oEPA
EPA/600/R-15/131 I September 2015 I www2.epa.gov/research
United States
Environmental Protection
Agency
Evaluation of the Inactivation of
Ricin Toxin on Surfaces Using Vapor
Phase Hydrogen Peroxide
Office of Research and Development
National Homeland Security Research Center
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ii
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Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development funded
the research described here under Contract Number EP-C-10-001 with Battelle. It has been subjected
to the Agency's review and has been approved for publication. Note that approval does not signify
that the contents necessarily reflect the views of the Agency. Mention of trade names, products, or
services does not convey official EPA approval, endorsement, or recommendation.
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:
U.S. Environmental Protection Agency (EPA)
Eletha Brady-Roberts
Leroy Mickelsen
Mike Nalipinski
Ramona Sherman
Joseph P. Wood
Battelle
Cover photo of mail sorting equipment: www.siemens.com/press
Cover photo of Castor beans: HediBougghanmi2014
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Executive Summary
The U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD) 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 vapor phase hydrogen peroxide (VPHP) in inactivating ricin toxin on indoor materials
representative of a mail sorting facility. The objective of this study was to provide an understanding
of the performance of VPHP to guide its use and implementation in HS applications. When assessing
options for decontamination following intentional release of ricin toxin, it is important to know the
extent to which factors such as VPHP concentration measured in parts per million (ppm) and
duration of exposure may impact the decontamination efficacy.
This investigation focused on the decontamination of eight types of materials representative of a mail
sorting facility: aluminum, industrial carpet, ceramic tile, neoprene rubber, optical plastic, paper,
stainless steel, and unpainted concrete. Decontamination efficacy tests were conducted using two
different VPHP generators - STERIS 1000ED and a Bioquell Clarus C - against two forms of ricin
toxin: a commercially-available purified version and a crude version prepared from castor beans.
Using a cell-based assay, decontamination efficacy was quantified in terms of percent (%) reduction
in the mass of bioactive ricin recovered from test coupons compared to the positive control (non-
decontaminated) coupons. Tests were conducted using cycles developed for both vapor-generating
technologies and by varying the time of the third phase (fumigant contact phase) of the
decontamination process. These data were utilized to assess the effect of these fumigation parameters
on decontamination efficacy.
Summary of Results
The STERIS and Bioquell VPHP generators used in this study have four similar user-defined phases
that comprise each decontamination test. The phase parameters developed for this testing were
unique to the VPHP test chamber utilized for this study and should not be directly applied to larger
spaces. Cycle development is required to obtain optimal conditions for each unique space to be
decontaminated. Phase 1 is a chamber conditioning phase in which injection lines and chamber
surfaces are warmed, and the chamber atmosphere is dehumidified to a cycle-defined level. Phase 1
set points remained consistent throughout testing for both generators. Phase 2 defined the hydrogen
peroxide injection rate, which varied from 2.5 to 3.8 g/min for 20 minutes. Phase 3 consisted of a
defined injection rate and delivery time, which varied from 2.5 to 3.8 g/min for 30 minutes to 16 hour
(hr) contact times over a total of 10 tests. Phase 4 (aeration phase) was allowed to run until the testing
chamber reached a concentration of VPHP that was <10 ppm. Table ES-1 shows the phase
parameters required to achieve greater than 99 % reduction on selected material types tested except
unpainted concrete at all operational parameters. Unpainted concrete was removed from testing due
to low recovery from control materials, which may have been a result of the caustic nature of this
material, affecting the bioactivity of the ricin.
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Table ES-1. Parameters Required to Achieve >99 % Reduction on All Materials*
Technology
Ricin
Form/Target
Mass
Avg
VPHP
ppm±SDb
Phase 1
Phase 2
Phase 3
Phase 4
Duration
(min)
Injection
Rate
(g/min)
Duration
(min)
Injection
Rate
(g/min)
Duration
(h:min)
Duration
(h:min)
Bioquell
Pure/250 |ig
279±45.0
15
3.8
20
1.0
8:00
11:41
Bioquell
Crude/250 |ig
301±37.8
15
3.8
20
1.0
16:00
6:26
Bioquell*
Pure/500 |ig
240±40.3
15
3.8
20
1.0
16:00
7:42
STERIS*
Pure/500 |ig
398±44.2
15
2.5
20
2.2
13:40
10:09
STERIS*
Crude/500 |ig
398±44.2
15
2.5
20
2.2
13:40
10:09
STERISt
Crude/500 |ig
392±18.5
15
2.5
20
2.2
13:40
4:47
* Limited materials tested were industrial carpet, optical plastic, paper, and stainless steel,
f Limited materials tested were neoprene rubber, aluminum, ceramic tile, and unpainted concrete.
a Detailed data from each test number can be referenced in Appendix A.
b Concentration of hydrogen peroxide measured in the vapor phase during Phases 2 and 3.
The data generated from this investigation suggest that VPHP reduces the bioactivity of both a
commercially-available purified form of ricin toxin, as well as a crude form produced by Battelle
from whole castor beans. The purified ricin, as well as the whole castor beans, were purchased from
Vector Laboratories (Vector Labs, Burlingame, CA). A Bioquell Clarus C Phase 3 contact time of 8
or 16 h was required to achieve greater than 99 % reduction of pure and crude ricin, respectively, on
all materials tested at target inoculation level of 250 micrograms (|ig). A Phase 3 contact time of 16
h was required on carpet, plastic, paper and stainless steel with an increased inoculum target of 500
|ig. STERIS 1000ED required Phase 3 contact time of 13 h 40 min and a modified injection rate of
2.2 g/m to achieve greater than 99 % reduction of pure and crude ricin toxin at the increased
inoculum target of 500 |ig.
Testing two VPHP technologies was not the original intent of the study. However, due to repeated
unforeseen system failures of the STERIS generator at an injection rate of 1.0 g/min (injection rate
failure), it was necessary to utilize a second technology (Bioquell) to complete testing. Mitigation of
this failure required the STERIS injection rates to be adjusted from 3.8 and 1.0 g/min to 2.5 and 2.2
g/min, respectively, and the exhaust connections slightly opened to allow for a dilution effect. The
dilution resulted in obtaining the targeted 400 ppm at a higher Phase 3 injection rate, allowing the
STERIS generator to be used again starting with Test 7.
VPHP appears to be an effective decontaminant against ricin toxin utilizing the STERIS 1000ED at a
targeted 400 ppm for 14 h of hydrogen peroxide injection (Phases 2 and 3) as well as with the
Bioquell Clarus C, targeting microcondensation for 8 or 16 h. In general, the crude form of ricin was
more difficult to inactivate on plastic and carpet (Tests 1-4, 6-8).
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Contents
Disclaimer iii
Acknowledgments iv
Executive Summary v
Abbreviations/Acronyms ix
1.0 Introduction 1
2.0 Technology Description and Test Matrices 1
2.1 Technology Description 1
2.2 Test Matrix 3
3.0 Test Procedures 4
3.1 Ricin Toxins 4
3.2 Test Materials 4
3.3 Inoculation of Coupons 5
3.4 Fumigation Description and Procedures 6
3.5 Coupon Extraction and Ricin Toxin Quantification 9
3.6 Decontamination Efficacy 12
3.7 Surface Damage 13
4.0 Quality Assurance/Quality Control 14
4.1 Equipment Calibration 14
4.2 QC Results 14
4.3 Audits 14
4.4 QA/QC Reporting 15
4.5 Data Review 15
5.0 Summary of Results and Discussion 16
5.1 Operational Parameters 16
5.2 Efficacy Comparison of Ricin Forms 18
5.3 Effects of STERIS VPHP efficacy for Pure and Crude Ricin 19
5.4 Effects of Bioquell VPHP Efficacy for Pure and Crude Ricin 23
5.5 Surface Damage to Materials 26
5.6 Summary and Conclusion 26
6.0 References 27
Appendix A Detailed Test Results 1
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Figures
Figure 2-2. Bioquell, Inc. Clarus™ C 2
Figure 2-1. STERIS, Inc. 1000ED 2
Figure 3-1. Coupon Types from Left to Right: Aluminum, Neoprene Rubber, Optically Clear
Acrylic, Stainless Steel, Industrial Carpet, Ceramic Tile, Unpainted Concrete, and Paper... 5
Figure 3-2. Liquid Inoculation of Coupon Using a Micropipette 6
Figure 3-3. Aerial Schematic of VPHP Test Chamber and Attached Fumigant Generator 7
Figure 3-4. Representative Graph of Bioquell and STERIS Decontamination Cycles 8
Figure 3-5. Visual Demonstration of MTT Assay on a Microplate 11
Figure 3-6. Example of Ricin Cytotoxic Profile with Corresponding Absorbance Measured Using
a Microplate Reader 11
Figure 5-1. Summary of Average Percent Reduction between Pure Ricin and Crude Ricin per
Material Type ± Standard Deviation 18
Figure 5-2. Summary of Average Percent Reduction for STERIS 1000ED VPHP Generator
between Pure Ricin and Crude Ricin per Material Type ± Standard Deviation 19
Figure 5-3. Summary of VPHP Efficacy (Tests 1 and 2) Results, by Material, Comparing Pure
and Crude Ricin ± 95% Confidence Interval 20
Figure 5-4. Summary of VPHP Efficacy (Tests 7 and 8) Results, by Material, Comparing Pure
and Crude Ricin ± 95% Confidence Interval 21
Figure 5-5. Summary of VPHP Efficacy (Tests 9 and 10) Results, by Material, Comparing
Pure and Crude Ricin ± 95% Confidence Interval 22
Figure 5-6. Summary of Average Percent Reduction for Bioquell Clarus C VPHP Generator
between Pure Ricin and Crude Ricin per Material Type ± Standard Deviation 23
Figure 5-7 Summary of VPHP Efficacy (Tests 3 and 4) Results, by Material, Comparing Pure
and Crude Ricin ± 95% Confidence Interval 24
Figure 5-8. Summary of VPHP Efficacy (Tests 5 and 6) Results, by Material, Comparing Pure
and Crude Ricin ± 95% Confidence Interval 25
Tables
Table ES-1. Parameters Required to Achieve >99 % Reduction on All Materials* vi
Table 2-1 VPHP Test Matrix 3
Table 3-1. Test Materials 5
Table 3-2. Average Dilution Factors per Coupon Material 12
Table 4-1. Performance Evaluation Audits 14
Table 5-1. Actual Fumigation Conditions for VPHP Tests 17
Table 5-2. Parameters Required to Achieve >99 % Reduction on All Materials 18
Table 5-3. Summary of Average Percent Reduction between Pure Ricin and Crude Ricin per
Material ± 95% Confidence Interval Type 19
Table A-l. Inactivation of Pure Ricin Toxin Using VPHPa A-l
Table A-2. Inactivation of Crude Ricin Toxin Using VPHPa A-3
List of Appendices
Appendix A Detailed Test Results A-l
viii
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Abbreviations/Acronyms
4-PL four-parameter logistic
BSC biological safety cabinet
CI confidence interval
cm centimeter(s)
°C degree(s) Celsius
E-beam electron beam
EPA U.S. Environmental Protection Agency
HEPA high efficiency particulate air
HSRP Homeland Security Research Program
HVAC heating, ventilation, and air conditioning
h hour
HS homeland security
IV intravenous injection
kg kilogram(s)
kGy kilogray(s)
L liter(s)
lb pound
LD5o median lethal dose; individual dose required to kill 50
percent of a population of test animals
LOD limit of detection
|ig microgram(s)
|iL microliter(s)
mg milligram(s)
mL milliliter(s)
mil thousandth of an inch
min minute(s)
MTT 3-(4,5-dimethlythiazol-2-yl)-2,5-diphenyltetrazolium bromide
NA not applicable
ng nanogram(s)
nm nanometer(s)
NHSRC National Homeland Security Research Center
ORD Office of research and development
PBS phosphate buffered saline
ppm part(s) per million
QA quality assurance
QC quality control
QMP Quality Management Plan
RH relative humidity
rpm revolution(s) per minute
SD standard deviation
SE standard error
SFW sterile filtered water (cell-culture grade)
T&E Technology and Evaluation
TSA technical systems audit
VPHP vapor phase hydrogen peroxide
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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 agents. With an emphasis on decontamination and
consequence management, water infrastructure protection, and threat and consequence assessment,
HSRP is working to develop tools and information that will help with the cleanup of chemical or
biological contaminants introduced into buildings or water systems.
In 2013, several letters containing ricin toxin were sent to various locations, including the White
House and the office of the New York City mayor according to the US Attorney's Office in a memo
dated June 28, 2013. These contaminated letters had the potential to contaminate the corresponding
mail sorting facilities and equipment, creating an exposure risk for those working in the area. Ricin
toxin is a highly toxic protein produced within the beans of the Ricinus communis plant. The median
lethal dose (LDso) in mice is 5 micrograms per kilogram (|ig/kg) via intravenous (IV) injection.(1)
Extrapolations have been made that indicate a human LDso exposure could be ~1 to 5 milligrams per
kg (mg/kg) IV.(1)
This investigation was conducted as a screening process in which the efficacy of vapor phase
hydrogen peroxide (VPHP) was tested against both a pure and a crude form of ricin toxin applied to
materials representative of a mail sorting facility (aluminum, industrial carpet, ceramic tile, neoprene
rubber, optical plastic, paper, stainless steel, and unpainted concrete) to provide efficacy data
assessing the suitability of VPHP as a decontaminant for ricin toxin. Decontamination efficacy was
quantified as a percent reduction in the mass of ricin toxin that induced cellular cytotoxicity
recovered from test coupons compared to the mass of toxin recovered from positive control coupons.
Lastly, these data provide a side-by-side efficacy comparison for the pure form of ricin as compared
to a likely real-world crude preparation that could be used to assess the suitability of using the
purified toxin for future fumigant decontamination investigations.
2.0 Technology Description and Test Matrices
2.1 Technology Description
Two commercially available VPHP technologies were used for testing, the STERIS VHP 1000ED
(Mentor, OH) generator (Figure 2-1) and the Bioquell Clarus C (Horsham, PA) generator (Figure 2-
2). These fumigant-generating technologies are advantageous for large-scale room decontamination
due to the ease of fumigant delivery, ease of distribution within the targeted space, and the relatively
low toxicity of the hydrogen peroxide in comparison to other fumigants such as chlorine dioxide,
ethylene oxide or formaldehyde.(2) Cycle development is required for each unique space or chamber
to ensure proper concentration delivery as well as equal distribution throughout the space.
1
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Each technology has four common phases for the delivery of the hydrogen peroxide into the target
enclosure to be decontaminated. These phases define how quickly and in what concentration the
hydrogen peroxide is delivered into the VPHP test chamber as well as how long the concentration is
maintained. Phase 1 is a chamber conditioning phase in which injection lines and chamber surfaces
are warmed up and the space is dehumidified to a cycle-defined level (e.g., <40 % relative humidity
[RH]). For this phase, the two technologies differ in that the STERIS generator dehumidifies to a
much lower RH during this phase compared to the Bioquell Clarus
C. The result of starting at a lower RH is the increased capacity to
carry the hydrogen peroxide in the vapor phase upon injection of
the STERIS 35 % Vaprox solution (Cat. No. PB006US proprietary
hydrogen peroxide) without condensation. The Bioquell generator
is designed so that any commercially-available 30 to 35 %
hydrogen peroxide (Cat. No. H325-4, Fisher Scientific) can be
injected until saturation is achieved and microcondensation forms
on all surfaces. Because condensation is the endpoint, the starting
RH is less critical. Phase 2 is defined by setting an injection rate of
the peroxide solution and time of delivery. The purpose of the
higher injection rate during this phase is to allow for a rapid
increase in concentration to the desired parts per million (ppm) Figure 2~h STERIS'Inc- WOOED
level (STERIS) or to achieve microcondensation (Bioquell). Phase 3 is defined by further setting an
injection rate and delivery time, but in most cases at a reduced injection rate to maintain the ppm or
microcondensation achieved in Phase 2. The fourth and final phase is aeration, in which the
hydrogen peroxide concentration is reduced catalytically to
water and oxygen until low or no measurable hydrogen peroxide
remains. A list of tested parameters is shown in Table 2-1.
Throughout the entirety of the run, the STERIS 1000ED unit
catalytically breaks down the delivered hydrogen peroxide upon
returning to the unit. The air is then transferred through a
desiccant chamber, dried, and passed through the vaporizer to
add additional hydrogen peroxide before injecting back into the
VPHP test chamber. At the conclusion of the run, a 3 hour (h)
regeneration cycle is required to heat the desiccant material to
remove collected moisture and make the desiccant material
ready for the next decontamination cycle.
In contrast, the Bioquell Clarus C generator uses a dual-loop
system in which air is recirculated through the vaporizer to Figure 2-2, Bioquell, Inc. Clarus™ C
continually add additional hydrogen peroxide into the system. During the aeration phase, the unit
switches to a second loop where the hydrogen peroxide is catalytically degraded into water and
oxygen and the moisture is removed via a refrigerated coil. The condensate is then pumped into a
waste collection bottle.
clarus
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Testing two VPHP technologies was not the original intent of the study. However, due to repeated
unforeseen system failures of the STERIS generator at an injection rate of 1.0 g/min (injection rate
failure), it was necessary to utilize a second technology (Bioquell) to complete testing.
2.2 Test Matrix
The test matrix for the VPHP fumigation tests is shown in Table 2-1. Tests 6 and 9 were evaluated
utilizing a downselected set of materials. This selection was made to maintain a representative
selection of porous and non porous materials while selecting some of the more difficult to
decontaminate materials such as carpet and plastic. Test 10 materials were selected to complete the
samples that had not been tested at the higher inoculum level.
Table 2-1 VPHP Test Matrix
Ricin
mass
Decontamination Parameters
Test
Materials
VPHP
Test
Phase 1
Phase 2
Phase 3
Number
lig
Technology
Chamber
Duration
Rate
Duration
Rate
Duration
Phase 4
min
g/m
min
g/m
h
1
15
3.8
20
1.0
0.5
Stainless
Rubber
Plastic
2
STERIS
15
3.8
20
1.0
4
3
Aluminum
Carpet
250
774 liter
15
3.8
20
1.0
4
4
Ceramic
Concrete
Paper
(L)
Class III
15
3.8
20
1.0
8
5
Bioquell
15
3.8
20
1.0
16
Stainless
6*
Plastic
Carpet
Paper
500
15
3.8
20
1.0
16
7
Stainless
Rubber
15
2.5
20
2.2
8
<10 ppm
Plastic
8
Aluminum
Carpet
Ceramic
Concrete
250
774L
Class III
15
2.5
20
2.2
13.7
Paper
with
Stainless
STERIS
HVAC
9*
Plastic
Carpet
Paper
500
15
2.5
20
2.2
13.7
Rubber
10*
Aluminum
Ceramic
Concrete
500
15
2.5
20
2.2
13.7
*Only four materials tested to allow for increased inoculum.
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3.0 Test Procedures
This section provides an overview of the procedures used for the bench-scale evaluation of VPHP to
inactivate both pure and crude forms of ricin toxin on eight different materials. Testing was
performed in accordance with a peer reviewed and EPA approved Test/Quality Assurance (QA)
Plan.
3.1 Ricin Toxins
Testing was conducted with a commercially available form of ricin toxin (Cat. No. L-1090: Ricin
communis agglutinin II, 5 mg per milliliter [mg/mL] protein concentration, Vector Laboratories,
Burlingame, CA), which was stored at 2 to 8 degrees Celsius (°C) and used as received. In
addition, a crude preparation of the toxin was extracted from whole castor beans obtained from
Vector Laboratories (Vector Laboratories, Inc., Burlingame, CA). The crude version of ricin
toxin was prepared in house according to Battelle methods derived from the scientific literature.
(4) Briefly, the whole castor beans were de-husked and homogenized into a slurry, precipitated
from the solution, dialyzed, and rinsed in sterile phosphate buffered saline (PBS). The final crude
ricin toxin was prepared in sterile PBS at an approximate concentration of 5 mg/mL, and stored
at 2 to 8 °C.
3.2 Test Materials
The test materials used for decontamination testing included aluminum, industrial carpet, ceramic
tile, neoprene rubber, optical plastic, paper, stainless steel, and unpainted concrete. Information on
these materials is presented in Table 3-1, and a picture of each is presented in Figure 3-1. Material
coupons were cut to uniform length and width (Table 3-1) from larger pieces of stock material.
Materials were prepared for testing by either sterilization via electron beam (E-beam) irradiation at
-200 kilogray (kGy; E-beam Services Inc., Lebanon, OH) or autoclaving at 121 °C for 15 minutes
(min). E-beam-irradiated material coupons were sealed in 6 mil (thousandth of an inch) Uline Poly
Tubing (Cat. No. S-2940, Uline, Chicago, IL), and autoclaved coupons were sealed in sterilization
pouches (Cat. No. 01-812-50, Fisher, Pittsburgh, PA) to preserve sterility until the coupons were
ready for use. Sterilization was intended to minimize contamination by microorganisms that might
interfere with the cell-based assay used to assess ricin bioactivity.
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Table 3-1. Test Materials
Material
Lot, Batch, or ASTM No., or
Observation
Manufacturer/
Supplier Name
Location
Approximate Coupon
Size, Width x Length x
Thickness
Material
Preparation
Stainless
Steel
Grade 304. gauge 12
Adept Products,
West Jefferson, OH
1.9 centimeters (cm) x 7.5
cm x 0.2 cm
Autoclave
Neoprene
Rubber
Nomnarking Neoprene Rubber
Part # 8837K214
McMaster Can-
Aurora, OH
1.9 cm x 7.5 cm x 0.3 cm
E-Beam
Optical
Grade
Plastic
Optically Clear Cast Acrylic Sheet
McMaster Item #8560K263
McMaster Can-
Aurora, OH
1.9 cm x 7.5 cm x 0.3 cm
E-Beam
Aluminum
Grade 2024
Adept Products,
West Jefferson. OH
1.9 cm x 7.5 cm x 0.2 cm
Autoclave
Carpet
Shaw Swizzle EcoWorx, Style: 10401
Color: Jacks
Shaw Industries.
Dal ton. GA
1.9 cm x 7.5 cm x 0.7 cm
E-Beam
Ceramic
Tile
Style Selections White Matte Ceramic
Floor Tile Item#: 437485
Lowes,
Milliard. OH
1.9 cm x 7.5 cm x 0.8 cm
Autoclave
Unpainted
Concrete
Cut Cinder Block
Lowes,
Milliard. OH
1.9 cm x 7.5 cm x 0.7 cm
Autoclave
Paper
Boise Aspen Laser Paper 24 pounds
(lb)
Part #BPL-2411-RC
Office Max,
Milliard. OH
1.9 cm x 7.5 cm x 0.3 cm
E-Beam
I 111 IU
Figure 3-1. Coupon Types from Left to Right: Aluminum, Neoprene Rubber, Optically Clear Acrylic,
Stainless Steel, Industrial Carpet, Ceramic Tile, Unpainted Concrete, and Paper
3.3 Inoculation of Coupons
Test and positive control coupons were placed on a flat surface within a Class II biological safety
cabinet (BSC) and inoculated individually with a target value of approximately 250 or 500 jig of
either the purified or crude ricin toxin. Actual delivered mass of toxin per material was determined
by cell-based bioassay. A 50 or 100 uL aliquot of a stock suspension of approximately 5 mg/mL was
dispensed using a micropipette and applied as a single or double streak across the coupon surface
(see Figure 3-2). This approach provided decreased drying times (-1.5 h) and a more uniform
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distribution of toxin across the coupon surface than would be obtained through a single drop of the
suspension. After inoculation, the coupons were transferred to a Class IE BSC and left undisturbed to
dry for approximately 1 h (or until visually dry) under ambient conditions, -22 °C and 40 % RH.
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, and environmental condition included were:
• Three test coupons (inoculated with ricin toxin and exposed to VPHP)
• Three dry time controls (inoculated with ricin toxin and extracted after 1 h drying time,
conducted for test one only)
• Three positive controls (inoculated with ricin toxin but not exposed to VPHP, stored at
ambient conditions)
• One laboratory blank (not inoculated and not exposed to VPHP)
• One procedural blank (not inoculated and exposed to VPHP).
Approximately 1 h post inoculation (or until materials were visually dry), coupons intended for
decontamination (including blanks) were transferred into the test chamber and exposed to the VPHP
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.
3.4 Fumigation Description and Procedures
Figure 3-3 shows a schematic of the VPHP test chamber and vapor generating system. Vapor phase
hydrogen peroxide decontamination testing was conducted within a test chamber comprised of a 774
L Class III BSC (The Baker Company, Sanford, ME) that was hard-ducted to the facility heating,
ventilation, and air conditioning (HVAC) filtered exhaust system. The VPHP test chamber was
modified with sensors capable of monitoring temperature, RH and VPHP concentration. A low-
speed fan was placed inside the test chamber to ensure a homogeneous distribution of VPHP
throughout. The STERIS and Bioquell generators were utilized based on availability and
performance throughout testing. The VPHP generators were connected to the test chamber, and the
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hydrogen peroxide delivered via supply and return hoses. In addition to each generator having
internal high efficiency particulate air (HEPA) filters, external in-line HEPA filters were used to
maintain containment and eliminate any potential contamination of the two technologies.
Main Chamber /— Test Sample Rack
Glove Ports
Trasfer Chamber
Supply Valve
Exhaust Va ve
Temp/%RH Datalogger
HEPA Filter
x— Mixing Fan
VPHP Concentration Probe
J L
HEPA Filter
Return Hose
Supply Hose
VPHP Generator
Figure 3-3. Aerial Schematic of VPHP Test Chamber and Attached Fumigant Generator
Temperature and RH were monitored and recorded every minute within the VPHP test chamber
using a U14 HOBO data logger (Cat. No. U12-12, Onset Corp., Bourne, MA), the hydrogen
peroxide concentrations were monitored using an ATI B-12 wet gas transmitter (Cat. No. B12-34-8-
2000-1, Analytical Technology Inc., Collegeville, PA), and the data were recorded by a UX120
HOBO data logger (Cat. No. UX120-006M, Onset Corp., Bourne, MA). During each test, inoculated
test samples were placed inside the VPHP test chamber and the chamber was sealed. The test
samples were allowed to dry for approximately 1 h (or until visually dry). Once dry, the controls
were removed by placing samples into a 9 L Lock & Lock® airtight control chamber (Cat. No.
HPL838 Lock & Lock, Farmers Branch, TX) and removed from the VPHP test chamber and placed
into a Class II BSC for the remainder of the test. Once the control samples were moved, the
predetermined decontamination cycle was performed. As previously stated, each technology has four
similar phases in common but differs in regards to the endpoint either being vapor phase or
microcondensation mode of decontamination.
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Figure 3-4 shows a representative graph of both a STERIS 1000ED and Bioquell Clarus C
decontamination run.
Bioquell® VPHP Cycle
700
600
500
400
300
200
100
0
1
Phase 1
Phase 2
Phase 3
Phase 4
¦Temp, °C
¦RH, %
¦PPM
STERIS VPHP Cycle
^ ^ ^ J?
Phase 1 Phase 2 Phase 3 Phase 4 Temp, °C RH, % PPM
Figure 3-4. Representative Graph of Bioquell and STERIS Decontamination Cycles
The VPHP test chamber heating, ventilation and air conditioning (HVAC) exhaust was utilized
during aeration to speed up the process (Phase 4). The test chamber was allowed to aerate until the
VPHP levels in the chamber reached <10 ppm. At this time, the samples were removed and
processed as described in Section 3.5.
8
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The control samples were held inside a 9 L Lock & Lock® airtight container at ambient laboratory
conditions for the duration of the experiment. The temperature and RH were not controlled within
this control chamber. The temperature and RH of the control chamber were measured and data
logged using a HOBO® data logger model U12 (Cat. No U12-11, Onset Corp., Bourne, MA).
As in previous studies,(2'5) multiple coupons of each material were inoculated with the ricin toxin and
placed on a wire rack inside the VPHP test chamber. Blank (i.e., not inoculated) and positive control
(i.e., inoculated but not decontaminated) samples were also prepared for each material and were
utilized with data from the test samples (inoculated and decontaminated) to determine
decontamination efficacy.
Ten VPHP decontamination tests were conducted at predetermined cycles as shown in Table 2-1.
Phase 1 was consistent throughout testing for both technologies at 15 min. Contact times and
injection rates for Bioquell Phase 2 were 3.8 g/min and 20 minutes. STERIS Phase 2 parameters
ranged from 3.8 to 2.5 g/min at 20 min. The Bioquell Phase 3 injection rate was 1.0 g/min while the
contact time ranged from 4 to 6 h. The STERIS Phase 3 injection rate varied from 1.0 to 2.2 g/min
and contact times ranged from 30 min to 13.7 h. Phase 4 parameters were the same for all tests at <
10 ppm VPHP. The change in injection rates for the STERIS generator was due to three failed
attempts at Test 3 (data not reported) in which the lower published injection rate limit for the
STERIS unit of 1.0 g/min prompted injection rate failure alarms. When in alarm mode, the STERIS
generator automatically aborted the cycle and initiated aeration mode (Phase 4). The Bioquell
generator was utilized for Tests 3 to 6 while the STERIS generator was used for Tests 1, 2, and 7 to
10.
3.5 Coupon Extraction and Ricin Toxin Quantification
After decontamination, test coupons, positive controls, and blanks were individually placed in 50 mL
polypropylene conical tubes containing 10 mL of sterile PBS for extraction. The vials were capped,
placed on their sides and agitated on an orbital shaker for 15 min at approximately 200 revolutions
per minute (rpm) at room temperature. Residual active toxin in the test and control coupon extracts
was determined using the bioassay approach described below.
The mechanism of action by which ricin toxin exerts its toxic effect is through inhibition of
protein synthesis within cells. Such inhibition of protein production leads to cell death.
Therefore, an in vitro cytotoxicity assay was used to evaluate the level of bioactive ricin toxin
extracted from both decontaminated and control material coupons. The bioassay used in this
evaluation for determining the cytotoxicity (concentration) of bioactive ricin toxin is based on
the 3-(4,5-dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT) assay developed by
Mosmann.(4) Cytotoxicity is reported as mass of bioactive toxin as determined using a reference
standard prepared from a purified form of ricin toxin.
To conduct this assay, Vero cells (kidney epithelial cells from the African green monkey) were
seeded in wells of a 96-well microplate at a density of approximately 2 x io4 cells/well. Cells
9
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were then incubated for approximately 18to30hat37±2°C under 95 % air and 5 % carbon
dioxide and exposed to the various coupon extracts (test, positive controls and blank controls) by
adding 100 |iL of extract or dilution to each well. Following 48 to 72 h exposure to sample
extracts, the cells were incubated in the presence of MTT, where mitochondrial enzymes convert
the yellow MTT to a purple formazan salt. The absorbance of this purple reaction product, read
at 570 nanometers (nm) using a SPECTRAmax PLUS384 microplate reader (Molecular Devices,
Sunnyvale, CA), is directly proportional to the number of living cells and inversely proportional
to the cytotoxic potential of ricin toxin (Figures 3-5 and 3-6). For all dilutions and sample
transfers into the individual wells of a 96-well plate, a micropipette was used in which the pipette
tip was replaced between wells to ensure that cross contamination did not occur.
To determine the concentration of ricin toxin from each test sample, a ricin toxin standard
(purified, Vector Laboratories, Inc., Burlingame, CA) was prepared from the commercial
purchased stock solution and assayed in parallel on each test plate. The ricin toxin stock solution
(purified) was used to prepare a seven-point standard curve of absorbance versus calculated mass
of ricin toxin protein. For each standard and test sample, absorbance values of the reference
wavelength (630 nm) were subtracted from the absorbance values at 570 nm for each well. For
each point used in generating the standard curve, the mean absorbance values (Y-axis) were
plotted against the concentration in nanograms (ng)/mL, and a four-parameter logistic (4-PL)
curve was generated by the SoftMax Pro Version 4.7 software included in the SPECTRAmax
microplate (Molecular Devices, Sunnyvale, CA) reader using the equation:
(max- min)
Y = 111111+ =- (1)
1 + (X/C)B K '
where:
Y = absorbance %:
X = concentration of ricin ng/niL;
max = Y-value of the asymptote at the low values of X % absorbance;
min = Y-value of the asymptote at the high values of X % absorbance:
B = value related] to the slope of the curve between the asymptotes;
C = X-value of the midpoint between max and min ng/niL
10
-------
Purple = cells alive;
little to no toxin
Increasing ricin
concentration
Yellow - cells dead;
abundant toxin
Figure 3-5. Visual Demonstration of MTT Assay on a Microplate
Ricin Standard Curve
Purple = aliye cells; little
\ to no toxin
Yellow = dead ceils
abundant toxin
0.1 1 10
Protein (ng/mL)
Figure 3-6. Example of Ricin Cytotoxic Profile with Corresponding Absorbance Measured Using a
Microplate Reader
11
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Throughout the study, the inherent cytotoxicity of coupon extracts from laboratory and
procedural blank coupons was assessed to determine a starting dilution that would mitigate any
potential confounding cytotoxic effects observed in the ricin bioassay. To account for this
potential for coupon extract-induced cytotoxicity in the ricin bioassay, the dilution factor of
coupon extracts exhibiting cytotoxicity of less than 20 % when compared to negative controls
(cell culture medium only) were selected as the starting dilution for all test samples. The dilution
scheme effectively baselined the cytotoxicity of the test coupons (see Table 3-2).
Table 3-2. Average Dilution Factors per Coupon Material
Material
Dilution Factors Required to "Zero Out" Coupon
Cytotoxicity
Stainless Steel
1:35
Neoprene Rubber
1:23
Optical Grade Plastic
1:6
Aluminum
1:10
Carpet
1:166
Ceramic Tile
1:27
Unpainted Concrete
1:58
Paper
1:34
3.6 Decontamination Efficacy
The performance, or efficacy, of VPHP was assessed by determining the mass of bioactive toxin
extracted from each test coupon after decontamination compared to the average mass of
bioactive toxin extracted from the positive control coupons.
Efficacy (% reduction) of a decontaminant for a test toxin/test condition on the 7th coupon material
was calculated as the difference between the mean control mass values and the individual test mass
values, i.e.:
(2)
Massc^Mas^, % = (j _ , 10„
Masscij \ Masscij j
where MassC; refers to the / individual mass values obtained from the positive control coupons, Mass*?
refers to the j individual mass values obtained from the corresponding test coupons, and the overbar
designates a mean value. In tests conducted under this plan, there were three positive controls and
three corresponding test coupons (i.e.,j = 3) for each coupon.
In samples where no bioactive toxin was observed in any of the three test coupon extracts after
decontamination, an adjusted limit of detection (LOD) value for that material was assigned. The
adjusted LOD was defined as mass of ricin toxin that corresponded to the lowest dilution factor
12
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in the standard curve. The assigning of adjusted LOD values for the test samples occurred when
the decontaminant was highly effective, and diluted sample values were below the linear range
of the 4-PL standard curve. In such cases, the final efficacy (adjusted LOD) for that material
was calculated by multiplying the lowest value assayed in the standard curve by the lowest
dilution factor for each test material based on coupon extract cytotoxicity limits (determined as
described in Section 3.5). The resultant ricin mass values were reported as greater than or equal
to (>) the adjusted LOD.
The variance of the mean percent reduction can be estimated using the Taylor series
approximation. Let S2q be the variance of the three positive control coupons, and let S2tj be the
variance of the three test coupons. Then the estimated standard error of percent reduction is:
where the number 3 represents the number j of coupons in both the control and test data sets.
Each efficacy result is reported as a mass value with an associated 95 % confidence interval (CI),
calculated as follows:
Significant differences in efficacy for the different test conditions and toxin types were assessed
using the 95 % CI 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 MASS as > LOD.
3.7 Surface Damage
The physical effect of VPHP on the materials was qualitatively monitored during the evaluation. This
approach provided a gross visual assessment of whether the decontaminant altered the appearance of
the test materials. The procedural blank (coupon that is decontaminated, but has no toxin applied)
was visually compared to a laboratory blank coupon (a coupon not exposed to the decontaminant and
having no toxin applied). Obvious visible damage might include structural damage, surface
degradation, discoloration, or other aesthetic impacts.
Masst,
Masse
Masst,
Masse,
* 100%.
(3)
95 % CI = Efficacy (% Mass Reduction) ± (1.96 x SE)
(4)
13
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4.0 Quality Assurance/Quality Control
QA/quality control (QC) procedures were performed in accordance with the Technology Testing and
Evaluation (T&E) Program Quality Management Plan (QMP) and the test/QA plan. The QA/QC
procedures and results are summarized below.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, microplate reader, BSCs) and monitoring devices (e.g.,
thermometer, hygrometer, VPHP sensor) used at the time of the evaluation were verified as being
certified, calibrated, or validated.
4.2 QC Results
QC efforts conducted during decontaminant testing included dry time control samples (inoculated,
dried for -1.5 h, and not decontaminated), procedural blanks (not inoculated, decontaminated),
laboratory blanks (not inoculated, not decontaminated), and inoculation control samples (analysis of
the stock toxin suspension).
Dry time control samples were run once during Test 1 to determine the loss of cytotoxicity over the
~1.5 h drying period. Percent recoveries ranged from 1.02 to 1.77 %. The amount of ricin recovered
from these controls was sufficient to determine % reduction due to the cytotoxicity assay standard
range of 0.1 to 10 ng. Outlier tests were not performed as this test was conducted only once.
All procedural and laboratory blanks met the acceptance criterion by the use of dilution to mitigate
inherent material specific cytotoxicity as previously discussed. Inoculation control samples were
taken from the purified and crude stock toxin suspension each day of testing and assayed against the
4-PL standard curve. Using a Grubbs outlier test, the inoculation control samples were assessed and
no outliers were found for target inoculum levels of 250 |ig. The increased inoculum target of 500
|ig was not assessable via this test as six replicates are required and only three tests were conducted
with 500 |ig inoculum.
4.3 Audits
4.3.1 Performance Evaluation A udit
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.
Table 4-1. Performance Evaluation Audits
Measurement
Audit
Procedure
Allowable
Tolerance
Actual
Tolerance
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10 %
± 0.00 % to 7.63 %
Time
Compared to independent clock
± 2 seconds/hour
0 seconds/hour
Temperature
Compared to independent calibrated thermometer
± 2 °C
± 0 to 0.3 °C
Relative Humidity
Compare to independent calibrated hygrometer
± 10 %
±1%
14
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4.3.2 Technical Systems Audit
Observations and findings from technical systems audits (TSAs) were documented and submitted to
the laboratory technical lead for response. TSAs were conducted on May 29, June 10, and June 20,
2014 to ensure that 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. One deviation was noted during the TSA.
4.3.3 Deviations
A deviation was prepared to address the finding in which no sterile filtered water (SFW) was
inoculated onto the blank coupons. In practice, this inoculation of coupons with diluent only had
been eliminated from previous work assignments, and its inclusion was an oversight in test/QA plan
preparation. An additional deviation from the test/QA plan included a change to the Phase 4 stopping
point from <1 ppm to <10 ppm. This change had little effect on overall testing and enabled sample
processing during normal business hours.
4.3.4 Data Quality Audit
At least 10 % of the data acquired during the evaluation were audited. A 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
verified. Only minor issues were noted with the data, mostly data transcription errors that were
corrected.
4.4 QA/QC Reporting
Each assessment and audit were documented in accordance with the test/QA plan and QMP. For
these tests, findings were noted (none significant) in the data quality audit, and no followup
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.
4.5 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 this report.
15
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5.0 Summary of Results and Discussion
The decontamination efficacy of two VPHP generators was evaluated against purified and crude ricin
toxin inoculated onto porous and nonporous material coupons. For the ten tests in this evaluation, the
decontamination cycles varied Phase 2 injection rates from 2.5 to 3.8 g/min for 20 min and varied
Phase 3 injection rates from 2.5 to 3.8 g/min from 30 min to 16 h contact times. The STERIS VPHP
cycle achieved a lower RH throughout the decontamination cycle, which enabled the hydrogen
peroxide to remain in the vapor phase for the entire decontamination cycle. Decontamination runs
using the Bioquell unit exhibited a much higher RH, which resulted in the formation of micro
condensation. This deposition of hydrogen peroxide on all surfaces within the VPHP test chamber
reduced the level of hydrogen peroxide measured in the vapor phase. Thus, the actual hydrogen
peroxide concentrations deposited onto the surface were assumed to be higher than in the vapor
phase, but were not measured. During the Bioquell aeration phase, a second increase of VPHP was
observed as a result of the condensed hydrogen peroxide evaporating off the surfaces VPHP test
chamber and returning to the vapor phase. In a larger chamber or room setting, size, complexity of
floor plan, and material compatibility must be considered and chamber specific cycle parameters
must be developed.
Testing two VPHP technologies was not the original intent of the study. However, due to repeated
unforeseen system failures of the STERIS generator at the lowest published injection rate of 1.0
g/min (injection rate failure), it was necessary to utilize a second technology (Bioquell) to complete
testing. Mitigation of this failure required the STERIS injection rates to be adjusted from 3.8 and 1.0
g/min in a sealed VPHP test chamber, to 2.5 and 2.2 g/min, respectively, and the exhaust connections
slightly opened to allow for a dilution effect. The dilution resulted in obtaining the targeted 400 ppm
at a higher Phase 3 injection rate, allowing the STERIS generator to be used again starting with Test
7.
VPHP appears to be an effective decontaminant against pure and crude forms of ricin toxin utilizing
the STERIS 1000ED at a targeted 400 ppm for 14 h of hydrogen peroxide injection (Phases 2 and 3,
Tests 9-10) as well as with the Bioquell Clarus C, targeting microcondensation for 8 or 16 h (Tests 4-
6). In some cases the test temperature/RH was notably higher than the control chamber. Additional
testing is needed to study the effect of increased temperature and RH on the inactivation of ricin
toxin in the absence of VPHP. Additional testing is also needed to confirm these data at higher
inoculum levels, as only three tests were completed using a targeted 500 |ig inoculation quantity.
Further testing is also needed to confirm the data presented here as well as to test additional surface
materials and combinations of cycle parameters including lower concentrations of VPHP and longer
contact times to potentially address application challenges when needed in larger area applications.
5.1 Operational Parameters
The temperature, RH, and VPHP concentrations during each test were controlled by each respective
generator technology, as described in Section 3.0. These VPHP generating technologies were set to
the target injection rates and contact times and initiated upon test sample readiness. Readings were
16
-------
taken once every minute for the duration of each test. The actual operational parameters for each test
are shown in Table 5-1 and reported as the average value ± standard deviation (SD).
Table 5-1. Actual Fumigation Conditions for VPHP Tests
Test
Number
Technology
VPHP Concentration
(ppm)
Temperature (°C)a
RH (%)a
Phase 4
Time
(h:min)
Target
Actual*
Fumigation
Actual*
Control
Actual*
Fumigation
Actual*
Control
Actual*
1
STERIS
400
480±175
25.1±3.75
20.3±0.50
26.6±19.2
30.7±1.61
17:41
2
STERIS
400
414±108
27.2±2.97
22.3±0.23
28.3±23.3
57.0±1.18
16:00
3
Bioquell
NA!
310±58.9
29.8±1.83
22.0±0.24
45.7±30.7
59.7±0.36
13:22
4
Bioquell
na!
279±45.0
30.4±2.67
21.7±0.13
82.2±9.87
60.5±0.41
11:41
5
Bioquell
na!
301±37.8
30.7±2.87
20.8±0.28
78.6±17.5
58.5±0.37
6:26
6
Bioquell
na!
240±40.3
29.8±3.51
21.6±0.69
70.3±24.1
53.9±1.54
7:42
7
STERIS
400
349±23.9
25.7±1.89
22.3±0.54
56.1±17.6
57.9±1.08
10:47
8
STERIS
400
387±21.7
25.5±1.78
22.4±0.53
62.6±9.01
53.5±1.17
9:09
9
STERIS
400
398±44.2
25.4±1.20
22.5±0.47
65.8±16.4
53.2±1.36
10:09
10
STERIS
400
392±18.5
25.1±1.68
21.8±0.41
70.3±13.6
56.6±1.01
4:47
* Data reported as average ± SD.
^ Bioquell technology targets micro condensation in lieu of ppm.
aNo defined temperatures or RH were targeted.
Table 5-2 shows the operational parameters required to achieve >99 % reduction on all material
types tested (aluminum, industrial carpet, ceramic tile, neoprene rubber, optical plastic, paper, and
stainless steel) except unpainted concrete (Tests 4-6, 9-10). Data for unpainted concrete were not
included in the summarized results due to little to no recoverable ricin toxin from positive control
samples. Although not evaluated, the caustic nature of this material may have affected the bioactivity
of the ricin. Actual operational parameters as measured were well within acceptable ranges and are
detailed above. The detailed decontamination efficacy results are provided in Appendix A. As seen in
Table 5-2, a Phase 3 contact time of 8 or 16 h was required to achieve >99 % reduction of pure and
crude ricin with the Bioquell Clarus C VPHP generator using an inoculum of -250 |ig. A Phase 3
contact time of 16 h was required to achieve >99 % reduction when the amount of inoculum was
increased to -500 |ig, but this contact time was only tested on industrial carpet, optical plastic, paper,
and stainless steel. These test materials were down selected to represent porous and non-porous
surfaces. STERIS 1000ED required a 13 h 40 min Phase 3 time with a target of 400 ppm to achieve
>99 % reduction on materials for the crude and pure form of ricin at an inoculum of-500 |ig.
17
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Table 5-2. Parameters Required to Achieve >99 % Reduction on All Materials
Technology
Ricin
Form/Target
Mass
lig
Avg
VPHP
ppm±SDbc
Phase 1
Phase 2
Phase 3
Phase 4
Duration
(min)
Injection
Rate
(g/m)
Duration
(min)
Injection
Rate (g/m)
Duration
(h:min)
Duration
(h:min)
Bioquell
Pure/250
279±45.0
15
3.8
20
1.0
8:00
11:41
Bioquell
Crude/250
301±37.8
15
3.8
20
1.0
16:00
6:26
Bioquell*
Pure/500
240±40.3
15
3.8
20
1.0
16:00
7:42
STERIS*
Pure/500
398±44.2
15
2.5
20
2.2
13:40
10:09
STERIS*
Crude/500
398±44.2
15
2.5
20
2.2
13:40
10:09
STERISt
Crude/500
392±18.5
15
2.5
20
2.2
13:40
4:47
* Materials tested were limited to industrial carpet, optical plastic, paper, and stainless steel,
f Materials tested were limited to neoprene rubber, aluminum, ceramic tile, and unpainted concrete.
a Detailed data from each test number can be referenced in Appendix A.
b Concentration of hydrogen peroxide measured in the vapor phase during Phases 2 and 3.
5.2 Efficacy Comparison of Ricin Forms
Results comparing the average percent reduction ± SD for the pure and crude ricin are shown in
Figure 5-1 and Table 5-3. These results are averages for all tests conducted using both VPHP
technologies, different inoculum amounts, and various testing conditions. In general, the results in
Table 5-3 show that little difference exists when comparing crude to pure ricin with percent reduction
ranging from 73.8 to 98.1 % (excluding concrete) for crude ricin, and 90.1 to 97.9 % (excluding
concrete) for pure ricin. When compared to the crude ricin, the pure ricin on neoprene rubber, optical
plastic, industrial carpet, and paper exhibited an average difference in efficacy ranging from -7.44 to
-16.3 percent. In contrast, on stainless steel, aluminium, and ceramic tile, the crude ricin was less
resistant to VPHP than pure ricin with average differences ranging from 0.10 to 1.03 percent. A
positive result indicates that the crude ricin was inactivated to a higher degree (less resistant) than
pure ricin.
Pure vs. Crude Percent Reduction
120%
IIIMIIilii
M •§ "S § & 's "S &
.s •§ ^ -g « 2 g (2
5 Pi ^ B °
K £ u U
<
¦ Pure Ricin ¦ Crude Ricin
Figure 5-1. Summary of Average Percent Reduction between Pure Ricin and Crude Ricin per Material
Type ± Standard Deviation
18
-------
Table 5-3. Summary of Average Percent Reduction between Pure Ricin and Crude Ricin per Material ±
95% Confidence Interval Type
Material Type
Average Percent Reduction
Difference (%)*
Pure Ricin (%)
Crude Ricin (%)
Stainless Steel
96.29 ±8.19
96.40 ± 6.05
±0.10
Neoprene Rubber
97.02 ±3.50
87.49 ±21.6
-9.53
Optical Plastic
97.92 ±2.49
90.48 ± 12.4
-7.44
Aluminum
97.77 ±2.99
98.10 ±3.40
±0.33
Industrial Carpet
90.13 ± 12.7
73.79 ±36.6
-16.34
Ceramic Tile
96.71 ±6.75
97.75 ±4.73
±1.03
Unpainted Concrete
31.26 ±40.7
56.08 ±38.6
±24.82
Paper
90.92 ± 20.6
83.03 ±30.9
-7.88
*Results shown as difference in average efficacy (percent reduction) ± standard deviation. A positive result indicates that the crude ricin was
inactivated to a higher degree than pure ricin.
TAveraged performed across all 10 tests.
5.3 Effects of STERIS VPHP efficacy for Pure and Crude Ricin
Results comparing the average percent reduction for the pure and crude ricin tested using the
STERIS 1000ED are shown in Figure 5-2. These results are averages including all tests performed
using the STERIS generator, different inoculum amounts and various testing conditions. Although
some significant differences between crude and pure ricin are shown in Figures 5-3 to 5-5 for
individual tests, the averages in Figure 5-2 show there is little to no difference in decontamination
efficacy when comparing the crude and pure forms of ricin when decontaminated with the STERIS
STERIS Pure vs. Crude Percent Reduction
liiiHiii
w 0) -rt B V -3 -ft D
^ -S £ .5 & b $
• 9 B X <-H r \ S-H Cl P~l
5 Pi ^ B °
K £ u U
<
¦ Pure Ricin ¦ Crude Ricin
Summary of Average Percent Reduction for STERIS 1000ED VPHP Generator between Pure
Ricin and Crude Ricin per Material Type ± Standard Deviation
The percent reduction results by material, for each test, are shown in Figures 5-3 through 5-5.
Differences in efficacy between the two ricin forms on a material are significant if the 95 % CIs of
the two efficacy results do not overlap. The STERIS 1000ED, when testing at suboptimal conditions
with crude ricin, was more difficult to inactivate on plastic, carpet, and paper shown in Figure 5-3
1000ED.
120%
Q
^ 100%
§ 80%
H 60%
§ 40%
§ 20%
0%
Figure 5-2.
19
-------
(Test 1 and 2). When Phase 2 contact times were increased for Tests 8 and 9 to 13 h 40 min, and the
inoculum was increased to target of 500 |ig (excluding concrete), a >99 percent reduction was
achieved on all materials inoculated with crude ricin and all materials inoculated with pure ricin
except industrial carpet (98.7 percent reduction). Detailed values for the decontamination efficacy
results are provided in Appendix A.
Test 1 (STERIS)
120%
100%
u
-H
a
-------
Test 7 (STERIS)
120%
100%
U 80%
-H
o
•§
u
Ph
60%
40%
20%
0%
.5
GO
1
£
.1
I
<
o
-------
120%
Test 9 (STERIS)
¦ Pure Ricin ¦ Crude Ricin
120%
100%
Test 10 (STERIS)
u 80%
-H
o
u
Ph
cN
60%
40%
20%
0%
j3
1
1
£
1
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-------
5.4 Effects of Bioquell VPHP Efficacy for Pure and Crude Ricin
Results comparing the average percent reduction for the pure and crude ricin tested against the
Bioquell Clarus C are shown in Figure 5-6. These results are averages including all tests performed
using the Bioquell generator, different inoculum amounts and various testing conditions. Although
some significant differences between crude and pure ricin are shown in Figures 5-7 to 5-8, the
averages in Figure 5-6 show there is little to no difference in decontamination efficacy when
comparing the crude and pure forms of ricin when decontaminated with the Bioquell Clarus C.
Bioquell Pure vs. Crude Percent Reduction
ilium
.a b .a y
n -2 .g .3 a a a £
« (5 ^ ° $ §
cw £ ° u
<
¦ Pure Ricin ¦ Crude Ricin
Summary of Average Percent Reduction for Bioquell Clarus C VPHP Generator between
Pure Ricin and Crude Ricin per Material Type ± Standard Deviation
The percent reduction results by material, for each test, are shown in Figures 5-7 and 5-8. Differences
in efficacy between two ricin forms on a material are significant if the 95 % CIs of the two efficacy
results do not overlap. When testing the Bioquell Clarus C, crude ricin was more difficult to
inactivate on rubber and plastic with a Phase 2 contact time of 4 hours (Test 3; Figure 5-7). When
Phase 2 contact times were increased to 8 h (Test 4; Figure 5-7), >99 percent inactivation of pure
ricin was achieved on all materials (except concrete) and all materials for crude ricin except paper
that exhibited a 98.1 percent reduction. Test 5 increased Phase 2 duration to 16 hours and (excluding
concrete) resulted in >99 percent reduction of crude ricin on all materials, while having a slightly
reduced efficacy for pure ricin on stainless and carpet (98.9 and 98.8, respectively). When the ricin
inoculum was increased to -500 |ig (Test 6; Figure 5-8), >99 percent reduction was achieved for
pure ricin on the limited number of materials tested (stainless steel, rubber, carpet, and paper) and
crude ricin for all materials except carpet that achieved 97.1 percent reduction. Detailed values for
the decontamination efficacy results are provided in Appendix A.
120%
Q
^ 100%
§ 80%
H 60%
§ 40%
§ 20%
0%
Figure 5-6.
23
-------
Test 3 (Bioquell)
120%
100%
o
-H
#o
"o
u
Ph
60%
40%
20%
0%
.5
GO
I
£
.1
I
<
o
-------
Test 5 (Bioquell)
120%
100%
u
-H
¦a
u
Ph
80%
60%
40%
20%
0%
.5
GO
1
.1
I
<
o
-------
5.5 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
VPHP 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 the eight material
surfaces after exposure to VPHP.
5.6 Summary and Conclusion
The data generated from this project demonstrate that VPHP reduces the bioactivity of both a
commercially-available purified form of ricin toxin, as well as a crude form produced from castor
beans. The Bioquell Clarus C generator with a contact time of 8 or 16 h demonstrated a greater than
99 % reduction of pure and crude ricin, respectively, on all materials tested at target inoculation level
of 250 micrograms (|ig). A contact time of 16 h was required for carpet, plastic, paper and stainless
steel with an increased inoculum target of 500 |ig. The STERIS 1000ED required a contact time of
13 h 40 min and a modified injection rate of 2.2 g/m to achieve greater than 99 % reduction of pure
and crude ricin toxin at the increased inoculum target of 500 |ig.
VPHP appears to be an effective decontaminant against ricin toxin utilizing the STERIS 1000ED at a
targeted 400 ppm for 14 h of hydrogen peroxide injection. Similarly the Bioquell Clarus C required
a time of 8 or 16 h depending on the material. In general, the crude form of ricin was more difficult
to inactivate on plastic and carpet.
Impact of Study
One of the primary goals of this project was to demonstrate the effectiveness of VPHP for
inactivating ricin on surfaces in a mail sorting machine. This work identified the operational
conditions necessary to inactivate ricin on a variety of surfaces, including those that are found in a
mail sorting machine. VPHP is compatible with most materials and will not damage high value items
such as mail sorting machines. VPHP is a viable option for the decontamination of mail sorting
machines that may have come into contact with the ricin toxin.
26
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6.0 References
1. BMBL. Biosafety in Microbiological Laboratories. 2009. 5th Edition, HHS Publication No.
(CDC) 21-1112 Revised December.
2. Rogers, J.V., C.L.K. Sabourin, Y.W. Choi, W.R. Richter, D.C. Rudnicki, K.B. Riggs, M.L.
Taylor and J. Chang. 2005. "Decontamination assessment of Bacillus anthracis, Bacillus
subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using hydrogen
peroxide gas generator. "Journal of Applied Microbiology (99): 739-748.
3. Lin T T-S and Li S S-L. Purification and Physicochemical Properties ofRicins and
Agglutinins from Ricinus communis. European Journal of Biochemistry, 105:453-459, 1980.
4. Mosmann, T. 1983. "Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays." Journal of Immunological Methods, (65): p. 55-63.
5. EPA. 2013. Evaluation of Ethylene Oxide for the Inactivation o/Bacillus anthracis. EPA
Technology Evaluation Report. EPA/13/R-13-220. December.
27
-------
Appendix A
Detailed Test Results
Efficacy Results
The detailed decontamination efficacy results for VPHP against pure and crude ricin toxin on eight
material types (glass, ceiling tile, carpet, painted wallboard paper, bare pine wood and unpainted
concrete) are shown in Tables A-l through A-3. Data highlighted green indicates >99% reduction.
Table A-l. Inactivation of Pure Ricin Toxin Using VPHPa
Test Parameters
Material
Inoculum
Mean Recovered Ricin ± SD
(|ig coupon)
" oReduction ±
Number
Technology Phase 2
Phase 3
(ug coupon)
Positive Control
Test Coupon
CI
Stainless Steel
49.953 ±24.671
12.648 ±3.041
74.68 ± 15.74
Neoprene Rubber
44.137 ± 11.554
3.307 ±0.563
92.51 ±2.65
Optical Plastic
82.850 ±2.048
2.893 ±0.507
96.51 ±0.70
3.8 g/min
STERIS 20 min
1.0 g/min
Aluminum
194.3
153.060 ±8.769
13.342 ±4.672
91.28 ±3.50
30 min
Industrial Carpet
87.611 ±45.841
5.402 ± 2.947
93.83 ±5.27
Ceramic Tile
55.444 ±17.159
10.929 ±2.872
80.29 ± 9.06
Unpainted Concrete
2.339 ±0.243
2.542 ±0.245
0.00 ± 17.44f
Paper
24.954 ± 11.307
15.758 ±3.625
36.85 ±36.31
Stainless Steel
85.883 ±2.925
0.849 ±0.204
99.01 ±0.27
Neoprene Rubber
60.891 ±21.002
4.571 ± 1.567
92.49 ±4.13
Optical Plastic
65.033 ± 10.721
4.247 ±0.504
93.47 ± 1.50
_ 3.8 g/min
1.0 g/min
Aluminum
152.5
61.795 ±44.901
2.129 ± 1.088
96.56 ±3.46
STERIS 20 min
4 hr
Industrial Carpet
119.899 ± 19.131
25.438 ±6.536
78.78 ± 7.26
Ceramic Tile
189.311 ± 19.775
6.191 ± 1.257
96.73 ±0.84
Unpainted Concrete
0.408 ±0.172
0.600 ±0.806
0.00 ± 234.28f
Paper
75.628 ±60.134
8.471 ± 1.545
88.80 ± 10.34
Stainless Steel
37.328 ± 12.339
0.037 ±0.001
99.90 ± 0.04
Neoprene Rubber
77.281 ±2.541
0.033 ± 0.035
99.96 ±0.05
Optical Plastic
76.303 ± 28.462
0.054 ±0.007
99.93 ± 0.03
3 Bioquell
20 nun
1.0 g/min
Aluminum
166.2
87.220 ± 15.103
0.033 ± 0.005
99.96 ±0.01
4 hr
Industrial Carpet
153.209 ±2.844
3.161 ±0.558
97.94 ±0.41
Ceramic Tile
27.027 ± 14.392
0.059 ±0.014
99.78 ±0.14
Unpainted Concrete
0.646 ±0.585
<0.049 ± 0.000e
92.43 ± 7.76
Paper
80.740 ± 8.564
0.568 ±0.187
99.30 ±0.28
Stainless Steel
123.007 ± 17.719
0.021 ±0.004
99.98 ±0.005
Neoprene Rubber
128.447± 33.110
0.076 ± 0.008
99.94 ±0.02
Optical Plastic
119.953 ±30.673
0.037 ±0.003
99.97 ±0.01
¦ „ 3.8 g/min
4 Bioquell „„ .
20 nun
1.0 g/min
8 hr
Aluminum
Industrial Carpet
283.0
50.979 ±22.141
122.050 ±3.790
0.070 ± 0.007
0.824 ±0.014
99.86 ±0.07
99.32 ±0.03
Ceramic Tile
63.487 ±20.364
0.040 ± 0.003
99.94 ±0.02
Unpainted Concrete
0.219 ±0.074
<0.195 ±0.000e
10.83 ±33.92
Paper
82.313 ±6.913
0.285 ±0.055
99.65 ± 0.08
3 Data are expressed as the mean (± SD) of the mass of toxin observed on three individual samples, and decontamination efficacy (percent reduction ± CI).
b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
d CI = confidence interval (± 1.96 x standard error [SE]).
e Data calculated based on LOD.
fNegative value reported as "0".
A-l
-------
Table A-l. Inactivation of Pure Ricin Toxin Using VPHPa (Continued)
Test
Test Parameters
Inoculum
Mean Recovered Ricin ± SD (|ig/coupon)
%Reduction ±
Number Technolo
gy
Phase 2
Phase 3
Material
(ug coupon)
Positive Control
Test Coupon
CI
Stainless Steel
153.615 ± 19.043
1.693 ±0.090
98.90 ±0.17
Neoprene Rubber
133.705 ±5.486
0.058 ±0.015
99.96 ±0.01
1.0
g/min
16 h
Optical Plastic
172.618 ±2.414
0.023 ± 0.004
99.99 ±0.002
5 Bioquell
3.8 g/min
Aluminum
322.0
143.040 ±70.931
0.045 ± 0.005
99.97 ±0.02
20 min
Industrial Carpet
193.877 ± 36.959
2.339 ±0.136
98.79 ± 0.27
Ceramic Tile
73.417 ±3.610
0.122 ±0.021
99.83 ± 0.03
Unpainted Concrete
1.064 ±0.297
0.276 ± 0.045
74.03 ±9.51
Paper
86.826 ± 15.870
0.062 ±0.018
99.93 ± 0.03
1.0
g/min
16 h
Stainless Steel
301.292 ± 37.582
0.049 ± 0.002
99.98 ±0.002
6 Bioquell
3.8 g/min
20 min
Neoprene Rubber
Industrial Carpet
553.7
317.757 ±26.133
341.690 ± 15.587
0.291 ± 0.048
1.437 ±0.115
99.91 ±0.019
99.58 ±0.044
Paper
189.273 ± 24.339
0.064 ±0.004
99.97 ±0.005
Stainless Steel
140.724 ±27.997
4.067 ±0.125
97.11 ±0.66
Neoprene Rubber
114.479 ±27.393
6.606 ± 0.422
94.23 ± 1.62
2.2
g/min
8 h
Optical Plastic
68.567 ±36.244
3.044 ±0.449
95.56 ±2.76
7 STERIS
2.5 g/min
20 min
Aluminum
Industrial Carpet
275.3
130.036 ±8.993
327.663 ± 45.399
4.184 ±0.201
66.432 ±3.060
96.78 ±0.31
79.73 ± 3.35
Ceramic Tile
152.556 ±22.895
0.259 ±0.144
99.83 ±0.11
Unpainted Concrete
2.130 ±0.513
17.708 ± 1.133
0 ± 235f
Paper
99.913 ±4.617
2.203 ±0.114
97.79 ±0.17
Stainless Steel
126.976 ±2.102
3.625 ±0.113
97.14 ± 0.11
Neoprene Rubber
122.773 ± 16.098
6.924 ±0.248
94.36 ±0.87
2.2
g/min
13.7 h
Optical Plastic
148.387 ±22.372
2.807 ± 1.595
98.11 ± 1.26
8 STERIS
2.5 g/min
20 min
Aluminum
Industrial Carpet
150.6
165.282 ± 13.294
187.116 ± 19.903
3.297 ±0.947
66.448 ± 12.573
98.01 ±0.67
64.49 ± 8.72
Ceramic Tile
Unpainted Concrete
Paper
128.016 ±72.227
0.360 ±0.158
83.039 ±4.516
3.082 ±0.084
1.282 ±0.092
2.928 ±0.141
97.59 ± 1.54
0 ± 179f
96.47 ±0.29
2.2
g/min
13.7 h
Stainless Steel
324.982 ± 16.358
0.231 ±0.042
99.93 ±0.015
9 STERIS
2.5 g/min
Optical Plastic
583.9
257.414 ±94.242
0.522 ±0.080
99.80 ±0.091
20 min
Industrial Carpet
329.371 ± 53.248
4.170 ±0.386
98.73 ±0.267
Paper
153.750 ±8.282
0.781 ± 0.278
99.49 ±0.207
Neoprene Rubber
229.901 ±76.867
0.348 ±0.114
99.85 ±0.080
10 STERIS
2.5 g/m
2.2 g/m
Aluminum
671.5
254.447 ±29.183
0.573 ± 0.024
99.77 ±0.031
20 min
13.7 h
Ceramic Tile
216.001 ±8.277
0.612 ±0.058
99.72 ±0.033
Unpainted Concrete
0.403 ± 0.083
0.109 ±0.014
72.77 ±7.397
aData are expressed as the mean (± SD) of the mass of toxin observed on three individual samples, and decontamination efficacy (percent reduction ± CI).
b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
d CI = confidence interval (± 1.96 x SE).
e Data calculated based on LOD.
fNegative value reported as "0".
A-2
-------
Table A-2. Inactivation of Crude Ricin Toxin Using VPHPa
Test
Test Parameters
Material
Inoculum
Mean Recovered Ricin ± SD
(|ig coupon)
" oReduction ±
Number
Technology
Phase 2
Phase 3
(ug coupon)
Positive Control
Test Coupon
CI
Stainless Steel
95.143 ± 3.458
16.980 ±4.885
82.15 ±5.86
Neoprene Rubber
82.833 ±27.268
32.559 ±23.246
60.69 ± 34.97
Optical Plastic
84.434 ±23.271
31.181 ±3.777
63.07 ± 12.58
1
STERIS
3.8 g/min
20 min
1.0 g/min
30 min
Aluminum
Industrial Carpet
Ceramic Tile
Unpainted Concrete
Paper
261.4
123.479 ± 15.888
196.871 ±36.817
119.181 ±26.517
1.840 ± 1.835
74.785 ± 22.401
12.263 ± 11.178
106.577 ± 14.901
16.447 ± 4.118
0.680 ±0.088
46.437 ±9.863
90.07 ± 10.35
45.86 ± 14.30
86.20 ±5.23
63.01 ±42.11
37.91 ±25.80
Stainless Steel
156.477 ± 18.253
3.185 ± 1.773
97.96 ± 1.31
Neoprene Rubber
117.420 ±40.827
2.157 ±0.850
98.16 ± 1.09
Optical Plastic
140.412 ±40.177
18.004 ± 1.483
87.18 ±4.32
2
STERIS
3.8 g/min
20 min
1.0 g/min
4 h
Aluminum
Industrial Carpet
Ceramic Tile
Unpainted Concrete
Paper
370.0
55.849 ±21.703
205.290 ±41.525
130.942 ± 22.749
2.123 ± 1.171
26.318 ±2.535
0.561 ±0.250
127.600 ±41.400
1.420 ±0.546
0.124 ±0.068
20.953 ± 2.470
98.99 ±0.67
37.84 ±26.89
98.92 ±0.52
94.18 ± 5.15
20.39 ± 13.72
Stainless Steel
152.013 ± 16.460
0.036 ± 0.046
99.98 ±0.03
Neoprene Rubber
44.526 ± 36.043
26.367 ± 1.685
40.78 ± 54.41
Optical Plastic
123.558 ± 14.486
14.705 ± 2.943
88.10 ± 3.12
3
Bioquell
3.8 g/min
1.0 g/min
Aluminum
217.0
159.760 ± 16.519
0.087 ±0.085
99.95 ±0.06
20 min
4 h
Industrial Carpet
179.894 ±41.481
4.845 ± 1.132
97.31 ± 1.00
Ceramic Tile
140.215 ±37.887
0.222 ±0.177
99.84 ± 0.15
Unpainted Concrete
0.353 ±0.005
0.194 ±0.123
45.13 ±39.44
Paper
140.594 ± 8.712
0.651 ±0.155
99.54 ± 0.13
Stainless Steel
154.701 ±52.434
0.285 ±0.055
99.82 ±0.08
Neoprene Rubber
165.291 ± 17.753
0.109 ±0.019
99.93 ±0.02
Optical Plastic
192.846 ± 13.827
3.513 ± 1.393
98.18 ±0.83
4
Bioquell
3.8 g/min
1.0 g/min
Aluminum
332.4
140.746 ±56.104
0.099 ± 0.003
99.93 ±0.03
20 min
8 h
Industrial Carpet
177.280 ±28.875
3.894 ± 1.491
97.80 ± 1.03
Ceramic Tile
44.841 ±3.013
0.042 ± 0.006
99.91 ±0.02
Unpainted Concrete
1.351 ±0.980
<0.195 ±0.000e
85.57 ± 11.85
Paper
25.883 ±2.586
0.482 ±0.252
98.14 ± 1.12
3 Data are expressed as the mean (± SD) of the mass of toxin observed on three individual samples, and decontamination efficacy (percent reduction ± CI).
b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
d CI = confidence interval (± 1.96 x SE).
e Data calculated based on LOD.
fNegative value reported as "0".
A-3
-------
Table A-2. Inactivation of Crude Ricin Toxin Using VPHPa (Continued)
Test Test Parameters Inoculum Mean Recovered Ricin ± SD (ng/coupon) o 0Reduction ±
Number Technology Phase 1
Phase 2
Material
(ug coupon)
Positive Control
Test Coupon
CI
Stainless Steel
122.233 ± 50.002
0.073 ±0.007
99.94 ±0.03
Neoprene Rubber
39.673 ± 11.048
0.256 ±0.062
99.35 ±.027
1.0
g/min
16 h
Optical Plastic
52.944 ±21.963
0.205 ±0.009
99.61 ±0.18
5 Bioquell 308/min
20 mm
Aluminum
Industrial Carpet
264.3
211.581 ± 11.293
103.768 ±3.337
0.073 ±0.007
<0.780 ± 0.000e
99.97 ±0.004
99.25 ±0.03
Ceramic Tile
96.938 ± 18.479
0.098 ±0.009
99.90 ±0.02
Unpainted Concrete
2.191 ±0.893
0.766 ± 0.070
65.04 ± 16.53
Paper
17.525 ±7.143
0.160 ±0.051
99.09 ±0.53
1.0
g/min
16 h
Stainless Steel
138.390 ±34.273
0.076 ±0.009
99.94 ±0.017
6 Bioquell
20 mm
Neoprene Rubber
528.7
191.177 ± 14.056
0.985 ± 1.278
99.48 ±0.758
Industrial Carpet
59.211 ±5.842
1.734 ±0.102
97.07 ±0.380
Paper
36.479 ± 11.937
0.067 ±0.017
99.82 ±0.087
Stainless Steel
37.559 ±2.940
3.269 ±0.124
91.31 ±0.86
Neoprene Rubber
73.161 ± 19.039
6.088 ±0.229
91.68 ±2.48
2.2
g/min
8 h
Optical Plastic
26.776 ±2.143
2.942 ±0.365
89.01 ± 1.83
2.5 g/min
STERIS 20 min
Aluminum
Industrial Carpet
294.5
36.323 ± 5.978
95.741 ± 27.082
1.099 ±0.172
9.839 ±2.111
96.98 ±0.78
89.72 ±4.13
Ceramic Tile
45.574 ± 11.803
1.063 ±0.433
97.67 ± 1.27
Unpainted Concrete
1.836 ±0.680
29.461 ± 2.936
0 ± 696f
Paper
117.354 ±22.002
3.741 ±0.384
96.81 ±0.77
Stainless Steel
7.557 ±2.106
0.481 ±0.050
93.63 ±2.15
Neoprene Rubber
8.526 ± 1.833
0.217 ±0.023
97.46 ± 0.69
Optical Plastic
12.913 ±3.972
0.152 ±0.049
98.82 ±0.59
2.5 g/min
2.2
g/min
13.7 h
Aluminum
124.1
17.367 ± 1.296
0.155 ±0.030
99.11 ±0.21
STERIS 20 min
Industrial Carpet
4.195 ±0.360
4.652 ±0.513
0± 17.5f
Ceramic Tile
30.955 ± 10.446
0.124 ± 0.017
99.60 ±0.17
Unpainted Concrete
0.159 ±0.047
0.282 ±0.224
0 ± 170f
Paper
17.966 ±9.894
0.689 ±0.066
96.16 ±2.43
2.2
g/min
13.7 h
Stainless Steel
324.122 ±63.312
0.533 ±0.104
99.84 ±0.051
2.5 g/min
STERIS 20 min
Optical Plastic
Industrial Carpet
579.8
341.860 ±76.185
291.982 ± 257.958
0.363 ±0.099
2.194 ±0.253
99.89 ±0.042
99.25 ±0.758
Paper
283.505 ±84.941
1.546 ±0.353
99.45 ±0.233
Neoprene Rubber
396.616 ± 19.176
0.382 ±0.016
99.90 ±0.007
2.5 g/m
STERIS 20 nun
2.2 g/m
13.7 h
Aluminum
Ceramic Tile
457.7
412.201 ± 19.875
457.101 ±35.529
0.827 ±0.223
0.318 ± 0.116
99.80 ±0.062
99.93 ±0.029
Unpainted Concrete
4.016 ±2.444
0.173 ±0.043
95.69 ±3.207
3 Data are expressed as the mean (± SD) of the mass of toxin observed on three individual samples, and decontamination efficacy (percent reduction ± CI).
b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
d CI = confidence interval (± 1.96 x SE).
e Data calculated based on LOD.
fNegative value reported as "0".
A-4
-------
&EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |