&EPA
United States
Environmental Protection
Agency
Freeze-Dried Vaccinia Virus
Persistence Testing and
Liquid Decontamination
Technology Evaluation
INVESTIGATION AND TECHNOLOGY
EVALUATION REPORT
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-09/139 November 2009 www.epa.gov/ord
INVESTIGATION AND TECHNOLOGY
EVALUATION REPORT
Freeze-Dried Vaccinia Virus Persistence
Testing and Liquid Decontamination
Technology Evaluation
By
Young W. Choi, Morgan Q. Shaw, James V. Rogers, Daniel J. Chappie, Michael L. Taylor,
Karen B. Riggs, and Zachary J. Willenberg
Battelle
505 King Avenue
Columbus, Ohio 43201
Joseph P. Wood
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
Office of Research and Development
National Homeland Security Research Center, Decontamination and Consequence Management Division
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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,
directed, and managed this investigation and technology evaluation through a
Blanket Purchase Agreement under General Services Administration contract number
GS23FOO11L-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.
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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,
EPA 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 ORD; it manages, coordinates,
and supports 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 clean up in the event of
such attacks.
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.
The evaluation reported herein was conducted by Battelle
as part of TTEP. Information on NHSRC and TTEP can be
found at http://www.epa.gov/nhsrc/ttep.html.
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct
the evaluation, analyze the data, and prepare this report. We also would like to thank
Dr. Timothy Dean of the EPA National Risk Management Research Laboratory -
Air Pollution Prevention and Control Division, Dr. Sarah Taft of the EPA NHSRC,
and Dr. Michael Ottlinger of the EPA Office of Emergency Management - National
Decontamination Team for their reviews of this report.
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Contents
Notice iv
Foreword v
Acknowledgments vi
Abbreviations/Acronyms ix
Executive Summary x
1.0 Introduction 1
2.0 Persistence Testing 3
2.1 Test Materials 3
2.2 Vaccinia Virus Recovery from Test Materials 3
2.2.1 Spiking the Coupons and Freeze-Drying 3
2.2.2 Vaccinia Virus Extraction and Quantification 3
2.2.3 Vaccinia Virus Recovery Results 3
2.3 Persistence Testing Approach 4
2.4 Test Results 6
2.4.1 Glass 6
2.4.2 Galvanized Metal 9
2.4.3 Painted Cinder Block 11
2.4.4 Industrial Carpet 14
2.4.5 Comparison of Persistence Testing Results 16
3.0 Decontamination Technology Evaluation 17
3.1 Technology Descriptions 17
3.2 Test Matrix 17
3.3 Technology Evaluation 18
4.0 Quality Assurance/Quality Control 21
4.1 Equipment Calibration 21
4.2 Audits 21
4.2.1 Performance Evaluation Audit 21
4.2.2 Technical Systems Audit 21
4.2.3 Data Quality Audit 21
4.3 QA/QC Reporting 21
4.4 Data Review 21
4.5 Deviations 21
5.0 Summary 23
5.1 Vaccinia Virus Persistence 23
5.2 Vaccinia Virus Liquid Decontamination 23
6.0 References 25
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Tables
Table ES-1. Summary of Persistence Test Conditions and Vaccinia Virus Recoveries xi
Table 2-1. Test Materials 3
Table 2-2. Vaccinia Virus Recoveries 4
Table 2-3. Persistence Tests Matrix 5
Table 2-4. Vaccinia Virus Persistence on Glass 7
Table 2-5. Vaccinia Virus Persistence on Galvanized Metal 9
Table 2-6. Vaccinia Virus Persistence on Painted Cinder Block 12
Table 2-7. Vaccinia Virus Persistence on Industrial Carpet 14
Table 2-8. Summary of Vaccinia Virus Persistence 16
Table 3-1. Decontamination Technology Evaluation Matrix 17
Table 3-2. Decontamination Efficacy against Vaccinia Virus on Galvanized Metal 18
Table 3-3. Decontamination Efficacy against Vaccinia Virus on Industrial Carpet 19
Table 3-4. Summary of Decontamination Efficacy against Vaccinia Virus 19
Table 4-1. Performance Evaluation Audit Results 21
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Abbreviations/Acronyms
AOAC Association of Official Analytical Chemists
ASTM American Society for Testing and Materials
ATCC American Type Culture Collection
°C degrees Celsius
CaCO3 calcium carbonate
cm centimeter
CO2 carbon dioxide
D/E broth Dey and Engley broth
EPA U.S. Environmental Protection Agency
g gram
MDCK Madin-Darby canine kidney
MEM minimum essential medium
mL milliliter
NHSRC National Homeland Security Research Center
ORD EPA Office of Research and Development
PBS phosphate-buffered saline
PFU plaque-forming unit
ppm parts per million
QA quality assurance
QC quality control
QMP quality management plan
RH relative humidity
rpm revolutions per minute
TSA technical systems audit
TTEP Technology Testing and Evaluation Program
uL microliter
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Executive Summary
The U.S. Environmental Protection Agency's (EPA) National
Homeland Security Research Center (NHSRC) Technology
Testing and Evaluation Program (TTEP) helps to protect
human health and the environment from adverse impacts of
terrorist acts by carrying out performance tests on homeland
security technologies. Under TTEP, the persistence of
viable vaccinia virus (surrogate for the variola virus, which
causes smallpox) that was freeze-dried on coupons prepared
from glass, galvanized metal, painted cinder block, and
industrial carpet was investigated. The efficacy of two
liquid decontamination technologies for inactivating the
vaccinia virus that was freeze-dried on coupons of galvanized
metal and industrial carpet was evaluated at room and low
temperatures.
For persistence testing, materials were "contaminated" by
spiking with a target level of 1 x 107 plaque-forming units
(PFUs) of the vaccinia virus per coupon and immediately
freeze-dried. The persistence of the vaccinia virus was
subsequently investigated for up to four test durations at
four environmental conditions comprised of variations
in temperature and relative humidity (RH). The four
environmental conditions (the four combinations of low/high
RH, and low and ambient temperature) were selected for
testing to investigate the effect of RH and temperature
on persistence, and are as follows:
• Room temperature, low RH
o Test durations: 14, 21, 28, and 42 days
• Room temperature, high RH
o Test durations: 1, 3, 7, and 14 days
• Low temperature, low RH
o Test durations: 14, 21, 28, and 56 days
• Low temperature, high RH
o Test durations: 7, 14, 21, and 42 days
Under the "room temperature, high RH" environmental
condition, the vaccinia virus remained viable for three days
on glass and one day on galvanized metal and painted cinder
block; the vaccinia virus was not detected on industrial
carpet after one day. Under the "low temperature, high RH"
environmental condition, the vaccinia virus remained viable
for at least seven days on all materials.
The vaccinia virus generally remained viable longer under
low RH conditions at both room and low temperatures
compared to the respective tests conducted at a high
RH. The vaccinia virus persisted on all four materials
for at least 14 days at the "room temperature, low RH"
environmental condition; the vaccinia virus persisted
for 42 days on glass, galvanized metal, and painted
cinder block. Under the "low temperature, low RH"
environmental condition, the vaccinia virus persisted on
all four materials for the 56-day duration. A summary
of the actual test conditions and PFU values recovered
from each of the materials is provided in Table ES-1.
The liquid decontamination technologies evaluated were
1% citric acid and hospital grade quaternary ammonium
salt (732 ppm) disinfectant; these common chemicals were
chosen because they are widely available for use. The
evaluation was conducted at room and low temperatures with
a 30-minute liquid decontaminant contact time. Both liquid
decontamination technologies were applied to galvanized
metal and industrial carpet coupons inoculated with the
vaccinia virus and freeze-dried.
Neither 1% citric acid nor 732 ppm quaternary ammonium
salt reduced the vaccinia virus to non-detectable levels. For
1% citric acid, mean log reductions in vaccinia virus PFUs
on galvanized metal were 3.2 at both the room and low
temperatures; on industrial carpet, log reductions were 2.6 at
room temperature and 2.5 at low temperature. When 732 ppm
quaternary ammonium salt was used, mean log reductions in
vaccinia virus PFUs on galvanized metal were 1.5 at both the
room and low temperature conditions; on industrial carpet the
log reductions were less than 1.0.
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Table ES-1. Summary of Persistence Test Conditions and Vaccinia Virus Recoveries3
Room Temperature, Low RH
14 Days
21 Days
28 Days
42 Days
Room Temperature, High RH
1 Day
3 Days
7 Days
14 Days
Low Temperature, Low RH
14 Days
21 Days
28 Days
56 Days
Low Temperature, High RH
7 Days
14 Days
21 Days
42 Days
22
23
23
23
21
23
23
21
7
8d
T
1
7
7
6e
7
10
1
1
1
89
93
98
89
7
8d
7e
1
90
101
95e
96
2.43 x 107
2.33 x 104
2.45 x 101
4.22 x 103
3.40 x 100c
6.93 x 103
ND
ND
6.04x 107
3.45 x 106
4.05 x 106
2.87 x 104
5.22 x 106
3.26x 103c
ND
ND
1.90x 107
1.49x 104
1.11 x 104
5.30 x 103
1.66x 101
ND
ND
ND
2.71 x 107
1.90x 106
3.04x 106
5.38 x 104
1.47 x 103c
ND
ND
ND
2.57x 107
5.46 x 10°
5.81 x 103
4.00 x 10-lc
2.55x 104
ND
ND
ND
6.27x 107
3.84x 106
3.06x 106
2.99x 104
7.68x 106
2.54x 105
1.49x 105
ND
4.34 x 106
ND
ND
ND
ND
Not Tested
ND
ND
1.24x 107
9.92x 105
1.07x 106
1.55x 101
1.01 x 106
1.32 x 105
3.72x 104
ND
a Vaccinia virus was applied to the coupons as an aqueous suspension, the coupons were freeze-dried, then placed in the environmental condition
for the test duration. Spike amounts ranged from 6.98 x 106 to 4.79 x 107 PFUs.
b Temperature and RH values are the means based on continuous monitoring at l-to-6-minute intervals.
c Vaccinia virus was not detected on some of the replicate test coupons; a value of 0 PFUs was used for non-detects in calculation of mean
vaccinia virus recovery.
d Temperature and RH were recorded for less than one day after test initiation due to unplanned shut-down/failure of data-loggers. No unusual
events occurred that would lead one to expect that the temperature and RH deviated from those of comparable tests.
e Temperature and RH were recorded for approximately 15 days after test initiation. No unusual events occurred that would lead one to expect that
the temperature and RH deviated from those of comparable tests.
ND = Not detected; the detection limit is 10 PFUs.
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1.0
Introduction
NHSRC's TTEP works in partnership with recognized testing
organizations; with stakeholder groups consisting of buyers,
vendor organizations, scientists, and permitters; and with
participation of individual technology developers in carrying
out performance tests on homeland security technologies.
In response to the needs of stakeholders, TTEP investigates
the natural persistence of biological and chemical agents
and 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 evaluation
design to produce useful performance information for each
evaluated technology.
Under TTEP, persistence of viable vaccinia virus was tested
after applying the vaccinia virus as an aqueous suspension
to the surface of a test material which was then freeze-dried.
The performance of liquid technologies for decontaminating
freeze-dried vaccinia virus on various materials was also
evaluated. The vaccinia virus is used in the smallpox
vaccine and is a commonly used surrogate for variola virus,
the etiological agent of smallpox. Variola virus is easily
transportable in a temperature-stable freeze-dried state, and
it is conceivable that variola virus might be aerosolized
to cause infection0}. The persistence of the vaccinia virus
was investigated under various environmental conditions,
after initially being freeze-dried on glass, galvanized metal,
painted cinder block, and industrial carpet. The ability of
two liquid decontamination technologies (1% citric acid
and 732 ppm quaternary ammonium salt) to inactivate the
vaccinia virus after the virus was initially freeze-dried on
galvanized metal and industrial carpet, and subsequently
held under room and low temperature conditions was also
evaluated.
The persistence investigation and liquid decontamination
technology evaluations were conducted according to a peer-
reviewed test/QA plan(2) that was developed according to
the requirements of the TTEP quality management plan
(QMP)(3). The persistence tests and decontamination
evaluations used the PFU assay with Vero (African Green
Monkey kidney) cells to quantify the viable vaccinia virus
extracted from test materials. This report documents the log
reductions in PFUs associated with a natural reduction in the
viable vaccinia virus under various environmental conditions
and the decrease in the viable vaccinia virus exposed to two
liquid decontamination technologies.
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2.0
Persistence Testing
2.1 Test Materials
Materials used for the vaccinia virus persistence testing
are described in Table 2-1. Test coupons were uniformly
cut to 1.9 cm width x 7.5 cm length from larger pieces of
material. Edges and damaged areas were avoided in cutting
test coupons. The coupons were then autoclaved or gamma-
irradiated (Table 2-1) and stored under sterile conditions
until use, which was intended to minimize microorganism
contamination of the coupons' surfaces. All of the materials
used for the vaccinia virus persistence testing have been used
in previous TTEP testing.
2.2 Vaccinia Virus Recovery from Test Materials
2,2,1 Spiking the Coupons and Freeze-Drying
Each coupon was spiked with a target level of 1 x 107 PFUs
of the vaccinia virus [American Type Culture Collection
(ATCC) VR119] per coupon (i.e., 100 uL of 1 x 108 PFUs/
mL stock suspension per coupon). Spiking of the coupons
was performed using a multichannel micropipette as two
rows of five droplets (10 uL per droplet) across the surface of
the test coupon.
After spiking, the coupons were frozen overnight at <-80°C
in preparation for the freeze-drying process. Using a VirTis
DBT Benchtop 3.5 L Freeze-Dryer, the spiked, frozen
coupons were freeze-dried until the samples were visibly dry
(approximately two to four hours).
2.2.2 Vaccinia Virus Extraction and Quantification
At the completion of the freeze-drying process (time zero),
the coupons were extracted and assayed for PFUs. The
extraction approach involved placing each individual coupon
in a 50 mL conical vial to which 10 mL of sterile extraction
buffer [i.e., phosphate-buffered saline (PBS)] was added.
The tubes were agitated on an orbital shaker for 15 minutes
at approximately 200 revolutions per minute (rpm) at room
temperature. Each coupon extract was then serially diluted
in PBS (10-fold dilutions through 10'7). An aliquot (0.1 mL)
of the undiluted extract and each serial dilution was plated
onto monolayers of Vero cells. The tissue culture plates
were rocked every 15 minutes for approximately one hour
at ~37°C, to allow adsorption of the vaccinia virus to the
Vero cells. The cultures were then overlaid with minimum
essential medium (MEM) containing 2% fetal bovine serum,
0.5% methylcellulose, and antibiotics. The cultures were
incubated for 72 ± 4 hours at 37 ± 2°C in 95% air and 5%
CO2. Following incubation, crystal violet dye was added to
the monolayers for 15 minutes, removed and the cells were
rinsed with PBS. Plaques were visualized as clearings in the
purple monolayer of Vero cells. The plaques were counted
manually and the number of PFUs/mL was determined by
multiplying the mean number of plaques per well by the
reciprocal of the dilution. The plaque assay has a detection
limit of 10 PFUs.
2.2.3 Vaccinia Virus Recovery Results
Prior to persistence testing, efforts were conducted to
determine if an acceptable level of the vaccinia virus (>1%
of the applied inoculum per the test/QA plan(2)) could be
recovered from the coupons. The coupons were spiked and
freeze-dried as described in the preceding sections. Then the
vaccinia virus was extracted from the coupons and analyzed
for PFUs. Initial determinations of vaccinia virus recoveries
were conducted with three replicate coupons per material.
As acceptable recoveries were obtained, final demonstrations
using five replicate coupons were used to obtain mean
Table 2-1. Test Materials
Glass
C1036
Brooks Brothers
1.9 cm x 7.5 cm
0.3cm
Autoclaved
Galvanized Metal
Painted Cinder
Block (concrete)
Industrial Carpet
Industry heating, ventilation,
and air conditioning standard
24 gauge galvanized steel
ASTM C90. Brush and roller
painted all sides. One coat
Martin Senour latex primer
(#71-1185) and one coat
Porter Paints latex semi-gloss
finish (#919)
ShawTek, EcoTek 6 Color:
mottled gray/dark brown/ black
Accurate 1 .9 cm x 7.5 cm
Fabrication
Wellnitz 1.9 cm x 7.5 cm
Shaw 1.9 cm x 7.5 cm
Industries, Inc.
0.06 cm Cleaned with
acetone;
autoclaved
0.5 cm Autoclaved
0.7 cm Gamma
Irradiation
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recovery and variance data. The vaccinia virus recovery
results are provided in Table 2-2. Acceptable vaccinia virus
recoveries were obtained for all materials used in persistence
testing (glass, galvanized metal, painted cinder block, and
industrial carpet). The percent vaccinia virus recoveries were
higher when three replicates were used as compared to five
replicates for glass, galvanized metal, and industrial carpet.
Testing was conducted on two different days, and the higher
inoculum associated with the five replicates contributed
to lower percent recoveries since comparable amounts of
vaccinia virus (PFUs) were recovered from the three and five
replicates per material.
Mean percent vaccinia virus recoveries were calculated based
on the mean of the replicate vaccinia virus percent recoveries
per material and test (tests with three replicates and five
replicates were conducted on different days) as:
Equation 2-1.
z
P, =—
O
x 100 %
n
Where:
PJk = mean percent vaccinia virus recovery for they*
material and &* test
Pijk = vaccinia virus recovered for the /'* replicate, 7*
material and A* test (in PFUs)
Q = inoculum amount (in PFUs)
'P ^
-^- xlOO%
.Q)
= sum of the replicate percent
vaccinia virus recoveries based on
the amount of vaccinia virus
recovered (Pijk) and the inoculum
amount (Q)
n = number of replicate test coupons (three or five).
If no PFUs were detected for a test coupon, a value of 0 was
substituted for the vaccinia virus recovery (P).
2.3 Persistence Testing Approach
The vaccinia virus was quantified from the liquid extracts
obtained from the test coupons (spiked coupons placed in
the exposure chamber with temperature, RH, and contact
time treatments) and positive controls (spiked coupons,
freeze-dried, and extracted at time zero [at the completion
of freeze-drying]). Spiking and freeze-drying of the coupons
and vaccinia virus extraction and quantification followed
the approach described in Section 2.2.1. The log reduction
in the vaccinia virus viability (quantified by determining
PFUs) was calculated as AW where TV is the mean number of
PFUs from the five positive controls of a given material and
environmental condition and N' is the number of PFUs from
each test coupon replicate of a given material, environmental
condition, and contact time. The log reduction in PFUs for
each individual test coupon (R) was calculated for each of the
five replicate test coupons of each material, environmental
condition, and contact time as:
Equation 2-2.
Where:
Rykl ~
R a = log reduction in PFUs for the /'* replicate test coupon,
7th material, F1 environmental condition,
and /* contact time
N.k = arithmetic mean PFU from the five positive controls
for the 7* material and &* environmental condition
N' = PFUs recovered on the /* replicate test coupon,
7th material, F1 environmental condition, and /*
contact time.
Table 2-2. Vaccinia Virus Recoveries
Glass
3 replicates
5 replicates
Galvanized Metal
3 replicates
5 replicates
Painted Cinder Block
3 replicates
5 replicates
Industrial Carpet
3 replicates
5 replicates
5.95 x 107
1.67 x 108
5.95 x 107
1.67 x 108
5.95 x 107
1.67 x 108
5.95 x 107
1.67 x 108
,; VvV
3/3
5/5
3/3
5/5
3/3
5/5
3/3
5/5
3S« ^f.'.yy,',, , "^ *^;'_ ,.lWi,
3.05±0.73x 107
2.37 ±0. 16 x 107
0.54±2.39x 107
1.90±0.66x 107
6. 26 ± 4.09 x 106
4.05 ± 0.48 x 106
1.52±0.13x 107
7.17±0.84x 106
ii, ', U ;S vv ' , f i i i i i
Si.'Si. < / (i i ^ t , j ,• ly i.
85 ± 12
24 ±0.97
90 ±40
57 ±4.0
18 ±6. 9
24 ±2. 9
43 ± 2.2
4.3 ±0.50
3 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.
b Data are expressed as mean + standard deviation.
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If no PFUs were detected for a test coupon (TV), the value
"1" was substituted for N'. Since the value 1 is greater than
the observed value of 0, the estimate with this substitution
becomes a lower bound for the true log reduction. Next, the
mean log reduction in PFUs (R) for the five replicate test
coupons of a given material and environmental condition is
calculated as:
Equation 2-3
Where:
i=l
Ijtd
n
R.u = mean log reduction in PFUs for they* material,
&* environmental condition, and Ith contact time
= sum of the log reductions in PFUs for each
'. individual test coupon (/') for they* material,
&* environmental condition, and /* contact time
n = number of test coupon replicates (five).
The test matrix and various test conditions that were utilized
for the vaccinia virus persistence testing are summarized in
Table 2-3. The environmental conditions included various
combinations of temperature and RH. Persistence was
measured for four types of test coupons: glass, galvanized
metal, painted cinder block, and industrial carpet, and the test
durations ranged from 1 to 56 days. Initial time points were
selected based on comparable data available in the literature
(i.e., Stone et al., 2006(4)), but subsequent time points were
adaptively chosen (i.e., shorter or longer durations) based
on the initial test results, and tests were not conducted
sequentially from the shortest to longest test duration. Please
note that when possible multiple tests were initiated on the
same day such that only one set of positive controls would be
used per material (i.e., the same inoculum stock was used for
all tests initiated on the same day).
Similar vaccinia virus (although not freeze-dried)
persistence testing was previously conducted under TTEP,
using galvanized metal and painted cinder block under
environmental conditions of ~20°C and 40%-70% RH, and
at 30°C at either high (>70%) or low RH (<40%) for up to 14
days(4). In that study, the vaccinia virus persisted longer when
exposed to low RH. The current testing was conducted at two
temperatures (room and low) and two RH levels (high and
low) to evaluate the influence of temperature and RH on the
persistence of the vaccinia virus following the freeze-drying
Table 2-3. Persistence Tests Matrix3
Room Temperature (22°C), Low RH (20%)
14 Days
21 Days
28 Days
42 Days
Room Temperature (22°C), High RH (70%)
1 Day
3 Days3
7 Days
14 Days
Low Temperature (10°C), Low RH (20%)
14 Days
21 Days
28 Days
56 Days
Low Temperature (10°C), High RH (70%)
7 Days
14 Days
21 Days
42 Days
21
22
23
23
21
23
22
21
7.
.9
.6
.0
.1
.3
.0
.8
.3
1
7.6b
7.0C
7.
6.
7.
0
9
5
6.1C
6.
6
20.44
21.58
22.15
22.42
20.94
22.54
21.32
19.46
6.36
5.90b
6.61c
6.64
5.77
7.14
5.80C
6.31
-25
-23
-24
-24
-23
-25
-23
-25
-24.
-25.
.48
.47
.41
.12
.54
.28
.33
.14
15
60b
- 24.87C
-24.
-23.
-23.
-25.
-23.
73
81
81
89C
50
10.1
1.0
1.0
1.0
89.2
93.0
97.9
89.1
7.2
7.7b
7.4C
1.0
90.4
100.6
94.8C
95.8
9.
1.
1.
1.
35
21
41.
51
00
00
00
.64
.53
50
39.29
6.37
5.68b
6.46c
1.
43
00
.91
90.39
37.
llc
46.21
-44.
-39.
-40.
-31.
-92
-97
34
25
33
57
.74
.51
- 100.88
-92
.80
-39.77
-39
.06
b
-40.51C
-39.
-93
02
.50
- 102.20
-97
-97
.34
.38
c
1 Test materials included glass, galvanized metal, painted cinder block, and industrial carpet, with the exception that industrial carpet was not
tested during the 3-day duration at room temperature, high RH.
D Temperature and RH were recorded for less than one day after test initiation.
: Temperature and RH were recorded for approximately 15 days after test initiation.
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process. The actual temperatures and RH levels associated
with each test are provided in Table 2-3.
Test coupons were placed inside sealed Lock&Lock™ plastic
storage containers. For low RH conditions, Drierite (W.A.
Hammond Drierite Co.) was added to the containers, and
for high RH conditions, 70% RH humidity beads (Heartfelt
Industries) were added to the containers. For testing at
the low temperature, the containers were placed inside
a refrigerator. No special manipulations for controlling
temperature were required for the room temperature testing.
Temperature and RH were measured and the data recorded
continuously at one-to-six-minute intervals with a HOBO
U10 data logger (Onset Computer Corporation), except as
noted on Table 2-3. Starting from ambient RH levels between
approximately 35-45%, the RH tended to rapidly decrease
during low RH tests and steadily increase during high RH
tests, due to the Drierite and humidity beads, respectively.
The range of RH values monitored included these relatively
short acclimation periods during which the RH level (starting
at ambient level) was decreasing or increasing within the
storage containers. As such, occasionally the range of RH
levels listed in Table 2-3 overlapped depending on the
starting ambient level.
2.4 Test Results
Persistence results for each material/environmental condition
combination are summarized in Tables 2-4 through 2-7.
Several tests (e.g., the 21-, 28-, and 42-day tests at the "room
temperature, low RH" environmental condition for glass
on Table 2-4) were initiated on the same day so the same
positive controls were applicable for each test. The amount
of PFUs in the inoculums (as quantified by spike controls and
presented as the inoculums for the positive and test coupons)
ranged from 6.98 x 106 to 4.79 x 107 PFUs per coupon due
to the varying amounts of the vaccinia virus liter propagated,
and all calculations were based on the recorded results. A
summary of vaccinia virus persistence results is provided in
Table 2-8.
2.4.1 Glass
The vaccinia virus persistence results on glass are
summarized in Table 2-4. With the exception of the "room
temperature, high RH" environmental condition, the
vaccinia virus remained viable for >14 days on glass. The
vaccinia virus was found to remain viable on glass (with
<3.9 mean log reductions in PFUs) for 42 days under the
"room temperature, low RH" environmental condition, 56
days under the "low temperature, low RH" environmental
condition, and seven days under the "low temperature, high
RH" environmental condition.
The vaccinia virus was least viable on glass with high RH
conditions at both room and low temperatures. For the "low
temperature, highRH" environmental condition, a 0.9 mean
log reduction in PFUs occurred following the 7-day duration,
while durations of 14, 21, and 42 days resulted in >5.6
mean log reductions in PFUs. At the "room temperature,
high RH" environmental condition, the vaccinia virus was
viable after one and three days but with >4.3 mean log
reductions in PFUs; the vaccinia virus was not detected at
the 7- and 14-day durations. At the "low temperature, low
RH" environmental condition, the virus recovery at 14 days
was 126% (no outliers were identified as being outside three
standard deviations of the mean). Recovery greater than
100% may be indicative of day-to-day variability within the
extraction procedure and assay. Test coupons showing greater
than 100% recovery or having a negative log reduction,
especially at the low temperature, low RH environmental
condition, are an indication of minimal inactivation of the
vaccinia virus and not necessarily viral propagation or an
error in measurement.
-------
Table 2-4. Vaccinia Virus Persistence on Glass
Room Temperature, Low RH
14 Days
Positive Control0
Test Coupond
Laboratory Blank6
Procedural Blank'
21 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
1 Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
3 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
3.72 x 107
3.72x 107
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
3.72 x 107
3.72 x 107
0
0
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
Room Temperature,
5/5
3/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
0/5
0/1
0/1
5/5
0/5
0/1
0/1
3.75±0.15x 107
2.43 ±0. 64 x 107
ND
ND
2.53± 1.49x 106
2.33 ± 2.34 x 104
ND
ND
2.53± 1.49x 106
2.45 ±0. 74 x 101
ND
ND
2.53± 1.49x 106
4.22 ±5. 83 x 103
ND
ND
High RH
2.53± 1.49x 106
3.40 ± 4.82 x 10° g
ND
ND
2.53± 1.49x 106
6.93 ± 10.1 x 103
ND
ND
2.53± 1.49x 106
NDS
ND
ND
3.75±0.15x 107
NDg
ND
ND
101 ±4.02
65.4 ± 17.1
-
-
36.3 ±21.4
0.33 ± 0.33
-
-
36.3 ±21.4
0.00 ±0.00
-
-
36.3 ±21.4
0.07 ±0.09
-
-
36.3 ±21.4
0.00 ± O.OOe
36.3 ±21.4
0.10±0.14
-
-
36.3 ±21.4
0.00 ± 0.006
-
-
101 ±4.02
0.00 ± 0.00g
-
-
_
0.2 ±0.1
-
-
_
2.6± 1.2
-
-
-
5.0 ±0.16
-
-
_
3.9± 1.4
-
-
_
6.1 ±0.5^
-
-
_
4.3 ±2.1
-
-
-
6.4 ±0. QS
-
-
_
7.6±0.0g
-
-
-------
Table 2-4. Vaccinia Virus Persistence on Glass (continued)
Low Temperature, Low RH
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
21 Days"
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
56 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.79 x
4.79 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
107
107
106
106
106
106
106
106
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
3.75
6.04
2.53
3.45
2.53
4.05
2.53
2.87
±
±
±
±
±
±
±
±
0.15
4.03
ND
ND
1.49
1.09
ND
ND
1.49
0.85
ND
ND
1.49
2.21
ND
ND
x
x
X
X
X
X
X
X
107
107
106
106
106
106
106
104
78.2
126
36.3
49.5
36.3
58.1
36.3
0.41
±3.12
±84.2
±21.4
± 15.6
±21.4
± 12.1
±21.4
±0.32
-0.2 ±0.2
-0.1 ±0.1
-0.2 ±0.1
2.0 ±0.3
Low Temperature, High RH
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.79 x 107
4.79 x 107
0
0
5/5
5/5
0/1
0/1
3.75±0.15x 107
5.22± 1.96 x 106
ND
ND
78.2 ±3.12
10.9 ±4.09
0.9 ±0.2
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
1/5
0/1
0/1
2.53± 1.49x 106
3.26 ± 7.29 x 103s
ND
ND
36.3 ±21.4
0.05±0.10g
5.6±
21 Days"
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
0/5
0/1
0/1
2.53± 1.49x 106
NDg
ND
ND
36.3 ±21.4
0.00±0.00g
6.4±0.0g
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
0/5
0/1
0/1
2.53± 1.49x 106
NDg
ND
ND
36.3 ±21.4
0.00 ± O.OQg
a PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls are inoculated, frozen overnight at <-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the
completion of freeze-drying).
d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.
e Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).
f Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.
g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for
non-detects in the calculation of mean log reduction.
h Temperature and RH were actually unknown.
ND = Not detected; the plaque assay detection limit is 10 PFUs.
"-" Not applicable.
-------
2.4.2 Galvanized Metal
The vaccinia vims persistence results on galvanized metal
are summarized in Table 2-5. Under both low RH conditions
(at room and low temperature), the vaccinia virus remained
viable for >42 days with the highest mean log reduction
in PFUs of 3.9 occurring after the 42-day duration at the
"room temperature, low RH" environmental condition. After
56 days in the "low temperature, low RH" environmental
condition, there was a 1.7 mean log reduction in PFUs.
The vaccinia virus did not remain as viable on galvanized
metal under high RH conditions (at room or low temperature)
compared to the low RH conditions, with >5.3 mean log
reductions over all test durations. The vaccinia virus was
only detected after one day in the "room temperature,
high RH" environmental condition and after 7 days in the
"low temperature, high RH" environmental condition on
galvanized metal.
Table 2-5. Vaccinia Virus Persistence on Galvanized Metal
Room Temperature, Low RH
14 Days
Positive Control0
Test Coupond
Laboratory Blank6
Procedural Blank'
21 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
3.72 x
3.72 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
107
107
106
106
106
106
106
106
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
1
1
2
1
2
1
2
5
.99
.90
.34
.49
.34
.11
.34
.30
±0.16x
± 0.49 x
ND
ND
± 1.09x
± 1.86x
ND
ND
± 1.09x
±0.64x
ND
ND
± 1.09x
±7.22x
ND
ND
107
107
106
104
106
104
106
103
53.4
51.0
33.6
0.21
33.6
0.16
33.6
0.08
±
±
±
±
±
±
±
±
4.36
13.2
15.6
0.27
15.6
0.09
15.6
0.10
0.0 ±0.1
3.4± 1.9
2.7± 1.1
3.9± 1.5
Room Temperature, High RH
1 Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
5/5
0/1
0/1
2.34 ± 1.09 x 106
1.66 ± 1.04x 101
ND
ND
33.6 ± 15.6
0.00 ±0.00
5.3 ±0.4
3 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
0/5
0/1
0/1
2.34 ± 1.09 x 106
NDg
ND
ND
33.6 ± 15.6
0.00±0.00g
6.4±0.0g
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
0/5
0/1
0/1
2.34 ± 1.09 x 106
NDg
ND
ND
33.6 ± 15.6
0.00±0.00g
6.4±0.0g
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
3.72x 107
3.72x 107
0
0
5/5 1.99 ±0.16 x 107
0/5 NDg
0/1 ND
0/1 ND
53.4 ±4.36
0.00±0.00g
7.3 ± 0.0g
-------
Table 2-5. Vaccinia Virus Persistence on Galvanized Metal (continued)
Low Temperature, Low RH
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
21 Daysh
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
56 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.79 x
4.79 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
107
107
106
106
106
106
106
106
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
1.99±
2.71 ±
2.34 ±
1.90±
2.34 ±
3.04 ±
2.34 ±
5.38 ±
0.16
0.42
ND
ND
1.09
0.42
ND
ND
1.09
0.77
ND
ND
1.09
2.64
ND
ND
x
x
x
x
x
x
x
x
107
107
106
106
106
106
106
104
41.5
56.6
33.6
27.2
33.6
43.6
33.6
0.77
±
±
-
-
±
±
-
-
±
±
-
-
±
±
-
-
3.39
8.77
15.6
6.02
15.6
11.0
15.6
0.38
_
-0.1 ±0.1
-
-
_
0.1 ±0.1
-
-
-
-0.1 ±0.1
-
-
-
1.7 ±0.3
-
-
Low Temperature, High RH
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.79 x 107
4.79 x 107
0
0
5/5
4/5
0/1
0/1
1.99±0.16x 107
1.47 ±3.28 x 103g
ND
ND
41.5 ±3.39
0.00 ±0.018
6.6±
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5 2.34 ± 1.09 x 106
0/5 NDg
0/1 ND
0/1 ND
33.6 ± 15.6
0.00 ± 0.00g
6.4±0.0g
21 Daysh
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
0/5
0/1
0/1
2.34 ± 1.09x 106
NDg
ND
ND
33.6 ± 15.6
0.00 ± 0.00g
6.4±0.0g
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
0/5
0/1
0/1
2.34 ± 1.09x 106
NDg
ND
ND
33.6 ± 15.6
0.00 ± 0.00g
-
-
_
6.4±0.0g
-
-
a PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls are inoculated, frozen overnight at <-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the
completion of freeze-drying).
d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.
e Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).
f Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.
g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for
non-detects in the calculation of mean log reduction.
h Temperature and RH were actually unknown.
ND = Not detected; the plaque assay detection limit is 10 PFUs.
"-" Not applicable.
-------
2.4.3 Painted Cinder Block
The vaccinia vims persistence results on painted cinder block
are summarized in Table 2-6. The vaccinia virus persisted
for at least 42 days under low RH conditions (at both room
and low temperatures). At the "low temperature, low RH"
environmental condition, the mean log reductions in PFUs
on painted cinder block were <2.2 for all test durations,
including the 56-day duration. The vaccinia virus was also
detected from all durations tested at the "room temperature,
low RH" environmental condition, but log reductions were
>4.2 at 21, 28, and 42 days.
The vaccinia virus did not persist as long on painted
cinder block under high RH conditions (room and low
temperatures), compared to the respective low RH conditions.
The vaccinia virus was detected after the 21-day duration,
but not the 42-day duration, under the "low temperature,
high RH" environmental condition. The vaccinia virus was
detected only at one day for the "room temperature, high
RH" environmental condition. At the "low temperature, low
RH" environmental condition, the virus recovery was 131%
(no outliers were identified as being outside three standard
deviations of the mean). Recovery greater than 100% may
be indicative of day-to-day variability within the extraction
procedure and assay.
-------
Table 2-6. Vaccinia Virus Persistence on Painted Cinder Block
Room Temperature, Low RH
14 Days
Positive Control0
Test Coupond
Laboratory Blank6
Procedural Blank'
21 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
3.72 x
3.72 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
107
107
106
106
106
106
106
106
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
3/5
0/1
0/1
3
2
3
5
3
5
3.
.85 ±
.57 ±
.15±
.46 ±
.15±
.81 ±
,15±
4.00 ±
0.25x
0.30 x
ND
ND
1.38x
5.60x
ND
ND
1.38x
8.00 x
ND
ND
1.38x
1.82x
ND
ND
107
107
106
10°
106
103
106
io-lg
104
69.2
45.1
0.00
45.1
0.08
45.1
0.00
±
±
-
-
±
±
-
-
±
±
-
-
±
±
-
-
6.66
7.94
19.8
0.00
19.8
0.11
19.8
0.00g
_
0.2 ±0.1
-
-
_
5.9 ±0.4
-
-
-
4.2 ± 1.7
-
-
_
6.6±0.1g
-
-
Room Temperature, High RH
1 Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
3 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
3.72x 107
3.72x 107
0
0
5/5
5/5
0/1
0/1
5/5
0/5
0/1
0/1
5/5
0/5
0/1
0/1
5/5
0/5
0/1
0/1
3.15± 1.38x 106
2.55± 1.17x 104
ND
ND
3.15± 1.38x 106
NDg
ND
ND
3.15± 1.38x 106
NDg
ND
ND
3.85 ±0. 25 x 107
NDg
ND
ND
45.1
0.37
45.1
0.00
45.1
0.00
104
0.00
± 19.8
±0.17
-
-
± 19.8
± 0.00g
-
-
± 19.8
± 0.00g
-
-
±6.66
± 0.00g
-
-
_
2.1 ±0.2
-
-
_
6.5±0.0g
-
-
-
6.5±0.0g
-
-
_
7.6±0.0g
-
-
-------
Table 2-6. Vaccinia Virus Persistence on Painted Cinder Block (continued)
Low Temperature, Low RH
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
21 Daysh
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
56 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.
4.
6.
6.
6.
6.
6.
6.
.79 x
.79 x
0
0
.98 x
.98 x
0
0
.98 x
.98 x
0
0
.98 x
.98 x
0
0
107
107
106
106
106
106
106
106
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
3
6
3
3
3
3
3
2
.85 ±
.27 ±
.15±
.84 ±
.15±
.06 ±
.15±
.99 ±
0.25x
3.35 x
ND
ND
1.38x
1.73x
ND
ND
1.38x
1.25x
ND
ND
1.38x
2.60x
ND
ND
107
107
106
106
106
106
106
104
80.4 ±
131 ±
45.1 ±
55.1 ±
45.1 ±
43.8 ±
45.1 ±
0.43 ±
5.17
70.0
19.8
24.8
19.8
18.0
19.8
0.37
-0.2 ±0.2
-0.1 ±0.2
0.0 ±0.2
2.2 ±0.4
Low Temperature, High RH
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
21 Daysh
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.
4.
6.
6.
6.
6.
6.
6.
.79 x
.79 x
0
0
.98 x
.98 x
0
0
.98 x
.98 x
0
0
.98 x
.98 x
0
0
107
107
106
106
106
106
106
106
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
0/5
0/1
0/1
3
7
3
2
3
1
3
.85
.68
.15
.54
.15
.49
.15
±0.25x
±9.37x
ND
ND
± 1.38x
± 1.60x
ND
ND
± 1.38x
± 1.17x
ND
ND
± 1.38x
NDg
ND
ND
107
106
106
105
106
105
106
80.4 ±
16.0 ±
-
-
45.1 ±
3.64 ±
-
-
45.1 ±
2.13±
-
-
45.1 ±
0.00±
-
-
5.17
19.6
19.8
2.29
19.8
1.68
19.8
0.00g
_
0.9 ±0.5
-
-
_
1.2 ±0.5
-
-
_
1.4 ±0.3
-
-
_
6.5±0.0g
-
-
a PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls are inoculated, frozen overnight at <-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the
completion of freeze-drying).
d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.
e Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).
f Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.
g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for
non-detects in the calculation of mean log reduction.
h Temperature and RH were actually unknown.
ND = Not detected; the plaque assay detection limit is 10 PFUs.
"-" Not applicable.
-------
2.4.4 Industrial Carpet
The vaccinia vims persistence results on industrial carpet are
summarized in Table 2-7. The vaccinia virus remained viable
longest under the "low temperature, low RH" environmental
condition in which <4.9 mean log reductions in PFUs were
observed after 56 days. Under the "room temperature, low
RH" environmental condition, the vaccinia virus remained
viable during the 14-day duration with a 0.2 mean log
reduction in PFUs, but was not detected at >21 days.
Table 2-7. Vaccinia Virus Persistence on Industrial Carpet
Under high RH conditions, the vaccinia virus was not
detected at room temperature after 1 day, but was viable at
the low temperature. The vaccinia virus was not detected
from any of the durations tested (including the shortest
duration of one day) at the "room temperature, high RH"
environmental condition. At the "low temperature, high RH"
environmental condition, the vaccinia virus was detected
at the 7-, 14-, and 21-day durations with <1.5 mean log
reductions in PFUs; the vaccinia virus was not detected at
42 days.
Room Temperature, Low RH
14 Days
Positive Control0
Test Coupond
Laboratory Blank6
Procedural Blank'
21 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
1 Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
3.72 x 107
3.72 x 107
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
6.98 x 106
6.98 x 106
0
0
3.72 x 107
3.72 x 107
0
0
5/5 6.89 ±
5/5 4.34 ±
0/1
0/1
5/5 1.13 ±
0/5
0/1
0/1
5/5 1.13±
0/5
0/1
0/1
5/5 1.13±
0/5
0/1
0/1
Room Temperature, High
5/5 1.13±
0/5
0/1
0/1
5/5 1.13±
0/5
0/1
0/1
5/5 6.89 ±
0/5
0/1
0/1
0.61 x 106
0.34 x 106
ND
ND
0.27x 106
NDg
ND
ND
0.27x 106
NDg
ND
ND
0.27x 106
NDg
ND
ND
RH
0.27x 106
NDg
ND
ND
0.27x 106
NDg
ND
ND
0.61 x 106
NDg
ND
ND
18.5 ± 1.65
11. 7 ±0.92
-
-
16.2 ±3. 81
0.00±0.00g
-
-
16.2 ±3. 81
0.00±0.00g
-
-
16.2 ±3. 81
0.00±0.00g
-
-
16.2 ±3. 81
0.00±0.00g
-
-
16.2 ±3. 81
0.00±0.00g
-
-
18.5 ± 1.65
0.00±0.00g
-
-
_
0.2 ±0.0
-
-
_
6.1 ±0.0g
-
-
-
6.1 ±0.0g
-
-
_
6.1 ±0.0g
-
-
_
6.1 ±0.0g
-
-
_
6.1 ±0.0g
-
-
-
6.8 ± 0.0g
-
-
-------
Table 2-7. Vaccinia Virus Persistence on Industrial Carpet (continued)
Low Temperature, Low RH
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
21 Daysh
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
28 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
56 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.79 x
4.79 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
6.98 x
6.98 x
0
0
107
107
106
106
106
106
106
106
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
6.89
1.24
1.13
9.92
1.13
1.07
1.13
1.55
±0.61
±0.51
ND
ND
±0.27
±7.96
ND
ND
±0.27
±0.19
ND
ND
±0.27
±0.35
ND
ND
x
x
x
x
x
x
x
x
106
107
106
105
106
105
106
101
14.
25.
16.
14.
16.
15.
16.
,4±
,8±
-
-
,2±
,2±
-
-
,2±
,4±
-
-
,2±
0.00 ±
-
-
1.28
10.6
3.81
11.4
3.81
2.74
3.81
0.00
_
-0.2 ±0.2
-
-
_
0.2 ±0.4
-
-
-
0.0 ±0.1
-
-
-
4.9 ±0.1
-
-
Low Temperature, High RH
7 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4.79 x 107
4.79 x 107
0
0
5/5
5/5
0/1
0/1
6.89 ±0.61 x 106
1.01 ±0.40x 106
ND
ND
14.4 ± 1.28
2.11 ±0.83
0.9 ±0.2
14 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
5/5
0/1
0/1
1.13±0.27x 106
1.32±0.40x 105
ND
ND
16.2 ±3.81
1.89 ±0.57
1.0±0.1
21 Days'1
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
5/5
0/1
0/1
1.13±0.27x 106
3.72 ±2.13 x 104
ND
ND
16.2 ±3.81
0.53 ±0.30
1.5 ±0.2
42 Days
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
6.98 x 106
6.98 x 106
0
0
5/5
0/5
0/1
0/1
1.13±0.27x 106
NDg
ND
ND
16.2 ±3.81
0.00 ± 0.00g
6.1 ±0.0g
a PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls are inoculated, frozen overnight at <-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the
completion of freeze-drying).
d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.
e Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).
f Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.
g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for
non-detects in the calculation of mean log reduction.
h Temperature and RH were actually unknown.
ND = Not detected; the plaque assay detection limit is 10 PFUs.
"-" Not applicable.
-------
2.4.5 Comparison of Persistence Testing Results
The vaccinia vims persistence results are summarized
in Table 2-8 by denoting the longest duration (days) that
the virus was detected and the shortest duration that the
virus was not detected to bracket the length of time that
the vaccinia virus remained viable after being freeze-
dried on the materials and then held under the various
environmental conditions. At the "room temperature, low
RH" environmental condition, the vaccinia virus persisted
for at least 42 days (the longest duration tested) on glass,
galvanized metal, and painted cinder block, but was only
detected after 14 days on industrial carpet. At the "room
temperature, high RH" environmental condition, the vaccinia
virus persisted three days or less on all four materials, and
at the "low temperature, low RH" environmental condition
the vaccinia virus persisted for at least 56 days (the longest
duration tested) for all materials. Under the "low temperature,
high RH" environmental condition, the vaccinia virus
persisted 14 days on glass, seven days on galvanized metal,
and 21 days on both painted cinder block and industrial
carpet. In general, for both the room and low temperature
tests, the virus remained viable longer at the lower RH.
Table 2-8. Summary of Vaccinia Virus Persistence
Glass
Room temperature, low RH
Room temperature, high RH
Low temperature, low RH
Low temperature, high RH
Galvanized Metal
Room temperature, low RH
Room temperature, high RH
Low temperature, low RH
Low temperature, high RH
Painted Cinder Block
Room temperature, low RH
Room temperature, high RH
Low temperature, low RH
Low temperature, high RH
Industrial Carpet
Room temperature, low RH
Room temperature, high RH
Low temperature, low RH
Low temperature, high RH
42
3
56
14
42
1
56
7
42
1
56
21
14
NA
56
21
•iiwi«-iiwi«wi :•/!, ,Jf, i, n i&i f.f<.i., A <•;'•'& <; a n i, .* K « ,ti, n ij i ,
-------
3.0
Decontamination Technology Evaluation
3.1 Technology Descriptions
The liquid decontamination technologies evaluated
consisted of:
• 1% Citric Acid
o 1% citric acid was prepared by adding 1 g citric acid,
anhydrous, (> 99.5% purity) to 99 mL hard water until
completely dissolved.
• 732 ppm Quaternary Ammonium Salt
o Hospital grade quaternary ammonium disinfectant
[w-alkyl dimethyl benzyl ammonium chloride (6.25%),
w-alkyl dimethyl ethylbenzyl ammonium chloride
(6.25%), inert ingredients (87.5%)] was purchased
from a local vendor and prepared per the vendor's
guidance (3/4 ounces of disinfectant added to 1 gallon
of hard-water) to obtain a solution containing 732 ppm
of the quaternary ammonium active ingredient. This
concentration was not independently verified in this
evaluation.
All preparations and dilutions were made using Association
of Official Analytical Chemists (AOAC) hard-water
prepared at 400 ppm hardness as CaCO3 (AOAC Official
Method 960.09, Germicidal and Detergent Sanitizing Action
of Disinfectants, Section E. Synthetic Hard Water, p. 11).
The liquid decontamination technologies were chosen
for testing because they are common chemicals used for
disinfection which are readily available. All decontamination
contact times were 30 minutes.
3.2 Test Matrix
Log reductions in the vaccinia virus were determined for
two materials (galvanized metal and industrial carpet)
following 30 minute contact with the liquid decontaminant
(1% citric acid and 732 ppm quaternary ammonium salt) at
room temperature (21°C) and low temperature (5°C). The
experimental treatments performed are shown in Table 3-1.
The evaluation of liquid decontamination technologies
utilized test coupons that were each spiked and freeze-dried
as described in Section 2.2.1 for persistence testing.
For the decontamination evaluation conducted at the lower
temperature, the coupons were placed in the refrigerator for
one hour, before adding the decontamination liquids (which
were also pre-cooled in the same refrigerator prior to use),
to equilibrate the materials to the test temperature. In the
decontamination evaluation spiked test coupons and control
coupons were inverted (spiked surface down) and placed into
separate 6 mL troughs each holding enough decontamination
liquid to cover the spiked surface of the coupon. At the end
of the 30-minute decontamination contact time, the coupons
were removed, neutralized with 4.5 mL of Dey and Engley
(D/E) broth, extracted to recover the vaccinia virus, and
extracts assayed as described in Section 2.2.2. In addition,
the trough solution was also analyzed to quantitate any of the
vaccinia virus that washed into the trough liquid. Since the
coupons were decontaminated via immersion in a liquid, RH
was not a relevant parameter to include in the test matrix.
Neutralization approaches for 1% citric acid and 732 ppm
quaternary ammonium salt followed the methods developed
from a previous TTEP evaluation of the efficacy of 1%
citric acid and 732 ppm quaternary ammonium salt against
the avian influenza H5N1 virus(5). Neutralization consisted
of 75% D/E broth for both 1% citric acid and 732 ppm
quaternary ammonium salt (i.e., 3 parts D/E broth to 1 part
decontamination liquid). During the decontamination
evaluation with the H5N1 virus, neutralized 1% citric acid
and 732 ppm quaternary ammonium salt were not cytotoxic
(>95% cell viability) to Madin-Darby canine kidney
(MDCK) cells (which were used to quantify the H5N1
virus)(5). Since MDCK and Vero cells are both mammalian
kidney epithelial cells, it was not expected (nor subsequently
observed) that the neutralized 1% citric acid or 732 ppm
quaternary ammonium salt would induce cytotoxicity in Vero
Table 3-1. Decontamination Technology Evaluation Matrix
1% Citric Acid
Galvanized metal
Galvanized metal
Industrial carpet
Industrial carpet
Room temperature (21°C)
Low temperature (5°C)
Room temperature (21°C)
Low temperature (5°C)
732 ppm Quaternary Ammonium Salt
Galvanized metal
Galvanized metal
Industrial carpet
Industrial carpet
Room temperature (21°C)
Low temperature (5°C)
Room temperature (21°C)
Low temperature (5°C)
-------
cells. In addition, H5N1 vims recoveries from galvanized
metal and soil exposed to neutralized 1% citric acid and
732 ppm quaternary ammonium salt were >2.60 x 105 tissue
culture infectious dose of 50%, so an appreciable amount
of the vaccinia virus was expected to be recovered from
the neutralized decontamination technologies. In fact, the
vaccinia virus has been shown to have comparable or less
susceptibility to antimicrobials than influenza viruses(6~8).
3.3 Technology Evaluation
The vaccinia virus recovery and log reduction in PFUs
for each decontamination liquid/environmental condition
combination are summarized in Table 3-2 for galvanized
metal and Table 3-3 for industrial carpet. Tables 3-2 and 3-3
also include the number of replicates for positive controls (5)
test coupons (5), laboratory blanks (1), and procedural
blanks (1). A summary of the log reductions obtained in all
decontamination liquid tests is provided in Table 3-4. For the
decontamination evaluations, the mean room temperature
was 21°C with a range of 20°C to 22°C and the mean low
temperature was 5°C with a range of 5°C to 6°C.
Table 3-2. Decontamination Efficacy against Vaccinia Virus on Galvanized Metal
None of the decontamination technologies evaluated reduced
the vaccinia virus on galvanized metal to non-detectable
levels (Table 3-2). For 1% citric acid, mean log reductions
in vaccinia virus PFUs on galvanized metal were 3.2 at both
the room and low temperatures. When 732 ppm quaternary
ammonium salt was used (at room and the low temperature),
mean log reductions in vaccinia virus PFUs were 1.5 on
galvanized metal.
None of the decontamination technologies evaluated reduced
the vaccinia virus on industrial carpet to non-detectable
levels (Table 3-3). For 1% citric acid, mean log reductions
in vaccinia virus PFUs on industrial carpet were 2.6 at room
temperature and 2.5 at low temperature. When 732 ppm
quaternary ammonium salt was used (at room and the low
temperature), mean log reductions in vaccinia virus PFUs on
industrial carpet were less than 1.0.
Room Temperature
1% Citric Acid
Positive Control0
Test Coupond
Laboratory Blank6
Procedural Blank'
732 ppm Quaternary Ammonium Salt
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.24 x
2.24 x
0
0
2.24 x
2.24 x
0
0
107
107
107
107
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
2
1
2
6
.00 ±
.23 ±
.00 ±
.16±
0.47
0.23
ND
ND
0.47
1.47
ND
ND
x
x
X
X
107
104
107
105
89.5
0.05
89.5
2.75
±20.9
±0.01
±20.9
±0.66
3. 2 ±0.1
1.5±0.1
Low Temperature
1% Citric Acid
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.60 x 107
2.60 x 107
0
0
5/5
5/5
0/1
0/1
2.09 ±0.15 x 107
1.37±0.15x 104
ND
ND
80.53 ± 5.66
0.05 ±0.01
3.2 ±0.1
732 ppm Quaternary Ammonium Salt
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.60 x 107
2.60 x 107
0
0
5/5
5/5
0/1
0/1
2.09 ±0.15 x 107
7.02± l.lOx 105
ND
ND
80.53 ± 5.66
2.70 ±0.42
1.5±0.1
a PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls are virus-inoculated coupons that are freeze-dried, placed into troughs containing PBS (but do not undergo decontamination),
exposed to the environmental condition, and extracted after the 30-minute contact time. Only one set of positive controls was used for each
environmental condition and both liquid decontaminants.
d Test coupons are virus-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the
environmental condition, and extracted after the 30-minute contact time.
e Laboratory blanks are non-inoculated coupons that are freeze-dried and extracted at time zero (at the completion of freeze-drying).
f Procedural blanks are non-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the
environmental condition, and extracted after the 30-minute contact time.
ND = Not detected; the detection limit is 10 PFUs.
"-" Not applicable.
-------
Table 3-3. Decontamination Efficacy against Vaccinia Virus on Industrial Carpet
Room Temperature
1% Citric Acid
Positive Control0
Test Coupond
Laboratory Blank6
Procedural Blank'
732 ppm Quaternary
Ammonium Salt
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.
2.
2.
2.
.24 x
.24 x
0
0
.24 x
.24 x
0
0
107
107
107
107
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5
1
5
9
.62 ±
.43 ±
.62 ±
.49 ±
0.39 x
0.26 x
ND
ND
0.39 x
5.86 x
ND
ND
106
104
106
105
25.1
0.06
25.1
4.24
± 1.75
±0.01
-
-
± 1.75
±2.62
-
-
_
2.6 ±0.1
-
-
_
0.8 ±0.2
-
-
Low Temperature
1% Citric Acid
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
732 ppm Quaternary
Ammonium Salt
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.
2.
2.
2.
.60 x
.60 x
0
0
.60 x
.60 x
0
0
107
107
107
107
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
5
1
5
6
.15±
.69 ±
.15±
.95 ±
0.51 x
0.39 x
ND
ND
0.51 x
0.94 x
ND
ND
106
104
106
105
19.82
0.07
19.82
2.67
± 1.97
±0.02
-
-
± 1.97
±0.36
-
-
_
2. 5 ±0.1
-
-
_
0.9 ±0.1
-
-
a PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls are virus-inoculated coupons that are freeze-dried, placed into troughs containing PBS (but do not undergo decontamination),
exposed to the environmental condition, and extracted after the 30-minute contact time. Only one set of positive controls was used for each
environmental condition and both liquid decontaminants.
d Test coupons are virus-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the
environmental condition, and extracted after the 30-minute contact time.
e Laboratory blanks are non-inoculated coupons that are freeze-dried and extracted at time zero (at the completion of freeze-drying).
f Procedural blanks are non-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the
environmental condition, and extracted after the 30-minute contact time.
ND = Not detected; the detection limit is 10 PFUs.
"-" Not applicable.
Table 3-4. Summary of Decontamination Efficacy against Vaccinia Virus
Galvanized Metal
Room Temperature
Low Temperature
3.2 ±0.1
3.2 ±0.1
1.5±0.1
1.5±0.1
Industrial Carpet
Room Temperature
Low Temperature
2.6 ±0.1
2.5 ±0.1
0.8 ±0.2
0.9 ±0.1
Data are expressed as mean + standard deviation.
-------
-------
4.0
Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were
performed in accordance with the test/QA plan(2) and the
TTEP QMP(3). QA/QC procedures are summarized below.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety
cabinets) used at the time of evaluation was verified as being
certified, calibrated, or validated.
4.2 Audits
4.2.1 Performance Evaluation Audit
No performance evaluation audit was performed for
biological agents and organisms because quantitative
standards for these biological materials do not exist.
Performance evaluation audits for analytical measurements
(e.g., temperature, RH, and contact time) were found to be
acceptable (allowable tolerances are from the test/QA plan(2)),
as indicated in Table 4-1.
4.2.2 Technical Systems Audit
Battelle QA staff conducted a technical systems audit (TSA)
on February 2, April 30, and May 4, 2009 to ensure that
the evaluation was being conducted in accordance with
the test/QA plan(2) and the QMP(3). 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. No items were noted during the course of the
TSA with this evaluation. TSA records were permanently
stored with the TTEP QA Manager.
4.2.3 Data Quality Audit
At least 10% of the data acquired during the persistence
testing and decontamination technology evaluation were
audited. A Battelle QA auditor traced the data from
the initial acquisition, through reduction and statistical
analysis, to final reporting to ensure the integrity of
the reported results. All calculations performed on
the data undergoing the audit were checked.
4.3 QA/QC Reporting
Each audit (i.e., the performance evaluation audit, TSA, and
data quality audit) was documented in accordance with the
QMP(3) for submission to the EPA (the NHSRC QA Manager
and the Task Order Project Officer). In addition, quality
control samples including laboratory blanks, procedural
blanks, and positive controls are reported along with the test
coupon results in Sections 2 and 3 for each persistence test
and decontamination evaluation conducted.
4.4 Data Review
Records and data generated from the persistence testing
and decontamination technology evaluation received a QC/
technical review. All data were recorded by Battelle staff. The
person performing the QC/technical review was not 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.
4.5 Deviations
Deviations from the test/QA plan(2) occasionally arose once
work in the laboratory was initiated. These deviations did
not adversely affect data quality as described below:
1. Alternative approaches other than those proposed in
the test/QA plan(2) were used for generating high RH
and low temperature conditions. Specifically, instead
of using a nebulizer to generate a high RH at the room
temperature condition, an RH beyond 70% could be
maintained by placing 70% humidity beads (Heartfelt
Industries) inside an airtight Lock&Lock™ plastic
storage container. Therefore the humidity beads were
used. For reducing temperature, rather than running
tubing from a re-circulating chiller through the glove
box, testing was conducted inside a refrigerator. Target
environmental conditions and acceptable measurement
tolerances were often not attained with these
alternative approaches as noted below.
Table 4-1. Performance Evaluation Audit Results
Temperature
Compare to independent calibrated thermometer value
±2°C
±0.2°C
RH
Compare to independent calibrated hygrometer value
±10%
±2%
Contact Time
Compare time to independent clock
2 seconds/hour
<1 second/hour
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2. Temperature and RH were found difficult to control
in some tests and were often outside of the allowable
test measurement target ranges (±2°C for temperature
and ±10% for RH) established in the test/QA plan(2),
as documented in Section 2.3. However, mean
temperature and RH levels indicate that the test
conditions of room and low temperatures and low
and high RH levels were attained. Although some of
the mean test temperatures deviated from the target
temperatures by a few °C, the temperature data and
associated PFU data remain valid and useful. Similarly,
although there were deviations from the target RH
levels, the RH and PFU data remain valid.
3. Continuous monitoring of temperature and RH was not
conducted during the entire vaccinia virus persistence
duration for the following tests (due to an unplanned
shut-down/failure of the data loggers):
- The 21-day test at low temperature and low RH
(monitoring was conducted for less than one day).
- The 28-day test at low temperature and low RH
(monitoring was conducted for approximately
15 days).
- The 21-day test at low temperature and high RH
(monitoring was conducted for approximately
15 days).
No unusual events occurred that would lead one to
expect that the temperature and RH of these tests
deviated from those of comparable tests. However, we
note that temperature and RH were difficult to control.
4. The target spike amount (inoculum) of the vaccinia
virus (1 x 107 PFUs per coupon or 1 x 108 PFUs per
mL) and the associated performance criterion of
±25% (7.5 x 106 to 1.25 x 107 PFUs per coupon), as
established in the test/QA plan(2), was sometimes not
attained during the method demonstration, persistence
testing, and liquid decontamination evaluation. The
amount of PFUs in the inoculums (as quantified by
spike controls and presented as the inoculums for the
positive and test coupons) ranged from 6.98 x 106 to
1.67 x 108 PFUs per coupon. These deviations did
not have any adverse impacts on the testing as an
appreciable amount of vaccinia virus was spiked to
quantify log reductions.
5. Method development testing associated with the
neutralization of the liquid decontaminants was
not conducted since neutralization methods for the
liquid decontaminants tested (1% citric acid and 732
ppm quaternary ammonium salt) were previously
established in the companion TTEP evaluation of
liquid decontamination technologies for the H5N1
virus(5).
6. Regarding the decontamination technology evaluation,
the positive controls (at the 30-minute contact time)
were exposed to PBS rather than being exposed only to
air, which better mimicked the actual application of the
decontamination liquid.
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5.0
Summary
5.1 Vaccinia Virus Persistence
The persistence of the vaccinia vims was investigated on four
materials (glass, galvanized metal, painted cinder block, and
industrial carpet), after being inoculated onto the materials
and freeze-dried, then held under four environmental
conditions (comprised of room and low temperatures and
low and high RH) for various durations. Under low RH
conditions at both room and low temperature, the vaccinia
virus persisted at least for 14 days on all materials. The
vaccinia virus persisted for 42 days on glass, galvanized
metal, and painted cinder block at the "room temperature,
low RH" environmental condition, and the vaccinia virus
persisted for 56 days on all four materials at the "low
temperature, low RH" environmental condition.
The persistence of the vaccinia virus was generally reduced
under high RH conditions at both room and low temperatures.
Under the "room temperature, high RH" environmental
condition, the vaccinia virus remained viable on glass for
three days and remained viable on galvanized metal and
painted cinder block for one day; the vaccinia virus was not
viable on industrial carpet at the one-day duration. Under
the "low temperature, high RH" environmental conditions,
the vaccinia virus was detected on glass after 14 days,
detected on galvanized metal after seven days, and detected
on painted cinder block and industrial carpet after 21 days.
5.2 Vaccinia Virus Liquid Decontamination
The efficacy of two liquid decontamination technologies
(1% citric acid and hospital grade quaternary ammonium
salt disinfectant at 732 ppm) was evaluated for the vaccinia
virus inoculated and freeze-dried on galvanized metal and
industrial carpet. Decontamination efficacies were assessed
at room and low temperatures with a 30-minute liquid
decontaminant contact time.
Neither liquid decontamination technology reduced the
vaccinia virus to non-detectable levels. For 1% citric acid,
mean log reductions in vaccinia virus on galvanized metal
were 3.2 at both the room and low temperatures; on industrial
carpet log reductions were 2.6 at room temperature and
2.5 at the low temperature. When 732 ppm quaternary
ammonium salt was used, mean log reductions in vaccinia
virus on galvanized metal were 1.5 at both the room and
low temperature conditions; on industrial carpet the log
reductions were less than 1.0.
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6.0
References
1. Fulginiti,V.A., A. Papier, J.M. Lane, J.M. Neff, and D.A. Henderson. 2003. Smallpox Vaccination: A Review, Parti.
Background, Vaccination Technique, Normal Vaccination and Revaccination, and Expected Normal Reactions. Clinical
Infectious Diseases, 37:241-250. http://www.journals.uchicago.edu/doi/pdf/10.1086/375824.
2. Technology Testing and Evaluation Program Test/QA Plan for Persistence Testing of Avian Influenza Viruses on Outdoor
Materials, Version 1, Battelle, Columbus, Ohio, March 2007.
3. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP), Version 1, Battelle,
Columbus, Ohio, January 2005.
4. Stone, H.J., J.V Rogers, EJ. Fleming, Y.W. Choi, J.D. Waugh, W.R. Richter, M.L. Taylor, K.B. Riggs, Z.J. Willenberg, R.T.
Krile, and S. Ryan. 2006. Investigation Report: Impact of Temperature and Humidity on the Persistence of Vaccinia Virus
andRicin Toxin on Indoor Surfaces. EPA/600/R-08/002.
5. Choi, Y.W., J.V. Rogers, D.J. Chappie, M.L. Taylor, K.B. Riggs, Z.J. Willenberg, and J.P. Wood. 2009. Investigation and
Technology Evaluation Report: Highly Pathogenic Avian Influenza H5N1 Virus Persistence Testing and Evaluation of
Liquid Decontamination Technologies. EPA/600/R-09/054.
6. Hamouda, T., A. Myc, B. Donovan, A.Y. Shih, J.D. Reuter, and J.R. Baker, Jr. 2001. A Novel Surfactant Nanoemulsion
with a Unique Non-Irritant Topical Antimicrobial Activity Against Bacteria, Enveloped Viruses and Fungi. Microbiological
Research, 156:1-7.
7. Sugimoto, Y. and S. Toyoshima. 1979. Na-Cocoyl-L-Arginine Ethyl Ester, DL-Pyroglutamic Acid Salt, As an Inactivator of
Hepatitis B Surface Antigen. Antimicrobial Agents and Chemotherapy, 16:329-332.
8. Kampf, G., J. Steinmann, and H. Rabenau. 2007. Suitability of Vaccinia Virus and Bovine Viral Diarrhea Virus (BVDV) for
Determining Activities of Three Commonly-Used Alcohol-Based Hand Rubs Against Enveloped Viruses. BMC Infectious
Diseases, 7:5.
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