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EPA/13/R-13/220
December 2013
Evaluation of Ethylene Oxide for the Inactivation of
Bacillus anthracis
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,
directed and managed this work through Contract Number EP-C-10-001 with Battelle. 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:
Shannon Serre, 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
919-541-3817
in
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Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
United States Environmental Protection Agency (EPA)
Jayson Griffin
Leroy Mickelsen
Eletha Brady-Roberts
Ramona Sherman
Joe Wood
Battelle Memorial Institute
IV
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Executive Summary
The U.S. Environmental Protection Agency, Office of Research and Development's Homeland
Security Research Program 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 was to determine the decontamination efficacy of ethylene oxide (EtO) in
inactivating Bacillus anthracis (causative agent for anthrax) spores on six material types
typically found in museums. The objective of this study was to provide an understanding of the
performance of EtO to guide its use and implementation in HS applications. In the assessment of
options for decontamination following the release of B. anthracis., it is important to know
whether and to what extent such factors can impact the decontamination efficacy.
This investigation focused on decontamination of six types of materials, including sensitive
materials such as those that may typically be found in museums. The materials tested include:
glass, bare pine wood, painted canvas, archival paper, silk fabric and carbon steel.
Decontamination efficacy tests were conducted with spores of B. anthracis or B. atrophaeus, the
latter organism included to assess its potential as a surrogate for future studies related to B.
anthracis and EtO. Additionally, the difference between EtO efficacy against B. atrophaeus
spores inoculated onto materials using a liquid suspension or dry method was assessed.
Decontamination efficacy was quantified in terms of log reduction (LR), based on the difference
in the number of bacterial spores recovered from the positive control coupons and test coupons.
Tests were conducted with varying temperatures, relative humidity (RH) levels, concentrations
of EtO, and contact times to assess the effect of these operational parameters on decontamination
efficacy. The goal of this research was to evaluate the efficacy of EtO on a variety of materials.
A LR of greater than 6 is considered to be effective ^; however, results where complete spore
inactivation was achieved is also reported. Inactivation or sterilization is the complete
elimination of microbial viability (-2\
Summary of Results
Various combinations of temperatures ranging from 30 °C to 50 °C, RH ranging from 30% to
75%, EtO concentrations ranging from 150 mg/L to 600 mg/L and contact times ranging from 45
minutes to 360 minutes were evaluated. The EtO gas decontamination technology provided a
>six log reduction of B. anthracis on all six coupon material types under the following
conditions:
• 50 °C, 50% RH, >600 mg/L EtO for > 180 minutes
• 50 °C, 60% RH, >300 mg/L EtO for >180 minutes
• 50 °C, 75% RH, >150 mg/L EtO for >180 minutes
• 37 °C, 75% RH, >300 mg/L EtO for >90 minutes
Although slightly less effective against B. atrophaeus, complete inactivation was achieved on all
six coupon types under the following conditions:
• 50 °C, 75% RH, >150 mg/L EtO for >180 minutes
• 37 °C, 75% RH, >300 mg/L EtO for >90 minutes
In general, B. anthracis was more difficult to inactivate on archival paper than B. atrophaeus.
The lowest LRs were observed on this coupon type. B. anthracis was easier to inactivate on glass
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and bare pine wood as these coupon types generally had higher LRs than the other four coupon
types. In contrast, B. atrophaeus was easier to inactivate on archival paper and bare pine wood.
Painted canvas and carbon steel were the most resistant to B. atrophaeus inactivation using EtO.
EtO fumigation against B. atrophaeus was evaluated using both liquid and dry coupon inoculum
methods. Only one test was completed for directly comparing the two methods at 50 °C, 50%
RH, and 150 mg/L EtO for a 45-minute contact time. Using these parameters, the spores
inoculated as a dried formulation were slightly easier, although not significantly, to inactivate
than spores applied to archival paper via liquid suspension.
EtO is an effective decontaminant against B. anthracis and B. atrophaeus under optimal
combinations of concentration, contact time, temperature, and RH.
VI
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Contents
Acknowledgments iv
Executive Summary v
Abbreviations/Acronyms ix
1.0 Introduction 1
2.0 Technology Description and Test Matrices 2
2.1 Technology Description 2
2.2 Test Matrix 2
3.0 Summary of Test Procedures 4
3.1 Biological Agent 4
3.2 Test Materials 4
3.3 Preparation of Coupons 5
3.4 EtO Fumigation Test and Control Chambers and Procedures 7
3.5 Coupon Extraction and Biological Agent Quantification 8
3.6 Decontamination Efficacy 8
3.7 Surface Damage 10
4.0 Quality Assurance/Quality Control 11
4.1 Audits 12
4.2 Test/Quality Assurance Plan Deviations 12
4.3 QA/QC Reporting 12
4.4 Data Review 12
5.0 Results 13
5.1 EtO Results 13
5.2 Surface Damage to Materials 25
6.0 Discussions and Conclusions 26
6.1 Effect of Temperature and Relative Humidity on Efficacy 26
6.2 Effect of Material Type on Efficacy 27
6.3 Effect of Inoculation Method on Efficacy 27
7.0 References 28
vn
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Figures
Figure 3-1 Coupon types from left to right: glass, carbon steel, silk fabric, archival
paper, painted canvas, and bare pine wood 5
Figure 3-2 Liquid inoculation of coupon using a micropipette 6
Figure 3-3 Schematic of EtO decontamination test chamber 7
Figure 5-1 Summary of decontamination efficacies for EtO fumigation testing on
glass 18
Figure 5-2 Summary of decontamination efficacies for EtO fumigation testing on
bare pine wood 19
Figure 5-3 Summary of decontamination efficacies for EtO fumigation testing on
painted canvas 20
Figure 5-4 Summary of decontamination efficacies for EtO fumigation testing on
archival paper 21
Figure 5-5 Summary of decontamination efficacies for EtO fumigation testing on
silk fabric 22
Figure 5-6 Summary of decontamination efficacies for EtO fumigation testing on
carbon steel 23
Figure 5-7 Oxidation on carbon steel coupons inoculated with B. atrophaeus 25
Tables
Table 2-1 EtO Test Matrix 3
Table 3-1 Test Materials 5
Table 4-1 Performance Evaluation Audits 11
Table 5-1 EtO Efficacy Against Bacillus anthracis Spores 14
Table 5-2 EtO Efficacy Against Bacillus atrophaeus Spores 16
Table 5-3 EtO Efficacy Against Bacillus atrophaeus Spores on Archival Paper 24
Vlll
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Abbreviations/Acronyms
ADC
ATCC
BBRC
BSC
CPU
CI
cm
°C
DNA
EPA
EtO
HC1
hr
kGy
L
LAL
LR
MDI
min
mg
mL
HL
NHSRC
ORD
PBSTx
PCR
QA
QC
RH
rpm
SD
SE
SEC
SFW
STS
TQAP
ISA
WA
aerosol deposition chamber
American Type Culture Collection
Battelle Biomedical Research Center
biological safety cabinet
colony-forming unit(s)
confidence interval
centimeter(s)
degree(s) Celsius
deoxyribonucleic acid
U.S. Environmental Protection Agency
ethylene oxide
hydrochloric acid
hour(s)
kiloGray
liter(s)
Limulus amebocyte lycate
log reduction
metered dose inhaler
minute(s)
milligram(s)
milliliter(s)
microliter(s)
National Homeland Security Research Center
EPA Office of Research and Development
phosphate-buffered saline + 0.1% Triton X-100
polymerase chain reaction
quality assurance
quality control
relative humidity
revolution(s) per minute
standard deviation
standard error
Sensor Electronics Corporation
sterile filtered water (cell-culture grade)
sodium thiosulfate
test/quality assurance plan
technical systems audit(s)
work assignment
IX
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1.0 Introduction
The U.S. Environmental Protection Agency (EPA), Office of Research and Development'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
agents. 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 or biological contaminants in buildings
or water systems, contain these contaminants, decontaminate buildings or water systems, and
facilitate the disposal of material resulting from cleanups.
EPA's HSRP evaluates the performance of innovative homeland security technologies 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 quality assurance (QA) protocols to ensure that data of known and high
quality are generated and that results are defensible. This program provides high-quality
information that is useful to decision makers in purchasing or applying the tested technologies.
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.
In this work, the efficacy of ethylene oxide (EtO) against Bacillus anthracis Ames and B.
atrophaeus spores applied to materials, including sensitive materials such as those that may
typically be found in museums or that could be sensitive to other types of decontaminants. The
materials included in this testing were: glass, bare pine wood, painted canvas, archival paper,
silk and carbon steel. Decontamination efficacy was determined based on the log reduction in
viable spores recovered from the inoculated samples (with and without exposure to ethylene
oxide). The goal of this research was to evaluate the efficacy of EtO on a variety of materials. A
LR of greater than 6 is considered to be effective (USEPA, 2010).
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2.0 Technology Description and Test Matrices
2.1 Technology Description
Ethylene oxide (EtO; Cat# 387614, Sigma Aldrich, St. Louis, MO, USA) is an organic
compound with the formula C2H4O. EtO is flammable at room temperature and is a carcinogenic,
mutagenic, irritant, and anesthetic gas with a misleadingly pleasant aroma. EtO is used to
sterilize many things except food, drugs and liquids. Because the gas leaves no residue and does
not damage the materials it contacts, EtO is widely used as a disinfectant and sterilant in
hospitals and the medical equipment industry to replace steam in the sterilization of heat-
sensitive tools such as disposable plastic syringes. This gas is a candidate that may possibly be
used to decontaminate and sterilize sensitive materials that might be found in museums such as
canvas paintings and fabrics in the event of a biological agent release.
2.2 Test Matrix
The test matrix for the EtO fumigation tests is shown in Table 2-1. The target temperature of
50 °C and target RH of 50% were based off standard EtO sterilization cycles developed by
Andersen Products (Haw River, NC, USA)*-4-*. B. anthracis and B. atrophaeus were tested on all
six material types for Tests 1 through 19. Test 20 was conducted using B. atrophaeus inoculated
onto archival paper only to compare the efficacy for coupons inoculated with dry versus liquid
inoculation methods.
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Table 2-1. EtO Test Matrix
Test
Number
1
2
3
4
5
6
7
8*
9
10
11
12
13
14
15
16
17
18
19
20f
Materials
Glass
Bare Pine Wood
Painted Canvas
Silk Fabric
Carbon Steel
Archival Paper
Target
Temperature
(°C; ±2 °C)
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
37
37
37
50
Target RH
(%; ±10%)
30
50
50
50
50
50
50
50
50
50
60
60
75
75
75
75
75
75
75
50
Target EtO
Concentration
(mg/L; ±10%)
300
150
150
150
300
300
300
300
300
600
300
600
150
300
300
300
300
300
300
150
Contact
Time (min)
180
45
90
180
45
90
180
180
360
180
180
360
180
45
90
180
45
90
180
45
Sample
Replicates
T=5
-5
B^-1
Bc=l
T=6
C=6
BT=1
Bc=l
T = Test Coupon
C = Positive Control Coupon
BT = Procedural Blank Coupon
Bc = Laboratory Blank Coupon
*Samples subjected to a 24 hour (hr) pre-humidification cycle
Samples inoculated withB. atrophaeus only; applied as a dry formulation as well as a liquid suspension
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3.0 Summary of Test Procedures
This section provides an overview of the procedures that were used for the bench-scale
evaluation of EtO to inactivate B. anthracis and B. atrophaeus spores on six different materials.
Testing was performed in accordance with the EPA approved Test/QA Plan for the Evaluation of
Ethylene Oxide for the Inactivation of Bacillus anthracis and associated amendments. The
test/QA plan provides additional procedural details that are not included in this report(3).
3.1 Biological Agent
The B. anthracis spores used for this testing were prepared from a qualified stock of the Ames
strain at the Battelle Biomedical Research Center (BBRC, Lot B21, West Jefferson, OH). All
spore lots were subject to a stringent characterization and qualification process required by
Battelle's standard operating procedure for spore production. Specifically, all spore lots were
characterized prior to use by observation of colony morphology, direct microscopic observation
of spore morphology and size and determination of percent refractivity and percent
encapsulation. In addition, the number of viable spores was determined by colony count and
expressed as colony forming units per milliliter (CFU/mL). Theoretically, once plated onto
bacterial growth media, each viable spore germinates and yields one CPU. Variations in the
expected colony phenotypes were recorded. Endotoxin concentration of each spore preparation
was determined by the Limulus amebocyte lysate (LAL) assay to assess whether contamination
from gram-negative bacteria occurred during the propagation and purification process of the
spores.
The B. atrophaeus spores (Lot 19076-03268) were obtained in powder form from Dugway
Proving Ground. No further activities were performed to verify the identity of the organism.
The B. anthracis stock spore suspensions were prepared in sterile filtered water (SFW) at an
approximate concentration of 1 x 109 CFU/mL and stored under refrigeration at 2 to 8 degrees
Celsius (°C). Similarly, the B. atrophaeus stock spore suspensions were prepared in sterile
phosphate-buffered saline containing 0.1% Triton X-100 surfactant (PBSTx; Sigma, St. Louis,
MO, USA) at the same concentration and stored at 2-8 °C.
3.2 Test Materials
Decontamination testing was conducted on six materials (glass, bare pine wood, painted canvas,
archival paper, silk fabric and carbon steel). Information on the materials used for testing is
presented in Table 3-1 and a picture of each is shown in Figure 3-1. Material coupons were cut to
uniform length and width from a larger piece of stock material. Materials were prepared for
testing either by sterilization via gamma irradiation at -40 kilogray (kGy; STERIS Isomedix
Services, Libertyville, IL, USA) or by autoclaving at 121 °C for 15 minutes. Gamma irradiated
material coupons were sealed in 6 mil Uline Poly Tubing (Uline, Chicago, IL, USA) and
autoclaved coupons were sealed in sterilization pouches (Fisher, Pittsburg, PA, USA) to preserve
sterility until the coupons were ready for use.
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Table 3-1. Test Materials
Material
Glass
Bare Pine
Wood
Painted
Canvas
Archival
Paper
Silk Fabric
Carbon
Steel
Lot, Batch, ASTM No.,
or Observation
C1036
Generic Molding
NA
10146,
Acquerello Portofmo Paper
Silk Dupioni,
Pewter Gray
ASTMA1008,
Grade CS, Type B
Manufacturer/
Supplier Name
Location
Brooks Brothers
Columbus, OH
Lowes
Columbus, OH
EPA
Dick Blick
Galesburg, IL
Joann Fabrics
Hudson, OH
Adept Products
West Jefferson,
OH
Approximate
Coupon Size,
width x length
1.9 centimeter
(cm)x7.5 cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
Approximate
Coupon
Thickness
0.3cm
0.5 cm
0.1 cm
0.1 cm
O.lcm
0.1 cm
Material
Preparation
Autoclave
Gamma
Irradiation
Gamma
Irradiation
Gamma
Irradiation
Gamma
Irradiation
Autoclave
NA = Not Applicable
Figure 3-1. Coupon types from left to right: glass, carbon steel, silk fabric, archival paper,
painted canvas and bare pine wood.
3.3 Preparation of Coupons
Test and positive control coupons were placed on a flat surface within a Class II biological safety
cabinet (BSC) and inoculated with approximately 1 x 108 CPU of viable B. anthracis or B.
atrophaeus spores per coupon. A 100 microliter (jiL) aliquot of a stock suspension of
approximately 1 x 109 CFU/mL was dispensed using a micropipette applied as 10 jiL droplets
across the coupon surface (see Figure 3-2). This approach provided a more uniform distribution
of spores across the coupon surface than would be obtained through a single drop of the
suspension. After inoculation, the coupons were left undisturbed overnight in a Class III BSC
(used for the containment of dry spores) to dry under ambient conditions, approximately 22 °C
and40%RH.
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Figure 3-2. Liquid inoculation of coupon using a micropipette.
The number and type of replicate coupons used for each combination of material, decontaminant,
concentration (Tests 1-19 only), and environmental condition included were:
• five test coupons (inoculated with B. anthracis or B. atrophaeus spores and exposed to
decontaminant)
• five positive controls (inoculated with B. anthracis or B. atrophaeus spores but not
exposed to decontaminant)
• one laboratory blank (inoculated only with sterile water and not exposed to the
decontaminant)
• one procedural blank (inoculated only with sterile water and exposed to the
decontaminant).
For Test 20, two inoculation methods (liquid and dry inoculation) were compared using B.
atrophaeus inoculated onto archival paper only. Archival paper was chosen as this material had
been the hardest to decontaminate and it was desirable to determine if the inoculation method
had a correlation with the LR. Six test and six positive controls were inoculated with liquid
suspension as described above. For dry inoculations, an additional six test and positive control
paper coupons were loaded into an aerosol deposition chamber (ADC) following an established
method.*^ A metered dose inhaler (MDI) of desired spore concentration was agitated using a
vortex mixer (set to high) for 30 seconds to mix the contents fully. The MDI was then inserted
into a stainless steel actuator which was connected to the ADC. The aerosol dose was
administered by depressing the MDI with one firm swift actuation and holding in the depressed
position for five seconds. The MDI actuator was decoupled from the lid, and the ADC remained
undisturbed on the coupon surface for >18 hours to allow gravitational deposition of the
particles. The coupons were inoculated at the EPA facility in Research Triangle Park, NC. The
coupons were then removed from the ADC, placed in 50 mL conical tubes and shipped to the
BBRC via common carrier. Laboratory and procedural blanks were not inoculated, but treated in
the same manner as the test or positive controls as detailed.
On the day following liquid spore inoculation, coupons intended for decontamination (including
blanks) were transferred into a test chamber and exposed to the EtO fumigant using the apparatus
and application conditions specified in Section 3.4. Control coupons were added to the control
chamber as described in Section 3.4. For Test 20, three positive controls were processed
6
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immediately to assess any potential loss of bacteria during transit from the EPA to the BBRC..
The remaining three controls were added to the control chamber and processed as described in
the following section.
3.4 EtO Fumigation Test and Control Chambers and Procedures
Figure 3-3 shows a schematic drawing of the EtO test chamber and containment system. EtO
decontamination testing was conducted inside a 23-L glass test chamber developed by Battelle.
As a means of secondary containment and laboratory personnel safety, this test chamber was
housed inside a BSC III cabinet. Once injected into the test chamber, the EtO gas was measured
continuously using an EtO Signature Process Gas Analyzer (Part No: 142-0597; Sensor
Electronics Corporation [SEC], Minneapolis, MN, USA) during the entire contact time. This
sensor was calibrated by SEC and was capable of measuring EtO gas between 0 and 2000
milligram/liter (mg/L). A low-speed fan was placed inside the test chamber to ensure a
homogeneous mixture of EtO was achieved. When required, temperature was controlled using a
heated/cooled water bath and RH was elevated using a Nafion (Permapure, Toms River, NJ,
USA) tube pervaporation system. Temperature and RH in the EtO test chamber was measured
using an HMT368 temperature and humidity probe (Vaisala, Inc, Woburn, MA, USA).
Temperature, RH and EtO concentration were controlled with a CNI-822 controller (Omega,
Stamford, CT, USA) and data were logged using the associated iLOG software.
Power Cord Omega Gas
Gas
^" "N
jr~w»n
Automated Valve ' /
S
Pressure Regulator '
n ~l
23L Test Chamber
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A 9-L Lock&Lock container (Lock&Lock, Farmers Branch, TX, USA) was used as the control
chamber. Fixed humidity point salts'-6-* were added as a slurry to a separate container placed in the
bottom of the EtO control chamber. Sodium chloride was used to control the RH at 75%,
potassium iodide for 60% RH, sodium bromide for 50% RH, and magnesium chloride was used
to control the RH at 30%. The control chamber was placed in an incubator (Thermo Scientific,
Waltham, MA, USA) for all tests and set to the appropriate temperature (i.e., 37 °C or 50 °C).
As in previous studies,(7) multiple inoculated coupons of each material were placed on a wire
rack inside the test or control chamber. Blank (i.e., not inoculated) and positive control (i.e.,
inoculated but not decontaminated) coupons were also prepared for each test material and were
utilized along with data from the test coupons (inoculated and decontaminated) to determine
decontamination efficacy. This procedure provides a highly controlled, reproducible approach to
assess sensitivity of the fumigation decontamination efficacy to temperature, RH, concentration
and contact time.
3.5 Coupon Extraction and Biological Agent Quantification
After fumigation, test coupons, positive controls, and blanks were placed in 50 mL
polypropylene conical vials containing 10 mL of sterile PBSTx. The vials were capped, placed
on their side and agitated on an orbital shaker for 15 minutes at approximately 200 revolutions
per minute (rpm) at room temperature.
Residual viable spores were determined using a dilution plating approach. Following extraction,
the extract was removed and a series of tenfold dilutions was prepared in SFW. An aliquot
(0.1 mL) of either the undiluted extract and/or each serial dilution was plated onto tryptic soy
agar in triplicate and incubated for 18-24 hours (hrs) at 35 -37 °C. Colonies were counted
manually and CFU/mL were determined by multiplying the average number of colonies per plate
by the reciprocal of the dilution. Dilution data representing the greatest number of individually
definable colonies were expressed as arithmetic mean ± standard deviation of the numbers of
CFU observed. Laboratory blanks controlled for sterility and procedural blanks controlled for
viable spores inadvertently introduced to test coupons. The blanks were spiked with an
equivalent amount of 0.1 mL of SFW. The target acceptance criterion was that extracts of
laboratory or procedural blanks were to contain no CFU.
After each decontamination test, the BSC III and the EtO test and control chambers were
thoroughly cleaned (using separate steps involving bleach, ethanol, water, then drying) following
procedures established under the BBRC Facility Safety Plan.
3.6 Decontamination Efficacy
The mean percent spore recovery from each coupon was calculated using results from positive
control coupons (inoculated, not decontaminated), by means of the following equation:
Mean % Recovery = [Mean CFUpc/CFUspike] x 100 (1)
-------
where Mean CFUpc is the mean number of CPU recovered from five replicate positive control
coupons of a single material, and CFUspike is the number of CPU spiked onto each of those
coupons. The value of CFUspike is known from enumeration of the stock spore suspension. Spore
recovery was calculated for B. anthracis or B. atrophaeus on each coupon, and the results are
included in Section 5.
The performance or efficacy of EtO was assessed by determining the number of viable
organisms remaining on each test coupon after decontamination. Those numbers were compared
to the number of viable organisms extracted from the positive control coupons.
The number of viable spores of B. anthracis or B. atrophaeus in extracts of test and positive
control coupons was determined to calculate efficacy of the decontaminant. Efficacy is defined
as the extent (as logic reduction, LR) to which viable spores extracted from test coupons after
decontamination were less numerous than the viable spores extracted from positive control
coupons. The logarithm of the CPU abundance from each coupon extract was determined, and
the mean of those logarithm values was then determined for each set of control and associated
test coupons, respectively. Efficacy of a decontaminant for a test organism/test condition on the
7th coupon material was calculated as the difference between those mean log values, i.e.:
Efficacy,= (\ogw CM/Cy)-(log10 CFUtv) (2)
where logic CFUcy refers to they individual logarithm values obtained from the positive control
coupons and logic CFUty refers to they individual logarithm values obtained from the
corresponding test coupons, and the overbar designates a mean value. In tests conducted under
this plan, there were five positive controls and five corresponding test coupons (i.e.,7 = 5) for
each coupon. Test 20 utilized six positive controls and six corresponding test coupons (i.e.,7 =
6).
In the case where no viable spores were found in any of the five test coupon extracts after
decontamination, a CPU abundance of 1 was assigned, resulting in a logic CPU of zero for that
material. This situation occurred when the decontaminant was highly effective, and no viable
spores were found on the decontaminated test coupons. In such cases, the final efficacy on that
material was reported as greater than or equal to (>) the value calculated by Equation 2.
The variances (i.e., the square of the standard deviation [SD]) of the logic CFUcy and logic
CFUtjj values were also calculated for both the control and test coupons (i.e., SD2Cy and
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The significance of differences in efficacy across different test conditions and spore types was
assessed based on the 95% confidence interval of each efficacy result. Differences in efficacy
were judged to be significant if the 95% CIs of the two efficacy results did not overlap. Any
results based on this formula are hereafter noted as significantly different. Note this comparison
is not applicable when the two efficacy results being compared are both reported with log
reductions as > some value.
3.7 Surface Damage
The physical effect of the decontaminants on the materials was also qualitatively monitored
during the evaluation. This approach provided a gross visual assessment of whether the
decontaminants altered the appearance of the test materials. The procedural blank (coupon that is
decontaminated, but has no spores applied) was visually compared to a laboratory blank coupon
(a coupon not exposed to the decontaminant and that has no spores applied). Obvious visible
damage might include structural damage, surface degradation, discoloration, or other aesthetic
impacts.
10
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4.0 Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the
Quality Management Plan (QMP) and the test/QA Plan (TQAP).(3) The QA/QC procedures and
results are summarized below.
4.1 Audits
4.1.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these experiments. Table 4-1 summarizes the performance evaluation audits that were
performed.
No performance evaluation audits were performed to confirm the concentration and purity of B.
anthracis or B. atrophaeus spores because quantitative standards do not exist for these
organisms. The control coupons and blanks support the spore measurements.
Table 4-1. Performance Evaluation Audits
Measurement
Volume of liquid from
micropipettes
Time
Temperature
Relative Humidity
EtO Concentration
Balance
Audit
Procedure
Gravimetric evaluation
Compared to independent clock
Compared to independent calibrated
thermometer
Compare to independent calibrated
hygrometer
SEC Gas Analyzer
Calibrated once annually by SEC and checked
by technician once prior to start of testing
Compared to independent calibrated
weight sets
Allowable
Tolerance
± 10%
± 2 sec/hr
±2°C
± 10%
± 10%
±0.5g
Actual
Tolerance
±0.15% to 2. 5%
0 sec/hr
± 1.02 °C
±3.14%
± 10%
±0.1g
4.1.2 Technical Systems Audit
Observations and findings from the technical systems audit (TSA) were documented and
submitted to the Battelle Work Assignment (WA) Leader for response. Battelle QA staff
conducted a TSA on October 30, 2012, to ensure that the tests were being conducted in
accordance with the appropriate 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 TSA required corrective action.
11
-------
4.1.3 Data Quality Audit
At least 10% of the data acquired during the evaluation were audited. A Battelle QA auditor
traced the data from the initial acquisition, through reduction and statistical analysis, to final
reporting to ensure the integrity of the reported results. All calculations performed on the data
undergoing the audit were checked. Minor data issues were noted and corrected before data were
used in reporting.
4.2 Test/Quality Assurance Plan Deviations
Section 2.1 of the TQAP states "Five positive controls and one procedural blank will be similarly
handled compared to the test coupons that undergo EtO efficacy testing. The positive controls
and laboratory blank will be placed in the control chamber and extracted/cultured at the
completion of the decontamination efficacy test." Test and control samples were inoculated on
October 29, 2012, and allowed to dry overnight for testing on October 30, 2012 (Test 2).
Laboratory blank coupons were inadvertently forgotten and were not run for this test. This is not
expected to have an impact on the data quality as previous blank coupons were negative.
Section 3.2 of the TQAP states "The temperature and RH of the control chamber (Lock
& Lock, Farmers Branch, TX, USA) will be measured with a thermometer/hygrometer (Fisher
Scientific Cat. No. S66283, Pittsburgh, PA, USA) and the data will be recorded using a HOBO
data logger (Onset Part No. U12-001, Bourne, MA, USA)." For Test 8 started on 1/30/13, the
HOBO was inadvertently not launched inside the control chamber, resulting in no temperature or
RH data at the end of the contact period. The parameters for Test 8 were as follows: 300 mg/L
EtO; 50 °C; 50% RH; 180 minute contact time with a 24-hour pre-humidification period.This is
not expected to have an impact on data quality as the temperature in the room did not vary
significantly.
4.3 QA/QC Reporting
Each assessment and audit was documented in accordance with the TQAP and QMP. For these
tests, findings were noted (none significant) in the data quality audit, but no follow-up corrective
action was necessary. The findings were mostly minor data transcription errors requiring some
recalculation of efficacy results, but none were gross errors in recording. QA/QC procedures
were performed in accordance with the TQAP.
4.4 Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in reports. The staff
member performing the QC/technical review was involved in the experiments and added his/her
initials and the date to a hard copy of the record being reviewed. This hard copy was returned to
the staff member who stored the record.
12
-------
5.0 Results
5.1 EtO Results
The efficacy of EtO against B. anthracis and B. atrophaeus was evaluated using the quantitative
method at concentrations ranging from 150 to 600 mg/L, temperatures ranging from 37 °C to 50
°C, and RH from 30% to 75%. Contact times ranged from 45 to 360 min. The results of these
tests are shown in Tables 5-1 and 5-2 and Figures 5-1 through 5-6.
Additionally, the efficacy of EtO against B. atrophaeus was evaluated using both liquid and dry
inoculum methods at a concentration of 150 mg/L EtO, 50 °C and 50% RH for a contact time of
45 minutes. These methods are described in Section 3.3. The results of this test are shown in
Table 5-3.
The temperature, RH, and EtO concentrations listed Tables 5-1, 5-2 and 5-3 are target values.
Actual values for temperature, RH and EtO concentration all fell within target tolerance ranges
specified in Table 2-1.
13
-------
Table 5-1. EtO Efficacy Against Bacillus anthracis11 Spores
Test Temp(°C)/ Concentration (mg/L) /
Number RH(%) Contact Time (min)
1 50/30 300/180
2 50/50 1 50/45
3 50/50 150/90
4 50/50 1 50/1 80
5 50/50 300/45
6 50/50 300/90
7 50/50 300/180
300/1 80
8 50/50 (with 24 Hour pre-
humidification)
9 50/50 300/360
10 50/50 600/180
Inoculum
Material (CFU/coupon)
Glass
Bare Pine Wood
Painted Canvas 7
7.60x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
9.07x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
PaintedCanvas ^^
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
9.37x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
8.63x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
7.17x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
9.20x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
1.17x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
1.10x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
9.07x10
Archival Paper
Silk Fabric
Carbon Steel
Mean Recovered^, anthracis (CFU/coupon)
Positive Control'
2.55 ± 0.78 xlO7
4.19 ± 0.52 xlO7
5.84±0.67xl07
4.05 ± 0.39 xlO7
1.37±0.24xl07
4.59 ± 0.73 xlO7
5.85±0.56xl07
7.83±1.34xl06
5.09±0.58xl07
4.10±0.40xl07
2.84 ± 0.28 xlO7
5. 79 ±1.21 xlO7
5.67±0.56xl07
4.35±1.32xl06
4.73±0.43xl07
3.93±0.53xl07
2.62 ±0.71 xlO7
3.69±1.59xl07
5. 55 ±0.81 xlO7
5.96±0.96xl06
8.03±0.56xl07
4.74±0.40xl07
2.20 ± 0.72 xlO7
4.99 ± 0.37 xlO7
2.37 ± 0.90 xlO7
8.93±4.28xl06
6.60±1.35xl07
3. 28 ±0.21 xlO7
2.59 ± 0.64 xlO7
2.59 ±2.21 xlO7
2.67 ± 0.93 xlO7
4.49±1.07xl06
6.66±1.20xl07
4.01 ±0.43xl07
2.36 ± 0.75 xlO7
3.96±1.16xl07
4.37±1.02xl07
4.15 ± 2.23 xlO6
6.26 ± 0.65 x 10
4.22 ± 0.73 xlO7
1.57±0.42xl07
4.19±1.23xl07
4.96 ± 0.80 xlO7
2.59 ±0.81 xlO6
8.11 ±0.57 xlO7
5.03±1.34xl07
2.38 ± 0.53 xlO7
1.09±0.19xl08
5. 25 ±0.67 xlO7
6.37±1.54xl06
9.58±0.76xl07
6.29 ± 0.84 xlO7
3.41 ±0.88xl07
1.09±0.17xl08
4.62 ± 0.52 xlO7
8.16±1.14xl06
8.51 ±0.92xl07
5.63±0.39xl07
2.41 ±0.94xl07
6.75 ± 0.92 xlO7
Test Coupon0
2.82±5.43xl03
3.96±4.42xl03
3.40±3.22xl03
7.11 ±2.89xlOs
1.43±0.93xl04
1.20±1.40xl03
9.70±5.98xl03
8.56±11.6xl03
4.00 ± 0.80 xlO4
3. 27 ±0.58x1 0s
5.13 ±2.01 xlO4
9.17±3.86xl03
6.61 ±11.2xl03
0.00 ±0.00
5.66±4.56xl03
1.96±1.29xl04
9.07 ± 9.47 xlO2
1.36±0.90xl04
4.99±8.65xl02
1.27±1.73xl02
2.74 ± 5.90x10'
7.19±3.21 xlO4
9.08 ±1 0.8 xlO2
6.05 ±10.1 xlO1
1.60±1.50xl02
4.72 ± 6.43x10'
3.39±5.72xl01
9.75 ± 2.23 xlO4
8.27±2.90xl02
5. 37 ±8. 34x10'
3. 39 ±4.66x10'
7. 46± 14.4x10
2.08 ± 4.43 xlO1
1.07±1.42xl03
2.73 ±4.30x10'
7. 46± 14.4x10
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
8.65±5.42xl03
0.00 ±0.00
0.00 ±0.00
2.14±2.72xl02
2.08 ±4.43x10'
0.00 ±0.00
4.20 ± 4.90 xlO2
3.00±4.70xl02
4.41 ±2.94x10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
2.10±2.33xl03
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Decontamination
Efficacy ± Cf1
4.77 ±0.91
4.26 ± 0.47
4. 54 ±0.59
1.78±0.14
3. 04 ±0.23
4.99 ±0.71
3. 89 ±0.35
3.31 ±0.59
3.11 ±0.09
2.10 ±0.07
2.77 ±0.17
3. 83 ±0.20
5. 68 ±1.82
26.62 ±0.12
4.65 ±1.50
3. 38 ±0.28
4.65 ± 0.47
3. 45 ±0.31
5. 84 ±1.07
5. 77 ±1.20
7. 48 ±0.83
2.86 ±0.21
4.85 ±0.79
6.86 ±1.02
5.61 ±0.93
6.09 ±1.01
7. 08 ±0.90
2.54 ±0.09
4.51 ±0.19
6.22 ± 0.96
6.64 ± 0.93
6.34 ±0.60
7. 42 ±0.79
5. 28 ±1.21
6.65 ±0.87
7. 28 ±0.61
>7. 63 ±0.09
>6.56±0.22
>7. 79 ±0.04
3. 78 ±0.33
>7. 18 ±0.11
>7.61 ±0.12
6.25 ±1.20
5. 99 ±0.79
£7.91 ±0.02
6.01 ±1.36
5. 53 ±0.99
7. 67 ±0.72
>7. 72 ±0.05
>6.79±0.11
>7. 98 ±0.03
4.68 ± 0.42
>7. 52 ±0.11
>8.03 ± 0.06
>7. 66 ±0.04
>6.91 ± 0.05
>7. 93 ±0.04
>7. 75 ±0.03
>7. 36 ±0.14
>7. 83 ±0.05
14
-------
Table 5-1. EtO Efficacy Against Bacillus anthracis Spores" (Continued)
Test Temp(°C)/
Number RH(%)
1 1 50/60
12 50/60
13 50/75
14 50/75
15 50/75
16 50/75
17 37/75
18 37/75
19 37/75
Concentration (mg/L) / . Inoculum
Contact Time (min) (CFU/coupon)
Glass
Bare Pine Wood
Painted Canvas s
300/180 1.08x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
600/360 1.35x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
150/180 9.73 xlO7
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
300/45 8.33x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
300/90 1.09x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 7
300/180 8.13x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
300/45 1.02x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 8
300/90 1.10x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas
300/180 . 1.18x10
Archival Paper
Silk Fabric
Carbon Steel
Mean Recovered^, anthracis (CFU/coupon)
Positive Control'
5.96±1.16xl07
6.27 ± 0.54 xlO6
7.36±0.85xl07
5.03±0.53xl07
2.69 ± 0.65 xlO7
4.74 ± 0.48 xlO7
4.11 ±0.86xl07
8.43±6.63xl06
9.66 ±0.71 xlO7
4.23 ± 0.90 xlO7
2.21 ±0.72xl07
2.85±1.12xl07
5.19± 2.07 xlO7
5.58±1.45xl06
7. 75 ±0.57 xlO7
4.09 ± 0.36 xlO7
1.80±0.67xl07
7.38±1.18xl07
2.88 ± 0.18 xlO7
7.00±0.87xl06
7.51 ±0.70xl07
4.40 ± 2.23 xlO7
2.02 ± 0.88 xlO7
3.63±0.53xl07
3.79±1.64xl07
5.29±0.97xl06
6.72±0.48xl07
4.54±0.45xl07
3.11 ±1.02xl07
1.31 ±0.10xl07
4.07 ±0.86xl07
5.62±2.72xl06
6.18 ±0.31 xlO7
3.81 ±0.35xl07
1.90±0.65xl07
2.99 ±0.31 xlO7
6.03 ± 0.92 xlO7
8.41 ±1.88xl06
6.07 ±0.72xl07
4.87±1.16xl07
3.12±0.47xl07
7.33±0.69xl07
5.41±1.23xl07
5.77±0.97xl06
6.02 ± 0.84 xlO7
3.35±0.52xl07
2.47 ± 0.87 xlO7
4.24 ± 0.58 xlO7
4.39 ± 0.49 xlO7
8.01 ±3.61 xlO6
5.17 ±0.52xl07
2.46 ± 0.68 xlO7
1.29±0.27xl07
3.04±0.48xl07
Test Coupon0
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
4.03 ± 5.43x10'
0.00 ±0.00
7.46±14.4xl01
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
3.27±6.57xl02
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
1.67±1.18xl02
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Decontamination
Efficacy ± Cf1
>7. 77 ±0.07
>6.80±0.03
>7. 86 ±0.04
>7. 70 ±0.04
>7. 42 ±0.11
>7. 67 ±0.04
>7.61 ±0.08
>6.85±0.23
>7. 98 ±0.03
>7. 62 ±0.08
>7. 33 ±0.12
>7. 42 ±0.17
>7. 69 ±0.14
>6.73±0.12
>7. 89 ±0.03
>7.61 ±0.03
>7. 23 ±0.14
>7. 86 ±0.06
6.43 ±0.86
>6.84±0.05
7. 57 ±0.60
>7. 59 ±0.24
>7. 27 ±0.16
>7. 56 ±0.05
>7. 54 ±0.19
>6.72±0.08
>7. 83 ±0.03
>7. 65 ±0.04
>7. 47 ±0.16
>7. 12 ±0.03
>7. 60 ±0.08
>6.71 ±0.20
>7. 79 ±0.02
>7. 58 ±0.04
>7. 26 ±0.12
>7.47 ±0.04
>7. 78 ±0.05
5. 55 ±1.20
>7. 78 ±0.05
>7. 68 ±0.10
>7. 49 ±0.06
5. 77 ±0.36
>7. 72 ±0.09
>6.76±0.07
>7. 78 ±0.05
>7. 52 ±0.06
>7. 37 ±0.14
>7. 62 ±0.06
>7. 64 ±0.04
>6.87±0.17
>7.71 ±0.04
>7. 37 ±0.12
>7. 10 ±0.08
>7. 48 ±0.06
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual coupons, the mean percent recovery
on those five coupons, and decontamination efficacy (log reduction).
Positive Controls = inoculated, not decontaminated coupons.
Test Coupons = inoculated, decontaminated coupons.
Laboratory Blank = not inoculated, not decontaminated coupon.
Procedural Blank = not inoculated, decontaminated coupon.
CI = confidence interval (± 1.96 x SE).
"-" Not Applicable.
15
-------
Table 5-2. EtO Efficacy Against Bacillus atrophaeus Spores"
Test Temp(°C)/ Concentration (mg/L) /
Number RH(%) Contact Time (min)
1 50/30 300/180
2 50/50 150/45
3 50/50 150/90
4 50/50 150/180
5 50/50 300/45
6 50/50 300/90
7 50/50 300/1 80
300/1 80
8 50/50 (with 24 Hour pre-
humidification)
9 50/50 300/360
10 50/50 600/180
Inoculum
(CFU/coupon)
Glass
Bare Pine Wood
Painted Canvas s
1.27 x 10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
1.04x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
1.21 xlO
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
1.25x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
1.22x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
1.10x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
1.14x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas ^^
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
1.08x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
1.08x10
Archival Paper
Silk Fabric
Carbon Steel
Mean Recovered^, anthracis (CFU/coupon)
Positive Control'
1.05±0.08xl08
5.04±1.58xl06
7.47±1.00xl07
6.69±1.26xl07
2.09 ± 2.07 xlO7
2.47 ± 0.90 xlO7
1.00±0.12xl08
4.53±1.30xl06
6.63±1.12xl07
3.82±2.06xl07
1.04±0.18xl07
3. 45 ±2.31 xlO7
9.95 ±1.71 xlO6
7. 88 ±6.21 xlO6
6.27±1.40xl07
5.15±0.38xl07
1.13±0.38xl07
1.11 ±0.11 xlO7
8.86±1.13xl07
9.53 ± 13.7 xlO6
7.10±0.68xl07
5.84±1.68xl07
1.39±0.89xl07
8.85±1.35xl06
9.86±1.65xl07
9.39 ± 4.65 xlO6
7.92±0.57xl07
5.26±0.44xl07
1.27±0.33xl07
1.49±0.44xl06
1.12±0.05xl08
8.13±1.47xl06
7.15±1.39xl07
5.02±0.97xl07
1.45±0.96xl07
4.21 ±2.03xl07
9.11 ±0.77 xlO7
5.39±1.60xl06
5.89±0.55xl07
4.25±0.52xl07
1.65±0.44xl07
1.94±0.93xl07
1.04±0.09xl08
2.07±1.16xl06
1.88±0.52xl07
1.37±0.20xl07
4.97 ± 2.23 xlO6
2.45 ±1. 54 x 10s
1.19±0.25xl08
1.27±0.48xl07
5.23±0.78xl07
4.29±0.45xl07
9.35 ± 4.50 xlO6
1.01 ±0.38xl07
1.04±0.16xl08
6.31 ±1.78xl06
6.99 ± 0.92 xlO7
4.31 ±0.94xl07
1.77±0.89xl07
2.63 ± 0.94 xlO7
Test Coupon0
3. 23 ±2.28x1 0s
3. 55 ±3. 61 xlO3
5.45±0.30xl04
4.07 ± 4.43 xlO2
1.51 ±0.99xl03
2.32 ±3. 67x1 0s
4.63 ± 0.84 xlO4
1.47±1.52xl02
1.04 ±0.23x1 0s
1.54 ±0.41 xlO3
6.05 ±6.80x10'
1.36±0.45xl04
1.41 ±1.06xlOs
0.00 ±0.00
7.33±2.50xlOs
4.08 ±8. 90x10'
1.15±2.18xl03
2.03±1.20xl04
6.25±1.83xl04
7.35±7.21 xlO1
6.67 ± 2.08 xlO4
8.53±4.22xl02
1.93±1.67xl02
2.53±1.53xl04
6.03±1.23xl03
1.80±1.86xl02
7.09±2.80xl04
2.89±1.23xl04
7.53±3.33xl02
6.69 ± 2.56 xlO3
4.65±1.75xl03
2.54 ± 2.72 xlO2
5.89±1.82xl04
7. 46± 14.4x10
1.45±2.43xl03
4.54 ± 2.66 xlO3
6.06±5.46xl04
3.20±3.41 xlO2
4.66±1.00xl04
3.53±1.98xl02
8.68±8.00x!0'
6.07 ± 2.43 xlO3
2.46 ± 2.22 xlO4
7. 40 ±14.31
3. 35 ±0.64x1 0s
0.00 ±0.00
3.42±7.42x!0'
8.89±8.26xl04
5.12±1.42xl03
1.07±1.44xl02
1.61 ±0.61 xlO4
4.33 ± 2.57 xlO2
1.00±0.97xl02
4.82 ± 2.05 xlO3
1.62±0.63xl03
3. 37 ±4.04x10'
4.11 ±0.44xl04
0.00 ±0.00
0.00 ±0.00
1.13±0.57xl04
Decontamination
Efficacy ± Cf1
2.65 ±0.39
3. 28 ±0.36
2.13 ±0.05
5. 40 ±0.40
4.12 ±0.40
2.44 ± 0.63
3.34 ±0.079
4.67 ± 0.42
2.. 81 ±0.11
4.35 ±0.25
5. 84 ±0.95
3. 34 ±0.31
2.94 ±0.30
>6.82±0.23
1.95±0.17
7. 25 ±0.90
5.31 ±1.47
2.79 ±0.22
3. 17 ±0.14
5. 25 ±0.87
3. 04 ±0.12
4.86 ±0.23
4.93 ±0.37
2.60 ±0.21
4.22 ±0.10
5. 17 ±0.96
3. 08 ±0.16
4.29 ±0.16
4.26 ± 0.23
3. 36 ±0.20
4.41 ±0.15
5. 36 ±1.25
3. 09 ±0.14
7. 39 ±0.60
4.38 ±0.64
3. 98 ±0.30
3. 36 ±0.45
4.53 ±0.59
3.11 ±0.10
5. 14 ±0.24
5. 65 ±0.81
3. 49 ±0.23
3. 78 ±0.37
5. 96 ±0.63
1.74±0.11
>7. 13 ±0.06
6.20 ± 0.90
0.64 ±0.61
4.37 ±0.13
5. 82 ±1.06
3. 53 ±0.13
5. 08 ±0.31
5. 07 ±0.39
3. 33 ±0.21
4.84 ±0.20
5. 78 ±0.83
3. 23 ±0.06
>7. 63 ±0.08
>7. 20 ±0.20
3. 38 ±0.23
16
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Table 5-2. EtO Efficacy Against Bacillus atrophaeus Spores" (Continued)
Test Temp(°C)/
Number RH(%)
1 1 50/60
12 50/60
13 50/75
14 50/75
15 50/75
16 50/75
17 37/75
18 37/75
19 37/75
Concentration (mg/L) / . Inoculum
Contact Time (min) (CFU/coupon)
Glass
Bare Pine Wood
Painted Canvas s
300/180 1.19x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
600/360 1.18x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
150/180 1.17x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
300/45 1.08x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
300/90 1.15x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
300/180 PaintedCanvas ^^
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas s
300/45 1.40x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas 8
300/90 1.12x10
Archival Paper
Silk Fabric
Carbon Steel
Glass
Bare Pine Wood
Painted Canvas K
300/180 1.23x10
Archival Paper
Silk Fabric
Carbon Steel
Mean Recovered^, anthracis (CFU/coupon)
Positive Control'
1.05±0.12xl08
4.48±5.13xl06
4.26 ± 0.79 xlO7
2.51 ±0.69xl07
1.77±0.68xl07
1.41 ±0.62xl07
1.02±0.24xl08
4.43 ± 2.27 xlO6
3. 74 ±0.41 xlO7
1.54±0.93xl07
5. 52 ±1.91 xlO6
2.44 ± 0.89 xlO7
9.70 ± 0.47 xlO7
1.40±0.42xl06
4.70±2.02xl07
2.09±0.80xl07
1.09±0.49xl07
1.08±0.22xl07
8.03±0.20xl07
6.19±1.43xl06
8.14±0.46xl07
5.32±1.14xl07
1.31 ±0.45xl07
1.61 ±0.69xl07
9.21 ±1.46xl07
6.78±5.39xl06
4.26±0.73xl07
1.01 ±0.52xl07
1.12±0.18xl07
1.28±0.75xl07
1.05±0.15xl07
3.49±1.07xl06
4.92 ± 0.57 xlO7
1.17±0.25xl07
6.69 ± 2.17 xlO6
4.94 ± 2.39 xlO7
7.40±1.42xl07
6.95 ± 2.50 xlO6
8.17±1.09xl07
4.84 ± 0.79 xlO7
1.07±0.32xl07
2.33±1.62xl07
1.01 ±0.07xl08
5.63±3.90xl06
5.56±0.94xl07
7.91 ±4.28xl06
1.03±0.44xl07
9.07 ±2.91 xlO6
1.12±0.02xl07
3.60±0.53xl06
7.39±1.52xl07
1.80±0.96xl07
5.33±2.48xl07
1.46±0.37xl07
Test Coupon0
4.05 ± 2.64 xlO3
4.72 ± 6.43 xlO1
8.01 ±0.39x10"
0.00 ±0.00
8.01 ±8.97x10'
5.29±3.03xl03
1.31 ±0.56xl03
0.00 ±0.00
9.91 ±3.84xl03
1.20 ±1.01 xlO2
4.04 ±4.30x10'
2.11 ±1.68xl03
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
7. 07 ±1.60x10"
2.66 ± 2.06 xlO2
2.37±1.39x!0"
9.21 ±2.19xl03
1.01 ±0.27xl03
1.50 ±0.22x10"
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Decontamination
Efficacy ± Cf1
4.47 ± 0.22
5. 64 ±1.06
2.72 ±0.07
>7. 38 ±0.11
5.71 ±0.80
3. 49 ±0.40
4.91 ±0.18
>6.60±0.19
3. 60 ±0.16
5. 47 ±0.89
5. 65 ±0.88
3. 16 ±0.39
>7. 99 ±0.02
>6.13±0.12
>7. 63 ±0.19
>7. 30 ±0.14
>7.01 ±0.14
>7. 03 ±0.08
>7. 90 ±0.01
>6.78±0.09
>7.91 ±0.02
>7. 72 ±0.09
>7. 09 ±0.14
>7.18±0.16
>7. 96 ±0.06
>6.75±0.24
>7. 62 ±0.07
>6.96±0.19
>7. 05 ±0.07
>7. 02 ±0.29
>8. 02 ±0.05
>6.52±0.13
>7. 69 ±0.04
>7. 06 ±0.08
>6.81 ±0.11
>6.66±0.15
3. 02 ±0.12
4. 54 ±0.43
3. 64 ±0.34
3. 73 ±0.13
4.02 ±0.15
3. 12 ±0.26
>8. 00 ±0.03
>6.66 ± 0.27
>7. 74 ±0.06
>6.86±0.17
>6.98±0.17
>6.94±0.13
>8. 05 ±0.01
>6.55±0.06
>7. 86 ±0.08
>7. 22 ±0.17
>7. 67 ±0.24
>7.15±0.10
Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual coupons, the mean percent recovery
on those five coupons, and decontamination efficacy (log reduction).
Positive Controls = inoculated, not decontaminated coupons.
Test Coupons = inoculated, decontaminated coupons.
CI = confidence interval (± 1.96 x SE).
17
-------
EtO Fumigation vs. Glass
9
8
7 -
6
S
0
••e 5 -
•5
«
3
2
1
*...**
T
1
I
,
1
I
I
i
i
i
'
I
i
r
I
*
1 I
ll
•I
:- i *x ^i
300 150 150 150 300 300 300 300 300 600 300 600 150 300 300 300
1 (180) I 1 1
l^JL
* *
* *- *-
i i
•
i
-i
• 1 mifltrnch
— B. atrophmnis
-
300 300 300
45) (90) (180) (45) (90) (180) (180)f (3dO) (180)11(180) (180)11(180) (45) (90) (180)11(45) (90) (180)1
r T i
50°C 50°C 50°C 50°C
30% RH 50% RH 60% RH 75% RH
mg L EtO (Minutes Contact Time)
T
37°O
75 »» RH
*Complete inactivation achieved.
^ Samples pre-humidified @ 50% RH for 24 hours prior to fumigation
Figure 5-1. Summary of decontamination efficacies for EtO fumigation testing on glass.
18
-------
9
8 -
7 -
6
3
Log Reducti
4- 'Jl
3
2
1
0
EtO Fumigation vs. Bare Pine Wood
I~
I
I
T
•
J
•'
.
•
1
I
*
*
1
•
c
*
I
1 !
I
<* =i
I
I
1=
*
I
300 150 150 150 300 300 300 300 300 600 300 dOO 150 300 300 300
rjl
r
:*
X
j — B. fflititmcis
~
— B. atrophartis
'
300 300 300
1(180)11(45) (90) (180) (45) (90) (180) (ISO)f (360) (180)11(180) (180)1 1 (ISO) (45) (90) (180)1 1(45) (90) (180)1
Y
y y y
50°C 50°C 50°C 50°C
30% RH 50% RH (50% RH 75% RH
ing LEtO (Minutes Contact Time)
I
37 °C'/
75% RH
*Complete inactivation achieved.
t Samples pre-humidified @ 50% RH for 24 hours prior to fumigation
Figure 5-2. Summary of decontamination efficacies for EtO fumigation testing on bare pine wood.
19
-------
9
-
7 -
6
g
1 5 -
1
bf "^
q
hJ
3 -
2 -
1
0
EtO Fumigation vs. Painted Canvas
i
I
I
I
I
I
I
I
•
T
1
• 1
1
I
I -
1 *
I
H
1 I
r- B. an tl tracts
~
<— B. ati-ophaens
300 150 150 150 300 300 300 300 300 600 300 (300 150 300 300 300 300 300 300
1 (ISO) 1 1 (
50 °C
30°o RH
45) (PO) (180) (45) (PO) (180) (180)f (3fiO) (180)1 1(180) (180)11 (180) (45) (PO) (180)1 1(45) (90) (180)1
1 T T T
50°CV 50°C 50°C 37°C.<
50»oRH 60»oRH 75% RH 75% RH
rag L EtO (Minutes Contact Time)
*Complete inactivation achieved.
^ Samples pre-humidified @ 50% RH for 24 hours prior to fumigation
Figure 5-3. Summary of decontamination efficacies for EtO fumigation testing on painted canvas.
20
-------
8
-
6
I
'€ 5 -
I
61.'
O
EtO Fumigation vs. Archival Paper
* *
I T *
300 150 150 150 300 300 300 300 300 ISOO 300 (300 150 300 300 300 300 300 300
(180) I I (45) (PO) (180) (45) (PO) (180) (ISO)f (3fiO) (180) I I (180) (180) 11 (180) (45) (90) (180)1 I (45) (PO) (180) I
I (180) I I
50 °C
30«o RH
Ml.
50 °C
50°o RH
50°C<
dO«. RH
50 °C
75% RH
ing L EtO (Minutes Contact Time)
*- B. an timid <>
•— B. fitropltaeus
*Complete inactivation achieved.
t Samples pre-humidified @ 50% RH for 24 hours prior to fumigation
Figure 5-4. Summary of decontamination efficacies for EtO fumigation testing on archival paper.
21
-------
9
8 -
7 -
6
g
1 5
£
M "*
o
hJ
3 -
2 -
1
0
EtO Fumigation vs. Silk Fabric
*
1
i
I
T
1
T
•^
1
"
••
. *
I
1
1
I
I
,
*
X
*
I
300 150 150 150 300 300 300 300 300 dOO 300 dOO 150 300 300 300
1 (180) 1 1 (45
1
50 °C
30°o RH
*
1 I* *i
H !
i
,-,
^- B. an tf tracts
•— B. atrophaeux
300 300 300
) (PO) (180) (45) (PO) (180) (180)f (3dO) (180)1 1(180) (180)11 (180) (45) (PO) (180)1 1(45) (PO) (180)1
Y Y Y
50°C 50°C 50°C
50»o RH dO»o RH 75»o RH
rag L EtO (Minutes Contact Time)
Y
37°C/
75»oRH
*Complete inactivation achieved.
t Samples pre-humidified @ 50% RH for 24 hours prior to fumigation
Figure 5-5. Summary of decontamination efficacies for EtO fumigation testing on silk fabric.
22
-------
9
7 -
6
g
'€ 5
^4
HI, 4
o
hJ
3 -
2 -
1
0
E tO Fumigation vs. Carbon Steel
I
M
I
I
t
I
(
*
I
I
«
'
c
I
I
*
i
i
I*
*
1 * *
* T *I
I
T
1
1
•
1 p— ]$ fiMtJtwici?
~
•— B. atrophattis
300 150 150 150 300 300 300 300 300 COO 300 (300 150 300 300 300 300 300 300
1(180)11(45) (PO) (180) (45) (90) (180) (180)t (360) (180)11(180) (180)11(180) (45) (PO) (180)1 1(45) (PO) (180)1
T T f Y ¥
50°C7 50°O 50°C 50°C 37°C<
30»oRH 50»«RH 60»oRH 75% RH 75% RH
rag LEtO (Minutes Contact Time)
*Complete inactivation achieved.
^ Samples pre-humidified @ 50% RH for 24 hours prior to fumigation
Figure 5-6. Summary of decontamination efficacies for EtO fumigation testing on carbon steel.
23
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Table 5-3. EtO Efficacy against Bacillus atrophaeus Spores" on Archival Paper
Test
Number
20
Concentration (mg/L) /
Contact Time (min)
150/45
Temp(°C)/
RH(%)
50/50
Material
Archival Paper
Inoculation
Type
Liquid
Dry
Inoculum
(CFU/coupon)
l.lSxlO5
t
t
MeanRecoveredS. atrophaeus
Positive Control1"
1.62±0.21xl04
7.51 ± 1.18xl04
3.58±3.14xl05*
6.41 ± 0.77 xlO5
(CFU/coupon)
Test Coupon0
3.95±1.50xl02
1.67±1.72xl03
Decontamination
±cr1
Efficacy
1.64±0.15
1.97±0.59
2.44 ±0.74*
2.90±0.59
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on six individual coupons, the mean percent recovery on those six coupons, and decontamination efficacy (log reduction).
b Positive Controls = inoculated, not decontaminated coupons.
c Test Coupons = inoculated, decontaminated coupons.
11 CI = confidence interval (± 1.96 x SE).
t Samples (N=3) immediately processed, not placed in control chamber. Inoculation concentration is unknown.
* Samples (N=3) added to control chamber for the 45 minute contact time. Inoculation concentration is unknown.
* Average of all six coupons.
24
-------
5.2 Surface Damage to Materials
At the end of each decontamination test, the procedural blanks were visually compared to the
laboratory blanks, and test coupons were visually compared to positive controls, to assess any
impact EtO and/or the test conditions may have had on each material type. Based on the visual
appearance of the decontaminated coupons, there were no apparent changes in the color,
reflectivity, or roughness of five of the six material surfaces after being exposed to EtO. A
noticeable change was observed on carbon steel after inoculation with B. atrophaeus (Figure 6-
7). The inoculation spots appear to have oxidized and this apparent oxidation was observed with
and without the presence of EtO. Due to the noticeable difference in both test and positive
control coupons (the apparent oxidation was not observed on blank coupons), the apparent
oxidation was due to the inoculation material rather than due to contact with the EtO fumigant.
The carrier buffer of the B. atrophaeus spores (PBSTx) and the high RH may have contributed to
the oxidation of the coupon.
Figure 5-7. Oxidation on carbon steel coupons inoculated with B. atrophaeus.
25
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6.0 Discussions and Conclusions
6.1 Effects of Temperature and Relative Humidity on Efficacy
The tests performed under this evaluation highlight the importance of achieving a proper RH and
temperature to achieve the maximum efficacy of EtO. A single test was conducted at 30% RH
and low LRs were achieved. Using these parameters (50 °C, 30% RH, 300 mg/L EtO for 180
minutes), LRs of 1.78 (archival paper) to 4.99 (carbon steel) were observed for B. anthracis and
LRs of 2.13 (painted canvas) to 5.40 (archival paper) were observed for B. atrophaeus. These
LRs are below the target of LR > 6 to demonstrate a process as a sporicidal decontaminant based
upon quantitative testing; the conditions described above were not suitable to achieve sporicidal
decontaminant requirements.
The effects of 50 °C and 50% RH were assessed next. At least a 6 LR was achieved against B.
anthracis on all materials at 50 °C and 50% RH using 600 mg/L EtO for a contact time of 180
minutes; further complete inactivation (no viable spores recovered on test coupons) was
achieved. This efficacy was also demonstrated at 300 mg/L EtO for 180 and 360 minutes against
all materials types except archival paper. In contrast, EtO was not efficacious against B.
atrophaeus at 50 °C and 50% RH for up to 600 mg/L EtO and 360 minutes (the highest
combination of concentration and time tested), with the exception of archival paper and silk
fabric where a few instances of LRs >6.20 were observed. These data suggest that B. atrophaeus
is more difficult to inactive than B. anthracis when testing with EtO under these conditions.
When the RH was raised to 60%, at least a 6 LR (and complete inactivation) was achieved
against B. anthracis at both conditions tested (300 mg/L EtO for 180 minutes and 600 mg/L EtO
for 360 minutes) and for all six coupon types with LRs ranging from >6.80 to >7.98. However,
LRs ranged from 2.72 (painted canvas) to >7.38 (archival paper) at 300 mg/L EtO for 180
minutes and 3.16 (carbon steel) to >6.60 (bare pine wood) at 600 mg/L EtO and 360 minutes
against B. atrophaeus. These data suggest that EtO is a less efficacious decontaminant against B.
atrophaeus at these test conditions.
At 50 °C and 75% RH, EtO was effective against both B. anthracis and B. atrophaeus., even
when using only 150 mg/L EtO for a contact time of 180 minutes (the lowest combination
tested). A least a 6 LR was achieved on all materials at all parameters tested. Further, complete
inactivation was achieved on all materials with the exception of of glass (6.43 LR) and painted
canvas (7.57 LR) at 300 mg/L EtO for 45 minutes against B. anthracis. Although complete
inactivation was not achieved on these two materials, LRs were still >6 LR, suggesting a high
efficacy.
In general, lowering the temperature from 50 °C to 37 °C resulted in decreased efficacy for B.
atrophaeus on all materials at 45 min. Lower EtO efficacy against B. anthracis at 45 min with
the same temperature decrease was observed for only bare pine wood and carbon steel.
EtO is an effective decontaminant against B. anthracis under optimal combinations of
concentration, contact time, temperature, and RH. At a minimum, the following combinations of
parameters should be achieved for EtO to be effective against glass, bare pine wood, painted
canvas, archival paper, silk fabric and carbon steel:
26
-------
• 50 °C, 50% RH, >600 mg/L EtO for > 180 minutes
• 50 °C, 60% RH, >300 mg/L EtO for >180 minutes
• 50 °C, 75% RH, >150 mg/L EtO for >180 minutes
• 37 °C, 75% RH, >300 mg/L EtO for >90 minutes
Additionally, although less effective against B. atrophaeus, a greater than 6 LR was achieved on
all six coupon types under the following conditions:
• 50 °C, 75% RH, >150 mg/L EtO for >180 minutes
• 37 °C, 75% RH, >300 mg/L EtO for >90 minutes
In general, as the RH increases, so does efficacy. The amount of EtO and the contact time may
decrease as the RH increases and still be efficacious. B. atrophaeus should be considered a
suitable surrogate for B. anthracis when testing at >37 °C and >75% RH.
6.2 Effects of Material Type on Efficacy
In general, B. anthracis was the most resistant to EtO decontamination when the B. anthracis
was inoculated on archival paper. LRs ranged from 1.78 to 6.01 on this coupon type in instances
where complete inactivation was not achieved (Tests 1-9). B. anthracis on glass and bare pine
wood was the least resistant to EtO decontamination as these coupon types exhibited higher LRs
than the other four coupon types. LRs ranged from 3.89 to 6.64 and 3.31 to 6.09 for glass and
bare pine wood, respectively, when complete inactivation was not achieved (Tests 1-6, 8).
In contrast, archival paper (3.73 to 7.39 LR) and bare pine wood (3.28-5.96 LR) were the least
resistant to B. atrophaeus decontamination, while painted canvas (1.74 to 3.63 LR) and carbon
steel (0.64 to 3.98 LR) were the most resistant to B. atrophaeus decontamination using EtO.
6.3 Effects of Inoculation Method on Efficacy
Although not significant, when comparing inoculation methods on archival paper, a dry
inoculation method was easier to decontaminate with EtO than a liquid inoculation method (2.44
vs 1.64 LR). Several factors could contribute to this difference and more testing at different
parameters and with different materials should be completed in order to fully understand the
differences, if any.
Fewer organisms were recovered from the three dry-inoculated positive controls that were
processed immediately than the three held in the control chamber for the 45 minute contact time
(Table 6-3). This difference in recovery is interesting to note and may be due to the RH in the
chamber slightly re-hydrating the spores, promoting higher recovery rates. More testing would
need to be completed to assess the effect of RH on this recovery fully.
27
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7.0 References
1. USEPA, Determining the Efficacy of Liquids and Fumigants in Systematic Decontamination
Studies for Bacillus anthracis Using Multiple Test Methods. US EPA Report 600/R-10/088,
December 2010.
2. Borland WAN. Borland's illustrated medical dictionary (standard version). In: Borland
WE, editor, 29th ed., Philadelphia, W.B. Saunders & Co, 2088, 2000.
3. Test/Quality Assurance Plan for The Evaluation of Ethylene Oxide for the Inactivation of
Bacillus anthracis, Version 1; April 2012 and Amendment 1, October, 2012. (Available
upon request by contacting the EPA Project Leader).
4. Anderson Products, Inc., website accessed August 28, 2013, www.anpro.com.
5. Calfee, M.W., S.B. Lee, S.P. Ryan, "A rapid and repeatable method to deposit
bioaerosols on material surfaces, Journal of Microbiological Methods, 92, 375-380,
2013.
6. Standard Practice for Maintaining Constant Relative Humidity by Means of Aqueous
Solutions. ASTM International, October, 2006.
7. Rogers, J.V., C.L. Sabourin, Y.W. Choi, W.R. Richter, B.C. Rudnicki, K.B. Riggs, M.L.
Taylor, and J. Chang, 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.
28
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Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Offce of Research and Development (8101R)
Washington, DC 20460
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