&EPA
United States
Environmental Protection
Agency
TEST/QA PLAN FOR
Evaluating Liquid and Foam Sporicidal
Spray Decontaminants
Office of Research and Development
National Homeland Security
Research Center
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EPA/600/R-06/066
Test/QA Plan
for
Evaluating Liquid and Foam Sporicidal Spray Decontaminants
March 2006
Battelle
505 King Avenue
Columbus, OH 43201
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A PROJECT MANAGEMENT
Al TITLE AND APPROVAL PAGE
EPA/Battelle Approval of Test/QA Plan
for
Evaluating Liquid and Foam Sporicidal Spray Decontaminants
March 2006
JoseplP, Waod '
USEPA Order Project Officer
Date
Eletba Brady-Roberts
NHSRC Quality 4ssurancc Manager
/
Date
Karen Riggs
BattcOe TTF.P Manager
Date
>' Willenljerg
Battdle Quality -issnrancc Maftager
Date
.J
BATTELLE
505 King Avenue
Columbus, OH 43201
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A2 TABLE OF CONTENTS
Page
A PROJECT MANAGEMENT 2
Al Title and Approval Page 2
A2 Table of Contents 3
A3 Distribution List 6
A4 Technology Evaluation Organization 7
A5 Problem Definition/Background 11
A6 Technology Evaluation Description and Schedule 13
A7 Quality Obj ectives 14
A8 Special Training/Certification 19
A9 Documentation and Records 21
B MEASUREMENT AND DATA ACQUISITION 23
B1 Experimental Design 23
B2 Methods Requirements and Procedures 32
B3 Sample Handling and Custody Requirements 40
B4 Analytical Methods Requirements 40
B5 Quality Control Requirements 40
B6 Instrument/Equipment Testing, Inspection, and Maintenance Requirements 42
B7 Instrument Calibration and Frequency 42
B8 Inspection/Acceptance Requirements for Supplies and Consumables 43
B9 Data Acquisition Requirements 43
BIO Data Management 43
C ASSESSMENT/OVERSIGHT 46
C1 Assessments and Response Actions 46
C2 Reports to Management 47
D DATA VALIDATION AND USABILITY 48
Dl Data Review, Validation, and Verification Requirements 48
D2 Validation and Verification Methods 48
D3 Reconciliation with Data Quality Objectives 48
E REFERENCES 50
APPENDIX
Detailed Description of the Decontamination Spraying System and Performance Parameter
Evaluation Al
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LIST OF TABLES
Table 1. Critical Data Quality Objectives 15
Table 2. Matrix of Materials, Organisms, and Treatments 25
Table 3. Default Spray Parameters 30
Table 4. Material Characteristics 31
Table 5. Quality Control Sample Requirements 41
Table 6. Measurement Parameters and Data Quality Indicators 42
LIST OF FIGURES
Figure 1. Organization Chart for the Spray Decontamination Evaluation 7
Figure 2. MREF Data Report Form BioDecon-018-01 for the Liquid Decontamination
Spraying System 28
Figure 3. Compact Glove Box for Sporicidal Decontamination Technology Evaluation 29
Figure 4. Flow Chart of Spray Decontamination Evaluation 39
Figure A-1. Flow Diagram of Spray System A5
Figure A-2. Sprayer Set-up Inside the Compact Glove Box A6
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LIST OF ACRONYMS
ANOVA
AOAC
ASTM
ATCC
BSC
BSL
BWD
C
CDC
CFR
CPU
cm
DL
EPA
FDA
GM
GS
in
1C
ISO
mL
,iL
MREF
NHSRC
NIST
PBS
PC
PW
QA
QC
QMP
rpm
SD
SOP
STS
TOPO
ISA
TTEP
two-way analysis of variance
AOAC International (formerly Association of Official Analytical
Chemists)
American Society for Testing and Materials
American Type Culture Collection
biosafety cabinet
biosafety level
bare wood
Celsius
Centers for Disease Control and Prevention
Code of Federal Regulations
colony forming unit
centimeter
decorative laminate
U.S. Environmental Protection Agency
Food and Drug Administration
galvanized metal ductwork
glass
inch
industrial grade carpet
International Organization for Standardization
milliliter
microliter
Medical Research and Evaluation Facility
National Homeland Security Research Center
National Institute of Standards and Technology
phosphate-buffered saline
painted concrete cinder block
painted wallboard paper
quality assurance
quality control
Quality Management Plan
revolutions per minute
standard deviation
standard operating procedure
sodium thiosulfate
Task Order Project Officer
technical systems audit
Technology Testing and Evaluation Program
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A3 DISTRIBUTION LIST
Ms. Karen Riggs
Mr. Zachary Willenberg
Ms. Elisha Morrison
Dr. James Rogers
Battelle
505 King Avenue
Columbus, OH 43201-2693
Dr. Michael Taylor
Battelle
10300 Alliance Rd, Ste 155
Cincinnati, OH 45242
Mr. Eric Koglin
USEPA National Homeland Security
Research Center
944 East Harmon Avenue
Las Vegas, NV 89119
Ms. Eletha Brady-Roberts
USEPA National Homeland Security
Research Center
26 W. Martin Luther King Drive
Cincinnati, OH 45268
Mr. Joseph P. Wood
U.S. Environmental Protection Agency
U.S. EPAMailroom, E343-06
Research Triangle Park, NC 27711
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A4 TECHNOLOGY EVALUATION ORGANIZATION
The technology evaluation will be performed by Battelle under the direction of the U.S.
Environmental Protection Agency's (EPA) National Homeland Security Research Center
(NHSRC) through the Technology Testing and Evaluation Program (TTEP). The majority of the
information in this test/quality assurance (QA) plan is based on a previously approved test/QA
plan by the Task Order Project Officer (TOPO). The organization chart in Figure 1 shows the
individuals from Battelle, the vendor(s), and EPA who will have responsibilities in the
technology evaluation. The responsibilities of these organizations and individuals are
summarized in the following subsections.
NHSRC
QA Manager
E. Brady-Roberts
Battelle TTEP
Manager
K. Riggs
Building Decontamination
Technology Area Leader
M. Taylor
EPA
Task Order
Project Officer
J. Wood
Battelle
Management
Battelle
QA Manager
Z. Willenberg
Laboratory Test
Coordinator
J. Roaers
Vendor
Representative
Figure 1. Organization Chart for the Spray Decontamination Evaluation
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A4.1 Battelle
Dr. Michael Taylor is Battelle's Building Decontamination Technology Area Leader and
Task Order Leader for this technology evaluation. He will have overall responsibility for
ensuring that the technical, schedule, and cost goals established for testing and evaluation is met,
and that the procedures employed for the evaluation are consistent with TTEP guidelines.
Dr. Taylor will serve as the primary interface for the TOPO. Dr. Taylor's responsibilities are to
• Ensure that TTEP procedures are being followed
• Select the appropriate laboratory or location for the evaluation
• Prepare the draft test/QA plan and evaluation reports
• Establish a test schedule
• Revise this test/QA plan and evaluation reports in response to reviewers' comments
• Keep the Battelle TTEP Manager informed of the progress and difficulties in
planning and conducting the evaluation
• Coordinate with the Battelle Quality Assurance Manager for the technical and
performance audits as required by Battelle or EPA Quality Management staff
• Have overall responsibility for ensuring that this test/QA plan is followed
• Respond to any issues raised in assessment reports and audits, including instituting
corrective action as necessary
• Establish a budget and schedule for the technology evaluation and direct the effort to
ensure that budget and schedule are met
• Coordinate distribution of the final test/QA plan and evaluation reports.
Ms. Karen Riggs is Battelle's TTEP Manager. As such, Ms. Riggs will
• Maintain communication with the EPA TTEP Program Manager on all aspects of the
program
• Monitor adherence to budgets and schedules in this work
• Provide the TOPO with monthly technical and financial progress reports
• Review and approve the draft and final test/QA plan
• Review the draft evaluation reports
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• Ensure that necessary Battelle resources, including staff and facilities, are committed
to the technology evaluation
• Ensure that vendor confidentiality is maintained
• Support Dr. Taylor in responding to any issues raised in assessment reports and audits
• Issue a stop work order if audits indicate that data quality is being compromised.
Mr. Zachary Willenberg is Battelle's Quality Assurance Manager for TTEP. As such,
Mr. Willenberg will
• Review and approve the draft and final test/QA plan
• Maintain communication with EPA Quality Management staff for this program
• Conduct a technical systems audit (TSA) at least once during the technology
evaluation
• Audit at least 10% of the evaluation data
• Prepare and distribute an assessment report for each audit
• Verify implementation of any necessary corrective action
• Notify Battelle's TTEP Manager to issue a stop work order if internal audits indicate
that data quality is being compromised. Notify the Task Order Leader if such an order
is requested
• Provide a summary of the Q A/quality control (QC) activities and results for the
evaluation reports
• Review the draft evaluation reports
• Ensure that all quality procedures specified in this test/QA plan and in the TTEP
Quality Management Plan[1] (QMP) are followed
• Maintain training records.
Dr. James Rogers is Battelle's Laboratory Test Coordinator for this evaluation. His
responsibilities are to
• Coordinate with vendor representatives to facilitate the performance of the evaluation
• Assist in preparing the draft test/QA plan
• Arrange for using the test facility and establishing evaluation schedules
• Arrange for the availability of qualified staff to conduct the evaluation
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• Assure that the evaluation is conducted in accordance with this test/QA plan
• Provide input into revision of this test/QA plan and evaluation report in response to
reviewers' comments
• Update the Battelle TTEP Manager and Task Order Leader on progress and
difficulties in planning and conducting the evaluation
• Coordinate with the Battelle Quality Assurance Manager for the performance of
TSAs as required by Battelle or EPA Quality Management staff.
Dr. Stephen R. Rohrer is the Battelle Biosafety Officer for this project.
A4.2 Vendors
Vendors of the sporicidal decontamination technologies may
• Provide input for preparing the draft test/QA plan
• Review this test/QA plan and approve the final version prior to the evaluation of their
technology
• Sign a vendor agreement specifying the respective responsibilities of the vendor and
of Battelle in the evaluation
• Provide information on the quantitative response of their sporicidal decontamination
technologies to aid in planning the evaluation
• Provide additional equipment (if applicable) used for their decontamination
technologies in the technology evaluation
• Train Battelle and/or test facility staff in implementing their sporicidal
decontamination technologies
• If available, provide information regarding contact time, spray distance, and spray
deposition requirements
• Provide support, if needed, in using their sporicidal decontamination technologies
during testing
• Review their respective draft evaluation reports.
A4.3 EPA
Mr. Eric Koglin is the EPA TTEP Program Manager for the EPA contract with Battelle,
"Testing and Evaluation of Homeland Security-Related Technologies for the Measurement,
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Sampling, Removal, and Decontamination of Chemical and Biological Agents" under which
TTEP has been established.
Mr. Joseph Wood is the EPA TOPO for TTEP Task Order 1113. As such, Mr. Wood will
• Have overall responsibility for directing the evaluation process
• Review the draft test/QA plan
• Approve the final test/QA plan and any subsequent versions
• Review the draft evaluation reports
• Oversee the EPA review process on the draft test/QA plan and evaluation reports
• Coordinate submission of evaluation reports for final EPA approval.
Ms. Eletha Brady-Roberts is the NHSRC QA Manager for TTEP. As such, Ms. Brady-
Roberts will
• Review and approve the draft test/QA plan and any subsequent versions
• Perform, at her option, one external TSA during the technology evaluation
• Notify the EPA TOPO to contact the Battelle TTEP Manager to issue a stop work
order if an external audit indicates that data quality is being compromised
• Prepare and distribute an assessment report summarizing the results of the external
audit, if one is performed
• Review the draft evaluation reports.
A5 PROBLEM DEFINITION/BACKGROUND
Among its responsibilities related to homeland security, EPA has the goal of identifying
methods and equipment that can be used for decontaminating both indoor and outdoor
environments following a terrorist attack using chemical or biological agents. In January 2003,
EPA established the NHSRC to manage, coordinate, and support a wide variety of homeland
security research and technical assistance efforts. The NHSRC is, through TTEP, conducting
tests to evaluate the performance of both developmental and commercially available products,
methods, and equipment for decontaminating porous (e.g., carpet) and non-porous (e.g., glass)
surfaces contaminated with biological and chemical agents.
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One or more biological agents (e.g., spores, vegetative cells, biotoxins) may be released
in or around a building during a terrorist attack. Indoor surfaces (e.g., carpet, laminate, concrete)
representing those found in a typical office building or mass transit station have been selected for
use in evaluating the decontamination technology. The indoor surfaces selected include both
porous and non-porous materials (see Section B1.3).
The purpose of this evaluation is to generate objective performance data that can
subsequently be used by building and facility managers, first responders, groups responsible for
building decontamination, and other technology buyers and users to make informed purchase and
application decisions. All potential users need unbiased, high quality, objective third-party data
and information to assess how well the available decontamination tools will meet their
performance objectives while protecting human health and the environment. All testing and
evaluation conducted through the TTEP is under the direction of EPA and is subject to the TTEP
QMP.[1] In performing each evaluation, Battelle will follow the general procedures described in
the QMP[1] and Battelle has (as stipulated in the QMP) developed this test/QA plan. This test/QA
plan has been prepared for the evaluation of sporicidal liquid or foam decontamination
technologies that are applied with a spray applicator. The appendix of this test/QA plan contains
a detailed description of the semi-automated spray system used to evaluate spray
decontamination technologies. The appendix also includes a description of how the semi-
automated spray system is used to identify spray-applied sterilants that have sporicidal activity
and therefore should be considered for evaluation.
The objective of this test/QA plan is to describe procedures to determine the efficacy of
decontamination technologies for killing the biological agent, Bacillus anthracis, Ames spores,
or surrogate spores on a range of representative surfaces typical of those found in or around a
public building, with the ultimate goal of providing technologies for restoring the building to a
usable state. Decontamination of personnel or large equipment items (e.g., manufacturing
equipment) is not covered in this test/QA plan. Decontamination technology testing and
evaluation are being performed to generate data indicative of the technology performance or
efficacy. For the evaluation conducted under this test/QA plan, performance is quantitatively
assessed by sampling and analysis of viable spores before and after using the decontamination
technology. The body of this test/QA plan provides the general framework under which this
decontamination technology class will be evaluated.
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A6 TECHNOLOGY EVALUATION DESCRIPTION AND SCHEDULE
This test/QA plan focuses on the evaluation of commercially available technologies for
decontaminating indoor surfaces in or around a typical office building or mass transit station.
This plan specifically focuses on decontamination in the building environment, in the context of
use by personnel responsible for decontamination of the area after a terrorist attack. The overall
objective of the evaluation called for under this plan is to determine the efficacy of sporicidal
decontamination technologies for inactivating biological agents in or on typical indoor surfaces.
Each technology will be evaluated by careful monitoring of contact time and temperature. For
this evaluation, the performance of each of the decontamination technologies will be evaluated as
described in Section B2.2.5.
A technology to be evaluated under this test/QA plan is a sodium hypochlorite
formulation, which has been pH-adjusted with 5% acetic acid to 7.0 ± 0.2, according to the
procedure outlined on the U.S. EPA Web site
(http://www.epa.gov/pesticides/factsheets/chemicals/bleachfactsheet.htm). This amended bleach
will be used as the baseline formulation for comparative testing of commercial or developmental
technologies. Control coupons will be sprayed with a benign liquid (e.g., sterile water or
phosphate-buffered saline [PBS]). Evaluations of additional commercial or developmental liquid
or foam technologies under this test/QA plan may be described in an amendment to this test/QA
plan.
The performance of technologies for decontaminating indoor surfaces spiked with a
biological agent (B. anthracis Ames) or surrogates will be assessed at temperatures and relative
humidity representative of those that could be found or established in a building decontamination
situation.
The performance parameters by which the decontamination technologies will be
evaluated under this plan include
• Log kill or efficacy
• Residual viable microorganisms (qualitative)
• Surface damage caused by the decontamination technology (qualitative).
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A brief qualitative assessment of overall ease of preparation, application, handling, and storage
will be included in the final report.
The decontamination evaluation to be conducted under this plan is limited to a biological
warfare agent and surrogates in or on individual samples (test coupons) of building materials.
Applications of decontaminants and subsequent evaluations involving the biological warfare
agent will be performed in the Medical Research and Evaluation Facility (MREF) Biosafety
Level 3 (BSL-3) facility.
The evaluation described in this test/QA plan is expected to begin 2 to 4 weeks after this
test/QA plan has been approved. Two trials are required. Each trial includes applying spores
(spiking) to test coupons (day 1); decontaminating test coupons, extracting spores, and dilution
plating (day 2); and counting colonies (day 3). The two trials required to complete all testing for
a single decontamination technology require two weeks of elapsed laboratory time.
A7 QUALITY OBJECTIVES
The performance parameters to be evaluated under this test/QA plan include:
• Quantitative assessment of decontamination efficacy of sporicidal spray
decontaminants
• Qualitative assessment of residual viable organisms on test surfaces
• Changes in appearance of test coupons based upon visual observation.
The quantitative assessment of decontamination efficacy at a given temperature and
contact time is impacted by uncertainty in four measurements: the volume of stock suspension
spiked onto coupons, the number of viable spores in the stock suspension and in the coupon
extracts, the temperature, and the contact time. Critical data required to achieve performance
objectives are summarized in Table 1.
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Table 1. Critical Data Quality Objectives
Data Required
Application
volume
Viable spores
Temperature
Time
Method
Micropipette
Manual count
National Institute of
Standards and Technology
(NIST) traceable
certification (±0.4°C @
25°C) and/or a NIST-
traceable thermometer/
hygrometer
Data logger manufacturer's
specifications indicate time
accuracy of 6 1 seconds/
month (±0.000023%)
Unit
mL, micro-
liter (uL)
CPU
°C
Hour
Acceptable
Uncertainty in
Data
±5%
±10%
(controls)
±2°C
±0.05%
(2 seconds/
hour)
Corrective Action
Replace with calibrated
and sufficiently
accurate micropipette
Provide training; test
performance; re-count
questionable plates
Replace with calibrated
and sufficiently
accurate thermometer
and note variance in
study file
Replace with calibrated
and sufficiently
accurate clock; note
variance in study file
Quality control requirements are summarized in Table 5 in Section B5. Two of the critical QC
measurements are the spore density (colony-forming units [CFU]/milliliter [mL] in the stock
suspension) and recovery (mean percentage of spores extracted from a material compared to
spores applied to the coupon). The number of colony-forming spores spiked onto coupons will
be acceptable if the spore density measured for the spike controls (shown in Table 5) is within ±
25% of the target level (approximately 1 x 109 spores per 1 mL). Recoveries will be acceptable if
they are >1% and <300% of the spores applied to the coupon. Recoveries below 10% or greater
than 150% will be discussed with the TOPO prior to decontamination testing.
A7.1 Recovery
The percent recovery of viable spores from each test sample (control and
decontaminated) will be determined in order to ascertain the differential number of spores
recovered from test coupons following initial spiking and completing the testing process. Here,
recovery is defined as the number of viable CPUs extracted from each test and control coupon
relative to the number of CPUs in the inoculum used to spike each coupon.
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The number of CPU spiked onto the control coupons is calculated as:
CPU spike/coupon = spore density (CFU/mL) x 0.1 mL spike (1)
The number of CPU extracted from a coupon is calculated as:
CPU extracted coupon = (mean CPU plate count x 1/dilution factor) (2)
x [(volume extraction buffer) + (volume sprayed deconcr)]
Where
deconcr is the total decontaminant on the coupon and run-off; this value is determined
from the spray and weigh test (see appendix). Recovery will be calculated for they'th
control coupon (an individual test coupon) within the rth test material (a specific test
material) as:
Recovery.. = —— (3)
Where
x. are the CPU values in extract samples for the rth of six control coupons within the
rth test material after the drying period
x.. are the CPU values spiked onto they'th replicate coupon of the rth test material
The percent recovery data will be discussed with the TOPO and at the TOPO's discretion the
following statistical analysis of the results will be performed. To determine the differential
recoveries (to assess whether the type of test material influences recovery) of spores from
various coupon types, statistical methods will be employed. The methods for calculating the
differential recovery and assessing outliers are summarized below.
The recovery data for each agent will be fit to a one-way analysis of variance (ANOVA) model
of the form:
Recovery,, = 0W + r(r)j + £(r}l] (4)
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Where
H(r} is the overall mean recovery obtained for spores of a specific type spiked onto a
specific test material
r(r). is the average effect on mean recovery due to the /'th test material
e(r>:/. are the error terms for they'th replicate of the /'th test material group; the errors are
assumed to be N(0,o )
Model diagnostics will be examined to determine whether there are any difficulties with
outliers or the model assumptions of constant variance and normality of the residuals. If the data
are not adequate for the model, appropriate transformations or more general statistical models
(e.g., non-parametric) will be considered. The Grubbs test[2] will be used to identify outliers.
Outliers will be further investigated; but, unless an error in recording or processing the data can
be identified, the outlier will be excluded in the final analysis and noted in the report. No more
than one outlier can be excluded for results to be acceptable. More than one outlier will result in
the samples being rerun.
The recovery results from the coupon testing will be a matrix table in which each entry
shows the mean percentage of recovery of viable spores, along with a 95% confidence interval
for each surface material.
Statistical analysis will consist of pairwise comparisons of recovery percentages between
materials. Both point estimates and corresponding p-values will be produced for each
comparison. The modeling and analysis will be carried out with PROC Mixed in SAS v9.2.
A7.2 Efficacy
The number of CPU of B. anthracis Ames or a surrogate in extracts of control and test
coupons will be determined. The first step in calculating overall decontamination efficacy is to
calculate decontamination efficacy for each coupon in a given set of replicates. Differential
efficacy is defined as the extent (by log reduction) to which the viable spores extracted from the
test coupons after the decontamination treatment were less than the viable spores extracted from
positive control coupons that were exposed only to water spray at the same temperature and
dwell time as the treatment. Efficacy will be calculated for each test coupon for each test
material as:
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Efficacy.. = Iog10 (xy )- Iog10 (x(t}l] ) (5)
Where
Xy is the arithmetic mean of the CPU values of the control coupons of the rth test
material
x(t}ij are the measured CPU values on theyth replicate coupon of the rth test material
The efficacy data will be discussed with the TOPO and at the TOPO's discretion the
following statistical analysis of the results will be performed. To calculate the differential
efficacy for a specific type of spores on a specific type of test material, statistical methods will be
employed. The efficacy data will be fit to a one-way ANOVA of the form:
Efficacy. . = /n+Ti + eff ^
Where
^t is the overall mean recovery
T. is the average effect on mean recovery due to the rth test material
£y are the error terms for theyth replicate of the rth test material group; the errors are
9
assumed to be N(0, a )
B. anthracis Ames and surrogates will be combined under one ANOVA to facilitate
comparisons among the spore types. A main effect for specific spore type will be added to this
model to compare mean efficacy differences due to the spore type.
Model diagnostics will be examined to determine whether there are any difficulties with
outliers or the model assumptions of constant variance and normality of the residuals. If the data
are not adequate for the model, appropriate transformations or more general statistical models
(e.g., non-parametric) will be considered. The Grubbs test[2] will be used to identify outliers.
Outliers will be further investigated; but, unless an error in recording or processing the data can
be identified, the outlier will be excluded in the final analysis and noted in the report.
The primary decontamination efficacy results from the coupon testing will be a matrix
table in which each entry shows the mean log reduction in viable spores.
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Statistical analysis will consist of comparing whether the differential efficacy of the
decontamination treatment at a particular temperature, contact time, and test material was
statistically significantly different from zero. Additional comparisons will be made of mean
efficacy between materials. Both point estimates and corresponding p-values will be produced
for each comparison. The modeling and analysis will be carried out with PROC Mixed in SAS
v9.2.
Cases may exist in which a very small number of CPUs are found on the replicate
coupons after a particular treatment. In these cases, the data may be modeled using methods
consistent with rare events, such as Poisson distributions.
Laboratory blanks will control for sterility, and procedural blanks will control for viable
spores inadvertently introduced to test coupons. The procedural blanks will be spiked with an
equivalent amount of 0.1 mL of "stock suspension" that does not contain the biological agent or
surrogate. As noted in Table 5, there can be no CPU from extracts of laboratory or procedural
blanks for the corresponding test results to be accepted.
A8 SPECIAL TRAINING/CERTIFICATION
The evaluation will be conducted at Battelle's laboratories. The MREF, in West
Jefferson, Ohio, has chemical and biological surety agent laboratories certified for the use of
chemical and biological warfare agents. Battelle test facilities at 505 King Avenue in Columbus,
Ohio, may be used for decontamination of biological agents where a BSL-2 is sufficient. The
Battelle Eastern Science and Technology Center in Aberdeen, Maryland, has both BSL-3 and
BSL-2 facilities and could be used if necessary, depending on the availability and capability of
the facilities. Alternative facilities would only be used if all the requirements for safety, security,
and testing capability established by this plan were met.
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A8.1 General Site Description
Sporicidal decontamination technologies will be evaluated at Battelle's MREF. The
evaluation will be performed in accordance with Battelle's facility-specific methods and the
standard operating procedures (SOPs) that are cited where appropriate throughout this test/QA
plan.
The MREF specializes in research, development, testing, and evaluation of medical
countermeasures against highly pathogenic biological and highly toxic chemical materials. This
facility is one of a very limited number of U.S. laboratories capable of studying aerosolized
etiological agents in animal models under BSL-3 containment. This facility maintains state-of-
the-art equipment and professional and technical staffing expertise to safely conduct testing and
evaluation of hazardous biological materials under the Food and Drug Administration's (FDA)
Good Laboratory Practices Guidelines (21 Code of Federal Regulations [CFR] Part 58). The
MREF operates in compliance with all applicable federal, state, and local laws and regulations,
including U.S. Army regulations, and is routinely inspected by personnel from the appropriate
government agency. Battelle operates the MREF in compliance with requirements contained in
32 CFR 626 and 627, Biological Defense Research Programs. The MREF is International
Organization for Standardization (ISO) 9001 certified, accredited by the American Association
for the Accreditation of Laboratory Animal Care, and inspected by and compliant with
regulations of the U.S. Department of Agriculture, FDA, Drug Enforcement Agency, Ohio EPA,
U.S. Army Safety Team, U.S. Army Inspector General, U.S. Army Medical Research Institute of
Chemical Defense Safety and Chemical Operations Branch, U.S. Army Medical Research and
Materiel Command Office of Animal Care and Use Review, Madison County Health
Department, and Battelle's Institutional Animal Care and Use Committee. The MREF is licensed
to ship, receive, and handle select agents, as defined by the Centers for Disease Control and
Prevention (CDC).
Testing outlined in this test/QA plan will be performed in the MREF BSL-3 facility,
which was completed in 1995 and expanded to 31,000 square feet in 2002. The containment area
within the facility is designed to meet or exceed the BSL-3 facility guidelines published by the
CDC and National Institute of Health entitled Biosafety in Microbiological andBiomedical
Laboratories^ Included are seven BSL-3 microbiology laboratories that contain multiple
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Class III biosafety cabinets (BSCs) and two autoclaves. Additional laboratories within this area
include multiple microbiology laboratories equipped with Class II BSCs. Test procedures at the
MREF are governed by established SOPs that are specified by facility, number, and title.
A8.2 Training
Because of the hazardous materials involved in this technology evaluation,
documentation of proper training and certification of the test personnel is mandatory before
testing takes place. The Battelle Quality Assurance Manager, or designee, must assure that
documentation of such training is in place for all evaluation personnel before allowing evaluation
to proceed.
All participants in this evaluation (i.e., Battelle, EPA, and vendor staff) will adhere to the
security, health, and safety requirements of the Battelle facility in which testing will be
performed. Vendor staff may offer instruction to Battelle evaluation personnel using their
decontamination technology, but will not be the technology users during the evaluation. To the
extent allowed by the test facility, vendor staff may observe, but may not conduct, any of the
technology evaluation activities identified in this test/QA plan.
Access to restricted areas of the test facility will be limited to staff who have met all the
necessary training and security requirements. The existing access restrictions of the test facility
will be followed, i.e., no departure from standard procedures will be needed for this evaluation.
All visiting staff at the test facility will be given a site-specific safety briefing prior to the start of
any test activities. This briefing will include a description of emergency operating procedures
and the identification and location and operation of safety equipment (e.g., fire alarms, fire
extinguishers, eyewashes, exits). Evaluation procedures must follow all safety practices of the
test facility at all times. Any report of unsafe practices in this evaluation, by those involved in the
evaluation or by other observers, shall be grounds for stopping the evaluation until the Quality
Assurance Manager and evaluation personnel are satisfied that unsafe practices have been
corrected.
A9 DOCUMENTATION AND RECORDS
Documentation of training related to technology testing, field-testing, data analysis, and
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reporting is maintained for all Battelle technical staff in training files at their respective
locations. The Battelle Quality Assurance Manager may verify the presence of appropriate
training records prior to the start of testing. If Battelle staff operate and/or maintain a vendor-
owned decontamination system during the technology evaluation, the vendor will be required to
train those staff prior to the start of testing. Battelle will document this training with a consent
form, signed by the vendor, that states which Battelle staff have been trained on their technology.
Battelle technical staff will have a minimum of a bachelor's degree in science/engineering or
have equivalent work experience.
As stated in Section 5.1.1 of the TTEP QMP,[1] program requirements and the technology
evaluation records needed to reconstruct evaluation activities and verify that reported data were
collected in a quality manner reconciled to the QMP will be retained for at least seven years after
final payment under the Blank Purchase Agreement for the TTEP. These records consist of
• Test/QA plan
• Chain-of-custody forms
• Laboratory record books
• Data collection forms
• Electronic files (both raw data and spreadsheets)
• Technology evaluation report
• Quality assessment reports.
All of these records will be maintained by the Task Order Leader or designee (with the
exception of the quality assessment reports) during the evaluation and transferred to Battelle's
Records Management Office for storage at the conclusion of the evaluation. All written records
must be in ink. Any corrections to entries, or changes in recorded data, must be made with a
single line through the original entry. The correction is then to be entered, initialed, and dated by
the person making the correction.
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B MEASUREMENT AND DATA ACQUISITION
Bl EXPERIMENTAL DESIGN
Bl.l General Test Design
This test/QA plan specifies procedures for bench-scale testing to evaluate sprayed
liquid/foam sporicidal decontaminants under specified operating conditions and ambient
conditions for decontaminating porous and non-porous surfaces consisting of small pieces (i.e.,
test coupons) of building materials to which a biological agent or surrogates have been added.
Treatments for a given biological agent and building material will be defined in terms of
the organism and material. Inactivation will refer to the log reduction in biological agent or
surrogate compared to the respective controls. Differential efficacy of inactivation for various
indoor surface materials may also be determined.
A pretest-posttest control group design will be used for each material and biological
agent or surrogate:
R Oi X O2
R Oi O3
where time passes from left to right and
R signifies random selection of the test coupons for control, experiment, and type of
biological organism.
O represents the mean of measurement of the spores extracted from the coupons, and
X represents the experimental variable, in this case the decontamination process
At a given point in time, the effect of the experimental variable is (62 -Oi)-(O3 -Oi), or
simplified, (62 -Os).
The experimental design will allow the following null (Ho) and alternate (HA) hypotheses
to be statistically tested:
HO '• Rrreatment ~ Rcontrol = 0
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HA '• RTreatment ~ Rcontrol > 0
Where:
^Treatment is the geometric mean reduction in viable spores extracted from coupons in the
treatment group
^control is the geometric mean reduction in viable spores extracted from the positive
control coupons
The experimental design will allow testing the hypothesis (Ho) that there is no significant
difference (p < 0.05) in the log reduction in viable spores between the treatment and the positive
control groups or the alternate hypothesis (HA) that a significant difference exists between the
treatment and positive control groups. Treatment will be defined in terms of the quantity of
viable agent or surrogate, identity of the decontaminant, operational implementation of the
decontaminant (e.g., concentrations, contact time), sprayer conditions (e.g., spray distance, air
pressure, spray time), and ambient conditions (e.g., temperature, humidity). Decontamination
efficacy will be calculated as described in Section A7.
Fourteen replicate coupons—six spiked test coupons [spiked, decontaminated], six
positive controls [spiked, not decontaminated], one laboratory blank [not spiked, not
decontaminated], one procedural blank [not spiked, decontaminated]—will be included for each
coupon material and each biological agent tested (see the test matrix in Table 2). Unique sample
identification codes will link each coupon to corresponding MREF standard data report forms;
for example, MREF Data Report Form BioDecon-018-01 for the Liquid Decontamination
Spraying System (Figure 2).
Practical constraints prevent all of the coupons from simultaneous decontamination.
Therefore, randomly selected coupons will be decontaminated in each of two decontamination
periods (trials).
-------
Table 2. Matrix of Materials, Organisms, and Treatments
Organism
B. anthracis (Ames)
B. anthracis (Ames)
B. anthracis (Ames)
B. anthracis (Ames)
B. anthracis (Ames)
B. anthracis (Ames)
B. anthracis (Ames)
B. anthracis (Sterne)
B. anthracis (Sterne)
B. anthracis (Sterne)
Material
Carpet
Bare wood
Glass
Decorative
laminate
Galvanized
metal
Painted
wallboard
paper
Painted
concrete block
Carpet
Bare wood
Glass
Test Coupon
(spiked,
decontaminated)
(n=6)
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Reference Method
(spiked,
decontaminated with
amended bleach)
(n=6)
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Positive Control
Coupons (spiked,
not
decontaminated)
(n=6)
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Laboratory Blank
(not spiked, not
decontaminated)
(n=l)
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Procedural
Blank
(not spiked,
decontaminated)
(n=l)
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
OQ
O
O
-------
Organism
B. anthracis (Sterne)
B. anthracis (Sterne)
B. anthracis (Sterne)
B. anthracis (Sterne)
B. subtilis
B. subtilis
B. subtilis
B. subtilis
B. subtilis
B. subtilis
Material
Decorative
laminate
Galvanized
metal
Painted
wallboard
paper
Painted
concrete block
Carpet
Bare wood
Glass
Decorative
laminate
Galvanized
metal
Painted
wallboard
paper
Test Coupon
(spiked,
decontaminated)
(n=6)
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Reference Method
(spiked,
decontaminated with
amended bleach)
(n=6)
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Positive Control
Coupons (spiked,
not
decontaminated)
(n=6)
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Laboratory Blank
(not spiked, not
decontaminated)
(n=l)
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Procedural
Blank
(not spiked,
decontaminated)
(n=l)
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
OQ
O
O
-------
Organism
B. subtilis
G. stearothermophilus
G. stearothermophilus
G. stearothermophilus
G. stearothermophilus
G. stearothermophilus
G. stearothermophilus
G. stearothermophilus
Material
Painted
concrete block
Carpet
Bare wood
Glass
Decorative
laminate
Galvanized
metal
Painted
wallboard
paper
Painted
concrete block
Test Coupon
(spiked,
decontaminated)
(n=6)
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Reference Method
(spiked,
decontaminated with
amended bleach)
(n=6)
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Six sample IDs, °C,
%RH, test date
Positive Control
Coupons (spiked,
not
decontaminated)
(n=6)
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Six sample IDs,
°C, %RH, test
date
Laboratory Blank
(not spiked, not
decontaminated)
(n=l)
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Sample ID, °C, %RH,
test date
Procedural
Blank
(not spiked,
decontaminated)
(n=l)
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
Sample ID, °C,
%RH, test date
o
o
I.
3
OQ
ft>
n>
O ~-
O fa
-------
535-CG667913
Liquid Decon Spraying System
Performed By/Date:
QC/Tech Reviewed By/Date:
Sample ID
Rug Spray Blank
Concrete Spray Blank
Rug 1 H20
Rug 2 H20
Rug 3 H20
Rug 4 H20
Rug 5 H2O
Rug 6 H2O
Concrete 1 H2O
Concrete 2 H2O
Concrete 3 H2O
Concrete 4 H2O
Concrete 5 H2O
Concrete 6 H2O
Rug 1 NaOCI
Rug 2 NaOCI
Rug 3 NaOCI
Rug 4 NaOCI
Rug 5 NaOCI
Rug 6 NaOCI
Concrete 1 NaOCI
Concrete 2 NaOCI
Concrete 3 NaOCI
Concrete 4 NaOCI
Concrete 5 NaOCI
Concrete 6 NaOCI
Liquid Decon Sprayer
Time (sec)
Distance (in)
Pressure (psi)
Contact Time
Start Time
End Time
Total (min)
% RH*
•Relative humidity recorded immediately prior to each spray replicate
q
O
o
3'
CJQ
ft
2. O
Figure 2. MREF Data Report Form BioDecon-018-01 for the Liquid Decontamination Spraying System
O ^
.
O
ON
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B1.2 Scale of Testing, Testing Apparatus
The liquid decontamination spraying system developed at the Battelle MREF will be used
inside Compact Glove Box 830-ABC (Plas Labs, Inc., Lansing, MI; see Figure 3) to contain the
decontamination spray and biological agents. This test chamber is 71 centimeters (cm) wide by
59 cm deep by 74 cm high (28 inches [in] x 23 in x 29 in) and has outer dimensions of 110 cm
(width) by 61 cm (depth) by 79 cm (height) (43 in x 24 in x 31 in). The chamber has a total
volume of 317 liters (11.2 cubic feet). The test chamber also has atop opening of 43 cm by
58 cm (17 in x 23 in) and an attached transfer chamber that is 30 cm (12 in) long and an inner
diameter of 28 cm (11 in). Glove ports, integral to
test cnamber, are available for working in the
glove box. The glove box will be modified with
high efficiency particulate air-filtered vent valves to
relieve potential pressure buildup during spraying.
The decontaminant will be administered as a spray
Figure 3. Compact Glove Box for onto the test surfaces at a ^ time' distance' and
Sporicidal Decontamination contact time recommended by the product
Technology Evaluation „ .„ -,11^1
manufacturer, if available. If these spray parameters
are not provided by the manufacturer, then default parameters will be implemented; for example,
a spray time of 10 seconds, a distance of 30 cm (12 in), and a contact time of 10 minutes. These
default spray time and distance parameters are derived from the recommended default
parameters given in the Association of Analytical Communities (AOAC) 961.02 Germicidal
Spray Products as Disinfectants (Table 3). Test coupons will be approximately 1.9 cm by 7.5 cm;
multiple coupons of each material will be spiked with the agent/surrogate, placed into the test
chamber on the spraying system, and sprayed with the decontaminant. Laboratory blank (i.e., not
spiked, not decontaminated) and positive control (i.e., spiked, not decontaminated) coupons will
also be prepared for each test material, and results obtained for these coupons will be used with
the data resulting from the analyses of post-treatment samples to calculate decontamination
efficacy. This evaluation methodology is a highly controlled, reproducible approach to assess
decontamination efficacy that simulates a realistic, small-scale spray application of the
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decontamination technology. A detailed description of the spraying system is provided in the
appendix.
Table 3. Default Spray Parameters
Parameter
Spray Timea
Spray Distance21
Contact Timea
Spray Air Pressure
Setting
10 seconds
30 cm (12 in)
10 minutes
40 pounds per
square inch
a AOAC 961.02, Section C.
bMay be subject to change.
B1.3 Test Surfaces
Various structural, decorative, and functional surfaces typically found inside an office
building or a mass transit station will be used to evaluate sporicidal decontamination
technologies. The surface materials that will be used include both non-porous and porous
surfaces. Test coupons (typically measuring 1.9 cm by 7.5 cm) will be prepared from larger
pieces of stock materials. The representativeness and uniformity of the test materials are critical
to assuring reliable evaluation results. Representativeness means that the materials are typical of
those used in buildings in terms of quality, surface characteristics, structural integrity, etc.
Uniformity means that all test pieces are essentially equivalent for evaluation purposes.
Representativeness will be assured by selecting test materials that meet industry standards or
specifications for indoor use and by obtaining those materials from appropriate suppliers.
Uniformity will be maintained by obtaining a large enough quantity of material that multiple test
samples with uniform characteristics can be prepared (e.g., test coupons will be cut from the
interior rather than the edge of a large piece of material) or by using standardized coupons where
available. Details of the test surfaces listed below are shown in Table 4.
Non-porous materials:
• Decorative laminate (DL)
• Galvanized metal ductwork (GM)
• Glass (GS)
Porous materials:
• Industrial-grade carpet (1C)
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• Painted (latex, semi-gloss) concrete cinder block (PC).
• Painted (latex, flat) wallboard paper (PW)
• Bare wood (pine lumber) (BWD)
B1.4 Biological Agents and Surrogates
The biological agent to be used under this test/QA plan was selected based on an
evaluation of potential threats to buildings.[4'5] The evaluation considered availability, lethality,
potential delivery pathways, and persistence of potential agents.
The biological agent used in evaluating the sporicidal decontamination technology will
be Bacillus anthracis Ames strain spores. Biological surrogates will be used to establish
correlations between the decontamination efficacy of surrogates and agents. To provide
correlations with the B. anthracis results, the surrogates B. anthracis Sterne strain (animal
vaccine strain), B. subtilis (American Type Culture Collection [ATCC] 19659), and
Geobacillus stearothermophilus (ATCC 12980) will be used. B. anthracis spores and surrogate
spores will be prepared and characterized according to MREF SOPs.[6"10]
Table 4. Material Characteristics
Material
DL
GM
GS
1C
PC
PW
BWD
Lot, Batch, or
ASTM No., or
Observation
Laminate/ Formica/
White Matte Finish
Industry Standard
24-Gauge Galvanized
Steel
C1036, 1/8 in thick
ShawTek, EcoTek 6.
Color: mottled
gray /dark brown/
black
American Society for
Testing and Materials
C90
05-16-03; Set-E-493;
Roll-3
Screen Molding, % in
thick (Pine Wood)
Manufacturer/
Supplier Name
Solid Surface
Design
Accurate
Fabrication
Brooks Brothers
Shaw Industries,
Inc
Wellnitz
United States
Gypsum
Company
Kingswood
Lumber
Approximate
Coupon Size,
L x W, inches
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
1.9 cm x 7.5 cm
Material Preparation
None; sterilized with gamma
irradiation
Autoclaved; cleaned with acetone
Autoclaved; cleaned with acetone
Sterilized with gamma irradiation
Brush and roller painted all sides.
One coat Martin Senour latex
primer (#7 1-1185) and one coat
Porter Paints latex semi-gloss
finish (#919); autoclaved.
Roller painted on one side using
Martin Senour Paints. One primer
(#71-1185) and two finish (flat,
#70-1001) coats; sterilized with
gamma irradiation.
Sterilized with gamma irradiation
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B. subtilis and G. stearothermophilus have commonly been used as surrogates for
B. anthracis in decontamination technology testing; B. anthracis Sterne has not been typically
used for decontamination testing. The G. stearothermophilus surrogate exhibits comparatively
high resistance to various sporicidal decontaminants. The B. subtilis (ATCC 19659) surrogate is
the most commonly used surrogate for B. anthracis.
Spores will be prepared according to established MREF procedures.[11'12] Working stock
suspensions of each spore type will be prepared at a target density of approximately
IxlO9 CFU/milliliter (mL).
B1.5 Temperature and Relative Humidity Conditions
During testing, the temperature and the relative humidity will be maintained at ambient
conditions (i.e., 20 to 26°C and <70% relative humidity). When using the spraying system,
relative humidity may increase due to aerosolizing the decontaminant. If this occurs, the glove
box will be evacuated using a vacuum pump to reduce the humidity to below 70%. Temperature
and relative humidity will be monitored using a calibrated thermometer/hygrometer.
B1.6 Surface Damage
Following decontamination of the test surfaces prepared as described in Section B2.2,
each test surface will be examined visually to establish whether use of the decontamination
approach caused any obvious damage to the surface. Surface damage will be observed before
extraction. The test surface will be allowed to dry before inspection for damage. Visual
inspection of the surface will then take place through side-by-side comparison of the
decontaminated test surface and control coupons of the same test material. Differences in color,
reflectivity, and roughness will be assessed qualitatively; and observations will be made by the
evaluation staff and recorded. Observed damage will be confirmed by a second evaluator.
B2 METHODS REQUIREMENTS AND PROCEDURES
B2.1 Agents
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B. anthracis (Ames and Sterne), B. subtilis (ATCC 19659), and G. stearothermophilus
(ATCC 12980) spores will be prepared according to established MREF procedures.[6'7] The
SOPs used for production also provide for identification and purity. Strains will be confirmed by
third-party genotyping. Identity of organisms received from other sources will be confirmed in
documentation from the source of the organism. Working stock suspensions of each spore type
will be prepared at a target density of approximately IxlO9 CFU/mL.
B2.2 Coupon-Scale Testing
B2.2.1 Preparation of Test Materials
Each of the test coupons will be cut to 1.9 cm by 7.5 cm from the interior of a large piece
of test material. Edges and damaged areas will be avoided when cutting test coupons. The
coupons will be sterilized prior to use. A methods demonstration will determine an appropriate
method of sterilization for each material type. Autoclaving, either using wet heat or dry heat, will
be used where possible to sterilize these materials. Other non-chemical sterilization methods
(e.g., gamma irradiation) will be evaluated for materials that cannot be sterilized by autoclaving.
Packaging and storage conditions will preserve sterility until the coupons are ready for use. To
prevent contamination of test surfaces, aseptic technique, following Battelle policies and
guidelines[8"10] will be exercised during all phases of handling the test coupons.
The test coupons will be visually inspected prior to spiking with the biological agents.
Coupons with anomalies on the test surface will not be used. An anomaly is any obvious
difference in a coupon when compared with similar coupons, such as an unpainted concrete
surface, an oil smudge on a metal surface, or chipped laminate. Coupons will be used once. On
each evaluation day, each coupon will be assigned a unique identifier code by the evaluation
staff. The identifier code will be placed on the coupons, vials, and plates in indelible ink. Prior to
applying the biological agent or surrogate, the surface of each coupon will be disinfected by
wiping with 70% isopropanol to minimize contamination by microorganisms other than those
being evaluated.
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B2.2.2 Spiking of Biological Agents onto Test Coupons
Biological agent/surrogates will be spiked onto test coupons in an appropriate BSC
(BSC-III or BSC-II) according to established MREF procedures.[8"12] Spiked coupons will be
prepared fresh for each day of experimental work. Test coupons will be placed flat in the BSC
and spiked at approximately 1 x 108 CPU per coupon. A 100-jiL aliquot of a stock suspension
(approximately 1 x 109 CFU/mL) of spores will be dispensed (using a micropipette) as small
droplets across the surface of the test coupon. After spiking with biological agent or surrogate
suspension, the test coupons will remain undisturbed overnight in the BSC-III (B. anthracis
Ames) or BSC-II (B. anthracis Sterne, B. subtilis, G. stearothermophilus) to dry.
B2.2.3 Confirmation of Spore Density in Stock Suspensions
To confirm the spore density (number of spores per volume) of biological agents and
surrogates, the respective stock spore suspensions used to spike the coupons will be re-
enumerated on each day of use. An aliquot (0.1 mL) of the stock suspension and each serial
dilution to 10"7 will be plated onto tryptic soy agar plates and incubated overnight at 35°C to
37°C for B. anthracis and B. subtilis and at 55°C to 60°C for G. stearothermophilus. Plates will
be enumerated within 18 to 24 hours of plating as described in the MREF SOPs.[13'14] The
number of CFU/mL will be determined by multiplying the average number of colonies per plate
by the reciprocal of the dilution.
B2.2.4 Application of the Decontaminant and Monitoring of Test Procedures
On the day following spiking, test coupons intended for decontamination (including
blanks and controls) will be transferred into the glove box (test chamber) where the spraying
system is located. The decontaminant will be applied in accordance with the vendor's
instructions (if available) with respect to spray distance and contact time. The decontaminant
contact time, spray distance, and temperature will be controlled and monitored/recorded. The
spraying system to be used for this testing automatically controls for parameters such as spray
distance, liquid decontaminant flow rate, air pressure for spraying, and product deposition,
thereby eliminating differences in these parameters that may be associated with human error.
Where applicable, the respective numeric values for each of these parameters will be recorded
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for each decontaminant tested. Spray parameters can readily be adjusted to meet vendor
recommendations or requirements. The design of the spraying system enables the user to adjust
any necessary parameters to ensure the accuracy and repeatability of sprays. Following
decontamination, the test chamber will be cleared using the vendor-supplied method for
neutralizing the decontamination reagent. If no instructions for neutralization are provided, the
test chamber will be cleaned following procedures established under the Battelle MREF Facility
Safety Plan.[12]
B2.2.5 Determination of Decontamination Efficacy
The performance or efficacy of the sporicidal decontaminants will be assessed by
determining the number of viable organisms remaining on the test coupons, as well as the
number of viable organisms in the liquid run-off after decontamination. These data will be
compared with the number of viable organisms extracted from the control coupons sprayed with
a benign liquid (e.g., sterile water or PBS).
This test/QA plan includes application of laboratory methods in novel combinations of
test organisms, materials, decontamination technologies, and neutralization methods. Therefore,
before executing this test/QA plan, a methods demonstration based on a modification of a testing
scheme for neutralization described in American Society for Testing and Materials (ASTM) E
1054-02,[15] will be used to determine (1) decontamination technology effectiveness (add spores
to decontamination liquid; determine CFU without neutralization), (2) neutralizer and/or dilution
effectiveness at terminating decontamination (add spores to decontamination liquid; determine
CFU with neutralization), (3) neutralizer toxicity (add spores to neutralizer; determine CFU), and
(4) decontamination control effectiveness (add spores to extraction buffer without neutralizer;
determine CFU). One modification is that approximately 1 x 108 CFU will be used for testing
instead of the 30 to 100 CFU/mL outlined in ASTM E 1054-02. The increased concentration of
CFU is anticipated to provide better sensitivity for this neutralization evaluation. For this test/QA
plan, it is anticipated that sodium thiosulfate (STS) will be used as the neutralizer for baseline
comparative technology, amended bleach. It is known that STS can inhibit bacterial growth;
therefore, when evaluating the use of STS as a neutralizer, step 3 (described above) may not be
performed. Findings will be discussed with the TOPO and will also guide the selection of the
neutralization method. The neutralization results will be summarized in the final report as mean
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(±SD) total spores recovered and percent neutralization efficacy. Neutralization efficiency will
be calculated by dividing the total spores recovered from test samples (e.g., spores +
decontaminant + neutralizer in extraction buffer) by the total number of spores recovered from
the controls (spores added to extraction buffer without decontaminant or neutralizer) and
expressed as a percentage.
In the methods demonstration, whether dilution in the extraction step is sufficient to
terminate decontamination will be determined, or whether adding a neutralizer to the extraction
fluid can be used to terminate decontamination. If neutralization can be accomplished in the
extraction step, the replicate test coupons and a procedural blank that was decontaminated will
be transferred aseptically to sterile 50-mL conical tubes after decontamination. For each type of
biological agent, the decontaminated, control, and blank coupons (except the procedural blank
used for evaluating obvious damage) will be placed individually in conical vials containing
10 mL of sterile extraction buffer to which, if necessary, a neutralizer to stop the
decontamination has been added. The tubes will be agitated on an orbital shaker for 15 minutes
at approximately 200 revolutions per minute (rpm) at room temperature.
If the method demonstration shows that neutralization, separate from extraction, is
needed, the test coupons and procedural blanks will be removed from the decontamination fluid
and placed into an individual container or well holding sufficient neutralizer to cover the spiked
surface of the coupon. The spiked surface will remain in contact with the neutralizer for the
period determined by the method demonstration. The neutralizer contact time will be monitored
and recorded.
The neutralized coupons will then be transferred individually into sterile 50-mL conical
vials containing 10 mL of sterile PBS extraction buffer. The tubes will be agitated on an orbital
shaker for 15 minutes at approximately 200 rpm at room temperature.
For spore extraction, the tubes will be agitated for 15 minutes on an orbital shaker at
approximately 200 rpm at room temperature. Following extraction, 1 mL of the coupon extract
(plus any decontaminant run-off from the coupon) will be removed, and a series of dilutions
through 10"7 will be prepared in sterile water. An aliquot (0.1 mL) of the undiluted extract (and
coupon run-off) and each serial dilution will be plated onto tryptic soy agar plates and incubated
overnight at 35°C to 37°C for B. anthracis and B. subtilis and at 55°C to 60°C for G.
stearothermophilus. Plates will be enumerated within 18 to 24 hours of plating as described in
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MREF SOPs.(13'14) The number of CFU/mL will be determined by multiplying the average
number of colonies per plate by the reciprocal of the dilution. The volume of neutralizer will be
included in the determination of the total CPU, where CFU/mL will be multiplied by the volume
of extraction buffer plus neutralizer to determine total CPU. Dilution data representing the
greatest number of individually definable colonies will be expressed as mean ± standard
deviation of the numbers of CPU observed. Figure 4 shows the spray decontamination evaluation
procedure.
Decontamination efficacy will be calculated as discussed in Section A7. The efficacy
calculations will be performed using the total of the spores extracted from the test coupons as
well as any collected decontaminant run-off from the coupons.
Based on previous decontamination studies, it is assumed that 100% recovery of spores
from the spiked test coupons will not be achieved; therefore, viable spores may remain on the
test coupons. A qualitative assessment will be performed to determine whether viable spores
remain on the decontaminated test coupons. Following the extraction process described above,
each coupon will be transferred into a sterile 50-mL conical tube containing 20 mL of sterile
tryptic soy broth culture medium. These vials will be cultured at the appropriate temperature for
B. anthracis or surrogates to encourage viable spore germination and subsequent proliferation of
vegetative bacteria.
At 1 and 7 days post-decontamination, the tubes will be visually assessed qualitatively for
viability. A cloudy culture medium may indicate "growth" of viable spores, vegetative cells, or
other microorganisms. A clear culture medium indicates "no growth," consistent with a complete
kill of all microorganisms.
B2.2.6 Observation of Surface Damage
Following application of the decontaminants, each test surface will be examined visually
to establish whether the decontaminant caused any obvious damage to the surface. Observation
of surface damage will be performed immediately after the designated contact time with the
decontaminant, but before post-decontamination sampling to assess efficacy. Visual inspection
of the surface will take place through side-by-side comparison of the decontaminated test surface
and the control coupons of the same test material. Differences in color, reflectivity, and
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roughness will be assessed qualitatively, and observations will be recorded by the evaluation
staff and recorded.
B2.2.7 Observation of Ease of Use
A non-critical qualitative assessment will be made in the final report to document the
ease of preparation, application, handling, and storage that was experienced. Shelf life, if any,
provided by the manufacturer will be noted in the final report.
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Clip Coupons in
Place
Inoculate
Implement Spray
Unclip & Seal Coupon
in Tube
Hold for Contact Time
Assay Coupon and
Collected Run-off
Neutralize & Extract
(10 mL Neutralizing Broth)
Dilution Plating
Enumeration
Figure 4. Flow Chart of Spray Decontamination Evaluation
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B3 SAMPLE HANDLING AND CUSTODY REQUIREMENTS
Testing will occur within a secure area. The test materials in labeled containers will be
delivered directly to the testing laboratory where coupons are spiked. Test coupons will be
spiked with biological agent/surrogates in an appropriate BSC (BSC-III or BSC-II) according to
established MREF procedures^8"121 Each coupon will be assigned a unique identifier code by the
evaluation staff for traceability.
B4 ANALYTICAL METHODS REQUIREMENTS
No analytical methods are required in this evaluation.
B5 QUALITY CONTROL REQUIREMENTS
Quantitative standards do not exist for biological agents and surrogates. The quantitative
measurement that is critical to this evaluation is a differential measurement; that is, the test
coupons are spiked with spores from the same batch and, subsequently, the coupons are treated
with the decontamination technology. The number of viable CPUs enumerated on
decontaminated coupons and the coupons that were spiked in the same fashion but not
decontaminated are used in calculating log kill (or efficacy) (see Section B2.2.5). The mean ±SD
of the log kill values will be calculated for each coupon type and biological agent combination.
The QC samples that are analyzed in each group (see Table 2) include six test coupons (spiked,
decontaminated), six positive controls, one laboratory blank, and one procedural blank. In
addition, one TSA plate will be evaluated for sterility with each group; and the measurement of
spore density, described in Section B2.2.3, will be used as a QC for growth on the medium.
For each liter of sterile tryptic soy broth prepared, one vial containing 10 mL of broth that
is not spiked (negative control) and one vial containing 10 mL of broth spiked with a positive
control organism will be incubated at 35 to 37°C for 7 days. Additionally, tryptic soy broth will
be visually examined for cloudiness or contamination prior to use.
The QC samples, acceptance criteria, and corrective actions are provided in Table 5.
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Table 5. Quality Control Sample Requirements
QC Sample
Spike Control
Laboratory Blank (coupon
spiked with diluent without
biological agent and not
subjected to the
decontamination treatment)
Procedural Blank (coupon
spiked with diluent without
biological agent and
subjected to the
decontamination treatment)
Positive Control
(coupon spiked with
biological agent but not
subjected to the
decontamination)
Test Coupons
(spiked and subjected to
decontamination)
Blank tryptic soy agar
plate (plate incubated, but
not spiked)
Growth Control
(tryptic soy agar plate
incubated after spiking
with organisms)
Blank Tryptic Soy Broth
(autoclaved tube of
medium, not spiked
incubated for seven days)
Positive Control, Tryptic
Soy Broth, (autoclaved
tube of spiked medium
incubated for seven days)
Information
Provided
Calculated value of spore
density in stock
suspensions.
Controls for sterility of
the coupon material.
Controls for
contamination during
decontamination
treatment.
Controls for recovery
and confounds arising
from history impacting
spore viability; controls
for special causes.
Replicate coupons
control for special
causes.
Controls for sterility of
plates.
Controls for ability to
support growth.
Controls for sterility.
Controls for ability to
support growth.
Acceptance
Criteria
±25% of target spike
level of 1 09 spores per
mL (108 spores per
0.1 mL).
No observed CPU.
No observed CPU.
Mean CPU >1% and
<300% of spiked
spores (108 spores per
0.1 mL in target
spike);
Grubbs outlier test
with no more than one
outlier.
Grubbs outlier test
with no more than one
outlier.
No observed growth
following incubation
In the verification of
spore density,
described in B2.2.3,
±50% of nominal
spore density
observed.
No observed growth
following 7 -day
incubation.
Growth observed
following 7 -day
incubation.
Corrective Action3
Reject results; prepare stock
suspensions meeting target spore
density level in spike control.
Reject results; identify and
remove source of contamination.
Reject results; identify and
remove source of contamination.
Mean CPU <1% or >300%
recovery = reject results;
evaluate/exclude values for
outliers.
Evaluate/exclude values for
outliers.
Incubate additional 10 plates. If
any additional growth is
observed, reject results from the
lot.
Incubate additional 10 plates,
including 5 from a different lot.
If significant differences in
growth are observed between the
lots, reject results from the lot
that is not supporting growth.
If remaining batch appears clear,
re-autoclave the remaining batch
and retest; if remaining batch
appears cloudy do not use that
batch.
Determine whether the medium
batch or lot is causing no
growth. Replace medium to
achieve positive growth.
"Testing results will be discussed with the TOPO prior to implementing corrective action
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B6 INSTRUMENT/EQUIPMENT TESTING, INSPECTION, AND MAINTENANCE
REQUIREMENTS
The equipment needed for the evaluation is listed in Table 6 with the data quality
indicators and corrective actions that will be taken in the event of substandard performance. All
instruments utilized in the technology evaluation will be calibrated as stipulated by the
manufacturer or, at a minimum, annually.
Table 6. Measurement Parameters and Data Quality Indicators
Parameter
Temperature
Relative Humidity
Pressure
Micropipette
Time
Volume of
Decontaminant
Measurement Method
NIST-traceable thermo
hygrometer
NIST-traceable
hygrometer
Certified gauge
All micropipettes will
be certified as
calibrated at time of use
Compare to U.S. Naval
Observatory time
values
Pipette
Data Quality Indicators
Compare against calibrated thermometer
before and after evaluation testing, agree
±10%
Compare against calibrated hygrometer
before and after evaluation testing, agree
±10%
Compare against NIST-traceable
calibrated gauge before and after
evaluation testing, agree ± 10%
±1%; pipettes are recalibrated by
gravimetric evaluation of pipette
performance to manufacturer's
specifications every 6 months by supplier
(Rainin Instruments)
±14 seconds per day, evaluate
semiannually
Check calibration gravimetrically over the
range of volumes used before and after
evaluation testing, agree ±5%
Corrective
Action
Replace
Replace
Replace
Recalibrate
Replace
Recalibrate
B7 INSTRUMENT CALD3RATION AND FREQUENCY
The equipment needed for the evaluation will be maintained and operated according to
the quality requirements and documentation of the evaluation facility. All equipment used at the
time of evaluation will be verified as being certified, calibrated, or validated. Calibration of
instruments will be done at the frequency specified in Section B6 and checked according to
Table 6. Any deficiencies will be noted. The instrument will be adjusted to meet calibration
tolerances and recalibrated. If tolerances are not met after recalibration, additional corrective
action will be taken, including replacement of the equipment.
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B8 INSPECTION/ACCEPTANCE REQUIREMENTS FOR SUPPLIES AND
CONSUMABLES
Supplies and consumables will be acquired from reputable sources. The source and purity
of reagent-grade chemicals and standards will be documented and documentation retained in the
file. Supplies and consumables will be examined for evidence of tampering or damage upon
receipt and prior to use, as appropriate. In addition, expiration dates will be noted and recorded.
Solutions will be prepared following MREF protocols and will be documented in reagent
preparation forms. These forms include preparation instructions, suppliers, catalog numbers, lot
number expiration dates for components, calculated and actual amounts used, and specific
equipment used with calibration information. A lot number and expiration date is assigned to
each reagent. All documents are initialed and dated.
Tryptic soy agar plates are manufactured by Remel (Lenexa, KS). Each box of tryptic soy
agar plates includes a certification of lot number and expiration date. Tryptic soy agar plates will
be examined for evidence of tampering or damage upon receipt and prior to use, as appropriate.
B9 DATA ACQUISITION REQUIREMENTS
No data needed for this project implementation are obtained from non-measurement
sources such as computer databases, programs, literature files, or historical databases.
BIO DATA MANAGEMENT
Data acquisition during the evaluation includes proper recording of the procedures used
to assure consistency in the evaluation and adherence to this test/QA plan; documenting
sampling/evaluation conditions; recording observations regarding the condition of the surface of
each coupon before and after the decontamination process; and recording efficacy results and
evaluation conditions. Data will be acquired by the Battelle testing staff manually and recorded
immediately in a consistent format throughout the evaluation. All written records will be in ink,
and any corrections to recorded data will be made with a single line through the original entry.
The correction will then be entered, initialed, and dated by the person making the correction.
Any non-obvious correction will include a reason for the correction. Strict confidentiality of
evaluation data will be maintained.
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B10.1 Efficacy Calculations
Prior to analyzing data for the inactivation of biological agents from indoor surface
materials, a separate analysis will be conducted to determine the recovery of viable spores from
the time of initial spiking of the control coupon to the completion of the drying period. Recovery
will be calculated as described in Section A7.1 to indicate the number of CPUs extracted from
the coupons after the drying time relative to the number of CPUs spiked onto the control coupon.
An ANOVA will be fitted to the data as described in Section A7.1. Model diagnostics
will be examined to assess whether there are any difficulties with outliers or the model
assumptions of constant variance and normality of the residuals. If the data are not adequate for
the model, appropriate transformations or more general statistical models (e.g., non-parametric)
will be considered. The Grubbs test[2] will be used to identify outliers. Unless an error in
recording or processing the data can be identified and/or corrected, the outlier will be excluded in
the final analysis and noted in the report.
Once final statistical models have been fit, pre-planned comparisons will be performed.
This will consist of comparing whether there are statistically significant differences in recovery
between pairs of materials. The overall error rate for the set of comparisons will be controlled at
5%.
To measure the effects of decontamination on the inactivation of biological agents,
decontamination efficacy will be calculated (as described in Section A7.2) to indicate the relative
reduction in viable spores achieved by the decontamination technology.[12'14'15] In cases where
no (or very small numbers of) CPU remain after decontamination, modeling will be conducted
using Poisson distribution theory.
The efficacy data will be fit to an ANOVA to evaluate the differential efficacy due to test
material on log reduction (described in Section A7.2). There will be a separate ANOVA for each
of B. anthracis (Ames), B. anthracis (Sterne), B. subtilis, and G. stearothermophilus. The B.
anthracis (Ames), B. anthracis (Sterne), B. subtilis, and G. stearothermophilus will be combined
under one ANOVA to facilitate pre-planned comparisons among the spore types.
Model diagnostics will be examined to assess whether there are any difficulties with
outliers or the model assumptions of constant variance and normality of the residuals. If the data
are not adequate for the model, appropriate transformations or more general statistical models
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(e.g., non-parametric) will be considered. The Grubbs test[2] will be used to identify outliers.
Unless an error in recording or processing the data can be identified and/or corrected, the outlier
will be excluded in the final analysis and noted in the report.
Statistical analysis will consist of performing a set of pre-planned comparisons. Once
final statistical models have been fit, the main effects and interactions will be examined to
determine if they are statistically significant. Based on these results, appropriate estimates will be
determined for the set of pre-planned comparisons. They include
• Comparing whether the efficacy of the decontamination treatment at a particular
temperature and contact time was statistically significantly different from zero for
each surface material
• Comparing the estimated mean decontamination efficacy to determine whether
significant differences exist among the seven surface materials
• Comparing the calculated mean decontamination efficacy for B. anthracis Ames to B.
anthracis Sterne, B. subtilis, and G. stearothermophilus to determine whether
significant differences exist between anthrax agents and the surrogate.
The overall error rate for these pre-planned comparisons will be controlled at 5%.
The evaluation results will be compiled in a report. The report will briefly describe TTEP and the
evaluation procedures utilized, as well as all evaluation data and observations. The preparation of
the draft report, review of the draft report, revision of the draft report, final approval, and
distribution of the final report will be conducted as stated in the QMP.
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C ASSESSMENT/OVERSIGHT
Cl ASSESSMENT AND RESPONSE ACTIONS
Battelle's Quality Assurance Manager will audit at least 10% of the evaluation data.
Battelle's Quality Assurance Manager will trace the data from initial acquisition, through
reduction and statistical comparisons, to final reporting. All data calculations will be checked.
Cl.l Technical Systems Audit
Battelle's Quality Assurance Manager or designee will perform at least one TSA during
the evaluation. The TSA is to ensure that the evaluation is performed in accordance with the
TTEP QMP,[1] the test/QA plan, and any SOPs to ensure that the necessary QA/QC procedures
are implemented. Battelle's Quality Assurance Manager may review evaluation methods,
compare test procedures to those specified in this test/QA plan, and review data acquisition and
handling procedures. Battelle's Quality Assurance Manager will prepare a TSA report, and the
findings must be addressed either by modifications of test procedures or by documentation in the
evaluation records and final report. At EPA's discretion, EPA QA staff may also conduct an
independent on-site TSA during the evaluation. The EPA TSA findings will be communicated to
evaluation staff at the time of the audit and documented in a TSA report. These findings must be
addressed as stated above.
C1.2 Performance Evaluation Audits
No performance evaluation audit will be performed for biological agents and surrogates,
as quantitative standards for these biological materials do not exist. The confirmation procedure,
controls, blanks, outlier test, and method validation efforts will be the basis of support for
biological evaluation results. Calibration of instruments used for measuring temperature,
humidity, pressure, and flow velocity will be monitored according to the processes and schedule
identified in Table 6 and documented. As a performance check of the spraying system, a
gravimetric assessment of the total amount of decontaminant deposited on each coupon and
collected in the run-off will be performed (see Appendix A).
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C1.3 Data Quality Audit
The Battelle Quality Assurance Manager will audit at least 10 percent of the evaluation
data. The Quality Assurance Manager will trace the data from initial acquisition, through
reduction and statistical comparisons, and to final reporting. All data analysis calculations will
be checked.
C2 REPORTS TO MANAGEMENT
Each assessment and audit will be documented in accordance with Section 9.5 in the
TTEP QMP[1] Assessment reports will include the following:
• Identification of any adverse findings or potential problems
• Space for response to adverse findings or potential problems
• Possible recommendations for resolving problems
• Citation of any noteworthy practices that may be of use to others
• Confirmation that solutions have been implemented and are effective.
During the course of any assessment or audit, Battelle's Quality Assurance Manager will identify
to the technical staff performing experimental activities any immediate corrective action that
should be taken. If serious quality problems exist, Battelle's Quality Assurance Manager will
contact the TTEP Manager to request a stop work order. Once the assessment report has been
prepared, the Building Decontamination Technology Area Leader or Task Order Leader will
ensure that a response is provided for each adverse finding or potential problem and will
implement any necessary follow-up corrective action. Battelle's Quality Assurance Manager will
ensure that follow-up corrective action has been taken.
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D DATA VALIDATION AND USABILITY
Dl DATA REVIEW, VALIDATION, AND VERIFICATION REQUIREMENTS
Records generated during the evaluation will receive a QC/technical review before being
used to evaluate or report results. This review will be performed by a Battelle technical staff
member other than the person who originally generated the record. Evaluation staff will be
consulted as needed to clarify any issues about the data records. The review will be documented
by the person performing it by adding his/her initials and date to a hard copy of the record being
reviewed. This hard copy will then be returned to the Battelle staff member who generated or
will be storing the record.
D2 VALIDATION AND VERIFICATION METHODS
To ensure that data generated meet the goals of the evaluation, a number of data
validation procedures will be performed. Section C of this test/QA plan provides a description of
the validation safeguards employed for evaluations. Data validation efforts include completing
QC activities and performing TSA audits. An audit of data quality will be conducted by the
Battelle Quality Assurance Manager to ensure that data review, verification, and validation
procedures were completed and to assure the overall quality of the data.
Data verification is conducted as part of data review. Handwritten data will be visually
inspected to ensure that all entries were properly recorded or transcribed and that any erroneous
entries were properly noted (i.e., single line through the entry, with an error code and the initials
of the recorder and date of entry). All calculations used to transform the data will be reviewed to
ensure their accuracy and appropriateness. Calculations performed manually will be reviewed
and repeated using a handheld calculator or commercial software (e.g., Excel). Calculations
performed using standard commercial software will be reviewed by inspecting the equations
used for the calculations and verifying selected calculations by handheld calculator.
D3 RECONCILIATION WITH DATA QUALITY OBJECTIVES
The data comparisons described in Section B2.2.5 will be conducted separately for each
sporicidal decontaminant undergoing evaluation. Separate evaluation reports will then be
prepared, each addressing one sporicidal decontaminant. Each evaluation report will present the
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test data, as well as the results of the evaluation of those data. The evaluation reports will briefly
describe the TTEP program and will present the procedures used in the evaluation. These
sections will be common to each report. The results of the technology evaluation will then be
stated quantitatively. The preparation of draft evaluation reports, review of reports by vendors
and others, revision of reports, final approval, and distribution of the reports will be conducted as
stated in the program QMP.[1]
Data obtained will be assessed by comparing them with the data quality objectives
contained in Section A7. In the case of the data collected for calculating efficacy, the data quality
objective will not be met if the positive controls (spiked, not decontaminated) fail to yield
extracts that contain viable organisms. In cases where no detectable growth occurs (such as when
decontamination reagent contacts the spiked controls), the test must be repeated.
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E REFERENCES
1. Battelle, Quality Management Plan (QMP)for the Technology Testing and Evaluation
Program (TTEP). 2005.
2. Grubbs, F., "Procedures for detecting outlying observations in samples." Technometrics,
1969. 11(1): p. 1-21.
3. Biosafety in Microbiological andBiomedical Laboratories. 1999, Centers for Disease
Control and Prevention and National Institute of Health: Washington, D.C.
4. Raber, E., Jin, A., Noonan, K., McGuire, R., and Kirvel, R.D., "Decontamination Issues
for Chemical and Biological Warfare Agents: How Clean is Clean Enough?" Int. J.
Environ. Health Res., 2001(1 l):p. 128-148.
5. Battelle, Decontamination Technology Testing and Evaluation: Task 1: Technology
Identification and Selection, prepared for the U.S. EPA under Task Order 1113 by
Battelle. 2004.
6. Battelle, Battelle MREF SOP Number: MREF. X-074, Standard Operating Procedure
(SOP) for the Production of Bacillus anthracis Spores: West Jefferson, Ohio.
7. Battelle, Battelle MREF SOP Number MREF.X-093, Standard Operating Procedure for
the Production o/"Bacillus anthracis Spores in a Small Per mentor.
8. Battelle, FSP Annex 5 to Appendix B, Guidelines for Safe Handling and Storage of
Etiologic Agents at the MREF. Columbus, Ohio.
9. Battelle, Battelle MREF Facility Safety Plan (FSP) Annex 6 to Appendix B of the FSP,
"Guidelines for the Control of Etiologic Agents in the MREF Biofacility."
10. Battelle, FSP Annex 7 to Appendix B, "Guidelines for Disinfection/Decontamination of
Etiological Agents at the MREF Biofacilities." West Jefferson, Ohio.
11. Battelle, Battelle MREF Facility Safety Plan Annex 12 to Appendix B, "Guidelines for the
Use of Class II and Class III Biological Safety Cabinets in the MREF Biofacility."
12. Battelle, Facility Safety Plan (FSP) for the Medical Research and Evaluation Facility
(MREF) Biofacility.
13. Battelle, Battelle MREF SOP Number: MREF. X-112, Standard Operating Procedure
(SOP) for Interpreting and Calculating Enumeration Data for Biological
Decontamination Testing. West Jefferson, Ohio.
14. Battelle, Battelle MREF SOP Number: MREF. X-054, Standard Operating Procedure
(SOP) for the Enumeration ofBL-2 andBL-3 Bacterial Samples via the Spread Plate
Technique. West Jefferson, Ohio.
15. ASTM, Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents.
ASTMInternational Designation E 1054-02. ASTM International Designation E, 2002.
1054-02.
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APPENDIX
DETAILED DESCRIPTION OF THE DECONTAMINATION SPRAYING SYSTEM AND
PERFORMANCE PARAMETER EVALUATION
Liquid Decontamination Spraying System Developed at the Battelle Medical Research and
Evaluation Facility
General Description:
The liquid decontamination spraying system enables the user to precisely control time of
spray, air pressure, product deposition, and spray distance. Moreover, the design of this sprayer
enables the user to easily adjust the setting(s) for one or all of these parameters to accommodate
the physical properties of the liquid/foam product being tested. Such precise control of these
parameters minimizes the potential for human error associated with spraying, enables control of
product deposition, and minimizes sample-to-sample variability. This spray methodology may
enhance the comparability of data obtained in different laboratories that are each following the
same testing protocol.
Specific Description Controlled Parameters
The main features/components of this spraying system include
• Aluminum and stainless steel construction
• Adjustable coupon holder for variable spray distance
• Pneumatic spray nozzle
• Liquid reservoir for decontaminants
• Needle valves (pressure and liquid flow)
• Pressure gauge
• Digital timer
• Pneumatic valve.
The aluminum and stainless steel construction was chosen to minimize the potential
damaging effects (e.g., oxidation, corrosion) of some of the decontaminants used during testing.
By sliding the base of the coupon holder using a tongue and groove design, the coupon holder
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easily adjusts for spray distances of 6 to 12 inches from the tip of the spray nozzle to the coupon
surface. The coupons are held in place on the coupon holder so that they are oriented vertically.
The pneumatic spray nozzle is positioned to spray the decontaminants in a conical pattern
perpendicular to the coupon surface. A pneumatic nozzle was chosen to achieve precise,
remotely activated on/off spray control. The tip of the nozzle can be easily removed and
exchanged with an alternate tip that provides a different spray pattern. The spray nozzle is
connected to two stainless steel lines—one line for the decontaminant tested and one line for the
pressurized air (supplied by a compressed air cylinder). The decontaminant is contained in a
stainless steel reservoir, where the decontaminant is fed to the spray nozzle by gravity. In the
nozzle, the liquid decontaminant is mixed with pressurized air fed from the second inlet line,
resulting in an atomized spray coming out of the nozzle. All lines, valves, and spray nozzles in
contact with decontaminant or PBS are flushed with sterile water followed by air between each
solution change.
The pressurized air feeding the nozzle is controlled in two ways. First, the air flow to the
nozzle (on/off) is controlled by the use of a pneumatic valve that is opened and closed using a
digital timer. The pressurized air line feeding the sprayer is connected to the pneumatic valve
that is normally in the "closed" position (zero air flow to the nozzle). This pneumatic valve is
connected to a digital timer that can be set to operate from 0.1 second to minutes. When the
digital timer is set for the appropriate spray time, the timer is activated, which opens the
pneumatic valve allowing air to flow to the spray nozzle. Once the timer counts down from the
set time to zero, the timer stops, thereby returning the pneumatic valve to the "closed" position.
The second control feature is the adjustable needle valve that regulates the air pressure to the
spray nozzle. A pressure gauge between the needle valve and the nozzle enables monitoring of
the air pressure (in psi) that is distributed to the spray nozzle. By turning the needle valve, the
pressure to the nozzle can be adjusted without the need to adjust air pressure at the air source.
Figure A-l is a flow diagram of the spray system. Figure A-2 shows photographs of side and top
views of the spray system set-up inside the compact glove box.
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Use of the Liquid Decontamination Spraying System for Assessing Retention of
Liquid/Foam Decontaminant in Test Material and Screening Sterilant and Disinfectant
Formulation for Sporicidal Activity
The retention of a spray-applied liquid formulation on a porous or non-porous test
material can be determined by performing a "spray and weigh" test using a minimum of one
porous and one non-porous material. For this test, the combined weight (grams) of the target test
coupon and conical vial beneath the coupon will be recorded prior to and after spraying. The
difference in weight (grams) between the before and after spraying represents the mass of the
liquid spray deposited on the surface and the collected run-off in the conical vial. Five periods of
spraying will be evaluated: 1, 5, 10, 15, and 20 seconds (N = 4/time period). The time periods
are controlled automatically by the spraying system, eliminating the element of human error.
The distance of the spray from the nozzle to the coupon surface will be 12 inches. The coupons
will be suspended vertically, and the spray stream will be 90 degrees to the surface of the
coupons. Eight replicate samples will be sprayed during each time period. The average and
standard deviation of the deposited spray and run-off during each period will be determined and
plotted on a graph. The correlation coefficient (R2) will also be calculated for the total recovery.
On each testing day, a performance check of the spraying system will be performed for each
decontaminant. This performance evaluation will consist of a spray and weigh (described above)
for a specified time period (e.g., 10 seconds) at a distance of 12 inches. The total decontaminant
mass from this spray will be plotted against the spray and weigh profile for the specific
decontaminant determined above. If the total mass from this performance test is within ±10% of
the previously determined spray and weigh profile, the results will be considered acceptable. If
the total mass from this performance test is >10% of the pre-determined spray and weigh profile,
the results will be considered unacceptable. If unacceptable, the spraying system will be
adjusted and the spray and weigh test repeated. Once acceptable results are obtained, the
decontamination testing will be initiated.
The sporicidal effects of spray-applied liquid/foam technologies such as sterilants or
disinfectants can be rapidly screened using the liquid decontamination spray system as follows:
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1. Apply 1 x lO8^. anthracis Ames spores in aqueous suspension to each of eight
glass coupons.
2. Allow to stand overnight to dry.
3. Apply liquid/foam formulation to four coupons (retain four as positive controls)
using technology supplied by or purchased from the vendor. The automated spray
apparatus will be used to apply the liquid/foam.
4. Using extraction and culture techniques described in the test/QA plan for
verifying hydrogen peroxide vapor technologies,[1] quantitate viable and
extractable spores.
5. Express results as log kill calculated using Equation 5 in the test/QA plan for
evaluating sporicidal spray decontaminants.
For each spray time (1, 5, 10, 15, and 20 seconds) and coupon material, data will be expressed as
mean (±SD) total mass sprayed decontaminant and summarized in the final report. The
correlation coefficient for total recovery as a function of the five time periods will also be
reported. For each technology evaluated, the calculated decontamination efficacy will be
reported with respect to the spray time, total mass deposited (per spray & weigh), and contact
time.
At the discretion of the TOPO and pending time/resource availability, additional "spray &
weigh" analyses will be performed on selected technologies and coupon test materials. This
analysis will differentiate between the mass of sprayed technology deposited on the coupon from
the mass that is collected as run-off. For each coupon test material and technology evaluated,
data will be expressed as mean (±SD) total mass sprayed decontaminant deposited on the coupon
and mean (±SD) total mass sprayed decontaminant collected as run-off.
References
1. Test/Quality Assurance Plan for Verification of Hydrogen Peroxide Vapor Technologies for
Decontaminating Indoor Surfaces Contaminated with Biological or Chemical Agents, July
21, 2003. Prepared and submitted to EPA as called for in Contract #GS-23F-0011L/BPA
2C-R903-NBLX/SIN 871-4/Order 1103.
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Drain Liquid
t
Display air pressure (psi)
to nozzle
Digital timer activated; sprayer "on";
pneumatic valve open
Digital timer not activated; sprayer "off";
pneumatic valve closed
Figure A-l. Flow Diagram of Spray System
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Figure A-2. Sprayer Set-up Inside the Compact Glove Box
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