&EPA
United States
Environmental Protection
Agency
Impact of Temperature and
Humidity on the Persistence of
Vaccinia Virus and Ricin Toxin on
Indoor Surfaces
INVESTIGATION REPORT
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-08/002
October 2006
Investigation Report
Impact of Temperature and
Humidity on the Persistence of
Ricin Toxin and Vaccinia Virus
on Indoor Surfaces
By
Harry J. Stone, James V. Rogers, Emily J. Fleming,
Young W. Choi, Jack D. Waugh, William R. Richter,
Michael L. Taylor, Karen B. Riggs, Zachary J.
Willenberg, and Robert T. Krile
Battelle
505 King Avenue
Columbus, Ohio 43201
Shawn Ryan
Task Order Project Officer
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development's
National Homeland Security Research Center (NHSRC), funded and managed this investigation through
a Blanket Purchase Agreement (BPA) under General Services Administration contract number
GS23F0011L-3 with Battelle. This report has been peer and administratively reviewed and has been
approved for publication as an EPA document. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use of a specific product.
11
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the nation's
air, water, and land resources. Under a mandate of national environmental laws, the Agency strives to
formulate and implement actions leading to a compatible balance between human activities and the
ability of natural systems to support and nurture life. To meet this mandate, the EPA's Office of
Research and Development (ORD) provides data and scientific support that can be used to solve
environmental problems and to build the scientific knowledge base needed to manage our ecological
resources wisely, to understand how pollutants affect our health, and to prevent or reduce environmental
risks.
In September 2002, EPA announced the formation of the National Homeland Security Research Center
(NHSRC). The NHSRC is part of the Office of Research and Development; it manages, coordinates,
supports, and conducts a variety of research and technical assistance efforts. These efforts are designed
to provide appropriate, affordable, effective, and validated technologies and methods for addressing
risks posed by chemical, biological, and radiological terrorist attacks. Research focuses on enhancing
our ability to detect, contain, and decontaminate in the event of such attacks.
NHSRC's team of world renowned scientists and engineers is dedicated to understanding the terrorist
threat, communicating the risks, and mitigating the results of attacks. Guided by the roadmap set forth in
EPA's Strategic Plan for Homeland Security, NHSRC ensures rapid production and distribution of
security related products.
The NHSRC has developed the Technology Testing and Evaluation Program (TTEP) in an effort to
provide reliable information regarding the performance of homeland security related technologies. TTEP
provides independent, quality assured performance information that is useful to decision makers in
purchasing or applying the tested technologies. It provides potential users with unbiased, third-party
information that can supplement vendor-provided information. Stakeholder involvement ensures that
user needs and perspectives are incorporated into the test design so that useful performance information
is produced for each of the tested technologies. The technology categories of interest include detection
and monitoring, water treatment, air purification, decontamination, and computer modeling tools for use
by those responsible for protecting buildings, drinking water supplies and infrastructure and for
decontaminating structures and the outdoor environment. Additionally, environmental persistence
information is also important for containment and decontamination decisions.
The U.S. EPA, through its Office of Research and Development's NHSRC, funded and managed this
investigation through a Blanket Purchase Agreement (BPA) under General Services Administration
contract number GS23F0011L-3 with Battelle. Information on NHSRC and TTEP can be found at
http://www.epa.gov/ordnhsrc/index.htm.
in
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the investigation,
analyze the data, and prepare this report. We also would like to thank Dr. Vipin K. Rastogi (Edgewood
Chemical Biological Center), Dr. Emily Snyder (EPA/NHSRC), and Mr. Joe Wood (EPA/NHSRC) for
their reviews of this report.
IV
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Contents
Notice ii
Foreword iii
Acknowledgments iv
Abbreviations/Acronyms viii
Executive Summary ix
1.0 Introduction 1
2.0 Investigation Procedures 3
2.1 Experimental Design 3
2.2 Test Chamber 4
2.3 Test Surfaces 5
2.4 Biological Agent and Surrogate 5
2.5 Application of Biological Agent to Test Coupons 6
2.6 Test Procedure 7
2.7 Determination of Percent Recovery and Persistence 7
2.8 Determination of Ricin Renaturation 12
3.0 Quality Assurance/Quality Control 13
3.1 Equipment Calibration 13
3.2 Audits 13
3.2.1 Performance Evaluation Audit 13
3.2.2 Technical Systems Audit 13
3.2.3 Data Quality Audit 13
3.3 QA/QC Reporting 13
3.4 Data Review 14
4.0 Test Results 15
4.1 Recovery of Ricin and Impact of Environmental Conditions on Persistence
of Ricin 15
4.1.1 Ricin on Galvanized Metal 16
4.1.2 Ricin on Painted Concrete 19
4.2 Recovery of Vaccinia and Impact of Environmental Conditions on
Persistence of Vaccinia 21
4.2.1 Vaccinia on Galvanized Metal 22
4.2.2 Vaccinia on Painted Concrete 25
4.2.3 Effect of Temperature on Persistence of Ricin and Vaccinia 27
5.0 Summary 28
6.0 References 30
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Appendix A: Report of Temperature and RH Measurement Data A-l
Appendix B: Report of Methods Development and Demonstration B-l
Figures
Figure 2-1. Test Chamber (left) and Test Chamber in Incubator (right) 4
Figure 2-2. Multichannel Micropipette 6
Figure 2-3. Visual Demonstration of MTT Assay on a Micropiate 9
Figure 2-4. Example of Ricin Standard Curve from this Investigation 9
Figure 4-1. Persistence of Ricin on Galvanized Metal 17
Figure 4-2. Mean Attenuation (Percent Reduction): Ricin on Galvanized Metal 18
Figure 4-3. Persistence of Ricin on Painted Concrete 20
Figure 4-4. Mean Attenuation: Ricin on Painted Concrete 21
Figure 4-5. Persistence of Vaccinia on Galvanized Metal 23
Figure 4-6. Mean Attenuation: Vaccinia on Galvanized Metal 24
Figure 4-7. Persistence of Vaccinia on Painted Concrete 26
Figure 4-8. Mean Attenuation: Vaccinia on Painted Concrete 27
Figure A-l. Temperature and RH Measurements for Ricin Trial 1 Test Chambers:
Ambient (Left) and High RH (Right) Conditions A-2
Figure A-2. Temperature and RH Measurements for Ricin Trial 2 Test Chambers:
Ambient (Left) and LowRH (Right) Conditions A-3
Figure A-3: Temperature and RH Measurements for Vaccinia Trial 1 Test Chambers:
Ambient (Left) and High RH (Right) Conditions A-3
Figure A-4: Temperature and RH Measurements for Vaccinia Trial 2 Test Chambers:
Ambient (Left) and Low RH (Right) Conditions A-4
VI
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Tables
Table 2-1. Test Matrix and Measured Parameters 3
Table 2-2. Material Characteristics 5
Table 4-1. Ricin Extraction Efficiencies 16
Table 4-2. Mean Recovery of Ricin Cytotoxicity on Galvanized Metal 17
Table 4-3. Mean Attenuation: Ricin on Galvanized Metal (Shown as Percent Reduction) 18
Table 4-4. Mean Recovery of Ricin Cytotoxicity on Painted Concrete 19
Table 4-5. Mean Attenuation: Ricin on Painted Concrete (Shown as Percent Reduction) 20
Table 4-6. Vaccinia Extraction Efficiency 22
Table 4-7. Mean Vaccinia Recovery from Galvanized Metal 23
Table 4-8. Mean Attenuation: Vaccinia on Galvanized Metal (Shown as Log Reduction) 24
Table 4-9. Mean Vaccinia Recovery from Painted Concrete 25
Table 4-10. Mean Attenuation: Vaccinia on Painted Concrete (Shown as Log Reduction) 26
Table 5-1. Ricin or Vaccinia Recovery from Different Materials under Varying RH Conditions at
Constant Temperature (Temperature = 30°C) 28
Table 5-2. Day 3 Ricin or Vaccinia Recovery from Different Materials, Ambient
Conditions 28
Table 5-3. Mean Attenuation: Ricin or Vaccinia on Different Building Materials under Varying
RH Conditions 29
Table B-l. Ricin Recoveries (jig) upon Immediate or Delayed Extraction B-3
vn
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Abbreviations/Acronyms
ASTM
ATCC
BSCII
°C
CDC
CFR
C102
cm
C02
d
EPA
h
HVAC
1
L
mL
mm
mg
MTT
n
nm
NHSRC
ORD
PBS
PFU
QA
QC
QMP
RH
SD
ISA
TTEP
TO
Hg
vs.
w
American Society for Testing and Materials
American Type Culture Collection
Class II biological safety cabinet
degrees Celsius
Centers for Disease Control and Prevention
Code of Federal Regulations
chlorine dioxide
centimeter
carbon dioxide
depth
U.S. Environmental Protection Agency
height
heating, ventilation, and air conditioning
length
liter
milliliter
microgram
microliter
millimeter
milligram
3-(4,5-dimethylthiazol-2-yl)-2, 5,-diphenyl tetrazolium bromide
number of observation (or replicate samples)
nanometer
National Homeland Security Research Center
Office of Research and Development
phosphate buffer solution
plaque-forming unit
quality assurance
quality control
quality management plan
relative humidity
standard deviation
technical systems audit
Technology Testing and Evaluation Program
Time zero
microgram
versus
width
Vlll
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Executive Summary
The U.S. EPA's NHSRC TTEP is helping to protect human health and the environment from adverse
impacts resulting from acts of terror by carrying out performance tests on homeland security technologies.
Under TTEP1, the persistence of biological agents on indoor building materials under various
temperature and humidity conditions, consistent with conditions that might be achieved using a heating,
ventilation, and air conditioning (HVAC) system, was investigated. The objective of the study was to
determine the attenuation of vaccinia virus viability, a surrogate for smallpox virus (variola), and the
cytotoxicity of ricin toxin on indoor building materials due to prolonged exposure to ambient conditions
[20°C, relative humidity (RH) 40 - 70%], or at 30°C with high (>70%) or low (<40%) RH. It is
important to know whether environmental conditions can impact the persistence of biological agents in
order to properly structure first-responder assessment and plan and interpret results from use of
fumigant, liquid or foam decontamination technologies. Further, decontamination of indoor surfaces
following intentional release of biological agents may potentially be enhanced by manipulating
environmental conditions, such as temperature or RH, if an effect on persistence of biological organisms
or toxicity of toxins is observed.
Ricin toxin and vaccinia virus were selected based upon a review of available information and other on-
going research and assessment efforts; the selection represent biological agents with a range of reported
environmental persistence. Both vaccinia and variola viruses are species in the genus Orthopoxvirus.
There is a 96% identity at the nucleotide level between vaccinia and variola^ Given the high levels of
similarity between these viruses, decontamination of variola is expected to be similar to that observed
for vaccinia.
Coupons of galvanized metal or painted concrete were inoculated with the respective agent being
investigated and placed in test conditions of either ambient (~20°C and 40%-70% RH), or at 30°C at
either high (>70%) or low RH (<40%). Thus, persistence of the agents on the materials was determined
at the three environmental conditions as a function of time up to 14 days. Persistence of a biological
agent exposed to known environmental conditions can be evaluated by comparing the temporal
reduction in toxin bioactivity or organism viability following inoculation onto indoor building material
coupons. The attenuation, or reduction in persistence, can be represented as a log reduction (for vaccinia
virus) or percent reduction (for ricin toxin) in the amount of viable organisms or bioactive toxin
extracted from coupons after a treatment (environmental conditions) compared to extractions one hour
after inoculating the coupons.
The results showed that the bioactivity of both ricin and vaccinia persisted on galvanized metal
ductwork and painted concrete for up to 14 days under low RH conditions. The results also indicated
that RH impacts the persistence of both ricin and vaccinia virus; both agents were more persistent when
maintained at low RH rather than at high RH. In each case, the persistence was dependent upon the type
of material onto which the biological organisms or toxin was applied. Furthermore, the results indicate
that, depending on the type of contaminated substrate, elevated RH may be useful for inactivating ricin
or vaccinia virus before the application of a decontamination technology. Because of the close
relationship between vaccinia and variola, the results will likely be applicable to variola; however,
because of differences between variola and vaccinia, in the event of variola contamination, the
persistence should be verified, rather than assumed.
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The Q-fever causative agent, Coxiella burnetii., was also selected for this study. However, the C. burnetii
strain used in this testing resulted in quantification challenges that could not be addressed within the
scope of the project, while still achieving the overall project objectives for ricin toxin and vaccinia virus.
Therefore, C. burnetii was removed from the test matrix; details of the challenges and attempts to
overcome them are provided in Appendix B to this report.
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1.0 Introduction
The U.S. EPA's NHSRC is helping to protect human health and the environment from adverse impacts
resulting from acts of terror. With an emphasis on decontamination and consequence management, water
infrastructure protection, and threat and consequence assessment, NHRSC is working to develop tools and
information that will help detect the introduction of chemical, biological, or radiological contaminants in
buildings or water systems, contain these agents, decontaminate buildings and/or water systems, and dispose
of materials resulting from cleanups.
NHSRC's TTEP works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, scientists, and government regulators; and with participation of
individual technology developers in carrying out performance tests of homeland security technologies. In
response to the needs of stakeholders, TTEP evaluates the performance of innovative homeland security
technologies by developing test plans, conducting evaluations, collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality assurance (QA)
protocols to ensure the generation of high quality data and defensible results. TTEP provides unbiased, third-
party information supplementary to vendor-provided information that is useful to decision makers in
purchasing or applying the evaluated technologies. Stakeholder involvement ensures that user needs and
perspectives are incorporated into the testing design to produce useful performance information for each
technology investigated.
The objective of the study was to determine the persistence of biological organisms or toxin on indoor
building materials under various temperature and RH conditions, consistent with conditions that might
be achieved using an HVAC system. The attenuation of the persistence was investigated as a function of
biological agent type, building material surface, and environmental conditions. The work was motivated
by the need to assess the impact of environmental conditions on the persistence (or attenuation) of
biological agents in order to properly structure testing and interpret results from use of decontamination
technologies, in addition to developing an understanding of the persistence of biological agents on
surfaces in indoor environments, Further, decontamination of indoor surfaces following intentional
release of biological agents may potentially be enhanced by manipulating environmental conditions,
such as raising the temperature and raising or lowering RH, if an effect on persistence of biological
organisms or toxicity of toxins is observed.
C. burnetii, ricin toxin, and vaccinia virus were selected to represent a broad range of biological agents
with reported environmental persistence. C. burnetii, the causative agent of Q-Fever, was selected as a
representative of the bacterial order Rickettsiae that is reported to be persistent. Rickettsiae include a
number of important human pathogens that cause diseases such as Rocky Mountain Spotted Fever and
Typhus. However, the C. burnetii strain used in this testing resulted in quantification challenges that
could not be addressed within the scope of the project, while still achieving the overall project objectives
for ricin toxin and vaccinia virus. Therefore, C. burnetii was removed from the test matrix; details of the
challenges and attempts to overcome them are provided in Appendix B to this report. Ricin toxin was
selected as a high-profile protein toxin with a history of terrorist use. Vaccinia virus was selected as a
1
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surrogate for variola virus, the causative agent for smallpox. Smallpox is a potential biological agent of
concern because of the extremely grave consequences that would be expected from its use.
The investigation of the persistence of the biological agents or surrogates on indoor building materials
was conducted according to a peer-reviewed test/QA plan[2] that was developed according to the
requirements of the quality management plan (QMP)[3] for the TTEP program. The test plan outlines an
investigation of the persistence of the selected agents on various materials. Bioactivity indicating
persistence was measured as the number of viable vaccinia virus determined as plaque-forming units
(PFU) or the cytotoxicity of ricin toxin remaining on the coupons after exposure to the specified test
conditions. In this investigation, coupons of galvanized metal ductwork or painted concrete were
inoculated with the ricin toxin or vaccinia virus and placed in test conditions of either ambient (~20°C
and 40%-70% RH), or at 30°C at either high (>70%) or low RH (<40%). Mean attenuation in
persistence at the treatment conditions was measured as the percent reduction in ricin cytotoxicity or log
reduction in viral viability.
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2.0 Investigation Procedures
2.1 Experimental Design
This report provides results for the investigation of the persistence of biological organisms or toxin
exposed to various temperature and RH conditions that might be observed or easily achieved in a
building. Persistence was measured on two indoor surfaces, galvanized metal ductwork and painted
concrete, typical of those that might require decontamination after a deliberate release of biological
agent inside of a public building or subway system. The experimental treatments performed are shown
in Table 2-1. The independent test parameters measured included temperature, RH, and time. The
dependent variable was the extracted residual PFU or mass of cytotoxic ricin.
Statistical comparisons were conducted to determine whether the treatment mean reduction in viable
virus or cytotoxic ricin exceeded the control mean reduction by an amount that was statistically
significant at a 95% confidence level.
Table 2-1. Test Matrix and Measured Parameters
Agent
Indoor Surface Material Environmental Conditions Time (Days) Dependent Variable
Ricin toxin
Ricin toxin
Ricin toxin
Ricin toxin
Ricin toxin
Ricin toxin
Vaccinia virus
Vaccinia virus
Vaccinia virus
Vaccinia virus
Vaccinia virus
Vaccinia virus
Galvanized metal ductwork
Galvanized metal ductwork
Galvanized metal ductwork
Painted concrete
Painted concrete
Painted concrete
Galvanized metal ductwork
Galvanized metal ductwork
Galvanized metal ductwork
Painted concrete
Painted concrete
Painted concrete
30°C; <40% RH
30°C; >70% RH
~20°C; -40% RH (ambient)
30°C; <40% RH
30°C; >70% RH
~20°C; -40% RH (ambient)
30°C; <40% RH
30°C; >70% RH
~20°C; -40% RH (ambient)
30°C; <40% RH
30°C; >70% RH
~20°C; -40% RH (ambient)
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
0,1, 3, 9, and 14
Cytotoxic mass
Cytotoxic mass
Cytotoxic mass
Cytotoxic mass
Cytotoxic mass
Cytotoxic mass
PFU
PFU
PFU
PFU
PFU
PFU
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2.2 Test Chamber
The persistence of biological agent on various indoor building materials was investigated at three
chamber temperature and RH conditions: "ambient" (40%-70% RH at ~20°C), "low RH" (<40% RH at
30°C), and "high RH" (>70% RH at 30°C). Test coupons (described in Section 2.3) were transferred into
the test chamber for experimental treatment after being inoculated with biological agent as described in
Section 2.5, below. Plastic Lock&Lock™ containers (Target®) were used as test chambers as shown in
Figure 2-1. The Lock&Lock plastic storage container had dimensions of 25 cm width (w) x 18 cm depth
(d)x 17 cm height (h).
Low RH (<40%) was maintained in the test chambers using calcium sulfate desiccants (W. A. Hammond
Drierite Co.). The W. A. Hammond Drierite Co. web site (www.Drierite.com) reports that the National
Bureau of Standards has verified that the moisture remaining in gases dried with Drierite at 25°C-30°C
is 0.005 mg/L (<1% RH). The desiccant could therefore reduce the %RH below the upper limit of 40%
RH. High RH (>70%) was generated by enclosing wet paper towels in the test chamber. The elevated
temperature (30°C) was maintained by placing the test chamber inside of an incubator.
Temperature and RH were recorded using a HOBO U10 Data Logger (in Figure 2-1, note cable coming
out of the sealed test chamber). Manufacturer's specifications indicate time accuracy of 61
seconds/month at 25°C (77°F), temperature accuracy of ± 0.4°C @ 25°C (± 0.7°F @ 77°F) and RH
accuracy of ±3.5% from 25% to 85% over the range of 15°C to 45°C (59°F to 113°F). A VWR®
Traceable™ hygrometer/thermometer with a temperature accuracy of ±1°C and RH accuracy of ±4%
(between 20% and 80%) was included in the test chamber to enable environmental conditions to be
independently verified.
Figure 2-1. Test Chamber (left) and Test Chamber in Incubator (right)
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2.3 Test Surfaces
The indoor surface materials, shown in Table 2-2, included both nonporous (galvanized metal ductwork)
and porous (painted concrete) surfaces. Test coupons were cut to 1.9 cm length (1) x 7.5 cm w in size
from a larger piece of test material. Edges and damaged areas were avoided in cutting test coupons. The
galvanized metal ductwork coupons were 0.6 mm thick. The painted concrete coupons were 0.7 cm
thick. The test coupons were visually inspected prior to being inoculated with the biological agents.
Coupons with anomalies on the test (application) surface were rejected from use.
On each day of testing, each coupon was assigned and marked with a unique identifier code for
traceability. Prior to the application of the biological agent or surrogate, the coupons were autoclaved
and the surface of each coupon was wiped with 70% isopropanol or ethanol. Principles of aseptic
technique following Battelle guidance ^' 4"6] was exercised during all phases of handling the test
coupons.
Table 2-2. Material Characteristics
Material
Galvanized
metal
ductwork
Painted
concrete,
cinder block
Lot, Batch, or
ASTM No., or
Observation
Industry HVAC
standard 24-gauge
galvanized steel
ASTM C90
Manufacturer/
Supplier Name
Accurate
Fabrication
Wellnitz
Approximate
Coupon Size,
1 x w, cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
Material Preparation
Cleaned with acetone; autoclaved
Brush and roller painted all sides. One
coat Martin Senour latex primer (#71-
1 185) and one coat Porter Paints latex
semi-gloss finish (#919); autoclaved
2.4 Biological Agent and Surrogate
The biological agent and surrogate used in the testing were ricin toxin (Vector Laboratories L-1090,
product specification: Ricin communis agglutinin II, 5 mg/mL protein concentration) and vaccinia virus
(American Type Culture Collection (ATCC) VR1 19, an orthopox virus closely related to Variola major,
the causative agent for smallpox). The biological agent and surrogate were selected based on an
evaluation of potential threats to buildings and discussions with and approval by EPA. Biological agent
was used according to the Centers for Disease Control and Prevention (CDC) Select Agents Program
(42 Code of Federal Regulations (CFR) Part 73) and the Biological Defense Research Program (32 CFR
626 and 627) in adherence with the Battelle Medical Research and Evaluation Facility Safety
A ricin stock solution was prepared by transferring a 100 jiL aliquot of ricin toxin (5 mg/mL), as
described below in Section 2.7, into a sterile 50 mL conical vial to which 10.0 mL of sterile phosphate-
buffered saline (PBS) was added and agitated on an orbital shaker for 15 minutes at approximately 200
rpm at room temperature (approximately 22°C). The cytotoxicity of the stock solution was determined
using the bioassay methods for the respective biological agents specified in Section 2.7.
The vaccinia stock used in this study was propagated by inoculating a monolayer of Vero cells with
vaccinia virus at a multiplicity of infection ranging from 0.01 to 1.0%. Cultures were maintained in an
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incubator at 37°C ± 2°C under a 95% air, 5% CO2 mixture until 90-100% cytopathic effects were
observed. Infected cells were harvested and subjected to a rapid freeze/thaw cycle. Cellular debris was
removed by centrifugation at 800 to 1,000 x g. The resulting supernatant (containing virus) was
harvested and stored in 1.0 mL aliquots, approximately 1.0 x 108 PFU/mL, at < -70°C until used. The
resulting supernatant, vaccinia in complete cell culture medium containing 5-10% fetal bovine serum,
was not filter sterilized because the virus is propagated in sterile tissue culture. All manipulations are
performed with sterile technique. The PFU/mL is determined after the virus has been aliquoted by
randomly selecting and assaying vials of the stock samples. The samples are assayed using a standard
plaque assay (described in Section B2.7) to determine the viral titer of the entire lot.
2.5 Application of Biological Agent to Test Coupons
The functional types of coupons included in this investigation are test coupons (five at each non-zero
time point), control coupons (five at time zero [TO]), laboratory blank coupons (one at TO) and
procedural blank coupons (one at each non-zero time point). Test coupons are inoculated with biological
agent and subjected to specified temperature and RH conditions for the specified time; the test coupons
are then extracted and analyzed. Positive control coupons are inoculated with biological agent and
extracted and analyzed at TO. Laboratory blank coupons are not inoculated and are extracted and
analyzed at TO. Procedural blank coupons are not inoculated and are extracted and analyzed along with
test coupons at each time point.
The ricin toxin stock solution was applied onto the test coupons, lying flat, at approximately 25 jig per
test and positive control coupon. A 5 jiL aliquot of a stock suspension (5 mg/mL of ricin) was dispensed
using a micropipette as a streak across the surface of the test coupon. The ricin toxin application was
performed in a Class II Biological Safety Cabinet (BSC II).[8]
Application of vaccinia virus to the test coupons was performed in a BSC II in accordance with internal
safety guidelines).^ Test and positive control coupons were placed lying flat in the cabinet and
. .
inoculated at challenge levels of vaccinia virus at approximately 1x10 PFU per coupon. A 100 jiL
aliquot of a stock suspension (approximately 1x10 PFU/mL) of virus was dispensed using a
multichannel micropipette (shown in Figure 2-2) as two rows of five 10 |jL droplets on the surface of
the test coupon.
Figure 2-2. Multichannel Micropipette
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After being inoculated with ricin toxin or vaccinia virus, the test coupons were left undisturbed for one
hour at ambient laboratory (or BSC II) temperature and RH and then placed into test conditions of
controlled temperature and RH, in accordance with the test matrix shown in Table 2-1.
2.6 Test Procedure
Two 14-day trial runs were used for each of the two agents investigated to complete the study at the
three environmental conditions. In the first run for each agent, the ambient and high RH (at 30°C)
conditions were investigated. In the second run for each agent, the coupons were treated at the ambient
and low RH (at 30°C) conditions. Therefore, for each run, two test chambers were used concurrently
(one for the ambient condition and one for the high or low RH condition). A total of five replicate
coupons of each type treated at the same conditions were used for each time point investigated at each
condition. The galvanized metal and concrete replicate coupons treated at the ambient conditions were
split between the first and second runs. Two galvanized metal coupons and three concrete coupons were
included for each time point in the first run at the ambient conditions. Three galvanized metal and two
concrete coupons for each time point were treated at the ambient conditions in the second run. One
blank coupon (inoculated only with PBS) of each type per time point at each condition was used in each
of the runs. Five positive controls, extracted after the one hour drying period at Day 0, of each coupon
type were used for each test condition. At the desired time points within each trial run, the appropriate
coupons (test and blank) of each material type were removed from each test chamber. The brief opening
and closing of the test chamber was observed to have minimal impact on the internal temperature and
RH.
2.7 Determination of Percent Recovery and Persistence
The testing investigated persistence of biological agent by measuring the amount of residual biological
agent on test coupons over time. The percent recovery, shown in Section 4, was determined by
measuring and comparing the amount of viable or cytotoxic biological agent recovered from control
coupons one hour after being inoculated to the amount of biological agent applied based upon analysis
of the stock suspension or solution. Persistence was determined by measuring the amount of residual
viable or cytotoxic biological agent on inoculated coupons over time. Attenuation in persistence was
calculated by determining the reduction in persistence at each time point compared to the TO
measurement (positive control coupons). For extraction of ricin toxin and vaccinia virus, the test,
control, and blank coupons were placed individually in a sterile 50 mL conical vial to which 10.0 mL of
sterile PBS was added. The tubes were agitated on an orbital shaker for 15 minutes at approximately 200
rpm at room temperature. This extraction method has been used in previous work.[9"11]
The amount of cytotoxic ricin persisting on test coupons over time was determined using the MTT (3-
(4,5-dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide) bioassay developed by Mosmann.[12] The
mechanism of action by which ricin exerts its toxic effect is through inhibition of protein synthesis
within cells and activation of enzymes inducing apoptosis. Such inhibition of protein production and
apoptosis lead to cell death. Therefore, an in vitro cytotoxicity assay, described below, was used to
evaluate the mass of cytotoxic ricin in the applied volume of extraction fluid. The MTT assay is an
indirect measure of ricin bioactivity. The MTT assay measures loss of mitochondrial activity arising
from cell death as a result of direct impacts of ricin. Other cytotoxic substances or those toxic to
mitochondrial activity could create false positives, result in an overestimate of ricin concentration, or
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reduce the sensitivity of the assay. The extracts of negative control coupons were used to calculate
background cytotoxicity. Dilutions, that reduce assay sensitivity, are used to "zero-out" background
cytotoxicity.
Vero (African Green Monkey kidney) cells were seeded in wells of a 96-well microplate at a density of
approximately 2 x 104 cells/well. Cells were cultured for 18-30 hours at 37°C ± 2°C under 95% air and
5% CC>2 and exposed to the coupon extracts. Following extraction of ricin from coupons, 1.0 mL of the
PBS extracts of the ricin test and control coupons were removed and an 8-point series of two-fold
dilutions were prepared in complete cell culture medium. An aliquot (100 |jL) of the undiluted extract
and each serial dilution was plated onto confluent Vero cells in the 96-well plate. For quantitation, a
parallel standard curve was prepared using three-fold serial dilutions starting from 10 ng/mL and plated
onto confluent Vero cells in the 96-well plate. Following exposure to extracts for 48-72 hours at 37°C ±
2°C in a humidified 5% CC>2 atmosphere, the cells were incubated in the presence of MTT (Promega,
Madison, WI) for four hours, where mitochondrial enzymes within cells convert the yellow MTT to a
purple formazan salt. The reaction was terminated at the end of the four-hour exposure to MTT by
adding 100 jiL of a Solubilizati on/Stop Solution (Promega). The plate was incubated overnight (for up
to 24 hours) at 37°C ± 2°C in a humidified atmosphere to solublilize the formazan crystals as specified
by Promega Technical Bulletin: CellTiter 96™ non-Radioactive Cell Proliferation Assay. '13^ A
Molecular Devices SPECTRAmax microplate reader was used to measure the absorbance at 570 nm
wavelength using a reference wavelength of 630-750 nm. For each standard and test sample, absorbance
values of the reference wavelength (630-750 nm) are subtracted from the absorbance values at 570 nm
for each well. Absorbance values are directly proportional to the number of viable cells present in the
sample well. For each standard, the mean absorbance values (Y-axis) are plotted against the
concentration in ng/mL, and a four-parameter logistic curve is generated by the software included in the
SPECTRAmax microplate reader using the equation:
^ T. (max-min)
Equation 1 . / = minH
where:
Y = absorbance %;
X = concentration of ricin ng/mL;
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/mL
The amount of formazan produced is inversely proportional to the cytotoxic potential of ricin (Figures 2-
3 and 2-4). To determine the concentration of ricin toxin from each test sample (i.e., the mass of ricin
toxin extracted in a specified volume of extraction fluid), the ricin toxin stock solution (purified;
purchased from Vector Laboratories, Burlingame, CA) was assayed in parallel and used to prepare a
standard curve of absorbance versus mass of ricin protein. The absorbance of samples of extracts were
plotted onto the standard curve to determine the mass of cytotoxic ricin in the sample. In cases where
samples were outside of the linear range, appropriate dilutions were made and the assay was repeated.
The limits of quantitation were dependent on the specific dilution and standard curve, but at the highest
dilutions were in the range of 0.041 to 0.062 ug. The mass of cytotoxic ricin and percent recovery were
calculated.
-------
Purple = cells alive;
little to no toxin
Increasing ricin
concentration
Yellow = cells dead;
abundant toxin
Figure 2-3. Visual Demonstration of MTT Assay on a Microplate
Standard Curve
Purple = alive
little to no
Yellow = dead cells;
abundant toxin
0.4
0.01
0.1
Concentration (ng/mL)
Figure 2-4. Example of Ricin Standard Curve from this Investigation
-------
The amount of vaccinia virus persisting on test coupons over time was determined using a plaque assay
approach. Following extraction, 1.0 mL of the PBS extract was removed and a series of dilutions up to
10~7 were prepared in sterile water. A 100-jiL volume of the undiluted and each serial dilution was
plated onto Vero (African Green Monkey kidney) cell monolayers, prepared as described above, and
allowed to adsorb for one hour. Following inoculation and adsorption of virus to the Vero cells, 1.0 mL
of 0.7% methylcellulose (containing fetal bovine serum and antibiotics) was added to each well of the
six-well plate. Plates were incubated for 44-48 hours at 37 ± 2°C under 95% air and 5% CO2. The
methylcellulose was removed and 2.0 mL of 0.13% crystal violet was added and cells incubated for 30
minutes. The crystal violet was removed, cells washed with PBS, and plaques visualized and counted.
Percent recovery for each biological agent on each test material, shown in Section 4, was determined
prior to investigating persistence. Percent recovery (mean ± standard deviation [SD]) was calculated for
each type of test material inoculated with each biological agent by dividing the mass of cytotoxic ricin
extracted from control coupons or number of viable vaccinia PFU by the number of biological
organisms or ricin mass applied. The quantity applied is determined during the confirmation of initial
titer described in Section 2.5.
Percent recovery (% R) is calculated as:
(x^
Equation 2. %R=\— x 100
UJ
where x is the number of viable vaccinia PFU or mass of cytotoxic ricin recovered from the control
coupons, and A is the number of viable vaccinia virus or mass of cytotoxic ricin applied to the control
coupons.
Persistence was the measurement of the amount of vaccinia virus (as PFUs) or cytotoxicity of ricin
recovered from each coupon at each time point. Attenuation of the biological agent was calculated as the
log reduction (mean ± SD) in viable vaccinia virus or percent reduction in cytotoxic ricin over the
observed time course for each testing scenario. The higher the attenuation value, the less persistent is the
biological agent on the test material with a given treatment. Attenuation was calculated for they'th test
coupon within the rth combination of treatment factors, e.g., high RH on Day 3 of treatment. For
vaccinia virus, attenuation was calculated as a log reduction according to the equation:
Equation 3. Attenuatim.. = log „ f 0 - log „ \x..
where:
xr
= arithmetic mean of the number of viable virus extracted from positive control coupons;
x.. = number of viable virus extracted from they'th replicate coupon of the rth treatment.
For ricin toxin, attenuation was calculated as a percent reduction according to the equation:
10
-------
X..
Equation 4. Attenuation . . = —^- x 100%
V xn
where:
xr
= arithmetic mean of the mass (mg) of cytotoxic ricin extracted from positive control
coupons;
x..
V = mass (mg) of cytotoxic ricin extracted from they'th replicate coupon of the rth treatment.
A total of five replicate coupons of each material type at each time point were analyzed for each agent.
The attenuation data for each agent and type of material, calculated as shown in Equation 3 or 4, were fit
to a one-way analysis of variance (ANOVA) model of form:
Equation 5. Attenuaticn^ = ju + tt + ep.
where:
H = overall mean attenuation;
' = average effect on the mean attenuation due to the rth treatment gl Iroup/day combination;
£
ij= error terms for they'th replicate of the rth treatment group; the errors are assumed to be
normally distributed.
After fitting each model, residuals were examined to assess normality and the adequacy of assuming a
common variance. These model diagnostics supported the appropriateness of the statistical models. The
modeling and analysis were carried out with PROC Mixed in SAS v9.1.
A mean and range of the attenuation values was determined for each material, agent, and environmental
condition combination at each time period. Persistence curves were developed by graphing reduction in
viable organisms (PFU) or toxin mass over time. Persistence curves were developed for the two
controlled temperature and RH conditions and for ambient conditions for each agent and material.
Statistical analysis consisted of performing a set of pre-planned comparisons. These included:
• Comparing whether the attenuation at a particular RH and day was statistically significantly
different from zero
• For the high and low RH treatments, comparing the estimated mean attenuation on each day to
the corresponding attenuation estimate for the ambient RH treatment to determine if there was a
statistically significant difference between RH treatments within each day.
The overall error rate for the set of comparisons was controlled to be no more than 5% for each material
and agent.
For the evaluation of ricin on galvanized metal, there are separate comparisons of ambient to zero for
each run. Also, the comparison of high and low RH to ambient can only be done within each run so that
the high RH to ambient comparison is based on the results of the ambient treatment coupons tested in
11
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the same run as the high RH coupons and the low RH to ambient comparison is based on the results of
the other set of ambient treatment coupons tested in the same run as the low RH coupons.
In some of the treatment groups for vaccinia, no virus was observed on any of the replicate coupons.
Since a zero value for a virus count leads to an undefined attenuation value, these treatment groups and
their data were excluded from the statistical model. However, it was still possible to report attenuation
for these treatment groups based on the following observations:
• A conservative estimate of the mean attenuation can be formed by replacing the value of zero
PFUs for each coupon with the value one.
• A discrete distribution analysis indicated the average true number of virus PFUs in the five
separate coupon extracts is less than 5% likely to be over 10 PFUs. Therefore, a lower 95%
bound on attenuation was determined for these treatments by replacing the observed number of
PFUs for the coupons (i.e., zero) with the value 10.
2.8 Determination of Ricin Renaturation
Ricin, a protein, is known to denature at high temperatures. Denaturation is associated with a loss of
biological activity. It is less certain whether ricin can renature and, thereby, regain biological activity
when returned to ambient temperature and RH. If bioactivity loss is observed under test, but not under
ambient conditions, an additional test would be run to evaluate the potential for ricin renaturation.
For the investigation of potential renaturation, ricin would be applied to test coupons as described in
Section B2.5. These coupons would be incubated at 30°C and controlled RH for a period of time shown
to result in a loss of cytotoxicity. The coupons would then be returned to ambient temperature and RH
for 24 hours. After the ambient 24-hour incubation, attenuation would be determined as described in
Section 2.6. A decline in attenuation would suggest that renaturation may have occurred.
12
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3.0 Quality Assurance/Quality Control
QA/quality control (QC) procedures were performed in accordance with the program QMP and the
test/QA plan)[2] for this investigation. QA/QC procedures are summarized below.
3.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety cabinets) used at the time of investigation was
verified as being certified, calibrated, or validated.
3.2 Audits
3.2.1 Performance Evaluation Audit
No performance evaluation audit was performed for biological agents and surrogates because
quantitative standards for these biological materials do not exist. The confirmation procedure, controls,
blanks, and method validation efforts support the biological evaluation results.
3.2.2 Technical Systems Audit
Battelle Q A staff conducted a technical systems audit (TS A) on June 9, 2005 to ensure the investigation
was being conducted in accordance with the test/QA plan^ and the QMP.)^ As part of the TSA, test
procedures were compared to those specified in the test/QA plan; and data acquisition and handling
procedures were reviewed. Observations and findings from the TSA were documented and submitted to
the Battelle Task Order Leader for response. None of the findings of the TSA required corrective action.
TSA records were permanently stored with the TTEP QA Manager.
3.2.3 Data Quality Audit
At least 10% of the data acquired during the investigation were audited. A Battelle QA auditor traced the
data from the initial acquisition, through reduction and statistical analysis, to final reporting to ensure
the integrity of the reported results. All calculations performed on the data undergoing the audit were
checked.
3.3 QA/QC Reporting
Each audit was documented in accordance with the QMPPl The results of the TSA were submitted to
the EPA.
13
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One deviation was documented, in compliance with the QMP,[3], and reported to the EPA Task Order
Project Officer. The test/QA plan specified that dilutions through 10~7 would be analyzed. In some cases,
dilutions outside of the useful range, based on expected concentrations of biological agent, were not run.
This saved time and money without reducing useful data.
3.4 Data Review
Records and data generated in the investigation received a QC technical review and a QA review before
they were used to calculate, or report results. All data were recorded by Battelle staff. The person
performing the review was involved in the experiments and added his/her initials and the date to a hard
copy of the record being reviewed. This hard copy was returned to the Battelle staff member who stored
the record.
14
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4.0 Test Results
Persistence of a biological agent exposed to known environmental conditions can be gauged by
measuring the reduction in the cytotoxicity or viable organisms extracted from inoculated coupons over
time. Attenuation is the reduction in amount of cytotoxicity or viable organisms extracted from coupons
after a treatment (environmental conditions) compared to extractions of inoculated coupons (held at
ambient conditions) one hour after inoculating the coupons. Thus, high attenuation indicates low
persistence. The test results presented here show the impact of temperature and RH conditions on the
persistence over time of ricin toxin or vaccinia virus extracted from inoculated galvanized metal or
painted concrete. The environmental conditions investigated were ambient (~20°C, 40% - 70% RH,
high RH (30°C, >70% RH), and low RH (30°C, <40% RH). The high and low RH conditions also were
investigated to assess the impact of higher temperature on persistence (or attenuation). The temperature
and RH throughout the trial runs are reported in Appendix A.
4.1 Recovery of Ricin and Impact of Environmental Conditions on Persistence of Ricin
A 25-|ig amount of total protein in the ricin stock solution was inoculated onto the coupons of indoor
building materials and allowed to dry for one hour under ambient conditions. The ricin-inoculated
coupons were then placed into controlled environmental conditions. After the one hour drying time and
at specified intervals, ricin was extracted from the coupons and the bioactivity was measured using a
cytotoxicity assay. The ricin bioactivity was reported as cytotoxicity in micrograms protein equivalent to
the cytotoxicity of a given mass of protein in the ricin stock solution.
Table 4-1 summarizes the extraction efficiency from different test material coupons by calculating the
recovery of cytotoxic ricin extracted after one hour drying time (TO positive control coupons) as a
percentage of the cytotoxic ricin applied (measured in the application control). The test matrix for ricin
(see Table 2-1) was conducted using two experimental trials (or runs). In Run 1, ambient and high RH
conditions were investigated. In Run 2, ambient and low RH conditions were run. Measurements at
different times (replicates) are referenced as different runs (Run 1 or Run 2).
The recovery for ricin is generally high, although the results for galvanized metal in the first run were
substantially lower than all other recovery averages. The variability in galvanized metal results between
Runs 1 and 2 may be attributed to different RH conditions in the BSC II during the initial one hour of
drying on the different days. There was high RH (-70%) experienced during drying in the BSC II on
Day 0 of the first run. The RH condition during drying on the day of the second run was approximately
40-50%. High RH, as shown in Table 4-2 below, results in about a 78% attenuation of ricin on
galvanized metal in the first day subsequent to drying. The lower recovery after high RH drying
conditions in the BSC II may result from higher rates of loss occurring during the one hour drying time
when RH during drying is moderate to low.
15
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Table 4-1. Ricin Extraction Efficiencies
Material Run Observations (n) Recovery (%)
Mean SD
Galvanized metal
Painted concrete
1
2
1
2
8
7
7
8
24.34
79.10
98.82
81.84
7.34
18.32
25.81
10.16
4.1.1 Ricin on Galvanized Metal
Table 4-2 and Figure 4-1 summarize the cytotoxicity of ricin extracted from galvanized metal under the
specified temperature and RH conditions. The average (and corresponding joint 95% intervals from the
statistical model) of the replicate (n) positive control samples are reported under "Mean Ricin
Cytotoxicity (ug) after Drying". The values are reported as the average (in units of ug) of cytotoxicity
recovered from the replicate coupons at the specific time point indicated; the numbers in parentheses are
the confidence interval endpoints for the averages as determined from the statistical model fit to the
data. The confidence intervals are individually of 99.8% confidence to control the chance of error at 5%
for the total number of statistical comparisons made. Cytotoxic ricin extracted from galvanized metal
declined over the 14 day period at all RH and temperature conditions investigated. However, there were
significant differences between the ricin from coupons maintained in the low RH condition compared to
those at the ambient or high RH conditions. Under the low RH condition, recovery on Day 14 was 14%
of the amount recovered on positive control coupons after drying. In ambient or high RH conditions, the
cytotoxic ricin recovered on Day 14 was <0.3% of the amount recovered on positive control coupons
after drying. A blank coupon was analyzed for each material at each time point and little cytotoxicity
was measured (cytotoxicity equal to 0.001 - 0.009 ug of cytotoxic ricin was observed for galvanized
metal).
The amount of ricin remaining on the coupons in Run 1 maintained at ambient conditions was
significantly lower on Day 1 than the amount of ricin remaining on coupons on Day 1 in Run 2
maintained at ambient conditions. This unexpected difference is attributed to the difference in RH
during the drying time. Specifically, after inoculation, coupons remained undisturbed at the ambient
conditions in the BSC II for one hour ("drying time") before being transferred into controlled
temperature and RH conditions. In the test/QA plan the RH during the drying time was not specified and
controlled. The drying was to be done under ambient conditions. The RH at ambient conditions during
drying on different days could be different. The ambient RH during the drying time was observed
qualitatively to have been higher on the day of Run 1 than on the day of Run 2. This difference in RH
during the drying time may have impacted ricin persistence on galvanized metal. The direction of
change (higher RH is associated with increased attenuation of bioactive ricin) is consistent with our
other observations.
16
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Table 4-2. Mean Recovery of Ricin Cytotoxicity on Galvanized Metal"
Mean Ricin
Run Cytotoxicity (jig) after Treatment
Drying1"
1 Ambient RH
(20°C, RH 40%-
6 086C 70%)
1 (4-04'8R132) HighRH
n (30°C, RH >70%)
2 Ambient RH
(20°C,RH40%-
70%)
(17.589,21.963) '
1 n=7 Low RH
(30°C, RH < 40%)
Mean Ricin Cytotoxicity (jig) after Treatment
Dayl Day 3 Day 9 Day 14
1.672
(0,5.013)
n=3
1.344
(0,3.932)
n=5
7.615
(3.523,11.706)
n=2
8.996
(6.408,11.584)
n=5
0.572
(0,3.913)
n=3
1.963
(0,4.551)
n=5
0.633
(0,4.725)
n=2
4.750
(2.162,7.338)
n=5
0.007
(0,3.348)
n=3
0.376
(0,2.963)
n=5
0.006
(0,4.098)
n=2
4.865
(2.277,7.453)
n=5
0.005
(0,3.346)
n=3
0.014
(0,2.602)
n=5
0.013
(0,4.105)
n=2
2.802
(0.214,5.39)
n=5
a Initial inoculation of 25 \ig protein
b Ambient BSC II conditions for one hour
0 High ambient RH conditions (—70%) were qualitatively observed during the one hour drying time before the coupons were placed into
controlled conditions
O)
>
+J
'u
o
o
100
Average Run 2 Recovery After 1 Hour Drying = 19.8 ug
0.01
0.001
Average Run 1 Recovery After 1 Hour Drying = 6.1 ug
0
Day
10
12
14
16
-OAmbient RH (Run 1) -•- High RH (Run 1) -O-Ambient RH (Run 2) -•- Low RH (Run 2)
Figure 4-1. Persistence of Ricin on Galvanized Metal
17
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Table 4-3 and Figure 4-2 show that at ambient to high RH condition, the decrease in cytotoxic ricin
extracted from galvanized metal after Day 14 (compared to the ricin extracted after one hour drying)
corresponds to a 99.77 to 99.93% reduction. In contrast, cytotoxic ricin exhibited less attenuation
(85.83% reduction on Day 14) at the low RH condition. Because ricin cytotoxicity was lost at ambient
conditions, no renaturation testing was performed.
Table 4-3. Mean Attenuation: Ricin on Galvanized Metal" (Shown as Percent Reduction)
Mean Attenuation of Ricin Cytotoxicity (% Reduction)
Run Treatment Day 1 Day 3 Day 9 Day 14
1 Ambient RH (20°C, RH 40 - 70%) 72.53
90.59 b
99.88
99.92
High RH (30°C, RH >70%)
77.91'
2 Ambient RH (20°C, RH 40 - 70%) 61.50'
67.75 b
96.80'
93.83
99.97
99.77b
99.93
Low RH (30°C, RH < 40%)
54.51 b
75.98'
75.40
bc
85.83
a Initial inoculation of 25 |ig protein; percent reduction was calculated from a basis of Day 0 (after one hour drying time)
b Attenuation significantly different from zero (P < 0.0021)
0 Attenuation significantly different from ambient within same day and run (P < 0.0021)
0
D-High
2
RH
(Run
1)
4
-•-Low
6
RH
(Run
Day
10
2) -0- Ambient RH
12
(Run 2)-9-
14
LowRH
16
(Run 2)
Figure 4-2. Mean Attenuation (Percent Reduction): Ricin on Galvanized Metal
18
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4.1.2 Ricin on Painted Concrete
Table 4-4 and Figure 4-3 summarize the extractable cytotoxic ricin from painted concrete at the three
treatment conditions. As for the galvanized metal, the values in the table and reported in the figure are
averages (in units of ug) of cytotoxity recovered from the replicate coupons at the specific time point
indicated; the numbers in parentheses in Table 4-4 are the statistical model based confidence intervals
(joint 95% confidence for all intervals) of the replicates. After one hour drying there were no significant
differences between extractable ricin in Run 1 and Run 2; therefore, these data were combined and are
shown as results after one hour in Table 4-4. There was no significant loss of ricin (compared to ricin
extracted after one hour drying) after Day 14 at ambient (20°C) or low RH at 30°C. However, there is a
statistically significant reduction in extractable ricin from painted concrete incubated at high RH and
30°C on Days 9 and 14.
A blank coupon was analyzed for each material at each time point and little cytotoxicity was measured
(0.005 - 0.011 jig protein cytotoxicity for painted concrete).
Table 4-4. Mean Recovery of Ricin Cytotoxicity on Painted Concrete"
Mean Ricin
Run Cytotoxicity (ug)
after Drying1"
1,2
1 22.441
(19.301,25.582)
n=15
2
Treatment
Ambient RH
(20°C, RH 40%-
70%)
HighRH
(30°C, RH >70%)
LowRH
(30°C, RH <40%)
Mean
Dayl
15.337
(9.898,20.776)
n=5
16.093
(10.654,21.532)
n=5
13.871
(8.432,19.310)
n=5
Ricin Cytotoxicity (jig) after Treatment
Day 3 Day 9 Day 14
16.033
(10.594,21.472)
n=5
19.264
(13.825,24.703)
n=5
17.950
(12.511,23.389)
n=5
18.398
(12.959,23.837)
n=5
10.008
(4.569,15.447)
n=5
16.538
(11.099,21.977)
n=5
19.017
(13.578,24.456)
n=5
6.339
(0.9,11.778)
n=5
16.375
(10.936,21.814)
n=5
a Initial inoculation of 25 |ig protein
b Ambient BSC II conditions for one hour
19
-------
u>
100 -r
Inoculation
= 25|jg
Average Recovery After 1 Hour Drying = 22.4 ug
0.001
Day
10
12
14
16
•High RH (Run 1)
-LowRH (Run 2)
-A-Ambient RH (Run 1,2)
Figure 4-3. Persistence of Ricin on Painted Concrete
The percent reduction values for ricin extracted from painted concrete are shown in Table 4-5 and
Figure 4-4. On Day 1, small but significant decreases in ricin extracted from painted concrete were
observed for the ambient RH (31.7%), the high RH (28.3%) and the low RH (38.2%) conditions; on Day
3, the average extracted ricin continued to be less than that compared to the ricin extracted after one
hour drying, but only the ambient RH reduction (28.6%) was statistically significant. For Days 9 and 14,
the reduction at high RH increased to 55.4 and 71.8%, respectively; significantly higher than the
baseline condition as well as significantly higher than the corresponding ambient RH reductions (18.0%
at Day 9 and 15.3% at Day 14). The reductions at ambient RH and low RH on Days 9 and 14 were not
statistically significant when compared to the ricin extracted after one hour drying. The statistical
significance is determined from the ANOVA model fit to the extracted cytotoxicity data. A Bonferroni
adjustment was employed to control the overall error rate for all statistical comparisons at 5%. With 20
total comparisons made for the ricin on painted concrete; this required that each comparison individually
have a p-value of 0.05 divided by 20, or 0.0025, to be identified as statistically significant. There is no
more than a 5% chance that the set of statistically significant comparisons contains an erroneous
conclusion of significance by chance.
Table 4-5. Mean Attenuation: Ricin on Painted Concrete11 (Shown as Percent Reduction)
Run
Treatment
Mean Attenuation of Ricin Cytotoxicity (% Reduction)
Dayl Day 3 Day 9 Day 14
1,2
1
Ambient RH (20°C, RH 40 - 70%)
High RH (30°C, RH >70%)
Low RH (30°C, RH <40%)
31.66 b
28.29 b
38.19b
28.56 b
14.16
20.02
18.02
55.40 bc
26.31
15.26
71.75bc
27.03
a Initial inoculation of 25 ng protein; percent reduction was calculated from a basis of Day 0 (after one hour drying time)
b Attenuation significantly different from zero (P < 0.0025)
20
-------
0 Attenuation significantly different from ambient within this day (P < 0.0025)
0
-m-
2
High RH
(Run
4
1)
6
-•- Low RH
8
Day
(Run 2)
10
12
-A- Ambient RH
14
(Runs
1
1,2)
Figure 4-4. Mean Attenuation: Ricin on Painted Concrete
The attenuation results for ricin on painted concrete indicate that the impact of temperature and RH are
much less than observed for galvanized metal. The Day 1 attenuation results for all conditions, as well
as the results for Ambient RH at Day 3 are significantly greater than zero, but the Ambient RH and low
RH attenuation falls to below significant levels at Days 9 and 14. By contrast, the high RH attenuation
increases through Days 9 and 14, but still exhibits lower final attenuation than was seen on galvanized
metal. Generally, cytotoxic ricin was persistent on painted concrete throughout the two week test period
regardless of the treatment. Since the cytotoxicity of ricin remained high, no testing for renaturation was
performed.
4.2 Recovery of Vaccinia and Impact of Environmental Conditions on Persistence of Vaccinia
Approximately 7 x 107 PFU vaccinia virus (vaccinia) were inoculated onto each test coupon of the
indoor building materials and allowed to dry for one hour under ambient BSC II conditions. The
vaccinia-inoculated coupons were then placed into the test chambers with controlled temperature and
RH. After the one hour drying time and at specified intervals, vaccinia was extracted from the coupons,
inoculated onto Vero (African Green Monkey kidney) cell monolayers, and quantified using a standard
21
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plaque assay. A blank coupon was analyzed for each material at each time point. No viable vaccinia
were detected on any of the galvanized metal or painted concrete blank coupons.
Table 4-6 summarizes the extraction efficiency from different test material coupons by calculating the
recovery of number of viable virus extracted after one hour drying time (TO positive control coupons) as
a percentage of the viable virus applied (measured in the application control). Measurements at different
times (replicates) are referenced as different runs (Run 1 or Run 2).
Less than 20% of the number of vaccinia inoculated onto the coupon were extracted and viable after one
hour drying. The recovery percentages for vaccinia are low for galvanized metal but are consistent
between the two runs. The percentages for painted concrete are higher than for galvanized metal but still
are low on an absolute basis. To demonstrate this low recovery was not due to poor extraction, vaccinia
was inoculated onto the coupons and immediately extracted. Recovery with immediate extraction of
these samples ranged from 90-100% (data not shown). The low levels of viable virus after one hour
drying are consistent with the continuing loss of viability observed in subsequent time periods at
ambient or high RH. Under high RH or ambient conditions, viable vaccinia exhibited a 3 log reduction
in the first day (see discussion below).
Table 4-6. Vaccinia Extraction Efficiency
Material
Galvanized metal
Painted concrete
Run
1
2
1
2
Observations
7
8
8
7
%
Recovery
4.52
6.65
10.64
19.19
SD
2.40
1.89
0.83
3.93
4.2.1 Vaccinia on Galvanized Metal
Table 4-7 and Figure 4-5 summarize the results for vaccinia recovered from galvanized metal
maintained under the test temperature and RH conditions. The data are reported as mean PFUs
recovered from the five replicate coupons (n = 5) at the specified time points; the SDs are reported in
parentheses. At ambient and high RH conditions no viable virus was extracted from any of the five
replicate coupons at Days 3, 9, and 14. At low RH, a comparatively high level of extractable virus (>105
PFU) was recovered from on the coupons after Day 14. The temperature and RH data for these runs are
reported in Appendix A.
22
-------
Table 4-7. Mean Vaccinia Recovery from Galvanized Metal"
Mean Vaccinia
Run Recovery (PFU) after Treatment
Drying1"
1, 2 Ambient RH
(20°C, RH 40 - 70%)
1 3 548E+06 High RR
(1.767E+06) (30°C,RH>70%)
n=15
2 LowRH
(30°C, RH <40%)
Mean Vaccinia Recovery (PFU) after Treatment
Dayl Day 3 Day 9 Day 14
2.27E+03
(1.75E+03)
n=5
4.06E+04
(6.39E+04)
n=5
1.52E+06
(6.60E+05)
n=5
O.OOE+00C
(O.OOE+00)
n=5
O.OOE+00C
(O.OOE+00)
n=5
8.67E+05
(3.00E+05)
n=5
O.OOE+00C
(O.OOE+00)
n=5
O.OOE+00C
(O.OOE+00)
n=5
4.81E+05
(6.97E+04)
n=5
O.OOE+00C
(O.OOE+00)
n=5
O.OOE+00C
(O.OOE+00)
n=5
5.10E+05
(1.29E+05)
n=5
a Average initial inoculation of 6.76E+07
b Ambient BSC II conditions for one hour
0 Zero organisms observed in extracts from all coupons
1.0E+08 *
1.0E+07
1.
1.C
1.C
1.C
1.0E+01
1.
Average Inoculation = 6.8x107 PFU
Average Recovery After 1 Hour Drying = 3.5x10 PFU
No observed virus for any coupons at Days 3, 9 and 14 for
the ambient and high temp/high humidity treatments; plotted
values show 1 PFU
0
-•-High
RH
2
(Run
1)
4
6
-•- Low RH
8
Day
(Run 2)
10
12
-A- Ambient
RH
14
(Runs 1
16
2)
Figure 4-5. Persistence of Vaccinia on Galvanized Metal
The data in Table 4-8 and Figure 4-6 indicate that in ambient to high RH conditions the decrease in
viable vaccinia extracted from galvanized metal at and after three days corresponds to a minimum 6.6
log reduction (conservative estimate based on replacing observation of no viable vaccinia PFU observed
with a value of one vaccinia PFU). In contrast, vaccinia exhibits a much higher persistence (<1 log
reduction at Day 14) at low RH conditions.
23
-------
Table 4-8. Mean Attenuation: Vaccinia on Galvanized Metal" (Shown as Log Reduction)
Run
Treatment
Dayl
Mean Attenuation after Treatment
Day 3 Day 9 Day 14
1 2 Ambient RH
(20°C, RH 40 - 70%)
1 High RH
(30°C, RH >70%)
2 LowRH
(30°C, RH <40%)
3.323b
,bc
2.548
0.393C
>6.550d
>6.550d
0.635b
>6.550d
>6.550d
0.872b
>6.550d
>6.550d
0.857b
a Average initial inoculation of 6.76E+07; log reduction was calculated from a basis of Day 0 (after one hour drying time)
b Attenuation significantly different from zero (P < 0.05)
0 Attenuation significantly different from ambient within this day (P < 0.05)
d Attenuation not calculable because all 5 replicate coupons had zero observed virus PFUs; minimum attenuation is estimated by assuming
one organism observed; 95% confidence that attenuation is >5.550 (i.e., less than or equal to 10 PFU in coupon extract) and hence it is
statistically significantly different from zero
7 -.
R
0 D
03
Q c;
E °
o
D
- 0
.= o
(0
0 »
•C ^
3
•o
O
o n
Fi..........-..-..-.......-............Fi.............................-ji
*
,l' After Day 1 , no remaining virus found on any of the
// five replicate coupons for either the ambient or hiqh
// temp/high humidity treatments; plotted values are a
•'•' minimum assuming a mean of 1 CPU
A '
t
t
*
• -+ " "
024
-•- 'High RH (Run 1)
6 rP
Day
-•- Low RH (Run 2)
10 12 14 11
- A- 'Ambient RH (Runs 1, 2)
Figure 4-6. Mean Attenuation: Vaccinia on Galvanized Metal
The attenuation in the persistence of vaccinia on galvanized metal was significantly greater than zero at
Day 1 for both the ambient and high temperature/high RH treatments. On Days 3, 9, and 14, no
remaining viable virus (0 PFU) was found for either of these treatments. Because only a sample of the
extract was tested for viable virus, the observed mean value of 0 PFU from every coupon was
determined to indicate (at 95% confidence) at least a 5.5 log reduction of viable virus in the total extract.
This suggests there may be a rapid decrease and complete loss of viable virus from galvanized metal
upon exposure to ambient to high temperature and RH. Vaccinia persisted at low RH (at high
24
-------
temperature), although the observed attenuation was statistically significantly than zero after Day 1.
However, the mean attenuation at low RH was less than 1 log reduction at Day 14.
4.2.2 Vaccinia on Painted Concrete
Table 4-9 and Figure 4-7 summarize the results for vaccinia virus recovered from painted concrete under
the test and control temperature and RH conditions. The extraction of vaccinia after one hour drying was
replicated at times designated as Run 1 or Run 2. There were no significant differences in the recovery
after the one hour drying period, as shown in Table 4-6, between these runs. Therefore, the overall
average number of PFU is shown as the one hour mean value. Less than 15% of the PFU of vaccinia
inoculated onto the coupons were extracted and viable after one hour drying. The low levels of viable
virus after one hour drying are consistent with the continuing loss of viability that is observed in
subsequent time periods at ambient or high RH. Under high RH conditions, a 3 log reduction in viable
vaccinia was observed within the first day. To demonstrate this low recovery was not due to poor
extraction, vaccinia was inoculated onto the coupons and immediately extracted. Recovery from these
samples ranged from 90-100% (data not shown).
At low RH, >106 PFU of extractable virus was recovered from the coupons after Day 14. Approximately
103 PFU of viable vaccinia were extracted from coupons after being maintained at ambient conditions
for Day 14. In contrast, at high temperature and RH no viable vaccinia was extracted from any of the
five replicate coupons at Days 9 and 14. Thus, a trend in persistence with RH is evident.
Table 4-9. Mean Vaccinia Recovery from Painted Concrete"
Mean Vaccinia
Run Recovery (PFU) Treatment
after Drying15
1, 2 Ambient RH
(20°C, RH 40 -
70%)
1 9.285E+06 High RH
(1.562E+06) (30°C, RH
n=15 >70%)
2 Low RH
(30°C, RH
<40%)
Mean Vaccinia Recovery (PFU) after Treatment
Dayl Day 3 Day 9 Day 14
2.63E+06
(1.18E+06)
n=5
2.16E+03
(1.30E+03)
n=5
7.54E+06
(1.967E+06)
n=5
6.00E+05
(5.28E+05)
n=5
2.40E+02
(1.67E+02)
n=5
6.27E+06
(2.24E+06)
n=5
2.68E+04
(2.07E+04)
n=5
O.OOE+00C
(O.OOE+00)
n=5
1.30E+06
(8.80E+05)
n=5
1.712E+03
(9.092E+02)
n=5
O.OOE+00C
(O.OOE+00)
n=5
3.19E+06
(1.35E+06)
n=5
a Average initial inoculation of 7.103E+07
b Ambient BSC conditions for one hour
0 Zero organisms observed in extracts from all coupons
25
-------
OE+08 *
OE+07
1.0E+01
1.0E+00
Average Inoculation = 7.1x107
Average Recovery After 1 Hour Drying = 9.3x10 PFU
No observed virus at Days 9 and 14 for the high
humiditv treatment: clotted values show 1 PFU
-•-
0
High
RH
2
(Run
1)
4 6
-•- Low RH
8
Day
(Run 2)
10
12
-A- Ambient
RH
14
(Runs
11
1,2)
Figure 4-7. Persistence of Vaccinia on Painted Concrete
Table 4-10 and Figure 4-8 provide results for mean attenuation in the persistence of vaccinia virus on
painted concrete at the three treatment conditions. Because only a sample of the extract was tested for
viable virus, in cases where the observed mean value of 0 PFU was observed from every coupon, a
lower 95% bound on attenuation was determined. At high temperature and RH conditions the decrease
in viable vaccinia extracted from galvanized metal at Days 9 and 14 corresponds to a mean reduction of
approximately 7 log (assumes 1 PFU rather than 0 actually observed) and a 95% confidence of at least a
6 log reduction. In contrast, at low RH conditions little viability of vaccinia is lost (-0.5 log reduction
after Day 14).
Table 4-10. Mean Attenuation: Vaccinia on Painted Concrete" (Shown as Log Reduction)
Run
1,2
1
Treatment
Ambient RH
(20°C, RH 40 - 70%)
HighRH
(30°C, RH >70%)
Dayl
0.587b
3.836bc
Mean Attenuation after Treatment
Day 3 Day 9
1.360b 2.814b
4.736bc > 6.968d
Day 14
3.828b
>6.968d
LowRH
(30°C, RH <40%)
0.1036
0.189C
0.909
,bc
0.504
be
3 Average initial inoculation of 7.103E+07
b Attenuation significantly different from zero (P < 0.05)
0 Attenuation significantly different from ambient within this day (P < 0.05)
26
-------
Attenuation not calculable because all five replicate coupons had zero observed virus PFUs; minimum log reduction is
estimated by assuming one organism observed; 95% confidence that attenuation is >5.968 and hence it is statistically
significantly different from zero
3 Attenuation not significantly different from zero (P > 0.05)
7 -r
On Days 9 and 14, no remaining virus is found on coupons for the high humidity
treatment; plotted values are minimum efficacy assuming mean of 1 CPU
01]
o
-J
024
-•-High RH (Run 1)
6 8
Day
-•- Low RH (Run 2)
10 12 14
-A- Ambient RH (Runs
1
1,2)
Figure 4-8. Mean Attenuation: Vaccinia on Painted Concrete
The attenuation in the persistence of viable vaccinia on painted concrete subjected to alternate
temperature and RH conditions were similar to those for galvanized metal. The high temperature/high
RH treatment promoted a decrease in viable vaccinia, with no extractable virus (PFU) observed at Days
9 or 14. At the ambient treatment conditions, significant reductions in vaccinia were observed increasing
from Day 1; however, detectable quantity of viable virus was extracted at Day 14. Viable vaccinia was
more persistent at low RH conditions with less than a 1 log attenuation throughout the test. At low RH,
the attenuation at Days 9 and 14 were significantly lower than the ambient treatment.
4.2.3 Effect of Temperature on Persistence ofRicin and Vaccinia
This investigation did not show, but cannot rule out, modest effects from temperature differences that
are masked by the large effects of RH.
27
-------
5.0 Summary
The results show that RH, indoor material type, and time after application of the agent to the indoor
material affect the persistence of cytotoxic ricin and viable vaccinia virus. The results summarized in
Table 5-1 show that the RH affects the persistence of cytotoxic ricin and viable vaccinia virus. Both
ricin toxin and viable vaccinia virus have greater persistence on galvanized metal and painted concrete
for up to Day 14 under low RH conditions than high RH conditions.
Table 5-1. Ricin or Vaccinia Recovery from Different Materials under Varying RH Conditions at
Constant Temperature (Temperature = 30°C)
Biological Agent or
Surrogate
Ricin toxin
Vaccinia virus
Building
Material
Galvanized
metal
Painted
concrete
Galvanized
metal
Painted
concrete
RH
>70%
<40%
>70%
<40%
>70%
<40%
>70%
<40%
Amount Inoculated onto
Coupons
25 ug cytotoxic protein
25 ug cytotoxic protein
25 ug cytotoxic protein
25 ug cytotoxic protein
7 x 107 PFU
7 x 107 PFU
7 x 107 PFU
7 x 107 PFU
Day 14 after Inoculation onto
Coupons
0.0 ug cytotoxic protein
2.8 ug cytotoxic protein
6.3 ug cytotoxic protein
16.4 ug cytotoxic protein
OPFU
5 x 105 PFU
OPFU
3 x 106 PFU
The results also suggest that the rate of loss of cytotoxic ricin and viable vaccinia virus (or, stated
differently, persistence at a given point in time) depends on the type of material onto which the
biological agent is applied. Table 5-2, for example, shows that under ambient conditions residual
amounts of both ricin and vaccinia measured on Day 3 are very different for the different building
materials.
Table 5-2. Day 3 Ricin or Vaccinia Recovery from Different Materials, Ambient Conditions
Biological Agent or Surrogate
Ricin toxin
Vaccinia virus
Building Day 3 after Inoculation onto
Material Coupons
Galvanized metal
Painted concrete
Galvanized metal
Painted concrete
0.6 ug cytotoxic protein
16.0 ug cytotoxic protein
OPFU
6 x 105 PFU
28
-------
Table 5-3 shows that, depending on the type of indoor material, elevated RH may be useful for lowering
the cytotoxicity of ricin before the application of a decontamination technology. Ricin is most persistent,
for both galvanized metal and painted concrete, at low RH conditions. Ricin was persistent on painted
concrete retaining substantial extractable cytotoxicity after Day 14, even at high RH. For ricin on
galvanized metal, little or no ricin cytotoxicity (above the background coupon cytotoxicity) was
observed by Day 14 at high RH.
Elevated RH may be useful for inactivating vaccinia virus before the application of a decontamination
technology. Vaccinia virus persisted on painted concrete or galvanized metal at Day 14 at low RH.
However at high RH, no viable vaccinia virus was extracted on Day 3 or after from galvanized metal, or
on Day 9 or 14 from painted concrete. Based on the results of this investigation, it is possible that
elevated RH may have a comparable impact on the viability of Variola or other pox viruses on metal and
painted concrete substrates.
Table 5-3. Mean Attenuation: Ricin or Vaccinia on Different Building Materials under Varying
RH Conditions
Bioagent
Ricin
Ricin
Vaccinia
Vaccinia
Material Treatment Providing Highest Attenuation Day 14 Mean Reduction
Galvanized metal
Painted concrete
Galvanized metal
Painted concrete
Ambient (20°C) or high RH (30°C)
HighRH(30°C)
Ambient (20°C) or high RH (30°C)
HighRH(30°C)
99.9% (ambient RH), 99.8% (high RH)
71.8%
>6.5 (no viable virus)
>6.9 (no viable virus)
Because substrates and environmental conditions investigated here show differential impact on the
cytotoxicity of the ricin or viability of the vaccinia, it is clear that these factors must be considered and
controlled in evaluation of decontamination technologies (e.g., for control conditions).
This investigation did not show, but cannot rule out, modest effects on persistence from temperature
differences that could be masked by the large effects of RH.
No results are presented for C. burnetii. Although C. burnetii were observed microscopically to be
present in the Vero (African Green Monkey) kidney cells used for the plaque assay, plaques were not
observed after inoculating either Vero kidney cell monolayer or a mouse fibroblast monolayer and
incubation under appropriate culture conditions for ten days. Therefore, the quantitative impact of
environmental conditions on C. burnetii persistence and the attenuation of controlling RH to kill C.
burnetii could not be determined.
29
-------
6.0 References
1. Aguado, B., IP. Selmes, and G.L. Smith, "Nucleotide sequence of21.kbp of variola major virus
strain Harvey and comparison with vaccinia virus. " Journal of General Virology, 1992(73): p.
2887-2902.
2. Battelle, Technology Testing and Evaluation Program Test/QA Plan for Evaluation of
Persistence ofCoxiella burnetii (Q-Fever Agent), Vaccinia Virus (Cowpox Agent) andRicin
Toxin on Contaminated Indoor Surfaces Using Elevated Temperature and Humidity Control for
Decontamination. March 2005.
3. Battelle, Quality Management Plan (QMP) for the Technology Testing and Evaluation Program
(TTEP); Version 2. January 2006.
4. Battelle, MREFFacility Safety Plan Annex 12 to Appendix B, "Guidelines for the Use of Class II
and Class III Biological Safety Cabinets in the MREF Biofacility. " July 2006.
5. Battelle, FSP Annex 5 to Appendix B, "Guidelines for Safe Handling and Storage ofEtiologic
Agents at the MREF." July 2006.
6. Battelle, FSP Annex 7 to Appendix B, "Guidelines for Disinfection/Decontamination of
Etiological Agents at the MREF Biofacilities." July 2006.
7. Battelle, Facility Safety Plan (FSP) for the Medical Research and Evaluation Facility (MREF).
July 2006.
8. Battelle, "Guidelines for the Use of Class II and Class III Biological Safety Cabinet in the MREF
Biofacility", in MREF Facility Safety Plan Annex 12 to Appendix B. July 2006.
9. Battelle, MREF Method No. 104/Microbiology, "Coupon Testing of Decontamination
Formulations against Bacillus spp. Spores. " May 2004.
10. Battelle, MREF Method No. 106/Microbiology, "Methodfor the Plaque Assay for Detection and
Quantitation of Viral Particles. May 2004.
11. Gutierrez, B., W., M, Rabinovitch, M., Columbo M., "Coxiella burnetii in a Rab7-Labeled
Compartment with Auto Phagic Characteristics." m Infection and Immunology. 2002. p. 5816-
5821.
12. Mosmann, T., "Rapid colorimetric assay for cellular growth and survival: application to
proliferation andcytotoxicity assays." Journal of Immunological Methods, 1983(65): p. 55-63.
13. CellTiter 96™ non-Radioactive Cell Proliferation Assay. Promega - Technical Bulletin, 2007.
30
-------
Appendix A
Report of Temperature and RH Measurement Data
A-l
-------
The temperature and RH for the each of the test chambers used in all four trial runs are reported in this
appendix. For each trial run, two test chambers were used; one chamber for the ambient treatment and
one for the alternate condition. A total of two runs were made for each of the two agents investigated. In
Run 1 for each agent, the ambient (~20°C, 40%-70% RH) and high RH (30°C, >70% RH) conditions
were investigated. In the second runs, the ambient (~20°C, 40%-70% RH) and low RH (30°C, <40%
RH) treatments were performed. The temperature and RH data for each chamber in each of the four runs
are presented graphically below for the entire 14 day run durations.
RicinRun 1:
For the ambient condition, the mean temperature and RH were 21.2°C and 59.7%, respectively. The SDs
were 0.6 and 0.7, respectively. The ranges were 18.2°C-22.1°C and 55.4%-61.3% RH. For the High RH
condition, the mean temperature and RH were 31.7°C and 89.4%, respectively. The SDs were 0.6 and
7.9, respectively. The ranges were 27.4°C-33.4°C and 59.0%-99.6% RH.
30
G"
-------
Ambient Conditions (Trial Run 2)
Low RH Conditions (Trial Run 2)
30-
P~
-------
Ambient Conditions (Trial Run 2)
Low RH Conditions (Trial Run 2)
35-
30-
G
0) 25-
s
-------
Appendix B
Report of Methods Development and Demonstration
B-l
-------
Method development and demonstration performed under the test/QA plan1, but not described in the
report are summarized in this appendix.
Demonstration of Methods for Measuring Biological Activity of C. burnetii
o
A method was needed in order to quantify C. burnetii. The method of Gutierrez et al. (2002) was tried.
A dilution series of C. burnetii (Nine Mile strain, phase II, plaque-purified clone 4) was inoculated onto
Vero (African Green Monkey) kidney cell, C. burnetii were observed microscopically to be present in
the cells of the monolayers. However, plaques, necessary for quantitation of the organisms, were not
observed at any dilution after 10-day incubation.
The plaque assay that was done with the Vero cells was repeated with a slower growing normal
fibroblast culture. The idea was that the slower-growing fibroblasts would enable resolution of plaques
caused by C. burnetii infection. Repeating the approach using normal mouse fibroblast monolayers
yielded similar results to those observed with Vero cells; although C. burnetii were observed
microscopically to be present in the cells of the monolayers, plaques were not observed.
Flow cytometry was considered as another approach for quantifying C. burnetii. The conceptual
approach was to use flow cytometry, with or without sorting, as a means to quantify cells discriminated
with the Baclight™ (Invitrogen) Live/Dead® stain. Cell sorting was dropped from consideration because
of the amount of method development and validation that would be required.
The proposed use of the Baclight stain was dropped from consideration because of reported over-
estimation of viability. Additional fluorescent probes were considered that assess viability in a variety of
organisms by measuring glucose uptake in live cells. For example, live/dead discrimination might be
done with 2-NBDG [2-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino)-2-deoxyglucose] and propidium
iodide, or just live cells could be indicated with 2-NBDG. A rapid method for using the fluorescent
probes was envisioned for quantifying living cells. The approach would use a florescence microplate
reader to measure the fluorescence from the living cells extracted from a sample. The fluorescence
would be calibrated against flow cytometer data to allow the number of viable cells to be quantified.
However, the fluorescent probe method had not been tested with C. burnetii and the use of a calibrated
microplate reader was a novel and untested idea. Limited method development showed that the
fluorescent probe worked, however the dynamic range was too small. Substantial method development
might be required.
In summary, a variety of approaches for quantifying C. burnetii were considered and some tried, but
none were found sufficient without development beyond the scope of this investigation. Therefore, the
quantitative impact of environmental conditions on persistence of C. burnetii could not be determined.
Demonstration of Methods for Improving Recovery from Coupons
Recoveries of ricin from galvanized metal coupons, shown in Table 4-1, after one hour were
consistently low (6 - 20 jig of cytotoxic protein). The coupons used in this testing were more than a year
old. To attempt to improve recoveries, coupons of new galvanized metal were tested. Three galvanized
metal and three painted concrete coupons were inoculated with 25 jig cytotoxic ricin and handled in the
same manner as described in the methods section of this report. Extractions were performed
B-2
-------
immediately after inoculating the coupon (<10 seconds) or after setting undisturbed for one hour.
Analysis was performed as described in the report. As shown in Table A-l, all or nearly all of the ricin
was extracted from the painted concrete at <10 seconds and at one hour. In contrast, 1.3% or less of the
cytotoxic ricin applied was extracted from galvanized metal at <10 seconds or at one hour. The
galvanized metal used in this method development was new (in contrast to aged galvanized metal that
was used in the persistence test. Recoveries of ricin were not improved by immediate recovery from new
galvanized metal. Recoveries from the new galvanized metal, shown in Table B-l, (1.3% or less) were
lower than from aged galvanized metal after one hour (24.4% and 79.2% from Run 1 and Run 2,
respectively, shown in Section 4.1.1).
The mechanism of loss of cytotoxic ricin, whether from reaction with the galvanized metal or adsorption
to the galvanized metal, was not determined. A possible explanation is that the reactive zinc in the new
galvanized metal is having a greater deactivation effect on the ricin than the aged metal with a zinc
carbonate patina. Zinc (the coating of galvanized metal) is highly reactive. It goes through a series of
reactions as it ages, first reacting with oxygen in the air to form zinc oxide; then reacting with moisture
to form zinc hydroxide; and finally the zinc oxides and hydroxides react with carbon dioxide to form
zinc carbonate. Zinc oxide has been shown to inhibit enzymes and Zn(+2) has been shown to inhibit
ricin cytotoxic effects, e.g., apoptosis. See, for example, Tamura, T., Sadakata, N., Oda, T., and
Muramatsu, T. (2002) Role of zinc ions in ricin-induced apoptosis in U937 cells. Toxicol. Lett. 132,
141-151. Therefore, the new galvanized metal with more of the zinc and zinc oxide may deactivate ricin
more readily than zinc carbonate.
Table B-l. Ricin Recoveries (jig) upon Immediate or Delayed Extraction
Dry time
<10 sec
One hour
Trial 1
Galvanized
metal, jig (n=3)
0.002
0
Concrete,
jig (n=3)
25.864
25.211
Trial 2
Galvanized
metal, ug (n=3)
0.053
0
Concrete,
Jig (n=3)
29.383
24.553
Trial3
Galvanized
metal, ug (n=3)
0.255
0.317
Concrete,
Jig (n=3)
29.987
23.492
B-3
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