EPA 600/R-14/405 I May 2015 I www.epa.gov/research
United States
Environmental Protection
Agency
               Comparative Efficacy of
               Sporicidal  Technologies for the
               Decontamination of Bacillus
               anthracis, B. atrophaeus, and
               Clostridium difficile Spores on
               Building Materials
Office of Research and Development
National Homeland Security Research Center

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                                                    EPA/600/R-14/405
                                                    May 2015
 Comparative Efficacy of Sporicidal Technologies for the
Decontamination of Bacillus anthracis, B. atrophaeus, and
    Clostridium difficile Spores on Building Materials
             U.S. Environmental Protection Agency
               Research Triangle Park, NC 27711
                            11

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                                     Disclaimer

The  U.S.  Environmental Protection  Agency  (EPA), through its  Office  of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), funded and
directed this work through  an Interagency Agreement DW-21-9234401-0/1 with Edgewood
Chemical Biological Center (ECBC). This report has been peer and administratively reviewed and
has been approved for publication as an EPA document. The views expressed in this report are
those of the authors and do not necessarily reflect the views or policies of the Agency. Mention of
trade names or commercial products does not constitute endorsement or recommendation for use
of a specific product.
Questions concerning this document or its application should be addressed to:


M. Worth Calfee, Ph.D.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-7600
                                         in

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                               Acknowledgments
Contributions  of the  following individuals  and organization  to  this  report are gratefully
acknowledged:
United States Army - Edgewood Chemical & Biological Center
      Dr. Vipin Rastogi
      Lisa S. Smith

United States Environmental Protection Agency (EPA)
      Dr. Worth Calfee, Office of Research & Development (PI)

Peer reviewers
      Dr. Doris Betancourt - US EPA, Office of Research and Development
      Dr. Frank Schaeffer - US EPA, Office of Research and Development
      John Archer - US EPA, Office of Research and Development
                                        IV

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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. This report summarizes the data generated under an interagency program
between Edgewood Chemical Biological Center (ECBC) and EPA,  entitled  "Comparing the
Efficacy   of Liquid  and  Fumigant  Technologies for  the  Decontamination  of Bacillus
anthracis  Spores and Bacillus atrophaeus Spores on Building Materials".
Comparative  sensitivity  (or  resistance)  of  the spores  of  Bacillus  anthracis (Ames),
Clostridium difficile (American Type Culture Collection (ATCC) 43498),  and B. atrophaeus
(Dugway Proving Ground-prepared ATCC  9372) to three commercial sporicidal  technologies
(vaporous hydrogen  peroxide (VHP),  chlorine dioxide gas  (CD),  and pH-amended liquid
bleach)  was evaluated.  Comparative  decontamination  efficacy  of these  technologies  has
previously been evaluated  for building  interiors by  the  US  EPA's  Office  of  Research
Development. However, until now,  no direct  side-by-side  laboratory  efficacy  studies  had
been  conducted to compare the  relative resistance of the Dugway B. atrophaeus spores (also
known as  Bacillus atrophaeus or B.g.) to the resistance of Bacillus anthracis Ames spores or C.
difficile spores.  The main objective of this study was to evaluate the validity of using Dugway-
prepared B.  atrophaeus spores as a surrogate for spores of the  Ames strain of B.  anthracis in
decontamination testing. A surrogate is considered suitable if its resistance to the test chemical
is equal to or slightly greater than the resistance of the organism being modeled.  A secondary
objective  was to  determine  the relative  resistances of B. anthracis.,  B.  atrophaeus., and C.
difficile.   Understanding the relative chemical  resistance of Bacillus spores and C. difficile
spores will  enable prediction of sporicide performance against Bacillus spores based upon the
vast body  of hospital disinfection/decontamination data generated for C. difficile.
Small-size coupons of glass and pinewood  (2x5  cm) were  inoculated with ~7 logs of spores
contained  in a 50-jiL aliquot of a spore  suspension. Spore recoveries from glass coupons ranged
between 10 and 25 % for C. difficile and 40 and 70 % for the two Bacillus spore types. The spore
recovery from pinewood was significantly lower. For the two Bacillus spore types, the recoveries
ranged between 25 and 40 %, for C. difficile the recoveries were typically ~5 % of the inoculum.
Overall, >6 logs of spores were recovered from glass and pinewood for all three spore types, thus
meeting the criteria for a 6 log dynamic range and allowing  demonstration of decontamination
efficacies  up to "6 log reduction". Sporicidal  efficacy results demonstrate that  for all  three
technologies, B. atrophaeus spores  showed a resistance to decontamination comparable to the B.
anthracis Ames spores on both glass and pinewood surfaces.  Interestingly, while the C. difficile
spores kill profile by bleach  and CD gas was comparable to the  other spore types on both glass
and pinewood,  sensitivity of this spore type to VHP was different on glass  vs. pinewood.  Log
reduction (LR) values against C. difficile spores on glass coupons were <5, compared to >6.5 or
near-complete kill at high dosages (2-3  hour exposure with 150 parts per million (ppm)) for the
two Bacillus spore types. The efficacy  data for pinewood were more variable, and the kill was
incomplete (only three-four LR even after three hours (h) of exposure). Taken together, the data
strongly support the contention that B.  atrophaeus spores serve as a suitable  surrogate  for the
pathogenic B. anthracis Ames spores  when  using these three decontamination technologies.
Finally, the  data are also consistent with the conclusion that sporicidal technologies are  quite
effective against spores of C. difficile. Additional work needs to be done to confirm our observation
that VHP (450 parts per million by volume (ppmv)-h) on glass is only partially effective  against

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C. difficile spores, and if true, this partial effectiveness may have significant influence on infection
control in medical treatment facilities and spread of this hospital-acquired infection.
Summary of Results


pH-Amended Bleach
When used as a sporicidal decontaminant, the recommended concentration of pH-amended bleach
is 6000 ppm (US EPA, 2011). In this study (due to biosafety concerns), instead of spraying the
bleach solutions, the coupons were covered with a 1-mL aliquot for 2, 5, 10, and 20 minutes. The
tests were performed in the  summer of 2012 (Phase 1) and were limited to two spore types, B.
anthracis Ames and B.  atrophaeus, and in the summer/fall  of  2013 (Phase 2). In addition to
Bacillus spores, spores of C. difficile were also included in Phase 2. After the intended contact
times, the excess liquid and the coupon  were immediately pipetted/transferred into a 20-mL
recovery medium containing 0.5 %  sodium thiosulfate.  Efficacy values were  >6.5 LR even
within a two-min exposure on glass coupons and barely 2  to 3 LR even after a 20-min exposure
on pinewood.

Chlorine Dioxide
CD gas was used at a target concentration of 3000 ppmv, and coupons were exposed for 0.5, 1, 2,
and 2.5 h. On glass coupons, LR values were >6.5 for all three spore types even with a 0.5 h (1500-
ppmv-h dosage) exposure.  No significant difference in the kill profile of the three spore types was
evident, although minor differences were noted on the glass coupons. However, the LR values of
5 to 6 were observed after only 2.5 h exposure (7500-ppmv-h) when inoculated pinewood coupons
were evaluated. Similar sporicidal efficacy of CD gas on pinewood has been observed previously,
and this surface was concluded to  be hard-to-decontaminate  with this fumigant (Rastogi et al.,
2010).
Vapor Hydrogen peroxide
VHP was used at a target concentration of 150 ppmv, and the coupons were exposed for 0.5, 1,
2, and 3 h. On glass coupons, high efficacy (7 LR for Ames and 4.5 LR for C. difficile) was
observed at sub-lethal exposure times for Ames and C. difficile spores, but LR values of 2-5 were
observed for B. atrophaeus spores at similar exposure times.  At high dosage (450-ppmv-h), the
LR values against C. difficile spores were significantly lower compared to the other two Bacillus
spore types. The efficacy measured as LR was <5 and kill kinetics were less rapid than the other
two sporicidal agents.


Comparative  Sporicidal Efficacy against Spores ofB. anthracis andB. atrophaeus
For all three technologies tested at two different times (summer of 2012 and summer/fall of 2013),
B. atrophaeus spores were as resistant to kill as spores  of B.  anthracis Ames.  No  significant
difference  was  observed  in  relative  sensitivities of  the  two  spore  types  to the  tested
technologies. In some cases, B. atrophaeus spores were  slightly more resistant than  the spores
of B. anthracis Ames. Consequently, the data suggest that B. atrophaeus spores  are appropriate
surrogates for B. anthracis Ames spores for the three tested technologies.
                                           vi

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Comparative Sporicidal Efficacy against Spores ofC. difficile andB. atrophaeus/B. anthracis
With the exception of pH-amended bleach on glass, spores of C. difficile demonstrated comparable
resistance (or sensitivity) to  all three  technologies relative to Bacillus species spores on both
surface types. On glass, at fractional kill levels, C. difficile spores exhibited greater resistance than
the other two spore types, for all three technologies tested.
                                            vn

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                                      Contents
Disclaimer  	iii
Acknowledgments	iv
Executive Summary	v
Abbreviations/Acronyms	x
    1.0  Introduction	1
    2.0  Technology Descriptions and Test Matrices	2
       2.1   Technology Descriptions	2
       2.2   Test Matrices for Decontamination Technologies	2
    3.0  Summary of Test Procedures	4
       3.1   Biological Agent	4
       3.2   Test Material Surfaces	5
       3.3   Spore Stock Preparation and Coupon Inoculation	6
       3.4   Decontamination with pH-Adjusted Bleach	6
       3.5   Decontamination with Fumigants, CD Gas and VHP	7
       3.6   Spore Extraction and Quantification	7
       3.7   Recovery Efficiency	8
       3.8   Decontamination Efficacy	8
    4.0  Quality Assurance/Quality Control	9
       4.1   Instrument/Equipment Testing, Inspection, and Maintenance	9
       4.2   Equipment Calibration	13
       4.3   QC Results	13
       4.4   Audits	13
       4.4.1   Performance Evaluation Audit	13
       4.4.2   Technical Systems Audit	14
       4.4.3   Data Quality Audit	14
    5.0  Results and Performance of Sporicidal Fumigants and Disinfectants	15
       5.1   Spore QA/QC	15
       5.2   Spore Recovery	15
       5.3   Sporicidal Efficacy	17
       5.3.1   pH-Amended Bleach	17
       5.3.2   VHP	17
       5.3.3   CD Gas	18
       5.3.4   Overall Results (Phase 1)	19
       5.4   Spore Recovery	20
       5.5   Sporicidal Efficacy	20
       5.5.1   pH-Amended Bleach	20
       5.5.2   CD Gas	23
       5.5.3   VHP	24
       5.6   Summary of Phase 1 and Phase 2 Results	24
    6.0  Discussion and Conclusions	26
       6.1   Comparing Efficacy against B. anthracis and B. atrophaeus	26
       6.2   Comparing Efficacy against Spores of Bacillus species and C. difficile	26
    References	27
                                          Vlll

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                                      Figures
Figure 3.1.  C. difficile colonies on a plate (A) and spores (B)	5
Figure 3.2. Anaerobic jar with GasPak	5
Figure 5.1. Spore recovery from glass and pinewood coupons during Phase 1	16
Figure 5.2. Efficacy of pH-Amended Bleach (6000 ppm)	17
Figure 5.3. Efficacy of VHP (150 ppmv)	18
Figure 5.4. Efficacy of CD (3000 ppmv)	19
Figure 5.6. Efficacy of pH-Amended Bleach on Three Spore Types on Glass Coupons	22
Figure 5.7. Efficacy of pH-Amended Bleach on Three Spore Types on Pinewood Coupons	22
Figure 5.8. Efficacy of CD Gas on Three Spore Types on Glass Coupons	23
Figure 5.9. Efficacy of CD Gas on Three Spore Types on Pinewood Coupons	23
Figure 5.10. Efficacy of VHP on Three Spore Types on Glass Coupons	24
Figure 5.11. Efficacy of VHP on Three Spore Types on Pinewood Coupons	25
                                       Tables
Table 2.1 Decontamination Technology Descriptions	2
Table 2.2 Test Matrix for Three Decontamination Technologies	3
Table 3.1 Test Materials	6
Table 4.1 Data Quality Objectives for Test Measurements	10
Table 4.2 Sample Performance Criteria	12
Table 4.3 Performance Evaluation Audits	13
Table 5.1 QA/QC of Three Spore Types	15
                                         IX

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Abbreviations/Acronyms
ATCC
B. anthracis
B. atrophaeus
BHI-HT
BSC
BSL
°C
C. difficile
CBR
cm
CRP
CSI
CD
CPU
DNA
ft2
ft3
EPA
ECBC
FIFRA
g
h
HS
HSRP
L
LR
M
m3
mg
min
mL
uL
NaOCl
NaOH
NHSRC
NIST
ORD
PCR
ppm
ppmv
PO
PLC
psi
QA
QC
QMP
American Type Culture Collection
Bacillus anthracis (Ames strain)
Bacillus atrophaeus
rain heart infusion agar with horse blood and taurocholate
biological safety cabinet
bio -safety level
degree(s) Celsius
Clostridium difficile
chemical, biological, and radiological
centimeter
Critical Reagent Program
ClorDiSys, Inc.
chlorine dioxide
colony forming unit(s)
deoxyribonucleic acid
square foot/feet
cubic foot/feet
U.S. Environmental Protection Agency
Edgewood Chemical Biological Center
Federal Insecticide, Fungicide, Rodenticide Act
gram
hour(s)
homeland security
Homeland Security Research Program
liter(s)
log reduction
molarity
cubic meter(s)
milligram(s)
minute(s)
milliliter(s)
microliter(s)
sodium hypochlorite
sodium hydroxide
National Homeland Security Research Center
National Institute of Standards and Technology
Office of Research and Development
polymerase chain reaction
part(s) per million
part(s) per million by volume
Project Officer
programmable logic controller
pounds per square inch
quality assurance
quality control
quality management plan

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RH
SD
sec
SOP
STS
TSA
VHP
relative humidity
standard deviation
second(s)
Standardized Operating Procedure
sodium thiosulfate
tryptic soy agar
vaporous hydrogen peroxide
XI

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                                  1.0   Introduction

The U.S.  Environmental Protection Agency's (EPA's) Homeland Security Research Program
(HSRP) is helping protect human health and the environment from adverse impacts resulting from
the release  of chemical, biological, or  radiological  (CBR) agents.  With  an  emphasis on
decontamination and consequence management,  water infrastructure protection, and threat and
consequence assessment, the HSRP is working to develop tools  and information that will help
detect and quantify the intentional release of chemical or biological contaminants in buildings,
water systems, or the outdoor environment; contain these contaminants; decontaminate buildings,
water systems or the outdoor environment; and facilitate the treatment and disposal of hazardous
materials resulting from remediation/cleanup activities.
As part of the above program, 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 the HSRP's EPA Program Office and Regional partners, conducting tests, collecting and
analyzing data, and preparing peer-reviewed reports. All evaluations are conducted in accordance
with rigorous quality assurance (QA) protocols to ensure that high quality data are generated and
that the results are defensible. EPA provides high-quality information that is useful to decision
makers in responding and implementing appropriate technologies to mitigate consequences to the
public resulting from CBR incidents.
The purpose of this study was to evaluate the decontamination efficacy of three decontamination
approaches for three spore types (B. anthracis Ames, B. atrophaeus, and Clostridium difficile).
Comparative efficacy data  for B. anthracis and B.  atrophaeus were gathered to evaluate the
suitability of B. atrophaeus as a surrogate for B. anthracis in decontamination  studies. Such a
comparative body of information is  critical  in evaluating candidate technologies in  field- and
building-scale studies, where no greater than a bio-safety level (BSL)-l spore-forming organism
can be used. The spores of B. anthracis and B.  atrophaeus are  quite different  with  respect to
hydrophobicity  and presence of an exosporium, which is present in B. anthracis Ames and not
present in B. atrophaeus. It is therefore imperative that a comparative set of data be available for
the test lead in selecting appropriate surrogates for future studies.
Comparative efficacy data for B. anthracis and C. difficile were gathered in attempts to bridge the
hospital disinfection-related and homeland security-related bodies of literature.  For example,
when  data from the current study are considered together with results from previous C. difficile
studies,  predictions of the effectiveness of technologies widely used in hospitals against B.
anthracis may be possible. These predictions may prove to be valuable for future decontamination
research in either area.  C. difficile infection is the most common cause of nosocomial infectious
diarrhea (Cloud and Kelly, 2007; McFarland et al., 2007).
During Phase  1  of this investigation, sporicidal efficacy was determined for bleach, chlorine
dioxide (CD) gas, and vaporous hydrogen peroxide (VHP) against spores of B. atrophaeus and the
pathogenic agent  it is used to model, B. anthracis Ames. In Phase 2, C. difficile (spores) were
added, as a third organism, to the test matrix. Additional decontamination trials were conducted
with the three spore-forming organisms to evaluate the efficacy of the  candidate technologies
against an important hospital clinical pathogen (C. difficile) and the two Bacillus species.

                                            1

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                2.0    Technology Descriptions and Test Matrices
2.1  Technology Descriptions
Table 2.1 summarizes the three  decontamination technologies  evaluated in this investigation.
Information is provided on the manufacturer, product name, chemical components and active
ingredients. Note that Ultra Clorox® Germicidal Bleach is registered as a disinfectant, but the pH-
amended  solution is not.  Further  details on  the chemical  composition,  preparation,  and
decontamination application procedures are provided in Section 3.
Table 2.1   Decontamination Technology Descriptions

Decontaminant
Chlorine Dioxide

VHP




pH-Amended
Bleach


Product Name,
Vendor, and
Location
CD-Gas ClorDisys
Trenton, NJ
Vaporous
hydrogen peroxide
STERIS Corp.
Mentor, OH
Ultra
Clorox®Germicidal
Bleach
Clorox®
Professional
Products Co.
Oakland, CA
Active Ingredients
and Sporicidal
Chemical
Chlorine dioxide gas

Vaporized form of
hydrogen peroxide



Sodium hypochlorite,
hypochlorous acid



Components
3000 ppmv chlorine dioxide gas
150 ppmv vaporous hydrogen
peroxide
35 % VHP and balance water
vapors

Sodium hypochlorite 6.15 %,
sodium hydroxide <1 %; diluted
with sterile distilled water;
with 5 % acetic acid added to
adjust the pH to 6. 8 -7. 2

EPA
Registration
80802-1

58779-4




67619-8
(Clorox®
disinfectant)


Chlorine dioxide (CD) was selected for testing because CD has been demonstrated to be effective
against B. anthracis on building materials (Canter, 2005, Canter et al., 2005; Rastogi et al., 2009,
2010). Furthermore, CD gas has been proven to be virucidal, bactericidal, and sporicidal (Beuchat
et al., 2004; Fukuyama et al., 1986; Rastogi et al., 2009, 2010). VHP was selected for its proven
sporicidal  effect (Canter, 2005) and its recent evaluations in building-scale studies (U.S.-EPA
BOTE project, 2013). pH-Amended bleach (with pH 7.0+0.1) was selected for testing because this
decontaminant has been  demonstrated to be effective against B.  anthracis on many  surface
materials, is readily available, and easily prepared using off-the-shelf chemicals.  It has also been
evaluated in recent building-scale studies (U.S.-EPA BOTE project, 2013).
2.2  Test Matrices for Decontamination Technologies
In general, the operating test conditions selected (e.g., contact time, concentration) were based on
previous similar B. anthracis  efficacy  tests (as described above),  as  well  as manufacturers'
recommended parameters.
The test matrices for the three technologies (pH-amended bleach, VHP, and CD gas) are shown in
Table 2.2. Working stocks of each spore type were diluted to -2-5 x 108 spores/mL in 0.01 %

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Tween® 80. In general, glass and pinewood coupons were inoculated with a 50-jiL aliquot as seven
droplets across  the surface. The spores were allowed to dry overnight in a Class II bio-safety
cabinet (BSC) under ambient conditions.  The inoculated coupons were used within 24 hours of
drying. The coupons were covered with a 1-mL aliquot of pH-amended bleach and after the desired
contact times (2, 5,  10, and 20  minutes  (min) ),  the excess liquid and the coupon were both
transferred to a tube  containing 20 mL of 0.01% Tween® 80 solution containing 0.5 % sodium
thiosulfate (STS) for quenching the active moiety. For evaluating the gases, the inoculated coupons
were placed in Petri plates and exposed in a 0.2265- cubic meter (m3) fumigation chamber to CD
gas (3000 ppmv) or VHP (150 ppmv) as previously described in detail (Rastogi et al., 2009, 2010).
A set of four coupons in a Petri plate was withdrawn after each exposure time (0.5, 1, 2, and 2.5
h) for CD gas and (0.5,  1, 2, and 3 h) for VHP. The coupons were aerated for 5 min in the Class II
BSC before being transferred into 20 mL of 0.01 % Tween® 80 solution.
Table 2.2   Test Matrix for Three Decontamination Technologies
Phase
#
Biological
Agent
Coupon types
Concentration of fumigant or
volume applied
Contact Time (h)
          B. anthracis
            Ames
          B. atrophaeus
Glass and pinewood
    Four each
   150 ppmv VHP
   3000 ppmv CD
lmLof6000ppmpH-
   amended bleach
 0.5, 1,2, and 3 h
0.5, 1,2, and 2.5 h
2, 5, 10, and 20 min
          B. anthracis
             Ames
          B. atrophaeus
           C. difficile
Glass and pinewood
    Four each
   150 ppmv VHP
   3000 ppmv CD
lmLof6000ppmpH-
   amended bleach
 0.5, 1,2, and 3 h
0.5, 1,2, and 2.5 h
2, 5, 10, and 20 min

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                        3.0    Summary of Test Procedures

Test procedures were performed in accordance with an approved Quality Assurance Project Plan
(QAPP)  for  "Comparing  the Efficacy  of  Liquid  and  Fumigant  Technologies  for  the
Decontamination of Bacillus anthracis  Spores and Bacillus atrophaeus Spores  on Building
Materials".

3.1  Biological Agent
The B. anthracis spores used for this testing were prepared from a documented stock of the Ames
strain in a BSL-3  facility located in building E-3150  of the U.S. Army's Edgewood Chemical
Biological Center (ECBC, Aberdeen Proving Ground, Edgewood, MD). The Ames  strain was
procured from Critical Reagent Program (CRP) Unified Culture Collection at Fort Detrick, MD)
and is maintained and characterized by stringent procedures internal to ECBC. The spores were
prepared on large sporulation plates as per the procedure outlined in the QAPP, and spore stocks
were maintained in 0.01 % Tween® 80. Specifically, the spore lots were characterized prior to use
by colony morphology, direct microscopic observation and determination of percent refractivity
and percent vegetative bacterial cells. In addition, the number of viable spores was determined by
colony count and expressed as colony forming units per milliliter (CFU/mL). Genotypic identity
of the frozen stock was confirmed by genomic deoxyribonucleic acid (DNA)  extraction  and
amplification  of chromosomal and plasmid-borne markers by polymerase chain reaction (PCR).
Prior to each testing,  spore quality was  determined by three approaches: >95 %  spores, heat
resistance to 65 degrees Celsius (°C) (<0.2-log difference after 30 min) and HC1 resistance (<2-
log reduction following 2 min exposure and 2-6 log reduction after 5 min exposure to 2.5-N HC1;
U.S. EPA Protocols, 2014).
The B. atrophaeus spores (American Type Culture Collection [ATCC] 9372) were procured from
Dugway Proving Ground, UT, as dry powder. One gram of spores was suspended in 40 mL of 0.01
% Tween® 80 and  enumerated (by dilution plating) to determine the  titer. Working stocks were
prepared by appropriate dilution using 0.01 % Tween® 80 and enumerated. The spore stock was
characterized by approaches similar to those used and described above for the B. anthracis Ames
spore stock. This stock was also found to be resistant to HC1 and heat.
The C. difficile (ATCC 43498) spores were initially procured from a commercial source (ATS
Lab, Eagan, MN). However, the titer of the spores  procured  from the  ATS laboratory was
inadequate, so the spores were therefore prepared in-house according to the ASTM E2839 protocol
(ASTM 2011), with one exception. All protocol steps were followed with the exception of the final
step of spore purification using HistoDenz, which was  omitted. The rationale for omission of the
last step was twofold:  a) the crop harvested from the plate was 90-95 % spores; and b) no similar
purification was performed for the other two Bacillus spore species, and a  comparable spore
quality of C. difficile was thought to be the most appropriate to include C. difficile in  this study.
C. difficile is a BSL-2 bacterial strain (Figure  3.1 A and B).  C. difficile cells were grown in
reinforced clostridial broth (ATCC medium 2107) at 37 °C  in an anaerobic environment (80 %
nitrogen, 10 % carbon dioxide, and 10 % hydrogen). Anaerobic jars containing GasPak (BD,
Franklin Lakes, NJ; GasPak 150 system, catalog number 260628) were used to culture the viable
spores (Figure 3.2). Colonies were grown on BHI-HT agar plates (brain heart  infusion agar with

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horse blood and taurocholate, AS-6463; Anaerobe Systems, Morgan Hill, CA). Single colonies
were  visible after 48-72 h of incubation. The spore  quality was determined using  the same
approaches used for the other two spore types, heat resistance and HC1 resistance (<2 log reduction
after 10-min exposure to 2.5N HC1).
The working stocks of all three spore types were prepared in 0.01 % Tween® 80 solution stored at
2 to 8 °C, and used within four weeks after preparation.
               Figure 3.1.  C. difficile colonies on a plate (A) and spores (B)
                         Figure 3.2. Anaerobic jar with GasPak
3.2  Test Material Surfaces
Information on the coupons and sterilization approaches used for testing is summarized in Table
3.1. Coupons were cut to uniform length and width from a large piece of stock material. Coupons
were prepared for testing by sterilization via autoclaving using a Getinge Vacuum Steam Sterilizer
(Model # 533LS, Goteborg, Sweden). The selected materials, shown in Table 3.1, were based on
both cost-effectiveness and minimization of physical alterations of the material. Autoclaving was

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done at a minimum of 15  pounds per square inch (psi) and 121 °C for 30 min. Sterilization is
intended to minimize contamination by microorganisms other than those being evaluated.
                               Table 3.1   Test Materials
Material
Glass
Pinewood
Lot, Batch, or
ASTM No., or
Observation
Wire-reinforced
glass
Structural Wood,
Hem-fir, type II
Manufacturer
Supplier Name,
Location
McMaster Carr,
Elmhurst, IL
Bowater,
Greenville, SC
Approximate
Coupon Size,
width x length
2x5 cm
1.5 x 1.5 cm
Material
Preparation
Autoclave
Autoclave
3.3  Spore Stock Preparation and Coupon Inoculation

Working spore stocks with a target titer of 2-5 x 108 spores/mL were prepared in 0.01 % Tween®
80. The stocks were used within four weeks of preparation, and new working stocks were prepared
each month. The spore stocks were stored in a refrigerator at 2-8 °C and thoroughly mixed before
use. An aliquot of 50 jiL was inoculated across the surface by transferring seven droplets. The
inoculated coupons were dried  in a Class IIBSC overnight under ambient conditions (a range of
18-25 °C and 20-45 % relative humidity [RH]). The dried coupons were used within 24 h. For each
data point, a set of four test replicates was included. Controls included a negative coupon with no
inoculation and a positive control that was not exposed to sporicidal agent.
3.4  Decontamination with pH-Adjusted Bleach
Bleach was prepared and pH-adjusted (as per the internal protocol) fresh on the day of testing. The
pH-amended bleach consisted of bleach diluted in water with its pH adjusted by addition of acetic
acid. Specifically, Ultra Clorox® Germicidal Bleach was used, which contains 6.15 % by weight
sodium hypochlorite (NaOCl) and <1.0 % sodium hydroxide  (NaOH) in aqueous solution. This
product has a pH between 11 and 12 and a density of 1.08 to 1.11 grams (g)/mL. The pH was
adjusted to 6.5 - 7.0 by the addition of 5 % acetic acid. The primary active decontaminating agent
in this final solution is hypochlorous acid. The recipe for preparation of pH-amended bleach for
use as a decontaminant was as follows:
   •   Prepare 5 % acetic acid  solution by diluting 50 mL of glacial acetic acid to 1 L with sterile
       distilled water in a volumetric flask.
   .   Mix eight parts sterile distilled water, one part Ultra Clorox® Germicidal Bleach, and add
       an adequate volume of 5 % acetic acid to adjust the pH to 6.8 - 7.2.

The pH-amended bleach decontamination procedure was conducted as follows: a pipette was used
to dispense a 1-mL aliquot of pH-amended bleach to the surface  of four replicate coupons.  No
reapplication of bleach was administered.  Following the desired contact  times (2, 5, 10, and 20
min), the excess liquid and the coupon were both transferred to a tube containing 20 mL of 0.01
% Tween® 80 solution containing 0.5 % sodium thiosulfate for quenching the active moiety and
                                           6

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subsequent spore recovery procedures. The spores were recovered by ten-min sonication and two-
min vortexing. Recoveries from test and control coupons were then determined by dilution-plating
onto tryptic soy agar (TSA) plates.
3.5  Decontamination with Fumigants, CD Gas and VHP
The test chamber used in these fumigation studies was 0.2265 m3 (8 cubic feet [ft3]) in dimension
(2 ft x 2 ft x 2ft). The test chamber was constructed by ClorDiSys, Inc. (CSI) from 316-grade
stainless steel. The vendor supplied the qualification documentation for performance of the test
chamber. The chamber had one window for observation, which was completely covered when
used with CD gas, since CD is light sensitive. This system has been safely utilized to test a wide
variety of decontamination gases and vapors. The  chamber was equipped with temperature, RH,
pressure, and CD sensors. Additionally, the test chamber was equipped with five antechambers for
easy access  and removal of Petri plates containing inoculated coupons. Each antechamber had
inner and outer airlock doors.
After fumigation with 3000-ppmv CD gas or 150-ppmv VHP for  a pre-specified time, one Petri
plate was placed in one of the antechambers by opening and closing of the inner door. The Petri
plate was removed by opening the outer door without affecting the exposure of other coupons. The
chamber was filled with CD at a flow  rate of 20 L/min, and  the concentration of CD was
maintained near constant by a programmable logic controller (PLC) sensor regulator installed in
the generator. A circulation fan installed in the chamber mimicked  the air circulation produced by
fans in a commercial large-room decontamination process. Air circulation in the chamber ensured
that the gas/vapor was evenly distributed over the exposed surfaces. The chamber temperature and
RH were programmed, controlled, and monitored. For CD testing, exposure was performed at
target settings of 75 % RH and 24 °C (75 °F). For VHP testing, RH was reduced to <35 %, and the
vapor concentration was set at 150 ppmv. The chamber was operated in 'exhaust on' mode when
using VHP vapors. After the specified exposure time, each Petri  plate was withdrawn from the
chamber through the antechamber. The coupons were aerated in a  Class IIBSC for one-two min,
following which each coupon was transferred into a tube containing 20 mL of 0.01 % Tween® 80
solution.
3.6  Spore Extraction and Quantification
For coupons inoculated with B. anthracis Ames and B. atrophaeus, the tubes containing control
or test coupons were vortexed for two min. However, in the case of C. difficile, the tubes containing
control and test coupons were sonicated for 10 ten min (Bransonic Sonicator, procedures described
in Rastogi et al., 2009, 2010) and vortexed for two min. For all three spore types, the spores were
diluted tenfold in 0.01 % Tween® 80 to 10"4 for positive controls and to 10"1 for the test samples
prior to plating. A 100-jiL aliquot was plated on two TSA Petri plates at 10"3 and 10"4 dilutions
(control sets) and from 10° and 10"1 dilutions (test samples). Plates were incubated at 37 °C for 18-
24 h to allow colonies to grow. Plates from test samples were  left for another day to allow for
growth of slow growing injured but viable spores. Colony forming units (CFU) were counted,
averaged from two replicate plates, and transformed into log (CFU) for each replicate coupon.
Averages were computed for each set of four replicate coupons.

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3.7  Recovery Efficiency
The mean percent spore recovery from positive control samples was calculated using results from
positive control samples (inoculated, but not decontaminated), by means of the following equation:
                  Mean % Recovery = [Mean CFUpc/CFUtiter] x 100
where Mean CFUpc is the mean number of CPU recovered from four replicate positive control
samples of a single material, and  CFUtiter is the number of CPU inoculated onto each of those
samples. The value of CFUtiter is determined from the working stock on the day of testing.  Spore
recovery was calculated for each of the two coupon types with each of the three spore types.


3.8  Decontamination Efficacy
Sporicidal efficacy was  computed by subtracting the mean log (CFU) of the test from the log
(CFU) of the control values, according  to the equation below.  Standard deviations were also
computed to assess the variability.
       Efficacy = LogwCFUc - LogioCFUt,
             CFUc = total colony forming units from control coupons, and
             CFUt = total colony forming units recovered from test coupons.

Since the amount of spore contamination on surfaces and the types of surfaces needing treatment
following an actual contamination incident are expected to vary widely, it is impossible to evaluate
all conditions (spore load, waste acceptance  criteria,  etc.) likely to be  encountered  during
decontamination activities.  To address this challenge and allow comparison across sporicidal
products  or  decontamination methods,  a  consistent  challenge   is  posed  to  evaluate
effectiveness. For example, a 7- log spore challenge (inoculation of material coupon surfaces with
~ 5 x 107 spores) was used across all tests and materials. Consistent with sporicidal efficacy tests
used to register sporicides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA),
the current study utilized the  generally  accepted criterion of 6- LR to consider an approach
effective. Recovery of no viable spores following treatment was considered highly effective.

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                     4.0   Quality Assurance/Quality Control

QA/QC procedures were performed in accordance with the Quality Management Plan (QMP) and
the test/QA Plan. The QA/QC procedures and results are summarized below.
In the measurement of decontamination  efficacy, experimental error or variability could be
introduced by inaccurate measurement of volumes of suspensions of bacteria being applied. Also
inaccurate measurement  of test parameters (e.g.,  disinfection solution concentration) being
manipulated in the testing can contribute to error in decontamination efficacy.  The data quality
objectives for test  measurements  provided  in  Table  4.1 limit the  error introduced into  the
evaluation.  Quantification of B.  anthmcis, B. atrophaeus or C. difficile in this evaluation does
not involve  the use of analytical  measurement  devices. Rather, the CPU were determined by
dilution plating and recorded. Sample performance criteria  are shown in Table 4.2. Standard
operating procedures  (SOPs) implemented by  qualified, trained, and  experienced  personnel
were used to ensure data collection consistency.
4.1  Instrument/Equipment Testing, Inspection, and Maintenance
A maintenance schedule for laboratory equipment was required. The equipment needed for the
evaluation was maintained and operated according to the quality requirements and documentation
of the evaluation facility.  Equipment includes BSCs, pipettes,  incubators, and orbital shakers.
However, there were no critical experimental parameters that must be calibrated for the BSCs and
orbital shaker equipment. Pipettes were calibrated every six  months. Only properly functioning
equipment was used; any observed malfunction was documented and appropriate maintenance or
replacement of malfunctioning equipment was performed. Daily, the laboratory staff checked the
temperature of the incubator and the results of the daily check of the incubator were entered into a
facilities data collection  form.  The incubators  were calibrated semi-annually on a schedule
maintained by the ECBC.
Prior  to use and following the frequency specified in  Table 4.1, all calibrated equipment was
checked by the user to verify that the equipment was within calibration. This information was also
documented on data sheets that included  equipment name, serial number, model number,  date
calibration was performed, and date calibration was due. The test personnel manually recorded
this information by initialing and dating at time of verification.
The facility  has  SOPs for the calibration  of all  instruments. A list of all instruments requiring
calibration is maintained in a database and calibrations are  scheduled by designated staff.  All
instruments  used at the time of experimentation were verified as being certified, calibrated, or
validated.  Calibration of  instruments  was done at  the frequency  shown in  Table 4.1. Any
deficiencies  were noted,  and the  instrument was  adjusted to meet  calibration tolerances and
recalibrated within 24 h or replaced. If allowable tolerances were not being met after recalibration,
additional corrective action was taken, including the replacement of the instrument.

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Table 4.1 Data Quality Objectives for Test Measurements
Test Measurement
Specifications

Parameter
to be
Measured




Volume
(i.e., spike
or dilution
volume)





Weight







PH






Unit





^L





g







PH
unit






Allowable
Test
Measurement
Tolerance





± 10 %





±0.1







±0.1 pHunit





Instrument Specification

Instrument





Micro-
pipette





Balance







pH meter






Instrument
Calibration/
Certification

Micropipettes
were verified as
calibrated at time
of use by
supplier- pipettes
are recalibrated
by gravimetric
evaluation of
pipette
performance by
supplier
Balances are
calibrated
monthly and
annually serviced
under a PMA
Perform two-
point calibration
with standard
buffers that
bracket targeted
pH every time of
use. Percent
slope value
generated by pH
meter must be >
90 % to pass
calibration

Instrument
Calibration
Frequency





Every six
months





Every 12
months







Prior to use






Expected
Instrument
Tolerance





± 10 %





±0.1g







±0.1pH
unit





Corrective
Action if
Expected
Instrument
Tolerance
Unattained



Replace with
calibrated and
sufficiently
accurate
micropipette



Replace with
calibrated and
sufficiently
accurate
balance





Recalibrate
instrument





                                       10

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Table 4-1 (Continued)
Test Measurement Specifications

Parameter
to be
Measured





RHin
chamber



Temperature




Time


Colony


Unit





o/o



°c




h

Colony
Forming
Unit
(CPU)

Allowable
Test
Measurement
Tolerance





± 20 % full
scale



±2°C




two seconds
(sec)/h


100% of
colonies must
be counted

Instrument Specification

Instrument





Hygrometer



Thermo-
meter




Timer


QCount™


Instrument
Calibration/
Certification

NIST*-
traceable

certification
and/or checked
against NIST-
hygrometer

Checked
against NIST-
traceable
thermometer



Check against
NIST-traceable
standard


Calibrated
once a year


Instrument
Calibration
Frequency





Once per
quarter



Once prior
to testing




Once per
quarter


Once per
month


Expected
Instrument
Tolerance


± 0.5 %
from 25 %

to 95 %
over the
range of 5
°C to 55 °C


± 0.5 °C at
25 °C




two sec/h-

one - two
small
colonies per
plate
Corrective
Action if
Expected
Instrument
Tolerance
Unattained

Replace with
calibrated

and
sufficiently
accurate
hygrometer
Replace with
calibrated
and
sufficiently
accurate
thermometer
Replace with
calibrated
and
sufficiently
accurate
timer

Re-Calibrate
Instrument

*NIST = National Institute for Standards and Technology
                                                     11

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Table 4.2 Sample Performance Criteria


Sample

Spike Control -
direct analysis of
the B. anthracis, B.
atrophaeus, and C.
difficile stock
suspension
Procedural Blank -
not inoculated with
either B. anthracis,

B. atrophaeus, or C.

difficile a
Positive Control -
inoculated with
either B. anthracis,
B. atrophaeus, or C.
difficile (not
exposed to
disinfectant but
extracted)
Test Coupon-
inoculated with

either B. anthracis,
B. atrophaeus, or C.
difficile, exposed to
the disinfectant and

extracted at non-
zero time points

Number of
Samples


One sample each
day of use at time
zero


One at time -zero
(after overnight

drying)




Three included per
disinfectant
concentration and
material




Five at each non-
zero time point per
disinfectant
concentration and
material



Information
Provided


Calculate value of
CPU in stock
suspensions


Controls for
sterility and cross

contamination





Controls for percent
recovery





Replicate coupons
that yield results
impacted by test
conditions




Performance
Criteria


± 1 log (1 x 107 to 1
x 109 CFU/mL)




No observed CPU






Mean CPU > 5 %
and< 150% of
spike control



CPU value outside

three standard
deviations (SDs) of
the mean will be
evaluated as an

outlier

Corrective Action
if Performance
Criteria are not
Attained
Discuss results with
the PO; identify and
correct the cause of
incorrect bacterial
levels in the stock
suspension(s)

Discuss results with
the PO; identify and
remove source of

contamination




Discuss results with
thePO





Discuss results with
thePO



1 Blank = Laboratory blanks were extracted at time zero and procedural blanks were placed in the test chamber and extracted at the same time as
             the samples exposed to disinfectant.
                                                      12

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4.2  Equipment Calibration
All equipment (e.g., pipettes, incubators, BSCs) and  monitoring devices (e.g., thermometer,
hygrometer) used at the time of evaluation were verified as being either certified, calibrated, or
validated.
4.3  QC Results
QC efforts conducted during decontaminant testing included positive control samples (inoculated,
not decontaminated), procedural blanks (not inoculated, decontaminated), laboratory blank (not
inoculated, not decontaminated),  and inoculation control samples (analysis  of the stock spore
suspension).
All positive control results were within the target recovery range of 1 to 150 % of the inoculated
spores, and all procedural and laboratory blanks met the criterion of no observed CPU for all three
organisms.
Inoculation control samples were taken from the spore suspension on the day of testing and serially
diluted, plated, and counted to establish the spore density used to inoculate the samples. The spore
density levels met the QA target criterion of 2 x  108 CFU/mL (±1 log) for all tests.
 4.4 Audits
       4.4.1   Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these experiments. Table 4.3 summarizes the performance evaluation audits that were performed.
No performance evaluation audits were performed to confirm the concentration of B. anthracis, B.
atrophaeus, or C. difficile spores. Unlike chemical analytes, commercially available quantitative
standards do  not  exist for these organisms.  The control samples  and  blanks support the spore
measurements.
Table 4.3 Performance
Measurement
Volume of liquid from
micropipettes
Time
Temperature
Relative Humidity
Balance
Evaluation Audits
Audit
Procedure
Gravimetric evaluation
Compared to independent clock
Compared to independent calibrated
thermometer
Compare to independent calibrated
hygrometer
Compared to independent calibrated
weight sets

Allowable
Tolerance
± 10 %
± two sec/h
±2°C
± 10 %
±0.5g

Actual
Tolerance
± 0.57 %
0 sec/h
±0.36°C
±2%
±0.03g
                                           13

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       4,4,2   Technical Systems Audit
Observations and findings from the technical systems audit were documented and submitted to the
laboratory staff lead. These audits were conducted to ensure that the tests were being conducted in
accordance with the test/QA plan and QMP. As part of the audit, test procedures were compared
to those specified in the test/QA plan and data acquisition and handling procedures were reviewed.
None of the findings of the audits required corrective action.
       4.4.3   Data Quality Audit
All of the data acquired during the evaluation were audited.  The data were traced from the initial
acquisition, through reduction and statistical analysis, to final reporting, to ensure the integrity of
the reported results. All calculations performed on the data undergoing the audit were checked.
                                            14

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   5.0   Results and Performance of Sporicidal Fumigants and Disinfectants
5.1  Spore QA/QC
To determine the quality of spore working stock preparations prior to testing, aliquots of spores
were heat-treated at 65 °C for 30 min and enumerated before and after treatment. The CFU/mL in
control and treated samples were  comparable (<20 % difference; <0.1 log SD); Table 5.1). The
microscopic analysis of spores showed >95 % phase-bright spores and high titer (~109/mL). The
working stocks of spores with an  approximate titer of 2 x 108/mL were prepared by appropriate
dilution with 0.01 % Tween® 80.
                        Table 5.1 QA/QC of Three Spore Types
Sample ID
B. anthracis Ames
B. atrophaeus
C. difficile
Control51
8.89
8.93
9.63
Heat-Shock13
8.88
8.75
9.52
Difference (%)c
1
15
20
             a. Values represent log (CPU) averages based on two replicate measurements.
             b. Spore stocks were exposed to 65 °C for 30 min and then were enumerated.
             c. These were computed from total CPU from control and heat-treated samples.
                 Phase 1 (B. anthracis Ames and B. atrophaeus)

5.2  Spore Recovery
Prior to decontamination testing, spore recovery was determined for glass and pinewood coupons.
The spores were inoculated as an aliquot of 50 jiL deposited in droplets of seven jiL spread over
a 2 x 5-cm surface area. The spores were dried overnight at room temperature in a Class II biosafety
cabinet. The dried inoculated coupons were transferred into tubes containing 20 mL of 0.01 %
Tween® 80 and vortexed for two min. The spores were enumerated by dilution plating on TSA
plates. Figure 5.1 summarizes the spore recovery results. While >7 log spores were recovered from
glass coupons, the spore recovery from pinewood coupons was <7 logs. In general, the spore
recovery of B.  atrophaeus (60 % from glass and 10  % from pinewood) was  less than the spore
recovery of B. anthracis Ames (>90 % from glass and 50 % from pinewood). However, recoveries
from all  materials exceeded 6 logs, allowing  demonstration of up to 6 LR in decontaminant
efficacy.
                                          15

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                                     A.
Spores Inoculated vs Recovered from Glass and Pinewood (Phase 1)



5*
M 4
0)
° 3-










1

T












I







T







T

1









	


Titer Glass Pinewood Titer Glass Pinewood
B. anthracisAmes B. atrophaeus
Spore Types and Positive Controls
                                     B
100

 90

 80

 70
        60
        50

        40

        30

        20

        10

         0
                         Spore Recovery from Glass and Pinewood
                                                  . atrophaeus
                                                  . anthracisAmes
                        Glass
                                                     Pinewood
                                    Coupon Types
Figure 5.1. Spore recovery from glass and pinewood coupons during Phase 1.
                   A. Log spores; and B. Percent Recovery
                                     16

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5.3  Sporicidal Efficacy
       5.3,1   pH-Amended Bleach
The bleach efficacy results are summarized in Figure 5.2. On glass coupons, with the exception of
the survival of a small number of B. atrophaeus spores at early time points, both spore types were
completely inactivated within 10-20 min of exposure to  6000 ppm of pH-amended bleach. In
contrast, 
-------
       5.3.3   CD Gas
Efficacy results for the CD tests  are  summarized  in Figure 5.4.  On  glass, complete kill was
achieved for both spore types within 30 min of exposure to CD gas. In contrast, on pinewood ~2-
logs of B. atrophaeus spores were recovered after two h of exposure to 3000-ppmv CD gas, while
no viable spores of B. anthracis Ames were recovered. Higher variability under these moderately
effective (fractional kill) conditions was evident. From these data, the two spore types demonstrate
similar sensitivities (or resistance) to CD gas, with B. atrophaeus being slightly more resistant than
B. anthracis.
                        Sporicidal Efficacy of VHP @ 150 ppm Concentration agamst Bacillus
                                 atrophaeus and B. anthracis fames Spores
               _
               O
               O)  3
               O
                                                         DB. atrophaeus Glass
                                                         QB. atrophaeus Pinewood
                                                         •B. anthracis Ames Glass
                                                         •B. anthracis Ames Pinewood
                        70=0         T1=75       72 = 150       73 = 300       74 = 450
                            CT Values (Derived with 30,60,120, and 180 min Exposure Times)
                           Figure 5.3. Efficacy of VHP (150 ppmv)
                                               18

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                       Sporicidal Efficacy of CD Gas @ 3000 ppm Concentration againstBacillus
                                 atrophaeusand B. anthracis (AMES) Spores
              _
             o
             O)   3
             O
                                  DB.atrophaeus Glass
                                                    DB.atrophaeusPinewood
                       T0 = 0       71=1500      72 = 3000       73 = 6000       74 = 7500
                           CT Values (Derived with 30,60,120, and 150 min Exposure Times)
                          Figure 5.4. Efficacy of CD (3000 ppmv)

       5.3.4  Overall Results (Phase 1)
Numerous conditions can affect decontamination efficacy. During the current tests, an inoculation
density of 1 logs per 10 square centimeters (cm2) (8-9 logs/square foot [ft2]) is  a  significant
challenge. At this inoculation density, spore layering is likely, resulting in challenges to sporicide
penetration  and limits to mass transfer rates. While prediction of the spore concentration on
surfaces expected to be  encountered following an actual release is impossible, laboratory tests
utilizing a coupon inoculation procedure with a lower spore titer may have produced differing
results. Nonetheless, the  chosen inoculum titer and coupon size were selected to maximize ease of
testing, ability to replicate experimental runs,  while maintaining the 6 log dynamic range of
possible efficacy results.  Further, slight differences in the preparatory conditions of the two strains
may have introduced unintended variability  or  differences  in  chemical  resistance.   Spore
preparation  purity may  impact spore  resistance to chemicals, yet these effects  remain largely
unknown. Similar to results of previous studies (Rastogi et al., 2009, 1010; Tomasino et al., 2010),
high variability in decontamination efficacy was observed at mid efficacy (fractional kill) levels.
For Phase 1, the trends in efficacy data for B. citrophaeus spores and B. anthracis Ames spores are
similar in their response to the three tested technologies. The data appear to support the conclusions
that the two spore types  are  similar with respect to resistance to pH-amended bleach, VHP, and
CD gas  exposure. The  data therefore  support  the use of  B.  atrophaeus as a surrogate for B.
anthracis Ames in decontamination studies.
Future tests may consider a broader set of surfaces with numerous inoculation densities (higher
and/or lower). The tests  conducted here were performed with suspension deposition; additional
tests with dry spore deposition may also be of interest for future studies.
                                             19

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           Phase 2 (B. anthracis Ames, B. atrophaeus and C. difficile)

5.4  Spore Recovery
In Phase 2, the work was extended to include spores of C. difficile, in addition to the two Bacillus
species used in Phase 1. The results are summarized in Figure 5.5 for both coupon types and the
three spore types. As seen in Figure  5.5,  spore recovery values were > 6 logs,  and the percent
recoveries were 20-65 % from glass  for all three spore types.  Spore recoveries from pinewood
were >30 % for Ames and <10 % for the other spore types. The overall trend appeared to support
the conclusion that compared to the two Bacillus spore types, C. difficile spore  recoveries were
significantly lower (Figure 5.5-A). In the case of the C. difficile tests, ten min  sonication, in
addition to two min vortexing, was included to improve spore recoveries.
5.5  Sporicidal Efficacy
The sporicidal efficacy was determined for pH-amended bleach, CD gas, and VHP as in Phase 1.
To avoid cross-contamination, C.  difficile work was conducted in a separate dedicated Class II
biosafety cabinet. The sporicidal efficacy results are summarized in the following sections.
       5,5,1   pH-Amended Bleach
Spores were exposed for 2, 5, 10,  and 20 min, followed by spore extraction from the control set
and test set. Figures 5.6  and  5.7  summarize  the efficacy results on glass  and pinewood,
respectively. Results were  similar to the results observed during Phase 1, as all spore types
demonstrated similar and rapid kill kinetics within two min of exposure, and near complete-kill by
5 through 20 min of exposure (Figure 5.6). Figure 5.7 summarizes the kinetics  of spore kill on
pinewood. As seen in Phase 1, once again the two Bacillus spore types and C. difficile spores were
comparably resistant to bleach. Only 2 to 3 LR was observed for all three spore  types even after
20 min exposure to bleach.
                                           20

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100

 90

 80

 70

 60

 50

 40

 30

 20

 10

 0
                           A.
                 Recovery of Spores from Glass and Pinewood
                        Glass DPinewood
       B. anthracisAmes
                            C. difficile
                            Spore Types
                                              B. atrophaeus
                           B
9
8
7
6
£ 5
O
§ 4
3
2
1
0
Spore Recovery of Three Spore Types from Glass and Pinewood







	
T 1


T

a B, anthracisAmes a c. difficile a B, atrophaeus












I


T T















—
TITER GLASS PINEWOOD
COUPON TYPES
Figure 5.5. Spore Recovery from Glass and Pinewood
        A. Percent recoveries and B. Log (CPU)
                           21

-------
Average Log Reduction of Three Spore Types on Glass Effected by
6000-ppm Bleach

7 -
6.
Z c
0 5
LOGREDUCT
J CO J^

0 -







	



T1


OB. anthracisAmes o C. difficile OB. atrophaem








— i —

	

T2
TIME






























T3 T4
(2, 5, 10, 20 Minutes)

  Figure 5.6. Efficacy of pH-Amended Bleach on Three Spore Types on Glass Coupons
         o


         g  4
         UJ
         a:

         O
         O  3
                     Average Log Reduction of Three Spore Types on Pinewood Effected by

                                       6000-ppm Bleach
                           nB. anthracisAmes n C. difficile  nB. atrophaeus
                     T1
                                     T2             T3



                                     TIME (2, 5,10, 20 Minutes)
                                                                    T4
Figure 5.7. Efficacy of pH-Amended Bleach on Three Spore Types on Pinewood Coupons
                                          22

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       5.5.2   CD Gas
 On glass coupons (Figure 5.8), the kill kinetics for all three spore types were comparable, and >6-
LR was observed within 30 min of CD gas exposure. This result is similar to the results observed
in Phase 1 for CD gas and the two Bacillus spore types. The spore kill kinetics on pinewood (Figure
5.9) were slower (3-4 LR up to two hours of exposure). On pinewood, even though the LR values
were slightly lower (1.0) after 2  and 2.5  hours for Ames spores, overall  the data support the
contention that the three spore types are comparably resistant (or  sensitive) to CD gas.
Average Log Reduction of Three Spore Types on Glass Effected by
3000-ppmCD


Z c
0 5
1-
o
3 A
Q 4
111
o:
8 o
0 3
1 -












T1


























o B. anthracis Ames a c. difficile o B. atrophaeus






T2
TIM
E(0.5



,1,2,2
5 Ho
T3
urs)









T4










          Figure 5.8. Efficacy of CD Gas on Three Spore Types on Glass Coupons
                         Average Log Reduction of Three Spore Types on Pinewood Effected by
                                          3000-ppmCD
                O
                §  4
                m
                o:
                o


n B. anthracis Ames o c. difficile o B. atrophaeus








































—
                          T1
                                       T2            T3
                                       TIME (0.5,1,2, 2.5 Hours)
                                                                 T4
       Figure 5.9. Efficacy of CD Gas on Three Spore Types on Pinewood Coupons
                                           23

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       5.5.3   VHP
 The sporicidal efficacy in terms of log reduction on glass coupons is summarized in Figure 5.10.
As seen in the figure, >6 LR was observed for both Bacillus spore types after 2 h of exposure to
VHP. At fractional kill levels, i.e., 30 min exposure to VHP, B. atrophaeus spores appear to be
slightly more resistant to VHP than B.  anthracis Ames spores. As for C. difficile spores, even
though 4 LR was observed within 30 min of exposure, no further kill was observed on prolonged
exposure up to a period of 3 h. Dosages required for complete kill of the two Bacillus spores appear
to be inadequate to achieve the same level of kill for C. difficile spores.
On pinewood coupons (Figure 5.11), only 3 to 4 LR was observed for the two Bacillus species
spore types, even after 3 h of exposure to VHP. The B. atrophaeus spores appear to be more
resistant than the Ames spores  at fractional kill  level, i.e., 30 min of exposure to  VHP. VHP
efficacy appears to be equally or slightly more effective against spores of C. difficile, i.e., 5 LR
after 3 h of exposure.
Average Log Reduction of Three Spore Types on Glass Effected by
150-ppmVHP


g
i-
O "5
u 3
Q
LU
a: .
o 4
o
2 -











OB. anthracis Ames nc. difficile &B. atrophaeus









	


























T1 T2 T3
TIME (0.5, 1,2, 3 Hours)










T4

          Figure 5.10. Efficacy of VHP on Three Spore Types on Glass Coupons

5.6  Summary of Phase 1 and Phase 2 Results

The sporicidal efficacy testing for three technologies, pH-amended bleach, CD gas, and VHP, was
performed in two phases. Phase 1 was conducted in the summer of 2012 with two Bacillus spore
types, and Phase 2 was conducted in the summer/fall of 2013 with two Bacillus spore types plus
spores of C. difficile. For each technology, four dosages derived from a fixed concentration with
four  exposure times were selected to observe fractional kill and high  kill. High variability at
fractional  kill exposures  resulting from high inoculation  density, spore layering, and  poor
penetration of fumigant/disinfectant was observed, but these results are consistent with other
studies.
                                           24

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For pH-amended bleach, in both phases, the disinfectant was very effective on glass surfaces. In
contrast, this technology (in both phases) was found to be less effective on pinewood. The efficacy
data suggest the similarity  in resistance of the two spore types, B.  anthracis Ames and B.
atrophaeus. Therefore, for bleach-based decontaminations, B. atrophaeus spores could serve as an
appropriate surrogate for B. anthracis Ames. C. difficile demonstrated slightly higher resistance to
pH-amended bleach on  glass  surfaces. These data  suggest  that  hypochlorous  acid-based
technologies that have previously demonstrated effectiveness against C. difficile may have even
greater efficacy against B. anthracis for hard, nonporous materials.
                        Average Log Reduction of Three Spore Types on Pinewood Effected by
                                           150-ppmVHP
                              n B. anthracis Ames n C. difficile  n B. atrophaeus
                        T1
                                       T2             T3

                                       TIME (0.5,1,2, 3 Hours)
                                                                     T4
         Figure 5.11. Efficacy of VHP on Three Spore Types on Pinewood Coupons

The efficacy of CD gas in both phases also demonstrates comparable effectiveness against spores
of B. anthracis Ames and B. atrophaeus on glass coupons. Within 30 min of exposure, the LR
values of >6 logs were observed. Overall, spore kill was more difficult on pinewood surfaces, but
comparable sensitivity of the three spore types was evident in this study. Again, the data support
the use of B. atrophaeus as a surrogate for B. anthracis in decontamination studies using CD, and
previously  demonstrated  CD technologies  for C. difficile may be promising for B. anthracis kill.
For VHP, variability in the efficacy at the fractional level was evident in both phases. On glass
coupons, high dosages resulted in >6 LR for spores of both Bacillus species, but at fractional levels,
B. atrophaeus appeared to be just as resistant (Phase 1) or slightly more resistant (Phase 2) than B.
anthracis Ames. Interestingly, high dosages of VHP against C.  difficile spores were only partially
efficacious (LR ~5 for both materials). On  pinewood coupons, high dosages in both phases appear
to be partially efficacious, LR of only 3 to 4 for both Bacillus species. Overall, both Bacillus spore
types demonstrated similar sensitivities to  VHP, in both phases.
                                            25

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                         6.0   Discussion and Conclusions

The main objective of the current study was to assess relative resistance (or sensitivity) of spores
of B. atrophaeus (common surrogate for B. anthracis) and the spores of pathogenic B. anthracis
Ames to three sporicidal technologies.  Spores of the two Bacillus species were exposed to four
dosages  of pH-amended bleach, CD,  and VHP.  These technologies have commonly been
employed in a number of previous  cleanup  efforts  and have been evaluated in recent building
cleanup demonstrations (US-EPA BOTE,  2013). The three technologies have been found to be
very effective against both Bacillus species spore types on various surfaces, although numerous
conditions  can affect their efficacy.  The choice of a given technology for decontamination must
therefore consider the type of material surface being  decontaminated.


 6.1 Comparing Efficacy against B. anthracis and B. atrophaeus
Are the two spore types significantly different in their sensitivity to decontamination technologies?
Difference in sensitivity to decontamination technologies has been the premise for some critics to
challenge studies using surrogate B. atrophaeus spores.  Presence of  an exosporium on Ames
spores and their hydrophobicity could render such spores more resistant than the surrogate spores.
If the surrogate spores were more sensitive than Ames spores, one would predict a faster kill rate
and a significantly lower dosage required  to achieve a complete kill of the surrogate organism,
Bacillus  atrophaeus.  The present  study  was designed to address and investigate just these
predictions. The results  show a comparable  kill rate and similar dosages required to achieve
complete kill by all three decontamination technologies. The data therefore strongly favor the
conclusion that the two spore types are comparable in their resistance to the three decontamination
technologies evaluated.  Taken together, the data support the use of B. atrophaeus spores as a
surrogate for B. anthracis Ames in decontamination  studies with these three technologies.
6.2  Comparing Efficacy against Spores of Bacillus species and C difficile
How different are C. difficile spores in their sensitivity to sporicidal chemicals relative to the other
two Bacillus spore types? In general, the spores of C. difficile appear to adhere to the glass surface
more tightly than the other two spore types. The spores of C. difficile also appear to be more
resistant to pH-amended bleach at fractional kill levels. Complete kill even after a 20-min exposure
time was not observed. These data suggest that hypochlorous acid-based technologies that have
previously demonstrated effectiveness against C. difficile may have even greater efficacy against
B. anthracis for hard,  nonporous  materials.    Spores  of C. difficile appear to  demonstrate
comparable resistance (or sensitivity) to the other two sporicidal technologies, i.e., CD gas and
VHP.  Considering the data collectively, technologies that have previously shown high efficacy
against C. difficile spores may perform well against Bacillus spores.  These data may allow
targeted testing of other sporicidal decontaminants against Bacillus anthracis spores, based upon
the results of C. difficile tests reported in hospital disinfection-oriented peer-reviewed journals.
                                           26

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References
   ASTM E2839-11. 2011. Standard test method for production of Clostridium difficile spores for
         use in efficacy evaluation of antimicrobials agents, pp 1-5. ASTM International, West
         Conshohocken, PA.

   Beuchat, L.R., C.A. Pettigrew, M.E. Tremblay, BJ. Roselle, and AJ. Scouten. 2004. Lethality
         of chlorine,  chlorine dioxide,  and  a commercial fruit and vegetable sanitizer to
         vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. J.
         FoodProt. 67: 1702-1708.

   Canter, D.A. 2005. Remediating anthrax-contaminated sites: learning from the past to protect
         the future. Chem. Health Safety. 12: 13-19.

   Canter, D.A., D. Gunning, P. Rodgers, L. O'Connor, C. Traunero, and CJ. Kempter. 2005.
         Remediation of Bacillus anthracis contamination in the U.S. Department of Justice mail
         facility. Biosecur. Bioterror. 3; 119-127.

   Cloud, J. and C.P. Kelly. 2007. Update on Clostridium difficile-associated disease. Curr. Opin.
         Gasteroenterol. 23: 4-9.

   Fukuyama, M.Y., H. Tan, W.B. Wheeler, and C.I. Wei.  1986. Reactions of aqueous chlorine
         and chlorine  dioxide with model food compounds. Environ. Health Perspect.  69: 267-
         274.

   McFarland, L.V., H.W.  Beneda, I.E. Clarridge, and GJ. Rungi. 2007. Implications  of the
         changing face of Clostridium difficile disease for health-care practitioners. Am. J. Infect.
         Control. 35:237-53.

   Rastogi, V.K., L. Wallace, L.S.  Smith, S.P. Ryan, and B. Martin. 2009. Quantitative method to
         determine sporicidal decontamination of building surfaces by gaseous fumigants, and
         issues related to lab oratory-scale studies. Appl. Environ. Microbiol. 75: 3688-3694.

   Rastogi, V.K., SP Ryan, L Wallace, L.S. Smith, S.S. Shah, and G.B. Martin. 2010. Systematic
         evaluation of chlorine dioxide efficacy in decontamination of building interior surfaces
         contaminated with anthrax spores. Appl. Environ. Microbiol. 76: 3343-3351.

   Tomasino, S.F., Rastogi, V.K., Wallace, L., Smith, L.S., Hamilton, M.A., andR.M. Pines. 2010.
         Use  of alternative carrier materials in AOAC  Official Method 2008.05, Efficacy of
         liquid sporicides against  spores  of  Bacillus subtilis on a hard, nonporous surface,
         quantitative three-step method. JAOAC Int. 93: 259-276.

   U.S.  EPA. 2011. Systematic investigation of liquid and fumigant decontamination efficacy
         against biological agents deposited on test coupons of common indoor materials. U.S.
         EPA 600/R-l 1/076.
                                           27

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U.S. EPA. 2013. Bio-response Operational Testing and Evaluation (BOTE) Project - Phase 1:
       Decontamination assessment. U.S. EPA, EPA/600/R-13/168.

U.S. EPA Protocols. 2014. http://www.epa.gov/pesticides/methods/atmpa2z.htm

Wood, J.P., Calfee, M.W., Clayton, M., Griffm-Gatchalian, N., and A. Touati 2011. Optimizing
       acidified bleach solutions to improve sporicidal efficacy on building materials.  Lett.
       Appl. Microbiol., 53: 668-672.
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