate,
Ix 108 spores
applied
Cellulose Insulation,
Ix 108 spores
applied
Particle Board,
Ix 108 spores
applied
Industrial Carpet,
Ix 108 spores
applied
[C102],
Contact Time,
CT
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
3000 ppmv, 3 hr,
9000 ppmv-hr
Temperature
Range
24 - 25 °C
24 - 25 °C
24 - 25 °C
24 - 25 °C
24 - 25 °C
24 - 25 °C
24 - 25 °C
24 - 25 °C
30 - 32 °C
30 - 32 °C
30 - 32 °C
30 - 32 °C
30 - 32 °C
30 - 32 °C
30 - 32 °C
RH
Range
85 - 95%
85 - 95%
85 - 95%
85 - 95%
85 - 95%
85 - 95%
85 - 95%
85 - 95%
74 - 89%
74 - 89%
74 - 89%
74 - 89%
74 - 89%
74 - 89%
74 - 89%
Control
Mean CFU
(SD)
4.70 x 106
(1.13xl06)
3.89 xlO7
(LlSxlO7)
4.68 xlO6
(2.00 x 106)
3.58 xlO7
(LlSxlO7)
4.83 x 107
(2.13xl07)
3.53 x 106
(3.25 x 106)t
4.31 xlO7
(8.43 x 106)
3.90 x 107
(9.24 x 106)
3.42 x 106
(6.98 x 105)
5.29 x 107
(2.33 x 107)
9.19 xlO6
(1.35xl07)
1.07 x 108
(9.03 x 107)
1.17xl08
(1.59xl08)
4.02 x 107
(1.39xl07)
5.42 x 107
(2.64 x 107)
Decon
Mean CFU
(SD)*
0
6.61 x 102
(1.48 xlO3)
[1 +, 4 -]
0
0
5.07 x 102
(3.24xl02)
[5+]
0
0
0
0
0
0
0
0
0
0
SD not calculated when no spores are recovered from any test coupon.
^Two of the five control coupons had no recovered agent.
61
-------
Table 4-5. Continued
Trial
5
6
6
6
6
6
6
6
6
B. anthracis Ames
Plate Glass,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Painted Concrete,
Ix 108 spores
applied
Galvanized Metal,
Ix 108 spores
Applied
Decorative Laminate,
Ix 108 spores
Applied
Cellulose Insulation,
Ix 108 spores
Applied
Particle Board,
Ix 108 spores
Applied
Industrial Carpet,
Ix 108 spores
Applied
Plate Glass,
Ix 108 spores
Applied
^ x x T' Temperature
Contact Time, „
CT Range
3000 ppmv,
3 hr, 30 - 32 °C
9000 ppmv-hr
3000 ppmv,
20 min,
1000 ppmv-hr ^
3000 ppmv,
20 min,
1000 ppmv-hr ^
3000 ppmv,
20 min,
1000 ppmv-hr
3000 ppmv,
20 min,
1000 ppmv-hr
3000 ppmv,
4 hr, 25 - 27 °C
12,000 ppmv-hr
3000 ppmv,
20 min, 25-27°C
1000 ppmv-hr
3000 ppmv,
20 min, 25 - 27 °C
1000 ppmv-hr
3000 ppmv,
20 min, 25 - 27 °C
1000 ppmv-hr
RH
Range
74 - 89%
83 - 97%
83 - 97%
83 - 97%
83 - 97%
83 - 97%
83 - 97%
83 - 97%
83 - 97%
Control Test
Mean CFU Mean CFU
(SD) (SD)*
(3.82xl07)
4.01 x 106
(1.63 x 106)
8.13 xlO7
(2.82 x 107)
2.35 xlO7
(1.92xl07)
7.63 x 107
(2.23 x 107)
9.07 x 107
(LlSxlO7)
4.55 xlO7
(2.27 x 107)
6.27 x 107
(3.64xl06)
7.32 xlO7
(8.02 x 106)
0
0
0
0
0
0
0
0
0
SD not calculated when no spores are recovered from any test coupon.
^Two of the five control coupons had no recovered agent.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
62
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Table 4-6. Mean Log Reduction of B. anthracis Ames Spores after ClOi Fumigation
at Various Temperatures and Contact Times
CT,
Trial ppmv-
hr
[C1O2] Time,
ppmv
hr
Material
Mean Log Reduction (95% Confidence
Interval) and p-value"
4 9000 3000
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Plate Glass
>6.66 (5/5) p=0.0135
>6.87 (4/5) p=0.0135
>6.64 (5/5) p=0.0135
>7.53 (5/5) p=0.0135
5.03 (4.67, 5.39) p<0.0001
Control Data Failed Acceptance Criteria*
>7.63 (5/5) p=0.0135
>7.58 (5/5) p=0.0135
5 9000 3000
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Plate Glass
>6.53 (5/5) p=0.0135
>7.69 (5/5) p=0.0135
>6.54 (5/5) p=0.0135
>7.92 (5/5) p=0.0135
>7.73 (5/5) p=0.0135
>7.58 (5/5) p=0.0135
>7.69 (5/5) p=0.0135
>7.75 (5/5) p=0.0135
6 1000 3000
12,000 3000
Glass (small)
Painted Concrete
Galvanized Metal
0.33 Decorative Laminate
Particle Board
Industrial Carpet
Plate Glass
4 Cellulose Insulation
>6.58 (5/5) p=0.0135
>7.89 (5/5) p=0.0135
>7.26 (5/5) p=0.0135
>7.87 (5/5) p=0.0135
>7.62 (5/5) p=0.0135
>7.80 (5/5) p=0.0135
>7.86 (5/5) p=0.0135
>7.95 (5/5) p=0.0135
*Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons. Confidence intervals
and p-values are from two sample t-tests comparing logs of control and treated coupons. Bolded results are statistically
significant at 0.05 level.
^One or more of treated coupons had no recovered agent (the exact number is shown in parentheses as test coupons
with no growth/total test coupons). The reported ">x" value is the mean log reduction with one CPU substituted for all
zero recovery coupon values to permit calculation of the log. For these trials, the test of statistical significance is a non-
parametric Kolmogorov-Smimov test where a p-value less than 0.05 indicate statistically significantly greater reduction
in the treated group than in the controls.
'Two of the five control coupons as well as all five of the treated coupons had no recovered agent.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
The efficacy of C1O2 fumigation against
B. anthracis Ames spores on seven types
of material coupons (all types except
cellulose insulation) was evaluated at a
lower CT of 1000 ppmv-hr (3000 ppmv
for 0.33 hr) (25 - 27 °C and 83 - 97%
RH). At the CT of 1000 ppmv-hr, no
CPU were observed on any fumigated
material. These results correspond to a
>6 log reduction in viable spores for all
fumigated materials.
Because C1O2 did not kill all spores at a
CT of 9000 ppmv-hr (at 24 - 25 °C), a
63
-------
higher CT of C1O2 fumigation was
applied against B. anthracis Ames
spores on cellulose insulation as an extra
test in Trial 6. At the higher CT, 12,000
ppmv-hr (3000 ppmv for 4 hr), at 25 - 27
°C and 83 - 97% RH, no CPU were
observed on fumigated cellulose
insulation coupons. These results
correspond to a >7 log reduction in
viable spores for fumigated cellulose
insulation coupons. Note that increased
efficacy was observed both at a higher
temperature (30 - 32 °C), a higher RH
range, and a higher CT.
Test coupons were in sealed vials in the
test chamber. The test coupons were
removed from the vials and exposed to
the fumigant for the specified contact
time. For example, a 1-hr contact time
would begin by removing the test
coupon from the sealed vial one hr
before the termination of the fumigation.
Controls were used to check whether
C1O2 could leak into the sealed vials in
the test chamber containing the test
coupons before the contact period began
(test coupons removed from the sealed
vials). These controls included both
positive control coupons and a
potassium iodide buffer ("impinger
solution") chemical indicator solution.
Time zero positive control coupons were
in sealed vials in the test chamber for the
full fumigation cycle and removed from
the chamber without opening the vials;
there was no exposure of the TO coupons
to the fumigant. The positive control
coupons were extracted and cultured to
determine the CPU indicative of viable
spores. No loss of viable spores (CPU)
from TO coupons was observed,
confirming that spores in the sealed vials
were not exposed to C1O2 fumigation
prior to the vials being opened.
Also, a sealed vial of potassium iodide
buffer was placed into the test chamber
for a full fumigation cycle at 3000 ppmv
C1O2. After the exposure, the potassium
iodide solution was then shaken and
checked for any color change that would
be expected if C1O2 leaked into the vial.
No detectable C1O2 was found in the
potassium iodide buffer, again
confirming that there was no leakage of
C1O2 into the sealed vials. The sealed
vials were identical to those used to
protect test coupons from C1O2 before
the vials were opened and the test
coupons removed to begin the contact
time.
4.1.5 Investigation of Effect of
Temperature on CIO2 Efficacy
Efficacy of fumigation with C1O2 was
compared at two temperatures. Except
for cellulose insulation, no viable spores
(zero CPU) were recovered from a
replicate set of test coupons after
fumigation at a CT of 9000 ppmv-hr at
either 24 °C (Trial 4) or 30 °C (Trial 5).
While it was possible to compare the
control and decontamination results
within such a test (with no recovered
CPU) to determine a conservative
estimate of effectiveness, the
comparison of two such decontamination
treatments to each other will yield an
indeterminate result.
Only for cellulose insulation could a
comparison be made. Cellulose
insulation decontaminated with 3000
ppmv for 3 hr at 24 °C in Trial 4
exhibited a mean reduction of 5.03 log
of B. anthracis Ames. In Trial 5, with
3000 ppmv for 3 hr at 32 °C, all the
decontaminated cellulose insulation
coupons exhibited zero remaining CPU,
resulting in a minimum 7.73 mean log
64
-------
reduction. The higher temperature trial
was thus more efficacious by a minimum
2.70 log than the lower temperature trial
and this difference was statistically
significant (p=0.0135), as shown in
Table 4-7. The results of this comparison
must be interpreted with caution, though,
since the difference between two test
conditions may be due to differences in
the RH range or trial-to-trial test
differences as well as true differences in
decontamination effectiveness.
Table 4-7. Statistical Comparison of ClOi Efficacy against B. anthracis Ames Spores
on Cellulose Insulation at Two Temperatures
Comparison
Difference in Mean Log Reduction in
30°C and 24°C and p-value'
Trial 5 Cellulose at 30 °C vs. Trial 4 Cellulose at 24 °C
>2.70 p=0.0135
*Difference in mean efficacy at 30 °C and 24 °C. Confidence intervals and p-values are from two sample t-tests
comparing mean efficacy at 30 °C and 24 °C. Bolded results are statistically significant at 0.05 level.
4.1.6 Investigation of Effect of Organic
Burden, Spore Types, andRH on
CIO 2 Efficacy
In cooperation with EPA, the U.S. Army
Research ECBC has performed parallel
fumigation studies, but with differences:
EPA/ECBC used a different strain of B.
anthracis, specifically NNR1A1, used
different methods of spore preparation,
different types of material coupons,
different sizes of coupons, different RH
control range, and added organic burden.
In the series of trials shown in Tables 4-
8 and 4-9, the following questions were
addressed:
• Does the choice of strain of B.
anthracis (Ames orNNRlAl)
impact efficacy results?
• Does lowering the RH from a
range of 80 - 84% to 75 - 77%
impact efficacy of C1O2
fumigation?
• Does the addition of organic
burden to the B. anthracis Ames
spore preparation in the
EPA/ECBC methods change the
decontamination efficacy of C1O2
fumigation?
To evaluate the impact of low levels of
added organic burden, the efficacy of
C1O2 fumigation was evaluated against
B. anthracis Ames spores on I-beam
steel, bare pine wood, and ceiling tile
coupons using the quantitative method.
Conditions were a CT of 3000 ppmv-hr
(3000 ppmv for 1 hr), 27 °C, and 82 -
84% RH. In Trial 4c, no additional
organic burden was added to the stock
spore suspension. In Trial 4d, 0.5% fetal
bovine serum was added to the stock
spore suspension. No significant
differences between spores with and
without added organic burden were
observed. Both with and without added
organic burden, about 400 spores were
recovered from coupons of ceiling tile
and no viable spores were recovered
after treatment from coupons of I-beam
steel and ceiling tile.
To evaluate the impact of slightly (about
5%) lower RH, the efficacy of C1O2
fumigation was evaluated against B.
anthracis Ames spores on ceiling tile
coupons using the quantitative method.
Conditions in Trial 4e were 3000 ppmv-
hr CT (3000 ppmv for 1 hr), 24 - 26 °C
and 71 - 77% RH. The Trial 4e results at
65
-------
lower RH could be compared to the
results from Trial 4d at higher RH and
with added organic burden. No moisture
was present in the test chamber at either
the higher or lower RH conditions. At
the lower RH the gloves did not feel
"slick" as they do when wet.
Table 4-8. Mean C1O2 Fumigation of B. anthrads Ames and NNR1A1 Spores CFU
with and without Added Organic Burden
B. anthrads Ames (Trial 4c)
I-Beam Steel
Bare Pine Wood
Ceiling Tile
B. anthrads Ames + 0.5%
Fetal Bovine Serum (Trial 4d)
I-Beam Steel
Bare Pine Wood
Ceiling Tile
B. anthrads + 0.5% Fetal
Bovine Serum (Trial 4e)
I-Beam Steel (NNR1A1 strain)
Bare Pine Wood (NNR1A1
strain)
Ceiling Tile (NNR1A1 strain)
Ceiling Tile (Ames strain)
[C102],
Contact Time,
CT
3000 ppmv, 1 hr,
3000 ppmv-hr
3000 ppmv, 1 hr,
3000 ppmv-hr
3000 ppmv, 1 hr,
3000 ppmv-hr
[C102],
Contact Time,
CT
3000 ppmv, 1 hr,
3000 ppmv-hr
3000 ppmv, 1 hr,
3000 ppmv-hr
3000 ppmv, 1 hr,
3000 ppmv-hr
[C102],
Contact Time,
CT
3000 ppmv, 1 hr,
3000 ppmv-hr
3000 ppmv, 1 hr,
3000 ppmv-hr
3000 ppmv, 1 hr,
3000 ppmv-hr
3000 ppmv, 1 hr,
3000 ppmv-hr
Temperature
26 °C
26 °C
26 °C
Temperature
27 °C
27 °C
27 °C
Temperature
24 - 26 °C
24 - 26 °C
24 - 26 °C
24 - 26 °C
RH
80%
80%
80%
RH
82-
84%
82-
84%
82-
84%
RH
71-
77%
71 -
77%
71-
77%
71-
77%
Control
Mean CFU
(SD)
3.87x10'
(LlSxlO7)
1.27 x 107
(8.04 x 106)
1.98 x 107
(7.29 x 106)
Control
Mean
(SD)
6.56 x 106*
(1.46 xlO7)
1.41 xlO7
(7.18xl06)
1.58 x 107
(6.55 x 106)
Control
Mean
(SD)
7.97 x 105
(2.24 x 105)
1.27 x 10s
(8.90 x 105)
1.20 x 106
(4.47 x 105)
7.70 x 106
(3.28xl06)
Test
Mean CFU
(SD)f
0
4.35 x 102
(9.70 x 102)
[1+]
0
Test
Mean (SD)
0
4.35 xlO2
(9.70 x 102)
[1+]
0
Test
Mean (SD)
4.74 x 101
(1.04 xlO2)
[1+]
9.00 x 103
(1.04 x 102)
2.02 xlO3
(1.36 xlO3)
3.36 x 104
(3.36 xlO4)
[3+]
*Two of the five control coupons as well as all five of the treated coupons had no recovered agent.
f SD not calculated when no spores are recovered from any test coupon.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
66
-------
Table 4-9. Mean Log Reduction of ClOi Fumigation of B. anthracis Ames and
NNR1A1 Spores with and without Added Organic Burden
„ . , Biological
Tnal A *
Agent
4c Ames strain
, Ames + organic
burden
Ames strain
4e
NNR1A1 strain
[C102],
Temperature
RH
3000 ppmv
25 °C
80%
3000 ppmv
27 °C
82 - 84%
3000 ppmv
24 - 26 °C
71-77%
3000 ppmv
24 - 26 °C
75 - 77%
Time, __ , . ,
, Material
hr
I-Beam Steel
Bare Pine
Wood
Ceiling Tile
I-Beam Steel
1 Bare Pine
Wood
Ceiling Tile
1 Ceiling Tile
I-Beam Steel
1 Bare Pine
Wood
Ceiling Tile
Mean Log Reduction (95%
Confidence Interval) and p-
value*'
>7.57 (5/5) p=0.0135
>6.38 (4/5) p=0.0135
>7.27 (5/5) p=0.0135
Control Data Failed Acceptance
Criteria*
>6.44 (4/5) p=0.0135
>7.17 (5/5) p=0.0135
>4.02 (2/5) p=0.0135
>5.41 (4/5) p=0.0135
2.38 (1.65, 3.11) p=0.0004
2.82 (2.44, 3.21) p<0.0001
"Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons. Confidence intervals
and p-values are from two sample t-tests comparing logs of control and treated coupons. Bolded results are
statistically significant at 0.05 level.
' One or more treated coupons had no recovered agent (the exact number is shown in parentheses as test coupons with
no growth/total test coupons). The reported ">x" value is the mean log reduction with one CPU substituted for all zero
recovery coupon values to permit calculation of the log. For these trials, the test of statistical significance is a
nonparametric Kolmogorov-Smirnov test where a p-value less than 0.05 indicate statistically significantly greater
reduction in the treated group than in the controls.
*Two of the five control coupons as well as all five of the treated coupons had no recovered agent.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
Trials 4c and 4d showed that low levels
of organic burden added to spores had
no impact on C1O2 fumigation efficacy
under the conditions tested. There was
no statistically significant observed
effect of adding 0.5% fetal bovine serum
to the stock solution of B. anthracis
Ames spores before applying spores to
bare pine wood or ceiling tile coupons
prior to fumigation with C1O2 at 3000
ppmv-hr. However, with complete
decontamination (zero CPU recovered
after fumigation) it cannot be assumed
that with alternate treatment conditions
(lower CT) the organic burden would not
impact the efficacy results. Because of
failure of control coupons to meet
acceptance criteria (two control coupons
showed no growth), the decontamination
efficacy of C1O2 against spores on I-
beam steel was not determined.
In cases where treatments being
compared both result in complete
decontamination, comparisons of
efficacy are indeterminate. Most of the
conditions for Trials 4c and 4d resulted
in complete decontamination (zero CPU
recovered after fumigation) and
comparisons are therefore indeterminate.
The exceptions are the two Trial 4e
results with decontamination of
67
-------
NNR1 Al on ceiling tile and on bare pine
wood shown in Table 4-10. Within Trial
4e, the mean decontamination efficacy
against B. anthracis Ames for ceiling tile
was not significantly different from
NNR1A1; data variability associated
with the mean log reduction of B.
anthracis Ames for ceiling tile
potentially inhibited a statistically
significant difference from being
detected.
(The differences in efficacy attributed to
the B. anthracis spore strain [Ames
versus NNR1 Al] might, alternatively, be
attributable to differences in spore
preparation methods used for the two
types of spores. The potential impact of
differences in the methods used to
prepare spores is an alternative source of
variability in spore resilience that merits
further investigation. In either case, the
efficacy observed for the B. anthracis
Ames spore preparation was not
significantly different from efficacy
observed for the B. anthracis NNR1 Al
spore preparation.)
However, comparing the log reduction in
B. anthracis Ames on ceiling tile in Trial
4d (higher RH) with B. anthracis Ames
on ceiling tile in Trial 4e (lower RH)
showed that efficacy was about 3.3 log
less at the lower RH. Thus, RH rather
than the strain of B. anthracis appears to
account for the difference in efficacy
between the EPA/ECBC results and the
Battelle results. The tighter RH range
has a significant impact on efficacy and
is a parameter that must be controlled
and adequately monitored to
appropriately explain efficacy testing
results.
Shown in Table 4-9, efficacy was
reduced at the lower RH. Viable spores
were recovered from three of the five
ceiling tile coupons at the lower RH
(average recovery about 3000 spores). In
contrast, no viable spores were
recovered from any ceiling tile coupons
at the higher RH in Trial 4d. At the high
RH, a 7.2 log reduction of viable B.
anthracis Ames spores from ceiling tile
was observed. However, a 4.0 log
reduction was observed at the low RH.
Further, after a 3000 ppmv-hr
fumigation at the lower RH in Trial 4e,
viable B. anthracis NNR1A1 spores
were recovered from one of five coupons
of I-beam steel and, on average,
thousands of viable spores were
recovered from all bare pine wood and
ceiling tile coupons. The efficacy of
C1O2 at the lower RH (71 - 77%) ranged
from a 2.4 log kill on bare pine wood to
>5.4 log kill on steel I-beam.
Shown in Table 4-10, compared to B.
anthracis Ames in Trial 4d (high RH),
the mean decontamination efficacy for
the B. anthracis NNR1 Al strain on bare
pine wood and ceiling tile was estimated
to be at least 4.1 and 4.4 log less,
respectively, at the lower RH in Trial 4e.
In both cases, the difference was
statistically significant.
These trials comparing the EPA/Battelle
testing and the EPA/ECBC testing were
intended only to identify the cause(s) of
differences observed in the two test
approaches at one CT treatment. Based
on this limited investigation, differences
in results between EPA/ECBC testing
and the EPA/Battelle testing at the
specific CT were not shown to be due to
the difference in organic burden nor the
type of spore/spore preparation.
However a significant difference was
found for differences in RH. The higher
68
-------
RH, 80% and higher, resulted in much
higher efficacy (mean log reduction). A
potential explanation is that at RH of
80% and higher, condensation tends to
be observed. Absorption of C1O2 into the
condensate may convert the fumigation
into a liquid decontamination with
different properties. The results showing
the extreme impact of RH on the
efficacy of C1O2 are supported by
additional studies conducted by
EPA/ECBC.[17] These results should not
be generalized to infer that the addition
of organic burden, alternative spore
strains, or alternative spore preparations
has no effect on efficacy results.
Table 4-10. Statistical Comparison of ClOi Fumigation of B. anthracis Ames and
NNR1A1 Spores with and without Added Organic Burden
Comparison
Difference in Mean Log
Reduction and p-value"
Trial 4e Ceiling Tile (B. anthracis Ames) vs. Trial 4e Ceiling Tile
(B. anthracis ~~ ~ "
Not significant
Trial 4d Bare Pine Wood (B. anthracis Ames + 0.5% FBS Organic
burden) vs. Trial 4e Bare Pine Wood (B. anthracis NNR1A1)
>4.06 p=0.0135
Trial 4d Ceiling Tile (B. anthracis Ames + 0.5% FBS Organic
burden, high RH) vs. Trial 4e Ceiling Tile (B. anthracis NNR1A1
+ 0.5% FBS Organic burden, lower RH)
>4.35 p=0.0135
'Difference in mean efficacy of paired test conditions. Confidence intervals and p-values are from two
sample t-tests comparing mean efficacy results from two test conditions. Bolded results are statistically
significant at 0.05 level.
' One or more treated coupons had no recovered agent (the exact number is shown in parentheses as test
coupons with no growth/total test coupons). The reported ">x" value is the mean log reduction with one
CPU substituted for all zero recovery coupon values to permit calculation of the log. For these trials, the
test of statistical significance is a nonparametric Kolmogorov-Smirnov test where a p-value less than 0.05
indicates statistically significantly greater reduction in the treated group than in the controls.
4.1.7 CIO2Fumigation ofRicin at Two
CTs
Trials 9 and 10 evaluated the efficacy of
C1O2 fumigation at two CT values
against ricin toxin (-25 jig per test
coupon) applied to seven types of
material coupons. The two CT values
tested were 500 ppmv-hr (1500 ppmv for
20 min) and 100 ppmv-hr (200 ppmv for
30 min). During fumigation the
temperature was maintained at 23 - 25
°C and the RH was controlled at 80 -
84%. The test parameters and results are
shown in Tables 4-11 and 4-12. A C1O2
fumigation level of 500 ppmv-hr
resulted in >99.1% geometric mean
reduction in ricin recovered from all
materials except cellulose insulation
within 20 min; a 92.7% reduction in
ricin from cellulose insulation was
observed.
A C1O2 fumigation level of 100 ppmv-hr
resulted in >99% geometric mean
reduction in ricin recovered from glass,
painted concrete, and decorative
laminate; lower levels of
decontamination were observed for
galvanized metal (98.5% reduction),
cellulose insulation (93.4% reduction),
particle board (95.8% reduction), and
industrial carpet (98.3% reduction).
69
-------
Comparing the ricin removal for the 100
and 500 ppmv-hr test conditions showed
that the results were not significant for
three materials. Smal but statistically
significant differences were observed for
the other four materials. In two cases the
recoveries at the higher CT showed that
the decontamination was more
efficacious; in the other two cases
decontamination at the lower CT were
slightly more efficacious. These small
differences are likely due to trial to trial
differences such as differences in the
recovered mass of ricin from the positive
control coupons in the two trials, rather
than a difference that could be attributed
to differences in operational conditions.
70
-------
Table 4-11. Ricin Cytotoxicity with and without ClOi Fumigation at Various CTs
Trial
9
9
9
9
9
9
9
10
10
10
10
10
10
10
,„ , . , Contact Time
Matenal
mm
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
20
20
20
20
20
20
20
30
30
30
30
30
30
30
, [C102],
ppmv
1500
1500
1500
1500
1500
1500
1500
200
200
200
200
200
200
200
Temperature,
°C
23
23
23
23
23
23
23
23
23
23
23
23
23
23
-25
-25
-25
-25
-25
-25
-25
-25
-25
-25
-25
-25
-25
-25
RH,
%
80
80
80
80-
80-
80-
80-
80-
80-
80-
80-
80-
80-
80-
-84
-84
-84
84%
84%
84%
84%
84%
84%
84%
84%
84%
84%
84%
TO Control
Mean
(SD)^g
2.56
(1.17)
17.58
(4.02)
2.97
(0.56)
0.70
(0.69)
2.33
(0.87)
1.24
(0.67)
19.36
(3.34)
4.25
(2.33)
25.50
(2.83)
2.46
(0.95)
9.29
(1.91)
4.70
(0.71)
3.52
(1.45)
30.22
(2.84)
20 or 30 min
Control
Mean
(SD),ng
4.88
(1.62)
27.10
(5.13)
1.92
(1.20)
1.21
(1.25)
3.22
(1.54)
4.75
(0.80)
21.46
(4.80)
4.92
(0.97)
33.70
(1.74)
1.02
(0.33)
4.47
(1.29)
4.24
(1.42)
4.52
(1.41)
26.33
(8.15)
Test
Mean
(SDXfig
0.01
(0.002)
0.01
(0.002)
0.01
(0.009)
0.01
(0.001)
0.20
(0.07)
0.03
(0.005)
0.09
(0.03)
0.01
(0.002)
0.01
(0.001)
0.01
(0.002)
0.01
(0.001)
0.27
(0.07)
0.21
(0.11)
0.62
(0.69)
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
71
-------
Table 4-12. Geometric Mean Percent Reduction of Ricin Cytotoxicity with ClOi
Fumigation at Various CTs
Trial
9
9
9
9
9
9
9
10
10
10
10
10
10
10
CT,
ppmv-hr
500
500
500
500
500
500
500
100
100
100
100
100
100
100
[C102]
ppmv
1500
1500
1500
1500
1500
1500
1500
200
200
200
200
200
200
200
Contact
Time, min
20
20
20
20
20
20
20
30
30
30
30
30
30
30
Material
Glass (small)
Painted Concrete
Galvanized Metal
Decorative
Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Glass (small)
Painted Concrete
Galvanized Metal
Decorative
Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Mean % Reduction
(95% Confidence Interval) and p- Value*
99.84% ( 99.76% , 99.90% ) p=<0.0001
99.96% ( 99.95% , 99.97% ) p=<0.0001
99.46% ( 97.51% , 99.88% ) p=<0.0001
99.13% ( 97.79% , 99.66% ) p=<0.0001
92.74% ( 81.69% , 97.12% ) p=0.0003
99.46% ( 99.29% , 99.58% ) p=<0.0001
99.61% ( 99.43% , 99.74% ) p=<0.0001
99.75% ( 99.67% , 99.81% ) p=<0.0001
99.97% ( 99.97% , 99.97% ) p=<0.0001
98.49% ( 97.92% , 98.91% ) p=<0.0001
99.69% ( 99.59% , 99.76% ) p=<0.0001
93.42% ( 89.26% , 95.97% ) p=<0.0001
95.81% ( 92.17% , 97.75% ) p=<0.0001
98.31% ( 95.23% , 99.40% ) p=<0.0001
"Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons. This result was
transformed to percentage. Confidence intervals and p-values are from two sample t-tests comparing logs of control
and treated coupons. Bolded results are statistically significant at 0.05 level.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
4.1.8 CIO2 Fumigation of Vaccinia
Virus at Two CTs
Trials 13 and 14, shown in Tables 4-13
and 4-14, evaluated the efficacy of C1O2
fumigation against vaccinia virus at two
CT values. Trial 13 used a fumigation
CT of 500 ppmv-hr (1500 ppmv for 20
min) against vaccinia virus applied to
seven types of coupons. Trial 14 used a
fumigation CT of 125 ppmv-hr (250
ppmv for 30 min) against vaccinia virus
applied to six types of coupons. During
fumigation the temperature was
maintained at 24 ± 2 °C and the RH was
controlled at 80 ± 5% (full scale). The
C1O2 fumigation levels of 500 ppmv-hr
and 125 ppmv-hr each resulted in no
plaque-forming virus being recovered
from any coupons. For coupons with
high recovery efficiencies, the complete
inactivation of viable virus equated to >6
log inactivation after the fumigation.
The vaccinia results exhibited complete
inactivation for the decontaminated
coupons within every test, with each test
result compared to its respective controls
yielding a statistically significant mean
log reduction. Due to the complete
decontamination observed in all tests, no
statistical comparison can be made
between the two concentration and time
conditions for each material.
72
-------
4.1.9 Summary of Findings from the
CIO2 Fumigation Investigation
• C1O2 fumigation was efficacious
for the decontamination of B.
anthracis Ames spores (and
surrogates B. anthracis NNR1A1
and B. subtilis), ricin, and
vaccinia virus from all of a broad
range of indoor building
materials tested.
• A CT of 1000 ppmv-hr of C1O2
resulted in a >7 log reduction in
viable B. anthracis Ames spores
(no CPU detected) from six of
the seven building materials
tested (all except compressed
cellulose insulation) at 24 - 27 °C
and >80% RH.
• Compressed cellulose insulation
required 12,000 ppmv-hr at 24 -
27 °C and >80% RH or 9,000
ppmv-hr at elevated temperature
of 30 - 32 °C and 74 - 79% RH
for a >7 log reduction in B.
anthracis Ames spores (no CPU
detected). This limited
investigation suggests that
raising the temperature may
increase efficacy. While the
temperature impact was
statistically significant, further
testing is necessary to draw a
firm conclusion.
• High RH (82 - 84%) appears to
be more efficacious than lower
RH (75 - 77%).
• Whether a CT of 2,250 was
generated at lower concentrations
for a longer time (750 ppmv, 3
hr) or a higher concentration for
a shorter time (3000 ppmv, 0.75
hr) did not change the observed
>7 log reduction efficacy against
B. anthracis Ames on painted
concrete; the extrapolate of this
result to other concentration -
contact time combinations is not
clear from this work.
A CT of 500 ppmv-hr of C1O2
resulted in a geometric mean
reduction of ricin of >99.1%
from all materials tested except
cellulose insulation which
exhibited a 92.7% reduction.
A CT of 125 ppmv-hr of C1O2
resulted in no vaccinia virus
being recovered from any coupon
of any of the seven materials
tested.
73
-------
Table 4-13. Vaccinia PFU with and without C1O2 Fumigation at Various CTs
Trial
13
13
13
13
13
13
13
14
14
14
14
14
14
14
,„ , . , Contact Time
Matenal
mm
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
20
20
20
20
20
20
20
30
30
30
30
30
30
30
, [C102],
ppmv
1500
1500
1500
1500
1500
1500
1500
250
250
250
250
250
250
250
Temperature,
°C
22.7
22.7
22.7
22.7
22.7
22.7
22.7
22.1
22.1
22.1
22.1
22.1
22.1
22.1
-23
-23
-23
-23
-23
-23
-23
-22
-22
-22
-22
-22
-22
-22
.7
.7
.7
.7
.7
.7
.7
.4
.4
.4
.4
.4
.4
.4
RH,
%
80
80
80
80
80
80
80
75
75
75
75
75
75
75
-83
-83
-83
-83
-83
-83
-83
-78
-78
-78
-78
-78
-78
-78
TO Control ,n „ . „ . ,
T.» T.T^TT 20 or 30 mm Control
(1^7 Mean PFU (SD)
3.93 x 106
(4.26 x 105)
7.61 x W6
(4.42 x 105)
5.19X107
(1.45 xlO7)
3.97xio7
(1.02 xlO7)
7.34 x 105
(1.44 xlO5)
i.75xl03
(1.69 xlO3)
4.22 x 105
(3.13xl05)
3.73 x 104
(6.65 x 103)
4.5ixio5
(4.63 x 104)
3.85 x 105
(1.62 xlO5)
3.86xio5
(7.54 x 104)
5.28 x 105
(5.43 x 104)
1.07 xlO5
(1.31xl04)
2.95 x 105
(1.43 x 105)
2.29 xlO6
(1.05 x 106)
5.67 xlO6
(1.74 xlO6)
1.07x10'
(3.12xl06)
2.09x10'
(8.44 x 106)
8.41 x 10"
(2.51xl05)
1.81x10"
(2.07 x 103)
2.25 x 10"
7.91 x 104)
3.22 xlO4
(3.12xl03)
3.25 xlO5
(5.45 x 104)
2.95 x 105
(9.11xl04)
2.32 xlO5
(1.53 x 104)
3.67 xlO5
(3.12xl05)
LlOxlO5
(1.30 xlO4)
2.98x10"
(8.74 x 104)
C1O2 Test
Mean PFU*
0
0
0
0
0
0
0
0
0
0
0
0
0
0
* SD not calculated when no viruses are recovered from any test coupon. Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
74
-------
Table 4-14. Mean Log Reduction of Vaccinia PFU with ClOi Fumigation at Various CTs
Trial
13
13
13
13
13
13
13
14
14
14
14
14
14
14
CT,
ppmv-hr
125
125
125
125
125
125
125
500
500
500
500
500
500
500
[C102],
ppmv
250
250
250
250
250
250
250
1500
1500
1500
1500
1500
1500
1500
Contact
Time, min
30
30
30
30
30
30
30
20
20
20
20
20
20
20
Material
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Mean Log Reduction (95% Confidence
Interval) and p-Value"
>4.51 (5/5) p=0.0135
>5.51,(5/5) p=0.0135
>5.34 (5/5) p=0.0135
>5.36 (5/5) p=0.0135
>5.43 (5/5) p=0.0135
>5.04 (5/5) p=0.0135
>5.45 (5/5) p=0.0135
>6.32 (5/5) p=0.0135
>6.74 (5/5) p=0.0135
>7.02 (5/5) p=0.0135
>7.30 (5/5) p=0.0135
>5.91 (5/5) p=0.0135
>2.32 (5/5) p=0.0135
>5.33 (5/5) p=0.0135
"Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons. Confidence intervals and p-values are from two sample t-tests comparing logs of
control and treated coupons. Bolded results are statistically significant at 0.05 level.
HOne or more treated coupons had no recovered agent (the exact number is shown in parentheses as test coupons with no growth/total test coupons). The reported ">x" value is the
mean log reduction with one PFU substituted for all zero recovery coupon values to permit calculation of the log. For these trials, the test of statistical significance is a
nonparametric Kolmogorov-Smirnov test where a p-value less than 0.05 indicates statistically significantly greater reduction in the treated group than in the controls.
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
75
-------
4.2 MeBr Fumigation Results
4.2.1 Results for MeBr Fumigation of
B. anthracis on Glass and
Ceiling Tile at Various CT
Values (36 °C)
The efficacy of MeBr against B.
anthracis Ames spores on glass and
ceiling tile was evaluated using the
quantitative method at CT values
ranging from 315 to 2250 mg/L-hr at
75% RH and 36 °C. (Results are
reported in mg/L rather than ppmv to be
consistent with the historical precedent
in previous efficacy studies of reporting
MeBr concentrations as mg/L or g/L.)
The results of these tests are shown in
Tables 4-15 and 4-16 and Figure 4-1. A
sigmoid log reduction vs. CT curve was
observed. Complete kill of spores (0
CPU; >6 log reduction) from glass and
ceiling tile was observed at a CT value
of 2520 mg/L-hr. Low within-trial
variability and high between-trial
variability was observed along the
sloped portion of the CT curve. Because
within-trial variability was low, the
observed uncertainty along the slope
may suggest an unknown and
uncontrolled variable that impacts
efficacy in the transition range from
about 1 log to >6 log reduction in viable
spores.
Figure 4-1 shows that, for a given CT, a
low concentration (53 mg/L) and higher
contact time was generally less
efficacious than a higher concentration
(105, 212, or 320 mg/L) with a lower
contact time for decontamination of B.
anthracis Ames spores from glass or
ceiling tile coupons.
Table 4-16 shows that, for B. anthracis
Ames spores, at CT values of 795 mg/L-
hr or higher (75% RH and 36 °C)
significant log reductions were
consistently observed on glass and
ceiling tile. At CT values >795 mg/L-hr
and <1260 mg/L-hr the log reductions
were small (1.07 - 3.67), but significant.
76
-------
Table 4-15. Results of Varying MeBr CT (36 °C and 75% RH)
Trial
1
1
2
2
o
J
o
J
4
4
5
5
6
6
7
7
B. anthracis Ames
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
[MeBr] Range
(mg/L),
Contact Time,
CT
103 -117 mg/L
24 hr
-2520 mg/L-hr
103 -117 mg/L
24 hr
-2520 mg/L-hr
99 -114 mg/L
12 hr
-1260 mg/L-hr
99 -114 mg/L
12 hr
-1260 mg/L-hr
98 -110 mg/L
3hr
-3 15 mg/L-hr
98 -110 mg/L
3hr
-3 15 mg/L-hr
103 - 109 mg/L
6hr
-630 mg/L-hr
103 - 109 mg/L
6hr
-630 mg/L-hr
102-1 10 mg/L
9hr
-945 mg/L-hr
102-1 10 mg/L
9hr
-945 mg/L-hr
201-216 mg/L
6hr
-1272 mg/L-hr
201-216 mg/L
6hr
-1272 mg/L-hr
199-218 mg/L
9hr
-1908 mg/L-hr
199-218 mg/L
9hr
-1908 mg/L-hr
Temperature
Range,
°C
36-38
36-38
35-37
35-37
35-36
35-36
35-36
35-36
35-36
35-36
35-37
35-37
35-37
35-37
RH
Range,
%
73-80
73-80
70-76
70-76
70-76
70-76
70-76
70-76
71-76
71-76
72-79
72-79
72-75
72-75
Control
Mean CFU
(SD)
3.05 xlO6
(2.51xl05)
1.13xl07
(1.46 xlO6)
2.93 xlO5
(1.68 xlO5)
1.08 xlO7
(2.37 x 106)
4.05 x 106
(4.52 x 105)
2.06 xlO7
(9.34 x 106)
5.46 xlO6
(1.58 xlO6)
1.57xl07
(6.90 x 106)
4.78 xlO6
(5.06 x 105)
1.93xl07
(3.97xl06)
4.39 xlO6
(7.65 x 105)
9.01 xlO6
(9.32 x 105)
4.04 xlO6
(5.94 x 105)
7.77 xlO6
(7.86 x 105)
Decon
Mean CFU
(SD)t
0
0
0
1.32X101
(1.81 xlO1)
3.52xl06
(4.73 x 105)
6.83 x 106
(3.55 x 106)
2.29 xlO6
(9.54 x 105)
9.85 xlO6
(2.22 x 106)
4.34 xlO5
(1.97 xlO5)
1.68 x 106
(7.00 x 105)
3.13xl04
(2.79 x 104)
3.57 x 105
(8.87 x 104)
0
1.34 xlO1
(3.00 xlO1)
^SD not calculated when no spores are recovered from any test coupon.
Note [MeBr] is concentration of MeBr in the chamber atmosphere, in mg/L.
77
-------
Table 4-15. Continued
[MeBr] Range _ , „„
( /n Temperature RH
Trial B. anthracis Ames „ , ™. J.' Range, Range,
Contact Time, Jf 0/6
CT °C %
8
8
9
9
10
10
11
11
12
12
13
13
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
Glass (small),
Ix 107 spores
applied
Ceiling tile,
Ix 108 spores
applied
201-212 mg/L
7hr
-1484 mg/L-hr
201-212 mg/L
7hr
-1484 mg/L-hr
200-2 12 mg/L
7hr
-1484 mg/L-hr
200-2 12 mg/L
7hr
-1484 mg/L-hr
50-54 mg/L
15 hr
-795 mg/L-hr
50-54 mg/L
15 hr
-795 mg/L-hr
52-56 mg/L
18 hr
-954 mg/L-hr
52-56 mg/L
18 hr
-954 mg/L-hr
5 1-55 mg/L
24 hr
-1272 mg/L-hr
5 1-55 mg/L
24 hr
-1272 mg/L-hr
102-108 mg/L
12 hr
-1250 mg/L-hr
102-108 mg/L
12 hr
-1250 mg/L-hr
35-37 71-77
35-37 71-77
35-36 36-43
35-36 36-43
35-37 70-75
35-37 70-75
35-37 71-77
35-37 71-77
35-37 71-77
35-37 71-77
35-37 71-74
35-37 71-74
Control
Mean CFU
(SD)
2.08 x 107
(7.54 x 106)
5.15xl07
(9.16xl06)
9.38 xlO6
(5.64 x 105)
3.02 x 107
(2.77 x 106)
6.37 x 106
(4.24 x 105)
5.07 x 107
(3.69xl06)
5.92 x 106
(8.86 x 105)
3.16xl07
(6.91 x 106)
8.31xl06
(1.26 xlO6)
5.22 x 107
(9.18xl06)
3.26 x 106
(4.55 x 105)
9.73 x 106
(2.92 x 105)
Decon
Mean CFU
(SD)*
8.47 xlO2
(1.14xl02)
3.39 xlO4
(5.91 x 103)
8.21 xlO3
(8.53 x 102)
7.25 xlO4
(1.01 x 104)
1.90 xlO5
(1.13xl05)
2.03 xlO6
(2.18xl05)
2.33 xlO3
(1.82 xlO3)
3.98xl05
(1.06 xlO5)
0
2.95 xlO3
(1.42 xlO3)
3.44 xlO4
(7.42 x 103)
3.50xl05
(6.01 x 104)
^SD not calculated when no spores are recovered from any test coupon.
Note [MeBr] is concentration of MeBr in the chamber atmosphere, in mg/L.
78
-------
Table 4-16. Analysis of Results of Varying MeBr CT (36 °C and 75% RH) for Mean
Log Reduction of B. anthracis Ames
Trial
3
4
10
5
11
14
13
2
6
12
8
17
16
15
7
1
Nominal
CT,
mg/L-hr
315
630
795
945
954
1260
1260
1260
1272
1272
1484
1575
1890
1908
1908
2520
Time,
hr
3
6
15
9
18
12
12
12
6
24
7
15
18
9
9
24
Actual
[MeBr],
mg/L
98-110
103-109
50-54
102-110
52-56
102-108
102 - 108
99-114
201-216
51-55
201-212
102-109
99-105
198-210
199-218
103-117
Mean Log Reduction (95% Confidence Interval)
and p-value*
Glass Ceiling tile
Not significant
0.40 (0.15, 0.65)
p=0.0094
1.59 (1.32, 1.86)
p=0.0002
1.07 (0.89, 1.25)
p<0.0001
3.67 (2.95, 4.39)
0.64 (0.53, 0.75)
p<0.0001
1.98 (1.87, 2.09)
p<0.0001
> 6.46 (5/5)"
p=0.0135
2.49 (1.70, 3.27)
p=0.0017
> 6.92 (5/5)"
p=0.0135
4.36 (4.16, 4.56)
p<0.0001
>6.50 (5/5)"
p=0.0135
>6.67 (5/5)"
p=0.0135
>5.75 (4/5)"
p=0.0135
> 6.60 (5/5)"
p=0.0135
> 6.48 (5/5)"
p=0.0135
0.54 (0.08, 1.00)
p=0.0356
Not significant
1.40 (1.34, 1.46)
p<0.0001
1.09 (0.86, 1.31)
p<0.0001
1.91 (1.75, 2.06)
p<0.0001
1.26 (1.04, 1.4675)
PO.0001
1.45 (1.37, 1.53)
pO.OOOl
> 6.42 (3/5)'"
PO.0135
1.41 (1.29, 1.54)
p<0.0001
4.33 (3.93, 4.74)
p<0.0001
3.18 (3.07, 3.29)
p<0.0001
4.73 (3.98, 5.48)
PO.0001
>7.11 (5/5)'
p=0.0135
2.72 (1.92, 3.53)
p=0.0013
> 6.52 (4/5)'"
p=0.0135
> 7.05 (5/5)'
p=0.0135
Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons. Confidence intervals
and p-values are from two sample t-tests comparing logs of control and treated coupons. Bolded results are statistically
significant at 0.05 level.
H One or more treated coupons had no recovered agent (the exact number is shown in parentheses as test coupons with
no growth/total test coupons). The reported ">x" value is the mean log reduction with one CPU substituted for all zero
recovery coupons values to permit calculation of the log. For these trials, the test of statistical significance is a
nonparametric Kolmogorov-Smirnov test where a p-value less than 0.05 indicates statistically significantly greater
reduction in the treated group than in the controls.
Note [MeBr] is concentration of MeBr in the chamber atmosphere, in mg/L.
79
-------
•#— 53 mg/L, Glass
-•-53 mg/L, Tile
•vv-105 rng/L, Glass
^^105 mg/L, Tile
•*-212. rng/L, Glass
*-320 rng/L, Tile
500 1000 1500 2000
CT, mg/L-Hr
2500
3000
Figure 4-1. Mean B. anthracis log reduction from glass and ceiling tile vs. CT at
various concentrations of MeBr.
4.2.2 Results for MeBr Fumigation of
B. anthracis on Various
Materials at Various CT Values
(36 °C, 75% RH)
The efficacy of MeBr fumigation against
B. anthracis Ames spores on five
additional types of materials was
evaluated using the quantitative method
at four contact times. The fumigation
was performed at 75% RH and 36 °C at
105 or 212 mg/L MeBr. The results of
these tests are shown in Table 4-17 and
Figure 4-2. For all materials, little or no
significant log reduction (<2 log) was
observed at CT of 1260 mg/L-hr.
Greater than 6 log reduction was
observed for B. anthracis on all
materials except cellulose at a CT of
1900 (1890-1908) mg/L, whether by 18-
hr contact with 105 mg/L MeBr or 9 hr
contact with 212 mg/L MeBr.
As shown in Figure 4-3, very similar
results were observed in log reduction at
a CT of about -1900 mg/L-hr whether
105 mg/L for 18 hr or 212 mg/L for 9 hr
was used for the fumigation. For
cellulose, no significant difference was
found between the two CTs. For tile, the
lower concentration, longer dwell time
gave statistically significantly better
mean log reduction (at least 2.37 log
more) than the higher concentration,
shorter dwell time. However, this result
appears to reflect trial to trial variability
rather than a difference attributable to
the approach used to generate the CT.
For all other materials (glass, painted
concrete, laminate, galvanized metal,
and carpet), there was complete kill for
at least one coupon of each of the two
CT values; no statistical comparison
could be performed.
80
-------
1200
1400
1600
CT, mg/L-Hr
1800
» Painted Concrete
• Cellulose
—.i—Laminate
X Galvanized Metal
Carpet
Glass
Tile
B. subtilis, Glass
2000
Figure 4-2. Mean B. anthracis (B. subtilis on glass) log reduction from various
materials vs. CT.
81
-------
Table 4-17. Results of Contact Time Series of MeBr Fumigation (36 °C and 75%
RH) of B. anthracis Spores on Various Building Materials
Contact Mean Log Reduction (95% Confidence Interval) and
Trial mg/L_'jjr mg/L Time> Painted Cellulose Decorative Galvanized
hr Concrete Insulation Laminate Metal
0.81
14 1260 102-108 12 ^65'
pO.OOOl
>6.86
17 1575 102-109 15 ^L.
p=0.0135
>7.37
16 1890 99-105 18 (4/5)'
p=0.0135
>7.13
15 1908 198-210 9 (4/5)'
p=0.0135
Not
significant
3.47(3.11,
3.82)
pO.OOOl
4.68
(3.90, 5.47)
pO.OOOl
>5.44
(1/5)'
p=0.0135
0.90 (0.71,
1.28)
pO.OOOl
>5.41
(1/5)'
p=0.0135
>7.32
(5/5)'
p=0.0135
>6.69
(3/5)'
p=0.0135
1.72
(1.16,2.29)
p=0.0018
>7.6983
(5/5)'
p=0.0135
>6.93
(3/5)'
p=0.0135
>6.32
(3/5)'
p=0.0135
p-Value*
Indust.
Carpet
0.97
(0.63,
1.31)
p=0.0004
4.06
(3.39,
4.74)
pO.OOOl
>7.30
(4/5)'
p=0.0135
>7.06
(4/5)"p=
0.0135
"Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons. Confidence intervals
and p-values are from two sample t-tests comparing logs of control and treated coupons. Bolded results are statistically
significant at 0.05 level.
H One or more treated coupons had no recovered agent (the exact number is shown in parentheses as test coupons with
no growth/total test coupons). The reported ">x" value is the mean log reduction with one CFU substituted for all zero
recovery coupons values to permit calculation of the log. For these trials, the test of statistical significance is a
nonparametric Kolmogorov-Smirnov test where a p-value less than 0.05 indicates statistically significantly greater
reduction in the treated group than in the controls.
Note [MeBr] is concentration of MeBr in the chamber atmosphere, in mg/L.
82
-------
D105 mg/L, 18 hr
• 212 mg/L, 9 hr
'' B.s. refers to 5. subtilis
Figure 4-3. Mean B. anthracis log reduction from various materials
(-1900 mg/L-hr).
¥. 2.3 Results for MeBr Fumigation of
B. anthracis on Various
Materials at 2 CT Values (25 °C
and36°C, 75% RH)
Two trials were performed to evaluate
whether lower temperature would
significantly lower the efficacy of MeBr
fumigation against B. anthracis Ames
spores on eight types of materials.
Efficacy was evaluated using the
quantitative method at two contact times,
i.e., 9 and 24 hr. The fumigation was
performed at 75% RH and at the lower
temperature of 25 °C at 212 mg/L MeBr.
The results of these tests are shown in
Table 4-18 and Figure 4-4. (Testing
results at 36 °C are included for
comparison.)
Except for carpet, the decontamination
efficacy was much lower at 25 °C than
at 36 °C. For all materials except carpet,
low levels of log reduction (<4 log) were
observed at a CT of 1908 mg/L-hr at the
low temperature of 25 °C. In contrast,
log reductions at the higher temperature
of 36 °C were >5.4 for all materials.
Except for cellulose and tile, the log
reductions at the higher temperature
were >6.3.
Greater than a 6 log reduction was
observed for B. anthracis on all
materials except ceiling tile at a CT of
5088 mg/L generated with a 24-hr
contact at 212 mg/L MeBr.
83
-------
Table 4-18. Results of 9 hr and 24 hr Contact Time of MeBr Fumigation (75% RH)
of B. anthracis on Various Building Materials at a Target of 320 mg/L
Trial
18
15
19
CT, Temp.,
mg/L-hr °C
1908
(212 mg/L, 25
9hr)
1908
(212 mg/L, 36
9hr)
5088
(212 mg/L, 25
24 hr)
Painted
Concrete
2.20
(0.05)
>7.13
(1.38)
>7.92
(0.00)
Cellulose
Insulation
2.33
(0.28)
>5.47
(1.24)
6.02
(1.40)
Mean Log Reduction (SD)*
Decor. Galv. Indust.
Laminate Metal Carpet
>7.61
(0.00)
>6.69
(1.24)
>7.64
(0.00)
2.15
(0.12)
>6.35
(1.85)
>7.61
(0.00)
>7.71
(0.00)
>7.06
(1.28)
>7.78
(0.00)
Glass
2.55
(0.08)
>6.51
(0.00)
>7.70
(0.00)
Ceiling
Tile
2.85
(0.02)
4.74
(0.72)
4.55
(0.35)
Wood
3.52
(0.54)
NA
7.64
(0.00)
Mean log reduction is mean of logs of control coupons minus mean of logs of treated coupons.
1908ppm-Hr 5088 ppm-Hr
CT, mg/L-Hr
Painted Concrete
Cellulose
Laminate
Galvanized Metal
Carpet
Glass
Tile
Wood
Figure 4-4. Mean B. anthracis log reduction from various materials vs. CT (25 °C,
75% RH, [MeBr] = 212 mg/L, contact times = 9 and 24 hr).
4.2.4 Results for MeBr Fumigation of
B. subtilis on Glass at Various
CT Values
B. subtilis was prepared and inoculated
onto the small glass coupons in a manner
identical to that used for B. anthracis
and included in all of the test conditions.
As shown in Figure 4-5 and in Table 4-
19, significant differences were observed
between decontamination efficacy of
MeBr fumigation against B. anthracis
Ames and B. subtilis on glass. The
difference in efficacy was very large
(>5.48 log reduction difference) in the
CT range of 1260 - 2880 mg/L-hr.
84
-------
Table 4-19. Difference in Log Reduction of B. anthracis Ames and B. subtilis on
Glass at Various CTs
Difference in Mean Log Reduction
and p-Value*
CT, mg/L-hr Bacterium
Glass
~ B-™thracis Ames0.41(0.25,0.57)
„ VSA',. P5'48 (4/5)
B ,'.,. p=0.047
B. subtilis r
B. anthracis Ames , , . ,-,-,.
1890 vs. >6.64(5/5)
B ,'.,. p=0.047
B. subtilis r
B. anthracis Ames - „
1890 vs >3.»
B Ll- P=°-°47
B. subtilis
'Difference in mean efficacy of paired test conditions. Confidence intervals and p-values are from two
sample t-tests comparing mean efficacy results from two test conditions. Bolded results are statistically
significant at 0.05 level.
' One or more treated coupons had no recovered agent (the exact number is shown in parentheses as test
coupons with no growth/total test coupons). The reported ">x" value is the mean log reduction with one
CPU substituted for all zero recovery coupons values to permit calculation of the log. For these trials, the
test of statistical significance is a nonparametric Kolmogorov-Smirnov test where a p-value less than 0.05
indicates statistically significantly greater reduction in the treated group than in the controls.
As shown in Figure 4-5, little or no anthracis Ames on glass at a CT of 1575
efficacy of MeBr against B. subtilis on or greater CT with no viable spores
glass was observed in the CT range or observed at CTs of 1890 mg/L-hr (105
1260-1908 mg/L-hr. In contrast, a high mg/L for 18 hr) or 1908 mg/L-hr (212
level of efficacy was observed against B. mg/L for 9 hr).
85
-------
8.00
7.00
6.00
5'°°
o>
4.00
3.00
2.00
1260
1575 1890
CT, mg/L-Hr
190E
Figure 4-5. Mean log reduction of B. anthracis on various materials and B. subtilis
spores on glass at various CTs.
4.2.5 Results for MeBr Fumigation of
B. anthracis on Glass and
Ceiling Tile at Alternate RH
Conditions
A trial was performed to evaluate
whether, at a given CT, efficacy would
be different at a low RH (40%) during
fumigation compared to a higher RH
(75%) during fumigation. The results,
shown in Table 4-20, indicated that there
was a significant log reduction observed
(compared to the controls) using MeBr
at both RH conditions. The results also
show that efficacy was higher at 75%
RH than at 40% RH. However, as noted
above, high between-trial variability was
observed in the transition region of the
CT curve between the low kill to
complete kill. Because this was a single
run with a relatively small, but
significant, difference between the log
reductions observed at the two RH
conditions, no conclusions are
conclusively drawn.
86
-------
Table 4-20. Significance of Differences in Log Reduction Arising from Percent RH
at 212 mg/L MeBr and 7-Hr Contact Time at 37 °C
Trial
CT
RH
Mean Log Reduction (SD)
Glass Ceiling Tile
1484
1484
75%
40%
4.39 (0.06)
3.19(0.08)
3.06 (0.04)
2.62 (0.06)
Statistical significance of difference between
75% RH and 40% RH
p< 0.0001
p< 0.0001
4.2.6 Results for MeBr Fumigation of
B. subtilis on Suture Loops at
Various CT Values
Suture loops, prepared and inoculated
with B. anthracis spores as described for
B. subtilis spores in AOAC method
966.04,[13] were included in the
fumigation tests. The use of the
qualitative suture loop test was included
to provide a point of comparison
between results from this investigation
using B. anthracis spores and the results
from AOAC 966.04 qualitative testing
by others using B. subtilis spores as a
surrogate.
After fumigation for a specific contact
time, suture loops were placed into TSB
and incubated for seven days. The tubes
containing the inoculated suture loops
were checked for cloudiness, indicating
growth, on days one and seven. As
shown in Table 4-21, at a CT of 1260
mg/L-hr (105 mg/L x 12 hr) the
inoculated suture loops all resulted in
cloudy media indicating growth of
viable spores after fumigation. In
contrast, after inoculated suture loops
were exposed to MeBr at a CT of 1575
mg/L-hr (105 mg/L x 15 hr) or greater,
no cloudiness was observed in the
incubated medium consistent with
complete kill of the spores. Positive and
negative controls exhibited expected
growth and no growth, respectively.
Table 4-21. Qualitative Spore Viability Test
105 mg/L
Contact Time
12 hr 15 hr 18 hr
212 mg/L
9hr
Suture Loop Positive Control (n=5)
Suture Loop Decontamination Test Coupons (n=5)
Positive Control
Negative Control
"-" indicates that all coupons at the specified time and treatment exhibited no growth in TSB.
"+" indicates that all three coupons at the specified time and treatment exhibited growth in TSB.
Note [MeBr] is concentration of MeBr in the chamber atmosphere, in mg/L.
87
-------
In Table 4-22, the log reduction in
recovered spores from glass and ceiling
tile after decontamination at a given CT
is shown, along with the corresponding
number of silk suture loop carriers that
were positive for growth of B. anthracis
Ames spores after the decontamination.
The qualitative results from the suture
loops and the log reduction from both
glass and ceiling tile showed more
growth of spores at the lower CT and
less growth at the higher CT. The log
reduction from glass is 5.75 or greater
when no bacterial growth is observed
from any of the five replicate silk suture
loops included in the test. However,
ceiling tile exhibited a log reduction as
low as 2.73 when all silk suture loops
were exhibiting no growth. Suture loop
decontamination efficacy appeared to be
more similar to the decontamination of
nonporous than porous materials; i.e.,
silk suture loops appeared to be easier to
decontaminate than the porous material.
Table 4-22. Comparison of Results for Log Reduction and Qualitative Spore
Viability Test at Various CT Conditions
CT, mg/L
Log Reduction
Glass Ceiling Tile
Silk Suture Loops
Exhibiting No
Growth/ Total
1260
1575
1890
1908
0.64
5.75
6.67
6.51
1.26
2.73
7.15
4.74
5/5
0/5
0/5
0/5
4.2.7 Results for MeBr Fumigation of
B. atrophaeus on Stainless Steel
in Tyvek* Packaging at Various
CT Values
For all MeBr fumigation trials, Bis (B.
atrophaeus on stainless steel in Tyvek
packaging [Apex Labs]) were included.
At all CTs investigated, viable B.
atrophaeus spores were detected in the
qualitative viability test; fumigation did
not kill all of the B. atrophaeus spores
inside the Tyvek packaging.
4.2.8 Summary of Findings from the
MeBr Investigation
• MeBr fumigation was
efficacious for the
decontamination of B.
anthracis Ames from all of a
broad range of indoor
building materials tested.
ACT of 1890mg/L-hr
resulted in a >6 log reduction
in viable B. anthracis Ames
spores from all building
materials except the
compressed cellulose
insulation and, in one trial
only, from ceiling tile.
Whether a given CT was
generated at lower
concentrations for a longer
time (105 mg/L for 18 hr) or
a higher concentration for a
shorter time (212 mg/L - 9
hr) did not appear to impact
the efficacy against B.
anthracis; this result may not
be generalized to lower
concentrations of MeBr (53
mg/L) or to other
88
-------
concentration and contact
time combinations based
upon these data alone.
• The CT range that resulted in
a 1 to 6 log reduction in
viable B. anthracis Ames
spores exhibited little or no
efficacy against B. subtilis on
glass; B. subtilis on glass
appears to be a conservative
surrogate for B. anthracis
Ames in MeBr fumigation.
• A small, but significant,
difference in efficacy
between 75% and 40% RH
was observed. Because of the
small difference in a single
trial, no conclusions are
drawn. However, even if the
difference was caused by RH,
the difference was so small as
to have limited practical
significance.
• When tested at a higher
temperature (36 °C vs. 25
°C), mean log reductions in
B. anthracis were
considerably higher on the
painted concrete, cellulose,
galvanized metal, glass, and
ceiling tile (CT of 1908
mg/L-hr MeBr).
• No viable spores were
detected on silk suture loops
after exposure to a MeBr CT
of 1575 mg/L-hr. These
results suggest that B.
anthracis spores on silk
suture loops are easier to kill
than on any other material
tested except galvanized
metal.
• The qualitative results from
the suture loops and the log
reduction from both glass and
ceiling tile show more growth
of spores at the lower CT and
less growth at the higher CT.
The log reduction from glass
is 5.75 or greater when no
bacterial growth is observed
from any of the five replicate
silk suture loops included in
the test.
4.3 HP Fumigation
4.3.1 Results of Varying HP CT at 22
°C
The efficacies of HP fumigation against
B. anthracis Ames spores on nine
materials at varying CT values are
shown in Table 4-23 and Figures 4-6 and
4-7. No viable B. anthracis spores were
recovered from carpet, painted concrete,
glass, aluminum, keyboard, laminate,
ductwork, and ceiling tile at exposures
of 120 min to the 500 ppmv HP
fumigation cycle. Decontamination of
wood was more difficult than other
materials: B. anthracis spores were still
recovered from wood after 120-min
exposure to the 500 ppmv HP
fumigation cycle. No viable B. anthracis
spores were recovered from wood at an
exposure of 240 min to the 500 ppmv
HP fumigation cycle.
With the 200-250 ppmv HP fumigation
cycle, shown in Table 4-24, B. anthracis
spores were not recovered from
laminate, ductwork, painted concrete,
glass, and ceiling tile after 120 min of
exposure, but B. anthracis spores were
recovered from carpet and wood after
the entire 240-min fumigation cycle. For
perspective, VHP® sterilant when used
with a STERIS VHP® generator for
sterilization of exposed pre-cleaned dry
porous and non-porous surfaces,
specifies a sterilization phase with a
89
-------
minimum of 250 ppm[v] of VHP® up to 4,000 ft3 [113,000 L].
sterilant for 90 min in sealed enclosures
90
-------
Table 4-23. STERIS VHP® Fumigation Results for B. anthrads (500 ppmv)
„ . , Contact Time, mm _ _ , . ,
Tnal „„ ' Matenal
CT, ppmv-hr
c -i, A j Mean Recovered B. anthrads (CFU/coupon)*
(CPU/coupon) P°si'ivft _ Test „
^ ^ ' Control7 Coupon11
Mean Log
Reduction*1
500 ppmv Fumigation Cycle
1 30
250
1 60
500
1 120
1000
1 240
2000
1 (Full cycle)
240
2000
2a 30
250
2a 60
500
Finished aluminum
Computer keyboard keys
Industrial carpet
Painted joint tape
Finished aluminum
Computer keyboard keys
Industrial carpet
Painted joint tape
Finished aluminum
Computer keyboard keys
Industrial carpet
Painted joint tape
Finished aluminum
Computer keyboard keys
Industrial carpet
Painted joint tape
Finished aluminum
Computer keyboard keys
Industrial carpet
Painted joint tape
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
4.87 xlO8* 7.30 ± 4.03 x 10* *
4.87xl08§ 3.96±3.29xl08
4.87xl08§ 7.70 ± 4.24 xlO8*
4.87xl08§ 5.42 ± 1.72 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
4.87 x 108
7.30 ± 4.03 xlO8*
3.96±3.29xl08
7.70 ± 4.24 xlO8*
5.42 ± 1.72 xlO8
7.30 ± 4.03 xlO8*
3.96±3.29xl08
7.70 ± 4.24 xlO8*
5.42 ± 1.72 xlO8
7.30 ± 4.03 xlO8*
3.96±3.29xl08
7.70 ± 4.24 xlO8*
5.42 ± 1.72 xlO8
7.30 ± 4.03 xlO8*
3.96±3.29xl08
7.70 ± 4.24 xlO8*
5.42 ± 1.72 xlO8
2.80 xlO8* 2.61 ±1.58x10"
2.80xl08§ 1.67 ± 1.04 x 108
2.80xl08§ 2.13 ± 2.39 x 108
2.80xl08§ 2.26 ± 1.60 x 108
2.80 xlO8* 2.61 ±1.58x10"
2.80xl08§ 1.67 ± 1.04 x 108
2.80xl08§ 2.13 ± 2.39 x 108
2.80xl08§ 2.26 ± 1.60 x 108
0
0
2.73 ± 4.06 xlO5
0
0
0
1.85 ± 2.56 xlO4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.74 ± 1.41 xlO5
1.20 ± 2.68 xlO2
0
0
6.67 ±1 1.6 xlO3
0
8.86 ±0.00
8.60 ±0.00
4.31 ±1.21
8.73 ±0.00
8.86 ±0.00
8.60 ±0.00
6.70 ±2.35
8.73 ±0.00
8.86 ±0.00
8.60 ±0.00
8.89 ±0.00
8.73 ±0.00
8.86 ±0.00
8.60 ±0.00
8.89 ±0.00
8.73 ±0.00
8.86 ±0.00
8.60 ±0.00
8.89 ±0.00
8.73 ±0.00
8.42 ±0.00
8.22 ±0.00
3.44 ±0.85
7.80 ±1.24
8.42 ±0.00
8.22 ±0.00
5. 57 ±1.69
8.35 ±0.00
Data are expressed as mean ± SD of five replicates.
t Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
# H
TO was lower than target recovery of >10% of spike amount. SD not calculated when no spores are recovered from any test coupon
91
-------
Table 4-23. Continued
Trial
2a
2a
2a
3a
3a
3a
3a
3a
Contact Time, min
CT, ppmv-hr
120
1000
240
2000
(Full cycle)
240
2000
30
250
60
500
120
1000
240
2000
(Full cycle)
240 min
2000
Material
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Pine wood
Plate glass
Ceiling tile
Pine wood
Plate glass
Ceiling tile
Pine wood
Plate glass
Ceiling tile
Pine wood
Plate glass
Ceiling tile
Pine wood
Plate glass
Ceiling tile
Spike
Amount
(CFU/coupon)
2.80 xlO8*
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
2.80xl08§
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
9.77 x 106
Mean Recovered B. anthracis
Positive Control
2.61 ±1.58x10"
1.67 ± 1.04 xlO8
2.13 ± 2.39 xlO8
2.26 ± 1.60 xlO8
2.61 ± 1.58 xlO8
1.67 ± 1.04 xlO8
2.13 ± 2.39 xlO8
2.26 ± 1.60 xlO8
2.61 ± 1.58 xlO8
1.67 ± 1.04 xlO8
2.13 ± 2.39 xlO8
2.26 ± 1.60 xlO8
5.47 ± 1.57 xlO5*
8.18 ± 10.5 xlO6
7.49 ± 1.40 xlO5*
5.47 ± 1.57 xlO5*
8.18 ± 10.5 xlO6
7.49 ± 1.40 xlO5*1
5.47 ± 1.57 xlO5*
8.18 ± 10.5 xlO6
7.49 ± 1.40 xlO5*
5.47 ± 1.57 xlO5*
8.18 ± 10.5 xlO6
7.49 ± 1.40 xlO5*
5.47 ± 1.57 xlO5*
8.18 ± 10.5 xlO6
7.49 ± 1.40 xlO5*
(CFU/coupon)*
Test Coupon"
0
0
0
0
0
0
0
0
0
0
0
0
1.82 ± 3.23 xlO4
2.73 ± 3.52 xlO2
0
4.51 ± 4.41 xlO3
0
0
7.19 ± 8.28 xlO2
0
0
0
0
0
0
0
0
Mean Log
Reduction*1
8.42 ±0.00
8.22 ±0.00
8.33 ±0.00
8.35 ±0.00
8.42 ± 0.00
8.22 ±0.00
8.33 ±0.00
8.35 ±0.00
8.42 ± 0.00
8.22 ±0.00
8.33 ±0.00
8.35 ±0.00
1.95 ±0.66
5.40 ±1.42
5. 87 ±0.00
2.97 ±1.69
6.91 ±0.00
5. 87 ±0.00
3. 51 ±1.31
6.91 ±0.00
5. 87 ±0.00
5.74 ±0.00
6.91 ±0.00
5. 87 ±0.00
5.74 ±0.00
6.91 ±0.00
5. 87 ±0.00
Data are expressed as mean ± SD of five replicates.
t Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
*TO was lower than target recovery of >10% of spike amount.
'SD not calculated when no spores are recovered from any test coupon
92
-------
8 -
7
c 5
o D
-^
u c
3 3
-u
« ,
OC *
tug
O 3
_i a
/ / —
x" / X -*-
/ ^ / ^
A^^ V^ :t
^^^~*~-^ -*-
4—^^
250 500 1000 2000
HP, ppmv
Finished Aluminum
Keyboard Keys
Industrial Carpet
Painted Joint Tape
Decorative Laminate
Galvanized Metal
Pine Wood
Plate Glass
Ceiling Tile
Figure 4-6. Mean 5. anthracis spores log reduction from various materials vs.
hydrogen peroxide CT (500 ppmv concentration) at 22 °C
93
-------
Table 4-24. STERIS VHP® Fumigation Results for B. anthrads Ames Spores (225 ppmv)
Trial
Contact Time, mm _ _ , . ,
„„ ' Material
CT, ppmv-hr
Spike Amount
(CFU/coupon)
Mean Recovered B. anthrads (CFU/coupon)*
Positive Test Mean Log
Control Coupon* Reduction*
200-250 ppmv Fumigation Cycle
2b
2b
2b
2b
2b
30
113
60
225
120
450
240
900
(Full cycle)
240
900
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
Decorative Laminate
Galvanized metal ductwork
Industrial carpet
Painted concrete block
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
6.93 x 106'
______
6.93 x 106'
6.93 xlO6'
6.93 xlO6'
3.22 ± 0.50 xlO6
5.25 ± 0.79 xlO6
6.17 ± 0.57 xlO6
6.73 ± 0.70 xlO6
3.22 ± 0.50 xlO6
5.25 ± 0.79 xlO6
6.17 ± 0.57 xlO6
6.73 ± 0.70 xlO6
3.22 ± 0.50 xlO6
5.25 ± 0.79 xlO6
6.17 ± 0.57 xlO6
6.73 ± 0.70 xlO6
3.22 ± 0.50 xlO6
5.25 ± 0.79 xlO6
6.17 ± 0.57 xlO6
6.73 ± 0.70 xlO6
3.22 ± 0.50 xlO6
5.25 ± 0.79 xlO6
6.17 ± 0.57 xlO6
6.73 ± 0.70 xlO6
1.34±3.00xl01
0
3.35±0.74xl05
1.65 ± 3.59 xlO3
0
0
1.11 ±2.01 xlO3
6.60 ±14.8x10°
0
0
2.00 ± 4.47 xlO1
0
0
0
2.00 ± 2.99 xlO1
0
0
0
1.34±3.001
0
6.14 ±0.82
6.72 ±0.00
1.27 ±0.09
5.28 ±1.63
6.51 ±0.00
6.72 ±0.00
5.06 ±1.67
6.52 ±0.68
6.51 ±0.00
6.72 ±0.00
6.39 ±0.89
6.83 ±0.00
6.51 ±0.00
6.72 ±0.00
6.12 ±0.92
6.83 ±0.00
6.51 ±0.00
6.72 ±0.00
6.42 ±0.82
6.83 ±0.00
* Data are expressed as mean ± SD of five replicates. SD not calculated when no spores are recovered from any test coupon.
t Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
Application was lower than the target 7.5 x 105 CFU/coupon.
* TO was lower than target recovery of >10% of spike amount.
94
-------
Table 4-24. Continued
Trial
3b
3b
3b
3b
3b
D2
D2
D2
D2
D2
Contact Time, mm _ _ , . , Spike Amount
„„ , Matenal ,/4V.TT/
CT, ppmv-hr (CFU/coupon)
30
113
60
225
Pine wood
Plate glass
Ceiling tile
Pine wood
Plate glass
Ceiling tile
120 Pine wood
450 Plate glass
Ceiling tile
240
900
Pine wood
Plate glass
Ceiling tile
(Full cycle) Pine wood
240 Plate glass
900 Ceiling tile
30
113
60
225
120
450
240
900
(Full cycle)
240
900
Particle board
Cellulose insulation
Particle board
Cellulose insulation
Particle board
Cellulose insulation
Particle board
Cellulose insulation
Particle board
Cellulose insulation
1.02 xlO7
1.02 xlO7
1.02 x 107
1.02 xlO7
1.02 xlO7
1.02 xlO7
I.02X107
1.02 xlO7
1.02 xlO7
1.02 x 107
1.02 xlO7
1.02 xlO7
I.02X107
1.02 xlO7
1.02 xlO7
l.llxlO8
l.llxlO8
l.llxlO8
l.llxlO8
l.llxlO8
l.llxlO8
l.llxlO8
l.llxlO8
l.llxlO8
l.llxlO8
Mean Recovered/?, anthracis (CFU/coupon)*
Positive Test Mean Log
Control Coupon* Reduction*
3.59±0.63xl05#
3.53±1.68xl06
7.62 ± 2.18 xlO5*
3.59±0.63xl05#
3.53±1.68xl06
7.62 ± 2.18 xlO5*
3. 59 ± 0.63 x 10" *
3.53±1.68xl06
7.62 ± 2.18 xlO5*
3. 59 ± 0.63 x 10" *
3.53±1.68xl06
7.62 ± 2.18 xlO5*
3. 59 ±0.63x10"*
3.53±1.68xl06
7.62 ± 2.18 xlO5*
6.28 ±2.22x10'
2.05 ± 0.97 xlO7
6.28 ±2.22x10'
2.05 ± 0.97 xlO7
6.28 ±2.22x10'
2.05 ± 0.97 xlO7
6.28 ± 2.22 xlO7
2.05 ± 0.97 xlO7
6.28 ± 2.22 xlO7
2.05 ± 0.97 xlO7
2.06 ± 1.60 xlO4
1.34±3.00xl01
3.32±4.08xl01
6.83 ± 10.9 xlO3
2.66 ± 5.95 xlO1
2.00 ± 2.99 xlO1
2.68 ± 0.60 x 103
0
0
1.61 ± 1.67 xlO3
0
0
2.73 ± 3.84 x 104
0
0
i.i4±i.o7xios
2.71 ± 1.95 xlO6
1.90 ± 2.81 x 10s
4.32 ± 3.41 xlO5
6.72 ± 2.37 x 105
1.72 ± 1.73 xlO5
8.92 ± 2.47 xlO4
1.41 ± 1.76 xlO4
2.24 ± 1.28 x It)4
3.23 ± 0.72 xlO3
1.35 ±0.35
6.18 ±0.82
5. 18 ±0.98
2.15 ±0.70
6.12 ±0.95
5.21 ±0.92
2.14 ±0.10
6.55 ±0.00
5.88 ±0.00
3.52 ±1.87
6.55 ±0.00
5.88 ±0.00
3.32 ±2.45
6.55 ±0.00
5.88 ±0.00
1.86 ±0.33
0.97 ±0.32
1.86 ±0.58
1.84 ±0.48
2.00 ±0.18
2.41 ±0.74
2.86 ±0.13
3.41 ±0.53
3.52 ±0.30
3.81±0.10
* Data are expressed as mean ± SD of five replicates. SD not calculated when no spores are recovered from any test coupon.
t Positive control coupons were spiked but not exposed to the fumigant.
* Test coupons were spiked and exposed to the fumigant for the contact time.
'Application was lower than the target 7.5 x 105 CFU/coupon.
* TO was lower than target recovery of >10% of spike amount.
95
-------
113
225
450
HP, ppmv
900 Full Cycle
900
« I n d u s tri a I Ca rp et
• Concrete
A Decorative Laminate
X Galvanized Metal
—*—Pine Wood
—•—Plate Glass
—I—Ceiling Tile
Particle Board
—— Cellulose
Figure 4-7. Mean B. anthracis log reduction from various materials vs. HP CT (225
ppmv concentration) at 22 °C.
4.3.2 Summary of Findings from the
HP Investigation
• HP fumigation was
efficacious for the
decontamination of B.
anthracis Ames spores from
all of a broad range of indoor
building materials tested.
• ACT of 2000 ppmv-hr
resulted in a >5.7 log
reduction with no viable B.
anthracis Ames spores
recovered from all building
materials after a 4 hr cycle at
500 ppmv. (Higher or lower
log reduction with no viable
spores recovered results
mathematically from the
differential recovery
efficiencies for control
coupons among material
types.) A CT of 2000 ppmv-
hr is much higher than the
associated HP label
condition. Use of HP at 250
ppmv for 1.5hr(aCTof375
ppmv-hr) is the treatment
specified on the STERIS
(R)
Vaprox HP sterilant
package insert for sporicidal
efficacy (sterilization) for
pre-cleaned enclosures. The
registration is for the liquid
only, not the vapor.
ACT of 500 ppmv-hr
resulted in a >6.9 log
reduction with no viable B.
anthracis Ames spores
recovered from all nonporous
building materials after a 1 hr
contact time at 500 ppmv
(slightly higher than the
associated HP label condition
of 375 ppmv-hr).
96
-------
5.0 Liquid Decontamination Technologies Test Results
5.1 pH-Amended Bleach
5.1.1 Qualitative Results for pH-
Amended Bleach
Decontamination of Bacillus
Species
The qualitative cycle fraction test results
are shown in Table 5-1. Three test
coupons, in contact with the pH-
amended bleach, and three control
coupons, in contact with PBS, were
included at each time point.
No viable B. anthracis Ames spores
were observed on any material in the
qualitative testing after contact with pH-
amended bleach for 10 min or longer at
22 °C. Viable B. anthracis Ames spores
were observed after contact with PBS at
all time points from 10 to 60 min, except
for painted concrete.
Painted concrete positive controls
exhibited no growth in TSB at Day 7.
An aliquot of each negative TSB painted
concrete control on Day 7 was plated
onto tryptic soy agar plates. All of the
painted concrete positive control
samples at all contact times exhibited
growth of B. anthracis Ames when
plated onto TSA; colonies were
observed with morphologies consistent
with the morphology of B. anthracis
Ames colonies. No growth in TSA was
observed for blank controls. Painted
concrete coupons exhibited a biostatic
effect in TSB as demonstrated by the
growth after subsequent plating of an
aliquot from the TSB that showed that
viable B. anthracis Ames spores were
present in the TSB of all positive
controls.
Table 5-1. Results from Qualitative Evaluation of pH-Amended Bleach
Decontamination of B. anthracis Ames
Day?
Material Decontaminated, Contact Time Positive Control, Contact Time (PBS)
10 min 20 min 40 min 60 min 10 min 20 min 40 min 60 min
Glass .... + + + +
Painted Concrete .... . (+*) . (+*) . (+*) . (+*)
Galvanized Metal .... + + + +
Decorative Laminate - - - - + + + +
"-" indicates that all three coupons at the specified time and treatment exhibited no growth in TSB.
"+" indicates that all three coupons at the specified time and treatment exhibited growth in TSB.
"(+*)" indicates that when an aliquot of the negative TSB from painted concrete controls was plated onto tryptic soy
agar plates on Day 7, all samples at the specified time and treatment exhibited growth of B. anthracis on the TSA
plates.
97
-------
5.7.2 Quantitative Results for pH-
amended Bleach
Decontamination of Bacillus
Species
The efficacy of pH-amended bleach
solutions (6,300 - 6,600 ppm total
chlorine at pH 6.51 - 6.93) against three
strains of B. anthracis spores (virulent
Ames, avirulentNNRlAl, and Vollum)
and B. subtilis was evaluated using the
quantitative method. The spore recovery
results for individual types of Bacillus
spores exposed to pH-amended bleach or
PBS for specified time periods are
shown in Tables 5-2 through 5-5 and
Figure 5-1. In addition, the extracted
coupons were placed individually into
TSB and incubated for seven days to
look for cloudiness that would indicate
the presence of residual viable spores on
the test coupon. In all cases where no
spores were observed using the
quantitative methodology, no residual
viable spores were detected using the
qualitative method. A summary of
efficacy of pH-amended bleach against
the various types of spores, reported as
log reduction, is shown in Table 5-6.
Efficacy was shown to be dependent on
the type of material onto which the
spores are inoculated. For all spore types
tested, viable spores were present on
carpet and particle board after exposure
to pH-am ended bleach for up to 10 min.
Viable B. subtilis spores, but not B.
anthracis Ames spores, were recovered
from carpet and particle board after a 30-
min exposure to pH-amended bleach.
For all spore types tested, no viable
spores were present on glass, galvanized
metal, or decorative laminate after
exposure to pH-amended bleach for 5
min or longer.
While strong similarities in efficacy
were observed across spore types,
differences were also observed as
indicated above. For example, viable B.
anthracis, Vollum and B. subtilis spores
were present on painted concrete after
exposure to pH-amended bleach for five
min; the other two strains of B.
anthracis, Ames and NNR1 Al, did not
have viable spores present after exposure
to pH-amended bleach for 5 min. As
another example of observed
differences, for coupons of all materials
tested, there were no viable spores
recovered after 30-min exposure of B.
anthracis Ames spores to pH-amended
bleach. In contrast, viable B. subtilis
spores were recovered from both carpet
and particle board after 30-min exposure
to pH-amended bleach.
98
-------
Table 5-2. B. anthracis Ames Spores CFU after Various pH-Amended Bleach
Contact Times
Material
Glass
Painted Concrete
Industrial Carpet
Galvanized Metal
Particle Board
Decorative Laminate
Material
Glass
Painted Concrete
Industrial Carpet
Galvanized Metal
Particle Board
Decorative Laminate
CFU Applied
to Coupons
1.07 xlO7
1.05 xlO8
9.97 xlO7
9.97 xlO7
9.90 xlO7
1.08 x 108
CFU Applied
to Coupons
1.07x10'
1.05 x 10"
9.97 xlO7
9.97x10'
9.90 xlO7
1.08 x 108
Mean CFU (SD) Recovered from
Positive Control Coupons at Specified Contact Time
5 min 10 min 30 min
7.65 x 106 5.03 x 106 8.43 x 106
(1.17xl06) (2.22 xlO5) (8.05 x 105)
1.27 xlO7 1.41 xlO7 9.59 x 106
(1.29 xlO6) (1.08 xlO6) (6.96 x 105)
5.13xl07 4.32 xlO7 3.57xl07
(2.43 x 106) (2.40 x 106) (2.43 x 106)
5.07x10' 4.97x10' 4.24x10'
(7.50 x 106) (6.95 x 106) (4.54 x 106)
3.79x10' 3.45x10' 2.55x10'
(8.94 x 106) (4.68 x 106) (9.92 x 106)
1.27 xlO7 1.71 xlO7 2.71 x 107
(3.12xl06) (1.49 xlO6) (4.90 x 106)
Mean CFU (SD)* Recovered from
Test Coupons at Specified Contact Time
5 min 10 min 30 min
000
000
6.56 x 104 2.22 x 104
(2.39 x 104) (2.89 x 104)
000
6.34 xlO2 5.92xl02
(7.21 xlO2) (1.07 xlO3)
000
*SD not calculated when no spores are recovered from any test coupon.
99
-------
Table 5-3. B. anthracis Vollum Spores CFU after Various pH-Amended Bleach
Contact Times
Mean CFU/Coupon (SD)
,„ , . , CFU Applied at 5-min Contact Time
Material r
Positive Control Coupons Test Coupons
Glass 1.09 x 107
Painted Concrete 1 . 1 0 x 1 08
Industrial Carpet 1 . 10 x 108
Galvanized Metal 1.09 x 108
Particle Board LlOxlO8
Decorative Laminate 1.09 x 108
6.12 xlO6
(4.88 x 105)
5.56 x l66
(5.02 x 105)
3.45 xlO7
(l.SlxlO6)
1.95 x lO7
(7.64 x 106)
1.51 xlO7
(9.14xl05)
3.07 xlO7
(2.36 x 106)
0
5.88 x 102
(3.41 x 102)
4.24 x 103
(6.25 x 102)
0
1.04 x 103
(5.21 x 102)
0
*SD not calculated when no spores are recovered from any test coupon
Table 5-4. B. anthracis NNR1A1 Spores CFU after pH-Amended Bleach Contact
Times
Material
Glass
Painted Concrete
Industrial Carpet
Galvanized Metal
Particle Board
Decorative
Laminate
Material
Particle Board
CFU Applied
to Coupons
4.17 xlO6
3.50 xlO7
2.93 x 107
4.17 xlO7
3.50 xlO7
4.17 xlO7
CFU Applied
to Coupons
2.93 x 107
Mean CFU/Coupon (SD)*
at 5-min Contact Time
Positive Control Coupons Test Coupons
7.89 xlO5
(1.63 x 105)
3.97xio6
(9.55 x 105)
8.58 xlO6
(1.09 xlO6)
7.54 x 106
(2.06 x 106)
8.17'xi6"6
(1.25 x 106)
8.07 xlO6
(1.96xl06)
Mean CFU/Coupon
at 10-min Contact
Positive Control Coupons
8.85 xlO6
(l.SlxlO6)
0
0
2.07 x 101
(S.lOxlO1)
0
8.83 x 102
(6.38 x 102)
0
(SD)*
Time
Test Coupons
3.49 xlO1
(7.80 x 101)
SD not calculated when no spores are recovered from any test coupon.
100
-------
Table 5-5. B. subtttis CFU after Various pH-Amended
Material
Glass
Painted Concrete
Industrial Carpet
Galvanized Metal
Particle Board
Decorative Laminate
Material
Glass
Painted Concrete
Industrial Carpet
Galvanized Metal
Particle Board
Decorative Laminate
CFU
Applied
to Coupons
LlOxlO7
1.08 x 108
1.06 x 108
1.06 x 108
1.06 x 108
1.00 xlO8
CFU
Applied
to Coupons
LlOxlO7
1.08 x 108
1.06 x 108
1.06 xlO8
1.06 x 108
1.00 xlO8
Bleach Contact Times
Mean CFU (SD)* Recovered from
Positive Control Coupons at Specified Contact Time
5 min 10 min 30 min
4.00 x 106
(9.16xl05)
3.41 xlO7
(1.89 xlO7)
4.54 xlO7
(6.65 x 106)
4.11 xlO7
(1.03 x 107)
3.22 xlO6
(1.32 xlO6)
6.41 x 107
(6.60 x 106)
Mean CFU
Test Coupons
5 min
0
1.39X101
(S.lOxlO1)
6.45 x 104
(1.48 xlO4)
0
3.83 x 103
(1.17xl03)
0
2.92 x 106
(2.51xl05)
3.99xl07
(6.75 x 106)
5.27 x 107
(8.34 x 106)
3.73 x 107
(7.19xl06)
3.43 x 106
(1.87 xlO6)
5.74 x 107
(5.30 x 106)
5.07 xlO5
(6.01 x 104)
3.03 x 107
(2.56 x 106)
3.77 xlO7
(3.37xl06)
3.40 xlO7
(9.56 x 106)
4.35 xlO7
(1.99xl06)
4.35 xlO7
(LlSxlO7)
(SD)* Recovered from
at Specified Contact Time
10 min 30 min
0
0
5.43 x 103
(1.31 xlO3)
0
1.33xl03
(9.11xl02)
0
0
0
2.90 xlO3
(1.39xl03)
0
9.04 xlO1
(3.96X101)
0
*SD not calculated when no spores are recovered from any test coupon
101
-------
9nn
.UU
8 00
7r\r\
.UU
S R nn
.2 D.UU
« c nn
-§ 5-°°
8 A nn
CL 4-.UU
o Q nn
j o.UU
2nn
.UU
'i nn
\ .UU
Onn
.UU n
(
* " — ^H5
±- --£ —• ^^ - a
/ ^
/
/
»
i
) 10 20 30 4
Contact Time (min)
0
-•—Carpet
-A- Glass
-Laminate
x uaivanizea
Metal
-•—Particle
Board
Figure 5-1. Decontamination efficacy of pH-amended bleach against B. anthracis
Ames spores at 22 °C.
A further comparison of the efficacy of
pH-amended bleach at a 5-min contact
time was conducted for the
decontamination of B. anthracis Ames
spores and three surrogates (B. anthracis
Vollum, B. anthracis NNR1A1, and B.
subtilis spores). The efficacy was
determined for each of the six indoor
building materials tested (both porous
and nonporous). As shown in Figure 5-2,
after a 5-min contact time with pH-
amended bleach the results were similar
for the biological agent (B. anthracis
Ames spores) and the three surrogates.
For the biological agent and the three
surrogates, no viable spores were
recovered from any of the hard,
nonporous building materials after a 5-
min exposure to pH-amended bleach.
Shown in Table 5-7, greater than a 6 log
reduction in viable B. anthracis spores
(Ames, Vollum, and NNR1A1) and B.
subtilis spores was observed from all
hard, nonporous building materials
(glass, galvanized metal, and decorative
laminate), except B. anthracis NNKlAl
spores on glass. While no viable B.
anthracis NNR1 Al spores were
recovered after a 5-min contact time,
only a 5.89 log reduction was observed
(maximum possible) because a lower
CFU inoculation of NNR1A1 spores (4.2
x 106) was applied to the coupon than
the 1.1 x 10 CFU of spores applied for
Ames and Vollum.
Likewise, for carpet and particle board, a
2.86 or greater log reduction in viable
spores was observed for the biological
agent and three surrogates after a 5-min
contact time. Viable spores of the
biological agent and three surrogates
were recovered from one or more of the
replicate coupons.
102
-------
A 30-min contact time with pH-amended
bleach resulted in a >6 log reduction in
viable B. anthracis Ames spores (no
CPU observed) from all building
materials tested. B. subtilis spores on
carpet and particle board after a 30-min
contact time with pH-amended bleach
only exhibited a 4.2 to 4.7 log reduction;
viable spores were still recovered. B.
subtilis appears to be a conservative
surrogate for B. anthracis Ames when
pH-amended bleach is used.
9
8
7 H
c
o
B
^
•a
a 4
O)
5
3 1
2
1
0
DS. anthracis Ames
• B. anthracis Vollum
OB. anthracis NNR1A1
HB. subtilis
Figure 5-2. Decontamination efficacy of pH-amended bleach against B. anthracis
spores and surrogates with 5-min contact time.
103
-------
Table 5-6. Summary of pH-Amended Bleach Decontamination of Various Bacillus
Species and Strains
Material
Glass
Painted
Concrete
Carpet
Galvanized
Metal
Particle Board
Decorative
Laminate
Spore
B. anthracis Ames
B. anthracis Vollum
B. anthracis NNR1A1
B. subtilis
B. anthracis Ames
B. anthracis Vollum
B. anthracis NNR1A1
B. subtilis
B. anthracis Ames
B. anthracis Vollum
B. anthracis NNR1A1
B. subtilis
B. anthracis Ames
B. anthracis Vollum
B. anthracis NNR1A1
B. subtilis
B. anthracis Ames
B. anthracis Vollum
B. anthracis NNR1A1
B. subtilis
B. anthracis Ames
B. anthracis Vollum
B. anthracis NNR1A1
B. subtilis
Mean Log Reduction (95% Confidence Interval), and
p- Value After Decontamination for Specified Contact Time
5 min 10 min 30 min
>6.88 p=0.014
>6.79 p=0.014
>5.89 p=0.014
>6.59 p=0.014
>7.10 p=0.014
4.03 (3.78, 4.28)
p<0.0001
>6.59 p=0.014
>7.04 p=0.014
2.92 (2.73, 3.11)
p<0.0001
3.92 (3.85, 4.00)
p<0.0001
6.26 p=0.014
2.86 (2.73, 2.98)
p<0.0001
>7.70 p=0.014
>7.26 p=0.014
>6.87 p=0.014
>7.60 p=0.014
5.06 (4.38, 5.73)
p<0.0001
4.22 (3.94, 4.50)
p<0.0001
4.11 (3.64, 4.58)
p<0.0001
2.91 (2.64, 3.17)
p<0.0001
>7.09 p=0.014
>7.49 p=0.014
>6.90 p=0.014
>7.81 p=0.014
>6.70 p=0.014
>6.46 p=0.014
>7.15 p=0.014
>7.60 p=0.014
>5.03 p=0.014
3.99 (3.85, 4.14)
p<0.0001
>7.69 p=0.014
>7.57 p=0.014
>6.32 p=0.014
3.41 (3.00, 3.82)
p<0.0001
>7.23 p=0.014
>7.76 p=0.014
>6.92 p=0.014
NA
NA
>5.70 p=0.014
>6.98 p=0.014
NA
NA
>7.48 p=0.014
>7.55 p=0.014
NA
NA
4.16 (3.91, 4.41)
p<0.0001
>7.63 p=0.014
NA
NA
>7.52 p=0.014
>7.38 p=0.014
NA
NA
4.67 (4.31, 5.04)
p<0.0001
>7.43 p=0.014
NA
NA
>7.63 p=0.014
NA = not applicable
104
-------
5.1.3 Quantitative Results for pH-
Amended Bleach Efficacy
Decontamination of Vaccinia
Virus
The efficacy of pH-amended bleach
(6,000 - 6,700 ppm total chlorine at pH
6.51 - 6.55) against vaccinia virus was
evaluated using the quantitative method
by counting PFU after a given treatment.
The recovery of viable vaccinia virus
exposed to pH-amended bleach
(decontamination) or PBS (positive
control) for a 5-min contact time is
shown in Table 5-7. The efficacy of the
5-min pH-am ended bleach treatment,
shown as log reduction, is included in
Table 5-7. Using the plaque assay, no
viable vaccinia virus was observed on
coupons of any material type after a 5-
min contact time with pH-amended
bleach.
Table 5-7. Vaccinia PFU after 5 Min pH-amended Bleach Contact Time
PFU
Material Applied
to Coupons
Glass 7.37 x 106
rPainte? 7.37 x 106
Concrete
Industrial 1()6
Carpet
Galvanized _ ,_ 1ft6
-\ r . -I / . J / X _LU
Metal
PRartit 7.37 x 106
Board
Decorative 1()6
Laminate
Mean PFU/
Positive
Control
Coupon (SD)
(0 Min)
1.61x10"
(7.86 x 104)
1.23 x 106
(7.86 x 105)
1.15xl05
(1.63 x 105)
9_45 x io5
(1.43 x 105)
1.53 x 103
(1.04 xlO3)
4.88 x 105
(1.20 xlO5)
Mean PFU/ __ TI^TT/T ^ Differential Log
n •*• r^ 4- i MeanPFU/Test _ , ,. s
Positive Control „ ,„_,. , Reduction
/^ /-CT»\ =• Coupon (SD) at „ , ,
Coupon (SD), 5- , . „ , ' Compared to
. „ , ' 5-mm Contact „ , ,
mm Contact „. # Controls,
Time with PBS ime Statistical p-value*'
6.32 x 104
(2.84 x 104)
1.89xl05
(2.81 x 104)
6.61 xlO4
(1.58 xlO4)
3.66'xio2
(6.16xl02)
3.59xl02
(3.86xl02)
2.15 xlO4
(1.46 xlO4)
o >4'76
p=0.0135
o >5'27
p=0.0135
o >4'81
p=0.0135
Indeterminate*
p=0.8186
0 >2'34
p=0.0135
o >4'20
p=0.0135
*Log reduction values are calculated by taking the mean of the Iog10 recovered values of the positive control coupons at
5 min contact time with PBS and subtracting the mean of the Iog10 recovered values of the 5 min treated coupons with 1
PFU substituted for any observed 0 values. In cases where only decontaminated coupons have observed zero values,
this procedure makes the log reduction a minimum value as indicated by the "> x" reported value.
HThe statistical result comes from comparing the five recovered values from the positive controls at 5 min contact time
to the five recovered values from the treated coupons (all zeros) using a nonparametric Kolmogorov-Smimov test.
Values less than 0.05 indicates the reduction due to the pH-amended bleach treatment for 5 min was superior to the
reduction of the controls for 5 min.
*With zero values in both the positive control group and the decontamination group, the estimated log reduction is
indeterminate.
*SD not calculated when no viable viruses are recovered from any test coupon.
105
-------
5.1.4 Summary of Findings from the
pH-Amended Bleach
Investigation
• Contact with pH-amended bleach
was efficacious (significant log
reduction was observed) for the
decontamination of B. anthracis
Ames spores from the six indoor
building materials tested (both
porous and nonporous); efficacy
increased with increasing contact
time.
• A contact time of 5 min with pH-
amended bleach at 22 °C resulted
in a >6 log reduction in viable B.
anthracis spores (Ames and
Vollum) and B. subtilis spores
from all hard, nonporous
building materials; no viable B.
anthracis NNR1 Al spores were
recovered after 5 min, but a 5.89
log reduction was observed. The
relatively lower log reduction
was due to a lower CPU
inoculation of NNR1A1 spores
(4.2 x 106) being applied to the
coupon rather than 1.1 x 107
CPU applied for Ames and
Vollum. Previous EPA testing
with spray-applied pH-amended
bleach (60 min contact time) was
also found to be effective against
B. anthracis and B. subtilis
spores on painted cinder block
(log reductions >7.0).(32)
• A contact time of 30 min with
pH-amended bleach at 22 °C
resulted in a >6 log reduction in
viable B. anthracis Ames spores
(no CPU observed) from all
building materials tested.
• In contrast to B. anthracis Ames
spores where no CPU were
observed on any material after a
contact time of 30 min or less at
22 °C, only a 4.2 to 4.7 log
reduction in B. subtilis spores on
carpet and particle board was
observed after 30 min contact
with pH-amended bleach at 22
°C.
• A contact time of 5 min with pH-
amended bleach at 22 °C resulted
in no PFU of vaccinia virus being
recovered from the six indoor
building materials tested (both
porous and nonporous).
5.2 Liquid C1O2
5.2.1 Results for Liquid CIO2
Decontamination of Bacillus
Species
The efficacy of liquid C1O2 was
evaluated when applied to B. anthracis
Ames spores and B. subtilis spores on
coupons of four building materials. The
four building materials (glass, industrial
carpet, particle board, and decorative
laminate) were selected from, and
identical to, coupons used in the pH-
amended bleach testing described in
Section 5.1. The recovery results for
individual types of Bacillus spores
exposed to liquid C1O2 or PBS for
specified time periods at 20 °C are
shown in Tables 5-8 and 5-9. In
addition, the extracted coupons were
placed individually into TSB and
incubated for seven days to look for
cloudiness that would indicate the
presence of residual viable spores on the
test coupon. In all cases where no spores
were observed using the quantitative
methodology, no residual viable spores
were detected using the qualitative
method. The efficacy of liquid C1O2
against B. anthracis Ames spores and B.
subtilis spores, reported as log reduction,
is shown in Table 5-10.
106
-------
Efficacy was shown to be dependent on
the type of material onto which the
spores were applied. For B. anthracis
and B. subtilis spores, viable spores were
recovered from particle board after
exposure to liquid C1O2 for 120 min at
22 °C. Likewise, viable B. anthracis
Ames and B. subtilis spores were
recovered from carpet after exposure to
liquid C1O2 for 30 min at 22 °C. Viable
B. subtilis spores, but not B. anthracis
Ames, were recovered from carpet after
a 120-min exposure to liquid C1O2 at 22
A complete kill of spores on glass,
industrial carpet, and decorative
laminate, i.e., no viable spores recovered
(quantitative analysis) or detected
(qualitative analysis), occurred at a
lower contact time with liquid C1O2 at
22 °C for B. anthracis Ames spores than
for B. subtilis spores.
Exterm C1O2 solution provided >6 log
reduction in recoverable viable B.
anthracis Ames spores, shown in Figure
5-3, dependent on the building material
and the contact time at 22 °C. No viable
B. anthracis Ames spores were
recovered from industrial carpet,
decorative laminate, or glass after 120
min exposure to the C1O2 solution at 22
°C; viable B. anthracis Ames spores
were recovered from particle board after
120 min exposure to C1O2 solution at 22
°C. Shown in Figure 5-4, Exterm C1O2
solution provided >6 log reduction in B.
subtilis spores (no viable spores
recovered) from glass and decorative
laminate (hard, nonporous surfaces) after
120 min exposure to C1O2 solution at 22
°C. Viable B. subtilis spores were
recovered from both industrial carpet
and particle board (porous surfaces) after
120 min exposure to C1O2 solution at 22
°C. In general, B. subtilis spores appear
to be able to survive a longer exposure to
C1O2 solution at 22 °C than B. anthracis
Ames spores.
Carpet
Particle Board
Glass
Laminate
20 40 60 80 100
Contact Time, min
120 140
107
-------
Figure 5-3. Decontamination efficacy of Exterm ClOi solution against B. anthracis
Ames spores at 22 °C.
Table 5-8. B. anthracis Ames spores CFU after Various Liquid ClOi Contact Times
Material
Glass
Industrial Carpet
Particle Board
Decorative
Laminate
Material
Glass
Industrial Carpet
Particle Board
Decorative
Laminate
B. anthracis
(Ames)
CFU Applied
to Coupons
1.09 x 107
1.09 x 108
l.llxlO8
1.03 x 108
B. anthracis
(Ames)
CFU Applied
to Coupons
1.09 x 107
1.09 x 108
l.llxlO8
1.03 x 108
Mean CFU (SD) Recovered from
Positive Control Coupons at Specified Contact Time with
PBS
10 min 30 min 120 min
2.08 x 106 1.39 x 106 8.57 x 105
(1.37xl06) (7.50 xlO5) 1.70 xlO5)
6.11 xlO7 4.50 xlO7 5.45 x 107
(1.00 xlO7) (1.26 xlO7) (1.73 x 107)
3.38xl07 3.93 x 107 3.66 x 107
(6.14 x 106) (6.35 x 106) (1.03 x 107)
2.07 xlO7 2.79 xlO7 2.69 x 107
(8.04 xlO6) (1.30 xlO7) (8.96 x 106)
Mean CFU (SD)* Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 30 min 120 min
000
6.11 xlO2 4.19 xlO1
(2.93 x 102) (5.92 x 101)
3.59xl03 1.28 xlO3 6.95 x 101
(6. 16 x 102) (5.32 x 102) (5.50 x 101)
6.65 x!0u
(1.49 xlO1) ° °
*SD not calculated when no viable virus are recovered from any test coupon.
108
-------
§> 3.00
_i
2.00
1.00
0.00
- Carpet
-Particle Board
-Glass
-Laminate
20 40 60 80 100
Contact Time, min
120
140
Figure 5-4. Decontamination efficacy of Exterm ClOi solution against B. subtilis
spores.
Table 5-9. B. subtilis CFU after Various Contact Times with Liquid C1O2
Material
Glass
Industrial Carpet
Particle Board
Decorative Laminate
Material
Glass
Industrial Carpet
Particle Board
Decorative Laminate
B. subtilis
CFU
Applied
to Coupons
1.01 xlO7
1.07 x 108
1.00 x 108
1.02 xlO8
B. subtilis
CFU Applied
to Coupons
1.01 xlO7
1.07 x 108
1.00 x 108
1.02 xlO8
Mean CFU (SD) Recovered from
Positive Control Coupons at Specified Contact Time with PBS
10 min 30 min 120 min
3.67 x 106
(7.55 x 105)
2.40 x 107
(6.48 x 106)
4.13 xlO6
(9.57 x 105)
4.27 x 107
(4.33 x 106)
4.00 x 106
(1.14xl06)
2.51 xlO7
(5.14xl06)
3.47 x 106
(3.95 x 105)
3.90 x 107
(4.02 x 106)
2.92 x 106
(1.49x10^)
3.48 xlO7
(6.85 x 106)
(1.03 x 106)
3.57xl07
(6.58 x 106)
Mean CFU (SD)* Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 30 min 120 min
1.44 x 103
(2.03 x 102)
1.39 xlO3
(1.23 x 103)
2.96 x 103
(5.03 x 102)
6.71 x 101
(1.32 x 102)
1.01 xlO3
(3.99 xlO2)
1.48 x 103
(1.80 xlO3)
1.91xl03
(3.91 x 102)
1.35X101
(3.02 x 101)
0
2^09'xlo2'
(2.38 x 102)
(1.12xl02)
0
*SD not calculated when no viable virus are recovered from any test coupon.
109
-------
Table 5-10. Log Reduction in Bacillus Species after Various Contact Times with
Liquid C1O2
Mean Log Reduction* (95% Confidence Interval'), p-value* After
Material Organism Decontamination for Specified Contact Time
10 min 30 min 120 min
Decorative
Laminate
Industrial
Carpet
Glass
Particle
Board
B. anthracis Ames
B. subtilis
B. anthracis Ames
B. subtilis
B. anthracis Ames
B. subtilis
B. anthracis Ames
B. subtilis
> 6.99 p=0.0135
> 6.83 p=0.0135
5.03 (4.81, 5.26)
p < 0.0001
4.44 (3.87, 5.01)
p < 0.0001
> 6.24 p=0.0135
3.40 (3.29, 3.51)
p < 0.0001
3.97 (3.86, 4.09)
p < 0.0001
3.14 (3.01, 3.27)
p < 0.0001
> 7.38 p=0.0135
> 7.22 p=0.0135
> 6.83 p=0.0135
4.58 (3.89, 5.27)
p < 0.0001
> 6.10 p=0.0135
3.62 (3.36, 3.89)
p < 0.0001
4.51 (4.30, 4.72)
p < 0.0001
3.27 (3.16, 3.37)
p < 0.0001
> 7.40 p=0.0135
> 7.55 p=0.0135
> 7.72 p=0.0135
>5.76 p=0.0135
> 5.93 p=0.0135
> 6.47 p=0.0135
> 6.04 p=0.0135
4.27 (4.05, 4.50)
p < 0.0001
*Reduction calculated as Iog10 of the arithmetic mean of five control coupons minus the Iog10 of the arithmetic mean of
the five decontaminated coupons. If decontaminated coupons showed zero organisms, the observed values are replaced
with one.
HApplies only for conditions where control and decontamination coupons had nonzero results.
tFor cases with measurable control and decontamination results, p-value is a result of t-test comparison of control to
decontamination with values less than or equal to 0.05 representing statistically significant decontamination effect. For
cases with complete kill observed on at least one coupon, the p-value is a result of the nonparametric Kolmogorov-
Smimov test of whether the residual organisms after decontamination are different from the controls. Values less than
or equal to 0.05, with positive mean log reductions, indicate that a positive and statistically significant reduction
occurred as a result of the decontamination treatment.
5.2.2 Results for Liquid CIO2
Decontamination of Vaccinia
Virus
The efficacy of liquid C1O2 against
vaccinia virus was evaluated using the
quantitative method by counting PFU
after a given treatment. The recovery of
viable vaccinia virus exposed to liquid
C1O2 (decontamination) or PBS (positive
control) for a 10-min contact time is
shown in Table 5-11. The efficacy of the
10-min liquid C1O2 treatment at 22 °C,
shown as log reduction, is included in
Table 5-11. Using the plaque assay, no
viable vaccinia virus was observed on
any coupon of any material type after a
10-min contact time with liquid C1O2 at
22 °C
110
-------
Table 5-11. Vaccinia PFU after 10-Min Contact with Liquid C1O2
Material
Glass
Industrial
Carpet
Particle
Board
Decorative
Laminate
Vaccinia
PFU
Applied
to
Coupons
1.14xl06
1.14xl07
1.05 x 107
1.14xl07
Mean (SD) PFU
Recovered from
Positive Control
Coupons, at 0
min (No Contact
Time)
3.91 xlO5
(1.36 xlO5)
2.37 x 105
(1.84 xlO5)
1.41 xlO3
(1.01 x 103)
8.41 x 105
(1.80 xlO5)
Mean (SD) PFU
Recovered from
Positive Control
Coupons, at 10
min Contact
Time with PBS
2.65 x 105
(l.SlxlO5)
5.61 xlO4
(3.31xl04)
9.52 x 102
(1.58xl03)
6.93 x 104
(1.24 xlO4)
Mean PFUJ
Recovered from
Coupons After
Decontamination
at 10 min
Contact Time
0
0
0
0
C1O2 Differential
Log Reduction
Compared to
Controls,
Statistical p-
Value*'
>5.36
p=0.0135
>4.38
p=0.0135
>2.54
p=0.0135
>4.84
p=0.0135
*Log reduction values are calculated by taking the mean of the Iog10 recovered values of the positive control coupons at
10 min contact time and subtracting the mean of the Iog10 recovered values of the 10 min treated coupons with 1 PFU
substituted for the observed 0 values. This procedure makes the log reduction a minimum value as indicated by the ">
x" reported value.
HThe statistical result comes from comparing the five recovered values from the positive controls at 10 min contact
time to the five recovered values from the treated coupons (all zeros) using a nonparametric Kolmogorov-Smimov
test. Values less than 0.05 indicates the reduction due to the C1O2 treatment for 10 min was superior to the reduction
of the controls for 10 min.
JSD not calculated when no viable viruses are recovered from any test coupon.
While approximately 107 PFU of
vaccinia virus were applied to the test
control coupons (except small glass), the
number of recoverable PFU on control
coupons drops by about 1 to 4 log
(depending on the material) within 10
min. Therefore, even with no
recoverable virus (0 PFU) from test
coupons, the log reduction attributable to
the treatment was moderate. For
example, about 2.5 for particle board.
Other unknown factors contribute to the
loss of viable virus in the absence of the
C1O2 treatment.
5.2.3 Summary of Findings from the
Liquid CIO2 Investigation
• Contact with liquid C1O2 at 22
°C was efficacious for the
decontamination of B. anthracis
Ames spores, B. subtilis spores,
and vaccinia virus from the four
indoor building materials tested
(glass, industrial carpet, particle
board, and decorative laminate,
although complete kills of B.
anthracis Ames spores on
particle board or B. subtilis
spores on carpet or particle board
did not occur even after 120 min
contact times).
A 10-min contact time with
liquid C1O2 at 22 °C resulted in a
>6 log reduction in viable B.
anthracis Ames spores on the
hard, nonporous building
materials (glass and decorative
laminate).
In contrast to B. anthracis Ames,
>6 log reduction in viable B.
subtilis spores on decorative
laminate and glass was not
observed at 10-min contact time
at 22 °C, but was observed after
111
-------
30-min and 120-min contact
times at 22 °C, respectively.
After a 120-min contact time at
22 °C, viable B. subtilis spores
were cultured from carpet and
particle board; at 120 min carpet
showed a >5.7 log reduction and
particle board showed a 4.3 log
reduction in viable B. subtilis
spores.
For these materials, B. subtilis
appears to be a conservative
surrogate for decontamination of
B. anthracis Ames spores using
liquid C1O2.
A 10-min contact time with
liquid C1O2 at 22 °C resulted in
no culturable vaccinia virus
being recovered (0 PFU) from
the four indoor building materials
tested (both porous and
nonporous).
5.3 Spor-Klenz® Ready-to-Use HP-
PA Solution
5.3.1 Qualitative Results for Spor-
KlenzR HP-PA Decontamination
of Bacillus Species
The qualitative cycle fractionation test
results are shown in Table 5-12.
Coupons were inoculated with 6.8 x 107
spores per coupon (slightly below the
specified target application range of 7.5
"7 o
x 10 - 1.25 x 10 ). Three test coupons,
in contact with the Spor-Klenz® HP-PA
solution were included at each time
point. Viable B. anthracis Ames spores
were observed after contact with Spor-
Klenz® HP-PA for up to 30 min. Some
of the replicate decorative laminate
coupons were negative for growth after
20-min and 30-min contact times with
Spor-Klenz® HP-PA at 20 °C. Viable B.
anthracis Ames spores were observed on
all positive control coupons after contact
with PBS for 5, 10, 20, or 30 min at 20
si-
Table 5-12. Results from Qualitative Evaluation of Spor-Klenzu HP-PA
Decontamination of B. anthracis Ames
Material
Day?
Decontaminated, Contact Time
5 min 10 min 20 min
30 min
Glass
Painted Concrete
Galvanized Metal
Decorative Laminate
Positive Control
Negative Control
"-" indicates that all three coupons at the specified time and treatment exhibited no growth in TSB.
"+" indicates that all three coupons at the specified time and treatment exhibited growth in TSB.
"+, +, -" indicates two coupons exhibited growth in TSB, one coupon did not.
"+, -, -" indicates one coupon exhibited growth in TSB, two coupon did not.
112
-------
5.3.2 Quantitative Results for Spor-
Klenz* HP-PA Decontamination
of Bacillus Species
The efficacy of Spor-Klenz® HP-PA
decontamination was evaluated when
applied to B. anthracis Ames spores on
coupons of six building materials and
when applied to B. subtilis on glass. The
six building materials (glass, painted
concrete, industrial grade carpet,
galvanized metal ductwork, particle
board, and decorative laminate) were
selected from, and to the extent feasible
identical to, coupons used in the pH-
amended bleach testing described in
Section 5.1. The recovery results from
various materials for individual types of
Bacillus spores exposed to Spor-Klenz®
HP-PA (for test coupons) or PBS (for
positive control and procedural blank
coupons) for specified time periods are
shown in Tables 5-13 and 5-14. The
efficacy of Spor-Klenz® HP-PA against
B. anthracis Ames and B. subtilis spores,
reported as log reduction, is shown in
Table 5-15 and Figure 5-5.
Efficacy was shown to be dependent on
the type of material onto which the
spores were applied. For both B.
anthracis Ames and B. subtilis, spores
were recovered from only one of five
glass coupons after 10-min contact time
with Spor-Klenz® HP-PA at 20 °C; no
spores were recovered from any glass
coupons after a 20-30 min contact time
at 20 °C. A complete kill of spores on
decorative laminate, i.e., no viable
spores were recovered (quantitative
analysis) or detected (qualitative
analysis), occurred at a 30-min contact
time with Spor-Klenz® HP-PA at 20 °C
for B. anthracis Ames spores. In
contrast, less than a 2-log reduction in
viable spores was observed after a 30-
min contact time with Spor-Klenz HP-
PA at 20 °C for B. anthracis Ames
spores on galvanized metal. Spor-Klenz
HP-PA efficacy was greater against B.
anthracis Ames spores on carpet than on
metal, but after a 30-min contact time
with Spor-Klenz® HP-PA at 20 °C less
than a 4-log reduction was observed.
®
113
-------
Table 5-13. B. anthracis Ames Spores CFU after Various Contact Times with Spor-
®
Klenzw HP-PA
Material
Glass
Painted Concrete
Indistrial Carpet
Galvanized Metal
Particle Board
Decorative
Laminate
Material
Glass
Painted Concrete
Carpet
Galvanized Metal
Particle Board
Decorative
Laminate
CFU Applied
to Coupons*
1.80 x 108
l.SOxlO8
1.80 x 108
2.00 x 108
2.00 x 108
2.00 x 108
CFU Applied to
Coupons*
1.80 x 108
l.SOxlO8
l.SOxlO8
2.00 x 108
2.00 x 108
2.00 x 108
Mean CFU (SD) Recovered from
Positive Control Coupons
7.96 xlO7
(9.10xl06)
____7_
(3.04 xlO7)
9.40x10'
(6.16xl06)
1.62 x 108
(2.05 x 107)
1.22 x 108
(1.48 xlO7)
1.26 x 108
(2.30 x 107)
Mean CFU (SD)T Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 20 min 30 min
6.00 x 10°
(1.34 xlO1)
1.97 x 106*
(3.25 x 106)
3.08xi6'5
(2.76 x 105)
i.ioxio7
(3.45 x 106)
1.03 x 105
(2.90 x 104)
1.92xl05
(5.12xl04)
0
^67'7'io5
(1.86 xlO6)
Y$2xW
(2.15xl04)
8;547Y5S
(3.47 x 106)
L"07x"io3
(1.01 x 103)
Data not valid
0
3.28 x 106
(6.57 x 106)
5.20 x 102
(5.97 x 102)
6.98 x 106
(4.52 x 106)
4.00 x 102
(5.87 x 102)
0
*Only three, rather than five, control coupons were included.
^SD not calculated when no viable spores (measured as CFU) were recovered from any test coupon.
*The number of spores inoculated onto the coupons was slightly above the specified range of 7.5 x 107 -1.25 x 108.
114
-------
Table 5-14. B. anthracis Ames and B. subtilis Spores CFU after Various Contact
Times with Spor-Klenz® HP-PA
Material
Glass
Material
Glass
Material
Glass
Material
Glass
B. anthracis (Ames)
CFU Applied
to Coupons
1.80 x 108
B. anthracis (Ames)
CFU Applied
to Coupons
1.80 x 108
B. subtilis
CFU Applied
to Coupons
1.20 x 108
B. subtilis
CFU Applied
to Coupons
1.20 x 108
Mean CFU (SD) Recovered from
Positive Control Coupons
7.96 x 107
(9.10xl06)
Mean CFU (SD)* Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 20 min 30 min
6.0x10°
(1.34 xlO1) ° °
Mean CFU (SD) Recovered from
Positive Control Coupons
7.16xl06
(3.45 x 106)
Mean CFU (SD)* Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 20 min 30 min
6.00 x 10°
(1.34 xlO1) ° °
SD not calculated when no viable spores (measured as CFU) were recovered from any test coupon.
t-The number of spores inoculated onto the coupons was slightly above the specified range of 7.5 x 107 -1.25 x 108).
Table 5-15. Log Reduction in Bacillus Species after Various Contact Times with
Spor-Klenz® HP-PA
Material
Glass
Painted
Concrete
Carpet
Galvanized
Metal
Particle
Board
Decorative
Laminate
Spore
B. anthracis Ames
B. subtilis
B. anthracis Ames
B. anthracis Ames
B. anthracis Ames
B. anthracis Ames
B. anthracis Ames
Mean Log
10 min
7.62
6.54
2.52
2.62
1.15
3.09
2.78
Reduction* After Decontamination
Contact Time
20 min
>7.91
>6.83
4.25
4.20
1.28
5.32
Data not valid
for Specified
30 min
>7.91
>6.83
2.42
6.26
1.46
6.89
>8.05
*Log reduction values are calculated by taking the mean of the Iog10 recovered values of the positive control coupons
and subtracting the mean of the Iog10 recovered values of the treated coupons at a given contact time with 1 CFU
substituted for the observed 0 values. This procedure makes the log reduction a minimum value as indicated by the ">
x" reported value.
115
-------
Log Reduction
9.00 -
8.00 -
7.00
ft 00
5.00
4.00 -
3.00
2.00
1.00 -
.
» Glass (B. anthracis)
— •— Painted Concrete
n -*- Carpet
*s^~
^^— Galvanized Metal
r_J " - m
— f— Particle Board
— • — Decorative Laminate
10 20 30
Contact Time, Min
Figure 5-5. Decontamination efficacy of Spor-KlenzR HP-PA solution against B.
anthracis Ames on various materials and B. subtilis spores on glass.
5.3.3 Summary of Findings from the
Spor-Klem® HP-PA
Investigation
• Contact with Spor-Klenz® HP-
PA exhibits variable efficacy for
the decontamination of B.
anthracis Ames spores from the
six indoor building materials
tested (both porous and
nonporous).
• A 30-min contact time with
Spor-Klenz® HP-PA at 20 °C
resulted in a >6 log reduction in
viable B. anthracis Ames spores
from all materials tested except
painted concrete and galvanized
metal.
• B subtilis spores on glass showed
the same pattern of
decontamination efficacy as B.
anthracis Ames spores on glass
and appears to be a good
surrogate for decontamination of
B anthracis Ames using Spor-
Klenz® HP-PA.
5.4 Oxonia ActiveR Solution
5.4.1 Quantitative Results for Oxonia
Active" Decontamination of
Bacillus Species
The efficacy of Oxonia Active®
decontamination was evaluated when
applied to B. anthracis Ames spores on
coupons of six building materials and
when applied to B. subtilis on glass. The
six building materials (glass, painted
concrete block, industrial grade carpet,
galvanized metal ductwork, particle
board, and decorative laminate) were
selected from, and to the extent feasible
identical to, coupons used in the pH-
amended bleach testing described in
Section 5.1. The recovery results for
individual types of Bacillus spores
exposed to Oxonia Active® (for test
coupons) or PBS (for positive controls)
for specified time periods are shown in
Tables 5-16 and 5-17. The efficacy of
Oxonia Active® against B. anthracis
Ames and B. subtilis spores, reported as
116
-------
log reduction, is shown in Table 5-18
and Figure 5-6.
Efficacy was shown to be dependent on
the type of material onto which the
spores were applied. No B. anthracis
Ames spores were recovered from any
galvanized metal or particle board
coupons after a 30- or 60-min contact
time at 20 °C. Viable B. anthracis spores
were recovered from one or more
coupons of glass, painted concrete block,
industrial grade carpet, and decorative
laminate after a 60-min contact time
with Oxonia Active® at 20 °C.
5. 4. 2 Summary of Findings from the
Oxonia Active® Investigation
Contact with Oxonia Active®
exhibits variable efficacy for the
decontamination of B. anthracis
Ames spores from the six indoor
building materials tested (both
porous and nonporous).
A 30-min contact time with
Oxonia Active® at 20 °C resulted
in a >6 log reduction in viable B.
anthracis Ames spores from all
materials tested except painted
concrete block. (At a 60-min
contact time at 20 °C, log
reduction observed for decorative
laminate was slightly less than 6
log [5.59]).
117
-------
Table 5-16. B. anthracis Ames Spores CFU after Various Oxonia ActiveR Contact
Times
Material
Glass
Painted Concrete
Industrial Carpet
Galvanized Metal
Particle Board
Decorative
Laminate
Material
Glass
Painted Concrete
Industrial Carpet
Galvanized Metal
Particle Board
Decorative
Laminate
B. anthracis
(Ames)
CFU Applied
to Coupons*
2.80 xlO8
2.30 xlO8
2.30 x 108
6.90 xlO8
1.50xl08
6.90 x 108
B. anthracis
(Ames)
CFU Applied
to Coupons*
2.80 xlO8
2.30 xlO8
2.30 x 108
6.90 xlO8
1.50xl08
6.90 x 108
Mean CFU (SD) Recovered from
Positive Control Coupons
1.18x10"
(1.30 xlO7)
1.01 xlO8
(5.57 x 107)
8.43 x 107t
(1.40 xlO7)
3.62 xlO7
(1.70 xlO7)
1.22x10"
(1.92 xlO7)
1.05 xlO7
(2.93 x 106)
Mean CFU (SD)* Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 30 min 60 min
8.26 x 102
(LlSxlO3)
1.39xl06
(2.70 x 106)
2.58 x 10"
(3.45 x 105)
LlSxlO4
(2.11 xlO4)
1.01 xlO3
(1.56xl03)
1.94xl02
(1.47 xlO2)
6.00 x 10"
(1.34 xlO1)
3.02X105
(6.69 x 105)
2.80 x 103
(5.72 x 103)
0
0
8.60 x lO2
(1.92 xlO3)
2.06 x 102
(3.04xl02)
2.54 x 10"
(4.77 x 105)
6.00 x 10"
(1.34 xlO1)
0
0
1.46 x 102
(1.50 xlO2)
*SD not calculated when no viable spores (measured as CFU) were recovered from any test coupon.
^Results based on only 3 positive control coupons (rather than 5).
* The number of spores inoculated onto the coupons was above the specified range of 7.5 x 107- 1.25 x
108).
118
-------
Table 5-17. B. anthracis Ames and B. subtilis Spores CFU after Various Oxonia
Active® Contact Times*
Material
Glass
Material
Glass
Material
Glass
Material
Glass
B. anthracis (Ames)
CFU Applied
to Coupons
2.80 x 108
B. anthracis (Ames)
CFU Applied
to Coupons
2.80 x 108
B. subtilis
CFU Applied
to Coupons
2.40 x 108
B. subtilis
CFU Applied
to Coupons
2.40 x 108
Mean CFU (SD) Recovered from
Positive Control Coupons
1.18x10"
(1.30 xlO7)
Mean CFU (SD) Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 20 min 30 min
8.26 x 102 6.00 x 10" 2.06 x 102
(LlSxlO3) (1.34 xlO1) (3.04 x 102)
Mean CFU (SD) Recovered from
Positive Control Coupons
1.40 xlO81
(7.07 x 106)
Mean CFU (SD) Recovered from Coupons
After Decontamination at Specified Contact Time
10 min 20 min 30 min
2.00 x 101* 9.40 x 10^ 9.80 x 102t
(4.47 xlO1) (1.57xl02) (9.63 x 102)
*The procedural blank showed a low level of B. subtilis spores.
Table 5-18. Log Reduction in Bacillus Species after Various Contact Times with
Oxonia Active®*
Mean Log Reduction* After Decontamination for Specified
Material Spore Contact Time
10 min 30 min 120 min
uiass
Painted
Concrete
Carpet
Galvanized
Metal
Particle Board
Decorative
Laminate
B. anthracis Ames
B. subtilis
B. anthracis Ames
B. anthracis Ames
B. anthracis Ames
B. anthracis Ames
B. anthracis Ames
6.37
7.76*
2.93
3.72
5.17
5.98
5.15
7.81
6.981
5.55
6.50
7.52
8.08
6.28
6.74
5.781
4.62
7.63
7.52
8.08
5.59
*Log reduction values are calculated by taking the mean of the Iog10 recovered values of the positive control coupons
and subtracting the mean of the Iog10 recovered values of the treated coupons at a given contact time with 1 CFU
substituted for the observed 0 values. This procedure makes the log reduction a minimum value as indicated by the ">
x" reported value.
*The procedural blank showed a low level of B. subtilis spores.
119
-------
111
DC
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
E
.
10 JO 60
Contact Time, Min
The procedural blank showed a low level of B. subtilis spores.
>—Glass (B. anthracis)
I— Painted Concrete
Carpet
•*—Galvanized Metal
It—Particle Board
t— Decorative Laminate
Glass (B. subtilis)*
®
Figure 5-6. Decontamination efficacy of Oxonia Active decontamination solution
against B. anthracis Ames on various materials and B. subtilis spores on glass.
120
-------
6.0 Summary
6.1 Summary of ClOi Fumigation
Results
The C1O2 fumigation results,
summarized in Table 6-1, show efficacy
against B. anthracis (Ames) spores, ricin
toxin, and vaccinia virus.
For B. anthracis, the efficacy was
dependent on the type of materials onto
which the spores were inoculated and the
strain. Shown in Table 6-1, C1O2
fumigation at a CT of 1000 ppmv-hr (25
- 27 °C and 83 - 97% RH) resulted in no
colony-forming unit (CPU) being
recovered off most types of coupons (>7
log reduction). Spores on compressed
cellulose insulation were the most
resistant to C1O2 fumigation, with viable
spores being recovered after exposure to
a CT of 9000 ppmv-hr (24 - 25 °C and
83 - 97% RH); after exposure to a CT of
12,000 ppmv-hr (25 - 27 °C and 83 -
97% RH) no viable spores were
recovered from compressed cellulose
insulation.
For C1O2 fumigation, results shown in
Table 6-2 suggest that increasing the
temperature and humidity increased the
efficacy of fumigation against B.
anthracis.
At 24 - 27°C, C1O2 efficacy against B.
anthracis on I-beam steel, bare pine
wood, and ceiling tile was higher at a
higher humidity (80 - 84% for C1O2)
than at a slightly lower humidity (75 -
77% for C1O2). At high relative humidity
condensation may occur. If liquid water
is present, the water may dissolve C1O2
from the air, effectively creating a liquid
decontamination rather than a true
fumigation.
121
-------
Table 6-1. Summary of Minimum ClOi Fumigation CT Resulting in 0 CFU of B. anthracis Ames Spores, <10% Bioactivity of
Ricin Applied, or 0 PFU of Vaccinia Being Recovered
Agent
Material
[C1O2], Contact time,
andCT
Temperature
RH
Biological Agent Recovery Results
and Efficacy
B. anthracis (Ames)
spores
(1 x 108 spores applied)
Painted Concrete
Galvanized Metal
Decorative Laminate
Particle Board
Industrial Carpet
Plate Glass
Glass (small with only Ix
107 spores applied)
3000 ppmv, 20 min,
1000 ppmv-hr
25 - 27 °C
83 - 97%
0 CFU extracted from any coupon
Log reduction: >7 (>6 for small glass)
B. anthracis (Ames)
spores
(1 x 108 CFU applied)
Ricin toxin
(25 ug applied)
Vaccinia virus
(2.04xl06-1.52xl08
PFU applied)
Cellulose Insulation
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Glass
3000 ppmv, 4 hr, op
12,000 ppmv-hr
200 ppmv, 30 min, 23 - 25 °C
100 ppmv-hr
250 ppmv, 30 min, ^ _ ^
125 ppmv-hr
OT mo/ 0 CFU extracted from any coupon
o J - y / /o T , _ _
Log reduction: >7
80-84% 93.4-100.0%
reduction compared to controls
0 PFU extracted from any coupon
„ _0o/ Log reduction: >4 to >5 (depending on
IJ - 10/0 „....,
recovery efficiencies)
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
122
-------
Table 6-2. Bacillus anthracis Ames Spores Fumigated with ClOi
Material
Glass (small)
Painted
Concrete
Galvanized
Metal
Decorative
Laminate
Particle Board
Industrial
Carpet
Plate Glass
Cellulose
Insulation
Painted
Concrete
Painted
Concrete
Glass (small)
Painted
Concrete
Galvanized
Metal
Decorative
Laminate
Particle Board
Industrial
Carpet
Plate Glass
Cellulose
Insulation
Temperature,
RH
24
83
24
83
24
83
24
83
24
83
24
83
24
83
24
83
24
81
24
72
30
74
30
74
30
74
30
74
30
74
30
74
30
74
30
74
-25°C
- 97%
-25°C
- 97%
-25°C
- 97%
-25°C
- 97%
-25°C
- 97%
-25°C
- 97%
-25°C
- 97%
-25°C
- 97%
-25°C
- 87%
-25°C
- 82%
-32°C
- 89%
-32°C
- 89%
-32°C
- 89%
-32°C
- 89%
-32°C
- 89%
-32°C
- 89%
-32°C
- 89%
-32°C
- 89%
[C102],
ppmv
3000
3000
3000
3000
3000
3000
3000
3000
1500
750
3000
3000
3000
3000
3000
3000
3000
3000
Decontaminated Coupons
Contact time, Contact time, Contact time,
0 CFU or 0 CFU or 0 CFU or
Viable Spores Viable Spores Viable Spores
0.33 hr,
OCFU
0.33 hr,
OCFU
0.33 hr,
OCFU
0.33 hr,
OCFU
0.33 hr,
OCFU
0.33 hr,
OCFU
0.33 hr,
OCFU
0.75 hr,
OCFU
1.5 hr,
OCFU
3hr,
OCFU
NA
NA
NA
NA
NA
NA
NA
NA
3hr,
OCFU
3hr,
OCFU
(4 of 5 coupons)
3hr,
OCFU
3hr,
OCFU
3hr,
Invalid data
3hr,
OCFU
3hr,
OCFU
3hr,
Viable spores
3hr,
OCFU
6hr,
OCFU
3hr,
OCFU
3hr,
OCFU
3hr,
OCFU
3hr,
OCFU
3hr,
OCFU
3hr,
OCFU
3hr,
OCFU
3hr,
OCFU
NA
NA
NA
NA
NA
NA
NA
4hr,
OCFU
6hr,
OCFU
12 hr,
OCFU
NA
NA
NA
NA
NA
NA
NA
NA
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
123
-------
Shown in Table 6-3, ricin was reduced by 93% or greater from all building materials at a
C1O2 fumigation CT of 100 - 300 ppmv-hr at 23 °C-25 °C and 80% -84% RH.
Table 6-3. Ricin Fumigated with C1O2
Material
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Glass (small)
Painted Concrete
Galvanized Metal
Decorative Laminate
Cellulose Insulation
Particle Board
Industrial Carpet
Contact
Time,
min
20
20
20
20
20
20
20
30
30
30
30
30
30
30
[C102],
ppmv
1500
1500
1500
1500
1500
1500
1500
200
200
200
200
200
200
200
Temperature
23
23
23
23
23
23
23
23
23
23
23
23
23
23
-25
__
__
__
__
__
__
-25
__
__
__
__
__
__
0
O
o
0
o
0
o
o
0
o
0
0
o
0
c
c
c
c
c
c
c
c
c
c
c
c
c
c
80
80
80
80
80
80
80
80
80
80
80
80
80
80
RH
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
- 84%
% Reduction
99.84
99^96
99^6
99Tl3
92/74
9946
99^61
99.75
99^97
9849
99^69
9342
9511
9831
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
Shown in Table 6-4, no viable vaccinia virus was recovered from any building material
tested after a C1O2 fumigation CT of 125 - 300 ppmv-hr at 22 - 24 °C and 75 - 83% RH.
Table 6-4. Vaccinia Virus Fumigated with ClOi
Decontaminated
Coupons
Material
Contact
Time,
min
[C102],
ppmv
Temperature,
°C
RH, %
0 PFU or
Viable Virus
Glass (small), Painted Concrete,
Galvanized Metal, Decorative
Laminate, Cellulose Insulation,
Particle Board, Industrial Carpet
20
1500
Glass (small), Painted Concrete,
Galvanized Metal, Decorative
Laminate, Cellulose Insulation,
Particle Board, Industrial Carpet
30
250
23 -24
22-22
80-83
OPFU
75-78
OPFU
Note [C1O2] is concentration of C1O2 in the chamber atmosphere, in ppmv.
124
-------
6.2 Summary of MeBr Fumigation
Results
Shown in Figures 6-1 and 6-2, MeBr
fumigation demonstrated up to complete
kill (>6 log reduction, no viable spores
recovered) of B. anthracis, dependent on
the building material, the concentration
of the fumigant, and the contact time.
Figure 6-1 shows that, for a given CT, a
low concentration (53 mg/L) and higher
contact time was generally less
efficacious than a higher concentration
(105, 212, or 320 mg/L) with a lower
contact time for decontamination of B.
anthracis Ames spores from glass or
ceiling tile coupons.
-53 mg/L, Glass
-53 mg/L, Tile
-105 mg/L, Glass
-105 mg/L, Tile
-212 mg/L, Glass
-320 mg/L, Tile
500 1000 1500 2000
CT, mg/L-Hr
2500
3000
Figure 6-1. Log reduction of B. anthracis Ames spores vs. MeBr CT (concentration x
time) at various MeBr concentrations and at 36 °C and 75% RH.
In Figure 6-2, the MeBr concentration
was 105 mg/L for all fumigations shown
except that 211 mg/L was used for the
1899 mg/L-hr (highest CT value). These
data show that the CT required for a six
log reduction in recoverable spores
(measured as CFU) depends on the type
of building material. Figure 6-2 also
shows a comparison of MeBr
decontamination efficacy for B.
anthracis Ames and B. subtilis spores at
various CT values. Equivalent numbers
of spores (~107 spores) of each species
were applied to identical small glass
coupons as single droplets of about 10
jiL each. The coupons were placed in a
test chamber and fumigated with MeBr
at various CTs. Little or no efficacy of
MeBr against B. subtilis was observed in
the CT range or 1260-1899 mg/L-hr. In
contrast, a high level of MeBr efficacy
was observed against B. anthracis Ames
spores at CTs of 1575 mg/L-hr or
greater; no viable B. anthracis spores
125
-------
were recovered at CTs of 1890 mg/L-hr
(105 mg/L for 18 hr) or 1899 mg/L-hr
(211 mg/L for 9 hr).
For all MeBr fumigation trials, Bis (B.
atrophaeus on stainless steel in Tyvek
packaging (Apex Labs)) were included.
At all CTs investigated, viable B.
atrophaeus spores were detected in the
qualitative viability test; fumigation did
not kill all of the B. atrophaeus spores
inside of the Tyvek packaging. These
results are consistent with the findings
for the increased resistance of B. subtilis
to MeBr.
MeBr showed efficacy to be impacted by
the RH of the test chamber. Specifically,
higher humidity (75%) exhibited higher
efficacy than lower humidity (40%).
Painted Concrete
Cellulose
Laminate
Galvanized Metal
Carpet
Glass
Tile
B. subtilis, Glass
1200
1400
1600
CT, tng/L-Hr
1800
2000
Figure 6-2. Log reduction of B. anthracis Ames and B. subtilis spores vs. MeBr CT
at various MeBr concentrations at 36 °C and 75% RH.
6.3 Summary of HP Fumigation
Results
Shown in Figure 6-3, FTP fumigation of
building materials contaminated with B.
anthracis resulted in efficacies up to >6
log reduction (no CFU) dependent on the
building material, the concentration of
the fumigant, and the contact time. The
FTP concentration was 500 ppmv for all
fumigations shown. After a 4-hr
fumigation at 500 ppmv, about a 6 log
reduction or higher was observed from
all building materials tested. Note that
HP at 250 ppmv for 1.5 hr (CT of only
375 ppmv-hr) is the treatment specified
on the STERIS Vaprox® HP sterilant
package insert for sporicidal efficacy
(sterilization) for pre-cleaned enclosures.
Note that the registration is for the
solution that is used to generate the
vapor.
126
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» Finished Aluminum
—•—Keyboard Keys
—*— Industrial Carpet
—w— Painted Joint Tape
—*— Decorative Laminate
250 ppm. 500ppmv 1000 ppmv 2000 ppmv
HP, ppmv-hr
Pine Wood
Plate Glass
Ceiling Tile
Figure 6-3. Log reduction of B. anthracis Ames spores vs. HP CT at 500 ppmv for
various contact times.
6.4 Summary of Results from
Decontamination of B. anthracis
with Liquid pH-Amended
Bleach (10% adjusted to
approximately pH 7)
Shown in Figure 6-4, pH-amended
bleach adjusted to pH 7 provided
complete kill of B. anthracis Ames
spores (0 CPU recovered, >6 log
reduction) dependent on the building
material and the contact time. Note,
previous EPA testing with pH-amended
bleach was also found to be effective (>7
log reduction) against B. anthracis
spores on painted cinder block when
applied as a spray (60-min contact
time).[16]
127
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9/1/1
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8 00
7.00
S £5 nn
.2 O.UU
u c nn
= 5.00
5 A nn
Q£ 4.UU
D)
X o nn
jj o.UU
2(1(1
.uu
1 (1(1
1 .UU
0(1(1
* * _^^-J[
^ >— ^^ ' B
/ ^
/
/
.UU Til
—•—Carpet
-•—Concrete
—A— Glass
-x— Laminate
-*— Galvanized
Metal
-•—Particle
Board
0 10 20 30 40
Contact Time (min)
Figure 6-4. Decontamination Efficacy of pH-amended Bleach against B. anthracis
Ames spores at 22 °C.
The efficacy of pH-amended bleach
against three strains of B. anthracis
spores (virulent Ames, avirulent
NNR1 Al, and virulent Vollum) and B.
subtilis was evaluated using the
quantitative method. The log reduction
in recovery of various types of viable
Bacillus spores on coupons exposed to
pH-am ended bleach or PBS for specified
time periods is shown in Figure 6-5. In
addition, the extracted coupons were
placed individually into TSB and
incubated for seven days to look for
cloudiness that would indicate the
presence of residual viable spores on the
test coupon. In all cases where no spores
were observed using the quantitative
methodology, no residual viable spores
were detected using the qualitative
method.
Efficacy was shown to be dependent on
the type of material onto which the
spores were applied. For all spore types
tested, viable spores were present on
carpet and particle board after exposure
to pH-amended bleach for five min or
longer. In contrast, for all spore types
tested, no viable spores were recovered
from glass, galvanized metal, or
decorative laminate after exposure to
pH-amended bleach for five min. While
strong similarities in efficacy were
observed across spore types, differences
were also observed. For example, viable
B. anthracis Vollum and B. subtilis
spores were present on painted concrete
after exposure to pH-amended bleach for
five min; the other two strains of B.
anthracis, Ames and NNR1 Al, did not
have viable spores present after exposure
to pH-amended bleach for five min. As
another example of observed
differences, for coupons of all materials
tested, after 30 min exposure of B.
anthracis Ames spores to pH-amended
bleach there were no viable spores
recovered from any type of material. In
contrast, viable B. subtilis spores were
recovered from both carpet and particle
board after 30 min exposure to pH-
amended bleach.
128
-------
3
2
1 -
%^~ ^
^ ^
/
^
¥
C
c
-
?
^
1
_
—
-
/
\^
*
p
^
-f
^
/
—
-
<2
5
x^
s^
^
_
—
-
.?
*v
J&
-.
\
—
-
<$
&
D 6. anthracis Ames
• 6. anthracis Vollum
FIR anthraric; A/A/P"/ A •/
BS. subtilis
-
-
Figure 6-5. Decontamination efficacy of pH-amended bleach against various B.
anthracis strains and B. subtilis at 22 °C.
6.5 Summary of Results from
Decontamination of B. anthracis
with Liquid Exterm ClOi
Solution
Exterm C1O2 solution provided >6 log
reduction in recovered viable B.
anthracis Ames spores dependent on the
building material and the contact time
(shown in Figure 6-6). No viable B.
anthracis Ames spores were recovered
from industrial carpet, decorative
laminate, or glass after 120 min
exposure to the C1O2 solution; viable B.
anthracis Ames spores were recovered
from particle board after 120 min
exposure to C1O2 solution. Shown in
Figure 6-7, Exterm C1O2 solution
provided >6 log reduction in recovered
viable B. subtilis spores (no viable
spores recovered) after 120 min
exposure to C1O2 solution. Viable B.
subtilis spores were recovered from both
carpet and particle board after 120 min
exposure to C1O2 solution. In general, B.
subtilis spores appear to be able to
survive a longer exposure to C1O2
solution than B. anthracis Ames spores.
129
-------
Carpet
Particle Board
Glass
Laminate
20 40 60 80 100
Contact Time, min
120
140
Figure 6-6. Decontamination efficacy of Exterm ClOi solution against B. anthracis
Ames spores at 22 °C.
2.00
1.00
0.00
- Carpet
-Particle Board
-Glass
-Laminate
20 40 60 80 100
Contact Time, min
120
140
Figure 6-7. Decontamination efficacy of Exterm ClOi solution against B. subtitis
spores at 22 °C.
130
-------
6.6
Summary of Results from
Decontamination of B. anthracis
®
with Liquid Spor-Klenzu HP-PA
Solution
Spor-Klenz HP-PA solution exhibited a
range of efficacies, up to >6 log
reduction in B. anthracis Ames spores
(no viable spores recovered), dependent
on the building material and contact time
(shown in Figure 6-8). No viable B.
anthracis Ames or B. subtilis spores
were recovered from any glass coupons
after a 20-30 min contact time. A
complete kill of spores on decorative
laminate, i.e., no viable spores recovered
or detected, occurred at a 30-min contact
time with Spor-Klenz® HP-PA for B.
anthracis Ames spores. In contrast, less
than a 2-log reduction in viable spores
was observed after a 30-min contact time
with Spor-Klenz® HP-PA for B.
anthracis Ames spores on galvanized
metal. Spor-Klenz® HP-PA efficacy was
greater against B. anthracis Ames spores
on carpet than on metal, but after a 30-
min contact time with Spor-Klenz HP-
PA less than a 4-log reduction was
observed.
Glass (B. anthracis)
Painted Concrete
Carpet
Galvanized Metal
Particle Board
D eco ra ti v e La m i n a te
Glass (B. subtilis)
10 20 30
Contact Time, Min
Figure 6-8. Decontamination efficacy of Spor-KlenzR HP-PA decontamination
solution against B. anthracis Ames on various materials and B. subtilis spores on
glass.
6.7
Summary of Results from
Decontamination of B. anthracis
with Liquid Oxonia ActiveR
s>
Oxonia Active exhibited a range of
efficacies, up to >6 log reduction in B.
anthracis Ames spores (no viable spores
recovered), dependent on the building
material and contact time (shown in
Figure 6-9). No B. anthracis Ames
spores were recovered from any
galvanized metal or particle board
coupons after a 30- or 60-min contact
time. Viable B. anthracis spores were
recovered from one or more coupons of
glass, painted concrete, carpet, and
131
-------
decorative laminate after a 60-min
contact time with Oxonia Active . The
efficacy of Oxonia Active® against B.
anthracis Ames and B. subtilis spores,
reported as log reduction, showed
similar efficacies. For both B. anthracis
Ames and B. subtilis spores, a >6 log
reduction was observed after a 10-min
contact time (except that only a 5.77 log
reduction was observed for B. subtilis
after 60 min). Small numbers of viable
spores were recovered from one or more
replicate coupons for both B. anthracis
Ames and B. subtilis spores at each
contact time (10, 30, and 60 min).
Decontamination using Oxonia Active®
against B. anthracis Ames and B. subtilis
spores, reported as log reduction,
showed similar efficacies. For both B.
anthracis Ames and B. subtilis spores, a
>6 log reduction was observed after a
10-min contact time. Small numbers of
viable spores were recovered from one
or more replicate coupons for both B.
anthracis Ames and B. subtilis spores at
each contact time (10, 20, and 30 min).
Glass (B. anthracis)
Painted Concrete
Carpet
Galvanized Metal
Particle Board
Decorative Laminate
Glass (B. subtilis}
10 ?.0
Contact Time, Min
60
®
Figure 6-9. Decontamination efficacy of Oxonia Active against B. anthracis Ames
on various materials and B. subtilis spores on glass.
132
-------
7.0 References
2008.
4.
Inglesby, T.V., et al., Anthrax as
a biological weapon: Medical
and public health management.
Journal of the American Medical
Association, 1999. 281(18): p.
1735-1745.
Rogers, J.V., Sabourin, C. L.,
Choi, Y. W., Richter, W. R.,
Rudnicki, D. C., Riggs, K. R.,
Taylor, M. L., Chang, J,
Decontamination assessment of
Bacillus anthracis, Bacillus
subtilis, and Geobacillus
stearothermophilus spores on
indoor surfaces using a hydrogen
peroxide gas generator. Journal
of Applied Microbiology,
2005(99): p. 739-748.
AOAC, AOAC 2008-05: Efficacy
of Liquid Sporicides Against
Spores of Bacillus subtilis on
Hard Nonparous and Porous
Surfaces; First Action 2008
Mosmann, T., "Rapid
colorimetric assay for cellular
growth and survival: application
to proliferation and cytotoxicity
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Immunological Methods,
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Aguado, B., IP. Selmes, and
G.L. Smith, "Nucleotide
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2902.
CDC, Guide F - Environmental
Control of Smallpox Virus.
March 2003.
Center for Disease Control,
American Society for
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Public Health Laboratories,
Appendix B: nasal specimens for
screen Bacillus anthracis., in
Basic Diagnostic Testing
Protocols for Level A
Laboratories for the Presumptive
Identification of Bacillus
anthracis. 2002.
Kolb, R.W. and R. Schneiter, The
germicidal and sporicidal
efficacy of methyl bromide for
Bacillus Anthracis. Journal of
Bacteriology, 1950. 59(3): p.
401-412.
Schade, J.E. and A.D. King, Jr.,
Methyl Bromide as a
Microbicidal Fumigantfor Tree
Nuts. Applied and Environmental
Microbiology, 1977. 33(5): p.
1184-1191.
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10. Fritze, D. and R. Pukall, 14.
Reclassification of bioindicator
strains Bacillus subtilis DSM 675
and Bacillus subtilis DSM 2277 15.
as Bacillus atrophaeus.
International Journal of
Systematic and Evolutionary
Microbiology, 2001. 51: p. 35-
37.
11. Weinberg, M.F., R.H.
Scheffrahn, and M. A.
Juergensmeyer, PART 1: Efficacy
of Methyl Bromide Gas against 16.
Bacillus anthracis and Allied
Bacterial Spores in Final Report:
Whole-Structure
Decontamination of Bacillus
Spores by Methyl Bromide
Fumigation, U.S. Environmental
Protection Agency, Small
Business Innovation Research
Phase II. 2004.
12. Weinberg, MJ. and R.H.
Scheffrahn, PART 2: Whole-
Structure Decontamination of
Bacterial Spores by Methyl
Bromide Fumigation in Final
Report: Whole-Structure 17.
Decontamination of Bacillus
Spores by Methyl Bromide
Fumigation. 2004, U.S.
Environmental Protection
Agency, Small Business
Innovation Research Phase II.
13. AOAC, AOAC Official Method
966.04; Sporicidal Activity of
Disinfectants Alternative
Method; First Action 2006. .
2006.
STERIS, VHP® 100
Biodecontamination System.
2002 [cited March 1, 2007];.
EPA. Anthrax spore
decontamination using bleach
(sodium hypochlorite). 2007
October 22, 2007 [cited 2010
April 29]; Available from:
http ://www. epa.gov/pesti cides/fa
ctsheets/chemicals/bleachfactshe
ethtm., accessed February 23,
2011.
EPA, Evaluation of Liquid and
Foam Technologies for the
Decontamination ofB. anthracis
andB. subtilis Spores on
Building and Outdoor Materials,
DioxiGuard™ (Frontier
Pharmaceutical), pH-Amended
Bleach, Calcium Polysulfide,
CASCAD™ Surface
Decontamination Foam (Allen-
Vanguard), Oxonia Active®
(Ecolab Inc.), MinncareR Cold
SterilantR (Minntech Corp.),
SanDes (DTI-SwedenAB). , in
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Rastogi, V.K., S.P. Ryan, L.
Wallace, L.S. Smith, S.S. Shah,
and G.B. Martin, Systematic
evaluation of the efficacy of
chlorine dioxide in
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interior surfaces contaminated
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3351.
134
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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