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EPA/600/R-14/229
September 2014
Evaluation of Methyl Iodide for the
Inactivation of Bacillus anthracis
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), funded, directed
and managed this work through Contract Number EP-C-10-001 with Battelle. This report has been
peer and administratively reviewed and has been approved for publication as an EPA document. The
views expressed in this report are those of the authors and do not necessarily reflect the views or
policies of the Agency. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use of a specific product.
Questions concerning this document or its application should be addressed to:
Shannon D. Serre, Ph.D.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-3817
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Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
U.S. Environmental Protection Agency (EPA)
Leroy Mickelsen
Michael Nalipinski
Eletha Brady-Roberts
Ramona Sherman
Joseph Wood
Battelle Memorial Institute
in
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Executive Summary
The U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD) is
striving to protect human health and the environment from adverse impacts resulting from acts of
terror by investigating the effectiveness and applicability of technologies for homeland security (HS)-
related applications. The purpose of this evaluation was to determine the decontamination efficacy of
methyl iodide (Mel) fumigant in inactivating Bacillus anthracis (causative agent for anthrax) spores
on indoor and outdoor materials. Mel has been proposed as an alternative to methyl bromide to
possibly expand the decontaminant capacity in the case of a wide area B. anthracis incident.
Although all pesticide registrations of methyl iodide products have been cancelled, the reagent is still
widely available. The objective of this study was to provide an understanding of the performance of
Mel to guide its use and implementation in HS applications. When assessing options for
decontamination following intentional release of B. anthracis., it is important to know the extent to
which factors may impact the decontamination efficacy. These tests were designed to be screening
tests to determine if Mel was effective at inactivating B. anthracis and to assess potential surrogates
for future work.
This evaluation focused on the decontamination of six types of common indoor and outdoor
materials: glass, ceiling tile, carpet, painted wallboard paper, bare pine wood and unpainted concrete.
Decontamination efficacy tests were conducted with spores of virulent B. anthracis Ames and non-
virulent strains (i.e.., B. atrophaeus and B. anthracis Sterne), the latter organisms included to assess
their potential as surrogates for future studies. Decontamination efficacy was quantified in terms of
log reduction (LR), based on the difference in the number of bacterial spores recovered from the
positive control coupons and test coupons. Tests in which a >6 log reduction (LR) value is considered
effective on the materials tested for a given set of fumigation conditions (Mel concentration, temperature,
and RH). Tests were conducted with various temperatures, relative humidity (RH) levels,
concentrations of Mel, and contact times to assess the effect of these fumigation operational
parameters on decontamination efficacy. An attempt was made to assess operational parameters that
are achievable in the field in the case of a wide area event. For example, higher temperatures such as
37 °C (99 °F) would be difficult to achieve for large buildings and may be practical only for smaller
items that can be placed inside heated chambers.
Summary of Results
Fourteen tests were conducted with Mel, with concentrations of 100, 200, 300 or 400 milligrams per
liter (mg/L). Additionally, the temperature during testing was either 25 or 37 °C, the RH either 45 or
70 % and the contact times ranged from 12 to 48 hours. Table ES-1 shows the contact time required
to achieve >6 LR on all material types tested and at all operational parameters.
IV
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Table ES-1. Contact Time Required to Achieve >6 LR on all Materials*
Target Mel
Concentration
(mg/L)
100
200
300
300
300
400
Target
Temperature
(°C)
25
25
25
25
37
37
Target
RH
(%)
70
70
45
70
70
70
Time (hours) Required to Achieve >6 LR on
All Materials
B.a. Ames
<48C
>12 and <24
>36
>12 and <24
>24 and <36
<12C
R atrophaem
b
—
-
>36
>36
>24
Ro. Sterne
>48
>24
>36
>12 and <24
<36C
--
Test Number
Reference3
14
11,13
10,12
6,8,9
1,2,5,7
3,4
* Materials tested were glass, ceiling tile, carpet, painted wallboard paper, bare pine wood and unpainted concrete.
a Contact times and microorganism tested may be variable between tests. Detailed data from each test number can be referenced in Tables A-l
through A-3 in Appendix A.
b"~" Not Tested.
0 < indicates that no experiment was conducted to assess efficacy less than the listed contact time and that >6 LR was achieved at this contact
time.
Conditions were found that were effective for all material types and spore type used. The data
generated from this evaluation suggest that B. atrophaeus may be a conservative surrogate for B.a.
Ames when assessing the decontamination efficacy of Mel. However, this organism was tested only at
300 and 400 mg/L Mel and at a high RH (70 %). More evaluations at lower concentrations of Mel, as
well as various temperatures and RH, are needed to fully assess B. atrophaeus as a potential surrogate
for B.a. Ames for use in Mel tests.
Additional testing was completed using B.a. Sterne as a potential surrogate for B.a. Ames. The data
collected in this evaluation show that B.a. Sterne is an appropriate surrogate for B.a. Ames when tested
at 100, 200, or 300 mg/L Mel, 25 or 37 °C, and 45 or 70 % RH. B.a Sterne was less resistant than B.a.
Ames at 45 % RH and may, therefore, not be a good surrogate for testing with Mel at these parameters.
At 70 % RH, increasing temperature does not significantly increase the efficacy of Mel against B. a.
Ames; however, the effect of temperature may be masked at time points >12 hours as high efficacy
was achieved (>6LR) in most instances. The efficacy of increasing temperature was not tested at 45 %
RH.
RH has a significant impact on the overall efficacy of Mel against B.a. Ames. An efficacy >6 LR was
achieved only on painted wallboard paper and unpainted concrete when tested at 45 % RH. However,
increasing the RH from 45 % to 75 % resulted in a significant increase in decontamination efficacy to
complete inactivation on most material types.
Increasing the Mel concentration from 200 to 300 mg/L had a significant impact on Mel efficacy at 25
°C and 70 % RH for 12 hours. However, this increase in concentration did not significantly increase
Mel efficacy at other combinations of parameters tested. Complete inactivation of B.a. Ames on all
materials was achieved when the Mel concentration was increased to 400 mg/L.
When increasing the contact time from 24 to 36 hours at either 200 or 300 mg/L Mel at either 45 or 70
% RH, no significant increase of Mel efficacy was observed. However, increasing the contact time
from 12 to 24 hours at 200 or 300 mg/L, 25 °C, and 70 % RH resulted in a significant increase of
efficacy of Mel against B.a. Ames.
Mel appears to be an effective decontaminant against B.a. Ames under specific combinations of
concentration, temperature, RH, and contact time. In general, elevated RH appears to reduce the need
for increased Mel and contact time to maintain efficacy. At 45 % RH, increasing the contact time did
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not significantly improve the efficacy of Mel against B.a. Ames. However, more testing at this RH
level is needed to confirm these data as only two tests were completed using this parameter. More
testing is also needed to confirm the data presented here as well as to test additional materials and
combinations of parameters.
VI
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Contents
Disclaimer ii
Acknowledgments iii
Executive Summary iv
Abbreviations/Acronyms x
1.0 Introduction 1
2.0 Technology Description and Test Matrices 2
2.1 Technology Description 2
2.2 Test Matrix 2
3.0 Test Procedures 3
3.1 Biological Agents 3
3.2 Test Materials 4
3.3 Preparation of Coupons 5
3.4 Coupon Extraction and Biological Agent Quantification 5
3.5 Decontamination Efficacy 6
3.6 Surface Damage 8
4.0 Fumigation Description and Procedures 9
5.0 Quality Assurance/Quality Control 13
5.1 Equipment Calibration 13
5.2 QC Results 13
5.2.1 Operational Parameters 13
5.3 Audits 14
5.3.1 Performance Evaluation Audit 14
5.3.2 Technical Systems Audit 14
5.3.3 Data Quality Audit 15
5.4 QA/QC Reporting 15
5.5 Data Review 15
6.0 Summary of Results and Discussion 16
6.1 Comparing Efficacy for the Different Species 16
6.2 Effects of Test Materials on Mel efficacy for B.a. Ames 17
6.3 Effects of Temperature on Efficacy of Mel against B. anthracis Ames 22
6.4 Effect of Relative Humidity on Efficacy of Mel against B. anthracis Ames 23
6.5 Effects of Mel Concentration on Efficacy against B. anthracis Ames 23
6.6 Effects of Contact Time on Efficacy of Mel against B. anthracis Ames 24
6.7 Surface Damage to Materials 25
6.8 Summary and Conclusion 25
7.0 References 27
Figures
Figure 3-1. Coupon types from left to right: ceiling tile, carpet, glass, painted wallboard paper,
bare pine wood, and unpainted concrete 4
Figure 3-2. Liquid inoculation of coupon using a micropipette 5
Figure 4-1. Schematic of Mel decontamination test chamber housed inside BSC III chamber.. 10
Figure 4-2. OMA-300 Gas Analyzer 11
vn
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Figure 6-1. Summary of Mel efficacy (Tests 1-4) results, by material, against B. anthracis
Ames 18
Figure 6-2. Summary of Mel efficacy (Tests 5-8) results, by material, against B. anthracis
Ames 19
Figure 6-3. Summary of Mel efficacy (Tests 9-12) results, by material, against B. anthracis
Ames 20
Figure 6-4. Summary of Mel efficacy (Tests 13-14) results, by material, against B. anthracis
Ames 21
Figure 6-5. Effect of temperature on Mel decontamination efficacy against B. anthracis Ames at
300 mg/LMel and 70% RH (average log reduction for all test materials) 23
Figure 6-6. Effect of relative humidity on Mel decontamination efficacy against B. anthracis
Ames (average log reduction for all test materials) 23
Figure 6-7. Summary of effects of increasing concentration on average Mel decontamination
efficacy for B.a. Ames (average log reduction for all test materials) 24
Figure 6-8. Summary of the effect of contact time on average Mel decontamination efficacy
against B. anthracis Ames (average log reduction for all test materials) 25
Figure C-l. Summary of Mel efficacy against B. anthracis Ames on glass and bare pine wood.
Results shown in average log reduction± CI 2
Figure C-2. Summary of Mel efficacy against B. anthracis Ames on ceiling tile and carpet.
Results shown in average log reduction ± CI 3
Figure C-3. Summary of Mel efficacy against B. anthracis Ames on painted wallboard paper and
unpainted concrete. Results shown in average log reduction ± CI 3
Tables
Table ES-1. Contact Time Required to Achieve >6 LR on all Materials* v
Table 2-1 Mel Test Matrix 2
Table 3-1. Test Materials 4
Table 5-1. Actual Fumigation Conditions for Mel Tests 14
Table 5-2. Performance Evaluation Audits 14
Table 6-1. Contact Time Required to Achieve >6 LR on all Materials* 16
Table 6-2. Summary of Average Differences in Efficacy between B.a. Ames and Avirulent
Strains* 17
Table 6-3. Summary of B.a. Ames Log Reductions by Material Type 22
Table A-l. Inactivation of B. anthracis Ames Spores using Methyl Iodidea 1
Table A-1. Inactivation of B. anthracis Ames Spores using Methyl Iodidea (Continued) 2
Table A-2. Inactivation of B. atrophaeus Spores using Methyl Iodide3 3
Table A-3. Inactivation of B. anthracis Sterne Spores using Methyl Iodide3 4
Table B-l. Difference in Mel Efficacy between B. anthracis Ames and.8. atrophaeus* 1
Table B-2. Difference in Mel Efficacy between B. anthracis Ames and B. anthracis Sterne* . 2
Table C-1. Effect of Increasing Temperature at High Relative Humidity on Mel Efficacy* 4
Table C-2. Effect of Increasing Relative Humidity at Low and High Temperatures on B.
anthracis Ames* 5
Table C-3. Effect of Increasing Mel Concentration at High Relative Humidity on B. anthracis
Ames* 6
Table C-4. Effect of Increasing Contact Time at High Relative Humidity on B. anthracis
Ames* 7
Vlll
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Table C-5. Effect of Increasing Contact Time at Low Relative Humidity on B. anthracis
Ames* 8
List of Appendices
Appendix A Detailed Test Results 1
Appendix B Comparing Efficacy for the Different Microorganisms 1
Appendix C Effects of Materials and Operational Parameters on Mel Efficacy 1
IX
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Abbreviations/Acronyms
B. a. Bacillus anthracis
E.g. Bacillus atrophaeus
BBRC Battelle Biomedical Research Center
BSC biological safety cabinet
CPU colony-forming unit(s)
CI confidence interval
cm centimeter(s)
°C degree(s) Celsius
DNA deoxyribonucleic acid
EPA U.S. Environmental Protection Agency
HC1 hydrochloric acid
HS homeland security
kGy kilogray(s)
L liter(s)
LAL Limulus Amebocyte Lysate
Ib pound(s)
LED light-emitting diode
LR log reduction
Mel methyl iodide
min minute(s)
mg milligram(s)
mL milliliter(s)
\\L microliter(s)
NA not applicable
NHSRC National Homeland Security Research Center
ORD EPA Office of Research and Development
PBST phosphate-buffered saline + 0.1% Triton X-100
PCR polymerase chain reaction
QA quality assurance
QC quality control
QMP Quality Management Plan
RH relative humidity
rpm revolution(s) per minute
SD standard deviation
SE standard error
SFW sterile filtered water (cell-culture grade)
T&E Technology and Evaluation
TSA technical systems audit
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1.0 Introduction
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
(HSRP) is helping protect human health and the environment from adverse impacts resulting from
the release of chemical, biological, or radiological agents. With an emphasis on decontamination and
consequence management, water infrastructure protection, and threat and consequence assessment,
the HSRP is working to develop tools and information that will help detect the intentional
introduction of chemical or biological contaminants into buildings or water systems; contain these
contaminants; decontaminate buildings, outdoor environments, or water systems; and facilitate the
disposal of material resulting from restoration activities.
This evaluation studied the efficacy of methyl iodide (Mel) against Bacillus anthracis (B.a.) Ames
and surrogate spores applied to indoor and outdoor materials (glass, ceiling tile, carpet, painted
wallboard paper, bare pine wood and unpainted concrete). Decontamination efficacy was determined
based on the log reduction (LR) in viable spores recovered from the inoculated samples (with and
without exposure to Mel). A decontaminant or fumigant technology is considered to be effective via
AOAC 966.04 if a 6 LR or greater is achieved on the materials tested for a given set of fumigation
conditions (Mel concentration, temperature, and RH)2.
Lastly, another objective was to obtain side-by-side efficacy data for B.a. Ames and other microbes
that could be used to assess the suitability of potential surrogates for B. a. Ames when decontaminating
with Mel.
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2.1
2.0 Technology Description and Test Matrices
Technology Description
Mel (99.5 %, lofma Chemical, Covington, KY) is a colorless liquid organic compound with a sweet
pungent odor and a boiling point of 42.4 degrees Celsius (°C). Although very few incidences
involving humans have been reported, Mel is considered a potential occupational carcinogen and
exhibits moderate to high acute toxicity for inhalation and ingestion.1 Mel has been used widely in
the past as a soil fumigant in place of methyl bromide. Hence, the utility of Mel for decontaminating
and sterilizing other materials commonly found in buildings such as the materials tested here (glass,
bare pine wood [i.e., wood trim], carpet, painted wallboard paper, unpainted concrete, and ceiling
tile) was of interest to EPA.
2.2 Test Matrix
The test matrix for the Mel fumigation tests is shown in Table 2-1. Each test was performed using six
materials inoculated with B. anthracis or a potential surrogate. Operational parameters were chosen
in an attempt to assess parameters that are achievable in the field in the case of a wide area event. For
example, higher temperatures such as 37 °C (99 degrees Fahenheit [°F]) would be difficult to
achieve for large buildings and may be practical only for smaller items that can be placed inside
heated chambers. Tests focused on the use of a lower temperature (i.e., 25 °C) that is more easily
achievable in larger applications.
Table 2-1 Mel Test Matrix
Test
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Materials
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
Microorganisms*
B. anthracis Ames
B. atrophaeus
B. anthracis Ames
B. anthracis Steme
Target Operational Parameters
Mel
Concentration
(mg/L)
300
300
400
400
300
300
300
300
300
300
200
300
200
100
Temperature
(°C)
37
37
37
37
37
25
37
25
25
25
25
25
25
25
Relative
Humidity
(RH,%)
70
70
70
70
70
70
70
70
70
45
70
45
70
70
Contact
Time
(hours)
24
12
24
12
36
36
36
24
12
24
24
36
12
48
*Test material coupons were inoculated with both microorganisms listed were inoculated with the organism listed on separate coupons.
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3.0 Test Procedures
This section provides an overview of the procedures used for the bench-scale evaluation of Mel to
inactivate B. anthracis Ames and potential surrogate spore species on six different materials. Testing
was performed in accordance with the peer-reviewed and EPA approved Test/Quality Assurance
(QA) Plan for the Evaluation of Methyl Iodide for the Inactivation of Bacillus anthracis and
associated Amendment I.2 The test/QA plan provides additional procedural details that are not
included in this report.
3.1 Biological Agents
The virulent B.a. spores used for this testing were prepared from a qualified stock of the Ames strain
at the Battelle Biomedical Research Center (BBRC, Lot B21, West Jefferson, OH) using a BioFlo
3000 fermenter (New Brunswick Scientific Co., Inc., Edison, NJ). The spore lot was subject to a
stringent characterization and qualification process required by the Battelle standard operating
procedure for spore production. Specifically, the spore lot was characterized prior to use by
observation of colony morphology, direct microscopic observation of spore morphology, and size
and determination of percent refractivity and percent encapsulation. In addition, the number of viable
spores was determined by colony count and expressed as colony forming units per milliliter
(CFU/mL). Theoretically, once plated onto bacterial growth media, each viable spore germinates and
can yield one CPU. Variations in the expected colony phenotypes were recorded. Endotoxin
concentration of each spore preparation was determined by the Limulus Amebocyte Lysate (LAL)
assay to assess whether contamination from gram-negative bacteria occurred during the propagation
and purification process of the spores. Genomic deoxyribonucleic acid (DNA) was extracted from
the spores and DNA fingerprinting by polymerase chain reaction (PCR) was done to confirm the
genotype. This work was confirmed by an independent third party. The virulence of the spore lot was
measured by challenging guinea pigs intradermally with a dilution series of spore suspensions, and
virulence was expressed as the intradermal median lethal dose. In addition, testing was conducted for
robustness of the spores via hydrochloric acid (HC1) resistance.
Using growth from a stock culture, B.a. Sterne 34f2 (Cat. No. 19104, Colorado Serum Company,
Denver, CO) was inoculated into 10 mL tubes of nutrient broth and incubated at 37 °C in a shaking
incubator (Model 3877, Labline Instruments, Kerala, India) for 24 ± 2 hours at approximately 150
revolutions per minutes (rpm). This culture was used to inoculate amended nutrient agar plates.
Plates were inoculated with 500 microliters (jiL) of the culture and spread with a sterile plate
spreader (KG-5P, Arben Bioscience Inc., Rochester, NY),. Plates were inverted (with no shaking)
and incubated at 37 °C for 12 to 14 days. Following incubation, plates were harvested by washing
with 10 mL sterile water and scraped into sterile tubes. The harvested spores were centrifuged at
5000 rpm and washed with water three times and resuspended in sterile water. The prepared spores
were examined via microscopy and determined to have >95 % refractile spores with <5 % cellular
debris. No further activities were performed to verify the identity of the organism.
The B. atrophaeus (B.g.) spores (Lot 19076-03268) were supplied in powder form by EPA,
originally obtained from Dugway Proving Ground (Tooele County, UT). No further activities were
performed to verify the identity of the organism.
Both B.a. stock spore suspensions were prepared in sterile filtered water (SFW) at an approximate
concentration of 1 x 109 CFU/mL and stored under refrigeration at 2 to 8 °C. Similarly, the B.
atrophaeus stock spore suspensions were prepared in sterile phosphate-buffered saline containing
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0.1% Triton X-100 surfactant (PBST; Sigma, St. Louis, MO) at the same concentration and stored at
2 to 8 °C. This buffer was chosen to be consistent with previous work conducted with the same E.g.
spores at EPA.
3.2 Test Materials
Decontamination testing was conducted on glass, ceiling tile, carpet, painted wallboard paper, bare
pine wood and unpainted concrete. Information on these materials is presented in Table 3-1, and a
picture of each is presented in Figure 3-1. Material coupons were cut to uniform length and width
from a larger piece of stock material. Materials were prepared for testing either by sterilization via
gamma irradiation at -40 kilograys (kGy; STERIS Isomedix Services, Libertyville, IL) or by
autoclaving at 121 °C for 15 minutes. Gamma-irradiated material coupons were sealed in 6 mil Uline
Poly Tubing (Uline, Chicago, IL) and autoclaved coupons were sealed in sterilization pouches
(Fisher, Pittsburgh, PA) to preserve sterility until the coupons were ready for use.
Table 3-1. Test Materials
Material
Glass
Ceiling
Tile
Carpet
Painted
Wallboard
Paper
Bare Pine
Wood
Unpainted
Concrete
Lot, Batch, or ASTM No., or
Observation
C1036
Armstrong® B513, classic fine textured
Shaw Swizzle EcoWorx, Style: 10401
Color: Jacks
Roller painted on one side using Martin
Senour Paints. One primer (#71-1 185) and
two finish (flat, #70-1001) coats
Generic molding
ASTM C90 cinder block
Manufacturer/
Supplier Name
Brooks Brothers,
Columbus, OH
Armstrong,
Columbus, OH
Shaw Industries,
Dalton, GA
United States
Gypsum
Company,
Chicago, IL
Lowes,
Columbus, OH
Wellnitz
Columbus, OH
Approximate Coupon
Size,
width x length x
thickness
1.9 centimeters (cm) x 7.5
cm x 0.3 cm
1.9 cm x 7.5 cm x 0.3 cm
1.9 cm x 7.5 cm x 0.3 cm
1.9 cm x 7.5 cm x 0.1 cm
1.9 cm x 7.5 cm x 0.3 cm
1.9 cm x 7.5 cm x 0.6 cm
Material
Preparation
Autoclave
Gamma Irradiation
Gamma Irradiation
Gamma Irradiation
Gamma Irradiation
Autoclave
Figure 3-1. Coupon types from left to right: ceiling tile, carpet, glass, painted wallboard
paper, bare pine wood, and unpainted concrete.
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3.3 Preparation of Coupons
Test and positive control coupons were placed on a flat surface within a Class II biological safety
cabinet (BSC) and inoculated with approximately 1 x 108 CPU of viable B.a. Ames (or indicated
surrogate) spores per coupon. Only one organism was inoculated on each individual coupon surface,
separate coupons were used for each organism. A 100 jiL aliquot of a stock suspension of
approximately 1 x 109 CFU/mL was dispensed using a micropipette applied as 10 jiL droplets across
the coupon surface (see Figure 3-2). This approach provided a more uniform distribution of spores
across the coupon surface than would be obtained through a single drop of the suspension. Although
application of the inoculum onto each material was uniform, the behavior of the inoculum droplets
was not. Droplets beaded on the surface of the glass (nonporous material) while they soaked into the
other porous materials after producing a liquid bead for a short period of time. The difference in the
behavior of the inoculum droplets on each material could lead to a variance in microorganism
distribution across coupons; however, this effect was not studied in this evaluation. After inoculation,
the coupons were transferred to a Class HI BSC and left undisturbed overnight to dry under ambient
conditions, approximately 22 °C and 40 % relative humidity (RH).
Figure 3-2. Liquid inoculation of coupon using a micropipette.
The number and type of replicate coupons used for each combination of material, decontaminant,
concentration, and environmental condition included:
• Five test coupons (inoculated with B. anthracis or surrogate spores and exposed to Mel)
• Five positive controls (inoculated with B. anthracis or surrogate spores but not exposed
to Mel)
• One laboratory blank (not inoculated and not exposed to Mel)
• One procedural blank (not inoculated and exposed to Mel).
On the day following liquid spore inoculation, coupons intended for decontamination (including
blanks) were transferred into a test chamber and exposed to the Mel fumigant using the apparatus
and application conditions specified in Section 4.0 of this report. Control coupons were added to the
control chamber as described in Section 4.0.
3.4 Coupon Extraction and Biological Agent Quantification
For sample extraction, test coupons, positive controls, and blanks were placed in 50 mL
polypropylene conical tubes containing 10 mL of sterile PBST. The vials were capped, placed on
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their side and agitated on an orbital shaker for 15 minutes (min) at approximately 200 rpm at room
temperature.
Residual viable spores were determined using a dilution plating approach. Following extraction, the
extract was removed, and a series of tenfold dilutions was prepared in sterile filtered water (SFW).
An aliquot (0.1 mL) of either the undiluted extract and/or each serial dilution was plated onto tryptic
soy agar in triplicate and incubated for 18 to 24 hours at 37 ±2 °C. Colonies were counted manually
and CFU/mL was determined by multiplying the average number of colonies per plate by the
reciprocal of the dilution. Dilution data representing the greatest number of individually definable
colonies were expressed as arithmetic mean ± standard deviation (SD) of the numbers of CFU
observed. Laboratory blanks controlled for sterility and procedural blanks controlled for viable
spores inadvertently introduced to test coupons. The target acceptance criterion was that extracts of
laboratory or procedural blanks were to contain no CFU.
After each decontamination test, the Mel test and control chambers were thoroughly cleaned (using
separate steps involving bleach, ethanol, water, then drying).
3.5 Decontamination Efficacy
The mean percent spore recovery from each coupon was calculated using results from positive
control coupons (inoculated, not decontaminated), by means of the following equation:
Mean % Recovery = [Mean CFUpc/CFUSpike] x 100 (1)
where Mean CFUpc is the mean number of CFU recovered from five replicate positive control
coupons of a single material, and CFUspjke is the number of CFU spiked onto each of those coupons.
The value of CFUspike was known from enumeration of the stock spore suspension. One aliquot of the
stock suspension was plated and enumerated on each day of testing to confirm CFUspike
concentration. Spore recovery was calculated for B.a. Ames or surrogate on each coupon, and the
results are included in Section 6 and Appendix A.
The performance or efficacy of Mel was assessed by determining the number of viable
organisms remaining on each test coupon after decontamination. Those numbers were compared
to the number of viable organisms extracted from the positive control coupons.
The number of viable spores of B.a. Ames or surrogate in extracts of test and positive control
coupons was determined to calculate efficacy of the decontaminant. Efficacy is defined as the extent
(as logic reduction or LR) to which viable spores extracted from test coupons after decontamination
were less numerous than the viable spores extracted from positive control coupons. The logarithm of
the CFU abundance from each coupon extract was determined, and the mean of those logarithm
values was then determined for each set of control and associated test coupons, respectively. Efficacy
of a decontaminant for a test organism/test condition on the /h coupon material was calculated as the
difference between those mean log values, i.e.:
Efficacy (LR) = (log 10 CFUcy ) - (log 10 CFUt t] ) (2)
where logic CFUcy refers to they individual logarithm values obtained from the positive control
coupons and logic CFUty refers to they individual logarithm values obtained from the individual
corresponding test coupons, and the overbar designates a mean value. In tests conducted under this
plan, there were five positive controls and five corresponding test coupons (i.e., j = 5) for each
coupon. A decontaminant or fumigant technology is considered to be effective via AOAC 966.04 if
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r\
a 6 LR or greater is achieved.
In the case where no viable spores were found in any of the five test coupon extracts after
decontamination, a CPU abundance of 1 was assigned, resulting in a logic CPU of 0 for that material.
This situation occurred when the decontaminant was highly effective, and no viable spores were
found on the decontaminated test coupons. In such cases, the final efficacy on that material was
reported as greater than or equal to (>) the value calculated by Equation 2.
The variances (i.e., the square of the SD) of the logio CFUcy and logio CFUty values were also
calculated for both the control and test coupons (i.e., S2Cy and 5%), and were used to calculate the
pooled standard error (SE) for the efficacy value calculated in Equation 2, as follows:
S2c S2t
where the number 5 again represents the number y' of coupons in both the control and test data sets.
Each efficacy result is reported as an LR value with an associated 95 % confidence interval (CI),
calculated as follows:
95 % CI = Efficacy (LR) ± (1.96 x SE) (4)
The significance of differences in efficacy across different test conditions and spore types was
assessed based on the 95 % CI of each efficacy result. Differences in efficacy were judged to be
significant if the 95 % CIs of the two efficacy results did not overlap. Any results based on this
formula are hereafter noted as significantly different. Note this comparison is not applicable when the
two efficacy results being compared are both reported with LRs as > some value.
The average difference in efficacy was determined when comparing the results of two tests and
reported as an LR value. This difference in efficacy was calculated as follows:
n
^ LRa,2 - LRa,l
Avg Diffeence in Eficacy (LI} - — (5)
n
where the letters a through n represent the material types, the number 1 represents B.a. Ames, and the
number 2 represents the avirulent (potential surrogate) microorganism for which results are being
compared. The letter n represents the number of materials tested, with n = 6 for Tests 1 through 8,
and equal to 4 for the remaining tests. When both values were >LR (indicating complete
inactivation), these were not included in the formula. A positive value indicates that the avirulent
organism was inactivated on average to a higher degree (i.e., it was less resistant) across the materials
tested compared to B.a. Ames.
In some instances, significant differences in average efficacy between tests were assessed with a
(6)
t-test using Microsoft® Excel, according to the formula below:
where and are the means of Tests 1 and 2, respectively. is the standard error of the
difference between Tests 1 and 2. This formula compares the averages of two tests to see if they are
-------
reliably different from each other. Using this formula, a p-value was assigned where indicated. If the
calculated p-value was <0.05, then the two sets of data were considered to be significantly different.
3.6 Surface Damage
The physical effect of Mel on the materials was qualitatively monitored during the evaluation. This
approach provided a gross visual assessment of whether the decontaminant altered the appearance of
the test materials. The procedural blank (coupon that is decontaminated, but has no spores applied)
was visually compared to a laboratory blank coupon (a coupon not exposed to the decontaminant and
having no spores applied). Obvious visible damage might include structural damage, surface
degradation, discoloration, or other aesthetic impacts.
-------
4.0 Fumigation Description and Procedures
Figure 4-1 shows a schematic drawing of the Mel test chamber and containment system. Mel
decontamination testing was conducted inside a 23-liter (L) glass test chamber. As a means of
secondary containment and laboratory personnel safety, this test chamber was housed inside a BSC
III cabinet.
Mel liquid was heated above the boiling point (-50 °C) to produce a vapor and injected into the test
chamber at the indicated target concentrations. Temperature was controlled using a
refrigerated/heated water bath, and RH was elevated using a Nafion tube pervaporation system
(Nafion, Toms River, NJ) (controlled using a water bath). Temperature and RH in the test chamber
were measured using an HMT368 temperature and humidity probe (Vaisala, Inc., Woburn, MA).
Temperature, RH, and Mel concentration were controlled with a CNI-822 controller (Omega
Engineering, Stamford, CT), and the data were recorded every minute during the contact time using
the associated iLOG software (Omega Engineering).
-------
Power Cord
Omega Gas
Controller
23L Test Chamber
ra
u
1'
_o
o
'CD
1/1
_ra
u
Automated Valve
Pressure Regulator
\
Omega RH
Controller
02-
O
O
Radiator & Fan
(EH
Mel Tank
o
Flow Cell
Heated Water
Bath/Temp
Controller
Humidification
System
WB = water bath
Indicates RH and temperature lines
Indicates Mel loop from chamber to flow cell
Indicates electrical lines to and from RH, temperature, and Mel concentration controllers
OMA-300 Mel
Analyzer
Figure 4-1. Schematic of Mel decontamination test chamber housed inside BSC III chamber.
10
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Liquid Mel was added to a 1 pound (Ib) lecture bottle and heated to approximately 50 °C and
pumped through a heated injection line to keep the compound in vapor form. Once injected into the
test chamber, the Mel vapor was measured continuously using an OMA-300 Gas Analyzer (Applied
Analytics, Houston, TX; Figure 4-2) during the entire contact time. This sensor was calibrated by
Applied Analytics for use with Mel up to 500 milligram/liter (mg/L) and displayed the percent Mel
concentration on a digital light-emitting diode (LED) display. A concentration of 1% Mel is equal to
approximately 58.1 mg/L. Mel vapor was added to the chamber, as necessary, to maintain the
specified concentration within ±10%. The OMA-300 meter included an air pump that pulled a vapor
sample from the test chamber through a flow cell at a controlled rate and exhausted the vapor back
into the test chamber (providing a continuous loop of fumigant). A low-speed fan was placed inside
the test chamber to ensure a homogeneous mixture of Mel was achieved.
Figure 4-2. OMA-300 Gas Analyzer.
®
A 9 L Lock & Lock airtight container (Lock & Lock, Farmers Branch, TX) served as the positive
control chamber. Fixed humidity point salts3 were added as a slurry to a separate container placed in
the bottom of the Mel positive control chamber. Sodium chloride was used to control the RH at 70 %
and potassium carbonate to control the RH at 45 %. The control chamber housing all positive control
coupons (five of each material-organism combination and blanks) was placed in an incubator (Innova
4230, Eppendorf, Enfield, CT) for all tests and set to the appropriate temperature (i.e., 25 or 37 °C).
The temperature and RH of the positive control chamber were measured and data logged using a
HOBO® data logger model U12-11 (Onset Computer Corporation, Cape Cod, MA).
As in previous studies,4'5 multiple coupons of each material were inoculated with the biological agent
and placed on a wire rack inside the test chamber. Only one agent was added to each coupon. Blank
(i.e., uninoculated) and positive control (i.e., inoculated but not decontaminated) samples were also
prepared for each material and were utilized with data from the test samples (inoculated and
decontaminated) to determine decontamination efficacy.
11
-------
The 14 Mel tests were conducted at target concentrations of 100, 200, 300, or 400 mg/L, as shown in
Table 2-1. Contact times ranged from 12 to 48 hours with target temperatures of either 25 or 37 °C
and target RH of either 45 or 70 %. During each test run, inoculated test coupons (five of each test
material-organism combination and blanks) were placed inside the Mel test chamber and the
chamber was sealed. The chamber was allowed sufficient time to equilibrate to the target temperature
and RH prior to start of the run. Once the temperature and RH were stable, Mel was slowly injected
into the chamber until the target concentration was reached. The test chamber remained sealed until
the end of the required contact time. At this time, the Mel was turned off, and the seal of the test
chamber broken by removing the lid. The test chamber and BSC HI were allowed to offgas until the
Mel levels in the chamber reached 0 %, or 0 mg/L, as read by the OMA-300 analyzer, which
happened within minutes of lid removal. At this time the samples were removed and processed as
stated in Section 3.4.
12
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5.0 Quality Assurance/Quality Control
QA/quality control (QC) procedures were performed in accordance with the Testing and Evaluation
(T&E) program Quality Management Plan (QMP) and the test/QA Plan. The QA/QC procedures
and results are summarized below.
5.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety cabinets) and monitoring devices (e.g.,
thermometer, hygrometer) used at the time of evaluation were verified as being certified, calibrated,
or validated.
5.2 QC Results
QC efforts conducted during decontaminant testing included positive control samples (inoculated,
not decontaminated), procedural blanks (not inoculated, decontaminated), laboratory blanks (not
inoculated, not decontaminated), and inoculation control samples (analysis of the stock spore
suspension).
All positive control results were within the target recovery range of 5 to 120 % of the inoculated
spores, except for some instances with ceiling tile, bare pine wood, and unpainted concrete. These
recoveries were as low as 2.47 %, 1.03 %, and 1.60 %, respectively. Low recoveries from these
coupon types are not uncommon due to the porosity of the materials. LRs of >6 logs were recovered
in all instances, even with the low recoveries. All procedural and laboratory blanks met the criterion
of no observed CPU for both organisms.
Inoculation control samples were taken from the spore suspension on the day of testing and serially
diluted, nutrient plated, and counted to establish the spore density used to inoculate the samples. The
spore density levels met the QA target criterion of 1 x 109 CFU/mL (±1 log) for all tests.
5.2.1 Operational Parameters
The temperature, RH, and Mel concentration during each test were controlled using Omega
controllers, as described in Section 4.0. These controllers were set to the target conditions and
allowed heat, RH, or Mel to be injected as needed to stay within target ranges of ±2 °C, ±20 % RH
and ±10 % Mel. Readings were taken once every minute for the duration of the contact time. The
actual operational parameters for each test are shown in Table 5-1 and reported as the average value
±SD.
The Mel tank ran out of liquid Mel approximately 20 hours into the 36-hour run for Test Number 7
(most likely due to an incomplete seal on the fumigation chamber door), resulting in no Mel vapor
being injected after this time and a slow decrease of Mel concentration in the fumigation chamber
over the remainder of the test. At the end of the 36-hour contact time, the concentration in the
chamber was approximately 122 mg/L Mel.
13
-------
Table 5-1. Actual Fumigation Conditions for Mel Tests
Test
Number
1
2
3
4
5
6
7§
8
9
10
11
12
13
14
Mel Concentration
(mg/L)
Target
300
300
400
400
300
300
300
300
300
300
200
300
200
100
Actual*
304.84 ± 5.73
316.19 ±17.32
401.26 ±0.23
406.51 ±16.10
305.61 ±6.57
305.45 ±4.95
258.01 ±0.55
308.67 ±11. 51
307.22 ±7.10
303. 95 ±4.98
200.47 ± 16.49
298.40 ±3. 19
203. 50 ±6.00
105. 32 ±8.60
Temperature (°C)
Target
37
37
37
37
37
25
37
25
25
25
25
25
25
25
Fumigation
Actual*
37.32 ±0.08
36.45 ±0.09
35. 16 ±0.69
37.31 ±0.06
37.07 ±0.08
25. 13 ±0.15
37.19±0.17
24.90 ±0.28
25.02 ±0.07
25.22 ±0.08
25. 19 ±0.09
25. 16 ±0.07
25.08 ±0.08
25. 32 ±0.24
Control
Actual*
36.89 ±0.90
36.87 ± 1.22
37.16 ±1.04
36.50 ± 1.36
37.08 ±0.84
24.63 ±0.14
37.18 ±0.41
24.77 ±0.21
24.58 ±0.23
24.91 ±0.29
24.70 ± 0.22
24.89 ±0.16
24.66 ± 0.23
24.77 ±0.14
RH (%)
Target
70
70
70
70
70
70
70
70
70
45
70
45
70
70
Fumigation
Actual*
65. 13 ±0.12
66.66 ±0.58
70.22 ±0.23
70.11 ±0.12
70.21 ±0.17
70.39 ±0.30
70.09 ±0.55
70.17 ±0.12
70.16±0.11
45. 84 ±0.45
70.16 ±0.19
47. 18 ±0.86
70.11 ±6.00
70.36 ±0.26
Control
Actual*
65.04 ±4. 14
64.69 ±3.06
66.00 ±3. 87
56.71 ±5.06
68.25 ±6.15
72.51 ±2.00
73.26 ±1.95
73.72 ±0.51
74.29 ± 0.47
60.02 ±5.80
65.22 ±5.99
52.01 ±2.25
73.05 ±0.42
74.98 ± 1.07
Contact
Time
(hours)1^
24
12
24
12
36
36
36
24
12
24
24
36
12
48
t
1 Data reported as average ± SD.
Contact time did not deviate from target during any test.
Mel tank ran out of liquid approximately 20 hours into the 36-hour run, resulting in a slow decrease in Mel concentration.
5.3 Audits
5.3.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these experiments. Table 5-2 summarizes the performance evaluation audits that were performed.
No performance evaluation audits were performed to confirm the concentration and purity of
B.a. or surrogate spores because quantitative standards do not exist for these organisms. The
control coupons and blanks support the spore measurements.
Table 5-2. Performance Evaluation Audits
Measurement
Volume of liquid from
micropipettes
Time
Temperature
Relative Humidity
Mel Concentration
Audit
Procedure
Gravimetric evaluation
Compared to independent clock
Compared to independent calibrated thermometer
Compare to independent calibrated hygrometer
Measure calibration gas
Allowable
Tolerance
± 10 %
± 2 seconds/hour
±2°C
± 20 %
± 10 %
Actual
Tolerance
±0.01% to 3. 17%
0 seconds/hour
± 0.00 °C
± 0.09 %
±0.01%
5.3.2 Technical Systems Audit
Observations and findings from the technical systems audit (TSA) were documented and submitted
to the laboratory staff leader for response. TSAs were conducted on February 5 and February 6, 2014
to ensure that the tests were being conducted in accordance with the appropriate test/QA plan and
14
-------
QMP. As part of the audit, test procedures were compared to those specified in the test/QA plan and
data acquisition and handling procedures were reviewed. None of the findings of the TSA required
corrective action.
5.3.3 Data Quality Audit
At least 10 % of the data acquired during the evaluation were audited. A QA auditor traced the data
from the initial acquisition, through reduction and statistical analysis, to final reporting to ensure the
integrity of the reported results. All calculations performed on the data undergoing the audit were
checked.
5.4 QA/QC Reporting
Each assessment and audit was documented in accordance with the test/QA plan and QMP. For these
tests, findings were noted (none significant) in the data quality audit, but no followup corrective
action was necessary. The findings were mostly minor data transcription errors requiring some
recalculation of efficacy results, but none were gross errors in recording. Copies of the assessment
reports were distributed to the EPA QA Manager and laboratory staff. QA/QC procedures were
performed in accordance with the test/QA plan.
5.5 Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in reports.
15
-------
6.0 Summary of Results and Discussion
The decontamination efficacy of Mel against virulent B.a. Ames and surrogates was evaluated at
target concentrations of 100, 200, 300, and 400 mg/L, target temperatures of 25 or 37 °C, target RH
of 45 or 70 %, and contact times ranging from 12 to 48 hours for a total of 14 tests. Table 6-1 shows
the contact time required to achieve > 6 LR (the level considered effective)2 on all material types
tested (glass, ceiling tile, carpet, painted wallboard paper, bare pine wood, and unpainted concrete)
and at all target operational parameters. Actual operational parameters as measured were well within
acceptable ranges (except for one deviation) and are detailed in Section 5. The detailed
decontamination efficacy results are found in Appendix A. As seen in the table, a contact time of 24
hours was required to achieve > 6 LR of B.a. Ames when fumigating at either 200 or 300 mg/L, 25
°C, and 70 % RH. Only 12 hours was required to achieve > 6 LR when the Mel concentration was
increased to 400 mg/L and temperature increased to 37 °C.
Table 6-1. Contact Time Required to Achieve >6 LR on all Materials*
Target Mel
Concentration
(mg/L)
100
200
300
300
300
400
Target
Temperature
(°C)
25
25
25
25
37
37
Target
RH
(%)
70
70
45
70
70
70
Time (hours) Required to Achieve >6 LR on All Materials
B.a. Ames
<48C
>12 and <24
>36
>12 and <24
>24 and <36
<12C
B. atrophaem
b
-
-
>36
>36
>24
B.a. Sterne
>48
>24
>36
>12 and <24
<36C
--
Test
Number
Reference3
14
11,13
10,12
6,8,9
1,2,5,7
3,4
* Materials tested were glass, ceiling tile, carpet, painted wallboard paper, bare pine wood and unpainted concrete.
a Contact times and microorganism tested may be variable between tests. Detailed data from each test number can be referenced in Tables A-l
through A-3 in Appendix A.
b"~" Not Tested.
0 < indicates that no experiment was conducted to assess efficacy less than the listed contact time and that >6 LR was achieved at this contact time.
6.1 Comparing Efficacy for the Different Species
Results comparing the average difference in decontamination efficacy for the microorganisms are
shown in Table 6-2. Testing was first conducted using B. atrophaeus as a potential surrogate for B.a.
Ames (Tests 1 through 6). The results showed that although B. atrophaeus is more resistant than B.a.
Ames to Mel exposure, resulting in average differences ranging from -1.21 to -6.77 LR. Therefore,
B. atrophaeus was eliminated from further testing, since a surrogate should be at least as resistant as the
virulent strain. Thus B.a. Sterne was subsequently tested as an additional potential surrogate.
The results in Table 6-2 (grouped by target test parameters) show that B.a. Sterne was more resistant
(lower decontamination efficacy) to Mel as compared to B.a. Ames at the high RH condition (70 %),
with the average difference in efficacy ranging from -0.18 to -5.17 LR. In contrast, at 45 % RH, B.a.
Sterne was less resistant to Mel than B.a. Ames in the two times tested with average differences of
0.33 and 0.95 LR.
16
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Table 6-2.
Strains*
Summary of Average Differences in Efficacy between B. a. Ames and Avirulent
Test
Number
2
1
5
7
9
8
6
10
12
4
3
11
13
14
Target Mel
Concentration
(mg/L)
300
300
300
300
300
300
300
300
300
400
400
200
200
100
Target
Temperature
(°C)
37
37
37
37
25
25
25
25
25
37
37
25
25
25
Target RH
(%)
70
70
70
70
70
70
70
45
45
70
70
70
70
70
Contact
Time
(hours)
12
24
36
36
12
24
36
24
36
12
24
24
12
24
Average Difference in Efficacy
B. atrophaeus
-5.53
-2.04
-1.21
-
—
-
-6.77
-
-
-5.47
-1.95
—
-
-
B.a. Sterne
a
-
-
-0.35
-5.17
-0.18
—
0.33
0.95
-
-
-1.97
-2.14
-1.63
* Results shown as average difference in efficacy (log reduction). A positive result indicates that the avirulent microorganism was inactivated to
a higher degree (less resistant) than B.a. Ames
a "-" Not Tested.
6.2 Effects of Test Materials on Mel efficacy for B.a. Ames
The LR results by material, for each test, are shown in the bar graphs in Figures 6-1 through 6-4.
Differences in efficacy between two materials are significant if the 95 % CIs of the two efficacy
results do not overlap. Table 6-3 shows the average LR for each of the six materials across all 14
tests. Further details on the decontamination efficacy results are found in Appendices A through
C.
17
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Test #1:300 mg/L Mel, 37 °C, 70 % RH, 24 hours
Test #2: 300 mg/L Mel, 37 °C, 70 % RH, 12 hours
Glass Ceiling Carpet Painted Bare
Tile Wallboard Pine
Paper Wood
Test #3: 400 mg/L Mel, 37 °C, 70 % RH, 24 hours
Unpainted
Concrete
Glass Ceiling Carpet Painted Bare
Tile Wallboard Pine
Paper Wood
Test #4: 400 mg/L Mel, 37 °C, 70 % RH, 12 hours
Unpainted
Concrete
Glass Ceiling Carpet Painted Bare Unpainted
Tile Wallboard Pine Concrete
Paper Wood
Glass Ceiling Carpet Painted Bare Unpainted
Hie Wallboard Pine Concrete
Paper Wood
(Results shown are average log reduction ± CI. *Complete inactivation achieved.)
Figure 6-1. Summary of Mel efficacy (Tests 1-4) results, by material, against B. anthracis Ames.
18
-------
Test #5:300 mg/L Mel, 37 °C, 70 % RH, 36 hours
Test #6: 300 mg/L Mel, 25 °C, 70 % RH, 36 hours
Glass
Unpainted
Concrete
Test #7: 300 mg/L Mel, 37 °C, 70 % RH, 36 hours
Test #8: 300 mg/L Mel, 25 °C, 70 % RH, 24 hours
Glass
Painted
\Vallboard
Paper
Bare
Pine
Wood
Unpainted
Concrete
(Results shown in average log reduction ± CI. *Complete inactivation achieved.)
Figure 6-2. Summary of Mel efficacy (Tests 5-8) results, by material, against B. anthracis Ames.
19
-------
Test #9:300 mg/L Mel, 25 °C, 70 % RH, 12 hours
Test #10: 300mg/L Mel, 25 °C, 45 % RH, 24 hours
Unpainted
Concrete
Test #11: 200 mg/L Mel, 25 °C, 70 % RH, 24 hours
Test #12: 300 mg/L Mel, 25 °C, 45 % RH, 36 hours
I
Glass
Unpainted
Concrete
Glass
Unpainted
Concrete
(Results shown in average log reduction ± CI. *Complete inactivation achieved.)
Figure 6-3. Summary of Mel efficacy (Tests 9-12) results, by material, against B. anthracis Ames.
20
-------
Test #13: 200 mg/L Mel, 25 °C, 70 % RH, 12 hours
Test #14:100 mg/L Mel, 25 °C, 70 % RH, 48 hours
Painted
Wallboard
Paper
Bare
Pine
Wood
Unpainted
Concrete
Figure
(Results shown in average log reduction ± CI. *Complete inactivation achieved.)
6-4. Summary of Mel efficacy (Tests 13-14) results, by material, against B. anthracis Ames.
21
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Table 6-3. Summary of B.a. Ames Log Reductions by Material Type
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
LR Across All Tests
Minimum
1.07
1.86
2.27
2.20
1.17
2.11
Maximum
7.70
7.21
8.03
7.96
6.91
7.37
Average ± SD
5.64 ±2.48
5.72 ±1.85
6.80 ±2.05
7.35 ±1.49
5.69 ±2.05
6.82 ±1.36
In general, glass, ceiling tile, and wood were the most difficult materials to decontaminate (exhibited
lower efficacy than the other three material types). In previous work, B. anthmcis deposited onto
glass and wood materials was the least resistant to fumigant decontamination4'5; however, this is the
first instance of Mel being tested on these material types so comparisons cannot reasonably be made.
Testing with MeBr showed similar results on wood coupons6.
6.3 Effects of Temperature on Efficacy of Mel against B. anthracis Ames
The decontamination efficacy of Mel against virulent B.a. was evaluated at target temperatures of 25
or 37 °C. These temperatures were tested at various combinations of RH, Mel concentration, and
contact time; the results are organized by test condition in Figure 6-5 to visualize the effect of
temperature. Additional analyses of the effect of temperature, including LR data for each specific
material, are included in Appendices A and C.
I25°C
i37°C
24 36
Contact Time (Hours)
36
(Test numbers shown at bottom of each bar. Results shown in average log reduction for all test materials ± standard
deviation.)
22
-------
Figure 6-5. Effect of temperature on Mel decontamination efficacy against B. anthracis
Ames at 300 mg/L Mel and 70% RH (average log reduction for all test materials).
At 70 % RH, temperature (while holding all other test variables constant) had no significant effect on
efficacy (based on t-test); however, it is difficult to assess the effects of temperature on Mel efficacy
due to the high level of efficacy obtained in all instances.
6.4 Effect of Relative Humidity on Efficacy of Mel against B. anthracis Ames
The decontamination efficacy of Mel against B.a. Ames was evaluated at target RHs of 45 or 70 %.
The actual %RH conditions for each test are shown in Section 5. These RH levels were tested at 25
°C, 300 mg/L Mel, and either 24 or 36 hours (Figure 6-6). Detailed tabulated results to assess the
effect of RH are summarized in Table C-2 of Appendix C. The average decontamination efficacy
across all materials increased significantly, based on t-test results, with increasing RH in both
instances.
1
•c
0
at
3
U
I45%RH
170 %RH
300, 25,24 300, 25,36
Mel Concentration (mg/L), Temperature (°C), Contact Time (Hours)
(Test numbers shown at bottom of each bar. Results shown in average log reduction for all test materials ± standard
deviation.)
Figure 6-6. Effect of relative humidity on Mel decontamination efficacy against B.
anthracis Ames (average log reduction for all test materials).
6.5 Effects of Mel Concentration on Efficacy against B. anthracis Ames
The decontamination efficacy of Mel against virulent B.a. was also evaluated at target concentrations
of 100, 200, 300, and 400 mg/L. Refer to Section 5 for the actual Mel concentrations achieved for
each test. These concentrations were tested at various combinations of temperature and RH. There
were four test conditions, all at 70 % RH, for which results could be compared to assess the effect of
increasing Mel concentration. These comparisons are shown in Figure 6-7 with detailed results for
each material presented in Table C-3 of Appendix C.
23
-------
12 00 ing L Mel
1300 ing L Mel
25.70.12 25,70.24 37,70,12
Temperature fC), %RH. Contact Time (Hours)
37,70,24
(Test numbers shown at bottom of each bar. Results shown in average log reduction for all test materials ± standard
deviation.)
Figure 6-7. Summary of effects of increasing concentration on average Mel
decontamination efficacy for B. a. Ames (average log reduction for all test materials).
In general, increasing the concentration from 200 to 300 mg/L, while keeping the RH at 70 %, did
not significantly increase the efficacy of Mel against B.a. Ames. Due to the high level of efficacy
obtained for three out of four conditions, it is difficult to assess the effect that concentration may have
on the efficacy of Mel. However, at 25 °C and a contact time of 12 hours, increasing the
concentration from 200 to 300 mg/L Mel did have a significant impact, based on t-test results, with
average LRs of 2.41 and 6.24, respectively.
6.6 Effects of Contact Time on Efficacy of Mel against B. anthracis Ames
The effect of increasing the contact time on the efficacy against B.a. Ames was also assessed. The
contact times tested ranged from 12 to 48 hours; there were five test conditions for which results
could be compared to assess the effect of increasing contact time at either 12, 24, or 36 hours with
RH of 45 or 70%. These comparisons are summarized in Figure 6-8 and presented in full detail in
Tables C-4 and C-5 of Appendix C.
24
-------
112 hours
12-1 hours
36 hours
300,25,45 200.25,-0 300,25, "0 300,3'. 70
Mel Coiiceutratiu (mg/L). Temperature (°C). °oRH
400.3', 7Q
(Test numbers shown at bottom of each bar). Results shown in average log reduction for all test materials ± standard
deviation.
Figure 6-8. Summary of the effect of contact time on average Mel decontamination
efficacy against B. anthracis Ames (average log reduction for all test materials).
There was no significant effect of increasing contact time from 24 to 36 hours on decontamination
efficacy when fumigating at 300 mg/L Mel and at 25 or 37 °C. However, when increasing the
contact time from 12 to 24 hours at either 200 or 300 mg/L Mel, 25 °C, and 70 % RH, there was a
significant increase in efficacy, based on t-test results.
6.7 Surface Damage to Materials
At the end of each decontamination test, the procedural blanks were visually compared to the
laboratory blanks, and test coupons were visually compared to positive controls to assess any impact
Mel may have had on each material type. Based on the visual appearance of the decontaminated
coupons, there were no apparent changes in the color, reflectivity, or roughness of the six material
surfaces after being exposed to Mel.
6.8 Summary and Conclusion
This evaluation focused on the decontamination of six types of common indoor and outdoor
materials: glass, ceiling tile, carpet, painted wallboard paper, bare pine wood and unpainted concrete.
Decontamination efficacy tests were conducted with spores of virulent B.a. Ames and non-virulent
strains (i.e., B. atrophaeus and B.a. Sterne), the latter organisms included to assess their potential as
surrogates for future studies with Mel. Tests were conducted with various temperatures, relative
humidity (RH) levels, concentrations of Mel, and contact times to assess the effect of these
fumigation operational parameters on decontamination efficacy.
Conditions were found that were effective for all material types and spore types used. The data
generated from this evaluation suggest that B. atrophaeus may be a conservative surrogate for B.a.
Ames when assessing the decontamination efficacy of Mel. The data collected in this evaluation
25
-------
show that B.a. Sterne is an appropriate surrogate for B.a. Ames when tested at 100, 200, or 300 mg/L
Mel, 25 or 37 °C, and 45 or 70 % RH. B.a Sterne was less resistant than B.a. Ames at 45 % RH and
may, therefore, not be a good surrogate for testing with Mel at these parameters.
RH has a significant impact on the overall efficacy of Mel against B.a. Ames. An efficacy >6 LR
was achieved only on painted wallboard paper and unpainted concrete when tested at 45 % RH.
However, increasing the RH from 45 % to 75 % resulted in a significant increase in decontamination
efficacy to complete inactivation on most material types.
Mel appears to be an effective decontaminant against B.a. Ames under specific combinations of
concentration, temperature, RH, and contact time. In general, elevated RH appears to reduce the need
for increased Mel and contact time to maintain efficacy. At 45 % RH, increasing the contact time did
not significantly improve the efficacy of Mel against B.a. Ames. Additional testing at the lower RH
level is needed to confirm these data as only two tests were completed using this parameter. More
testing is also needed to confirm the data presented here as well as to test additional materials and
combinations of parameters.
Impact of Study
This work provides information on the efficacy of Mel as a fumigant for decontamination of
common building materials that have been contaminated with B.a. spores. Such results may be useful
in the development of guidance to aid in deployment of Mel fumigation after a wide-area release of
B.a spores. This report also provides data to assist in selection of an avirulent surrogate for B.a.
Ames, for use in future field studies and additional lab-based investigations utilizing Mel.
26
-------
7.0 References
1. lofina Chemical. 2008. Methyl Iodide Material Safety Data Sheet. January.
2. Determining the Efficacy of Liquids and Fumigants in Systematic Decontamination
Studies for Bacillus anthracis Using Multiple Test Methods. US EPA Report 600/R-
10/088, December 2010.
3. ASTM International. 2006. Standard Practice for Maintaining Constant Relative
Humidity by Means of Aqueous Solutions. October.
4. EPA. 2013. Evaluation ofEthylene Oxide for the Inactivation o/"Bacillus anthracis. EPA
Technology Evaluation Report. EPA/13/R-13-220. December.
5. EPA. 2011. Systematic Investigation of Liquid and Fumigant Decontamination Efficacy
against Biological Agents Deposited on Test Coupons of Common Indoor Materials. US
EPA Report 600/R-l 1/076. August.
6. EPA, 2014, Methyl Bromide Decontamination of Indoor and Outdoor Materials
Contaminated with Bacillus anthracis Spores, US EPA Report 600/R-14/170, August.
27
-------
Appendix A
Detailed Test Results
Efficacy Results
The detailed decontamination efficacy results for methyl iodide against B.a. Ames, B. atrophaeus,
and B. a. Sterne on six material types (glass, ceiling tile, carpet, painted wallboard paper, bare pine
wood and unpainted concrete) are shown in Tables A-l through A-3. Zero CPU were observed on all
laboratory and procedural blanks.
Table A-l. Inactivation of B. anthracis Ames Spores using Methyl Iodide"
Target Parameters
Test
Number Concentration (mg/L) / Temp (°C) / Material
Contact Time (h) RH (%)
1 300/24
2 300/12
3 400/24
4 400/12
5 300/36
6 300/36
7 300/36
Glass
Ceiling Tile
Industrial Carpet
37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
25/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Inoculum
(CFU/counon)
Mean Recovered B. a. A]
(CFU/coupon)
Positive Control"
3.08
1.15
9.23
1.24xl08
3.83
4.04
1.81
3.97
2.13
8.33
l.llxlO8
9.03
5.85
2.26
3.49
1.21
8.59
1.60xl08
5.74
8.89
9.90
4.05
1.04
9.26
1.12xl08
6.84
6.59
1.80
3.19
9.46
9.03
9.53 xlO7
6.05
7.56
1.58
4.20
1.54
1.05
1.02 xlO8
6.71
7.95
1.82
2.69
7.69
7.17
9.67xl07
4.53
5.60
1.47
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.08 xlO7
0.31 xlO7
1.13xl07
0.74 xlO7
0.35 xlO6
1.13xl07
1.34 xlO7
2.62 xlO7
0.94 xlO7
0.35 xlO7
1.56 xlO6
1.55 xlO7
0.99 xlO7
0.16 xlO7
0.74 xlO7
0.86 xlO7
4.25 xlO6
4.27 xlO6
0.35 xlO7
0.21 xlO7
0.74 xlO7
2.02 xlO7
2.36 xlO6
1.87 xlO7
0.69 xlO7
1.21 xlO6
0.90 xlO7
0.94 xlO7
5.96 xlO6
0.99 xlO7
0.46xl07
0.26 xlO7
0.08 xlO8
0.63 xlO7
3.25 xlO6
0.89 xlO7
0.60 xlO7
1.65 xlO6
1.03 xlO7
0.26 xlO7
2.18 xlO6
0.43 xlO7
mes
Test Coupon0
2.55 ± 2.25 xlO3
4.21 ± 6.58 xlO2
0.00
0.00
0.00
0.00
±0.00
±0.00
±0.00
±0.00
4.76 ±4.51x10*
4.68±5.40xl02
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
±0.00
Decontamination
Efficacy ± CI"
4.56 ±
5.45 ±
>7.96 ±
>7.58 ±
>6.60±
>7.20 ±
3.51 ±
5.45 ±
>7.92 ±
>7.96 ±
>6.76 ±
>7.26 ±
>7.53 ±
>7.08 ±
>7.93 ±
>7.75 ±
>6.91 ±
>6.96±
>7.61 ±
>7.01 ±
>7.97±
>7.82 ±
>6.79 ±
>7.11±
>7.50 ±
>6.98±
>7.95 ±
>7.78 ±
>6.80±
>7.11±
>7.62 ±
>7.18±
>8.02±
>7.83 ±
>6.87±
>7.22 ±
>7.42 ±
>6.88±
>7.85 ±
>7.66 ±
>6.72 ±
>7.15±
0.90
1.32
0.05
0.07
0.03
0.22
1.04
1.41
0.04
0.01
0.10
0.29
0.09
0.05
0.03
0.06
0.18
0.18
0.03
0.08
0.03
0.12
0.15
0.33
0.09
0.05
0.04
0.06
0.24
0.31
0.04
0.07
0.03
0.04
0.15
0.19
0.09
0.07
0.06
0.02
0.16
0.12
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 x SE).
A-l
-------
Table A-l. Inactivation of B. anthracis Ames Spores using Methyl Iodide" (Continued)
Target Parameters
Number Concentration (mg/L) / Temp (°C) / Material
Contact Time (h) RH (%)
8 300/24
9 300/12
10 300/24
1 1 200/24
12 300/36
13 200/12
14 100/48
Glass
Ceiling Tile
Industrial Carpet
25/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
25/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
25/45
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
25/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
25/45
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
25/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
25/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Mean Recovered B. a. Ames
Inoculum (CFU/coupon)
(CFU/coupon)
Positive Control"
5.22 ±
1.66±
1.07±
9.30 xlO7
6.69 ±
1.35±
1.77±
5.28±
1.87±
1.32 xlO7
0.41 xlO7
0.06 xlO8
0.85 xlO7
0.68 xlO7
0.52 xlO7
0.95 xlO7
0.18 xlO7
1.07±0.08108
l.llxlO8
6.75 ± 1.04 xlO7
7.57 ±
2.18±
5.28±
1.34±
9.55 ±
1.05x10"
3.60 ±
6.47±
2.00 ±
4.50 ±
1.13±
8.21 ±
1.00 xlO8
5.46±
8.61 ±
2.19 ±
3.95 ±
1.66±
8.64 ±
1.13xl08
3.95 ±
6.19±
1.17±
5.61 ±
1.24±
1.01 ±
9.12xl07
5.07 ±
7.49 ±
2.20 ±
4.71 ±
8.62 ±
1.01 ±
1.26 xlO8
5.50 ±
5.52 ±
2.80 ±
2.11 xlO6
1.02 xlO7
1.43 xlO7
0.12 xlO7
1.79 xlO7
0.47 xlO7
0.89 xlO6
0.78 xlO7
0.80 xlO7
0.40 xlO7
0.75 xlO7
0.35 xlO7
3.09 xlO6
1.67 xlO7
0.57 xlO7
0.70 xlO7
1.01 xlO7
0.61 xlO7
1.02 xlO6
1.03 xlO7
LlOxlO7
0.12 xlO7
0.12 xlO8
0.32 xlO7
4.02 xlO6
1.04 xlO7
1.02 xlO7
2.22 xlO6
0.12 xlO8
1.17xl07
1.23 xlO6
1.92 xlO7
Test Coupon0
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.75 ±1.45x10'
0.00 ±0.00
1.99±3.44xl03
2.73 ± 1.99 xlO2
1.87±1.63xl02
0.00 ±0.00
0.94±1.19xl02
0.00 ±0.00
5.27±3.19xl06
2.46 ±0.79x10*
1.24±0.70xl05
0.00 ±0.00
5.85 ± 5.87 xlO5
0.00 ±0.00
2.08 ±4.43x10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
1.12 ±0.35 xlO6
8.83 ± 1.72 xlO3
4.53 ± 3.55 xlO3
0.00 ±0.00
1.68±0.47xl05
0.00 ±0.00
3.66 ± 3.95 xlO5
2.84 ± 3.05xl05
7.16 ± 4.36 xlO5
2.85 ± 6.06 xlO6
4.17±2.56xl03
0.86 ± 1.20 xlO6
2.08 ±4.43x10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
Decontamination
Efficacy ± Cl"
>7. 70 ±0.11
>7.21±0.09
>8.03±0.02
>7.82±0.05
6. 77 ±0.64
>7.23±0.12
5.77±1.61
4.98 ±0.41
5.96 ±0.45
>7.82±0.06
5.63 ±1.04
>7.30±0.18
1.07±0.31
2.75 ±0.13
2.94 ±0.25
>7.55±0.05
1.17±0.32
>7.28±0.14
7.25 ±0.79
>7.02±0.19
>7.91±0.04
>7. 74 ±0.02
>6.91±0.13
>7.26 ± 0.25
1.57±0.15
3.25 ±0.16
4.47 ±0.45
>7.59±0.06
1.58±0.13
>6.96±0.29
2.58 ±0.73
1.86 ±0.45
2.27 ±0.39
2.20 ±0.83
3.43 ±0.61
2.11±1.19
7.27 ±0.79
>6.92±0.11
>8.00±0.05
>7. 73 ±0.08
>6.73±0.10
>7.37±0.26
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 x SE).
A-2
-------
Table A-2. Inactivation of B. atrophaeus Spores using Methyl Iodide"
Test
Number
1
2
3
4
5
6
Target Parameters
Concentration (mg/L) / Temp (°C) / Material
Contact Time (h) RH (%)
Glass
Ceiling Tile
Industrial Carpet
300/24 37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
300/12 37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
400/24 37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
400/12 37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
300/36 37/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
300/36 25/70
Wallboard Paper
Pine Wood
Unpainted Concrete
Mean Recovered B. atrophaeus
Inoculum (CFU/coupon)
(CFU/coupon)
Positive Control"
1.08 ± 0.13x10*
3.89 ± 0.52 xlO6
5.53 ± 0.73 xlO7
1.24xl08
I.ll±0.31xl07
3.72 ± 1.59 xlO6
1.27±0.20xl06
7.14±1.16xl07
3.79 ± 0.72 xlO6
3.90 ± 0.81 xlO7
9.43 xlO7
1.47±0.38xl07
5.79±2.17xl06
1.51±0.77xl06
1.42±0.46xl08
4.13 ± 0.69 xlO6
5.47 ± 0.97 xlO7
1.50x10"
9.82 ± 2.19 xlO6
5.93 ± 2.36 xlO6
2.99 ± 1.88 xlO6
7.69±1.43xl07
8.91 ± 3.09 xlO6
5.19±1.41xl07
1.03x10"
1.15±0.60xl07
5.91 ± 1.80 xlO6
1.84±1.39xl06
7.91 ± 1.02 xlO7
3.10±1.07xl06
4.02±1.18xl07
1.24xl08
6.27 ± 1.69 xlO6
9.71 ± 14.6 xlO6
1.93 ±1.43 xlO6
I.ll±0.12xl08
6.52 ± 1.34 xlO6
6.89 ± 1.51 xlO7
1.06 xlO8
1.33 ± 0.28 xlO7
8.13±1.97xl06
1.78 ±1.61 xlO6
Test Coupon0
2.75 ± 1.39 xlO5
1.27±2.65xl02
4.84±l.llxl04
1.20 ±1.31 xlO3
4.70 ±5.52x10'
0.00 ±0.00
1.05 ± 0.30 xlO7
3.33 ± 3.41 xlO6
5.16±3.26xl06
1.48±0.43xl06
5.43 ±4.18x10*
5.12±2.23xl05
1.57 ±0.70x10*
0.00 ±0.00
3.40 ±1.73x10*
1.94±3.77xl02
0.00 ±0.00
0.00 ±0.00
5.22 ± 1.23 xlO6
1.25 ± 0.50 xlO5
2.25 ± 2.48 xlO5
6.00 ± 1.66 xlO5
1.05±1. 16x10*
2.43 ±1.23x10*
4.52±6.07xl03
0.00 ±0.00
0.00 ±0.00
2.34 ± 1.85 xlO2
0.00 ±0.00
0.00 ±0.00
3.29 ± 0.52 xlO7
1.20 ±0.18 xlO6
1.33 ± 0.50 xlO7
3.30 ± 1.82 xlO6
1.24±0.92xl06
3.63 ± 1.72 xlO5
Decontamination
Efficacy ± Cl"
2. 64 ±0.22
5.73 ±1.10
3.07±0.11
4. 16 ±0.44
5.44 ±0.91
>6. 10 ±0.06
0.84±0.13
0.22 ±0.38
1.00 ±0.40
1.00±0.16
2. 11 ±0.35
0.49 ±0.30
4.01 ±0.32
>6.61 ±0.06
3.25 ±0.24
5.48 ±0.91
>6.75±0.15
>6. 38 ±0.31
1.17±0.11
1.85 ±0.20
2.52 ±0.36
1.24 ±0.26
2.89 ±0.34
1.84 ±0.37
5. 15 ±1.44
>6.47±0.13
>7.59±0.11
4. 85 ±0.97
>6. 69 ±0.46
>6. 14 ±0.40
0.53 ±0.07
0.73 ±0.10
0.73 ±0.16
0.64 ±0.20
0.88 ±0.25
0.62 ±0.39
a Data are expressed as the mean (± SD) of the logs of the number of spores (CPU) observed on five individual samples and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 x SE).
A-3
-------
Table A-3. Inactivation of B. anthracis Sterne Spores using Methyl Iodide"
Target Parameters
Number Concentration (mg/L) / Temp (°C) /
Contact Time (h) RH (%)
7 300/36 37/70
8 300/24 25/70
9 300/12 25/70
10 300/24 25/45
1 1 200/24 25/70
12 300/36 25/45
13 200/12 25/70
14 100/48 25/70
Material
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Industrial Carpet
Wallboard Paper
Pine Wood
Unpainted Concrete
Mean Recovered B. a. Sterne
Inoculum (CFU/coupon)
(CFU/coupon)
Positive Control"
1.03 ± 0.50 xlO7
4.80 ± 1.08 xlO6
5.21 ± 0.79 xlO7
1.08 xlO8
2.72 ± 0.97 xlO7
7.52 ± 1.00 xlO6
3.80 ± 1.61 xlO6
1.85 ± 0.90 xlO7
9.28 ± 2.00 xlO6
7.15 ± 1.45 xlO7
1.09 xlO8
4.1 1± 0.66 xlO7
2.24 ± 0.78 xlO7
6.10±4.59xl06
1.38 ± 0.31 xlO7
1.12±0.27xl07
7.33 ± 0.94 xlO7
1.06 xlO8
3.51 ± 0.79 xlO7
2.62 ± 0.73 xlO6
6.76 ± 6.12 xlO6
5.38 ± 1.76 xlO6
8.29±1.17xl06
6.08 ± 0.84 xlO7
9.83 xlO7
3.91 ± 0.72 xlO7
1.96±0.68xl07
5.25 ± 0.93 xlO6
9.66 ± 2.55 xlO6
9.73 ± 2.58 xlO6
5.93 ± 0.81 xlO7
9.03 xlO7
4.13 ± 0.60 xlO7
1.40±0.38xl07
7.13 ± 4.95 xlO6
1.22±0.17xl07
1.05 ± 0.21 xlO7
6.52 ± 0.44 xlO7
8.97 xlO7
2.98 ± 0.47 xlO7
1.30 ± 0.32 xlO7
4.00 ± 2.05 xlO6
2.73 ± 1.20 xlO7
9.69 ± 1.22 xlO6
7.59±1.14xl07
9.20 xlO7
4.29 ± 0.50 xlO7
1.96 ± 0.69 xlO7
7.60 ± 2.86 xlO6
2.18±0.96xl07
5.86 ± 1.48 xlO6
5.64 ± 1.00 xlO7
9.67xl07
3.89 ± 0.48 xlO7
1.29 ± 0.36 xlO7
4.82 ± 1.80 xlO6
Test Coupon0
0.00 ±0.00
0.00 ±0.00
0.75 ±1.44 xlO1
0.00 ±0.00
0.75 ±1.44x10'
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
3.30 ±3.35 xlO6
1.42 ±1.36 xlO6
4.91 ± 2.66 xlO5
1.13±0.54xl07
3.56 ± 1.82 xlO5
1.65± 1.76xl06
7.19 ±5.60x10*
1.88 ±1.30x10*
5.08 ±5.16x10*
0.00 ±0.00
6.97 ±3.07x10*
0.00 ±0.00
0.00 ±0.00
3.09 ± 3.06xl03
4.73 ±7.27x10'
7.63 ±6.48x10*
0.65 ±1.44x10*
4.67 ± 5.75 xlO2
5.17±5.24xl03
4.17±3.07xl03
3.41 ± 7.43 xlO2
0.00 ±0.00
2.19±2.54xlO*
0.00 ±0.00
1.00 ± 0.35 xlO7
5.93 ± 1.31 xlO6
5.41 ± 1.08 xlO7
2.38 ± 0.95 xlO7
1.38 ± 0.20 xlO7
3.02 ± 1.38 xlO6
2. 74 ±5. 90x10'
2.04 ± 3.74 xlO2
0.86± 1.17xl03
2.93 ± 3.13xl03
6.54 ± 8.73 xlO2
1.41 ±2.94x10'
Decontamination
Efficacy ± Cl"
>6.97±0.19
>6. 67 ±0.09
7.41 ±0.60
>7.41±0.16
6. 57 ±0.60
>6.53±0.24
>7.23±0.15
>6. 96 ±0.08
>7.85±0.08
>7.61±0.06
>7.33±0.13
>6. 70 ±0.25
0.87 ±0.57
1.24 ±0.67
2.22 ±0.22
0.56 ±0.33
0.90 ±0.22
0.64 ±0.58
1.98 ±0.39
2.71 ±0.23
3.27 ±0.45
>7.59±0.07
2.47 ±0.22
>6.71±0.07
>6. 97 ±0.09
4.34 ±1.43
6.96 ±0.98
3.14±0.81
5.80 ±1.76
5.05 ±1.40
3.61 ±0.50
3.48 ±0.27
6.86 ±1.26
>7. 47 ±0.06
2.97 ±0.43
>6. 54 ±0.25
0.41 ±0.25
0.22 ±0.10
0.15 ±0.09
0.28 ±0.15
0.14±0.13
0.41 ±0.25
6.88 ±0.85
5.44±1.14
5.30 ±0.71
4.87 ±1.38
5.45 ±1.41
6.29 ±0.73
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples and decontamination
efficacy (log reduction).b Positive Controls = samples inoculated, not decontaminated.
c Test Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 x SE).
A-4
-------
Appendix B
Comparing Efficacy for the Different Microorganisms
Testing was first conducted using B. atrophaeus as a potential surrogate for B.a. Ames (Tests 1
through 6). The results showed that although B. atrophaeus is more resistant than B.a. Ames to
Mel exposure, therefore, B.a. Sterne was tested as an additional potential surrogate. The detailed
differences in efficacy by material type and test number are shown in Tables B-l and B-2.
Table B-l. Difference in Mel Efficacy between B. anthracis Ames and B. atrophaeus*
T Target Mel Target Target
,T . Concentration Temperature RH
Number (mg/L) (°C) (%)
1 300 37 70
2 300 37 70
3 400 37 70
4 400 37 70
5 300 37 70
6 300 25 70
Contact
Time Material Type
(hours)
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
36
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
36
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
B.a.
Ames
Efficacy
4.56
5.45
> 7.96
> 7.58
> 6.60
> 7.20
3.51
5.45
> 7.92
> 7.96
> 6.76
> 7.26
> 7.53
> 7.08
> 7.93
> 7.75
> 6.91
> 6.96
> 7.61
> 7.01
> 7.97
> 7.82
> 6.79
> 7.11
> 7.50
> 6.98
> 7.95
> 7.78
> 6.80
> 7.11
> 7.62
> 7.18
> 8.02
> 7.83
> 6.87
> 7.22
B. atrophaeus
Efficacy
2.64
5.73
3.07
4.16
5.44
> 6.10
0.84
0.22
1.00
1.00
2.11
0.49
4.01
> 6.61
3.25
5.48
> 6.75
> 6.38
1.17
1.85
2.52
1.24
2.89
1.84
5.15
> 6.47
> 7.59
4.85
> 6.69
> 6.14
0.53
0.73
0.73
0.64
0.88
0.62
Average
Difference
in Efficacy
-2.04
-5.53
-1.95
-5.47
-1.21
-6.77
* Results shown as efficacy (log reduction).
B-l
-------
Table B-2. Difference in Mel Efficacy between B. anthracis Ames and B. anthracis Sterne*
T Target Mel Target Target
,T . Concentration Temperature RH
Number (mg/L) (°C) (%)
7 300 37 70
8 300 25 70
9 300 25 70
10 300 25 45
11 200 25 70
12 300 25 45
13 200 25 70
14 100 25 70
Contact
Time Material Type
(hours)
Glass
Ceiling Tile
Carpet
36
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
24
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
36
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Glass
Ceiling Tile
Carpet
48
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
B.a.
Ames
Efficacy
> 7.42
> 6.88
> 7.85
> 7.66
> 6.72
> 7.15
> 7.70
> 7.21
> 8.03
> 7.82
6.77
> 7.23
5.77
4.98
5.96
> 7.82
5.63
> 7.30
1.07
2.75
2.94
> 7.55
1.17
> 7.28
7.25
> 7.02
> 7.91
> 7.74
> 6.91
> 7.26
1.57
3.25
4.47
> 7.59
1.58
> 6.96
2.58
1.86
2.27
2.20
3.43
2.11
7.27
> 6.92
> 8.00
> 7.73
> 6.73
> 7.37
B. atrophaeus
Efficacy
> 6.97
> 6.67
7.41
> 7.41
6.57
> 6.53
> 7.23
> 6.96
> 7.85
> 7.61
> 7.33
> 6.70
0.87
1.24
2.22
0.56
0.90
0.64
1.98
2.71
3.27
> 7.59
2.47
> 6.71
> 6.97
4.34
6.96
3.14
5.80
5.05
3.61
3.48
6.86
> 7.47
2.97
> 6.54
0.41
0.22
0.15
0.28
0.14
0.41
6.88
5.44
5.30
4.87
5.45
6.29
Average
Difference
in Efficacy
-0.35
-0.18
-5.17
0.33
-1.97
0.95
-2.14
-1.63
* Results shown as efficacy (log reduction).
B-2
-------
Appendix C
Effects of Materials and Operational Parameters on Mel Efficacy
Effects of Test Materials on Mel efficacy
Testing was conducted using six test materials: ceiling tile, carpet, glass, painted wallboard
paper, bare pine wood, and unpainted concrete. A summary of the results in terms of LR are
organized by operational parameters and can be seen in Figures C-l through C-3 below.
C-l
-------
Glass
Bare Pine Wood
5" 3
I 2
i
i
•8 «
% 5
d 4
t' 3
£ 2
w ,
I ° °
37 °C
70% EH
el C'ontvict Tiine (
25 °C
70% EH
25 °C
45% EH
37 °C
70% EH
25 °C
70% EH
in LL T Mel C'oiitvict Tiine i,houi's)
25 °C
45% EH
Figure C-l. Summary of Mel efficacy against B. anthracis Ames on glass and bare pine wood. Results shown in average log
reduction ± CI.
* Complete inactivation achieved
d -1
I;
0
Ceiling Tile
I
tttft
37 °C
70% EH
2.5 °C
70% EH
25 °C
45% EH
Carpet
mg L Mel Couraf t Tlinf (hours)
mg L Mel Contact Time (hours)
C-2
-------
w
4
Figure C-2. Summary of Mel efficacy against B. anthracis Ames on ceiling tile and carpet. Results shown in average log
reduction ± CI
Complete inactivation achieved
Painted Wallboard Paper
t
37 °C
70%RH
25 °C
70% EH
mgL Mfl Contact Time (hours)
r-> r-i
25 °C
45% EH
1 !
Unpainted Concrete
I
37 °C
70% EH
25 °C
70% EH
mgL Mfl Contact Time (horns)
25 °C
45% EH
Figure C-3. Summary of Mel efficacy against B. anthracis Ames on painted wallboard paper and unpainted concrete. Results
shown in average log reduction ± CI
* Complete inactivation achieved
C-3
-------
Effects of Temperature on Mel Efficacy against B.a. Ames
The decontamination efficacy of Mel against B.a. Ames was evaluated at target temperatures of 25 or
37 °C. These temperatures were tested at 70 % RH and 300 mg/L Mel and contact times of 12, 24,
and 36 hours. Results are summarized in Table C-l. The comparisons are made for two test
conditions that share the same fumigation parameters except temperature.
Table C-l. Effect of Increasing Temperature at High Relative Humidity on Mel Efficacy*
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 9"
Test 2
300 mg/L; 25 °C; I 300 mg/L; 37 °C;
70 % RH; 12 h | 70 % RH; 12 h
5.77
4.98
5.96
> 7.82
3.51
5.45
> 7.92
> 7.96
Average
Difference
in Efficacy
0.23
5.63 ! > 6.76 !
> 7.30
> 7.26
Tests
Testl
300 mg/L; 25 °C; I 300 mg/L; 37 °C;
70 % RH; 24 h | 70 % RH; 24 h
> 7.70
> 7.21
> 8.03
> 7.82
4.56
5.45
> 7.96
> 7.58
Average
Difference in
Efficacy
-0.90
6.77 ! > 6.60 !
> 7.23
> 7.20
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 6
300 mg/L; 25 °C;
70 % RH; 36 h
> 7.62
> 7.18
> 8.02
> 7.83
> 6.87
Tests
300 mg/L; 37 °C;
70 % RH; 36 h
> 7.50
> 6.98
> 7.95
> 7.78
> 6.80
Average
Difference
in Efficacy
-0.10
> 7.22 | > 7.11 |
Test 6
300 mg/L; 25 °C;
70 % RH; 36 h
> 7.62
> 7.18
> 8.02
> 7.83
> 6.87
Test?
300 mg/L; 37 °C;
70 % RH; 36 h
> 7.42
> 6.88
> 7.85
> 7.66
> 6.72
Average
Difference in
Efficacy
-0.18
> 7.22 | > 7.15 |
* Data are expressed as decontamination efficacy (log reduction).
a Parameters for each test listed in order of Mel concentration (mg/L), temperature (°C), % RH, and contact time (h).
C-4
-------
Effects of Relative Humidity on Efficacy of Mel against B. anthracis Ames
The decontamination efficacy of Mel against B. a. Ames was evaluated at target relative humidities of
45 or 70 %. The actual %RH conditions for each test are shown in Appendix A. These RH levels
were tested at various temperatures, Mel concentrations, and contact times and results are
summarized in Table C-2 below and discussed in Section 6.4. The comparisons are made for two test
conditions that share the same fumigation parameters except RH.
Table C-2. Effect of Increasing Relative Humidity at Low and High Temperatures on B.
anthracis Ames*
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 10"
300 mg/L; 25 °C;
45 % RH; 24 h
1.07
2.75
2.94
> 7.55
1.17
> 7.28
Tests
300 mg/L; 25 °C;
70 % RH; 24 h
Average
Difference
in Efficacy
> 7.70 !
> 7.21
> 8.03
> 7.82
6.77
> 7.23
3.67
Test 12
300 mg/L; 25 °C;
45 % RH; 36 h
Test 6
300 mg/L; 25 °C;
70 % RH; 36 h
Average
Difference in
Efficacy
1.57 ! > 7.62 !
3.25
4.47
> 7.59
1.58
> 6.96
> 7.18
> 8.02
> 7.83
> 6.87
> 7.22
3.22
* Data are expressed as decontamination efficacy (log reduction).
a Parameters for each test listed in order of Mel concentration (mg/L), temperature (°C), % RH, and contact time (h).
C-5
-------
Effects of Mel Concentration on Efficacy against B. anthracis Ames
The decontamination efficacy of Mel against virulent B. a. was also evaluated at target concentrations
of 100, 200, 300, and 400 mg/L. These concentrations were tested at various combinations of
temperature and RH. The results are summarized in Table C-3 below. The comparisons are made for
two test conditions which share the same fumigation parameters except Mel concentration.
Table C-3. Effect of Increasing Mel Concentration at High Relative Humidity on B.
anthracis Ames*
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 13"
200 mg/L; 25 °C;
70%RH; 12 h
2.58
1.86
2.27
2.20
3.43
2.11
Test 9
300 mg/L; 25 °C;
70% RH; 12 h
5.77
4.98
5.96
> 7.82
5.63
> 7.30
Average
Difference
in Efficacy
3.84
Test 11
200 mg/L; 25 °C;
70% RH; 24 h
7.25
> 7.05
> 7.91
> 7.74
> 6.91
> 7.26
Tests
300 mg/L; 25 °C;
70% RH; 24 h
> 7.70
> 7.21
> 8.03
> 7.82
6.77
> 7.23
Average
Difference in
Efficacy
0.11
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 2
300 mg/L; 37 °C;
70%RH; 12 h
3.51
5.45
> 7.92
> 7.96
> 6.76
> 7.26
Test 4
400 mg/L; 37 °C;
70% RH; 12 h
> 7.61
> 7.01
> 7.97
> 7.82
> 6.79
> 7.11
Average
Difference
in Efficacy
0.91
Testl
300 mg/L; 37 °C;
70% RH; 24 h
4.56
5.45
> 7.96
> 7.58
> 6.60
> 7.20
Tests
400 mg/L; 37 °C;
70% RH; 24 h
> 7.53
> 7.08
> 7.93
> 7.75
> 6.91
> 6.96
Average
Difference in
Efficacy
0.80
* Data are expressed as decontamination efficacy (log reduction).
a Parameters for each test listed in order of Mel concentration (mg/L), temperature (°C), % RH, and contact time (h).
C-6
-------
Effects of Contact Time on Efficacy of Mel against B. anthracis Ames
The effect of increasing the contact times for Mel at high % RH on the efficacy against B.a.
Ames was also assessed. The contact times tested ranged from 12 to 48 hours, and actual contact
times did not deviate from these targets. The results are summarized in Tables C-4 and C-5. The
comparisons are made for two test conditions which share the same fumigation parameters
except contact time.
Table C-4. Effect of Increasing Contact Time at High Relative Humidity on B. anthracis
Ames*
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 13"
200 mg/L; 25 °C;
70 % RH; 12 h
Test 11
200 mg/L; 25 °C;
70 % RH; 24 h
Average
Difference
in Efficacy
2.58 ! 7.25 !
1.86
2.27
2.20
3.43
2.11
> 7.05
> 7.91
> 7.74
> 6.91
> 7.26
4.95
Test 9
300 mg/L; 25 °C;
70 % RH; 12 h
Tests
300 mg/L; 25 °C;
70 % RH; 24 h
Average
Difference in
Efficacy
5.77 ! > 7.70 !
4.98
5.96
> 7.82
5.63
> 7.30
> 7.21
> 8.03
> 7.82
6.77
> 7.23
1.22
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 8 1 Test 6
300 mg/L; 25 °C;
70 % RH; 24 h
> 7.70
300 mg/L; 25 °C;
70 % RH; 36 h
> 7.62
> 7.21 ! > 7.18
> 8.03
> 7.82
6.77
> 7.23
> 8.02
> 7.83
> 6.87
> 7.22
Average
Difference
in Efficacy
0.00
Test 2 I Testl I
1
300 mg/L; 37 °C;
70 % RH; 12 h
3.51
300 mg/L; 37 °C;
70 % RH; 24 h
4.56
5.45 ! 5.45
> 7.92
> 7.96
> 6.76
> 7.26
> 7.96
> 7.58
> 6.60
> 7.20
Average
Difference in
Efficacy
0.08
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Testl
300 mg/L; 37 °C;
70 % RH; 24 h
4.56
Tests
300 mg/L; 37 °C;
70 % RH; 36 h
Average
Difference
in Efficacy
> 7.50 !
5.45 | > 6.98 |
> 7.96
> 7.58
> 6.60
> 7.20
> 7.95
> 7.78
> 6.80
> 7.11
0.80
Test 4
400 mg/L; 37 °C;
70 % RH; 12 h
Tests
400 mg/L; 37 °C;
70 % RH; 24 h
Average
Difference in
Efficacy
> 7.61 ! > 7.53 !
> 7.01 | > 7.08 |
> 7.97
> 7.82
> 6.79
> 7.11
> 7.93
> 7.75
> 6.91
> 6.96
-0.03
* Data are expressed as decontamination efficacy (log reduction).
a Parameters for each test listed in order of Mel concentration (mg/L), temperature (°C), %RH, and contact time (h).
C-7
-------
Table C-5. Effect of Increasing Contact Time at Low Relative Humidity on B. anthracis
Ames*
Material Type
Glass
Ceiling Tile
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
Test 10"
300 mg/L; 25 °C;
45 % RH; 24 h
1.07
2.75
2.94
> 7.55
1.17
> 7.28
Test 12
300 mg/L; 25 °C;
45 % RH; 36 h
1.57
3.25
4.47
> 7.59
1.58
> 6.96
Average
Difference
in Efficacy
0.44
* Data are expressed as decontamination efficacy (log reduction).
a Parameters for each test listed in order of Mel concentration (mg/L), temperature (°C), % RH, and contact time (h).
C-8
-------
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
------- |