United States
Environmental Protection
Agency
EPA 600/R-14/399 I October 2014 I www.epa.gov/research
Compatibility of Material and
Electronic Equipment with Ethylene
Oxide Fumigation
ASSESSMENT AND EVALUATION REPORT
i
i
PASSED
PASSED
Office of Research and Development
National Homeland Security Research Center
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EPA 600-R-14-399
October 2014
Compatibility of Material and Electronic Equipment
with Ethylene Oxide Fumigation
Assessment and Evaluation Report
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and directed this investigation through EP-C-09-
027 WAs 4-58 and 5-58 with ARCADIS U.S., Inc. This report has been peer and administratively
reviewed and has been approved for publication as an Environmental Protection Agency document. It
does not necessarily reflect the views of the Environmental Protection Agency. No official endorsement
should be inferred. This report includes photographs of commercially available products. The
photographs are included for purposes of illustration only and are not intended to imply that EPA
approves or endorses the product or its manufacturer. Environmental Protection Agency does not
endorse the purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to:
Shannon Serre
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109. T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-3817
Fax:919-541-0496
E-mail: serre.shannon@epa.gov
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Acknowledgments
This effort was directed by the principal investigator from the Office of Research and Development's
(ORD's) National Homeland Research Center (NHSRC), utilizing the support from the US Environmental
Protection Agency's (EPA's) Chemical, Biological, Radiological, and Nuclear (CBRN) Consequence
Management Advisory Division (CMAD) within the Office of Emergency Management (OEM). The
contributions of the entire team are acknowledged.
Project Team:
Shannon Serre (Principal Investigator)
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
R. Leroy Mickelsen
CBRN CMAD, OEM, Office of Solid Waste and Emergency Response, US EPA
Research Triangle Park, NC 27711
Jayson Griffin
CBRN CMAD, OEM, Office of Solid Waste and Emergency Response, US EPA
Research Triangle Park, NC 27711
This effort was completed under U.S. EPA contract #EP-C-09-027 with ARCADIS-US, Inc. The support
and efforts provided by ARCADIS-US, Inc. are gratefully acknowledged.
Ramona Sherman and Eletha Brady-Roberts (Quality Assurance)
NHSRC, ORD, US EPA
Cincinnati, OH 45220
Joan Bursey (Quality Assurance and Editorial Assistance)
NHSRC, ORD, US EPA
Research Triangle Park, NC 27711
The peer reviewers of this report are also acknowledged for their input to this product:
Joseph Wood, NHSRC, Office of Research and Development, US EPA
Michael Nalipinski, CBRN CMAD, OEM, Office of Solid Waste and Emergency Response, US EPA
Lawrence Kaelin, CBRN CMAD, OEM, Office of Solid Waste and Emergency Response, US EPA
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Table of Contents
Disclaimer ii
Acknowledgments iii
List of Tables viii
List of Acronyms and Abbreviations ix
Executive Summary xi
1 Project Description and Objectives 1
1.1 Purpose 1
1.2 Process 1
1.2.1 Overview of Compatibility Testing 2
1.2.1.1 Category 2 and 3 Materials Testing 2
1.2.1.2 Category 4 Materials Testing 2
1.3 Project Objectives 3
2 Materials and Methods 4
2.1 Subject Materials and Equipment 4
2.2 Laboratory Materials and Equipment 7
2.2.1 Ethylene Oxide Facility 7
2.2.2 RH/Temperature Measurement 9
2.2.3 Surface Roughness 11
2.2.4 Biological Indicators (Bis) 11
2.2.5 Spore Preparation 11
2.2.6 Inoculated Rubber Coupons 11
2.3 Test Preparation 13
2.4 Sampling Strategy 14
2.4.1 Frequency of Sampling/Monitoring Events 14
2.4.2 Testing Approach 15
2.5 Measurements 16
2.6 Sampling Procedures 16
2.6.1 Visual Inspection (Category 2-4) 16
2.6.2 Functionality Testing (Category 2-4) 17
2.6.2.1 Smoke Detectors 17
2.6.2.2 CO Detectors 17
2.6.2.3 Personal Digital Assistant (PDA) -Palm Pixi Plus 17
iv
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2.6.2.4 Fax/Phone/Copier Machine 17
2.6.2.5 Cell Phone 17
2.6.2.6 Data CD 18
2.6.2.7 Data DVD 18
2.6.2.8 USB Flash Drive 18
2.6.2.9 SD Memory Card 18
2.6.2.10 PCMD Test Configuration 18
2.6.3 Microbiology Methods 19
2.6.3.1 Coupon Spore Enumeration 19
2.6.3.2 Bl Analysis 19
3 Results 21
3.1 Category 2 Materials 21
3.1.1 Fumigation conditions 21
3.1.2 Visual Inspection 21
3.1.3 Functionality Testing 23
3.1.3.1 Smoke and Smoke/CO Detector Results 23
3.1.3.2 Surface Roughness Testing Results 24
3.2 Category 3 Materials 27
3.2.1 Visual Inspection 27
3.2.2 Functionality Testing 29
3.3 Category 4 Materials 30
3.3.1 Fumigation Conditions 30
3.3.2 Testing Difficulties 31
3.3.3 Visual Inspection 31
3.3.4 Functionality Testing 33
3.3.4.1 BIT 33
3.3.4.2 PC-Doctor® Functionality Testing Results 33
3.4 Fumigation Effectiveness 37
4 Quality Assurance 38
4.1 Sampling, Monitoring, and Analysis Equipment Calibration 38
4.2 Data Quality 38
4.3 QA/QC Checks 38
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4.4 Acceptance Criteria for Critical Measurements 40
4.5 Data Quality Audits 42
4.6 QA/QC Reporting 42
5 Conclusions 43
6 References 44
Appendix A: Andersen EOGas System Characterization 46
Appendix A 47
VI
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List of Figures
Figure 2-1. Test Configuration and Duty Cycles for BurnlnTest® Software 7
Figure 2-2. EOGas 333 Cabinet 8
Figure 2-3. EtO Concentration over Time in a Standard Cycle 9
Figure 2-4. MadgeTech RHTemp Sensor 10
Figure 2-5. 18-mm Stub Stage in Container 12
Figure 2-6. Location of MadgeTech device and rubber coupons within the computers (between
the CD drives and hard drives) 12
Figure 2-7. Location of Bis within the computers (inside the bottom panel) 13
Figure 3-1. Laser Printer Paper exposed to EtO (left and center) and not exposed (right) 22
Figure 3-2. Copper exposed to EtO (2Cu-01 and 2Cu-02 on left) and not exposed (2Cu-03 on
right) 22
Figure 3-3. Test Photograph 23
Figure 3-4. Average Surface Roughness Measurements of Test Coupons 24
Figure 3-5. Type 304 Stainless Steel Roughness Measurements 25
Figure 3-6. Type 316 Stainless Steel Roughness Measurements 26
Figure 3-7. Aluminum Roughness Measurements 26
Figure 3-8. Copper Roughness Measurements 27
Figure 3-9. Photo of digital photographs taken over a year after exposure. There was no
discernable difference between the control (2PH-03 on right) and test samples
(2PH-01 and-02) 28
Figure 3-10. Exposed (left) and unexposed (right) CD 10 months later 29
Figure 3-11. Inside SS04 (pre-fumigation) 31
Figure 3-12. Inside SS04 (two months post-fumigation) 32
Figure 3-13. SS04 back panel (pre-fumigation) 32
Figure 3-14. SS04 Back Panel (two months post-fumigation) 33
Figure 3-15. Test Condition 1 PCMD Scores Compared to Controls 34
Figure 3-16. Test Condition 2 PCMD Scores Compared to Controls 34
Figure 3-17. Test Condition 3 PCMD Scores Compared to Controls 35
Figure 3-18. Test Condition 4 PCMD Scores Compared to Controls 35
Figure 3-19. Average PC Doctor Scores over Testing Period 36
VII
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List of Tables
Table 2-1. Category 2 Materials 4
Table 2-2. Category 3 Materials and Equipment 5
Table 2-3. Category 4 (Personal Computer) Specifications 6
Table 2-4. Madgetech (RH and Temperature) Sensor Specifications 10
Table 2-5. Individual Bag Preparation 13
Table 2-6. Monitoring Methods for EtO fumigation 15
Table 2-7. Test Matrix T01 (Category 2 and 3) 15
Table 2-8. Test Matrix T02 (Category 4) 15
Table 2-9. Critical and Non-Critical Measurements 16
Table 3-1. Contents and Conditions of Category 2 and 3 EtO exposures 21
Table 3-2. Hexadecimal Color Comparison 23
Table 3-3. Smoke Detector Functionality Testing 24
Table 3-4. Average Roughness for Test Sets 25
Table 3-5. Category 3 Functionality Testing Results 29
Table 3-6. Final Test Matrix 30
Table 3-7. p-Values between PCMD Scores of all PC Subsets* 37
Table 3-8. CFU Counts and Log Reduction for All Biological Tests 37
Table 4-1. Sampling and Monitoring Equipment Calibration Frequency 38
Table 4-2. Quality Assurance (QA)/Quality Control (QC) Sample Acceptance Criteria 39
Table 4-3. Data Quality Indicators 40
VIM
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List of Acronyms and Abbreviations
AC
ACPI
Bl
BIT
CBRN
CD
CE
CFC
CPU
CMAD
CO
DPG
DQI
DQO
DVD
EPA
ESD
EtO
FM
HCFC
HSPD
HSRP
IS
LCD
LED
MDI
MP
NHSRC
OEM
ORD
OSHA
PBST
PC
PCMD
PDA
QA
QAPP
Alternating current
Advanced configuration and power interface
Biological indicator
BurnlnTest®
Chemical biological radiological and nuclear
Compact disc
European Commission
Chlorofluorocarbon
Colony Forming Units(s)
Consequence Management Advisory Division
Carbon monoxide
Dugway Proving Ground
Data Quality Indicator
Data Quality Objective
Digital versatile disc
U. S. Environmental Protection Agency
Electrostatic discharge
Ethylene oxide
Factory Mutual Research Corporation
Hydrochlorofluorocarbon
Homeland Security Presidential Directive
Homeland Security Research Program
Intrinsically safe
Liquid crystal display
Light-emitting diode
Metered Dose Inhaler
Megapixel
National Homeland Security Research Center
Office of Emergency Management
Office of Research and Development
Occupational Safety and Health Administration
Phosphate Buffered Saline with Tween® 20
Personal computer
PC Doctor
Personal Digital Assistant
Quality Assurance
Quality Assurance Project Plan
IX
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QC Quality Control
RH Relative humidity
RTD Resistance Temperature Detector
RTP Research Triangle Park
SD Secure Digital
SVGA Super video graphic array
TSA Tryptic Soy Agar
USB Universal Serial Bus
WACOR Work Assignment Contracting Officer Representative
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Executive Summary
The objective of the research presented in this report was to determine the material compatibility of
electronics and other materials to exposure with ethylene oxide gas (EtO) fumigation, a commercially
available fumigation technology used widely in the medical industry for sterilization and considered for
use to inactivate Bacillus anthracis spores. A secondary objective was to test the efficacy of EtO against a
Bacillus anthracis surrogate. The current study was designed to provide direct information on the impact
of EtO gas on sensitive electronic components and materials that can be viewed as surrogates for
sensitive materials and high-end equipment (e.g., medical devices and airport scanners) that use similar
types of components.
Bacillus atrophaeus, on commercial biological indicators and inoculated onto 18 mm rubber coupons, was
used as a surrogate for the lethal biological agent, Bacillus anthracis. Manufacturer-suggested
operational conditions were targeted for the study. The manufacturer-suggested fumigation method is an
11 g EtO cartridge activated within a specialized selectively permeable bag. The bag also contains
Humidichips®, which contain water to humidify the environment inside the bag, as the ventilation cabinet
containing the bag heats to the manufacturer-suggested temperature of 50 °C. As EtO is released from
the cartridge (EtO is very volatile), the EtO slowly permeates through the bag wall into the ventilation
cabinet over an 18-hour cycle. The cabinet removes the EtO through an abater, and the bag can be
retrieved safely at the conclusion of the 18-hour cycle. The study also investigated the use of a higher
EtO concentration with the use of an 18 g cartridge.
The study found that EtO was sporicidal with greater than 6 log reduction under all tested conditions.
Three different categories of materials were tested as surrogates for sensitive materials and high-end
equipment. Category 1 materials (household building materials) which were previously tested with
chlorine dioxide, hydrogen peroxide, and methyl bromide, were not included in this study. Because of size
limitations, EtO would not be considered as an option for decontamination of Category 1 materials.
Category 2 materials included construction materials of low surface area but high functionality within
a building. These construction materials included aluminum, copper, stainless steel, smoke detectors
with and without carbon monoxide (CO) alarms, laser-printed paper, InkJet-colored paper, and color
photographs.
Category 3 materials and equipment included pieces of small personal electronic equipment:
Personal Digital Assistants (PDAs), cell phones, fax/phone/copier machines, compact discs (CDs),
digital versatile discs (DVDs), Universal Serial Bus (USB) flash drives, and Secure Digital (SD)
memory cards.
Category 4 materials included desktop computers and monitors.
The effects of EtO on all tested materials was minimal, with no recorded visual impacts on any of the
materials. All fumigated electronic components maintained the same functionality as the control
equipment.
XI
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1 Project Description and Objectives
This project supports the mission of the U.S. Environmental Protection Agency's (EPA's) Office of
Research and Development (ORD), Homeland Security Research Program (HSRP) by providing
information pertinent to the decontamination of contaminated equipment and materials where the
contamination results from an act of terrorism. Under Homeland Security Presidential Directives (HSPDs)-
5, 7, 8, and 10, the EPA, in a coordinated effort with other federal agencies, is responsible for "developing
strategies, guidelines, and plans for decontamination of equipment, and facilities" to mitigate the risks of
contamination following a biological agent contamination incident.
EPA's National Homeland Security Research Center (NHSRC) aims to help EPA address the mission of
the HSRP by providing expertise and products that can be widely used to prevent, prepare for, and
recover from public health and environmental emergencies arising from terrorist threats and incidents.
One of the missions of NHSRC is to provide expertise and guidance on the selection and implementation
of decontamination methods and to provide the scientific basis for a significant reduction in the time, cost,
and complexity of decontamination events. Fumigation with ethylene oxide (EtO) for the decontamination
of certain materials and equipment contaminated with anthrax spores has been suggested as a safe
alternative to more harsh fumigants such as chlorine dioxide or hydrogen peroxide. Unlike hydrogen
peroxide and chlorine dioxide, EtO is not an oxidizing agent and kills organisms through alkylation.
Information on the compatibility of materials and equipment with typical EtO fumigation conditions
effective for anthrax spores has not been determined in a systematic, reproducible way. Future guidance
on selection and operation of decontamination technologies is dependent upon such information.
Data on the impact of fumigation with EtO on materials/equipment under sporicidal conditions relevant to
facility decontamination are needed to define the guidance further with respect to the selection and use of
fumigant technologies for small scale decontamination operations.
1.1 Purpose
The purpose of this study was to determine the impact of EtO fumigation on sensitive electronic
components and materials that can be viewed as surrogates for sensitive materials and high-end
equipment (e.g., medical devices and airport scanners) that use similar types of components.
Decontamination conditions used were those achievable by commercial equipment. This effort
investigated the impact on the physical appearance, properties, and functionality of the materials and
equipment, as appropriate.
1.2 Process
Category 2, 3, and 4 materials and equipment were tested before exposure to EtO. The equipment was
exposed to EtO according to the finalized test matrix. The diagnostic protocols (as outlined in Section 2.8)
were repeated on all materials and equipment after fumigation and monthly for a period of at least nine
months. The results of these testing protocols were used to evaluate the impact of fumigation on the
materials tested. Fumigations were conducted in High Bay Room H-222 and compatibility testing was
performed in Room E288 on EPA's Research Triangle Park, North Carolina, campus.
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1.2.1 Overview of Compatibility Testing
Compatibility testing was performed to monitor the short- and long-term effects of EtO fumigation on
materials and electronic equipment. Category 1 materials (household building materials) which were
previously tested with chlorine dioxide, hydrogen peroxide, and methyl bromide, were not included in this
study. EtO would be considered an option for decontamination of valuable items that might be damaged
by other fumigants and would not be considered as an option for decontamination of Category 1
materials.
1.2.1.1 Category 2 and 3 Materials Testing
Category 2 includes construction materials of low surface area but high functionality within a building.
These materials include aluminum, copper, stainless steel, smoke detectors with and without carbon
monoxide (CO) alarms, color laser-printed paper, color InkJet-printed paper, and color photographs. The
objective for this category of materials was to assess the visual and/or functionality (as appropriate)
impact of the fumigation process on the materials. Building materials were tested to better compare to
testing previously conducted with other fumigants [1, 2]. The impact was analyzed using visual
inspections under each set of fumigation conditions and functionalities, where appropriate. The visual
inspections were directed towards possible locations suspected of corrosion and possible material
defects due to the fumigation process. Printed documents and pictures were inspected for possible
alteration of their content. Inspection occurred at regular intervals over a ten-month period, with the
material stored under ambient laboratory conditions throughout that time period. The visual inspections
were documented in writing and by digital photography for each material before and after each
fumigation.
Category 3 materials and equipment include small pieces of personal electronic equipment. These pieces
of equipment included Personal Digital Assistants (PDAs), cell phones, fax/phone/copier machines,
compact discs (CDs), digital versatile discs (DVDs), Universal Serial Bus (USB) flash drives, and Secure
Digital (SD) memory cards. The objective for this category was to determine visual and functionality
impacts on the equipment as a function of time post-fumigation. The assessment of the impact was visual
inspection for aesthetic effects and evaluation of functionality pre-/post-fumigation. Inspection occurred at
regular intervals over a ten-month period, with the equipment stored at ambient laboratory conditions
throughout that time period. Visual inspections of the equipment were documented in writing and by
digital photographs. Further, the functionality of each piece of equipment was assessed comparatively
with similar equipment that was not subjected to the fumigant exposure.
1.2.1.2 Category 4 Materials Testing
Category 4 equipment included desktop computers and monitors. The objective for this category of
equipment (and materials) was to assess the impact of the fumigation conditions using visual inspection,
functionality testing, and a software personal computer (PC) diagnostic tool. The objective was to identify
components and specific parts of components that may be susceptible to corrosion due to the fumigation
process. This information can be used to make informed decisions about the compatibility of other
equipment that may have similar components (at least similar in operation) and can at least reduce
further testing or uncertainty in the field application. The equipment and materials included in this
category are as follows:
• Dell OptiPlex 790 Desktop Computer (see Table 2-3 for specifications)
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• Dell 19-inch flat panel monitor
• USB keyboard and mouse
• Computer and monitor power cords and connecting analog super video graphic array (SVGA)
cable
• USB flash drives in all USB ports
• Network loopback adapter
• Serial loopback adapter.
1.3 Project Objectives
The primary objective of the work was to assess the impact of sporicidal fumigation with EtO on materials
and electronic equipment. Specifically, the fumigation conditions of interest are those provided by the
commercially used Andersen EOGas 333 system. Visual appearance of all items was documented before
and after fumigation exposure. Some materials were not tested for functionality due to the multiplicity of
potential uses. Since EtO is explosive under the target conditions, the state of operation of all electrical
equipment was in the off state and de-energized for all test conditions involving EtO. Any electronic
equipment with capacitors would need to have the capacitors discharged prior to fumigation with EtO.
An additional primary objective in this study was to obtain an indication of the potential impact that the
local conditions inside the EtO bag may have on the effectiveness of the fumigation process to inactivate
anthrax spores potentially located within equipment. For this purpose, inoculated rubber stubs were used.
Under a previous study (Appendix A), rubber was determined to be the most resistant to EtO fumigation
due to apparent adsorption and desorption of the fumigant.
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2 Materials and Methods
2.1 Subject Materials and Equipment
Three categories of material and equipment were tested under the different fumigation conditions
discussed in detail below; the categories can be separated based upon the conditions of testing and
analysis performed to assess the impacts. Category 1 materials (building construction materials
comprising high surface-area within a volume) were not included in this effort. The Category 2, 3, and 4
materials and equipment tested are listed in Tables 2-1, 2-2, and 2-3, respectively.
Table 2-1. Category 2 Materials
Material
Type 3003 Aluminum
Alloy 101 Copper
Type 31 6 Stainless
Steel
Type 304 Stainless
Steel
Smoke Detector
Smoke Detector and
CO alarm
Laser-printed paper
InkJet-colored paper
Color Photograph
Description
Textured 0.0625-inch thick sheet
0.064-inch thick polished electrical
grade, 99.99% pure
0.0625-inch thick 2B finish
0.0625-inch thick #3 finish
Battery-powered ionization sensor
Electrochemical CO sensor,
Photoelectric sensing technology
Stack of 1 5 pages
Stack of 1 5 color pages
4-inch by 6-inch Kodak processing
Supplier/
Manufacturer
McMaster Carr
(Elmhurst, IL)
McMaster Carr
(Elmhurst, IL)
McMaster Carr
(Elmhurst, IL)
McMaster Carr
(Elmhurst, IL)
First Alert
(Aurora, IL)
First Alert
(Aurora, IL)
RTO-E340-PS
HP Color
LaserJet
(Palo Alto, CA)
HP DeskJet
932C
Palo Alto (CA)
Walgreens
(Springfield, IL)
Part
Number
88685K12
3350K19
9090K1 1
9085K1 1
SA304
SCO5CN
NA
NA
NA
Coupon/ Sample
Size
2-inch by 2-inch,
three pieces
2-inch by 2-inch,
three pieces
2-inch by 2-inch,
three pieces
2-inch by 2-inch,
three pieces
one piece
one piece
8-1/2-inch by 11 -inch
844-inch by 11 -inch
4-inch by 6-inch,
three pieces
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Table 2-2. Category 3 Materials and Equipment
Equipment
PDA
Cell Phone
Fax/Phone/Copier
Machine
Data CD
Data DVD
USB Flash Drive
SD Memory Card
Description
Handheld
Thin Flip Phone, 1.3 MP camera. 2.4"
liquid crystal display (LCD)
Plain-paper fax and copier with ten-
page auto document feeder and up to
50-sheet paper capacity. 512KB
memory stores up to 25 pages for out-
of-paper fax reception
Software CD
Standard 21 331 DVD Video
4 GB Flash Drive
4 GB SD Card
Manufacturer
Palm
(Sunnyvale,
CA)
Samsung
(Ridgefield
Park, NJ)
Brother
(Bartlett, TN)
Snap!
(Johns Creek,
GA)
Warner
Brothers
(Los Angeles,
CA)
Sandisk
(Milpitas, CA)
Kingston
(Fountain
Valley, CA)
Model Number
Pixi Plus
M400
Fax 575
01-0170-026-
000
Harry Potter and
the Sorcerer's
Stone DVD
SDCZ36-004G-
A11
SD4/4GB/SKLW
9643151
Sample Size
one piece
one piece
one piece
one piece
one piece
one piece
one piece
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Table 2-3. Category 4 (Personal Computer) Specifications
Base Unit:
Processor:
Memory:
Keyboard:
Monitor:
Video C and:
Hard Drive:
Operating System:
Operating System:
Mouse:
NIC:
CD-ROM or DVD-ROM Drive:
CO-ROM or DVD-ROM Drive:
CD-ROM or DVD-ROM Drive:
Sound Card:
Speakers:
Cable:
Cable:
Cable:
Cable:
Documentation Diskette:
Documentation Diskette:
Factory Installed Software:
Feature
Service:
Service:
QptiPlex790 Minitower Base.90 PSU (225-0782)
Opti 790. CORE iS 2500 Processor (3.3GHz. 6M) (317-6644)
4GB.Non-ECC, 1333MHz DDR3 2X2GB,Dell OptiPlex 990 (317-6987)
Dell USB Entry Keyboard, No Hot Keys, English, Opti Rex (331-2024)
Dell 19 inch Flat Panel Display.E191 t.Black,OptiPlex.Precision and Latitude (320-1762)
Integrated Video, HD Graphics 2000, Dell Optiplex 790 $320-2520}
250GB SATA G.OGb/s and 8MB Data Burst Cache.Dell OptiPlex (342-2453)
Windows 7 Professional, No Media, 64-bit. Optiplex, English (421-5606)
Windows 7 Label, Optiplex, Fixed Precision, Vostro Desktop (33.0-6228)
Dell MS111 USB Optical Mouse.OptiPlex and Fixed Precision {330-9458}
Intel Standard Manageability, Dell OptiPlex 790 (331 -2680)
16X DVD+J-RW SATA.Data Only, Dell OptiPlex 790 Desktop or Minitower.Black (318-0623)
Roxio Creator Starter.Media. Dell OptiPlex. Latitude and Precision Workstation (421-4540)
Cyberlink Power DVD 9.5.1, Media, Dell OptiPlex. Latitude and Precision Workstation (421-5095)
Heat Sink, Performance. Dell OptiPlex 790 Minitower (331-2023)
Internal Speaker, OPtiiplex 990 (31B-0319)
Enable Low Power Mode for EUP Compliance. Dell OptiPlex (330-7422)
Dell Data Protection Access.OptiPlex (421-5078)
OptiPlex 790 Minitower Up to 90 Percent Efficient Power Supply (318-0875)
Regulatory Label, Dell OptiPlex 790 Minitower (331-2689)
Power Cofd,125V,2M,G13,Dell OptiPtex (330-1711)
Documentation. English, Dell OptiPlex (331-2030)
Energy Star 5.0 Category C (209kWh TEC), EPEAT Gold. Dell E SMART Settings, Dell OptiPlex
790(331-2019)
No Resource DVD for Dell Optiplex, Latitude, Precision (313-3673)
Basic Hardware Service: Next Business Day Limited Onsite Service After Remote Diagnosis 2
Year Extended (938-7662)
Basic Hardware Service: Next Business Day Limited Onsite Service After Remote Diagnosis
Initial Year (951 -7510)
The Category 4 items, specifically the computers and monitors, were treated differently than the items
included in the other categories. The computers and monitors were removed from their original
packaging, labeled with a designated sample number, set up according to protocol, and tested for
functionality. This equipment was transported to and from the EtO facility in anti-static and anti-corrosion
bags (Corrosion Intercept Technology, http://www.staticintercept.com/CI_product.htm), specifically
designed to protect equipment from exposure to potentially damaging electrostatic charge or corrosive
gases. Computers and monitors remained energized and operated over the course of at least nine
months to continually assess delayed effects due to the test conditions at which they were treated. All
operations were done on an Electrostatic Discharge mat. Three control computers were used in this
testing.
Computers were kept in operation simulating a five-day work week using BurnlnTest®(BIT®), (Version 5.3
Pro, Passmark Software Pty. Ltd., Sydney, Australia). For each of five sequential days, BIT® was
programmed to run as follows:
• 50 % load for eight hours (Figure 2-1)
16 hours in the ACPI standardized S3 state (standby mode)
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• Computer is shut down and rebooted.
Test configuration and duty cycles
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Figure 2-1. Test Configuration and Duty Cycles for BurnlnTest Software
After five days of this sequence, the computer was programmed to enter the advanced configuration and
power interface (ACPI) standardized S3 state for 40 hours. After these 40 hours elapse, the test ends
with the BIT® screen displayed. Probably due to an unidentified programming bug or operator error, some
computers would only reboot 1-5 times instead of 6 during a testing cycle.
2.2 Laboratory Materials and Equipment
2.2.7 Ethylene Oxide Facility
The EtO fumigation facility, located in the EPA's Research Triangle Park (RTP) facility in High Bay Room
H222, includes a heated and aerated cabinet (EOGas 333, Andersen Products, Inc., Haw River, NC,
USA) which is used as an isolation chamber for the sterilization system (Figure 2-2).
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Figure 2-2. EOGas 333 Cabinet
The EOGas 333 has the following features:
33 ft capacity
100 % EtO, no chlorofluorocarbons (CFCs) or hydrochlorofluorocarbons (HCFCs)
Sterilizes and aerates in the same cabinet
Power outage backup protection
Digital display with load tracking function
EPA-registered; certifications to international standards
Complies with current Occupational Safety and Health Administration (OSHA) regulations for
personnel exposure
Economical gas-disposal emissions-abatement equipment available
All sterilizers available for either 110-V 60-Hz or 220-V 50-Hz.
The efficacy of EtO is dependent on both temperature and humidity during exposure. The cabinet was
used to control the temperature at 50 °C during EtO exposure. Wetted sponges were included inside the
permeable bags to moderate the relative humidity (RH) during exposure at or above the manufacturer-
recommended humidity of 50-60 percent.
The permeable bags and wetted sponges were contained in premade kits (AN1006, Andersen Products,
Inc., Haw River, NC, USA), which included 22-inch by 36-inch bags and 11 g EtO cartridges. Cartridges
containing 18 g EtO were also used for this project.
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Items for sterilization were de-energized (if applicable) and placed inside permeable bags with a cartridge
of EtO (11 g and 18 g were used in this testing), a wetted sponge or humidichip, and a chemical indicator
(AN-1087, Andersen Products, Inc., Haw River, NC, USA). The bag was then vacuum-sealed.
Once the cabinet reached the target conditions, bags were loaded into the cabinet. The cartridge was
then activated, and the cabinet 18-hour cycle began. The temperature of the objects in the cabinet
remained below the cabinet air temperature for a period of time due to the thermal mass. Typical
exposure conditions are shown in Figure 2-3.
Conditions in EtO Cycle
250
-50
-200
200 400 600
Minutes elapsed
1000
1200
•Temperature (°C)
•Humidity (%RH)
•EtO(mg/L)
Figure 2-3. EtO Concentration over Time in a Standard Cycle
The chemical indicator (placed in each bag) changed color in proportion to the dose of sterilant and was a
visual confirmation that the cycle operated as expected.
The EtO cabinet was used with an abater (ion exchange resin), which removed EtO from the exhaust of
the cabinet before venting to the hood air handling system.
2.2.2 RH/Temperature Measurement
RH and temperature were monitored using a MadgeTech RHTemp Sensor (MadgeTech, Warner, NH)
(Figure 2-4). These sensors were calibrated before use. At the conclusion of each fumigation, the sensors
underwent post-test calibration. The data reduction procedure involved downloading the data from the
sensor by using a USB cable and the Madgetech software (MadgeTech, Warner, NH).
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KHTemplOOOlS
Figure 2-4. MadgeTech RHTemp Sensor
The RHTemplOOOIS is Factory Mutual Research Corporation (FM)-approved as intrinsically safe for
hazardous environments. This certification makes the device well-suited for EtO sterilization,
environmental studies, and numerous other hostile environment applications. The real-time clock ensures
that all the data are time- and date-stamped. One RHTempI OOIS sensor was placed inside the bag and
another was placed inside the computer chassis for each Category 4 test. One MadgeTech device was
placed inside the monitor bag. For each Category 2 and 3 test, one sensor was placed in each bag. Table
2-4 lists the specifications of the MadgeTech device.
Table 2-4. Madgetech (RH and Temperature) Sensor Specifications
Temperature sensor
Temperature range
Temperature resolution
Temperature calibrated accuracy
Humidity sensor
Humidity range
Humidity resolution
Humidity calibrated accuracy
Memory
Reading rate
battery life
Material
Dimensions
IP Rating
Operating environment
Required interface package
Approvals
Resistance temperature detector (RTD)
-20 °C to +80 °C (-4 °F to +176 °F)
0.01 °C
±0.5°C(0°Cto55°C)
Capacitive digital humidity sensor
0 to 100 % RH (non-condensing)
0.1 %RH
±3 % RH maximum; ±2.0 % RH typical at 25 °C
16,350 readings per channel
1 second to 1 reading every 24 hours
two years typical at 25 °C, 15 minute reading intervals
316 stainless steel
1 .0" x 2.0" dia. (25.4 mm x 50.8 mm)
IP30
-20 °C to +80 °C, 0 to 95 % RH (non-condensing)
IFC400
CE, Intrinsically Safe (IS) rated
10
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2.2.3 Surface Roughness
A surface roughness tester (SRG-4000, Phase II Machine and Tool, Inc., Upper Saddle River, NJ, USA)
uses a diamond stylus to measure average surface roughness to the nearest 0.001 micron. The stylus is
pushed over the material surface, and the resistance to this action is measured. An average surface
roughness is calculated from this measurement. Three surface roughness measurements were taken
before and after exposure for each replicate Category 2 material coupon and recorded in the testing form.
2.2.4 Biological Indicators (Bis)
Commercial Bacillus atrophaeus biological indicators (Bis) (Catalog No. 1-6100, Mesa Laboratories, Inc.,
Lakewood, CO, USA) were used for all EtO fumigations. These are the standard Bl used to measure EtO
sterilization cycles. These Bis are paper strips inoculated with approximately 106 Bacillus atrophaeus
spores, inside a Tyvek® pouch.
All Bis were maintained in their sterile Tyvek® envelopes until transferred to the NHSRC Biocontaminant
Laboratory (Biolab) for analysis.
2.2.5 Spore Preparation
The test organism for this work was a powdered spore preparation of Bacillus subtilis and silicon dioxide
particles. The preparation was obtained from the U.S. Army Dugway Proving Ground (DPG) Life Science
Division. The preparation procedure is reported in Brown et al. [3]. Briefly, after 80 - 90 percent
sporulation, the suspension was centrifuged to generate a preparation of approximately 20 percent solids.
A preparation resulting in a powdered matrix containing approximately 1011 viable spores per gram was
prepared by dry blending and jet milling the dried spores with fumed silica particles (Degussa, Frankfurt
am Main, Germany). The powdered preparation was loaded into metered dose inhalers (MDIs) by DPG
according to a proprietary protocol. Control checks for each MDI were included in the batches of coupons
contaminated with a single MDI.
2.2.6 Inoculated Rubber Coupons
Rubber coupons were inoculated and included in each Category 4 fumigation bag to evaluate the efficacy
of each individual cycle following the procedures listed below. Circles (18-mm diameter) were punched
from 1/16-inch thick sheets of silicone rubber (Part #5787T11, McMaster-Carr, Atlanta, GA) and fastened
to 18 mm aluminum stubs (P/N 16119, Ted Pella, Inc., Redding, CA) using double-sided, adhesive-
backed tape (P/N 16084-8, Ted Pella, Inc., Redding, CA). The coupons were sterilized priorto use via an
autoclave. Test coupons were inoculated with Bacillus subtilis at approximately 2 x 108 colony forming
units (CPU) by placing the surface of the coupons a precise distance from the MDI during actuation by
placing the coupons on custom-built stages (shown in Figure 2-5) contained in autoclaved glassware. The
container consisted of a Petri dish on the bottom and a crystallization dish on top.
A procedural blank went through the same transfer process as the positives, but the inoculum was not
applied. Three positive coupons on a separate stage were also inoculated but were not fumigated. Three
negative rubber coupons remained in the dish in which they were sterilized and were not inoculated. The
six-sample stage was placed inside the computer chassis with the Bis to help determine efficacy under
the local conditions within the PC chassis.
11
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Figure 2-5. 18-mm Stub Stage in Container
Inoculated material coupons and Bis were placed inside the computer chassis to provide an indication of
the effectiveness of the fumigation within each computer and are shown in Figures 2-6 and 2-7.
Figure 2-6. Location of MadgeTech device and rubber coupons within the computers (between
the CD drives and hard drives)
12
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Figure 2-7. Location of Bis within the computers (inside the bottom panel)
2.3 Test Preparation
The testing chamber was conditioned prior to release of EtO inside the bag as described in Section 2.2.
Individual bag loading was dependent on materials to be tested and the conditions to be tested, as
specified by the test matrix. Table 2-5 lists the items included in each type of EtO fumigation cycle.
Table 2-5. Individual Bag Preparation
Cycle
Type
Category
2
Category
3
Category
4
Category
4
Category
4
Test
Condition
1
1
1
2
3
EtO
11 g
cartridge
ng
cartridge
ng
cartridge
11 g
cartridge
i« a
cartridge
Humidification
Two
Humidichips®
Two
Humidichips®
Two
Humidichips®
Wetted sponge
(w/18 g water)
Two
Humidichips®
Biologicals
Bis
Bis
Bis, rubber
test
coupons
Bis, rubber
test
coupons
Bis, rubber
test
coupons
Monitoring
Equipment
Madgetech
Madgetech
Madgetech
(one in each
bag)
Madgetech
(one in each
bag)
Madgetech
(one in each
bag)
Materials/
Equipment
Category 2
materials (as
they fit)
Category 3
materials (as
they fit)
One PC in PC
bag. Monitor,
keyboard and
mouse in
separate
bag.*
One PC in PC
bag. Monitor,
keyboard and
mouse in
separate
bag.*
One PC in PC
bag. Monitor,
keyboard and
mouse in
separate
bag.*
Chemical
Indicator
Yes
Yes
Yes
Yes
Yes
13
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Cycle
Type
Category
4
Test
Condition
4
EtO
NoEtO
Humidification
Two
Humidichips®
Biologicals
Bis, rubber
test
coupons
Monitoring
Equipment
Madgetech
(one in each
bag)
Materials/
Equipment
One PC in PC
bag. Monitor,
keyboard and
mouse in
separate
bag.*
Chemical
Indicator
Yes
* Both bags contain the EtO cartridge and humidification.
** Used for only one replicate.
Before fumigation of the Category 4 materials, it was necessary to open the computer chassis to insert
the RH/temperature sensor (MadgeTech, Warner, NH), Bis, and inoculated rubber coupons into each
desktop case. The inside of the desktop computers was digitally photographed. To maintain the integrity
of the computer by avoiding static electricity, an electrostatic discharge (ESD) work station was set up to
work on the computers in E288. All personnel were trained in operating these stations. In general, the
ESD work station consists of an ESD work mat, an electrostatic monitor, and ESD wrist bands. All
computers were inspected and operated (e.g., diagnostic testing, long-term operation of computers for
analysis of residual effects) on the certified ESD work stations. During operation of the computers, all
computers were energized using surge protectors.
2.4 Sampling Strategy
Local variations in temperature were expected, especially due to the thermal mass of the equipment or
materials. This variation in temperature also affects RH. Because RH is a critical parameter in the
effectiveness of the fumigant, the RH was checked by placing an RH/temperature sensor (MadgeTech,
Warner, NH) in each fumigation bag. Each sensor was checked against a standard RH meter for
calibration. For Category 2 and 3 testing, one sensor was placed in each bag. The sensors logged RH
and temperature in real time, and the data were downloaded after the fumigation event was complete.
The testing strategy for the impact of the fumigation processes on Category 4 material and electronic
equipment required monitoring the RH inside the bag (outside the computer chassis) and inside the
computers. The sampling locations of the temperature and RH meters were identical to avoid any bias in
the measurement. This positioning allowed direct comparisons between the bag and the localized RH
after correcting for individual sensor bias.
2.4.1 Frequency of Sampling/Monitoring Events
Table 2-6 provides information on methods, test locations, and frequency for the measurement
techniques used for this compatibility testing.
14
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Table 2-6. Monitoring Methods for EtO fumigation
Monitoring Method
Visual Inspection
RH/Temperature sensor
PC Doctor (PCMD)
testing
BIT
Test Location
E288
Inside bag
Inside Cat. 4 computers
E288
E288
Sampling
Rate
NA
NA
NA
NA
Scope
Effects of Fumigation
0-1 00 %RH, -20-80 °C
Computer Functionality
Hardware Compatibility
Computer Functionality
Hardware Compatibility
Frequency and
Duration
Monthly
Real-time six per
minute
Monthly
Weekly
2.4.2 Testing Approach
Two test matrices were used for the testing. Test Matrix T01 (Table 2-7) was used for Category 2 and 3
materials (combined) and Test Matrix T02 (Table 2-8) was used for Category 4 computers. The test
matrices were built around the main objective of this project; to assess the damage, if any, to material and
electronic equipment functionality after remediation of contaminated materials and equipment. Fumigated
equipment was compared to control equipment not exposed to fumigation conditions.
Table 2-7. Test Matrix T01 (Category 2 and 3)
Test
Condition
1
2
Treatment Conditions
EtO (1 1 g), two Humidichips® T=122
°F.
All de-energized.
Controls, no exposure
Description
Standard Andersen Cycle
Controls
Table 2-8. Test Matrix T02 (Category 4)
Test
Condition
1
2
3
4
5
Treatment Conditions
EtO (1 1 g), two Humidichips®, T=122 °F.
All de-energized.
EtO (1 1 g), wetted sponge (with 18 g
water), T=1 22 °F.
All de-energized.
EtO (18 g), two Humidichips® T=122 °F.
All de-energized.
No EtO, two Humidichips® T=122 °F.
Controls, no exposure
Objective
Baseline - Standard
Andersen Cycle
Effect of higher RH on
materials
Effect of higher EtO
Exposure
Effect of RH and
temperature alone
Controls
Test Materials
(Computer IDs)
SS 04-08
SS 09-10
SS 14-16
SS 11-13
SS 01-03
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2.5 Measurements
Table 2-9 lists the critical and non-critical measurements associated with this project.
Table 2-9. Critical and Non-Critical Measurements
Sample Type
Environmental
conditions inside
fumigation bags
Bis
Inoculated coupons
Category 2 Materials
Category 3 Materials
Category 4 Materials
Sample Purpose
Characterize T and RH
Indicative that sporicidal
conditions were met during
fumigation
Indicative that sporicidal
conditions were met during
fumigation
Measures impact of
fumigation
Measures impact of
fumigation
Measures impact of
fumigation
Critical Measurement
RH
Temperature
Growth/No growth
Incubation Temperature
Incubation Time
Incubation temperature
CPU counts
Extraction volume
Plated volume
Visual Inspection
Roughness Testing
Visual Inspection
Functionality Testing
PC-Doctor Tests
Non-Critical
Measurements
Incubation time
BIT results
2.6 Sampling Procedures
Test samples, materials and equipment were stored under RH/temperature-controlled indoor ambient
laboratory conditions until testing was performed. All samples, both test and control, were stored under
the same conditions prior to and after the fumigation event. Sampling activities for this study include
monitoring of environmental conditions (RH and temperature) and collecting microbiological samples for
sporicidal efficacy. Tested materials and equipment were photographed before and after exposure and
any visual changes were noted including color, legibility, and contrast.
2.6.1 Visual Inspection (Category 2-4)
Visual inspection focused on the possible effects of fumigation: a change in color or increase of corrosion.
Color change may also affect legibility. Digital photographs of each test material were taken prior to
fumigation. Digital photographs were taken after fumigation, with the fumigated sample next to a control
sample when practical. Some electronic equipment was partially dismantled on an approved ESD work
station to take digital photographs inside the casing. . The color of sections of digital photographs were
compared to and described by a computer-based hexadecimal color chart. Any problems with legibility or
contrast of materials before/after fumigation were recorded.
Photographs were taken of each material and device pre-exposure, immediately post-exposure, and then
monthly thereafter for a period often months for Category 2 and 3 materials. Category 4 materials were
only photographed periodically after Month 3, but no visual changes were noted. The purpose of this
16
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physical documentation was so that comparisons could be made over time, looking for changes such as
discoloration, corrosion, residue, and decrease in any device functionality.
2.6.2 Functionality Testing (Category 2-4)
All electronic equipment in Category 3 and 4 underwent functionality testing prior to and post-fumigation.
Smoke Detectors and Smoke/CO detectors from Category 2 also underwent functionality testing.
Functionality testing was conducted by running a predefined routine specific to each of the Category 3
and 4 items. These routines were documented for each item and maintained in the item's log book. For
the computer systems, PCMD Service Center 7.5 (PC Doctor, Reno, NV) was run to complete a hardware
and software diagnostic investigation. The results were stored digitally and converted to PC scores
throughout the testing period.
2.6.2.1 Smoke Detectors
Two functions of the smoke detectors were tested, the TEST/SILENT button and the smoke detection
system. The TEST/SILENT Button was pressed and held. The expected result was a loud repeating
three-beep horn pattern, the smoke light on, and light-emitting diode (LED) flashing once every second.
To test the smoke detection system, smoke detector tester aerosol (SmokeCheck™, Home Safeguard
Industries Fire and Safety Group LLC., Elk Grove Village, IL) was sprayed at a distance of 2 to 4 feet from
the vent on top of the detector for 1-2 seconds. The expected result was a loud, repeating three-beep
horn pattern, the smoke light on, and LED flashing once every second.
2.6.2.2 CO Detectors
The CO detector function on Smoke/CO alarms was tested using a CO detector test gas (CoCheck™,
Home Safeguard Industries Fire and Safety Group LLC., Elk Grove Village, IL). The test gas was sprayed
at the vent on top of the detector for 1 -2 seconds from a distance of 2 to 4 feet. The expected result was a
loud, repeating four-beep horn pattern, the CO light on, and LED flashing once every second.
2.6.2.3 Personal Digital Assistant (PDA) - Palm Pixi Plus
Two functions of the PDA were tested; transfer of files from the PDA to a personal computer, and transfer
of files from the personal computer to the PDA. The transfer was considered successful if the file could be
opened without noticeable corruption.
2.6.2.4 Fax/Phone /Copier Machine
The fax machine had to meet all of the following criteria to receive a PASS; otherwise, the unit failed:
• A fax was successfully transmitted. Successful transmission would be evident if a response was
faxed back.
• A fax was successfully received.
• Outgoing and incoming telephone calls were successfully connected.
2.6.2.5 Cell Phone
Prepaid cell phones were refilled as necessary. An outgoing call was placed from the cell phone. The
audibility of the ring tone was noted. The call (to a local phone) was answered and sound transmittal was
17
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recorded. An incoming call was received on the cell phone. The audibility of the ring tone was noted. The
call (from a local phone) was answered and sound transmittal was recorded. All buttons on the keypad
were also tested.
2.6.2.6 Data CD
The first ten seconds of each track were evaluated for audible glitches or skips.
2.6.2.7 Data DVD
The first ten seconds of each track were evaluated for audible or visual glitches or skips.
2.6.2.8 USB Flash Drive
Two functions of the USB flash drives were tested: transfer of files from the USB flash drives to a
personal computer and transfer of files from the personal computer to the USB flash drives. The transfer
was considered successful if the file could be opened without noticeable corruption.
2.6.2.9 SD Memory Card
Two functions of the SD Memory Card were tested; transfer of files from the SD Memory Card to a
personal computer and transfer of files from the personal computer to the SD Memory Card. The transfer
was considered successful if the file could be opened without noticeable corruption.
2.6.2.10 PCMD Test Configuration
Functionality of Category 4 computers was tested using PCMD Service Center (Version 7.5, PC-Doctor,
Rebo, NV, USA). The software identified and ran 96 distinct tests, including tests on the following
subsystems:
• System Board
• Random Access Memory
• Central Processing Unit
• Complementary metal-oxide-semiconductor
• Graphics Card
• Hard Drive
• CD/DVD Drive
• Audio Visual Interleave
• Monitor
• Keyboard
• Mouse
• Network Connections and Protocols
• Peripheral Component Interconnect Buses
• Standby/Hibernate Functions
• Serial Communications Ports
• Universal Serial Bus (USB)Ports
• Audio Board.
18
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PC Doctor testing was performed monthly to evaluate Category 4 equipment failures. All computers had
individual cables and equipment to prevent corrosion material being transferred from one computer to
another through reuse of cables or common equipment.
The PC-Doctor® Service Center™ 7.5 protocol was developed to have an industry-accepted standard
method of determining pass versus failure of the computer subsystems. PC-Doctor® Service Center™
7.5 functionality testing was conducted on each computer pre-fumigation, one day post-fumigation, then
monthly.
For every monthly test, a standard protocol called for each test to be performed once. If any particular test
failed the first time, the computer was tested a second time to correct for possible human error. A test that
failed the second time was labeled "Fail". If the test failed the first time but passed the second time, it was
labeled "Pass2". For tabulation, a score of 100 was assigned to each "Fail", while a "Pass2" received a
score of 1. During each pre- and post-fumigation testing period, a total PCMD score was assigned to
each computer based upon the number of tests that failed on the first or second attempt. A "Pass" results
in a score of 0. Scores are only evaluated relative to controls and to the results of previous fumigation
studies.
Statistical analysis was used to evaluate whether or not fumigated computers differed from control
computers.
2.6.3 Microbiology Meth ods
2.6.3.1 Coupon Spore Enumeration
The day after EtO exposure, 18 mm rubber coupons (test, procedural blank, and positive control) were
transferred aseptically into empty 50 ml sterile vials. The sample vials were then transported to the
NHSRC Biolab, where 10 ml of sterile Phosphate Buffered Saline plus Tween® 20 (PBST) was
aseptically added. The sample vials were then sonicated for 10 minutes using an 8510 Branson
(Danbury, CT) ultrasonic cleaner at 44 kHz and 250 Watts. The sonication step was immediately followed
by two continuous minutes of vortexing to further dislodge any viable spores. Each vial was briefly re-
vortexed immediately before any solution was withdrawn for analysis. The solution was subjected up to a
five-stage serial dilution. Each dilution (0.1 ml) was inoculated onto trypic soy agar (TSA) plates, spread
with sterile beads, and incubated at 35 ± 2 °C for 18-24 hours. Plates with 30-300 CFU were counted
manually. Any samples below countable criteria (30 CFU) on the primary dilution plates were filtered. The
filters were incubated at 35 ± 2 °C for 18-24 hours prior to manual enumeration.
Extraction and initial plating of biological samples was performed within five days. Samples were stored
under refrigeration until analyzed.
2.6.3.2 Bl Analysis
Bis were processed as advised by manufacturer's instructions. Each Bl was given a unique sampler
identifier prior to the start of any experimentation. These BIS were analyzed qualitatively by aseptically
transferring each individual sample into a sterile pre-labeled culture tube containing approximately 10 ml
of growth media (tryptic soy broth). The culture tubes containing the growth media and the Bl were
agitated using a vortex mixer and then placed into an incubator at an appropriate temperature for the
surrogate microbiological organism as found on the Bl (as advised by manufacturer's instructions).
19
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Following a seven- to ten-day incubation period, each of the culture tubes containing the growth media
and biological indicators was analyzed for turbidity and tested for growth using a direct plating technique.
Each of the samples were homogenized using a vortex mixer, and using a sterile pipette, 100 uL of liquid
was removed from the culture tubes and plated onto tryptic soy agar and incubated at the temperature-
time combination best suited for growth (as advised by manufacturer's instructions). The agar plates
were then analyzed visually and notes were made concerning any observed growth.
20
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3 Results
3.1 Category 2 Materials
3.1.1 Fumigation con ditions
Not all Category 2 or 3 materials could fit into any one exposure bag, so three separate exposures were
required. Conditions and contents of each bag are listed in Table 3-1. Fumigations were not sequential.
Table 3-1. Contents and Conditions of Category 2 and 3 EtO exposures
Fumigation
1
2
8
Treatment Conditions
EtO (1 1 g), two
Humidichips®, T=122°F.
All de-energized.
EtO(11g), two
Humidichips®, T=1 22 °F.
All de-energized.
EtO (1 1 g), two
Humidichips®, T=1 22 °F.
All de-energized.
Objective
Baseline:
Standard
Andersen
Cycle
Baseline:
Standard
Andersen
Cycle
Baseline:
Standard
Andersen
Cycle
Average
Fumigation
Conditions
RH: 51.8%
T: 46.8 °C
RH: 55.7%
T: 47.7 °C
RH: 59.6%
T: 42.3 °C
Category 2
Materials
Aluminum, type
304 and 316
stainless steel,
smoke detector,
laser-printed
paper, InkJet-
printed paper
Photographs,
smoke/CO
detector, laser-
printed paper,
InkJet-printed
paper
Category 3
Materials
Cellphones,
CDs, DVDs,
USB flashdrive,
SD memory card
Fax machine
PDA
Photographs were taken of each material pre-exposure, immediately post-exposure, and then monthly
thereafter for a period often months.
The purpose of this physical documentation was so that comparisons could be made overtime, looking
for changes such as discoloration, loss of functionality, corrosion, residue, and decrease in the quality or
readability of documents and photographs.
3.1.2 Visual Inspection
There was no recorded visual impact of the EtO fumigation on any Category 2 materials. No discoloration
was found, even in high quality photographs and InkJet- and laser-printed documents. Certain individual
colors in photographs were compared to and described by a computer-based hexadecimal color chart. No
differences were noted over the course of testing. Figures 3-1 and 3-2 show the minimal visual impact on
Category 2 materials (laser-printed paper and copper coupons) ten months post-fumigation.
21
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\ \ e
a
•l "50. x
Figure 3-1. Laser Printer Paper exposed to EtO (left and center) and not exposed (right)
Figure 3-2. Copper exposed to EtO (2Cu-01 and 2Cu-02 on left) and not exposed (2Cu-03 on
right)
The three peppers circled in Figure 3-3 were used to measure photograph fading by comparison to a
hexadecimal color chart before and after testing. Some of the results are shown in Table 3-2. No change
was apparent during testing.
22
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Figure 3-3. Test Photograph
Table 3-2. Hexadecimal Color Comparison
Test Date
Pre-fumigation (9/4/13)
Post-fumigation
(11/29/13)
End-testing (7/4/14)
Red Pepper Code
#E3170D
#E3170D
#E3170D
Orange Pepper Code
#FFBOOF
#FFBOOF
#FFBOOF
Green Pepper Code
#003300
#003300
#003300
3.1.3 Functionality Testing
Functionality testing for Category 2 materials consisted of operation of the smoke and CO Detectors and
material roughness testing, to determine if the surfaces of the metal coupons were affected by fumigation.
Metal coupons were tested for surface roughness monthly throughout the testing campaign. Results were
recorded for data analysis.
3.1.3.1 Smoke and Smoke/CO Detector Results
Table 3-3 shows the test results for the last month of testing. This table lists the tests required to pass
and indicates that the detectors both passed. Both detectors passed similarly for every month they were
tested.
23
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Table 3-3. Smoke Detector Functionality Testing
Category 2
Smoke Detector
1. Test/silent button and escape light are functional
2. Smoke alarm with escape light is functional
Smoke Detector / CO Alarm
1. Test/silent button with smoke LED and CO LED
2. Smoke alarm with smoke led,
3. CO alarm with CO LED
Material ID
2SD-01-T01
2SD-02-T01
2SD-03-T01
2SC-01-T01
2SC-02-T01
2SC-03-T01
PASS
X
X
X
X
X
X
FAIL
3.1.3.2 Surface Roughness Testing Results
Measurements were taken incorrectly for pre-test sampling, post-test sampling and Month 1 sampling.
The error was discovered before Month 2 sampling and the procedure was corrected. Therefore, the test
coupon measurements are compared only to the controls that were not fumigated. As seen in Figure 3-4,
there were a couple of clear outliers during testing. These measurements are clearly outliers because the
readings returning to normal the following month and are most likely due to small scratches on the
surface of the coupon. The coupon was not sampled in the exact same spot every time, and the
instrument covers only a fraction of the surface. As is apparent in the graphs below, there was no trend of
degradation in post-fumigation samples. Also, as seen in Table 3-4, there was no significant difference in
roughness between test (n=3) and control samples (a P-value of <0.05 would indicate a significant
difference). Figures 3-5 to 3-8 show the average surface roughness for each material compared to the
controls.
Average Surface Roughness Measurements of Test Coupons
•Type 304 stainless steel
•Type 316 stainless steel
•Aluminum
•Copper
4 6
Test Number
Figure 3-4. Average Surface Roughness Measurements of Test Coupons
24
-------
Table 3-4. Average Roughness for Test Sets
Test Set
Type 304 stainless
steel
Type 316 stainless
steel
Aluminum
Copper
Average Test
Coupon
Roughness((jm)
0.29
0.22
0.41
0.43
Average Control
Coupon
Roughness (urn)
0.22
0.21
0.44
0.23
P-Value
(control set
compared to test
set)
0.06
0.30
0.17
0.12
OQ
00
07
Oc
E~" n R
^
S. u'^
n 3
U.d ^
n 9
0.2 t
0.1
Type 304 (2S4) Stainless Steel Coupons
A
/ \
\
K ....*... •• 2S4 Controls
/H....;:::---*-^,. j* — ::-»
.^y =t^
468
Months after fumigation
Figure 3-5. Type 304 Stainless Steel Roughness Measurements
25
-------
0.3
0.25
0,'
E
^ n 1 c;
ro
OC
0.1
0.05
0
Type 316 (2S6) Stainless Steel Coupons
.••••Ox
^\ /*x/ \
>*=^
-^ ••
» 2S6
! 4 6 8
Months after fumigation
Figure 3-6. Type 316 Stainless Steel Roughness Measurements
07
Oc
Oc
'P' n A i
3
09
0.1
Aluminum (2AL) Coupons
P\ ..-••
A / \ X
5, o r\. u-...\.^-\
*T / Vs-«--r o
\ f \ / « 9AI
\ / V
468
Months after fumigation
Figure 3-7. Aluminum Roughness Measurements
26
-------
1.8
1 A
1 9
E"""1* 1
3
ra n R
0.6
0.4
0..
n
Copper (2CU) Coupons
m
A
/ \
/ s 'CU
\ •• 2CU Controls
0 o
468
Months after fumigation
Figure 3-8. Copper Roughness Measurements
3.2 Category 3 Materials
Fumigation conditions are listed in Section 3.1.1.
3.2.1 Visual Inspection
There was no recorded visual impact of fumigation on any Category 3 materials (cell phone, DVD and
CD). No discoloration was found in electronic displays. Figures 3-9 through 3-10 below show the minimal
visual impact on digital photographs and CDs, respectively.
27
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Figure 3-9. Photo of digital photographs taken over a year after exposure. There was no
discernable difference between the control (2PH-03 on right) and test samples (2PH-
01 and -02).
28
-------
Figure 3-10. Exposed (left) and unexposed (right) CD 10 months later
3.2.2 Functionality Testing
Category 3 materials showed no functionality failures over the course of testing. The test results shown in
Table 3-5 are from the last month of testing.
Table 3-5. Category 3 Functionality Testing Results
Category 3 Material
Cell Phone
1 . A call made from the phone was connected.
2. A call made to the phone was connected and the ringtone was audible.
3. All buttons on the key pad are functional.
PDA
1 . A data file can be transferred to the PDA and opened.
2. A data file can be transferred from the PDA and opened.
Fax/Phone/Copier
1 . A fax was transmitted.
2. A fax was received.
Material ID
3PE-01-T01
3PE-02-T01
3PE-03-T01
3PD-01-T01
3PD-02-T01
3PD-03-T01
3FA-01-T01
3FA-02-T01
3FA-03-T01
PASS
X
X
X
X
X
X
X
X
X
FAIL
29
-------
Category 3 Material
3. Outgoing and incoming calls were connected.
Data CD
1. The disk was readable.
2. The tracks were audible with no defects.
Data DVD
1. The disk was readable.
2. The tracks were visible and audible with no defects.
USB Flash Drive
1 . A data file can be transferred to the USB and opened.
2. A data file can be transferred from the USB and opened.
Memory Card
1 . A data file can be transferred to the memory card.
2. A data file can be transferred from the memory card and opened.
Material ID
3CD-01-T01
3CD-02-T01
3CD-03-T01
3DV-01-T01
3DV-02-T01
3DV-03-T01
4FD-01-T01
4FD-02-T01
4FD-03-T01
4SD-01-T01
4SD-02-T01
4SD-03-T01
PASS
X
9*
X
X
X
9*
X
X
X
X
X
X
FAIL
*3CD—02-T01 and 3DV-03-T01 went missing during testing
3.3 Category 4 Materials
3.3.1 Fumigation Conditions
Table 3-6 shows the average RH and temperature for each of the four controlled test conditions.
Table 3-6. Final Test Matrix
Test
Condition
1
2
3
4
5
Treatment Conditions
EtO (1 1 g), two Humidichips®, T=122
°F.
All de-energized.
EtO (1 1 g), wetted sponge (with 18 g
water), T=1 22 °F.
All de-energized.
EtO (18 g), two Humidichips®, T=122
°F.
All de-energized.
No EtO, two Humidichips®, T=122
°F.
Controls, no exposure
Objective
Baseline - Standard
Andersen Cycle
Effect of higher RH on
materials*
Effect of higher Ethylene
Oxide Exposure
Effect of RH and
temperature alone
Controls
Average
Fumigation
Conditions
RH: 57.6 %
T: 46.5 °C
RH: 57.9 %
T: 45.2 °C
RH: 53.8 %
T: 46.1 °C
RH: 55.7 %
T: 46.1 °C
Test Materials
(Computer IDs)
SS 04-08
SS 09-10*
SS 14-16
SS 11-13
SS 01-03
' This test was designed to show effects of higher RH, but the condition was not achieved
30
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3.3.2 Testing Difficulties
The test matrix originally proposed had to be modified during the course of testing due to difficulties
encountered during testing. Originally, the objective for the wetted sponge tests was to determine the
effect of higher RH during fumigation on the computers. This test was based on scoping testing
performed outside this effort. Unfortunately, this high RH condition could not be duplicated using EOGas
permeable bags. Actual test conditions are listed in Table 3-6.
3.3.3 Visual Inspection
There was no recorded visual impact of fumigation on any Category 4 materials. There was no evidence
of corrosion on any metal surfaces or edges. The lack of corrosion is especially important. Other
fumigation technologies {USEPA, 2010 #3074} have shown significant corrosion on many components,
especially unfinished metal edges. No discoloration was found in electronic displays. Figures 3-11
through 3-14 show the minimal visual impact on Category 4 materials. No visual impacts were seen after
three months, and although visual inspection continued, digital photographs were no longer documented
for the remainder of the testing period.
Figure 3-11. Inside SS04 (pre-fumigation)
31
-------
Figure 3-12. Inside SS04 (two months post-fumigation)
Figure 3-13. SS04 back panel (pre-fumigation)
32
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Figure 3-14. SS04 Back Panel (two months post-fumigation)
3.3.4 Functionality Testing
PCMD testing performed well, with one exception. Serial port tests (COM1) were sometimes excluded
from testing for an unknown reason. When this occurred, the software would not recognize the COM1
hardware even though the Windows operating system reported it was operating normally. Multiple
attempts were made to resolve the issue, but a resolution was not reached. While the problem persisted,
any PCs that failed to run the test would be subject to an additional test. For these PCs, the Mouse
Interactive Test was run with a serial mouse connected to the COM1 port to prove it was functional.
3.3.4.1 BIT
There were no BIT failures with the exception of those relating to failing DVD media (DVD media that had
defects from the factory). In the case of BIT DVD errors, replacing the media always fixed the error.
3.3.4.2 PC-Doctor® Functionality Testing Results
Results from PC Doctor® testing are shown in Figures 3-15 through 3-19, below. To tabulate PCMD
scores, a score of 100 was given to any failing test of the 96 conducted (subsystems are listed in Section
2.6.2.10). For any test that initially failed due to user error or bad media but that passed after correcting
the issue (a "Pass 2" result), a score of 1 was given. The number of failures and "Pass 2" results can
therefore be determined. For example, a score of 201 indicates two tests failed and one test was scored a
"Pass 2". Figure 3-19 shows the averages of each test set over the course of the period tested. Figures
3-15 to 3-18 show each subset of fumigated personal computer (PC) PCMD results as compared to the
controls. Test results of "0" (all tests passed) will not show up on these figures. Averaged scores simply
represent the average number of failures. Pass 2 results are barely accounted for mathematically since
33
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they are commonly a result of human error and not indicative of a larger issue. An average score of 335
indicates an average of 3.35 total failures.
Since only a single fumigation could be performed for one PC at a time (overnight), and some test
conditions were determined during the testing series, not all PCs were tested for the same amount of
time. All PCs were tested for a minimum of nine months.
Average PCMD Scores
300
250
OJ
8 200
uo
Q
§ 150
Q_
100
50
0
0 4 6 8 11 13 15 18 20 22 24 27 29 32 34 37 39 42 47 52
I Average Control
Weeks afterfumigation
I Average llg EtO, 2 humidichips, 122°F
Figure 3-15. Test Condition 1 PCMD Scores Compared to Controls
250
200
a, 150
O
LO
| 100
Q_
50
0
c
Average PCMD Scores
)
4 6 8 11 13 15 18 20 22 24 27 29 32 34 37 39 42 47 52
Weeks after fumigation
• Average Control • Average llg EtO, 18g Water, 122°F
Figure 3-16. Test Condition 2 PCMD Scores Compared to Controls
34
-------
250
9nn
g 150
o
u
LO
o 1 nn
u
Q_
50
0
(
Average PCMD Scores
)
* 6 8
Average (
11 13
Control
15 18 20 22 24 27 29 32 34 37 39 42 47 52
Weeks after fumigation
• Average llg EtO, 18g Water, 122°F
Figure 3-17. Test Condition 3 PCMD Scores Compared to Controls
250
Tnn
PCMD Score
h-* h-* h
Ln O Ln C
O O O O C
Average PCMD Scores
)
• A
1
4 6 8 11 13 15 18 20 22 24 27 29 32 34 37 39 42 47 52
Weeks afterfumigation
verage Control • Average No EtO , 2 humidichips, 122°F
Figure 3-18. Test Condition 4 PCMD Scores Compared to Controls
35
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Average Score over testing period
160
140
142
I Controls •NoEtO
I llg EtO, 18g water, 122F • llg EtO, 2 humidichips, 122F
118g EtO, 2 humidichips, 122F
Figure 3-19. Average PC Doctor Scores over Testing Period
There were very few failures for Category 4 machines. The test failure rate across all machines was 0.4
%. Approximately 85 % of the failures were related to the CD/DVD drive or a result of an incorrect
procedure with the audio loopback test. In some cases, new CD/DVD media would correct the error. Most
of these failures were intermittent. A PC would fail one month's CD/DVD test and pass the next month.
Some PCs experienced multiple persistent CD/DVD failures. Of the three PCs that experienced persistent
failures, one was a control. This result indicates that the failure may not be a product of fumigation at all.
The PCs used were susceptible to these failures even under normal usage conditions. Tests 83 and 84
required a very specific order of user interaction for the system to recognize the audio loopback cable.
Tests where this failed were most likely due to this procedure not being carried out correctly. There is an
identical test earlier in the procedure which never failed but uses different physical audio ports that are
automatically recognized and activated by the software. Tests 83 and 84 failed approximately 20 % of the
time across all PCs. The DVD tests (57, 58, and 59) failed approximately 10 % of the time across all PCs.
Since the control set had a higher average score, the failures witnessed were probably not a result of the
fumigation. Tests conducted without EtO should show only the effects of increased temperature and RH.
The results of tests without EtO are also consistent with fumigated test and control results. The p-values
in Table 3-7 suggest that there are significant differences between only a few data sets. The PC subsets
of Test Condition 2 (11 g EtO, 18 g water, 122 °F) Test Condition 4 (No EtO) resulted in the least failures
over the testing period. These p-values are significantly different from Test Condition 5 (Controls). While
the values are significantly different from the controls, it is likely not an effect of fumigation, since they
actually performed better than the controls. The only other significant difference was between Test
Condition 2 (11 g EtO, 18 g water, 122 °F) and Test Condition 1 (11 g EtO, two Humidichips®, 122 °F). As
was shown in the Table 3-6 and described previously, there was no actual measured difference in
fumigation conditions between these two subsets. The result is also unlikely to be any indication of an
effect of the fumigation. The computers procured for this testing experienced intermittent failures, making
it difficult to attribute these results to the fumigation conditions.
36
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Table 3-7. p-Values between PCMD Scores of all PC Subsets*
Subset Being Compared
Test Condition 5
Test Condition 5
Test Condition 5
Test Condition 5
Test Condition 1
Test Condition 1
Test Condition 1
Test Condition 4
Test Condition 4
Test Condition 3
Compared to Subset
Test Condition 1
Test Condition 2
Test Condition 4
Test Condition 3
Test Condition 2
Test Condition 4
Test Condition 3
Test Condition 2
Test Condition 3
Test Condition 4
p-Value
0.447
0.018
0.015
0.244
0.048
0.057
0.549
0.291
0.092
0.155
* Values <0.05 (shown in red) indicate a significant difference.
3.4 Fumigation Effectiveness
For all test conditions, the EtO fumigations achieved greater than six log reduction, or non-detect. Test
R14 was not filter-plated by the time of this report, and thus had a much higher detection limit. Though the
test samples were non-detect for this test, this delay resulted in a lower (5.17) log reduction. These
results are shown in Table 3-8, below. Non-detect results are shaded in yellow. Test R12 was the control
condition without EtO.
Table 3-8. CPU Counts and Log Reduction for All Biological Tests
Test Number
R01
R02
R03
R04
R05
R06
R07
R08
R09
RIO
R12 (control)
R14
R15
R16
Positive CPU
3. 64 x 10s
8.83 x 10s
3. 27 x 10s
7.67 x 10s
6.55 x 10s
1.32 xlO7
6. 13 x 10s*
7.19 x 10s
9.60 x 10s
1.18 xlO7
1.71 xlO7
7.36 x 10s
2.11 xlO7
2.28 xlO7
Test CPU
<1
<1
<2
<1
<1
<1
<2
<1
<1
<1
9.35 x 10s
50
<1
<1
Log Reduction
6.76
7.01
6.30
7.09
7.01
7.33
6.39
6.86
7.18
7.26
0.26
5.17
7.51
7.56
*Only four replicates for this test
All fumigated Bis exhibited no growth.
37
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4 Quality Assurance
This project was performed under an approved Category III Quality Assurance Project Plan titled The
Impact of Decontamination Technologies (Ethylene Oxide) on Materials and Equipment (July 2013).
4.1 Sampling, Monitoring, and Analysis Equipment Calibration
Accuracy and precision of the RH measurements were determined during pre- and post-test calibrations.
Temperature calibrations were performed on MadgeTech devices before use on this project. The
accuracy and precision of volumetric measurements were assessed through calibration checks performed
by the NHSRC Biolab. These calibration checks were performed before and after use of volume
dispensing devices on the project, or once every four months.
There were operating procedures for the maintenance and calibration of all laboratory and NHSRC Biolab
equipment. All equipment was verified as being certified calibrated or having the calibration validated by
the EPA RTP on-site Metrology Laboratory at the time of use. Calibration of instruments was done at the
frequency shown in Table 4-1. Any deficiencies were noted. The instrument was adjusted to meet
calibration tolerances and recalibrated within 24 hours. If tolerances were not met after recalibration,
additional corrective action was taken, possibly including recalibration or/and replacement of the
equipment.
Table 4-1. Sampling and Monitoring Equipment Calibration Frequency
Equipment
MadgeTech Sensors
SRG-4000
Roughness Tester
MadgeTech Sensors
Critical Measurement
Relative humidity inside the
bag
Roughness measurements
Temperature inside the bag
Calibration/Certification
RH calibrated monthly to a salt cell
calibrated Vaisala sensor.
Calibrated monthly before roughness
testing to a standard surface.
Devices were factory calibrated.
Calibration only required annually.
Expected Tolerance
+_ 3 % RH
+ 12%
+_0.5°C
4.2 Data Quality
The Quality Assurance Project Plan (QAPP) for this project was followed with deviations noted as follows:
Test Condition 1 (18 g EtO, two Humidichips®, T=122 °F) was performed in quintuplet instead of triplicate.
Also, all computers in Conditions 1 (SS 04-08) and 2 (SS 09-10) were fumigated with 11 g EtO instead of
the 18 g EtO cartridge. While the original test matrix used an 18 g EtO cartridge for all tests, because of
this error, it was decided to make the EtO exposure amount a variable as well. Category 4 materials were
photographed only periodically and at the end of testing after Month 3 because no visual changes had
been noted. Visual monitoring continued for the full ten months.
4.3 QA/QC Checks
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, CPU were enumerated
manually and recorded. Critical QC checks are shown in Table 4-2. The acceptance criteria were set at
the most stringent level that could be achieved routinely and are consistent with the data quality
38
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objectives described in Section 4.4. Positive controls and procedural blanks were included along with the
test samples in the experiments so that well-controlled quantitative values were obtained. Background
checks were also included as part of the standard protocol. Replicate coupons were included for each set
of test conditions. Qualified, trained, and experienced personnel ensured data collection consistency.
When necessary, training sessions were conducted by knowledgeable parties, and in-house practice runs
were used to gain expertise and proficiency prior to initiating the research.
The following Sample Acceptance Criteria were followed for this effort. Any deficiencies were noted and
reported to the WACOR.
Table 4-2. Quality Assurance (QA)/Quality Control (QC) Sample Acceptance Criteria
QC Sample
Procedural Blank
(coupon without
biological agent)
Inoculum Control
(100 uL spike of
inoculum into 10 ml_
PBST)
Blank plating of
microbiological supplies
Blank ISA Sterility
Control
(plate incubated, but not
inoculated)
Field Blank Samples
(Sample matrices
handled in sampling
area without contact
with surfaces)
Biological samples
Information Provided
Controls for sterility of
materials and methods
used in the sampling
procedure.
Initial contamination
level on the coupons
shows plate's ability to
support growth.
Controls for sterility of
supplies used in
dilution plating
Controls for sterility of
plates.
The level of
contamination present
during sampling
Number of CPU
Frequency
one per test
three
replicates
per
inoculation
day
three of each
supply per
plating event
Each plate
three per
sampling
event
Each
replicate
Acceptance Criteria
No observed CPU
For high inoculation
target loading of 10 CPU
per sample with a
standard deviation of <
0.5 log. (5x1 0s -5x1 07
CPU/sample);
Grubbs outlier test (or
equivalent).
No observed growth
following incubation
No observed growth
following incubation.
No observed growth
following incubation
Significant (reported)
growth is between 30
and 300 colonies per
plate. Replicate plates
must agree within 100 %.
Samples with fewer than
30 CPU on the undiluted
plate may be filter-plated
to reduce detection limit.
Corrective Action
Identify and remove
source of contamination.
Consult WACOR*
Outside target range:
discuss potential impact
on results with the
WACOR; correct loading
procedure for next test
and repeat depending on
decided impact.
Outlier: evaluate stability
of pipette.
Sterilize or dispose of
source of contamination.
Re-plate samples.
All plates are incubated
prior to use, all
contaminated plates will
be discarded.
Clean up environment.
Sterilize sampling
materials before use.
Replate.
*WACOR = Work Assignment Contracting Officer Representative
39
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4.4 Acceptance Criteria for Critical Measurements
The Data Quality Objectives (DQOs) define the critical measurements needed to address the stated
objectives and specify tolerable levels of potential errors associated with simulating the prescribed
decontamination environments. The following measurements were deemed to be critical to accomplish
part or all of the project objectives:
• Temperature
• RH
• Material inspection and electronic equipment functionality over time
• Growth/No Growth of the Bis CPU count.
The Data Quality Indicators (DQIs) listed in Table 4-3 are specific criteria used to quantify how well the
collected data met the DQOs. Failure to provide a measurement method or device that meets these goals
results in the rejection of results derived from the critical measurement. For instance, if the plated volume
of a sample is not known, then that sample is invalid. In contrast, for the real-time RH measurements,
some missing data would not invalidate a test. Visual inspection and operational testing of all categories
of materials were performed to determine if the materials maintained their pre-exposure physical and
functional characteristics throughout a ten-month observation period following an EtO decontamination
event. PC-Doctor Service Center 6, a commercially available software, was used to test the
functionality of each computer pre-exposure, immediately post-exposure, and then up to monthly
thereafter for a period of ten months looking to diagnose and detect computer component failures. If any
particular test failed the first time, the computer was tested a second time to correct for possible human
error. A test that failed the second time was labeled "Fail". If the test failed the first time, but passed the
second time, the test was labeled "Pass 2".
Table 4-3. Data Quality Indicators
Critical
Measurement
RH
Temperature
CPU counts
Measurement
Device
Vaisala Model
333 RH and
temperature
probe
Desired
Accuracy
±3%RH
± 0.5 °C
10 %of
significant data
will be
recounted by a
second person.
Counts must
agree within
10%
Achieved
Accuracy
±3%RH
±0.5°C**
Criteria
met
Desired
Precision
±15%RH
± 5.0 °C
10 %of
significant data
will be
recounted by a
second person.
Counts must
agree within
10%
Achieved
Precision*
±12%RH
± 5.0 °C
Criteria met
Detection
Limits
0 to 100%
RH
-20 °C to
80 °C
1CFU
Desired/
Achieved
Completeness
95%/93 %***
95 W93 %***
100^100%
Precision for RH and temperature is defined as deviation from target conditions (50 °C, 60 % RH).
This value was not checked with a post-testing calibration. Devices were calibrated by the manufacturer within a
year of use, and manufacturer calibrations are required annually.
There were no intermittent failures for RH/T sensors. However, two tests lost entire device readings. One
MadgeTech device failed for R03 inside the PC, and no data were collected. For test R05, only the monitor bag
MadgeTech device data were downloaded. PC bag and PC measurements were lost.
40
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EtO cartridges, Humidichips , and sponges were weighed before and after use for fumigation to ensure
exposures were consistent.
Visual inspections were performed before each test, at the end of each test, and at the start of each
month thereafter for a period often months from the date of the fumigation event. Written documentation
was augmented with high resolution digital photography. A comparison to the control materials/equipment
was performed. For electronic equipment, the ESD work station was used to inspect the interior of the
equipment.
Equipment functionality for electronic equipment was assessed before each test, at the end of each test,
and at the start of each month thereafter for a period often months from the date of fumigation. The
Category 4 equipment maintained in-house was set in operation mode on the ESD work station;
temperature and RH of the area was typical of an office environment. The Category 3 equipment was
maintained in the same area as the Category 4 equipment. The Category 3 equipment powered by
alternating current (AC) was maintained in operational mode continuously; the battery powered
equipment was turned on for functionality testing (e.g., maintained in the 'power off mode).
For Category 4 items, the functionality was assessed using PCMD, a diagnostic software program used
by PC manufacturers for pre-inspection of computer hardware and software. The results were stored in
hard copy and electronically using a USB memory stick for each computer, along with the report of the
visual inspection. The computers remained in the certified ESD work station area at a temperature and
RH typical of an office environment fora period often months following the date of fumigation. The
computers remained in the operational state. PCMD was run every month to assess changes in the
performance of the computers.
Fumigation effectiveness: The sporicidal effectiveness was assessed for localized hot spots inside the
computers, where the RH or temperature may be lower because of the thermal mass of the computers. A
set of three Bis was placed inside each computer case, and another set was placed inside the bag to
determine the effectiveness of the fumigation to inactivate the Bis as a function of location within the
chamber (i.e., in the bag compared to inside computers). The Bis provided a qualitative result of growth
or no growth after an incubation period of seven days. Inoculated rubber coupons were also enumerated
for efficacy quantification.
Plated volume critical measurement goals were met. All pipettes are calibrated yearly by an outside
contractor (Calibrate, Inc.).
Plates were analyzed quantitatively (CFU/plate) using a manual counting method. For each set of results
(per test), a second count was performed on 25 percent of the plates with significant data (data found to
be between 30-300 CFU). All second counts were found to be within 10 percent of the original count.
There are many QA/QC checks used to validate microbiological measurements. These checks include
samples that demonstrate the ability of the NHSRC Biolab to culture the test organism, as well as to
demonstrate that materials used in this effort do not themselves contain spores. The checks include:
• Negative control coupons: sterile coupons that remained in sterile packaging before biolab processing
41
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• Procedural blank coupon: sterile coupons that go through the inoculation procedure without actually
being inoculated and fumigated
• Positive control coupons: coupons inoculated but not fumigated.
4.5 Data Quality Audits
This project was assigned a QA Category III and did not require technical systems or performance
evaluation audits.
4.6 QA/QC Reporting
QA/QC procedures were performed in accordance with the QAPP for this investigation.
42
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5 Conclusions
EtO can be used safely on small electronic equipment and all materials tested in this study, with the use
of adequate engineering controls. Little to no impact was recorded for any materials or equipment tested.
Unfortunately, EtO is difficult to scale up due to the high temperature (122 °F) and RH requirements. In
addition, EtO is very toxic and flammable under the target conditions. EtO is not suitable for wide area
fumigations such as a building or in any environment where a flame might be present or possible. While
this work focused on using a commercial off the shelf system for fumigation, it is possible to use EtO in
chamber fumigations when the safety issues are considered (flammability).
43
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6 References
1. USEPA, Compatibility of Material and Electronic Equipment with Methyl Bromide and Chlorine
Dioxide Fumigation. EPA/600/R-12/664. October, 2012.
2. USEPA, Compatibility of Material and Electronic Equipment with Chlorine Dioxide Fumigation.
EPA/600/R-10/169. December, 2010.
3. Brown, G.S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.S., Boucher,
R.M., Tezak, M., Wilson, M.E., Rudolph, T., Evaluation of a Wipe Surface Sample Method for
Collection of Bacillus Spores from Nonporous Surfaces. Appl. Environ. Microbiol., 2007. 73(3): p.
706-710.
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45
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Appendix A: Andersen EOGas System Characterization
Taskl
The primary objective of this task was to characterize the exposure of materials during Andersen EOGas
ethylene oxide (EtO) sterilization cycles, including the relative humidity (RH), temperature, and total
exposure in terms of net EtO weight and exposure time. These tests were conducted using 5,11, and
18 g EtO cartridges and two different bag sizes:
• The AN1005 (EOGas 5) is a 18" x 24" bag
• The AN 1006 (EOGas 6) is a 22" x 36" bag
Electronic devices cannot be used during EtO exposure, so RH and temperature measurements were
done during a cycle without EtO. The manufacturer (Anderson Products, Inc.) targets 750 mg/liter*hours
at 50 °C and RH between 35 % and 80 %. Tests were performed with and without materials, using the
manufacturer's recommendations of one water saturated sponge (Humidichip®) per bag. The measured
RH during mock exposures using the EOGas 6 bag is shown in Figure 1.
90
80
70
60
^ 50
40
30
20
10
RH Inside Bag
•HOBO RH (%) Wood coupons
HOBO RH (%) Rubber coupons
HOBO RH (%} Canvas coupons
•HOBO RH (%) PVC Coupons
•HOBO RH (%) Stainless coupons
HOBO RH (%) Tile coupons
•HOBO RH (%) Paper coupons
12
15
18
time (hrs)
Figure 1. RH in EOGas 5 (small) and EOGas 6 (large) Bags with one Humidichip
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The temperature is very stable due to the control by the EOGas 333 sterilization cabinet. The average RH
and temperature are shown in Table 1. Some minimum temperatures shown are during ramp-up and are
not indicative of the exposure conditions.
Table 1. Measured Environmental Conditions inside Sterilizer Bags during Mock Exposures
Test
1A
1B
1C
1D
1E
1F
1G
1H
11
Bag
Sizes
18"x24"
22" x 36"
22" x 36"
22" x 36"
22" x 36"
22" x 36"
22" x 36"
22" x 36"
22" x 36"
Material
s in bag
None
None
Wood
coupons
Paper
coupons
Ceramic
(tile)
coupons
Stainless
coupons
PVC
coupons
Canvas
coupons
Rubber
coupons
RH (%)
Min
50.8
47.9
76.7
50.8
65.0
49.1
49.0
55.3
77.3
Avg
58.4
66.9
77.5
58.1
74.4
58.1
72.9
55.9
77.9
Max
59.8
68.1
83.6
60.4
78.3
58.9
74.8
57.5
83.0
Temperature (°C)
Min
23.3
27.0
49.4
48.0
48.2
46.4
49.2
34.4
45.3
Avg
49.4
49.3
50.7
50.1
50.0
49.6
50.5
48.0
48.8
Max
51.4
51.7
52.2
51.4
51.3
51.1
51.6
49.0
49.0
Figure 1 and Table 1 show that all environmental target conditions were met by the cabinet with one
Humidichip in the sterilizer bag.
The variation in RH seen in Figure 1 is not necessarily an effect of the materials present. Figure 2 shows
the same type of data for bags that contained no materials.
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0 L
Empty Bag RH and Temperature
Small bag Cabinet Temp (°C)
Small bag RH (%)
Large bag Cabinet Temp (°C)
Large bag RH (%)
time (hrs)
Figure 2. RH and Temperature in EOGas 5 (small) and EOGas 6 (large) bags with one Humidichip"
All things being equal, the RH in the smaller bag would be expected to be higher than the RH in the large
bag due to the higher ratio of volume to Humidichip®. There were large variations in RH for all tests,
suggesting that Humidichips® may have a large variation in water content.
Characterization of the EtO conditions inside the bag are difficult to determine directly. The EtO is
explosive in the exposure range and cannot be measured using electronic devices such as
electrochemical sensors. Use of extractive measurements would change the conditions by removing the
target gas. Therefore, exposure conditions were measured indirectly by measuring the EtO permeating
out of the sterilization bag. An FID was used to measure the exhaust EtO concentration. The FID was
initially calibrated with propane calibration gases, but this procedure failed to close the mass balance.
Other researchers had documented a significant response factor for EtO, and because EtO was the only
hydrocarbon expected in these tests, an EtO calibration gas was used in subsequent calibrations. All
results are reported in terms of ppm EtO, based on the EtO calibration gas. The response factor for EtO
was measured as roughly 0.5, meaning that 100 ppm EtO would read 50 ppm ethane.
By measuring the concentration in and flow out of the cabinet exhaust, a total mass of EtO release can be
calculated, and compared in turn to the total mass change of the EtO cartridge. An example graph of the
exhaust concentration and calculated mass release is shown in Figure 3.
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EO Concentration and Mass Released
250 4.5
10
FID Concentration
•Total EO released
Figure 3. Exhaust Concentration and Total Mass Released
Table 2 shows the mass balance (recovery) on baseline (empty) sterilization bags. The EO released is
based on the change in cartridge weight before and after exposure. The EO captured was measured in
the exhaust.
Table 2. Mass Balance of EtO from Sterilization Bags without Materials
Test
U
1K
1L
1M
1N
10
1P
Bag Size
18"x24"
22" x 36"
18"x24"
22" x 36"
18"x24"
22" x 36"
18"x24"
EO
Cartridge
5g
5g
5g
5g
ng
ng
ng
EO Released
(grams)
4.41
4.26
4.491
4.548
10.375
10.208
10.38
EO Captured
(grams)
4.465
4.244
4.464
4.532
10.575
10.903
10.434
% Recovery
101.3
99.6
99.4
99.7
101.9
106.8
100.5
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Test
1Q
1R
1S
1T
1U
Bag Size
22" x 36"
18"x24"
22" x 36"
18"x24"
22" x 36"
EO
Cartridge
119
I8g
18g
18g
18g
EO Released
(grams)
10.59
17.606
17.41
17.638
17.599
EO Captured
(grams)
10.303
18.225
18.649
17.9533
19.346
% Recovery
97.3
103.5
107.1
101.8
109.9
The percent recovery varied between 97 and 110 %. Integration of very low level concentrations over a
long period of time during the aeration phase can lead to positive or negative bias due to instrument drift.
Exposure time can be estimated in a variety of ways. Exposure begins immediately upon rupture of the
cartridge (and is detected as permeation in the exhaust within three minutes). At the conclusion of the 18-
hour cycle, there is still measureable EtO inside the sterilization bag (approximately 40 ppm). It is
unknown whether long durations at very low concentrations contribute materially to efficacy.
Determining the exposure concentrations is a more complicated affair. Sterilization bags are evacuated
before sealing. The final pressure is not measured, but it can be assumed that the major components of
the atmosphere during exposure are water vapor (from the RH provided by the Humidichip®) and EtO.
The partial pressure of the water vapor is known, but, as discussed above, the EtO concentration cannot
be directly determined. There are two processes determining the actual EtO concentration: release from
the cartridge and permeation out of the sterilization bag. To help characterize both permeation and
cartridge release rates, measurements were made using two different sized bags. The small bag (EOGas
5) is designed to reach target EtO concentration with a 5 gram cartridge, and the larger bag (EOGas 6) is
designed for use with an 11 or 18 gram cartridge. The smaller bag has less area for permeation and
should thus increase exposure within the bag as compared to the larger bag as illustrated in Figure 4
using the temporal EtO concentration traces during an 18-hour EtO cycle.
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5 g cartridge release
300 r
•Small bag
•Large bag
Figure 4. Small Bag vs. Large Bag with 5 g Releases
The cartridge release rate and amount for both of these tests is presumed to be identical (replicate 5
gram cartridges). The larger bag, with the larger surface area for permeation, released EtO more quickly,
thus resulting in a higher concentration in the exhuast. The small bag released the EtO more slowly,
increasing the contact time inside the bag.
Task 2
The two primary objectives of Task 2 were to determine any material demand commonly sterilized
materials may have for EtO, and to determine the efficacy of EtO on deactivation of spores on these
materials. Secondary objectives include determining any residue formed during sterilization and
determining conditions required to obtain a 6 or greater log reduction (LR) in active spores following the
EtO cycle.
Results show that rubber has the most significant material demand (see Figure 5 for different masses of
rubber). All materials that have any significant material demand adsorb EtO, and then desorb EtO later in
the cycle, resulting in complete recovery of EtO in all tests performed to date.
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Material Demand of Rubber
300
•Test 2ER2
Concentration(785 g
excess)
•5 g standard test
Concentration
•Test 2ER2 Mass (785
g excess)
Standard 5 g test
mass
time (hrs)
Figure 5. Exhaust Concentration of Rubber and Baseline Exposures
The upward trend seen in the exhaust concentration at the 15 hour mark in Figure 5 is unexpected, and
none of the measured exposure parameters suggest a cause. A shift in the bag, due to settling, for
instance, could cause a change in cartridge position or bag geometry, which could cause a spike in
permeation.
Table 4 shows the results of efficacy tests with material demand effects. These tests were performed by
inoculating coupons of the material with approximately 1x 106 Bacillus subtilis spores and placing them
inside a sterilization bag with a large amount of material. The amount of material, though arbitrary, was
determined more by volume than weight.
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Table 4. Material Demand Efficacy Tests
Material
Paper
PVC
Wood
Stainless
Rubber
Ceramic
Canvas
Excess
Material
(yes/no)
yes
yes
yes
yes
yes
yes
yes
Cartridge
Size
5g
5g
5g
5g
5g
5g
5g
RH
No Humidichip®
(<50 %)
No Humidichip®
(<20 %)
No Humidichip®
(<20 %)
No Humidichip®
(<20 %)
No Humidichip®
(<20 %)
No Humidichip®
(<20 %)
No Humidichip®
(<20 %)
Bag size
Large (22" x 36")
Large (22" x 36")
Large (22" x 36")
Large (22" x 36")
Large (22" x 36")
Large (22" x 36")
Large (22" x 36")
Sporicidal
Efficacy
Not detected
Not detected
Not detected
Not detected
Approximately
20 CPU
recovered
Not detected
Not detected
In the case of rubber, there is some evidence to suggest that the longer exposure time at lower
concentration (cause by material demand) is less sporicidal than the higher concentration of the exposure
as designed (baseline fumigation with little demand). The EOGas system is designed by the manufacturer
with a safety factor of 50 % more concentration*time exposure than is generally accepted as effective
(750 mg/liter*hour vs. 500 mg/liter*hour). All tests wherein Bacillus subtilis spores were detected after
exposure were performed at RH conditions below the manufacturer's recommendations, and with half of
the recommended EtO (5 gram cartridge in an 11 gram sterilization bag). These conditions had been
chosen to better define cusp requirements for sterilization.
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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