SC IEN

CE

United States

Environmental Protection

Agency

Office of Research and Development

National Homeland Security Research Center

Evaluation of Liquid and 

Foam Technologies for the 

Decontamination of B. anthracis 

and B. subtilis Spores on Building 

and Outdoor Materials

DioxiGuardTM (Frontier Pharmaceutical) 

pH-Amended Bleach Calcium Polysulfide 

CASCADTM Surface Decontamination 

 Foam (Allen-Vanguard) 

Oxonia Active® (Ecolab Inc.) 

Minncare® Cold Sterilant® (Minntech Corp.) 

SanDes (DTI-Sweden AB)

TEChNology EvaluaTIoN REPoRT 

EPA 600/R-09/150 | November 2009 | www.epa.gov/ord
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EPA/600/R-09/150 | November 2009 | www.epa.gov/ord 

TEChNology EvaluaTIoN REPoRT 

Evaluation of Liquid and Foam 

Technologies for the Decontamination 

of B. anthracis and B. subtilis Spores 

on Building and Outdoor Materials 

DioxiGuardTM (Frontier Pharmaceutical) 

pH-Amended Bleach

Calcium Polysulfide

CASCADTM Surface Decontamination 

Foam (Allen-Vanguard)

Oxonia Active® (Ecolab Inc.)

Minncare® Cold Sterilant (Minntech Corp.) 

SanDes (DTI-Sweden AB) 

Joseph P. Wood 

National homeland Security Research Center 

Office of Research and Development 

U.S. Environmental Protection Agency 

Mail Code E343-06 

Research Triangle Park, NC 27711 

Report prepared under contract # gS23F0011l-3 

Battelle Memorial Institute 

505 King Avenue 

Columbus, ohio 43201 

Office of Research and Development 

National Homeland Security Research Center, Decontamination and Consequence Management Division 
Notice
 

The U.S. Environmental Protection Agency (EPA), through its Office of Research and 

Development’s National Homeland Security Research Center (NHSRC), funded, directed, 

and managed this technology evaluation through a Blanket Purchase Agreement (BPA) 

under General Services Administration contract number GS23F0011L-3 with Battelle. This 

report has been peer and administratively reviewed and has been approved for publication 

as an EPa document. Mention of trade names or commercial products does not constitute 

endorsement or recommendation for use of a specific product. 

iv 
Foreword
 

The U.S. Environmental Protection Agency (EPA) is charged 

by Congress with protecting the nation’s air, water, and 

land resources. Under a mandate of national environmental 

laws, the Agency strives to formulate and implement actions 

leading to a compatible balance between human activities 

and the ability of natural systems to support and nurture 

life. To meet this mandate, the EPA’s Office of Research and 

Development (ORD) provides data and science support that 

can be used to solve environmental problems and to build the 

scientific knowledge base needed to manage our ecological 

resources wisely, to understand how pollutants affect our 

health, and to prevent or reduce environmental risks. 

In September 2002, EPa announced the formation of the 

National Homeland Security Research Center (NHSRC). The 

NHSRC, part of the Office of Research and Development, 

manages, coordinates, supports, and conducts a variety of 

research and technical assistance efforts. These efforts are 

designed to provide appropriate, affordable, effective, and 

validated technologies and methods for addressing risks 

posed by chemical, biological, and radiological terrorist 

attacks. Research focuses on enhancing our ability to detect, 

contain, and decontaminate in the event of such attacks. 

Guided by the roadmap set forth in EPA’s Strategic Plan for 

homeland Security, NhSRC ensures rapid production and 

distribution of security-related products. 

The NhSRC has created the Technology Testing and 

Evaluation Program (TTEP) in an effort to provide reliable 

information regarding the performance of homeland security 

related technologies. TTEP provides independent, quality 

assured performance information that is useful to decision 

makers in purchasing or applying the tested technologies. 

TTEP provides potential users with unbiased, thirdparty information that can supplement vendor-provided 

information. Stakeholder involvement ensures that user 

needs and perspectives are incorporated into the test design 

so that useful performance information is produced for each 

of the tested technologies. The technology categories of 

interest include detection and monitoring, water treatment, air 

purification, decontamination, and computer modeling tools 

for use by those responsible for protecting buildings, drinking 

water supplies and infrastructure, and for decontaminating 

structures and the outdoor environment. Additionally, 

environmental persistence information is also important for 

containment and decontamination decisions. 

The evaluation reported herein was conducted by Battelle as 

part of the TTEP program. Information on NhSRC and TTEP 

can be found at http://www.epa.gov/nhsrc/index.html. 

v 
Acknowledgments
 

The following Battelle staff contributed to this report: Thomas J. Kelly, young W. 

Choi, James v. Rogers, Karen B. Riggs, and Zachary J. Willenberg. The authors wish 

to acknowledge the support of all those who helped plan and conduct the evaluation, 

analyze the data, and prepare this report. We also would like to thank J. Carlton Kempter, 

Frank W. Schaefer III, and anthony Zimmer, all of the EPa, and Martin hamilton of 

Montana State University, for their reviews of the test/QA plan for this evaluation, and 

J. Carlton Kempter, Michael Ottlinger, and Worth Calfee of EPA for their reviews of this 

Technology Evaluation Report. 

vi 
 

 

 

 

 

 

 

Contents
 

Notice.............................................................................................................................................................................iv
	

Foreword.........................................................................................................................................................................v
	

acknowledgments..........................................................................................................................................................vi
 

Abbreviations/Acronyms ..............................................................................................................................................xii
	

Executive Summary.................................................................................................................................................... xiii
	

1.0 Introduction............................................................................................................................................................1
 

2.0 Technology Description.........................................................................................................................................3
 

3.0 Summary of Test Procedures .................................................................................................................................5
 

3.1 Preparation and analysis of Test Coupons...................................................................................................5
 

3.2 Decontamination Efficacy ............................................................................................................................6
 

3.3 Qualitative Assessment of Residual Spores .................................................................................................7
 

3.4 Qualitative Assessment of Surface Damage.................................................................................................7
 

4.0 Quality Assurance/Quality Control........................................................................................................................9
 

4.1 Equipment Calibration .................................................................................................................................9
 

4.2 QC Results....................................................................................................................................................9
 

4.3 audits ...........................................................................................................................................................9
 

4.3.1 Performance Evaluation Audit ...........................................................................................................9
 

4.3.2 Technical Systems audit ...................................................................................................................9
 

4.3.3 Data Quality Audit..............................................................................................................................9
 

4.4 Test/QA Plan Amendments and Deviations .................................................................................................9
 

4.5 QA/QC Reporting .......................................................................................................................................9
 

4.6 Data Review .................................................................................................................................................9
 

5.0 DioxiGuardTM (Frontier Pharmaceutical)Test Results.........................................................................................11
 

5.1 QC Results..................................................................................................................................................11
 

5.2 Decontamination Efficacy ..........................................................................................................................11
 

5.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms .............................................11
 

5.2.2 Qualitative Assessment of Residual Spores .....................................................................................12
 

5.3 Damage to Coupons ..................................................................................................................................14
 

5.4 other Factors ..............................................................................................................................................14
 

5.4.1 operator Control ..............................................................................................................................14
 

5.4.2 Technology Spray Deposition ..........................................................................................................16
 

5.4.3 Neutralization Methodology.............................................................................................................17
 

6.0 ph-amended Bleach Test Results .......................................................................................................................19
 

6.1 QC Results..................................................................................................................................................19
 

6.2 Decontamination Efficacy ..........................................................................................................................19
 

6.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms .............................................19
 

6.2.2 Qualitative Assessment of Residual Spores .....................................................................................19
 

6.3 Damage to Coupons ..................................................................................................................................22
 

6.4 other Factors ..............................................................................................................................................22
 

6.4.1 operator Control ..............................................................................................................................22
 

6.4.2 Technology Spray Deposition ..........................................................................................................23
 

6.4.3 Neutralization Methodology.............................................................................................................23
 

vii 
 

 

 

 

 

 

 

7.0 Calcium Polysulfide Test Results ........................................................................................................................25
 

7.1 QC Results..................................................................................................................................................25
 

7.2 Decontamination Efficacy ..........................................................................................................................25
 

7.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms .............................................25
 

7.2.2 Qualitative Assessment of Residual Spores .....................................................................................27
 

7.3 Damage to Coupons ..................................................................................................................................28
 

7.4 other Factors ..............................................................................................................................................29
 

7.4.1 operator Control ..............................................................................................................................29
 

7.4.2 Technology Spray Deposition ..........................................................................................................29
 

7.4.3 Neutralization Methodology.............................................................................................................29
 

8.0 CASCADTM SDF (Allen-Vanguard) Test Results ...............................................................................................31
 

8.1 QC Results..................................................................................................................................................31
 

8.2 Decontamination Efficacy ..........................................................................................................................31
 

8.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms .............................................31
 

8.2.2 Qualitative Assessment of Residual Spores .....................................................................................31
 

8.3 Damage to Coupons ..................................................................................................................................35
 

8.4 other Factors ..............................................................................................................................................35
 

8.4.1 operator Control ..............................................................................................................................35
 

8.4.2 Technology Spray Deposition ..........................................................................................................35
 

8.4.3 Neutralization Methodology.............................................................................................................36
 

9.0 Oxonia Active® (Ecolab) Test Results .................................................................................................................39
 

9.1 QC Results..................................................................................................................................................39
 

9.2 Decontamination Efficacy ..........................................................................................................................39
 

9.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms .............................................39
 

9.2.2 Qualitative Assessment of Residual Spores .....................................................................................42
 

9.3 Damage to Coupons ..................................................................................................................................44
 

9.4 other Factors ..............................................................................................................................................44
 

9.4.1 operator Control ..............................................................................................................................44
 

9.4.2 Technology Spray Deposition ..........................................................................................................44
 

9.4.3 Neutralization Methodology.............................................................................................................44
 

10.0 Minncare® Cold Sterilant (Minntech) Test Results..............................................................................................47
 

10.1 QC Results................................................................................................................................................47
 

10.2 Decontamination Efficacy ........................................................................................................................47
 

10.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms ...........................................47
 

10.2.2 Qualitative Assessment of Residual Spores ...................................................................................51
 

10.3 Damage to Coupons .................................................................................................................................51
 

10.4 other Factors ............................................................................................................................................51
 

10.4.1 operator Control ............................................................................................................................51
 

10.4.2 Technology Spray Deposition ........................................................................................................53
 

10.4.3 Neutralization Methodology...........................................................................................................53
 

11.0 SanDes (DTI-Sweden AB) Test Results ..............................................................................................................55
 

11.1 QC Results................................................................................................................................................55
 

11.2 Decontamination Efficacy ........................................................................................................................55
 

11.2.1 Quantitative Assessment of the Log Reduction of Viable Organisms ...........................................55
 

11.2.2 Qualitative Assessment of Residual Spores....................................................................................58
 

11.3 Damage to Coupons ................................................................................................................................60
 

11.4 other Factors ............................................................................................................................................60
 

11.4.1 operator Control ............................................................................................................................60
 

11.4.2 Technology Spray Deposition.........................................................................................................60
 

11.4.3 Neutralization Methodology...........................................................................................................60
 

viii 
 

 

12.0 Performance Summary ........................................................................................................................................63
 

12.1 DioxiGuardTM Results ..............................................................................................................................63
 

12.2 ph-amended Bleach Results ...................................................................................................................63
 

12.3 Calcium Polysulfide Results.....................................................................................................................63
 

12.4 CASCADTM SDF Results.........................................................................................................................64
 

12.5 Oxonia Active® Results ............................................................................................................................64
 

12.6 Minncare® Cold Sterilant Results.............................................................................................................64
 

12.7 SanDes Results.........................................................................................................................................64
 

13.0 References............................................................................................................................................................67
 

Appendices – Technology Descriptions and Applications Procedures for the Evaluated Decontaminants
	

a DioxiGuardTM Description and Application Procedure....................................................................................69
 

B pH-Amended Bleach Description and Appliciation Procedure........................................................................71
 

C Calcium Polysulfide Description and application Procedure ..........................................................................73
 

D CASCADTM SDF Description and application Procedure ..............................................................................75
 

E Oxonia Active® Description and application Procedure..................................................................................77
 

F Minncare® Cold Sterilant Description and application Procedure...................................................................79
 

g SanDes Description and application Procedure...............................................................................................81
 

ix 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tables
 

Table ES-1. Summary of Quantitative Efficacy by Decontaminant and Test Material ..........................................xiv
	

Table 5-3. Summary of Efficacy Values (Log Reduction) Obtained for 

Table 5-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 5-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 5-7. Neutralization Testing with Bacillus anthracis ames Spores for
 

Table 5-8. Neutralization Testing with Bacillus subtilis Spores for 

Table 6-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 2-1. Technology Information ........................................................................................................................3
 

Table 5-1. Inactivation of Bacillus anthracis Ames Spores—Frontier Pharmaceutical’s DioxiGuardTM ............12
 

Table 5-2. Inactivation of Bacillus subtilis Spores—Frontier Pharmaceutical’s DioxiGuardTM ..........................13
 

Frontier Pharmaceutical’s DioxiGuardTM ............................................................................................14
 

Bacillus anthracis Ames Spores—Frontier Pharmaceutical’s DioxiGuardTM.....................................15
 

Bacillus subtilis Spores—Frontier Pharmaceutical’s DioxiGuardTM...................................................16
 

Table 5-6. Deposition/Runoff Weight of Frontier Pharmaceutical’s DioxiGuardTM on Test Materials................16
 

Frontier Pharmaceutical’s DioxiGuardTM ............................................................................................17
 

Frontier Pharmaceutical’s DioxiGuardTM ............................................................................................17
 

Table 6-1. Inactivation of Bacillus anthracis ames Spores—ph-amended Bleach............................................20
 

Table 6-2. Inactivation of Bacillus subtilis Spores—ph-amended Bleach .........................................................21
 

Table 6-3. Summary of Efficacy Values (Log Reduction) Obtained for pH-Amended Bleach............................21
 

Bacillus anthracis ames Spores—ph-amended Bleach ....................................................................22
 

Table 6-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with 

Bacillus subtilis Spores—ph-amended Bleach..................................................................................22
 

Table 7-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 7-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 8-3. Summary of Efficacy Values (Log Reduction) Obtained for
	

Table 8-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 8-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 6-6, Deposition/Runoff Weight of pH-Amended Bleach on Test Materials ...............................................23
 

Table 6-7. Neutralization Testing with Bacillus anthracis ames Spores for ph-amended Bleach.....................24
 

Table 6-8. Neutralization Testing with Bacillus subtilis Spores for ph-amended Bleach...................................24
 

Table 7-1. Inactivation of Bacillus anthracis Ames Spores—5.8% Calcium Polysulfide....................................26
 

Table 7-2. Inactivation of Bacillus subtilis Spores—5.8% Calcium Polysulfide .................................................27
 

Table 7-3. Summary of Efficacy Values (Log Reduction) Obtained for 5.8% Calcium Polysulfide....................27
 

Bacillus anthracis ames Spores—5.8% Calcium Polysulfide ............................................................28
 

Bacillus subtilis Spores—5.8% Calcium Polysulfide..........................................................................28
 

Table 7-6. Deposition/Runoff Weight of 5.8% Calcium Polysulfide on Test Materials .......................................29
 

Table 7-7. Neutralization Testing with Bacillus anthracis Ames Spores for 5.8% Calcium Polysulfide.............30
 

Table 7-8. Neutralization Testing with Bacillus subtilis Spores for 5.8% Calcium Polysulfide...........................30
 

Table 8-1. Inactivation of Bacillus anthracis ames Spores—Allen-Vanguard’s CASCADTM SDF....................32
 

Table 8-2. Inactivation of Bacillus subtilis Spores—Allen-Vanguard’s CASCADTM SDF..................................33
 

Allen-Vanguard’s CASCADTM SDF....................................................................................................34
 

Bacillus anthracis Ames Spores—Allen-Vanguard’s CASCADTM SDF ............................................34
 

Bacillus subtilis Spores—Allen-Vanguard’s CASCADTM SDF ..........................................................35
 

x 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 8-6. Deposition/Runoff Weight of Allen-Vanguard’s CASCADTM SDF on Test Materials........................36
 

Table 8-7. Neutralization Testing with Bacillus anthracis ames Spores for 

Table 8-8. Neutralization Testing with Bacillus subtilis Spores for
 

Table 8-9. additional Neutralization Testing with Bacillus anthracis ames Spores for
 

Table 8-10. additional Neutralization Testing with Bacillus subtilis Spores for
 

Table 9-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 9-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 10-1. Inactivation of Bacillus anthracis Ames Spores—Minntech’s Minncare® Cold Sterilant
 

Table 10-2. Inactivation of Bacillus anthracis Ames Spores—Minntech’s Minncare® Cold Sterilant
 

Table 10-3. Inactivation of Bacillus subtilis Spores—Minntech’s Minncare® Cold Sterilant
 

Table 10-4. Inactivation of Bacillus subtilis Spores—Minntech’s Minncare® Cold Sterilant
 

Table 10-5. Summary of Efficacy Values (Log Reduction) Obtained for 

Table 10-6. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 10-7. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 10-9. Neutralization Testing with Bacillus anthracis ames Spores for
 

Table 11-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Table 11-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with
	

Allen-Vanguard’s CASCADTM SDF....................................................................................................37
 

Allen-Vanguard’s CASCADTM SDF....................................................................................................37
 

Allen-Vanguard’s CASCADTM SDF....................................................................................................37
 

Allen-Vanguard’s CASCADTM SDF....................................................................................................37
 

Table 9-1. Inactivation of Bacillus anthracis Ames Spores—Ecolab’s Oxonia Active®.....................................40
 

Table 9-2. Inactivation of Bacillus subtilis Spores—Ecolab’s Oxonia Active® ..................................................41
 

Table 9-3. Summary of Efficacy Values (Log Reduction) Obtained forEcolab’s Oxonia Active®......................42
 

Bacillus anthracis Ames Spores—Ecolab’s Oxonia Active® ..............................................................43
 

Bacillus subtilis Spores—Ecolab’s Oxonia Active® ............................................................................43
 

Table 9-6. Deposition/Runoff Weight of Ecolab’s Oxonia Active® on Test Materials .........................................44
 

Table 9-7. Neutralization Testing with Bacillus anthracis Ames Spores for Ecolab’s Oxonia Active® ...............45
 

Table 9-8. Neutralization Testing with Bacillus subtilis Spores for Ecolab’s Oxonia Active®.............................45
 

(10 minute contact time)......................................................................................................................48
 

(30 minute contact time)......................................................................................................................49
 

(10 minute contact time)......................................................................................................................50
 

(30 minute contact time)......................................................................................................................50
 

Minntech’s Minncare® Cold Sterilant..................................................................................................51
 

Bacillus anthracis Ames Spores—Minntech’s Minncare® Cold Sterilant...........................................52
 

Bacillus subtilis Spores—Minntech’s Minncare® Cold Sterilant ........................................................52
 

Table 10-8. Deposition/Runoff Weight of Minntech’s Minncare® Cold Sterilant on Test Materials......................53
 

Minntech’s Minncare® Cold Sterilant..................................................................................................54
 

Table 10-10. Neutralization Testing with Bacillus subtilis Spores for Minntech’s Minncare® Cold Sterilant .........54
 

Table 11-1. Inactivation of Bacillus anthracis Ames Spores—DTI-Sweden AB’s SanDes...................................56
 

Table 11-2. Inactivation of Bacillus subtilis Spores—DTI-Sweden AB’s SanDes ................................................57
 

Table 11-3. Summary of Efficacy Values (Log Reduction) Obtained for DTI-Sweden AB’s SanDes...................58
 

Bacillus anthracis Ames Spores—DTI-Sweden AB’s SanDes ...........................................................59
 

Bacillus subtilis Spores—DTI-Sweden AB’s SanDes.........................................................................59
 

Table 11-6. Deposition/Runoff Weight of DTI-Sweden AB’s SanDes on Test Materials ......................................60
 

Table 11-7. Neutralization Testing with Bacillus anthracis Ames Spores for DTI-Sweden AB’s SanDes............61
 

Table 11-8. Neutralization Testing with Bacillus subtilis Spores for DTI-Sweden AB’s SanDes..........................61
 

xi 
Abbreviations/Acronyms
 

aTCC american Type Culture Collection 

BBRC Battelle Biomedical Research Center 

BSC biosafety cabinet 

C Celsius 

CaSx calcium polysulfide 

CFU(s) colony-forming unit(s) 

CI confidence interval 

Clo2 chlorine dioxide 

cm centimeter 

D/E Dey/Engley 

EPA U.S. Environmental Protection Agency 

g gram 

h horizontal 

hr hour 

l liter 

min minute 

ml milliliter 

NhSRC National homeland Security Research Center 

NIST National Institute of Standards and Technology 

NS neutralization solution 

ORD U.S. EPA Office of Research and Development 

PBS phosphate-buffered saline 

ppm parts per million 

psi pounds per square inch 

QA quality assurance 

QC quality control 

QMP quality management plan 

RH relative humidity 

rpm revolutions per minute 

SD standard deviation 

SDF surface decontamination foam 

SE standard error 

SFW sterile filtered water (cell-culture grade) 

STS sodium thiosulfate 

TOPO Task Order Project Officer 

TSa technical systems audit 

TTEP Technology Testing and Evaluation Program 

v vertical 

wt weight 

xii 
Executive Summary
 

The U.S. Environmental Protection Agency’s (EPA) 

National Homeland Security Research Center (NHSRC) 

Technology Testing and Evaluation Program (TTEP) helps 

to protect human health and the environment from adverse 

impacts of terrorist acts by carrying out performance tests 

on homeland security technologies. TTEP recently evaluated 

the performance of liquid and foam decontamination 

technologies under vendor-specified application conditions 

to decontaminate test coupons prepared from the materials 

listed below. These materials include building materials 

typical of surfaces found in an office building or 

transportation terminal and outdoor materials such as soil 

that could become contaminated with biological agents. 

The first seven materials listed below were used as “indoor” 

surfaces, and the last four as “outdoor” surfaces, with bare 

wood and glass being common to the two sets of materials 

(in one case painted cinder block replaced glass as an 

outdoor material). For testing, each coupon was placed in 

an appropriate orientation (vertical or horizontal) for typical 

use of the material; for some materials either orientation 

may be appropriate but only one was chosen for testing. 

The orientation used in testing is indicated in the listing as 

vertical (v) or horizontal (h): 

■		 Industrial-grade carpet (h) 

■		 Decorative laminate (h) 

■		 Galvanized metal ductwork (v) 

■		 Painted (latex, flat) wallboard paper (v) 

■		 Painted (latex, semi-gloss) cinder block (v) 

■		 Bare wood (pine lumber) (v) 

■		 Glass (v) 

■		 Unpainted concrete (h) 

■		 Topsoil (h). 

Test coupons were 1.9 cm by 7.5 cm, except for topsoil 

which was prepared by filling a Parafilm®- lined 3.5 cm 

diameter by 1 cm deep petri dish with uncompacted soil. 

For testing, coupons were “contaminated” by spiking with 

the biological warfare agent, Bacillus anthracis ames, or 

a surrogate, B. subtilis (American Type Culture Collection 

[ATCC] 19659). The technologies evaluated for their ability 

to inactivate B. anthracis Ames or B. subtilis on test coupons 

of either the seven indoor or the four outdoor surface 

materials were: 

○		 Frontier Pharmaceutical’s DioxiGuardTM 

○		 pH-Amended bleach (Clorox® bleach diluted 

with sterile filtered water and 5% acetic 

acid to obtain pH-amended solution)
	

○		 Calcium polysulfide (lime sulfur) solution at 5.8% 

(wt/wt) (i.e., diluted with sterile filtered water by 

a factor of 5 from the original 29% solution)
	

○		 Allen-Vanguard’s CASCADTM Surface 

Decontamination Foam (SDF)
	

○		 Ecolab Inc.’s Oxonia Active® 

○		 Minntech Corp.’s Minncare® Cold Sterilant 

○		 DTI-Sweden AB’s SanDes. 

With the exception of pH-amended bleach and calcium 

polysulfide, each decontaminant was tested using the 

application apparatus and conditions provided by the 

respective vendor, and according to the vendor’s instructions. 

For pH-amended bleach and calcium polysulfide, no 

single vendor exists. Those two products were tested for 

decontamination of outdoor surfaces using a conventional 

hand-pumped household garden sprayer to apply the product. 

Technical descriptions and preparation and application 

procedures (including the spray device, contact time, 

and reapplication rate) for all the decontaminants tested 

are included as appendices to this report. Spray distance, 

humidity, and temperature were the same for all applications. 

The following performance characteristics of the 

decontamination technologies were evaluated: 

■		 Decontamination efficacy 

○ Quantitative assessment of the decontamination 

efficacy for viable organisms (log reduction)
	

○ Qualitative assessment for residual spores on the
	

test coupons 

■		 Qualitative assessment of material surface damage 

following decontamination. 

Summary results: 

Results for the seven decontaminants tested are summarized 

in the following paragraphs. Table ES-1 lists the quantitative 

efficacy results for all decontaminants on all test materials. 

DioxiGuardTM - This decontaminant was applied to the test 

coupons until they were fully wetted, and no reapplication 

was done. The total contact time before spore extraction 

was 10 minutes. Quantitative efficacy was 2.6 log reduction 

or less for B. anthracis and 0.87 log reduction or less for 

B. subtilis, on the seven indoor materials. All materials 

showed the presence of viable spores after decontamination, 

consistent with the quantitative efficacy results. No damage 

was observed on any of the materials from DioxiGuardTM 

immediately after quantitative efficacy testing, or seven 

days later after completion of the qualitative assessment for 

residual spores. 

ph-amended bleach - This decontaminant was applied to the 

test coupons until they were fully wetted, and the product was 

reapplied if coupons became dry (only one such reapplication 

was needed, on painted cinder block). The total contact time 

before spore extraction was 60 minutes. Quantitative efficacy 

for B. anthracis ames ranged from a log reduction of 7.31 

on painted cinder block, to 4.99 on unpainted concrete, to 

1.47 on topsoil and 0.81 on bare pine wood. Quantitative 

efficacy for B. subtilis ranged from a log reduction of ≥ 7.22 

on painted cinder block, to ≥ 5.63 on unpainted concrete, to 

xiii 
0.18 on topsoil and 0.68 on bare pine wood. Most materials 

showed the presence of viable spores after decontamination, 

except that no growth of B. anthracis or B. subtilis was found 

on painted cinder block, and none of B. subtilis was found 

on unpainted concrete; these results are consistent with the 

quantitative efficacy results. No damage was observed on 

any of the materials from ph-amended bleach immediately 

after quantitative efficacy testing, or seven days later after 

completion of the qualitative assessment for residual spores. 

Calcium polysulfide - This decontaminant was applied to the 

test coupons until they were fully wetted, and then reapplied 

30 minutes after the initial application. The total contact 

time before spore extraction was 60 minutes. Quantitative 

efficacy on the four outdoor materials was very low with 

both test organisms, with a maximum log reduction of 0.24 

for B. anthracis Ames (on unpainted concrete) and of 0.33 

for B. subtilis (on glass). All materials showed the qualitative 

presence of viable organisms after decontamination, 

consistent with the low efficacy results. Decontamination 

with calcium polysulfide left a grayish surface residue on 

glass and topsoil coupons; the presence of such a residue on 

bare wood and unpainted concrete could not be confirmed 

due to the surface characteristics of those materials. The 

residue remained on the glass surfaces throughout agitation 

for spore recovery and the subsequent seven-day qualitative 

assessment for residual spores. 

CASCADTM SDF - This decontaminant was applied to the 

test coupons until they were fully covered with the foam, 

and no reapplication was done. The total contact time before 

spore extraction was 30 minutes. Quantitative efficacy was 

greater than 7.0 log reduction for both B. anthracis and 

B. subtilis on five of the seven indoor materials. Lower 

efficacy values were found only on painted wallboard 

paper and bare pine wood. Efficacy results for B. anthracis 

and B. subtilis on painted wallboard paper were 4.82 and 

≥ 6.14 log reduction, respectively; on bare pine wood the 

corresponding efficacy results were 2.77 and 1.28 log 

reduction, respectively. Only those two materials showed 

the presence of viable organisms after decontamination, 

consistent with the quantitative efficacy results. The only 

materials damage observed from decontamination with 

CASCADTM SDF was that the top coat of paint peeled away 

from the primer coat on painted cinder block coupons. 

Oxonia Active® - This decontaminant was applied to 

the test coupons until they were fully wetted, and then 

reapplied every 10 minutes after the initial application. The 

total contact time before spore extraction was 60 minutes. 

Quantitative efficacy of Oxonia Active® was 7.0 log 

reduction or greater on six of the seven indoor test materials 

for B. anthracis and on five of those seven test materials for 

B. subtilis. Lower efficacy values were found only on bare 

pine wood and painted wallboard paper. Efficacy results for 

B. anthracis and B. subtilis on bare pine wood were 4.64 

and 5.15 log reduction, respectively; on painted wallboard 

paper the efficacy for B. subtilis was ≥ 6.69 log reduction. 

No viable spores were found on any decontaminated coupon 

after either one or seven days of incubation, consistent 

with the quantitative efficacy results. No visible damage 

was observed on any of the test materials after 60 minutes 

contact time with Oxonia Active®, or seven days later after 

completion of the qualitative assessment of residual spores. 

Minncare® Cold Sterilant - This decontaminant was applied 

to the test coupons until they were fully wetted, and no 

reapplication was done. The total contact time before spore 

extraction was 30 minutes for coupons of industrial-grade 

carpet, painted cinder block, and bare pine wood, and 

10 minutes for coupons of decorative laminate, galvanized 

metal ductwork, painted wallboard paper, and glass. 

Quantitative efficacy of Minncare® Cold Sterilant was 7.5 log 

reduction or greater on six of the seven indoor test materials 

for both B. anthracis and B. subtilis. Lower efficacy values 

were found only on bare pine wood, for which efficacy 

results for B. anthracis and B. subtilis were 5.40 and 6.00 

log reduction, respectively. No viable spores were found on 

any decontaminated coupon after either one or seven days of 

incubation, consistent with the quantitative efficacy results. 

No visible damage was observed on any of the test materials 

after either 10 or 30 minutes contact time with Minncare® 

Cold Sterilant, or seven days later after completion of the 

qualitative assessment of residual spores. 

SanDes - This decontaminant was applied to the test coupons 

until they were fully wetted, and reapplication was done at 

10, 20, 30, and 60 minutes after the initial application. The 

total contact time before spore extraction was 70 minutes. 

Quantitative efficacy of SanDes was less than 1.0 log 

reduction for six of the seven indoor materials for both 

B. anthracis and B. subtilis. The exceptions were for B. 

anthracis on glass (4.65 log reduction) and for B. subtilis 

on decorative laminate (1.37 log reduction). Viable spores 

were found on all decontaminated coupons after one day and 

seven days of incubation, consistent with the low quantitative 

efficacy results. No visible damage was observed on any of 

the test materials after 70 minutes contact time with SanDes, 

or seven days later after completion of the qualitative 

assessment of residual spores. 

xiv 
Table ES-1. Summary of Quantitative Efficacy by Decontaminant and Test Material 

Table ES-1a 

Test Material 

Quantitative Efficacy (log reduction) for Bacillus anthracis Ames /Bacillus subtilis 

DioxiGuardTM pH-Amended Bleach Calcium Polysulfide CASCADTM SDF 

Industrial-Grade Carpet 1.83 / 0.87 -- -­ 7.40 / ≥ 7.62 

Decorative Laminate 2.59 / 0.30 -­ -­ 7.40 / ≥ 7.30 

Galvanized Metal Ductwork 0.95 / -0.66 -­ -­ ≥ 7.59 / ≥ 7.60 

Painted Wallboard Paper 0.70 / 0.73 -­ -­ 4.82 / ≥ 6.14 

Painted Cinder Block 1.77 / -0.49 7.31 / ≥ 7.22 -­ ≥ 7.84 / ≥ 7.05 

Bare Pine Wood 0.75 / 0.31 0.81 / 0.68 0.05 / -0.12 2.77 / 1.28 

Glass 2.53 / 0.30 -­ -0.04 / 0.33 ≥ 7.85 / ≥ 7.51 

Unpainted Concrete -­ 4.99 / ≥ 5.63 0.24 / 0.12 -­

Topsoil -­ 1.47 / 0.18 0.21 / 0.21 -­

-- Decontaminant not tested with this material. 

Table ES-1b 

Test Material 

Quantitative Efficacy (log reduction) for Bacillus anthracis Ames /Bacillus subtilis 

Oxonia Active® Minncare® Cold Sterilant SanDes 

Industrial-Grade Carpet 7.00 / ≥ 7.42 ≥ 7.82 / ≥ 7.91 0.13 / 0.59 

Decorative Laminate ≥ 7.61 / ≥ 7.66 ≥ 7.58 / ≥ 7.87 0.18 / 1.37 

Galvanized Metal Ductwork ≥ 7.87 /≥ 7.64 ≥ 7.80 / ≥ 7.89 0.09 / 0.76 

Painted Wallboard Paper ≥ 7.42 / ≥ 6.69 ≥ 7.53 / ≥ 7.46 0.19 / 0.60 

Painted Cinder Block ≥ 7.86 / ≥ 7.29 ≥ 8.08 / ≥ 7.93 0.33 / 0.51 

Bare Pine Wood 4.64 / 5.15 5.40 / 6.00 0.39 / 0.65 

Glass ≥ 7.72 / ≥ 7.03 ≥ 7.75 / ≥ 7.95 4.65 / 0.22 

Unpainted Concrete -­ -­ -­

Topsoil -­ -­ -­

-- Decontaminant not tested with this material. 

xv 
xvi 
1.0 

Introduction
 

NHSRC’s TTEP works in partnership with recognized testing 

organizations; with stakeholder groups consisting of buyers, 

vendor organizations, scientists, engineers, and permitters; 

and with participation of individual technology developers 

in carrying out performance tests on homeland security 

technologies. In response to the needs of stakeholders, 

TTEP evaluates the performance of innovative homeland 

security technologies by developing test plans, conducting 

evaluations, collecting and analyzing data, and preparing 

peer-reviewed reports. All evaluations are conducted in 

accordance with rigorous quality assurance (QA) protocols 

to ensure the generation of high quality data and defensible 

results. TTEP provides unbiased, third-party information 

supplementary to vendor-provided information that is useful 

to decision makers in purchasing or applying the evaluated 

technologies. Stakeholder involvement ensures that user 

needs and perspectives are incorporated into the evaluation 

design to produce useful performance information for each 

evaluated technology. 

TTEP evaluated the performance of liquid and foam 

sporicidal decontamination technologies using vendorspecified application conditions. The primary objective 

of testing sporicidal decontamination technologies was 

to evaluate their ability to inactivate Bacillus anthracis 

(Ames) spores and Bacillus subtilis (ATCC 19659) spores 

on representative indoor or outdoor surface materials. These 

technologies were selected for testing based on existing 

information or data indicating potential sporicidal efficacy 

on building or outdoor materials. Such information or data 

could include EPa registration as a sterilant on hard nonporous surfaces, or data showing sporicidal efficacy on 

different materials or under different test conditions. 

The technologies, which were applied using vendor-specified 

procedures and evaluated on test coupons of either seven 

indoor or four outdoor surface materials, included the 

following: 

○		 Frontier Pharmaceutical’s DioxiGuardTM 

○		 pH-Amended bleach (Clorox® bleach diluted with 

certified cell-culture grade sterile filtered water (SFW) 

and 5% acetic acid to obtain pH-amended solution) 

○		 Calcium polysulfide (lime sulfur) solution at
	

5.8% (wt/wt) (i.e., diluted with SFW by a factor of
	

5 from the original 29% solution)
	

○		 Allen-Vanguard’s CASCADTM Surface 

Decontamination Foam (SDF)
	

○		 Ecolab Inc.’s Oxonia Active® 

○		 Minntech Corp.’s Minncare® Cold Sterilant 

○		 DTI-Sweden AB’s SanDes. 

Testing was performed using application procedures specified 

by each vendor, or (for pH-amended bleach and calcium 

polysulfide) developed by EPA and Battelle based on likely 

use of these decontaminants. The application procedures for 

all decontaminants are included as appendices to this report. 

The decontaminant test procedures are specified in a peerreviewed test/QA plan,(1) that was developed according to the 

requirements of the quality management plan (QMP) for the 

TTEP program.(2) The following performance characteristics 

of the decontamination technologies were evaluated: 

■		 Decontamination efficacy 

○ Quantitative assessment of the decontamination 

efficacy for viable organisms 

○ Qualitative assessment for residual spores 

■ Qualitative assessment of material surface damage 

following decontamination. 

1 
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2 
Table 2-1. Technology Information 

Product Vendor 

General 

Description/ 

Active Ingredients Components 

EPA 

Registrationa 

Contact 

Time 

(min) 

DioxiGuardTM Frontier 

Pharmaceutical 

Chlorine dioxide Sodium chlorite solution; acid solution; contains 

alcohol. 

None 10 

Bleach Clorox® Sodium 

hypochlorite, 

hypochlorous acid 

Sodium hypochlorite 5-6% (pH-amended by 

adding acetic acid 5%)b 

5813-1 

(disinfectant) 

60 

Calcium 

polysulfide 

(lime sulfur) 

VGS, Inc. Calcium polysulfide c 5.8% CaSx 769-558 

(fungicide, 

insecticide, 

miticide) 

60 

CASCADTM SDF Allen-Vanguard Hypochlorite Sodium myristyl sulfate 10-30%, sodium (C14-

16) olefin sulfonate 10-30%; ethanol denatured 

3-9%; alcohols (C10-16) 5-10%, sodium 

sulfate 3-7%; sodium xylene sulfonate 1-5%; 

proprietary mixture of sodium and ammonia salt 

along with co-solvent >9%; dichloroisocyanuric 

acid, sodium salt 48-85%; sodium tetraborate 

3-7%; sodium carbonate 10-15%. 

None 30 

Oxonia Active® Ecolab Inc. Peroxide/ 

peroxyacetic acid 

Hydrogen peroxide 27.5%, peroxyacetic acid 

5.8%. 

1677-129 

(sterilant, 

disinfectant, 

sanitizer) 

60 

Minncare® Cold 

Sterilant 

Minntech Corp. Peroxide/ 

peroxyacetic acid 

Stabilized mixture of 4.5% peroxyacetic acid, 

22% hydrogen peroxide, and acetic acid. 

52252-4 

(sterilant, 

disinfectant, 

sanitizer) 

10 or 

30d 

SanDes DTI-Sweden AB Chlorine dioxide 1,500 ppm ClO2. None 70 

 

a Registered with the EPA Office of Pesticide Programs (OPP). Registration indicates EPA/OPP has evaluated the antimicrobial pesticide to show its effectiveness and 

that it will not have unreasonable adverse effects on humans, the environment, and non-target species, and EPA/OPP has issued a registration or license for use in the 

United States. Note: No product is registered for use against B. anthracis. 

 

b Using procedure recommended by TTEP stakeholders, 5% acetic acid was added to the household bleach to obtain a pH-amended bleach solution. The solution was 

prepared using 9.4 parts SFW, 1 part commercial household bleach, and 1 part 5% glacial acetic acid to yield a solution having a mean pH of 6.81 ± 0.15 and a 

mean total chlorine content of 6,215 ± 212 ppm. This “pH-amended bleach” was evaluated for sporicidal activity. c

 Solution tested was a 1:5 dilution (with SFW) of commercially supplied 29.0% (wt/wt) product.

d

 10 minutes for decorative laminate, glass, wallboard paper, and metal ductwork; 30 minutes for carpet, cinder block, and bare wood. 

2.0 

Technology Description 

Table 2-1 lists the decontamination technologies and the was not confirmed in this evaluation. Detailed technology 

contact times used. The information on product composition descriptions and the application procedures used are included 

in Table 2-1 is based on vendor-provided information as appendices a to g. 

(except for pH-amended bleach and calcium polysulfide) and 

3 
Note that Clorox® bleach is registered as a disinfectant, but 

ph-amended bleach is not. 

Below are brief physical descriptions of the decontamination 

technologies (their form, appearance as received) and 

preparation instructions. greater detail on product 

composition, preparation, and application procedures is 

provided in Appendices A to G. 

■		 DioxiGuardTM – This two component product was 

mixed in equal volumes at the time of use. Component 

a was a sodium chlorite solution and component B 

was an acid solution. The vendor-provided applicator 

was a dual spray bottle containing the two component 

solutions in separate compartments within the bottle, 

and designed to deliver equal portions of the two 

reagent solutions through a single spray nozzle to 

produce the Clo2 decontaminant. 

■		 pH-Amended bleach – Clorox® bleach purchased in 

a one gallon container from a local retail store. The 

diluted, pH-adjusted final solution was applied using a 

hand-pressurized portable garden sprayer. 

■		 Calcium polysulfide (lime sulfur) − This product 

was a red clear liquid consisting of 29.0% by 

weight calcium polysulfide (CaSx) in water. This 

solution was diluted by a factor of five with SFW
	

to produce a 5.8% by weight solution for use in 

testing. The diluted solution was applied using 

a hand-pressurized portable garden sprayer. 

■		 CASCADTM SDF – One CASCADTM solution was 

prepared by diluting 31.2 g of GP2100 (decontaminant) 

to 300 ml with SFW, and the other solution was made 

by diluting 7.2 g of GPB-2100 (buffer) and 18 mL 

of GCE2000 (surfactant) to 300 mL with SFW. The 

application process used a dual spray bottle designed 

to deliver equal portions of the two solutions through 

a single spray nozzle equipped with a diffuser mesh to 

produce the foam. 

■		 Oxonia Active® – a decontaminant solution containing 

5,000 ppm peroxyacetic acid was prepared fresh daily 

by diluting 76 mL of Oxonia Active® to 1 l with SFW 

water. The diluted solution was applied using a handpressurized portable garden sprayer. 

■		 Minncare® – a 10% solution of Minncare® Cold 

Sterilant was prepared fresh shortly before use on each 

day of testing, by diluting 1 part of the Cold Sterilant 

with 9 parts of SFW. The 10% Cold Sterilant solution 

was applied to test coupons using a hand-held plastic 

spray bottle. 

■		 SanDes – This product was an aqueous solution of 

1,500 ppm Clo2, and was used without dilution. The 

product was applied to test coupons using a small pushbutton spray attachment that replaced the cap on a bottle 

of SanDes. 

4 
 

3.0 

Summary of Test Procedures
 

Test procedures were performed in accordance with the test/ 

QA plan(1) and are briefly summarized here. 

3.1 Preparation and Analysis of Test Coupons 

B. anthracis ames and B. subtilis spores were spiked onto 

test coupons in an appropriate biosafety cabinet (BSC-II or 

-III) according to established Battelle procedures.(3-8) Spiked 

coupons were prepared fresh for each day of experimental 

work, by placing coupons flat in the BSC and spiking at 

approximately 1 x 108 colony-forming units (CFUs) per 

coupon. This spiking was accomplished by dispensing a 

100-μL aliquot of a spore stock suspension (approximately 

1 x 109 CFUs/mL) using a micropipette as 10 droplets (each 

of 10 μL volume) across the surface of the coupon. This 

approach provided more uniform distribution of spores across 

the coupon surface than would be obtained through a single 

drop of the suspension. after spiking, the coupons remained 

undisturbed overnight in a BSC to dry. Except in testing 

of DioxiGuardTM with B. anthracis, and in testing of phamended bleach with both B. anthracis and B. subtilis, blank 

(unspiked) coupons were held in a separate cabinet from the 

spiked coupons, to avoid contamination of the blanks with 

spores during the drying period.
 

on the day following spiking, coupons intended for 

decontamination (including blanks and controls) were 

transferred into a glove box (test chamber) where the 

decontamination technology was applied using the apparatus 

and application conditions specified in the appendices 

of this report. The decontamination spray distance of 

30 cm (12 inches), humidity (< 70% relative humidity), 

and temperature (20 to 25 °C) were the same for all 

applications. For most decontaminants tested, the amount 

of decontaminant, contact time, spray pressure, application 

and reapplication procedures, etc., were as specified by the 

vendor. For pH-amended bleach and calcium polysulfide, 

these parameters were chosen by EPa and Battelle based on 

common use of these products and reasonable application 

procedures for small-scale evaluation. 

The materials used for test coupons were: 

■ Industrial-grade carpeta 

■ Decorative laminate 

■ Galvanized metal ductwork 

■ Painted (latex, flat) wallboard paper 

■ Painted (latex, semi-gloss) concrete cinder block 

■ Bare wood (pine lumber) 

■ Glass 

■ Unpainted concrete 

■ Topsoil. 

a Carpet used was treated with zinc omadine (a broad spectrum fungicidealgaecide) during manufacture. This treatment may affect test results on 

this material. 

With the exception of topsoil, test coupons were sterilized 

before use by gamma irradiation (carpet, laminate, wallboard 

paper, cinder block, bare wood) or autoclaving (metal 

ductwork, glass, unpainted concrete). 

The use of topsoil as a test coupon required development of 

techniques to assure adequate recovery of spiked B. anthracis 

or B. subtilis spores, and the absence of interference from 

native soil microorganisms in counting of recovered 

spores. a heat shock procedure was found to minimize 

interference by native microorganisms. Specifically, spiked 

or blank topsoil was extracted in phosphate-buffered 

saline (PBS) solution containing Triton X surfactant, and 

the recovered supernatant was heat-shocked in a water 

bath at 65 °C for one hour before being serially diluted 

and plated. Topsoil samples spiked with B. anthracis or 

B. subtilis spores each showed the presence of a single 

homogeneous species, with all colonies of uniform size 

and morphologically distinctive for the respective Bacillus 

species. Blank topsoil samples showed growth of colonies 

of other, native, Bacillus species, which were not seen with 

the spiked topsoil samples. Consequently, although topsoil 

blanks showed some growth, that growth did not occur 

with extracts of spiked topsoil, so no interference existed 

in terms of counting recovered spores. The mechanism by 

which growth of native Bacillus is suppressed in the extracts 

of spiked topsoil was not investigated, but may involve 

monopolization of nutrients by the large numbers of spiked 

spores. By this procedure, the recovery of spores spiked onto 

topsoil was found to be approximately 50% for B. anthracis 

and approximately 34% for B. subtilis. The heat shock 

procedure for use of topsoil differed from the procedure 

originally stated in the test/QA plan;(1) an appropriate 

amendment to the plan was prepared and approved before 

any testing with topsoil coupons was conducted. 

In all testing of each decontaminant, test coupons of those 

materials that are likely to be oriented horizontally in actual 

use (carpet, decorative laminate, topsoil, unpainted concrete) 

were placed flat in the BSC for decontamination, whereas 

coupons of materials likely to be oriented vertically (painted 

wallboard paper, glass, painted cinder block, bare wood, 

and metal ductwork) were held vertically in the BSC for 

decontamination. For some materials (e.g., metal ductwork) 

either a horizontal or vertical orientation could be realistic, 

but only one orientation was used in testing. Runoff of the 

decontaminant from each vertically oriented coupon was 

captured in a vial placed under the coupon and neutralized 

after the requisite contact time, as was the decontaminant 

5 
 

 

 

remaining on the coupon. Decontaminant pooled on top of 

horizontally positioned coupons was similarly captured and 

neutralized. 

Following decontamination, each coupon (along with any 

associated run-off or pooled decontaminant) was transferred 

aseptically to a sterile 50 mL conical vial containing 10 mL 

of extraction solution. All extraction solutions consisted 

primarily of sterile phosphate-buffered saline (PBS) solution 

with Triton X-100 surfactant (i.e., 99.9% PBS solution, 0.1% 

Triton X-100). In extraction of coupons used with a specific 

decontaminant, the PBS/Triton X-100 solution also included 

a neutralizer chosen (or recommended by the vendor) to stop 

the action of that decontaminant. The required concentration 

of each neutralizer was determined in trial runs for each 

decontaminant tested; results of those trial runs are shown 

in the respective results chapters (Chapters 5 to 11). With 

the exception of bare concrete, the coupons were then 

extracted by agitation on an orbital shaker for 15 minutes 

at approximately 200 revolutions per minute (rpm) at room 

temperature. For bare concrete, recovery of spores required 

an alternate procedure in which 45 minutes of sonication 

was used, instead of the period of agitation. For all coupons, 

following extraction 1 mL of the coupon extract was 

removed, and a series of dilutions through 10-7 was prepared 

in SFW. An aliquot (0.1 mL) of the undiluted extract and 

each serial dilution was then spread plated onto tryptic soy 

agar plates and incubated overnight at 35 to 37 °C. Plates 

were enumerated within 18 to 24 hours of plating. The 

number of CFus/ml was determined by multiplying the 

average number of colonies per plate by the reciprocal of the 

dilution, and accounting for the 0.1 mL volume of extract or 

dilution that was plated.
 

Before further decontamination tests, the test chamber was 

cleaned using the vendor-supplied method for neutralizing 

the decontamination reagent (see the appendices to this 

report). If no instructions for neutralization were provided, 

the test chamber was cleaned following procedures 

established under the Battelle Biomedical Research Center 

(BBRC) Facility Safety Plan.(5, 8) 

laboratory blanks controlled for sterility, and procedural 

blanks controlled for viable spores inadvertently introduced 

to test coupons. The procedural blanks were spiked with 

an equivalent amount of 0.1 mL of “stock suspension” that 

did not contain the biological agent or surrogate. To be 

considered acceptable for quantitative efficacy determination, 

extracts of laboratory or procedural blanks had to contain no 

CFU. The mean percent spore recovery from each coupon 

type was calculated using results from positive control 

coupons (spiked, not decontaminated (sprayed with deionized 

water instead of the decontaminant)), by means of the 

following equation: 

Mean % Recovery = [Mean CFUpc/CFuspike] × 100 (1) 

where Mean CFupc is the mean number of CFUs recovered 

from five replicate positive control coupons of a single type, 

and CFuspike is the number of CFus spiked onto each of those 

coupons. The value of CFUspike is known from enumeration 

of the stock spore suspension. Spore recovery was calculated 

for both B. anthracis and B. subtilis on each coupon type, and 

the results are included in Chapters 5 through 11. 

3.2 Decontamination Efficacy 

The performance or efficacy of the decontamination 

technology was assessed by determining the number of 

viable organisms remaining on each test coupon, and 

in any decontaminant run-off from the coupon, after 

decontamination. These data were compared with the number 

of viable organisms extracted from the positive control 

coupons sprayed with SFW, which was the matrix for the 

spore suspension used to spike the test coupons. 

The number of colony-forming units (CFUs) of B. anthracis 

or B. subtilis in extracts of test and positive control coupons 

was determined to calculate efficacy of the decontaminant. 

Efficacy is defined as the extent (as log10 reduction) by 

which viable spores extracted from test coupons after 

decontamination were less numerous than the viable spores 

extracted from positive control coupons subjected only to an 

inert SFW spray, at the same temperature and contact time 

as the decontaminant application. First, the logarithm of the 

CFU count value from each coupon extract was determined, 

and then the mean of those logarithm values was determined 

for each set of control and associated test coupons, 

respectively. Efficacy of a decontaminant for a test organism 

on the i

th coupon material was calculated as the difference 

between those mean log values, i.e.: 

(2) Efficacy  (log CFUcij ) - (log CFUtij ) 

where log CFUcij refers to the j individual logarithm values 

obtained from the positive control coupons and log CFUtij 

refers to the j individual logarithm values obtained from the 

corresponding test coupons, and the overbar designates a 

mean value. In tests conducted under this plan, there were 

five control and five corresponding test coupons (i.e., j = 5). 

In the case where no CFUs were found in a coupon extract, a 

CFu count of 1 was assigned, resulting in a log CFu of zero 

for that coupon. This situation occurred frequently when a 

decontaminant was highly effective, and no CFUs were found 

in the plated aliquot of extract from the decontaminated 

test coupons. In such cases, the final efficacy was reported 

as greater than or equal to (≥) the value calculated by 

Equation 2. 

The variances (i.e., the square of the standard deviation) of 

the log CFUcij and log CFutij values were also calculated for 

both the control and test coupons (i.e., S 2cij and S 2tij), and 

were used to calculate the pooled standard error (SE) for the 

efficacy value calculated in Equation 2, as follows: 

S 2 cij S 2tij (3) SE  

5 5 

6 
where the number 5 again represents the number j of coupons 

in both the control and test data sets. Thus each efficacy 

result is reported as a log reduction value with an associated 

SE value. 

The significance of differences in efficacy across different 

coupon materials and spore types was assessed based on the 

95% confidence interval of each efficacy result. The 95% 

confidence interval (CI) is: 

95% CI = Efficacy ± (1.96 × SE) (4) 

Differences in efficacy were judged to be significant if the 

95% CIs of the two efficacy results did not overlap. The 

efficacy results are presented in a series of tables in Chapters 

5 through 11 for each decontaminant technology by coupon 

material and spore type. 

3.3 Qualitative Assessment of Residual Spores 

Based on the results of previous decontamination 

studies,(9-12) spores might not be expected to be completely 

recovered from coupons by the extraction process. 

Therefore, viable spores might remain on the test coupons 

following decontamination and extraction. As in previous 

decontamination studies, a qualitative assessment was 

performed to determine whether viable spores remained 

on the test coupons after extraction, including both the 

decontaminated test coupons and the positive control 

coupons not subjected to decontamination. This qualitative 

assessment involved different conditions and a much longer 

growth period than the conditions and growth period used in 

the quantitative assessment of efficacy. The assessment was 

made to determine whether the decontaminated coupons with 

zero growth in the quantitative measurement also showed no 

growth in the qualitative method. 

To conduct the qualitative assessment, the test coupons 

from the quantitative assessment, following extraction, 

were transferred into tryptic soy broth culture medium and 

incubated for seven days at appropriate temperatures for 

growth. The culture media were visually inspected after 

one day and after seven days of incubation. A cloudy liquid 

culture after incubation indicated that viable organisms of 

some type remained on the coupon after decontamination 

and extraction. For liquid cultures in which cloudiness was 

observed, a loop of the liquid sample was streaked onto 

a tryptic soy agar plate and incubated under appropriate 

conditions for growth. after incubation the plates were 

examined to determine qualitatively (morphologic 

comparison performed visually) if the observed growth was 

a pure culture of the organism that was inoculated onto the 

coupons, a mixture of the inoculated organism and other 

endogenous organisms, or a mixture of organisms, such as 

molds and bacteria. Thus, by itself a cloudy appearance in the 

growth medium did not necessarily indicate the presence of 

residual viable organisms that had been spiked onto the test 

coupon. This morphological comparison is not definitive, but 

relies on the morphology observed being consistent with the 

distinctive morphology of the target Bacillus species. 

3.4 Qualitative Assessment of Surface Damage 

Trial runs were conducted before any testing with each 

decontaminant, using coupons that had not been spiked with 

spores. In these trial runs the decontaminant was applied 

exactly as specified in the test/QA plan, and measurements 

were made with multiple coupons of each material type to 

determine the amount of the decontaminant that remained 

on, or ran off from, each material. This information was used 

in the calculation of efficacy on each respective material, 

and in trial runs to determine the amount of neutralizing 

agent needed to stop the action of the decontaminant after 

the prescribed contact time. In addition, visual inspection of 

each coupon surface by two test personnel took place after 

the prescribed decontaminant contact time, through sideby-side comparison of the decontaminated test surface and 

control coupons of the same test material. Differences in 

color, reflectivity, and roughness were assessed qualitatively, 

and observations were recorded by the test personnel. The 

same inspection was conducted after the conclusion of the 

seven-day growth period that assessed qualitative efficacy 

(Section 3.3). 

7 
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8 
4.0 

Quality Assurance/Quality Control
 

Quality assurance/quality control (QC) procedures were 

performed in accordance with the program QMP(2) and the 

test/QA plan(1) for this evaluation, except as noted below. 

QA/QC procedures are summarized below. 

4.1 Equipment Calibration 

All equipment (e.g., pipettes, incubators, biological safety 

cabinets) and monitoring devices (e.g., temperature, relative 

humidity) used at the time of evaluation were verified as 

being certified, calibrated, or validated. 

4.2 QC Results 

Quality control efforts conducted during decontaminant 

testing included positive control coupons (spiked, 

not decontaminated), procedural blanks (not spiked, 

decontaminated), laboratory blanks (not spiked, not 

decontaminated), and spike control samples (analysis of the 

stock spore suspension). The results for these QC samples 

in each decontaminant evaluation are included in the results 

chapter for each respective decontaminant (i.e., see Chapters 

5 through 11). 

A common observation was relatively low recovery of 

spores from coupons of wood and unpainted concrete. 

However, such recoveries were sufficient to meet QA 

targets in nearly all cases, and allowed determination of 

efficacy up to ~6 log reduction. 

4.3 Audits 

4.3.1 Performance Evaluation Audit 

No performance evaluation audit was performed for 

B. anthracis ames or B. subtilis organisms because 

quantitative standards for these biological materials 

do not exist. 

4.3.2 Technical Systems Audit 

Battelle QA staff first conducted a technical systems audit 

(TSA) at the BBRC during DioxiGuardTM testing on 

February 7, 2008 to ensure that the evaluation was being 

conducted in accordance with the test/QA plan(1) and the 

QMP.(2) A second such TSA was conducted during various 

activities of the Oxonia Active® testing, on multiple days 

between November 5 and November 21, 2008. As part of 

the TSAs, test procedures were compared to those specified 

in the test/QA plan, and data acquisition and handling 

procedures were reviewed. Observations and findings from 

the TSas were documented and submitted to the Battelle 

Task Order Leader for response. No adverse findings resulted 

from these TSas. TSa records were permanently stored with 

the TTEP QA Manager. 

4.3.3 Data Quality Audit 

At least 10% of the data acquired during the evaluation were 

audited. A Battelle QA auditor traced the data from the initial 

acquisition, through reduction and statistical analysis, to final 

reporting to ensure the integrity of the reported results. all 

calculations performed on the data undergoing the audit were 

checked. 

4.4 Test/QA Plan Amendments and Deviations 

Two amendments to the test/QA plan were prepared, 

reviewed, approved, and distributed to all parties involved 

in this evaluation. One amendment established the heat 

shocking approach to be used with the soil test material, in 

place of the soil sterilization approach indicated in the test/ 

QA plan. The second amendment established sonication, 

rather than agitation, as the spore extraction procedure for 

unpainted concrete coupons. The TSas cited in Section 4.3.2 

showed that all test procedures followed the test/QA plan, 

i.e., no deviations were recorded. 

4.5 QA/QC Reporting 

Each audit was documented in accordance with the QMP.(2) 

The results of the audits were submitted to the EPA (i.e., to 

the NHSRC Quality Assurance Manager and the Task Order 

Project Officer (TOPO)). 

4.6 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. All 

data were recorded by Battelle staff. The person performing 

the QC/technical review was involved in the experiments and 

added his/her initials and the date to a hard copy of the record 

being reviewed. This hard copy was returned to the Battelle 

staff member who stored the record. 

9 
This page intentionally blank.

10 
5.0 

DioxiGuardTM (Frontier Pharmaceutical)
 

Test Results
 

5.1 QC Results 

In testing of DioxiGuardTM, all positive control results were 

well within the target recovery range of 1 to 150% of the 

spiked spores. For B. anthracis positive control recovery 

values ranged from 7 to 77%, with the lowest recovery (and 

the only recovery value below 50%) occurring on bare wood. 

For B. subtilis positive control recovery values ranged from 

3 to 42%, with recoveries below 10% on bare wood, painted 

wallboard, galvanized metal, and painted concrete. 

In testing of DioxiGuardTM, all procedural and laboratory 

blanks met the criterion of no observed CFUs in quantitative 

efficacy testing, with both B. anthracis and B. subtilis. 

However, in the qualitative assessment of residual spores, 

which involves a much longer nutrient growth period, growth 

was observed from procedural and laboratory blanks used 

in testing with B. anthracis. This finding suggested slight 

contamination of the blanks during overnight drying of all 

coupons in the test chamber. Modification of the drying 

procedure (i.e., placing procedural and laboratory blank 

coupons in a separate chamber before overnight drying of 

the coupons) was implemented for B. subtilis testing with 

DioxiGuardTM. As a result, no CFUs were observed from 

any of the procedural or laboratory blanks used in B. subtilis 

testing with DioxiGuardTM. 

Spike control samples were taken from the spore suspension 

on each day of testing, and serially diluted, nutrient 

plated, and counted to establish the spore density used 

to spike the coupons. This process takes approximately 

24 hours, so spore density is known after completion of 

each day’s testing. The target criterion is to maintain a 

spore suspension density of 1 × 109

/mL (± 25%), leading 

to a spike of 1 × 108 spores (± 25%) on each test coupon. 

The actual spike values for two days of B. anthracis 

testing were 1.79 × 108/coupon and 1.25 × 108/coupon, 

and for two days of B. subtilis testing the actual spike 

values were 1.30 × 108/coupon and 9.57 × 107/coupon. 

5.2 Decontamination Efficacy 

The decontamination efficacy of Frontier Pharmaceutical’s 

DioxiGuardTM was evaluated for B. anthracis ames 

and B. subtilis on seven indoor material surfaces. The 

following sections summarize the results found with this 

decontaminant. 

5.2.1 Quantitative Assessment of the Log Reduction of 

Viable Organisms 

The decontamination efficacy of Frontier Pharmaceutical’s 

DioxiGuardTM was approximately 2.6 log reduction or less 

on all materials, as shown for B. anthracis and B. subtilis 

in Tables 5-1 and 5-2, respectively, and summarized in 

Table 5-3. For each test material, Tables 5-1 and 5-2 show the 

number of CFus inoculated per coupon, the mean of the logs 

of the five observed spore counts in terms of CFUs found 

on both control and test coupons, the mean percent recovery 

(calculated using Equation 1), and the quantitative efficacy 

value and its 95% confidence interval, calculated using 

Equations 2 through 4. 

The highest efficacy of DioxiGuardTM with B. anthracis 

(over 2.5 log reduction) was seen on non-porous materials 

(decorative laminate, glass) (Table 5-1). Although galvanized 

metal is a non-porous material, efficacy for B. anthracis on 

that material (approximately one log reduction, Table 5-1) 

differed from the efficacy on the other non-porous materials. 

Intermediate efficacy for B. anthracis (i.e., approximately 

1.8 log reduction) was seen with industrial-grade carpet 

and painted concrete. The other porous materials (bare 

wood, wallboard paper), showed consistently lower efficacy 

with B. anthracis (less than 0.8 log reduction). During the 

DioxiGuardTM testing, the porous materials appeared wet 

while the DioxiGuardTM was being sprayed on, but then 

absorbed the DioxiGuardTM within a few seconds once the 

application stopped. Wetting or saturation could not be 

discerned with the industrial grade carpet due to its weave. 

Therefore, the DioxiGuardTM was continuously sprayed 

across the surfaces of the five replicates and blank for 

ten seconds as stated in the application procedure for this 

decontaminant (Appendix A).
	

The efficacy of DioxiGuardTM for B. subtilis was lower than 

for B. anthracis. As Table 5-2 shows, the highest efficacy 

for the B. subtilis was found with the industrial-grade 

carpet (0.87 log reduction). On metal ductwork and painted 

concrete, fewer B. subtilis spores were recovered from the 

control coupons than from the decontaminated coupons, 

leading to a negative result for efficacy.
	

Table 5-3 summarizes the efficacy results for DioxiGuardTM 

on all test materials. Bolded entries in the table indicate 

materials for which the efficacy results with B. subtilis are 

significantly different from those with B. anthracis. Efficacy 

results for B. subtilis on these materials were zero to 0.3 logs. 

As Tables 5-1 and 5-2 show, no CFUs were observed from 

extraction and plating of either the laboratory or procedural 

blanks for either organism in the quantitative efficacy testing. 

However, in the subsequent qualitative assessment of residual 

spores, the blank coupons from B. anthracis testing did 

exhibit some growth. These results are discussed in Section 

5.2.2 below. 

11 
Table 5-1. Inactivation of Bacillus anthracis Ames Sporesa

—Frontier Pharmaceutical’s DioxiGuardTM 

(10 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.79 x 108 

1.79 x 108 

0 

0 

8.07 ± 0.08 

6.24 ± 0.50 

0 

0 

66.3 ± 10.5 

1.8 ± 2.7 

0 

0 

-­

1.83 ± 0.45 

-­

-­

Decorative Laminate 

Positive Controlsb 1.79 x 108 8.04 ± 0.06 61.4 ± 8.1 -­

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.79 x 108 

0 

0 

5.45 ± 0.53 

0 

0 

0.26 ± 0.26 

0 

0 

2.59 ± 0.47 

-­

-­

Galvanized Metal Ductwork 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.25 x 108 

1.25 x 108 

0 

0 

7.98 ± 0.05 

7.03 ± 0.13 

0 

0 

76.8 ± 8.5 

8.9 ± 2.7 

0 

0 

-­

0.95 ± 0.12 

-­

-­

Painted Wallboard Paper 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.25 x 108 

1.25 x 108 

0 

0 

7.80 ± 0.10 

7.10 ± 0.07 

0 

0 

51.7 ± 11.4 

10.2 ± 1.7 

0 

0 

-­

0.70 ± 0.11 

-­

-­

Painted Cinder Block 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.25 x 108 

1.25 x 108 

0 

0 

7.98 ± 0.06 

6.21 ± 0.30 

0 

0 

76.9 ± 10.0 

1.6 ± 1.5 

0 

0 

-­

1.77 ± 0.27 

-­

-­

Bare Pine Wood 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.79 x 108 

1.79 x 108 

0 

0 

7.09 ± 0.12 

6.34 ± 0.47 

0 

0 

7.1 ± 1.7 

1.8 ± 1.4 

0 

0 

-­

0.75 ± 0.42 

-­

-­

Glass 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.79 x 108 

1.79 x 108 

0 

0 

8.01 ± 0.05 

5.48 ± 1.08 

0 

0 

57.7 ± 7.0 

1.7 ± 3.4 

0 

0 

-­

2.53 ± 0.95 

-­

-­ a Data are expressed as mean (± SD) total number of spores (CFUs) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“--” Not Applicable.
 

5.2.2 Qualitative Assessment of Residual Spores 

Results from the liquid culture growth assessment of coupons 

at one and seven days post-decontamination are provided 

in Tables 5-4 and 5-5 for coupons spiked with B. anthracis 

ames and B. subtilis spores, respectively. In this assessment, 

cultures showing positive growth (i.e., a cloudy growth 

medium) were applied to streak plates and the identity of the 

growing organism was checked by colony morphology. only 

B. anthracis colonies were found in cultures of coupons used 

with B. anthracis, and only B. subtilis colonies were found in 

cultures of coupons used with B. subtilis. 

Table 5-4 shows that almost all coupons of all materials 

showed positive growth for B. anthracis, including most 

of the blank coupons. The growth observed with the blank 

coupons is most likely due to slight contamination due 

to the proximity of these blanks to their B. anthracisinoculated replicates during post-spike drying in the test 

chamber. As noted in Section 5.1, once the positive results 

on the blanks were observed in the B. anthracis testing, a 

procedural change was made to avoid cross-contamination 

in the B. subtilis testing (i.e., the blanks were placed inside a 

different Class III BSC from the spore-inoculated materials 

for drying overnight). As Table 5-5 shows, no growth was 

observed on any of the blank coupons in the qualitative 

assessment of residual B. subtilis spores, though growth was 

observed on all the spiked coupons of all materials. 

12 
Table 5-2. Inactivation of Bacillus subtilis Sporesa

—Frontier Pharmaceutical’s DioxiGuardTM 

(10 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.30 x 108 

1.30 x 108 

0 

0 

7.31 ± 0.12 

6.44 ± 1.06 

0 

0 

16.0 ± 4.6 

9.7 ± 14.4 

0 

0 

-­

0.87 ± 0.94 

-­

-­

Decorative Laminate 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.30 x 108 

1.30 x 108 

0 

0 

7.67 ± 0.05 

7.38 ± 0.12 

0 

0 

36.3 ± 4.0 

18.9 ± 6.0 

0 

0 

-­

0.30 ± 0.12 

-­

-­

Galvanized Metal Ductwork 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

9.57 x 107 

9.57 x 107 

0 

0 

6.59 ± 0.07 

7.25 ± 0.25 

0 

0 

4.1 ± 0.7 

21.0 ± 10.7 

0 

0 

-­

-0.66 ± 0.23 

-­

-­

Painted Wallboard Paper 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

9.57 x 107 

9.57 x 107 

0 

0 

6.81 ± 0.20 

6.08 ± 0.01 

0 

0 

7.3 ± 2.7 

1.3 ± 0.04 

0 

0 

-­

0.73 ± 0.18 

-­

-­

Painted Cinder Block 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

9.57 x 107 

9.57 x 107 

0 

0 

6.81 ± 0.15 

7.35 ± 0.31 

0 

0 

7.1 ± 3.0 

22.8 ± 11.4 

0 

0 

-­

-0.49 ± 0.23 

-­

-­

Bare Pine Wood 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.30 x 108 

1.30 x 108 

0 

0 

6.56 ± 0.19 

6.25 ± 0.22 

0 

0 

3.0 ± 1.2 

1.5 ± 0.7 

0 

0 

-­

0.31 ± 0.26 

-­

-­

Glass 

Positive Controlsb 

Test Couponsc 

Laboratory Blankd 

Procedural Blanke 

1.30 x 108 

1.30 x 108 

0 

0 

7.73 ± 0.02 

7.43 ± 0.17 

0 

0 

41.8 ± 2.1 

17.5 ± 11.8 

0 

0 

-­

0.30 ± 0.15 

-­

-­

 

a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“--” Not Applicable.
 

13 
Table 5-3. Summary of Efficacy Values (Log Reduction) 

Obtained for Frontier Pharmaceutical’s DioxiGuardTM a 

Material B. anthracis Ames B. subtilis 

Industrial-Grade Carpet 1.83 0.87 

Decorative Laminate 2.59 0.30 

Galvanized Metal Ductwork 0.95 -0.66 

Painted Wallboard Paper 0.70 0.73 

Painted Cinder Block 1.77 -0.49 

Bare Pine Wood 0.75 0.31 

Glass 2.53 0.30 

a

 Numbers in bold are statistically different (p ≤ 0.05) from B. anthracis Ames 

The qualitative, liquid culture growth assessment results are 

consistent with the quantitative, observed efficacy results for 

all of the materials, except for the industrial-grade carpet, 

perhaps due to the antibacterial component (zinc omadine) 

in the carpet. For both B. anthracis and B. subtilis, this 

material exhibited only partial growth for the five replicate 

samples (both decontaminated with DioxiGuardTM and not 

decontaminated) after Day 1. After Day 7, however, all 

replicate samples were positive for growth. 

5.3 Damage to Coupons 

No visible damage was observed on any of the test materials 

after the 10 minute contact time with DioxiGuardTM in 

the quantitative efficacy testing, or seven days later after 

completion of the qualitative assessment of residual spores. 

5.4 Other Factors 

5.4.1 Operator Control 

On each day of testing, Frontier Pharmaceutical’s 

DioxiGuardTM was prepared by placing the spray nozzle 

onto the dual bottle, in which each half of the bottle 

contained one of the two DioxiGuardTM reagent solutions. 

Prior to each application, the DioxiGuardTM spray nozzle 

was primed by repeatedly spraying into an absorbent cloth 

to clear any air bubbles that may have formed between 

applications. after each application, the spray nozzle was 

removed from the bottle and any residual DioxiGuardTM was 

removed by repeated pulls on the trigger of the spray nozzle. 

The spray nozzle was then placed onto a dual bottle that 

contained only SFW to completely clean out the spray nozzle 

until its next use. 

all tests were conducted under ambient conditions inside 

a climate-controlled laboratory. The temperature inside the 

test chamber was equilibrated to the ambient laboratory 

temperature of approximately 22 °C. The RH inside the test 

chamber was monitored with a NIST-traceable hygrometer. 

Whenever the RH exceeded 40%, the dehumidification 

system attached to the testing chamber was actuated until 

the RH dropped below 40%. The dehumidifier was actuated 

only after the ten minute contact time with the DioxiGuardTM. 

Therefore, the testing chamber was always within 40% Rh 

prior to the decontamination of a new set of materials with 

DioxiGuardTM. 

14 
 

Table 5-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis Ames Spores— 

Frontier Pharmaceutical’s DioxiGuardTM 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

+ 

+ 

-

+ 

+ 

-

+ 

+ 

-

-

-

a 

-

b 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

Decorative Laminate 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

Galvanized Metal Ductwork 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

Painted Wallboard Paper 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

Painted Cinder Block 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

Bare Pine Wood 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

Glass 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. anthracis Ames spores) ); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. anthracis Ames spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. anthracis Ames spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth.
 

15 
 

Table 5-6. Deposition/Runoff Weight of Frontier 

Pharmaceuticals’ DioxiGuardTM 

on Test Materials 

Material 

Avg. Deposition/Runoff 

Weight (g) 

Industrial-Grade Carpet 0.19 

Decorative Laminate 0.10 

Galvanized Metal Ductwork 0.34 

Painted Wallboard Paper 0.07 

Painted Cinder Block 0.26 

Bare Pine Wood 0.26 

Glass 0.15 

Table 5-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus subtilis Spores— 

Frontier Pharmaceutical’s DioxiGuardTM 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

-

+ 

-

-

-

-

+ 

-

-

+ 

-

a 

-

b 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

-

-

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. subtilis spores) ); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. subtilis spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. subtilis spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth. 

5.4.2 Technology Spray Deposition 

Frontier Pharmaceutical’s DioxiGuardTM was applied 

according to the procedure included as Appendix A of this 

report. DioxiGuardTM was applied from a distance of 30 cm 

(12 inches) from the horizontally and vertically oriented 

materials until the materials appeared fully wetted. For most 

test materials only a few sprays from the dual bottle, over 

a few seconds, were required. The one exception was for 

the industrial-grade carpet. Since it was difficult to discern 

whether this material was wetted with DioxiGuardTM due to 

its weave, the carpet was, instead, wetted using several sprays 

over ten full seconds. No reapplication of the DioxiGuardTM 

was made on any coupon surface. after the ten minute 

contact time, each material coupon was placed in the tube 

that also served to collect excess decontaminant runoff. The 

horizontally and vertically oriented coupon materials stayed 

in their respective configurations for the duration of their ten 

minute contact times. 

To assess DioxiGuardTM deposition, triplicate coupons of 

each test material were weighed prior to application of 

DioxiGuardTM in trial runs, and these values were recorded. 

Then the triplicate coupons were sprayed with DioxiGuardTM 

until the triplicate coupons were fully wetted in their 

respective vertical or horizontal orientations, allowed a 

10 minute contact time, and then each coupon was weighed 

again. The pre-application weights were then subtracted from 

the post-application weights, and that difference was added to 

the weight of decontaminant runoff captured separately from 

each coupon. Table 5-6 summarizes the results, showing that 

the amount of DioxiGuardTM deposited on different materials 

ranged from 0.07 g to 0.34 g; the average deposited amount 

was approximately 0.2 g (or 200 μL). That average amount 

was used to determine the amount of sodium thiosulfate 

(STS) needed to effectively neutralize the DioxiGuardTM. 

16 
Table 5-7. Neutralization Testing with Bacillus anthracis Ames Spores for Frontier Pharmaceutical’s DioxiGuardTM 

Treatment Inoculum (CFUs) Total Observed CFUs % of Control 

DioxiGuardTM + Sporesa 1.37 x 108 0 0 

DioxiGuardTM + PBS + Triton X-100 + Sporesab 1.37 x 108 0 0 

PBS + Triton X-100 + Spores (Control)b 1.37 x 108 1.40 x 108 -

DioxiGuardTM + PBS + Triton X-100 + 0.0002% STS + Sporesab 1.37 x 108 0 0 

DioxiGuardTM + PBS + Triton X-100 + 0.002% STS + Sporesab 1.37 x 108 0 0 

DioxiGuardTM + PBS + Triton X-100 + 0.02% STS + Sporesab 1.37 x 108 1.25 x 108 89.3 

a

 DioxiGuardTM volume of 0.2 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with 

DioxiGuardTM = 10.2 mL (10 mL PBS+Triton +STS + 0.2 mL DioxiGuardTM). 

“-” Not Applicable. 

Table 5-8. Neutralization Testing with Bacillus subtilis Spores for Frontier Pharmaceutical’s DioxiGuardTM 

Treatment Inoculum (CFUs) Total Observed CFUs % of Control 

DioxiGuardTM + Sporesa 1.11 x 108 0 0 

DioxiGuardTM + PBS + Triton X-100 + Sporesab 1.11 x 108 0 0 

PBS + Triton X-100 + Spores (Control)b 1.11 x 108 1.14 x 108 -

DioxiGuardTM + PBS + Triton X-100 + 0.0002% STS + Sporesab 1.11 x 108 0 0 

DioxiGuardTM + PBS + Triton X-100 + 0.002% STS + Sporesab 1.11 x 108 0 0 

DioxiGuardTM + PBS + Triton X-100 + 0.02% STS + Sporesab 1.11 x 108 1.10 x 108 96.5 

a

 DioxiGuardTM volume of 0.2 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with 

DioxiGuardTM = 10.2 mL (10 mL PBS+Triton +STS + 0.2 mL DioxiGuardTM). 

“-” Not Applicable. 

5.4.3 Neutralization Methodology 

The vendor reported 190 ppm of ClO2 was present in the 

delivered DioxiGuardTM formulation. For testing this ClO2 

value was assumed to be correct to calculate the amount of 

STS needed to neutralize the DioxiGuardTM. That calculation 

was based on the formula weights for Clo2, the average 

mass of spray deposition on the test materials, and other 

factors. The target concentration of STS needed to effectively 

neutralize the DioxiGuardTM was thus calculated at 0.002% 

in the PBS/Triton X-100 extraction solution. This calculated 

STS concentration was coincidentally the same as that used 

in previous testing,(13) as a result of the higher nominal Clo2 

concentration (i.e., 190 ppm) and lower average deposited 

amount of DioxiGuardTM (i.e., 200 μL) in this evaluation. 

However, during the DioxiGuardTM neutralization trial 

conducted with B. anthracis and B. subtilis, the extraction 

media (PBS/Triton/0.002% STS) still showed significant 

kill after neutralization (i.e., the DioxiGuardTM was not 

effectively neutralized) (see Tables 5-7 and 5-8). The upper 

and lower limits for this neutralization trial were set at 

0.02% and 0.0002% STS, respectively, to provide a range of 

neutralization results. The upper limit STS concentration of 

0.02% neutralized the DioxiGuardTM, whereas the lower limit 

exhibited total kill (i.e., no neutralization of DioxiGuardTM). 

As result of these observations, the ClO2 concentrations 

from two recent shipments of DioxiGuardTM (saved after 

the B. anthracis and B. subtilis tests) were measured to try 

to explain why the calculated concentration of STS was 

ineffective. The DioxiGuardTM ClO2 concentration was 

measured by the procedure of titration with 0.1 N sodium 

thiosulfate(14, 15) and found to be over 400 ppm, more than 

twice the concentration indicated by the vendor. This result 

explains why the upper limit (0.02%) STS concentration 

successfully neutralized the DioxiGuardTM but the calculated 

target (0.002%) STS concentration failed to neutralize 

the DioxiGuardTM. The original target concentration of 

0.002% STS was used in tests described above with both 

B. anthracis and B. subtilis, so the potential exists for 

incomplete neutralization of DioxiGuardTM in those tests. 

Consequently, the efficacy results reported in Section 5.1 

must be considered as upper limits to the actual efficacy of 

DioxiGuardTM, in that the action of DioxiGuardTM may not 

have been promptly neutralized upon addition of the STS. 

17 
This page intentionally blank.

18 
6.0 

pH-Amended Bleach Test Results
 

6.1 QC Results 

In testing of ph-amended bleach with B. anthracis, percent 

recovery of inoculated spores from the positive control 

coupons ranged from about 9 to 77%, with the lowest 

recovery results on bare wood and unpainted concrete. For 

B. subtilis, positive control recovery values ranged from 

about 0.5 to 18%, also with the lowest recoveries on bare 

wood and unpainted concrete. All percent recovery values 

were well within the acceptable range of 1 to 150% stated 

in the test/QA plan, except for the value of 0.49% recovery 

found for B. subtilis on unpainted concrete. (In trial runs with 

this organism on this material, a recovery value of 1.22% had 

been found.) The EPA Task Order Project Officer (TOPO) 

was notified of this low recovery value, and he decided to 

retain the test results, i.e., testing was not repeated with this 

organism on this material. 

all procedural and laboratory blanks met the criterion of no 

observed CFUs in quantitative efficacy testing, with both 

B. anthracis and B. subtilis. In the qualitative assessment 

of residual spores, which involves a much longer nutrient 

growth period, growth was observed from the procedural and 

laboratory blank soil coupons used with both B. anthracis 

and B. subtilis, and from the laboratory blank coupons 

of bare pine wood and painted cinder block used with 

B. subtilis. This finding is discussed in Section 6.2.2. 

Preliminary tests indicated that extracts of blank soil samples 

(i.e., not spiked with B. anthracis or B. subtilis) showed 

the presence of several colony forming species. However, 

when spiked with B. anthracis or B. subtilis spores and 

extracted, each soil sample showed the presence of a single 

homogeneous species, with all colonies of uniform size 

and morphologically distinctive for the respective Bacillus 

species. Therefore, blank soil samples were deemed to be 

contaminated only if more than the one inoculated species 

was found in the extracts of inoculated soil samples. This 

approach was formalized by the approval of the test/QA plan 

amendment noted in Section 3.1. 

Spike control samples were taken from the spore suspension 

on each day of testing, and serially diluted, nutrient plated, 

and counted to establish the spore density used to spike the 

coupons. This process takes approximately 24 hours, so spore 

density is known after completion of each day’s testing. 

The target criterion is a spore suspension density of 1 × 109/ 

mL (± 25%), leading to a spike of 1 × 108 spores (± 25%) 

on each test coupon. The actual spike value for B. anthracis 

testing was 1.22 × 108/coupon, and for B. subtilis testing the 

actual spike value was 9.10 × 107/coupon. Thus all coupons 

received a spore spike that met the target criterion. 

6.2 Decontamination Efficacy 

The decontamination efficacy of pH-amended bleach was 

evaluated for B. anthracis and B. subtilis on four outdoor 

material surfaces. The following sections summarize the 

results found with this decontaminant. 

6.2.1 Quantitative Assessment of the Log Reduction of 

Viable Organisms 

The quantitative efficacy results for pH-amended bleach 

are presented in Tables 6-1 and 6-2. The decontamination 

efficacy of pH-amended bleach was highest for the painted 

cinder block (7.31 log reduction and ≥ 7.22 log reduction 

for B. anthracis ames and B. subtilis, respectively), and 

relatively high for unpainted concrete (4.99 and ≥ 5.63 log 

reduction, respectively), but was low for soil (1.47 and 

0.18 log reduction) and bare pine wood (0.81 and 0.68 log 

reduction). The porous bare pine wood tended to absorb some 

of the control application (i.e, SFW) and the decontaminant. 

The porous unpainted concrete also appeared to absorb the 

SFW and decontaminant, but allowed much higher efficacy 

than did the bare wood. 

Table 6-3 summarizes the quantitative efficacy results, and 

shows that the efficacy of pH-amended bleach for B. subtilis 

was similar to that for B. anthracis ames on most test 

materials. Only with soil as the test surface was the efficacy 

for B. subtilis significantly different (in this case, lower) than 

that for B. anthracis. 

6.2.2 Qualitative Assessment of Residual Spores 

Results from the liquid culture growth assessment of coupons 

at one and seven days post-decontamination are provided 

in Tables 6-4 and 6-5 for coupons spiked with B. anthracis 

ames and B. subtilis spores, respectively. In this assessment, 

cultures showing positive growth (i.e., a cloudy growth 

medium) were applied to streak plates and the identity of 

the growing organism was checked by colony morphology. 

only B. anthracis colonies were found in cultures of coupons 

inoculated with B. anthracis, and only B. subtilis colonies 

were found in cultures of coupons inoculated with B. subtilis. 

Table 6-4 shows that with B. anthracis, no growth was 

observed from decontaminated coupons of painted cinder 

block after either one or seven days’ incubation, but positive 

growth was observed with the other materials. Similarly, 

Table 6-5 shows that with B. subtilis, little to no growth was 

observed from decontaminated coupons of painted cinder 

block and unpainted concrete after either one or seven days’ 

incubation, but positive growth was observed with the other 

materials. These results are consistent with the quantitative 

19 
 

 

Table 6-1. Inactivation of Bacillus anthracis Ames Sporesa

 — pH-Amended Bleach (60 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Painted Cinder Block 

Positive Controlsb 1.22 x 108 7.96 ± 0.13 76.8 ± 21.2 -

Test Couponsc 1.22 x 108 0.65 ± 1.45 0.0003 ± 0.0006 7.31 ± 1.27 

Laboratory Blankd 0 0 0 -

Procedural Blanke 0 0 0 -

Bare Pine Wood 

Positive Controls 1.22 x 108 7.11 ± 0.11 10.8 ± 2.77 -

Test Coupons 1.22 x 108 6.29 ± 0.44 2.20 ± 1.48 0.81 ± 0.40 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Unpainted Concrete 

Positive Controls 1.22 x 108 7.04 ±0.14 9.32 ± 2.94 -

Test Coupons 1.22 x 108 2.05 ± 1.90 0.002 ± 0.003 4.99 ± 1.67 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Topsoil 

Positive Controls 1.22 x 108 7.87 ± 0.12 62.8 ± 16.1 -

Test Coupons 1.22 x 108 6.40 ± 0.95 4.76 ± 3.50 1.47 ± 0.84 

Laboratory Blank 0f 0 0 

Procedural Blank 0f 0 0 -

a Data are expressed as mean of the logs of total number of spores (CFU) observed on individual coupons, percent recovery, and decontamination efficacy (log 

reduction). 

CI = confidence interval (± 1.96 × SE).

b

 Inoculated, not decontaminated coupon (sprayed with SFW). 

c

 Inoculated, decontaminated coupon.

d

 Laboratory Blank = not inoculated, not decontaminated coupon. 

e

 Procedural Blank = not inoculated, decontaminated coupon.

f Endogenous organisms were found in uninoculated soil blanks; no organisms other than B. anthracis Ames or B. subtilis were found on inoculated coupons. 

“-” Not Applicable. 

efficacy observed on the test materials (Table 6-3). As noted and B. subtilis, including blanks, were stored in the same 

in Section 6.1, the laboratory and procedural blanks for BSC during the overnight drying of the spore-inoculated 

topsoil with both organisms, and the laboratory blanks for test coupons. Note that a few of the positive control coupons 

bare pine wood and painted cinder block with B. subtilis, of unpainted concrete showed no growth of B. anthracis or 

also showed positive growth at both one and seven days’ B. subtilis after one or seven days’ incubation (Tables 6-4 and 

incubation. This growth is likely to have resulted from 6-5), despite not being exposed to the decontaminant. This 

slight contamination of the blank coupons during storage, result may be an artifact of the low spore recovery achieved 

after inoculation of the test coupons. In this testing of ph- from this material. 

amended bleach, all coupons for both B. anthracis ames 

­

­

20 
Table 6-2. Inactivation of Bacillus subtilis Sporesa

 —pH-Amended Bleach (60 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Painted Cinder Block 

Positive Controlsb 9.10 x 107 7.22 ± 0.06 18.31 ± 2.89 -

Test Couponsc 

Laboratory Blankd 

9.10 x 107 

0 

0 ± 0.0 

0 

0 

0 

≥ 7.22 ± 0.06 

-

Procedural Blanke 0 0 0 -

Bare Pine Wood 

Positive Controls 9.10 x 107 6.23 ± 0.22 2.06 ± 1.05 -

Test Coupons 9.10 x 107 5.55 ± 0.53 0.64 ± 0.60 0.68 ± 0.50 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Unpainted Concrete 

Positive Controls 9.10 x 107 5.63 ± 0.16 0.49 ± 0.16 -

Test Coupons 9.10 x 107 0 ± 0.0 0 ≥ 5.63 ± 0.14 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Topsoil 

Positive Controls 9.10 x 107 7.03 ± 0.15 12.31 ± 4.33 -

Test Coupons 

Laboratory Blank 

Procedural Blank 

9.10 x 107 

0f 

0f 

6.85 ± 0.23 

0 

0 

8.53 ± 3.64 

0 

0 

0.18 ± 0.24 

-

-

 

a Data are expressed as mean of the logs of total number of spores (CFU) observed on individual coupons, percent recovery, and decontamination efficacy (log 

reduction). 

CI = confidence interval (± 1.96 × SE).

b

 Inoculated, not decontaminated coupon (sprayed with SFW). 

c

 Inoculated, decontaminated coupon.

d

 Laboratory Blank = not inoculated, not decontaminated coupon. 

e Procedural Blank = not inoculated, decontaminated coupon.

 

f Endogenous organisms were found in uninoculated soil blanks; no organisms other than B. anthracis Ames or B. subtilis were found on inoculated coupons. 

“-” Not Applicable. 

Table 6-3. Summary of Efficacy Values (Log Reduction) 

Obtained for pH-Amended Bleacha 

Material B. anthracis Ames B. subtilis 

Painted Cinder Block 7.31 ≥ 7.22 

Bare Pine Wood 0.81 0.68 

Unpainted Concrete 4.99 ≥ 5.63 

Topsoil 1.47 0.18 

a

Numbers in bold are statistically different (p ≤ 0.05) from B. anthracis Ames. 

­

21 
 

 

Table 6-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis Ames Spores— 

pH-Amended Bleach 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Painted Cinder Block 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

a 

-

b 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Unpainted Concrete 

Positive Controls - - + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Topsoil 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. anthracis Ames spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. anthracis Ames spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. anthracis Ames spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth.
 

Table 6-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus subtilis Spores— 

pH-Amended Bleach 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Painted Cinder Block 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

+a 

-

b 

+ 

+ 

+ 

+ 

+ 

-

+ 

-

+ 

-

+ 

-

Bare Pine Wood 

Positive Controls + + + + + + + + + + + -

Test Coupons + + + + + - + + + + + -

Unpainted Concrete 

Positive Controls - + + + - - - + + + - -

Test Coupons + - - - - - + - - - + -

Topsoil 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. subtilis spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. subtilis spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. subtilis spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth. 

6.3 Damage to Coupons 

No visible damage was observed on any of the four test 

materials with ph-amended bleach, either immediately 

after the 60-minute contact time or seven days after 

decontamination, at the conclusion of the qualitative 

efficacy test. 

6.4 Other Factors 

6.4.1 Operator Control 

The ph-amended bleach was prepared according to the 

procedure described in Appendix B, by mixing 9.4 parts 

SFW, 1 part commercial household bleach, and 1 part 5% 

acetic acid. The bleach used was Clorox® brand, obtained 

through a retail purchase, and the bottle was unopened until 

the first day of use. The actual resulting solution used for 

testing with B. anthracis had a ph of 6.6 and a total chlorine 

content of 6,800 ppm; the solution used for B. subtilis testing 

had a ph of 6.45 and a total chlorine content of 6,400 ppm. 

The ph-amended bleach was freshly prepared prior to each 

testing day (i.e., the preparation was assigned a one day 

shelf-life and excess was discarded at the end of the day). 

all trials were conducted under ambient conditions inside 

a climate-controlled laboratory. The temperature inside the 

testing chamber was equilibrated to the ambient laboratory 

temperature of approximately 22 °C. The RH was monitored 

with a NIST-traceable hygrometer. The chamber dehumidifier 

22 
was actuated only after the 60 min contact time with the 

control application (water) or pH-amended bleach. The RH 

in the test chamber never exceeded 70% during the 60 minute 

contact time. 

6.4.2 Technology Spray Deposition 

The ph-amended bleach was applied according to the 

procedure in Appendix B. The pH-amended bleach was 

applied 30 cm (12 inches) from the horizontally (soil and 

unpainted concrete) and vertically (bare pine wood and 

painted cinder block) oriented materials until the materials 

appeared saturated with liquid. A handheld garden sprayer 

was used to apply the control application (SFW) and pHamended bleach. This sprayer was slightly modified to 

accommodate a pressure gauge to ensure that the spray was 

applied using 4 to 6 psi pressure. Close observation of the 

respective material surfaces was made to ensure that they 

were thoroughly wetted (approximately 5 sec spray duration 

was needed to produce wetting across the surfaces of all 

five replicates and corresponding blank for each material 

type). Only one material, the painted cinder block, received 

a reapplication of the decontaminant at the 50 minute mark 

during the 60 minute contact time. a modest reapplication 

(approximately 2 sec spray duration) of the pH-amended 

bleach was done on that material, because the beaded liquid 

droplets visible on the painted surface appeared to have 

diminished substantially. after the 60 minute contact time, 

each material was placed in the 50 mL conical vial that also 

served to collect excess formulation run-off. The horizontally 

and vertically oriented materials stayed in their respective 

configurations throughout the 60 minute contact time. 

To assess ph-amended bleach deposition, triplicate coupons 

of each test material were weighed, and these values were 

recorded. Then the triplicate coupons were sprayed with phamended bleach until fully wetted in their respective vertical 

or horizontal orientations, allowed a 60 minute contact time, 

and then each coupon was weighed again. Painted cinder 

block required a single reapplication at 50 minutes into the 

60 minute contact time. The pre-application weights were 

then subtracted from the post-application weights, and that 

difference was added to the weight of decontaminant runoff 

captured separately from each coupon. Table 6-6 shows the 

results. The amount of ph-amended bleach deposited on 

bare pine wood, unpainted concrete, and soil coupons ranged 

from 0.20 g to 0.25 g; the ph-amended bleach deposited 

on painted cinder block coupons was 0.40 g, including 

the reapplication. The average of these values (0.27 g, or 

0.27 mL based on a density of 1.0) was used to estimate the 

amount of sodium thiosulfate (STS) needed to effectively 

neutralize the ph-amended bleach. 

Table 6-6. Deposition/Runoff Weight of pH-Amended 

Bleach on Test Materials 

Material 

Avg. Deposition/Runoff 

Weight (g) 

Painted Cinder Block 0.40 

Bare Pine Wood 0.20 

Unpainted Concrete 0.24 

Topsoil 0.25 

6.4.3 Neutralization Methodology 

Neutralization of the pH-amended bleach was achieved 

with STS. The STS concentration stated in Appendix B for 

neutralizing the ph-amended bleach was about 0.085% 

in the PBS/Triton X-100 extraction solution, based on 

an applied quantity in previous testing similar to those 

quantities noted above in Section 6.4.2. For performance 

of a neutralization panel of tests, the upper limit STS 

concentration was set at twice this target concentration 

(i.e., at 0.17% STS), and the lower limit was set at half 

this target concentration (i.e., at 0.042% STS). The results 

of the neutralization trials for B. anthracis and B. subtilis 

are shown in Tables 6-7 and 6-8, respectively. These 

tables show that all three tested concentrations of STS 

effectively neutralized the pH-amended bleach. The target 

concentration of 0.085% STS yielded a percent recovery 

of 97.6% for B. anthracis Ames. Similar results (i.e., a 

percent recovery of 93.6%) were found with B. subtilis at 

that same STS concentration. Based on the neutralization 

results from both organisms, 0.085% STS was chosen for 

neutralization of the ph-amended bleach in all tests. 

23 
Table 6-7. Neutralization Testing with Bacillus anthracis Ames Spores for pH-Amended Bleach 

Inoculum Total Observed 

Treatment (CFUs) CFUs % of Control 

pH-Amended Bleach + Sporesa 1.27 x 108 0 0 

pH-Amended Bleach + PBS + Triton X-100 + Sporesab 1.27 x 108 0 0 

PBS + Triton X-100 + Spores (Control)b 1.27 x 108 1.27 x 108 

pH-Amended Bleach + PBS + Triton X-100 + 0.17% STS + Sporesab 1.27 x 108 1.25 x 108 98.4 

pH-Amended Bleach + PBS + Triton X-100 + 0.085% STS + Sporesab 1.27 x 108 1.24 x 108 97.6 

pH-Amended Bleach + PBS + Triton X-100 + 0.042% STS + Sporesab 1.27 x 108 1.22 x 108 96.1 

a

 pH-Amended bleach volume of 0.27 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with pH-amended bleach = 10.27 mL (10 mL 

PBS+Triton +STS + 0.27 mL pH-amended bleach). 

“-” Not Applicable. 

Table 6-8. Neutralization Testing with Bacillus subtilis Spores for pH-Amended Bleach 

Inoculum Total Observed 

Treatment (CFUs) CFUs % of Control 

pH-Amended Bleach + Sporesa 9.80 x 107 0 0 

pH-Amended Bleach + PBS + Triton X-100 + Sporesab 9.80 x 107 0 0 

pH-Amended Bleach + Triton X-100 + Spores (Control)b 9.80 x 107 9.76 x 107 

pH-Amended Bleach + PBS + Triton X-100 + 0.17% STS + Sporesab 9.80 x 107 8.26 x 107 84.6 

pH-Amended Bleach + PBS + Triton X-100 + 0.085% STS + Sporesab 9.80 x 107 9.14 x 107 93.6 

pH-Amended Bleach + PBS + Triton X-100 + 0.042% STS + Sporesab 9.80 x 107 9.07 x 107 92.9 

a

 pH-Amended bleach volume of 0.27 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with pH-amended bleach = 10.27 mL (10 mL 

PBS+Triton +STS + 0.27 mL pH-amended bleach). 

“-” Not Applicable. 

­

­

24 
7.0 

Calcium Polysulfide Test Results
 

7.1 QC Results 

In testing of 5.8% calcium polysulfide (CaSx) solution with 

B. anthracis, percent recovery of inoculated spores from 

the positive control coupons ranged from about 18 to 87%, 

with the lowest recovery results on bare wood and unpainted 

concrete. For B. subtilis, positive control recovery values 

ranged from about 2 to 70%, also with the lowest recoveries 

on bare wood and unpainted concrete. All percent recovery 

values were well within the acceptable range of 1 to 150% 

stated in the test/QA plan. 

all procedural and laboratory blanks met the criterion of no 

observed CFUs in quantitative efficacy testing, with both 

B. anthracis and B. subtilis. In the qualitative assessment 

of residual spores, which involves a much longer nutrient 

growth period, growth was observed from the procedural and 

laboratory blank soil coupons used with both B. anthracis 

and B. subtilis. This finding is discussed in Section 7.2.2. 

Preliminary tests indicated that extracts of blank soil samples 

(i.e., not spiked with B. anthracis or B. subtilis) showed 

the presence of several colony forming species. However, 

when spiked with B. anthracis or B. subtilis spores and 

extracted, each soil sample showed the presence of a single 

homogeneous species, with all colonies of uniform size 

and morphologically distinctive for the respective Bacillus 

species. Therefore, blank soil samples were deemed to be 

contaminated only if more than the one inoculated species 

was found in the extracts of inoculated soil samples. This 

approach was formalized by the approval of the test/QA plan 

amendment noted in Section 3.1. 

Spike control samples were taken from the spore suspension 

on each day of testing, and serially diluted, nutrient plated, 

and counted to establish the spore density used to spike the 

coupons. This process takes approximately 24 hours, so spore 

density is known after completion of each day’s testing. 

The target criterion is a spore suspension density of 1 × 109

/ 

mL (± 25%), leading to a spike of 1 × 108 spores (± 25%) 

on each test coupon. The actual spike value for B. anthracis 

testing was 1.17 × 108/coupon, and for B. subtilis testing the 

actual spike value was 1.07 × 108/coupon. Thus all coupons 

received a spore spike that met the target criterion. 

7.2 Decontamination Efficacy 

The decontamination efficacy of CaSx was evaluated for 

B. anthracis and B. subtilis on four outdoor material surfaces. 

The following sections summarize the results found with this 

decontaminant. 

7.2.1 Quantitative Assessment of the Log Reduction of 

Viable Organisms 

The quantitative efficacy results for CaSx are presented 

in Tables 7-1 and 7-2. The decontamination efficacy of 

CaSx proved to be poor (< 0.4 log reduction for each of the 

materials for both Bacillus species). 

25 
Table 7-1. Inactivation of Bacillus anthracis Ames Sporesa

–5.8% Calcium Polysulfide (60 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs % Mean Recovery 

Decontamination 

Efficacy ± CI 

Bare Pine Wood 

Positive Controlsb 1.17 x 108 7.35 23.8 ± 16.6 -

Test Couponsc 

Laboratory Blankd 

1.17 x 108 

0 

7.30 

0 

25.0 ± 22.1 

0 

0.05 ± 0.47 

-

Procedural Blanke 0 0 0 -

Glass 

Positive Controls 1.17 x 108 7.70 43.3 ± 6.80 -

Test Coupons 1.17 x 108 7.74 47.8 ± 8.65 -0.04 ± 0.094 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Unpainted Concrete 

Positive Controls 1.17 x 108 7.21 18.4 ± 15.6 -

Test Coupons 1.17 x 108 6.96 12.3 ± 14.4 0.24 ± 0.50 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Topsoil 

Positive Controls 1.17 x 108 8.00 86.5 ± 8.94 -

Test Coupons 

Laboratory Blank 

Procedural Blank 

1.17 x 108 

0f 

0f 

7.80 

0 

0 

57.1 ± 20.1 

0 

0 

0.21 ± 0.17 

-

-

 

a Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and decontamination efficacy (log 

reduction). 

CI = confidence interval (± 1.96 × SE).

b

 Inoculated, not decontaminated coupon (sprayed with SFW). 

c

 Inoculated, decontaminated coupon.

d

 Laboratory Blank = not inoculated, not decontaminated coupon. 

e

 Procedural Blank = not inoculated, decontaminated coupon.

f Endogenous organisms were found in uninoculated soil blanks; no organisms other than B. anthracis Ames or B. subtilis were found on inoculated coupons. 

“-” Not Applicable. 

­

26 
Table 7-2. Inactivation of Bacillus subtilis Sporesa

–5.8% Calcium Polysulfide (60 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs % Mean Recovery 

Decontamination 

Efficacy ± CI 

Bare Pine Wood 

Positive Controlsb 1.07 x 108 6.31 2.11 ± 1.05 -

Test Couponsc 

Laboratory Blankd 

1.07 x 108 

0 

6.43 

0 

3.05 ± 2.12 

0 

-0.12 ± 0.32 

-

Procedural Blanke 0 0 0 -

Glass 

Positive Controls 1.07 x 108 7.62 38.9 ± 3.52 -

Test Coupons 1.07 x 108 7.28 19.6 ± 9.46 0.33 ± 0.18 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Unpainted Concrete 

Positive Controls 1.07 x 108 7.19 16.8 ± 10.1 -

Test Coupons 1.07 x 108 7.08 11.3 ± 1.75 0.12 ± 0.23 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Topsoil 

Positive Controls 1.07 x 108 7.87 70.1 ± 6.34 -

Test Coupons 

Laboratory Blank 

Procedural Blank 

1.07 x 108 

0f 

0f 

7.66 

0 

0 

43.8 ± 9.67 

0 

0 

0.21 ± 0.083 

-

-

 

a Data are expressed as mean of the logs of total number of spores (CFUs) observed on individual coupons, percent recovery, and decontamination efficacy 

(log reduction). 

CI = confidence interval (± 1.96 × SE).

b

 Inoculated, not decontaminated coupon (sprayed with SFW). 

c

 Inoculated, decontaminated coupon.

d

 Laboratory Blank = not inoculated, not decontaminated coupon. 

e

 Procedural Blank = not inoculated, decontaminated coupon.

f Endogenous organisms were found in uninoculated soil blanks; no organisms other than B. anthracis Ames or B. subtilis were found on inoculated coupons. 

“-” Not Applicable. 

Table 7-3. Summary of Efficacy Values (Log Reduction) 

Obtained for 5.8% Calcium Polysulfidea 

Materials B. anthracis Ames B. subtilis 

Bare Pine Wood 0.05 -0.12 

Glass -0.04 0.33 

Unpainted Concrete 0.24 0.12 

Topsoil 0.21 0.21 

a Numbers in bold are statistically different (p ≤ 0.05) from B. anthracis Ames. 

­

Table 7-3 summarizes the quantitative efficacy results, 

and shows that the efficacy of CaSx for B. subtilis was 

significantly different from the efficacy for B. anthracis ames 

only on the glass surface. The efficacy value of 0.33 log for 

B. subtilis on that material was the highest found for either 

organism on any of the test materials. 

7.2.2 Qualitative Assessment of Residual Spores 

Results from the liquid culture growth assessment of coupons 

at one and seven days post-decontamination are provided 

in Tables 7-4 and 7-5 for coupons spiked with B. anthracis 

ames and B. subtilis spores, respectively. In this assessment, 

cultures showing positive growth (i.e., a cloudy growth 

medium) were applied to streak plates and the identity of 

the growing organism was checked by colony morphology. 

only B. anthracis colonies were found in cultures of positive 

control and test coupons inoculated with B. anthracis, and 

only B. subtilis colonies were found in cultures of positive 

control and test coupons inoculated with B. subtilis. 

Tables 7-4 and 7-5 show that the positive controls and 

test coupons for all materials with both organisms were 

positive for growth. These results are consistent with the low 

quantitative efficacy values observed on the test materials 

(summarized in Table 7-3). As noted in Section 7.1 and 

shown in Tables 7-4 and 7-5, the laboratory and procedural 

blanks for topsoil with both organisms also showed positive 

growth at both one and seven days’ incubation. This growth 

is likely to have resulted from native organisms present on 

the test coupons, and has no impact on the test results. as 

stated above, topsoil coupons inoculated with B. anthracis 

or B. subtilis showed the presence of only the respective 

inoculated organism in the coupon cultures, at one and seven 

days post-decontamination. 

27 
 

 

7.3 Damage to Coupons perhaps due to the surface characteristics of the coupons 

A readily visible amount of grayish surface residue was themselves. The topsoil coupons exhibited a gray hue on 

observed on the glass coupons sprayed with 5.8% CaSx. This the surface of the material (uncompressed topsoil in 3.5 cm 

residue remained on the glass even after the agitation with petri dish). No such residues were observed on the respective 

spore extraction solution. Such a residue was not readily control coupons for glass and topsoil, indicating that the 

apparent on the bare wood or unpainted concrete coupons, residues were due to the CaSx decontaminant. 

Table 7-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis Ames 

Spores–5.8% Calcium Polysulfide 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Bare Pine Wood 

Positive Controls 

Test Coupons 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+a 

-

b 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

-

-

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Unpainted Concrete 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Topsoil 

Positive Controls + + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. anthracis Ames spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. anthracis Ames spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. anthracis Ames spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth.
 

Table 7-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus subtilis Spores– 

5.8% Calcium Polysulfide 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Bare Pine Wood 

Positive Controls 

Test Coupons 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+a 

-

b 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

-

-

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Unpainted Concrete 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Topsoil 

Positive Controls + + + + + + + + + + + 

Test Coupons + + + + + + + + + + + + 

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. subtilis spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. subtilis spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. subtilis spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth.
 

28 
Table 7-6. Deposition/Runoff Weight of 5.8% Calcium 

Polysulfide on Test Materials 

Avg. Deposition/Runoff 

Material Weight (g) 

Bare Pine Wood 1.49 

Glass 1.01 

Unpainted Concrete 0.38 

Topsoil 0.91 

7.4 Other Factors 

7.4.1 Operator Control 

The 5.8% CaSx was prepared according to the procedure 

described in Appendix C, by mixing 800 mL SFW with 

200 ml commercially obtained stock solution containing 

29% CaSx by weight. The CaSx was freshly prepared prior to 

each testing day (i.e., the preparation was assigned a one day 

shelf-life and excess was discarded at the end of the day). 

all trials were conducted under ambient conditions inside a 

climate-controlled laboratory. The temperature inside of the 

testing chamber was equilibrated to the ambient laboratory 

temperature of approximately 22 °C. All coupons were 

sprayed (SFW control or CaSx ) at the start of each exposure, 

the need for re-spraying was assessed every 10 min, the CaSx 

was re-sprayed at the 30 min mid-exposure time-point, and 

then the need for re-spraying was assessed every 10 min 

as before until 60 total minutes had elapsed since the first 

application. In practice, reapplication of CaSx took place only 

at the 30-min midpoint of the test. The Rh was monitored 

with a NIST-traceable hygrometer. The chamber dehumidifier 

was actuated only after the 60 minute contact time with 

the control application (SFW) or CaSx. The Rh in the test 

chamber exceeded 70% during the 60 minute contact time 

probably due to the additional spraying step at the 30 min 

mid-exposure time-point. 

7.4.2 Technology Spray Deposition 

The CaSx was applied according to the procedure in 

Appendix C. The CaSx was applied 30 cm (12 inches) 

from the horizontally (soil and unpainted concrete) and 

vertically (bare pine wood and glass) oriented materials 

until the materials appeared saturated with liquid. A new 

handheld garden sprayer was used to apply the control 

application (SFW) and CaSx. This sprayer was slightly 

modified to accommodate a pressure gauge to ensure that the 

spray was applied using 4 to 6 psi pressure, which resulted 

in a spray comparable to that from the vendor-provided 

applicator. Close observation of the respective material 

surfaces was made to ensure that they were thoroughly 

wetted (approximately 5 sec spray duration was needed to 

produce wetting across the surfaces of all five replicates 

and corresponding blank for each material type). After the 

60 minute contact time, each material was placed in the 50 

mL conical vial that also served to collect excess formulation 

run-off. The horizontally and vertically oriented materials 

stayed in their respective configurations throughout the 

60 minute contact time. 

To assess CaSx deposition, triplicate coupons of each test 

material were weighed and these weights were recorded. 

Then the triplicate coupons were sprayed with CaSx until 

the coupons were fully wetted in their respective vertical or 

horizontal orientations, the need for re-spraying was assessed 

every 10 min, the CaSx was sprayed again at the 30 min 

mid-exposure time-point, re-spraying was assessed every 

10 min as before, and then each coupon was weighed again 

after 60 min total time. The pre-application weights were 

then subtracted from the post-application weights, and that 

difference was added to the weight of decontaminant runoff 

captured separately from each coupon. as shown in Table 

7-6, the amount of CaSx deposited on bare pine wood, glass, 

and soil coupons ranged from 0.91 g to 1.49 g; the amount 

deposited on unpainted concrete coupons was lower at 0.38 g, 

including the reapplication at the 30 min mid-exposure timepoint. The average of these spray-and-weigh results (0.95 g, 

or 0.95 mL based on an approximate density of 1.0 for the 

diluted CaSx) was used to determine the concentration of the 

neutralizer (Dey/Engley (D/E) Broth) for testing. 

7.4.3 Neutralization Methodology 

The concentrations of D/E Broth tested as a neutralizer 

during the neutralization trials were 3%, 6%, 12.5%, 25%, 

50%, and 100% in the final extraction solution. That is, the 

extraction solutions tested ranged from 97% PBS/Triton 

X-100/3% D-E broth to 100% D-E broth. The results of the 

neutralization panel for B. anthracis are shown in Table 7-7. 

The results indicate that it did not matter which concentration 

of D/E Broth was used since each exhibited a high recovery 

percentage (> 96%) when compared to the control, and 

the ineffectiveness of CaSx gave no discrimination as to 

its neutralization by different D/E concentrations. For the 

B. subtilis spores (Table 7-8), 3% D/E Broth exhibited the 

highest recovery as a percent of control (108%), whereas the 

higher concentrations gave recoveries that ranged from 45 to 

62%. Therefore, 3% D/E Broth was chosen as the neutralizer 

for CaSx for both B. anthracis and B. subtilis. 

29 
Table 7-7. Neutralization Testing with Bacillus anthracis Ames Spores for 5.8% Calcium Polysulfide 

Treatment Inoculum (CFUs) Total Observed CFUs % of Control 

CaSx + Sporesa 1.19 x 108 1.04 x 108 114.1 

CaSx + PBS + Triton X-100 + Sporesab 1.19 x 108 9.76 x 107 106.7 

PBS + Triton X-100 + Spores (Control)b 1.19 x 108 9.15 x 107 -

CaSx + PBS + Triton X-100 + 100% D/E Broth + Sporesab 1.19 x 108 9.93 x 107 108.5 

CaSx + PBS + Triton X-100 + 50% D/E Broth + Sporesab 1.19 x 108 1.04 x 108 114.0 

CaSx + PBS + Triton X-100 + 25% D/E Broth + Sporesab 1.19 x 108 9.17 x 107 100.3 

CaSx + PBS + Triton X-100 + 12.5% D/E Broth + Sporesab 1.19 x 108 8.86 x 107 96.9 

CaSx + PBS + Triton X-100 + 6% D/E Broth + Sporesab 1.19 x 108 1.02 x 108 111.1 

CaSx + PBS + Triton X-100 + 3% D/E Broth + Sporesab 1.19 x 108 1.03 x 108 112.3 

a

 CaSx volume of 0.95 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of D/E broth; total volume for all samples with 

CaSx = 10.95 mL (10 mL PBS+Triton +D/E + 0.95 mL CaSx).

 “-” Not Applicable. 

Table 7-8. Neutralization Testing with Bacillus subtilis Spores for 5.8% Calcium Polysulfide 

Treatment Inoculum (CFUs) Total Observed CFUs % of Control 

CaSx + Sporesa 9.90 x 107 1.02 x 108 54.8 

CaSx + PBS + Triton X-100 + Sporesab 9.90 x 107 6.79 x 107 36.6 

PBS + Triton X-100 + Spores (Control)b 9.90 x 107 1.85 x 108 -

CaSx + PBS + Triton X-100 + 100% D/E Broth + Sporesab 9.90 x 107 9.28 x 107 50.1 

CaSx + PBS + Triton X-100 + 50% D/E Broth + Sporesab 9.90 x 107 9.27 x 107 50.0 

CaSx + PBS + Triton X-100 + 25% D/E Broth + Sporesab 9.90 x 107 9.34 x 107 50.4 

CaSx + PBS + Triton X-100 + 12.5% D/E Broth + Sporesab 9.90 x 107 8.26 x 107 44.6 

CaSx + PBS + Triton X-100 + 6% D/E Broth + Sporesab 9.90 x 107 1.15 x 108 62.0 

CaSx + PBS + Triton X-100 + 3% D/E Broth + Sporesab 9.90 x 107 2.01 x 108 108.5 

a

 CaSx volume of 0.95 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of D/E broth; total volume for all samples with 

CaSx = 10.95 mL (10 mL PBS+Triton +D/E + 0.95 mL CaSx). 

“-” Not Applicable. 

30 
8.0 

CASCADTM SDF (Allen-Vanguard) Test Results
 

8.1 QC Results 

In testing of CASCADTM SDF, all positive control results 

were within the target recovery range of 1 to 150% of the 

spiked spores. For B. anthracis positive control recovery 

values ranged from 35 to 145%, with the lowest recovery 

occurring on unpainted pine wood. For B. subtilis positive 

control recovery values ranged from 2 to 43%, with the 

lowest recoveries occurring on unpainted pine wood and 

painted wallboard paper. 

In testing of CASCADTM SDF, all procedural and laboratory 

blanks met the criterion of no observed CFUs in quantitative 

efficacy testing, with both B. anthracis and B. subtilis. also, 

no growth was observed for any procedural and laboratory 

blanks in the qualitative assessment of residual spores, 

which involves a much longer nutrient growth period. The 

industrial carpet exhibited the antimicrobial properties seen 

in previous testing (see Chapter 5) and initially inhibited the 

growth of the inoculated, non-decontaminated samples. This 

observation is further explained in Section 8.2.2. 

Spike control samples were taken from the spore suspension 

on each day of testing, and serially diluted, nutrient plated, 

and counted to establish the spore density used to spike the 

coupons. This process takes approximately 24 hours, so the 

spore density is known after completion of each day’s testing. 

The target criterion is to maintain a spore suspension density 

of 1 × 109/mL (± 25%), leading to a spike of 1 × 108 spores 

(± 25%) on each test coupon. The actual spike values for two 

days of B. anthracis testing were 9.40 × 107/coupon 

and 4.73 × 107/coupon, and for two days of B. subtilis 

testing the actual spike values were 1.18 × 108/coupon and 

1.04 × 108/coupon. Thus all spore spikes were well within

 the ±25% tolerance of the 1 x 108/coupon target, except 

for the 4.73 × 107/coupon spike on the second day of 

B. anthracis testing. This relatively low spike amount has 

no effect on data quality, as spore recoveries were good and 

efficacy of up to 7.7 logs could be determined. 

8.2 Decontamination Efficacy 

The decontamination efficacy of Allen-Vanguard’s 

CASCADTM SDF was evaluated for B. anthracis ames 

and B. subtilis on seven indoor material surfaces. The 

following sections summarize the results found with this 

decontaminant. 

8.2.1 Quantitative Assessment of the Log Reduction of 

Viable Organisms 

The decontamination efficacy of Allen-Vanguard’s 

CASCADTM SDF was greater than approximately 7.0 log 

reduction for five materials for both B. anthracis and 

B. subtilis, as shown in Tables 8-1 and 8-2, respectively, 

and summarized in Table 8-3. only painted wallboard paper 

and unpainted pine wood exhibited lower log reductions 

for both Bacillus species. Table 8-3 shows that efficacy 

results for B. subtilis were significantly different from 

results for B. anthracis for only two materials: painted 

cinder block (both results exceed 7 log reduction) and 

bare pine wood (the lowest efficacy results for each 

organism). In both these cases the efficacy values for 

B. subtilis were less than those for B. anthracis. 

8.2.2 Qualitative Assessment of Residual Spores 

Results from the liquid culture growth assessment of coupons 

at one and seven days post-decontamination are provided 

in Tables 8-4 and 8-5 for coupons spiked with B. anthracis 

ames and B. subtilis spores, respectively. In this assessment, 

cultures showing positive growth (i.e., a cloudy growth 

medium) were applied to streak plates and the identity of the 

growing organism was checked by colony morphology. only 

B. anthracis colonies were found in cultures of coupons used 

with B. anthracis, and only B. subtilis colonies were found in 

cultures of coupons used with B. subtilis. 

Tables 8-4 and 8-5 show results that are consistent with 

the efficacy of CASCADTM SDF. No decontaminated 

coupons of any material showed growth for B. anthracis 

or B. subtilis, with the exception of the two materials 

that had the lowest log reduction (painted wallboard 

paper and unpainted pine wood). All laboratory and 

procedural blanks were negative for growth. 

The qualitative, liquid culture growth assessment results are 

consistent with the quantitative, observed efficacy results for 

all of the materials, except perhaps for the industrial carpet. 

At one day of incubation, only two of five of the B. anthracisinoculated and three of five of the B. subtilis-inoculated, 

non-decontaminated industrial carpet positive controls 

were positive for growth, perhaps due to the antibacterial 

component (zinc omadine) present in this material. 

However, all inoculated, non-decontaminated industrial 

carpet positive control samples were positive for growth 

at the 7- day assessment, possibly due to the degradation 

of the antibacterial component over multiple days. 

31 
Table 8-1. Inactivation of Bacillus anthracis Ames Sporesa

—Allen-Vanguard’s CASCADTM SDF 

(30 minute contact time) 

Test Material 

Inoculum 

(CFUs) 

Mean of Logs of 

Observed CFUs 

Mean % 

Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlb 4.73 x 107 7.88 ± 0.08 144.5 ± 20.5 -

Decontaminatedc 4.73 x 107 0.48 ± 1.08 0 7.40 ± 0.95 

Laboratory Blankd 0 0 0 -

Procedural Blanke 0 0 0 -

Decorative Laminate 

Positive Control 4.73 x 107 7.72 ± 0.08 111.9 ± 22.0 -

Decontaminated 4.73 x 107 0.31 ± 0.70 0 7.40 ± 0.62 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Control 9.40 x 107 7.59 ± 0.04 41.9 ± 3.6 -

Decontaminated 9.40 x 107 0 0 ≥ 7.59 ± 0.03 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Painted Wallboard Paper 

Positive Control 4.73 x 107 7.67 ± 0.15 102.5 ± 34.3 -

Decontaminated 4.73 x 107 2.84 ± 1.76 0.02 ± 0.03 4.82 ± 1.55 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Painted Cinder Block 

Positive Control 9.40 x 107 7.84 ± 0.04 73.7 ± 6.8 -

Decontaminated 9.40 x 107 0 0 ≥ 7.84 ± 0.04 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Bare Pine Wood 

Positive Control 4.73 x 107 7.13 ± 0.28 34.8 ± 29.4 -

Decontaminated 4.73 x 107 4.36 ± 0.83 0.11 ± 0.10 2.77 ± 0.77 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 0 

Glass 

Positive Control 9.40 x 107 7.85 ± 0.14 77.9 ± 27.6 -

Decontaminated 9.40 x 107 0 0 ≥ 7.85 ± 0.12 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

a Data are expressed as mean (± SD) total number of spores (CFUs) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

­

­

­

32 
Table 8-2. Inactivation of Bacillus subtilis Sporesa

—Allen-Vanguard’s CASCADTM SDF 

(30 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs 

Mean % 

Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlb 1.04 x 108 7.62 ± 0.20 43.0 ± 14.8 -

Decontaminatedc 1.04 x 108 0 0 ≥ 7.62 ± 0.18 

Laboratory Blankd 0 0 0 -

Procedural Blanke 0 0 0 -

Decorative Laminate 

Positive Control 1.04 x 108 7.30 ± 0.21 21.1 ± 9.3 -

Decontaminated 1.04 x 108 0 0 ≥ 7.30 ± 0.19 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Control 1.18 x 108 7.60 ± 0.08 34.1 ± 6.0 -

Decontaminated 1.18 x 108 0 0 ≥ 7.60 ± 0.07 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Painted Wallboard Paper 

Positive Control 1.04 x 108 6.14 ± 0.27 1.6 ± 1.0 -

Decontaminated 1.04 x 108 0 0 ≥ 6.14 ± 0.24 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

Painted Cinder Block 

Positive Control 1.18 x 108 7.05 ± 0.25 10.7 ± 5.1 -

Decontaminated 1.18 x 108 0 0 ≥ 7.05 ± 0.22 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Bare Pine Wood 

Positive Control 1.04 x 108 6.22 ± 0.12 1.6 ± 0.4 -

Decontaminated 1.04 x 108 4.94 ± 0.45 0.1 ± 0.1 1.28 ± 0.41 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Glass 

Positive Control 1.18 x 108 7.51 ± 0.26 31.4 ± 16.2 -

Decontaminated 1.18 x 108 0 0 ≥ 7.51 ± 0.23 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

a Data are expressed as mean (± SD) total number of spores (CFUs) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

­

­

33 
Table 8-3. Summary of Efficacy Values (Log Reduction) Obtained for 

Allen-Vanguard’s CASCADTM SDFa 

Test Material B. anthracis Ames B. subtilis 

Industrial-Grade Carpet 7.40 ≥ 7.62 

Decorative Laminate 7.40 ≥ 7.30 

Galvanized Metal Ductwork ≥ 7.59 ≥ 7.60 

Painted Wallboard Paper 4.82 ≥ 6.14 

Painted Cinder Block ≥ 7.84 ≥ 7.05 

Bare Pine Wood 2.77 1.28 

Glass ≥ 7.85 ≥ 7.51 

a

 Numbers in bold are statistically different (p ≤ 0.05) from B. anthracis Ames. 

 

Table 8-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with 

Bacillus anthracis Ames Spores—Allen-Vanguard’s CASCADTM SDF 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

-

-

-

-

-

-

+ 

-

+ 

-

-

a 

-

b 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons + - + + + - + - + + + -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons - - + + - - + - + + + -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. anthracis Ames spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. anthracis Ames spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. anthracis Ames spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth.
 

34 
 

Table 8-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with 


Bacillus subtilis Spores—Allen-Vanguard’s CASCADTM SDF
 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 B1 S1 S2 S3 S4 S5 B1 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

+ 

-

-

-

+ 

-

+ 

-

-

-

-

a 

-

b 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons - + - - - - - + + - - -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons + - + - + - + + + - + -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. subtilis spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. subtilis spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. subtilis spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth. 


8.3 Damage to Coupons 

The only visible damage observed on the test materials after 

the 30 minute contact time with CASCADTM SDF in the 

quantitative efficacy testing was seen on the painted cinder 

block. The top-coat (semi-gloss latex paint) peeled away 

from the primer. This physical change was observed only 

after the extraction step, when small portions of paint floated 

in the extraction solution. The non-decontaminated painted 

cinder block did not undergo this physical change during 

extraction (i.e., the top-coat remained intact). 

8.4 Other Factors 

8.4.1 Operator Control 

on each day of testing, the two components of allenVanguard’s CASCADTM SDF were prepared according to 

the vendor’s instructions in Appendix D. The spray nozzle 

was then placed onto the dual bottle, in which each half of 

the bottle contained one of the two CASCADTM SDF reagent 

solutions. Prior to each application, the CASCADTM SDF 

spray nozzle was primed by repeatedly spraying into an 

absorbent cloth to clear any air bubbles that may have formed 

between applications. after each application, the spray nozzle 

was removed from the bottle and any residual CASCADTM 

SDF was removed by repeated pulls on the trigger of the 

spray nozzle. The spray nozzle was then placed onto a dual 

bottle that contained only SFW so as to completely clean out 

the spray nozzle until its next use. 

all tests were conducted at ambient conditions inside a 

climate-controlled laboratory. The temperature inside the 

test chamber was equilibrated to the ambient laboratory 

temperature of approximately 22 °C. The RH inside the test 

chamber was monitored with a NIST-traceable hygrometer. 

Whenever the RH exceeded 40%, the dehumidification 

system attached to the testing chamber was actuated until the 

RH dropped below 40%. The dehumidifier was actuated only 

after the prescribed contact time with the CASCADTM SDF. 

Therefore, the testing chamber Rh was always less than 40% 

prior to the decontamination of a new set of materials with 

CASCADTM SDF. 

8.4.2 Technology Spray Deposition 

Allen-Vanguard’s CASCADTM SDF was applied according 

to the procedure included as Appendix D of this report. 

CASCADTM SDF was applied from a distance of 12 inches 

from the horizontally and vertically oriented materials, with 

the aim of covering the materials with approximately a 

3/8 inch layer of foam. However, only the industrial carpet 

came close to the required thickness of foam, likely due 

to its dense weave which allowed the foam to accumulate. 

The CASCADTM SDF foam pooled and spilled over the 

edges of the other horizontal material (laminate) and only 

permitted accumulation of approximately 1/8 inch thickness 

of foam. None of the other materials (all vertically oriented) 

allowed for the 3/8 inch layer of foam to form since the 

35 
applied material almost immediately ran off, but a thin layer 

of foam remained on the materials. No reapplication of the 

CASCADTM SDF was made on any coupon surface. After 

the 30 minute contact time, each material coupon was placed 

in the tube that also served to collect excess decontaminant 

runoff. The horizontally and vertically oriented coupon 

materials stayed in their respective configurations for the 

duration of their 30 minute contact times. 

To assess CASCADTM SDF deposition, triplicate coupons 

of each test material were weighed prior to application of 

CASCADTM SDF in trial runs, and those weights were 

recorded. Then the triplicate coupons were sprayed with 

CASCADTM SDF until fully wetted in their respective 

vertical or horizontal orientations. After a 30 minute 

contact time, each coupon was then weighed again. The 

pre-application weights were then subtracted from the postapplication weights, and that difference was added to the 

weight of decontaminant runoff captured separately from 

each coupon. The average weights of deposition plus runoff 

for each of the test materials are shown in Table 8-6. 

Table 8-6. Deposition/Runoff Weight of Allen-Vanguard’s 

CASCADTM SDF on Test Materials 

Test Material 

Avg. Deposition/Runoff 

Weight (g) of Foam 

Industrial-Grade Carpet 3.35 

Decorative Laminate 0.69 

Galvanized Metal Ductwork 1.18 

Painted Wallboard Paper 0.82 

Painted Cinder Block 1.00 

Bare Pine Wood 1.15 

Glass 1.30 

The deposition/runoff weights for six of the seven materials 

in Table 8-6 ranged from 0.69 to 1.30 g, and averaged 

approximately 1.02 g. However, for industrial carpet the 

deposition weight was substantially larger (3.35 g). This 

difference was due to the ability of the carpet surface to 

retain a much greater depth of the foam. The density of the 

CASCADTM SDF deposited on the test coupons was not 

measured directly, but was estimated to be slightly greater 

than 1.0, based on the compositions of the two component 

solutions that produce the delivered foam (see Appendix D). 

As a result, the average volume of CASCADTM SDF 

deposited on six of the seven materials was estimated to be 

approximately 1.01 mL, and the volume deposited on carpet 

was estimated to be approximately 3.3 mL. These volumes 

were then used in trials to determine the amount of STS 

needed to neutralize the CASCADTM SDF (Section 8.4.3). 

8.4.3 Neutralization Methodology 

Neutralization of the CASCADTM SDF was achieved 

with STS. The concentrations of STS tested during the 

neutralization trial were 0.25, 0.50, and 1.00% in the 

PBS/Triton X-100 extraction solution. These test STS 

concentrations were based on historical data. a neutralization 

trial was done using the 1.01 mL of CASCADTM SDF that 

represents deposition on most of the test materials. The 

results of that neutralization trial with B. anthracis and 

B. subtilis are shown in Tables 8-7 and 8-8, respectively, and 

indicate that the 1% STS concentration was most effective 

in preventing the sporicidal action of CASCADTM SDF. 

Both the B. anthracis and B. subtilis results were considered 

in selecting the STS concentration to be used. on the basis 

of these results, 1% STS was used for neutralization of 

CASCADTM SDF in testing with both B. anthracis and 

B. subtilis. Subsequently, an additional neutralization panel 

was conducted to check whether 1% STS was effective at 

neutralizing the larger amount of CASCADTM SDF (3.3 mL) 

deposited on carpet coupons. The results of that neutralization 

panel are shown for the two organisms in Tables 8-9 

and 8-10, and confirmed that 1% STS was effective at 

neutralizing the CASCADTM SDF deposited on carpet. 

36 
Table 8-7. Neutralization Testing with Bacillus anthracis Ames Spores for Allen-Vanguard’s CASCADTM SDF 

Treatment Inoculum (CFU) Total Observed (CFU) % of Control 

CASCADTM SDF + Sporesa 9.40 x 107 0 0 

CASCADTM SDF + PBS + Triton X-100 + Sporesab 9.40 x 107 0 

PBS + Triton X-00 + Spores (Control) 9.40 x 107 4.84 x 108 

CASCADTM SDF + PBS + Triton X-100 + 0.25% STS + Sporesab 9.40 x 107 0 0 

CASCADTM SDF + PBS + Triton X-100 + 0.50% STS + Sporesab 9.40 x 107 0 0 

CASCADTM SDF + PBS + Triton X-100 + 1.00% STS + Sporesab 9.40 x 107 2.56 x 108 52.8 

a

 CASCADTM SDF volume of 1.01 mL corresponds to mean gravimetric deposition for foam application on most test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with CASCADTM 

SDF = 11.01 mL (10 mL PBS+Triton +STS + 1.01 mL CASCADTM SDF). 

“-” Not Applicable. 

Table 8-8. Neutralization Testing with Bacillus subtilis Spores for Allen-Vanguard’s CASCADTM SDF 

Treatment Inoculum (CFU) Total Observed (CFU) % of Control 

CASCADTM SDF + Sporesa 7.00 x 107 0 0 

CASCADTM SDF + PBS + Triton X-100 + Sporesab 7.00 x 107 0 

PBS + Triton X-100 + Spores (Control)b 7.00 x 107 9.31 x 107 

CASCADTM SDF + PBS + Triton X-100 + 0.25% STS + Sporesab 7.00 x 107 6.17 x 107 66.2 

CASCADTM SDF + PBS + Triton X-100 + 0.50% STS + Sporesab 7.00 x 107 9.76 x 107 104.9 

CASCADTM SDF + PBS + Triton X-100 + 1.00% STS + Sporesab 7.00 x 107 7.96 x 107 85.5 

a CASCADTM SDF volume of 1.01 mL corresponds to mean gravimetric deposition for foam application on most test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with CASCADTM 

SDF = 11.01 mL (10 mL PBS+Triton +STS + 1.01 mL CASCADTM SDF). 

“-” Not Applicable. 

Table 8-9. Additional Neutralization Testing with Bacillus anthracis Ames Spores for Allen-Vanguard’s 

CASCADTM SDF 

Treatment Inoculum (CFUs) Total Observed (CFUs) % of Control 

CASCADTM SDF + Sporesa 6.17 x 107 0 0 

CASCADTM SDF + PBS + Triton X-100 + Sporesab 6.17 x 107 0 0 

PBS + Triton X-100 + Spores (Control)b 6.17 x 107 1.18 x 108 

CASCADTM SDF + PBS + Triton X-100 + 1.00% STS + Sporesab 6.17 x 107 6.60 x 107 56.1 

a

 CASCADTM SDF volume of 3.3 mL corresponds to mean gravimetric deposition for foam application on carpet.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with 

CASCADTM SDF = 13.3 mL (10 mL PBS+Triton +STS + 3.3 mL CASCADTM SDF). 

“-” Not Applicable. 

Table 8-10. Additional Neutralization Testing with Bacillus subtilis Spores for Allen-Vanguard’s CASCADTM SDF 

Treatment Inoculum (CFUs) Total Observed (CFUs) % of Control 

CASCADTM SDF + Sporesa 8.57 x 107 0 0 

CASCADTM SDF + PBS + Triton X-100 + Sporesab 8.57 x 107 0 0 

PBS + Triton X-100 + Spores (Control)b 8.57 x 107 4.46 x 108 

CASCADTM SDF + PBS + Triton X-100 + 1.00% STS + Sporesab 8.57 x 107 6.46 x 108 145 

a

 CASCADTM SDF volume of 3.3 mL corresponds to mean gravimetric deposition for foam application on carpet.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with 

CASCADTM SDF = 13.3 mL (10 mL PBS+Triton +STS + 3.3 mL CASCADTM SDF). 

“-” Not Applicable. 

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38 
 

9.0 

Oxonia Active® (Ecolab) Test Results 

9.1 QC Results 

In testing of Oxonia Active®, all positive control results were 

within the target recovery range of 1 to 150% of the spiked 

spores. For B. anthracis positive control recovery values 

ranged from about 13 to 117%, with the lowest recovery 

occurring on unpainted pine wood. For B. subtilis positive 

control recovery values ranged from 3.4 to about 57%, with 

the lowest recoveries occurring on painted wallboard paper. 

In testing of Oxonia Active®, all procedural and laboratory 

blanks met the criterion of no observed CFUs in quantitative 

efficacy testing, with both B. anthracis and B. subtilis. No 

growth was also observed in the qualitative assessment of 

residual spores for all procedural and laboratory blanks, 

which involves a much longer nutrient growth period. Once 

again, the industrial carpet exhibited the antimicrobial 

properties seen in previous testing and initially inhibited the 

growth of the inoculated, non-decontaminated samples. This 

growth inhibition by the industrial carpet is further explained 

in Section 9.2.2. 

Spike control samples were taken from the spore suspension 

on each day of testing, and serially diluted, nutrient plated, 

and counted to establish the spore density used to spike the 

coupons. This process takes approximately 24 hours, so the 

spore density is known after completion of each day’s testing. 

The target criterion is to maintain a spore suspension density 

of 1 × 109

/mL (± 25%), leading to a spike of 1 × 108 spores 

(± 25%) on each test coupon. The actual spike values for two 

days of B. anthracis testing were 6.93 × 107/coupon and 

7.13 × 107/coupon, and for two days of B. subtilis testing 

the actual spike values were 7.80 × 107/coupon and 

1.74 × 108/coupon. The spike amounts outside the target 

range had no effect on data quality, as spore recoveries were 

good and efficacy exceeding seven logs could be determined. 

9.2 Decontamination Efficacy 

The decontamination efficacy of Oxonia Active® was 

evaluated for B. anthracis ames and B. subtilis on seven 

indoor material surfaces. The following sections summarize 

the results found with this decontaminant. 

9.2.1 Quantitative Assessment of the Log Reduction of 

Viable Organisms 

The efficacy results for Oxonia Active® with B. anthracis and 

B. subtilis are shown in Tables 9-1 and 9-2, respectively, and 

summarized in Table 9-3. The decontamination efficacy was 

7.0 log reduction or greater on six of the seven test materials 

for B. anthracis and on five of those materials for B. subtilis. 

For B. anthracis efficacy was 4.64 log reduction on bare pine 

wood, and for B. subtilis efficacy was 5.15 log reduction 

on bare pine wood and ≥ 6.69 log reduction on painted 

wallboard paper. 

39 
Table 9-1. Inactivation of Bacillus anthracis Ames Spores a

—Ecolab’s Oxonia Active® 

(60 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlb 7.13 x 107 7.92 ± 0.06 116.8 ± 17.6 -

Decontaminatedc 7.13 x 107 0.92 ± 2.05 0.01 ± 0.02 7.00 ± 1.79 

Laboratory Blankd 

Procedural Blanke 

0 

0 

0 

0 

0 

0 

-

-

Decorative Laminate 

Positive Control 7.13 x 107 7.61 ± 005 57.4 ± 7.1 -

Decontaminated 7.13 x 107 0 0 ≥ 7.61 ± 0.05 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Control 6.93 x 107 7.87 ± 0.07 107.0 ± 17.9 -

Decontaminated 6.93 x 107 0 0 ≥ 7.87 ± 0.06 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Painted Wallboard Paper 

Positive Control 7.13 x 107 7.42 ± 0.32 44.7± 30.7 -

Decontaminated 7.13 x 107 0 0 ≥ 7.42 ± 28 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

Painted Cinder Block 

Positive Control 6.93 x 107 7.86 ± 0.12 106.8 ± 26.8 -

Decontaminated 6.93 x 107 0 0 ≥ 7.86 ± 0.11 

Laboratory Blank 

Procedural Blank 

0 

0 

0 

0 

0 

0 

-

-

Bare Pine Wood 

Positive Control 7.13 x 107 6.95 ± 0.09 12.7 ± 2.8 -

Decontaminated 7.13 x 107 2.31 ± 2.14 0.01 ± 0.02 4.64 ± 1.87 

Laboratory Blank 

Procedural Blank 

0 

0 

0 

0 

0 

0 -

Glass 

Positive Control 6.93 x 107 7.72 ± 0.07 77.3 ± 11.5 -

Decontaminated 6.93 x 107 0 0 ≥ 7.72 ± 0.06 

Laboratory Blank 

Procedural Blank 

0 

0 

0 

0 

0 

0 

-

-

 

a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).
 

b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

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40 
Table 9-2. Inactivation of Bacillus subtilis Sporesa

—Ecolab’s Oxonia Active® (60 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlb 1.74 x 108 7.42 ± 0.13 15.5 ± 4.4 -

Decontaminatedc 1.74 x 108 0 0 ≥ 7.42 ± 0.12 

Laboratory Blankd 0 0 0 -

Procedural Blanke 0 0 0 -

Decorative Laminate 

Positive Control 1.74 x 108 7.66 ± .0.05 26.3 ± 2.9 -

Decontaminated 1.74 x 108 0 0 ≥ 7.66 ± 0.05 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Control 7.80 x 107 7.64 ± 0.06 56.9 ± 8.6 -

Decontaminated 7.80 x 107 0 0 ≥ 7.64 ± 0.06 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Painted Wallboard Paper 

Positive Control 1.74 x 108 6.69 ± 0.34 3.5 ± 2.4 -

Decontaminated 1.74 x 108 0 0 ≥ 6.69 ± 0.29 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

Painted Cinder Block 

Positive Control 7.80 x 107 7.29 ± 0.27 30.0 ± 21.6 -

Decontaminated 7.80 x 107 0 0 ≥ 7.29 ± 0.24 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Bare Pine Wood 

Positive Control 7.80 x 107 6.55 ± 0.27 5.3 ± 3.5 -

Decontaminated 7.80 x 107 1.40 ± 1.92 0.002 ± 0.003 5.15 ± 1.70 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Glass 

Positive Control 7.80 x 107 7.03 ± 0.15 14.4 ± 4.0 -

Decontaminated 7.80 x 107 0 0 ≥ 7.03 ± 0.13 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

a Data are expressed as mean (± SD) total number of spores (CFUs) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).
 

b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

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41 
Table 9-3. Summary of Efficacy Values (Log Reduction) Obtained for 

Ecolab’s Oxonia Active®a 

Test Material B. anthracis Ames B. subtilis 

Industrial-Grade Carpet 7.00 ≥ 7.42 

Decorative Laminate ≥ 7.61 ≥ 7.66 

Galvanized Metal Ductwork ≥ 7.87 ≥ 7.64 

Painted Wallboard Paper ≥ 7.42 ≥ 6.69 

Painted Cinder Block ≥ 7.86 ≥ 7.29 

Bare Pine Wood 4.64 5.15 

Glass ≥ 7.72 ≥ 7.03 

a

 Numbers in bold are statistically different (p ≤ 0.05) from B. anthracis Ames results. 

Table 9-3 shows that the efficacy values found for B. subtilis 

were significantly different from those found for B. anthracis 

on four materials; in all four cases the efficacy for B. subtilis 

was lower than that for B. anthracis. However, in all four of 

these cases, no viable spores of either organism were found 

on the decontaminated test coupons, i.e., Oxonia Active® 

achieved a complete kill of the inoculated spores, and the 

efficacy values are shown as “greater than or equal to” (≥) 

values. Thus the differences in efficacy seen in these four 

cases are due to limitations in the recovery of the two types 

of spores from these coupon materials (lower recoveries of 

B. subtilis), and cannot be attributed to actual differences in 

efficacy toward the two organisms. 

9.2.2 Qualitative Assessment of Residual Spores 

Results from the liquid culture growth assessment of coupons 

at one and seven days post-decontamination are provided 

in Tables 9-4 and 9-5 for coupons spiked with B. anthracis 

ames and B. subtilis spores, respectively. In this assessment, 

cultures showing positive growth (i.e., a cloudy growth 

medium) were applied to streak plates and the identity of the 

growing organism was checked by colony morphology. only 

B. anthracis colonies were found in cultures of coupons used 

with B. anthracis, and only B. subtilis colonies were found in 

cultures of coupons used with B. subtilis. 

The qualitative liquid culture growth assessment results 

in Tables 9-4 and 9-5 are consistent with the quantitative 

efficacy results, in that no growth was observed for 

B. anthracis or B. subtilis on any decontaminated test 

material. Growth was observed at both one and seven days 

on all non-decontaminated test materials, as expected, with 

the exception of the industrial carpet. For both B. anthracis 

and B. subtilis, only three of the five inoculated, nondecontaminated industrial carpet coupons were positive 

for growth after one day, perhaps due to the antibacterial 

component (zinc omadine) incorporated into this material. 

all inoculated, non-decontaminated industrial carpet 

coupons, however, were positive for growth at the seven 

day assessment, possibly due to the degradation of the 

antibacterial component over multiple days. 

42 
 

 

Table 9-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with
 

Bacillus anthracis Ames Spores—Ecolab’s Oxonia Active®
 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

-

-

-

-

+ 

-

+ 

-

+ 

-

-

a 

-

b 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. anthracis Ames spores) ); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. anthracis Ames spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. anthracis Ames spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth. 


Table 9-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with 

Bacillus subtilis Spores—Ecolab’s Oxonia Active® 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls - - + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. subtilis spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. subtilis spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. subtilis spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth. 


43 
Table 9-6. Deposition/Runoff Weight of Ecolab’s Oxonia 

Active® on Test Materials 

Test Material 

Avg. Deposition/Runoff 

Weight (g) 

Industrial-Grade Carpet 1.60 

Decorative Laminate 0.79 

Galvanized Metal Ductwork 0.94 

Painted Wallboard Paper 1.01 

Painted Cinder Block 1.14 

Bare Pine Wood 1.70 

Glass 0.97 

9.3 Damage to Coupons 

No visible damage was observed on any of the test materials 

after the 60 min contact time with Oxonia Active®. 

9.4 Other Factors 

9.4.1 Operator Control 

On each day of testing, Oxonia Active® was prepared 

according to the vendor’s explicit instructions as stated in 

Appendix E. After the Oxonia Active® was diluted in SFW, 

the product was tested to ensure that the active component 

(peroxyacetic acid) was within the range specified by the 

vendor. This check was done using a test kit also provided 

by the vendor (High Oxonia Active® Test Kit 322). All such 

checks showed the prepared solution to be in the correct 

range. The diluted Oxonia Active® was then transferred to a 

handheld garden sprayer modified with a pressure gauge to 

ensure that the spray was applied using 4 to 6 psi pressure. 

all tests were conducted at ambient conditions inside a 

climate-controlled laboratory. The temperature inside the 

test chamber was equilibrated to the ambient laboratory 

temperature of approximately 22 °C. The RH inside 

the test chamber was monitored with a NIST-traceable 

hygrometer. Whenever the RH reached 40%, as it did during 

reapplications of the Oxonia Active®, the dehumidification 

system attached to the testing chamber was actuated until the 

Rh dropped below 40%. Therefore, the testing chamber was 

always within 40% Rh during the decontamination of a set 

of material coupons with Oxonia Active®. 

9.4.2 Technology Spray Deposition 

Oxonia Active® was applied according to the procedure 

included as Appendix E of this report. Oxonia Active® was 

applied from a distance of 12 inches from the horizontally 

and vertically oriented materials until the materials were 

completely saturated. The respective material surfaces 

were closely observed to ensure that they were thoroughly 

wetted (approximately 3-5 sec spray duration was needed to 

produce wetting across the surfaces of all five replicates and 

corresponding blank for each material type). Reapplication of 

the Oxonia Active® was made on all coupon surfaces every 

10 minutes. after the 60 minute contact time, each material 

coupon was placed in its respective 50 mL collection vial 

that also served to collect excess decontaminant runoff. The 

horizontally and vertically oriented coupon materials stayed 

in their respective configurations for the duration of their 

60 minute contact times. 

To assess Oxonia Active® deposition, triplicate coupons 

of each test material were weighed prior to application 

of Oxonia Active® in trial runs, and those weights were 

recorded. Then the triplicate coupons were sprayed with 

Oxonia Active® until fully wetted in their respective 

vertical or horizontal orientations, Oxonia Active® was 

reapplied at 10-minute intervals, a 60 minute contact time 

was allowed, and then each coupon was weighed again. 

The pre-application weights were then subtracted from the 

post-application weights, and that difference was added 

to the weight of decontaminant runoff captured separately 

from each coupon. The average deposition/runoff weight 

of the Oxonia Active® for each of the test materials is 

shown in Table 9-6. The average deposited weight of 

Oxonia Active® over all the test materials was 1.16 g. 

That average mass of diluted Oxonia Active® (assumed 

to have a density of 1.0 g/mL) was used in neutralization 

tests to determine the amount of sodium thiosulfate (STS) 

needed to effectively neutralize the Oxonia Active®. 

9.4.3 Neutralization Methodology 

Neutralization of the Oxonia Active® was achieved with STS. 

The concentrations of STS used during the neutralization trial 

were 0.4, 0.8, and 1.6% in the PBS/Triton X-100 extraction 

solution. These STS concentrations were chosen for the trial 

based on historical data. The results of the neutralization 

panel are shown in Tables 9-7 and 9-8. From these results 

it was determined that a concentration of 0.4% STS in the 

extraction solution was sufficient for neutralization of Oxonia 

Active® for both B. anthracis and B. subtilis. 

44 
Table 9-7. Neutralization Testing with Bacillus anthracis Ames Spores for Ecolab’s Oxonia Active® 

Inoculum Total Observed 

Treatment (CFUs) (CFUs) % of Control 

Oxonia Active® + Sporesa 1.01 x 108 0 0 

Oxonia Active® + PBS + Triton X-100 + Sporesab 1.01 x 108 9.60 x 105 1.07 

PBS + Triton X-100 + Spores (Control)b 1.01 x 108 9.00 x 107 

Oxonia Active® + PBS + Triton X-100 + 1.6% STS + Sporesab 1.01 x 108 8.12 x 107 90.3 

Oxonia Active® + PBS + Triton X-100 + 0.80% STS + Sporesab 1.01 x 108 7.89 x 107 87.7 

Oxonia Active® + PBS + Triton X-100 + 0.4% STS + Sporesab 1.01 x 108 8.81 x 107 97.8 

a

 Oxonia Active® volume of 1.16 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with 

Oxonia Active® = 11.16 mL (10 mL of PBS/Triton X-100/STS + 1.16 mL Oxonia Active®). 

“-” Not Applicable. 

Table 9-8. Neutralization Testing with Bacillus subtilis Spores for Ecolab’s Oxonia Active® 

Inoculum Total Observed 

Treatment (CFUs) (CFUs) % of Control 

Oxonia Active® + Sporesa 9.73 x 107 0 0 

Oxonia Active® + PBS + Triton X-100 + Sporesb 9.73 x 107 0 0 

PBS + Triton X-100 + Spores (Control)c 9.73 x 107 1.73 x 108 

Oxonia Active® + PBS + Triton X-100 + 1.6% STS + Sporesb 9.73 x 107 1.39 x 108 80.3 

Oxonia Active® + PBS + Triton X-100 + 0.80% STS + Sporesb 9.73 x 107 1.20 x 108 69.2 

Oxonia Active® + PBS + Triton X-100 + 0.4% STS + Sporesb 9.73 x 107 1.90 x 108 110 

a

 Oxonia Active® volume of 1.16 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with 

Oxonia Active® = 11.16 mL (10 mL of PBS/Triton X-100/STS + 1.16 mL Oxonia Active®). 

“-” Not Applicable. 

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46 
10.0 

Minncare® Cold Sterilant (Minntech) 

10.1 QC Results 

During testing of Minncare® Cold Sterilant, all positive 

control results were well within the target recovery range 

of 1 to 150% of the spiked spores. For B. anthracis positive 

control recovery values ranged from 26 to 124%, with the 

lowest recovery occurring on bare pine wood. For B. subtilis 

positive control recovery values ranged from 5 to 93%, with 

the lowest recoveries occurring on bare pine wood. 

all procedural and laboratory blanks met the criterion of no 

observed CFUs in quantitative efficacy testing, with both 

B. anthracis and B. subtilis during Minncare® testing. No 

growth was also observed for any of the procedural and 

laboratory blanks in the qualitative assessment of residual 

spores, which involves a much longer nutrient growth period 

(up to 7 days). Once again, the industrial carpet exhibited the 

antimicrobial properties seen in previous testing and initially 

inhibited the growth of the inoculated, non-decontaminated 

samples. This inhibition of growth is further explained in 

Section 10.2.2. 

Spiked control samples were taken from the spore suspension 

on each day of testing, and serially diluted, plated on 

nutrient media, and counted to establish the spore density 

used to spike the coupons. This process takes approximately 

24 hours, so the spore density is known after completion of 

each day’s testing. The target criterion is to maintain a spore 

suspension density of 1 × 109/mL (± 25%), leading to a spike 

Test Results
 

of 1 × 108

 spores (± 25%) on each test coupon. The actual 

spike values for two days of B. anthracis testing were 

8.93 × 107/coupon and 9.63 × 107/coupon, and for one 

day of B. subtilis testing the actual spike value was 

9.87 × 107/coupon. 

10.2 Decontamination Efficacy 

The decontamination efficacy of Minncare® Cold Sterilant 

was evaluated for B. anthracis ames and B. subtilis on seven 

indoor material surfaces. The following sections summarize 

the results found with this decontaminant. 

10.2.1 Quantitative Assessment of the Log Reduction 

of Viable Organisms 

The decontamination efficacy of Minncare® Cold Sterilant 

was greater than approximately 7.5 log reduction for six 

materials for both B. anthracis (shown in Tables 10-1 and 

10-2) and B. subtilis (shown in Tables 10-3 and 10-4). Only 

bare pine wood exhibited log reductions lower than 7.5 for 

both organisms, with log reductions of 5.40 for B. anthracis 

and 6.00 for B. subtilis. Note that Tables 10-1 and 10-3 show 

results for those materials that were tested with a 10-minute 

contact time, and Tables 10-2 and 10-4 for those materials 

that were tested with a 30-minute contact time, as directed by 

the vendor of Minncare® Cold Sterilant. The efficacy results 

are summarized in Table 10-5. 

47 
Table 10-1. Inactivation of Bacillus anthracis Ames Sporesa

—Minntech’s Minncare® Cold Sterilant 

(10 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Decorative Laminate 

Positive Control 8.93 x 107 7.58 ± .0.06 42.6 ± 6.4 -

Decontaminated 8.93 x 107 0 0 ≥ 7.58 ± 0.06 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Controlb 8.93 x 107 7.80 ± 0.04 71.4 ± 6.4 -

Decontaminatedc 8.93 x 107 0 0 ≥ 7.80 ± 0.03 

Laboratory Blankd 

Procedural Blanke 

0 

0 

0 

0 

0 

0 -

Painted Wallboard Paper 

Positive Control 8.93 x 107 7.53 ± 0.08 38.7 ± 7.7 -

Decontaminated 8.93 x 107 0 0 ≥ 7.53 ± 0.07 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

Glass 

Positive Control 8.93 x 107 7.75 ± 0.03 63.2 ± 3.7 -

Decontaminated 8.93 x 107 0 0 ≥ 7.75 ± 0.02 

Laboratory Blank 

Procedural Blank 

0 

0 

0 

0 

0 

0 -

 

a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

­

­

48 
Table 10-2. Inactivation of Bacillus anthracis Ames Sporesa

—Minntech’s Minncare® Cold Sterilant 

(30 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlb 9.63 x 107 7.82 ± .0.13 71.7 ± 22.7 -

Decontaminatedc 9.63 x 107 0 0 ≥ 7.82 ± 0.11 

Laboratory Blankd 0 0 0 -

Procedural Blankc 0 0 0 -

Painted Cinder Block 

Positive Control 9.63 x 107 8.08 ± 0.04 124.0 ± 12.5 -

Decontaminated 9.63 x 107 0 0 ≥ 8.08 ± 0.04 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Bare Pine Wood 

Positive Control 9.63 x 107 7.28 ± 0.38 25.7 ± 16.9 -

Decontaminated 9.63 x 107 1.88 ± 1.83 0.0019 ± 0.0020 5.40 ± 1.60 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

a

 Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

­

49 
Table 10-3. Inactivation of Bacillus subtilis Sporesa

—Minntech’s Minncare® Cold Sterilant 

(10 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Decorative Laminate 

Positive Control 9.87 x 107 7.87 ± .0.07 76.7 ± 13.4 -

Decontaminated 9.87 x 107 0 0 ≥ 7.87 ± 0.06 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Controlb 9.87 x 107 7.89 ± 0.04 79.5 ± 7.4 -

Decontaminatedc 9.87 x 107 0 0 ≥ 7.89 ± 0.03 

Laboratory Blankd 0 0 0 

Procedural Blanke 0 0 0 -

Painted Wallboard Paper 

Positive Control 9.87 x 107 7.46 ± 0.17 30.8 ± 10.0 -

Decontaminated 9.87 x 107 0 0 ≥ 7.46 ± 0.15 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

Glass 

Positive Control 9.87 x 107 7.95 ± 0.10 93.1 ± 24.5 -

Decontaminated 9.87 x 107 0 0 ≥ 7.95 ± 0.09 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

 

a Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).
 

b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

Table 10-4. Inactivation of Bacillus subtilis Sporesa—Minntech’s Minncare® Cold Sterilant (30 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Controlb 9.87 x 107 7.91 ± .0.06 82.8 ± 12.0 -

Decontaminatedc 9.87 x 107 0 0 ≥ 7.91 ± 0.05 

Laboratory Blankd 0 0 0 -

Procedural Blankc 0 0 0 -

Painted Cinder Block 

Positive Control 9.87 x 107 7.93 ± 0.09 88.2 ± 18.8 -

Decontaminated 9.87 x 107 0 0 ≥ 7.93 ± 0.08 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Bare Pine Wood 

Positive Control 9.87 x 107 6.69 ± 0.13 5.1 ± 1.6 -

Decontaminated 9.87 x 107 0.69 ± 1.54 0.00056 ± 0.0012 6.00 ± 1.35 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

a

 Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).
 

b

 Inoculated, not decontaminated coupon (sprayed with SFW.
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

­

­

­

50 
Table 10-5. Summary of Efficacy Values (Log Reduction) Obtained for 

Minntech’s Minncare® Cold Sterilanta 

Material B. anthracis Ames B. subtilis 

Industrial-Grade Carpet ≥ 7.82b ≥ 7.91b 

Decorative Laminate ≥ 7.58c ≥ 7.87c 

Galvanized Metal Ductwork ≥ 7.80c ≥ 7.89c 

Painted Wallboard Paper ≥ 7.53c ≥ 7.46c 

Painted Cinder Block ≥ 8.08b ≥ 7.93b 

Bare Pine Wood 5.40b 6.00b 

Glass ≥ 7.75c ≥ 7.95c 

a

 Numbers in bold are statistically different (p ≤ 0.05) from B. anthracis Ames.
 b 30 minute contact time.
 c

 10 minute contact time.
 

Table 10-5 shows that the efficacy values found for 

B. subtilis were significantly different from those found 

for B. anthracis on four materials. However, in all four of 

these cases, no viable spores of either organism were found 

on the decontaminated test coupons, i.e., Minncare® Cold 

Sterilant achieved a complete kill of the inoculated spores, 

and the efficacy values are shown as “greater than or equal 

to” (≥ ) values. Thus the differences in efficacy seen in these 

four cases are due to limitations in the recovery of the two 

types of spores from these coupon materials, and cannot be 

attributed to actual differences in efficacy toward the two 

organisms. 

10.2.2 Qualitative Assessment of Residual Spores 

Results from the liquid culture growth assessment of coupons 

at 1 and 7 days post-decontamination are provided in 

Tables 10-6 and 10-7 for coupons spiked with B. anthracis 

ames and B. subtilis spores, respectively. In this assessment, 

cultures showing positive growth (i.e., a cloudy growth 

medium) were applied to streak plates, and the identity of 

the growing organism was checked by colony morphology. 

only B. anthracis colonies were found in cultures of 

coupons used with B. anthracis, and only B. subtilis colonies 

were found in cultures of coupons used with B. subtilis. 

The qualitative liquid culture growth assessment results in 

Tables 10-6 and 10-7 are consistent with the quantitative 

efficacy results, in that no growth was observed for 

B. anthracis or B. subtilis on any decontaminated test 

material except bare wood. Growth was observed at both 

one and seven days on all non-decontaminated test materials, 

as expected, with the exception of some coupons of the 

industrial carpet. For B. anthracis only three of the five 

inoculated, non-decontaminated industrial carpet coupons 

were positive for growth after one day, and for B. subtilis 

only two of the five inoculated, non-decontaminated 

industrial carpet coupons were positive for growth after one 

day. This result is likely due to the antibacterial component 

(zinc omadine) in this material. All inoculated, nondecontaminated industrial carpet coupons, however, were 

positive for growth at the seven day assessment, possibly 

due to the degradation of the antibacterial component over 

multiple days. No growth was observed from any of the 

blank coupons. 

10.3 Damage to Coupons 

No visible damage was observed on the test materials after 

either the 10 min or 30 min contact time with Minncare® 

Cold Sterilant, or seven days later after completion of the 

qualitative assessment of residual spores. 

10.4 Other Factors 

10.4.1 Operator Control 

on each day of testing, Minncare® Cold Sterilant was 

prepared according to the vendor’s explicit instructions as 

described in Appendix F. A 10 percent solution of Minncare® 

Cold Sterilant was prepared fresh before use on each day of 

testing by diluting one part of the concentrate with nine parts 

of SFW. This diluted solution was then transferred to a plastic 

handheld spray bottle and applied to the test coupons. The 

respective material surfaces were inspected closely to ensure 

that each was thoroughly wetted. After the required contact 

time, each coupon was placed in a 50 mL conical vial that 

also served to collect any pooled or runoff decontaminant. 

The horizontally and vertically oriented coupons stayed in 

their respective configurations throughout the contact time. 

51 
 

Table 10-7. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus subtilis Spores— 

Minntech’s Minncare® Cold Sterilant 

Day 1 Day 7 

Test Material S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls -

Test Coupons -

-

-

+ 

-

-

-

+ 

-

-

a 

b 

-

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Decorative Laminate 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Galvanized Metal Ductwork 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Painted Wallboard Paper 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Painted Cinder Block 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Bare Pine Wood 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

+ 

+ 

-

+ 

-

-

-

+ 

-

+ 

-

+ 

+ 

+ 

-

+ 

-

-

-

Glass 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. subtilis spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. subtilis spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. subtilis spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth. 


Table 10-6. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis Ames Spores— 

Minntech’s Minncare® Cold Sterilant 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

+ 

-

+ 

-

-

-

+ 

-

-

-

-

a 

-

b 

+ 

-

+ 

-

+ 

-

+ 

-

+ 

-

-

-

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons + - + + + - + - + + + -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons - - - - - - - - - - - -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. anthracis Ames spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. anthracis Ames spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. anthracis Ames spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth. 


52 
all tests were conducted under ambient conditions inside 

a climate-controlled laboratory. The temperature inside the 

test chamber was equilibrated to the ambient laboratory 

temperature of approximately 22 °C. The RH inside the test 

chamber was monitored with a NIST-traceable hygrometer. 

Whenever the RH exceeded 40%, the dehumidification 

system attached to the testing chamber was actuated until the 

Rh dropped below 40%. 

10.4.2 Technology Spray Deposition 

Minntech’s Minncare® Cold Sterilant was applied according 

to the procedure included as Appendix F of this report, from 

a distance of 12 inches from the horizontally and vertically 

oriented materials until the materials were completely wetted. 

No reapplication of the Minncare® Cold Sterilant was made. 

To assess Minncare® Cold Sterilant deposition, triplicate 

coupons of each test material were weighed prior to 

application in trial runs, and these weights were recorded. 

Then the triplicate coupons were sprayed with Minncare® 

Cold Sterilant until the coupons were fully wetted in their 

respective vertical or horizontal orientations, allowed 

the requisite 10 or 30 minute contact time, and then each 

coupon was weighed again. The pre-application weights 

were then subtracted from the post-application weights, and 

that difference was added to the weight of decontaminant 

runoff captured separately from each coupon. The average 

deposition/runoff weight of the Minncare® Cold Sterilant for 

each of the test materials is shown in Table 10-8. The average 

Table 10-8. Deposition/Runoff Weight of Minntech’s 

Minncare® Cold Sterilant on Test Materials 

Test Material 

Avg. Deposition/Runoff 

Weight (g) 

Industrial-Grade Carpet 0.18 

Decorative Laminate 0.21 

Galvanized Metal Ductwork 0.12 

Painted Wallboard Paper 0.11 

Painted Cinder Block 1.14 

Bare Pine Wood 0.12 

Glass 0.16 

deposition value over all materials (0.15 g) was then used in 

trial runs to estimate the amount of neutralization solution 

needed to effectively neutralize the Minncare® Cold Sterilant. 

10.4.3 Neutralization Methodology 

Neutralization of Minncare® Cold Sterilant was achieved 

with a vendor-specified neutralization solution (NS). The 

(NS) was prepared fresh prior to use on each day of testing 

by diluting 10 g of peptone, 1 g sodium thiosulfate, and 14 

g of potassium dihydrogen phosphate to 1 l with SFW. The 

ph was adjusted to 7 ± 0.5 with hCl and the solution was 

then autoclaved for 20 minutes at 121 °C. Once this solution 

cooled to room temperature, catalase was filter sterilized 

and added just before neutralization to achieve a catalase 

concentration of 0.005%. 

This NS was then mixed in differing proportions with the 

PBS/Triton X-100 solution to prepare extraction solutions 

for testing. The compositions of the solutions tested during 

the neutralization trials ranged from 8.5 ml PBS/Triton 

X-100 + 1.5 mL NS up to 7 mL PBS/Triton X-100 +3 mL 

NS for B. anthracis and from 9.75 mL PBS/TritonX-100 

+0.25 mL NS up to 9 mL PBS/Triton X-100 + 1 mL NS for 

B. subtilis. The results of the final neutralization trials are 

shown in Tables 10-9 and 10-10. on the basis of these trials, 

a neutralizer volume of 1.5 mL was used in testing with 

B. anthracis, and a volume of 0.5 mL was used in testing 

with B. subtilis. 

53 
Table 10-9. Neutralization Testing with Bacillus anthracis Ames Spores for Minntech’s Minncare® Cold Sterilant 

Treatment Inoculum (CFUs) Total Observed (CFUs) % of Control 

Minncare® + Sporesa (10 min contact) 6.23 x 107 0 0 

Minncare® + PBS + Triton X-100 + Sporesab (10 min contact) 6.23 x 107 0 0 

Minncare® + Sporesa

 (30 min contact) 6.23 x 107 0 0 

Minncare® + PBS + Triton X-100 + Sporesab (30 min contact) 6.23 x 107 0 0 

PBS + Triton X-100 + Spores (Control)b 6.23 x 107 7.06 x 107 -

Minncare® + PBS + Triton X-100 + 1.5 mL NSc

 + Sporesab 6.23 x 107 6.86 x 107 97.3 

Minncare® + PBS + Triton X-100 + 2.0 mL NSc

+ Sporesab 6.23 x 107 5.45 x 107 77.2 

Minncare® + PBS + Triton X-100 + 3.0 mL NSc

 + Sporesab 6.23 x 107 6.01 x 107 85.2 

a

 Minncare® Cold Sterilant volume of 0.15 mL corresponds to mean gravimetric deposition on test materials.


 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated volume of NS; total volume for all samples with Minncare® Cold Sterilant = 10.15 mL 


(10 mL of PBS/Triton X-100/NS + 0.15 mL Minncare Cold Sterilant). 

c

 NS = neutralization solution. 

“-” Not Applicable. 

Table 10-10. Neutralization Testing with Bacillus subtilis Spores for Minntech’s Minncare® Cold Sterilant 

Treatment Inoculum (CFUs) Total Observed (CFUs) % of Control 

Minncare®+ Sporesa (10 min contact) 8.77 x 107 0 0 

Minncare® + PBS + Triton X-100 + Sporesab (10 min contact) 8.77 x 107 0 0 

Minncare® + Sporesa

 (30 min contact) 8.77 x 107 0 0 

Minncare® + PBS + Triton X-100 + Sporesab (30 min contact) 8.77 x 107 0 0 

PBS + Triton X-100 + Spores (Control)b 8.77 x 107 9.41 x 107 -

Minncare® + PBS + Triton X-100 + 0.25 mL NSc

 + Sporesab 8.77 x 107 1.28 x 107 13.6 

Minncare® + PBS + Triton X-100 + 0.5 mL NSc

 + Sporesab 8.77 x 107 6.92 x 107 73.6 

Minncare® + PBS + Triton X-100 + 1.0 mL NSc

 + Sporesab 8.77 x 107 5.73 x 107 60.9 

a

 Minncare® Cold Sterilant volume of 0.15 mL corresponds to mean gravimetric deposition on test materials.


 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated volume of NS; total volume for all samples with Minncare® Cold Sterilant = 10.15 mL 


(10 mL of PBS/Triton X-100/NS + 0.15 mL Minncare® Cold Sterilant). c

 NS = neutralization solution. 

“-” Not Applicable. 

54 
11.0 

SanDes (DTI-Sweden AB) Test Results
 

11.1 QC Results 

In testing of SanDes, all positive control results were well 

within the target recovery range of 1 to 150% of the spiked 

spores. For B. anthracis positive control recovery values 

ranged from 12 to 115%, with the lowest recovery occurring 

on bare pine wood. For B. subtilis positive control recovery 

values ranged from 6 to 98%, with the lowest recoveries 

occurring on bare pine wood. 

In testing of SanDes, all procedural and laboratory blanks 

met the criterion of no observed CFUs in quantitative 

efficacy testing, with both B. anthracis and B. subtilis. No 

growth was also observed in the qualitative assessment of 

residual spores for all procedural and laboratory blanks, 

which involves a much longer nutrient growth period. Once 

again, the industrial carpet exhibited the antimicrobial 

properties seen in previous testing and initially inhibited 

the growth of the inoculated, non-decontaminated samples 

for the B. anthracis. This inhibition of growth is further 

explained in Section 11.2.2. 

Spike control samples were taken from the spore suspension 

on each day of testing, and serially diluted, nutrient plated, 

and counted to establish the spore density used to spike the 

coupons. This process takes approximately 24 hours, so the 

spore density is known after completion of each day’s testing. 

The target criterion is to maintain a spore suspension density 

of 1 × 109

/mL (± 25%), leading to a spike of 1 × 108 spores 

(± 25%) on each test coupon. The actual spike values for 

two days of B. anthracis testing were 7.37 × 107/coupon 

and 8.63 × 107/coupon, and for two days of B. subtilis 

testing the actual spike values were 8.87 × 107/coupon 

and 9.27 × 107/coupon. The B. anthracis spike value of 

7.37 x 107/coupon fell slightly outside the ± 25% target 

criterion, but this spike value was acceptable as spore 

recoveries were good and efficacy up to 7.87 logs could 

be determined. 

11.2 Decontamination Efficacy 

The decontamination efficacy of DTI-Sweden AB’s SanDes 

was evaluated for B. anthracis ames and B. subtilis on seven 

indoor material surfaces. The following sections summarize 

the results found with this decontaminant. 

11.2.1 Quantitative Assessment of the Log Reduction 

of Viable Organisms 

The decontamination efficacy of DTI-Sweden AB’s SanDes 

was less than 1.0 log reduction for six of the seven materials 

for both B. anthracis and B. subtilis. The exceptions were 

for B. anthracis (Ames) spores on glass (4.65 log reduction) 

and for B. subtilis spores on decorative laminate (1.37 log 

reduction), as shown in Tables 11-1 and 11-2, respectively, 

and summarized in Table 11-3. 

55 
Table 11-1. Inactivation of Bacillus anthracis Ames Sporesa

—DTI-Sweden AB’s SanDes (70 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Control 7.37 x 107 7.81 ± 0.08 89.7 ± 15.7 -

Decontaminated 7.37 x 107 7.68 ±0.06 65.9 ± 9.5 0.13 ± 0.09 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Decorative Laminate 

Positive Control 8.63 x 107 7.86 ± 0.04 84.3 ± 7.8 -

Decontaminated 8.63 x 107 7.68 ± 0.09 56.0 ± 12.5 0.18 ± 0.09 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Controlb 8.63 x 107 7.75 ± 0.13 66.9 ± 19.8 -

Decontaminatedc 8.63 x 107 7.65 ± 0.10 53.2 ± 11.9 0.09 ± 0.14 

Laboratory Blankd 0 0 0 -

Procedural Blanke 0 0 0 -

Painted Wallboard Paper 

Positive Control 8.63 x 107 7.86 ± 0.22 93.2 ± 45.8 -

Decontaminated 8.63 x 107 7.67 ± 0.09 55.6 ± 11.3 0.19 ± 0.21 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

Painted Cinder Block 

Positive Control 7.37 x 107 7.91 ± 0.07 111.4 ± 18.5 -

Decontaminated 7.37 x 107 7.58 ± 0.07 51.7 ± 8.4 0.33 ± 0.09 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Bare Pine Wood 

Positive Control 7.37 x 107 6.92 ± 0.21 12.2 ± 4.8 -

Decontaminated 7.37 x 107 6.53 ± 0.23 5.3 ± 3.4 0.39 ± 0.27 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Glass 

Positive Control 7.37 x 107 7.74 ± 0.09 75.7 ± 15.5 -

Decontaminated 7.37 x 107 3.09 ± 0.27 0.002 ± 0.001 4.65 ± 0.25 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

a

 Data are expressed as mean (± SD) total number of spores (CFU) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

­

­

56 
Table 11-2. Inactivation of Bacillus subtilis Sporesa

—DTI-Sweden AB’s SanDes (70 minute contact time) 

Test Material Inoculum (CFUs) 

Mean of Logs of 

Observed CFUs Mean % Recovery 

Decontamination 

Efficacy ± CI 

Industrial-Grade Carpet 

Positive Control 9.27 x 107 7.76 ± 0.27 72.5 ± 43.4 -

Decontaminated 9.27 x 107 7.17 ±0.03 15.9 ± 1.1 0.59 ± 0.24 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Decorative Laminate 

Positive Control 8.87 x 107 7.90 ± 0.19 97.7 ± 42.3 -

Decontaminated 8.87 x 107 6.53 ± 0.06 3.9 ± 0.5 1.37 ± 0.17 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Galvanized Metal Ductwork 

Positive Controlb 8.87 x 107 7.73 ± 0.26 70.9 ± 44.3 -

Decontaminatedc 8.87 x 107 6.98 ± 0.19 11.5 ± 4.4 0.76 ± 0.28 

Laboratory Blankd 0 0 0 -

Procedural Blanke 0 0 0 -

Painted Wallboard Paper 

Positive Control 8.87 x 107 7.36 ± 0.28 30.0 ± 16.8 -

Decontaminated 8.87 x 107 6.76 ± 0.09 6.6 ± 1.5 0.60 ± 0.25 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 

Painted Cinder Block 

Positive Control 9.27 x 107 7.76 ± 0.13 63.5 ± 16.2 -

Decontaminated 9.27 x 107 7.24 ± 0.03 18.9 ± 1.2 0.51 ± 0.12 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

Bare Pine Wood 

Positive Control 9.27 x 107 6.76 ± 0.07 6.3 ± 1.0 -

Decontaminated 9.27 x 107 6.11 ± 0.09 1.4 ± 0.4 0.65 ± 0.10 

Laboratory Blank 0 0 0 

Procedural Blank 0 0 0 -

Glass 

Positive Control 9.27 x 107 7.81 ± 0.18 74.3 ± 36.8 -

Decontaminated 9.27 x 107 7.58 ± 0.05 41.6 ± 5.0 0.22 ± 0.16 

Laboratory Blank 0 0 0 -

Procedural Blank 0 0 0 -

a

 Data are expressed as mean (± SD) total number of spores (CFUs) observed, percent recovery, and decontamination efficacy (log reduction).
 

CI = confidence interval (± 1.96 × SE).


b

 Inoculated, not decontaminated coupon (sprayed with SFW).
 

c

 Inoculated, decontaminated coupon.


d

 Laboratory Blank = not inoculated, not decontaminated coupon.
 

e

 Procedural Blank = not inoculated, decontaminated coupon.
 

“-” Not Applicable.
 

­

­

57 
Table 11-3. Summary of Efficacy Values (Log Reduction) 

Obtained for DTI-Sweden AB’s SanDesa 

Test Material B. anthracis Ames B. subtilis 

Industrial-Grade Carpet 0.13 0.59 

Decorative Laminate 0.18 1.37 

Galvanized Metal Ductwork 0.09 0.76 

Painted Wallboard Paper 0.19 0.60 

Painted Cinder Block 0.33 0.51 

Bare Pine Wood 0.39 0.65 

Glass 4.65 0.22 

 

a Numbers in bold are statistically different (p ≤ 0.05) from B. anthracis Ames 

result. 

Table 11-3 shows that for four of the seven materials, the 

efficacy results with B. subtilis were significantly different 

from the corresponding results with B. anthracis. In two such 

cases, both efficacy results were less than 1.0 log. The largest 

differences were for glass and decorative laminate, which 

as noted above resulted in the highest efficacy results for 

B. anthracis and B. subtilis, respectively. 

11.2.2 Qualitative Assessment of Residual Spores 

Results from the liquid culture growth assessment of coupons 

at one and seven days post-decontamination are provided in 

Tables 11-4 and 11-5 for coupons spiked with B. anthracis 

ames and B. subtilis spores, respectively. In this assessment, 

cultures showing positive growth (i.e., a cloudy growth 

medium) were applied to streak plates and the identity of the 

growing organism was checked by colony morphology. only 

B. anthracis colonies were found in cultures of coupons used 

with B. anthracis, and only B. subtilis colonies were found in 

cultures of coupons used with B. subtilis. 

Tables 11-4 and 11-5 are consistent with the relatively low 

efficacy of SanDes, in that all inoculated coupons of all 

materials showed growth for B. anthracis and B. subtilis. 

Blank (uninoculated) coupons showed no growth. 

These qualitative, liquid culture growth assessment results 

are consistent with the quantitative, observed efficacy 

results for all of the materials, except for the industrial 

carpet inoculated with B. anthracis. Only three of the five 

B. anthracis-inoculated, non-decontaminated industrial 

carpet positive controls were positive for growth at 

the 1 day assessment, perhaps due to the antibacterial 

component (zinc omadine) in this material. All inoculated, 

non-decontaminated industrial carpet positive control 

samples, however, were positive for growth at the seven 

day assessment, possibly due to the degradation of the 

antibacterial component over multiple days. 

58 
 

 

Table 11-4. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus anthracis Ames Spores— 

DTI-Sweden AB’s SanDes 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

+ 

-

-

-

-

+ 

+ 

-

+ 

+ 

-

a 

-

b 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

-

-

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. anthracis Ames spores) ); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. anthracis Ames spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. anthracis Ames spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth.
 

Table 11-5. Liquid Culture Assessment of Extracts from Coupons Inoculated with Bacillus subtilis Spores— 

DTI-Sweden AB’s SanDes 

Test Material 

Day 1 Day 7 

S1 S2 S3 S4 S5 Bl S1 S2 S3 S4 S5 Bl 

Industrial-Grade Carpet 

Positive Controls 

Test Coupons 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

-

+ 

+ 

-

a 

-

b 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

-

-

Decorative Laminate 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Galvanized Metal Ductwork 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Painted Wallboard Paper 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Painted Cinder Block 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Bare Pine Wood 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

Glass 

Positive Controls + + + + + - + + + + + -

Test Coupons + + + + + - + + + + + -

S1 to S5 = Sample 1 to Sample 5.
 

Bl = Blank (not inoculated with B. subtilis spores); a = laboratory blank, b = procedural blank.
 

Positive controls = coupons inoculated with B. subtilis spores, but not subjected to decontamination.
 

Test coupons = coupons inoculated with B. subtilis spores, and subjected to decontamination.
 

“+” = growth; “-” = no growth.
 

59 
Table 11-6. Deposition/Runoff Weight of DTI-Sweden 

AB’s SanDes on Test Materials 

Test Material 

Avg. Deposition/Runoff 

Weight (g) 

Industrial-Grade Carpet 0.04 

Decorative Laminate 0.02 

Galvanized Metal Ductwork 0.14 

Painted Wallboard Paper 0.13 

Painted Cinder Block 0.12 

Bare Pine Wood 0.12 

Glass 0.14 

11.3 Damage to Coupons 

No visible damage was observed on any of the test materials 

after the 70 min contact time with SanDes despite multiple 

applications of the decontaminant in that time period. 

11.4 Other Factors 

11.4.1 Operator Control 

DTI-Sweden AB’s SanDes was provided as a “ready-touse” formulation straight from the bottle. The product was 

provided in small bottles (30 mL), and the vendor provided 

a small push-button attachment for dispensing SanDes 

as a spray. This attachment produced a very fine spray, 

and numerous “pumps” were required to fully wet the 

surface of each test material. Because of the small volume 

of the SanDes bottles, and the multiple reapplications of 

SanDes during testing, a bottle was quickly depleted during 

application and had to be replaced. The SanDes spray 

attachment did a good job of dispersing the product, but 

it appeared that the 30 cm (12-inch) application distance 

exceeded the range at which the attachment would have 

wetted the testing material surfaces most effectively. Testing 

staff attempted to apply SanDes in as consistent a fashion 

as possible, but the spray attachment occasionally stuck in 

the down position, interrupting the back-and-forth motion 

needed to wet all six replicate coupons of a single test 

material (including the blank). Additional spray attachments 

were provided by the vendor to circumvent this problem. 

The respective material surfaces were observed closely to 

ensure that they were thoroughly wetted; approximately 

15 seconds of spray duration using the back-and-forth motion 

was needed to produce wetting across the surfaces of five 

replicate coupons and a corresponding blank coupon for each 

material type. 

all tests were conducted under ambient conditions inside 

a climate-controlled laboratory. The temperature inside the 

test chamber was equilibrated to the ambient laboratory 

temperature of approximately 22 °C. The RH inside the test 

chamber was monitored with a NIST-traceable hygrometer. 

Whenever the RH exceeded 40%, the dehumidification 

system attached to the testing chamber was actuated until the 

RH dropped below 40%. The dehumidifier was actuated only 

after the 70 minute contact time with the SanDes. Therefore, 

the testing chamber was always within 40% Rh prior to the 

decontamination of a new set of materials with SanDes. 

11.4.2 Technology Spray Deposition 

DTI-Sweden AB’s SanDes was applied according to the 

procedure included as Appendix G of this report. SanDes 

was applied from a distance of 30 cm (12 inches) from 

the horizontally and vertically oriented materials until the 

materials were fully wetted. Reapplication of the SanDes 

was made on all coupon surfaces at 10, 20, and 30 minutes 

after the initial application. at 60 minutes after the initial 

application, one more application of SanDes was made. After 

70 minutes total contact time since the initial application, 

each material coupon was placed in a tube that also served 

to collect decontaminant that had run off from or pooled on 

the coupon. The horizontally and vertically oriented coupon 

materials stayed in their respective configurations for the 

duration of their 70 minute contact times. 

To assess SanDes deposition, triplicate coupons of each test 

material were weighed prior to application of SanDes in trial 

runs, and these values were recorded. Then the triplicate 

coupons were sprayed with SanDes until fully wetted in 

their respective vertical or horizontal orientations, SanDes 

was reapplied as described above and allowed a 70 minute 

contact time, and then each coupon was weighed again. 

The pre-application weights were then subtracted from the 

post-application weights, and that difference was added to 

the weight of decontaminant runoff from or pooled on each 

coupon. The average deposition/runoff weight of SanDes 

from each of the test materials is shown in Table 11-6. The 

total averaged value of 0.10 g (density assumed = 1.0 g/mL) 

was then used in trials to determine the amount of sodium 

thiosulfate (STS) needed to effectively neutralize the SanDes. 

11.4.3 Neutralization Methodology 

Neutralization of the SanDes was achieved with STS. The 

concentrations of STS used during the neutralization panel 

were 2.0, 2.5, and 3.0% in the PBS/Triton X-100 extraction 

solution. These STS concentrations were chosen for the trial 

based on historical data. The results of the neutralization 

panel are shown in Tables 11-7 and 11-8. From these results 

a concentration of 2.0% STS in the extraction solution was 

determined to be sufficient for neutralization of SanDes for 

both B. anthracis and B. subtilis. 

60 
Table 11-7. Neutralization Testing with Bacillus anthracis Ames Spores for DTI-Sweden AB’s SanDes 

Total Observed 

Treatment Inoculum (CFUs) (CFUs) % of Control 

SanDes+ Sporesa 1.08 x 108 0 0 

SanDes + PBS + Triton X-100 + Sporesab 1.08 x 108 0 

PBS + Triton X-00 + Spores (Control)b 1.08 x 108 1.02 x 108 -

SanDes + PBS + Triton X-100 + 2.0% STS + Sporesab 1.08 x 108 1.02 x 108 100.0 

SanDes + PBS + Triton X-100 + 2.5% STS + Sporesab 1.08 x 108 9.69 x 107 95.3 

SanDes + PBS + Triton X-100 + 3.0% STS + Sporesab 1.08 x 108 9.86 x 107 97.0 

a

 SanDes volume of 0.10 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with SanDes = 10.1 mL (10 mL of PBS/Triton 

X-100/STS + 0.10 mL SanDes). 

“-” Not Applicable. 

Table 11-8. Neutralization Testing with Bacillus subtilis Spores for DTI-Sweden AB’s SanDes 

Total Observed 

Treatment Inoculum (CFUs) (CFUs) % of Control 

SanDes+ Sporesa 9.83 x 107 0 0 

SanDes + PBS + Triton X-100 + Sporesab 9.83 x 107 0 

PBS + Triton X-00 + Spores (Control)b 9.83 x 107 9.72 x 107 -

SanDes + PBS + Triton X-100 + 2.0% STS + Sporesab 9.83 x 107 9.83 x 107 101.1 

SanDes + PBS + Triton X-100 + 2.5% STS + Sporesab 9.83 x 107 9.34 x 107 96.1 

SanDes + PBS + Triton X-100 + 3.0% STS + Sporesab 9.83 x 107 9.72 x 107 100.0 

a

 SanDes volume of 0.10 mL corresponds to mean gravimetric deposition on test materials.

 

b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with SanDes = 10.1 mL 

(10 mL of PBS/Triton X-100/STS + 0.10 mL SanDes). 

“-” Not Applicable. 

61 
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62 
12.1 DioxiGuardTM Results 

■		 The quantitative decontamination efficacy of 

DioxiGuardTM was 2.6 log reduction or less for 

B. anthracis ames, and 0.87 log reduction or less for 

B. subtilis, on the seven test materials. Efficacy values 

above about 1.8 log reduction for B. anthracis were 

seen only with relatively non-porous materials (glass, 

laminate, painted concrete) and with carpet. 

■ Significant differences between efficacy values for 

B. subtilis and B. anthracis were found with non-porous 

materials (glass, laminate, painted concrete, metal 

ductwork), due primarily to the low efficacy values 

found with B. subtilis on these materials (i.e., zero to 

0.3 log reduction). 

■		 In the qualitative tests, coupons of all material 

types showed the presence of viable organisms after 

decontamination, consistent with the quantitative 

efficacy results. Morphological analysis confirmed that 

the growth observed indicated only B. anthracis ames 

or B. subtilis, respectively, from the spiked coupons. 

■		 In the qualitative tests growth was also observed with 

blank coupons of all material types used in testing 

with B. anthracis, although no CFus were found in 

the quantitative efficacy tests, indicating minimal 

contamination of the blanks. This result was attributed 

to contamination of blank materials within the test 

chamber during overnight drying of coupons spiked 

with B. anthracis. The drying procedure was changed 

for testing with B. subtilis (blanks were removed from 

the test chamber before overnight drying of spiked 

coupons) and no growth was observed subsequently 

with any blank coupons. 

■ No visible damage was observed on any of the test 

materials after the 10 minute contact time with 

DioxiGuardTM in the quantitative efficacy testing, or 

seven days later after completion of the qualitative 

assessment of residual spores. 

12.2 pH-Amended Bleach Results 

■		 The quantitative efficacy of pH-amended bleach was 

highest for the painted cinder block (7.31 log reduction 

and ≥ 7.22 log reduction for B. anthracis ames and 

B. subtilis, respectively), and relatively high for 

unpainted concrete (4.99 and ≥ 5.63 log reduction, 

respectively), but was low for topsoil (1.47 and 0.18 log 

reduction) and bare pine wood (0.81 and 0.68 log 

reduction). 

12.0 

Performance Summary
 

■ A significant difference between efficacy values for 

B. subtilis and B. anthracis was found only with topsoil 

as the test surface, with the efficacy for B. subtilis 

(0.18 log reduction) significantly lower than that for 

B. anthracis (1.47 log reduction). 

■		 In the qualitative tests most material types showed the 

presence of viable organisms after decontamination. 

However, no viable organisms of either B. anthracis or 

B. subtilis were found on painted cinder block, and none 

of B. subtilis were found on unpainted concrete. These 

results are consistent with the quantitative efficacy 

results for this decontaminant. Morphological analysis 

confirmed that the growth observed indicated only 

B. anthracis ames or B. subtilis, respectively, from the 

spiked coupons. 

■ In the qualitative tests growth was also observed with 

the laboratory and procedural blanks for topsoil with 

both B. anthracis and B. subtilis, and with the laboratory 

blanks for bare pine wood and painted cinder block with 

B. subtilis, although no CFu were found on these blanks 

in the quantitative efficacy tests. This growth is likely 

to have resulted from slight contamination of the blank 

coupons in the test chamber during the overnight drying 

of the spore-inoculated test coupons. 

■ No visible damage was observed on any of the test 

materials after the 60 minute contact time with phamended bleach in the quantitative efficacy testing, 

or seven days later after completion of the qualitative 

assessment of residual spores. 

12.3 Calcium Polysulfide Results 

■		 The quantitative efficacy of the 5.8% by weight 

CaSx solution was very low, achieving maximum log 

reductions of only 0.24 for B. anthracis ames and 0.33 

for B. subtilis. 

■		 A significant difference between efficacy values for 

B. subtilis and B. anthracis was found only with glass 

as the test surface, with the efficacy for B. subtilis 

(0.33 log reduction) significantly higher than that for 

B. anthracis (-0.04 log reduction). 

■		 In the qualitative tests coupons of all material types 

showed the presence of viable organisms after 

decontamination, consistent with the quantitative 

efficacy results. Morphological analysis confirmed that 

the growth observed indicated only B. anthracis ames 

or B. subtilis, respectively, from the spiked coupons. 

63 
■ A grayish residue was observed on glass and topsoil 

coupons after decontamination. That residue was not 

removed from the glass by the agitation used for spore 

extraction, or by the culturing process used in the 

seven-day qualitative test for viable spores. The surface 

characteristics of bare wood and unpainted concrete 

coupons made it impossible to discern whether a similar 

residue was also present on those materials. 

12.4 CASCADTM SDF Results 

■		 The quantitative efficacy of CASCADTM SDF exceeded 

7.0 log reduction for both B. anthracis and B. subtilis 

on five of the seven test materials. Lower efficacy 

values were found only on painted wallboard paper and 

bare pine wood. Efficacy results for B. anthracis and 

B. subtilis on painted wallboard paper were 4.82 and ≥ 

6.14 log reduction, respectively; on bare pine wood the 

corresponding efficacy results were 2.77 and 1.28 log 

reduction, respectively. 

■ Significant differences between efficacy values for 

B. subtilis and B. anthracis were found only with 

painted cinder block and bare pine wood as the test 

surfaces. With painted cinder block, no viable spores 

of either organism were found after decontamination 

(i.e., the efficacy values were both reported as 

“≥” values). Thus the difference in efficacy values 

on that material is due to different efficiencies of 

recovery of the two spore types, and cannot be 

attributed to an actual difference in the efficacy of 

CASCADTM SDF. On bare pine wood, the efficacy 

for B. anthracis was significantly higher than that 

for B. subtilis (2.77 vs. 1.28 log reduction). 

■ In the qualitative tests, only painted wallboard paper 

and bare pine wood showed the presence of viable 

organisms after decontamination, consistent with the 

quantitative efficacy results. Morphological analysis 

confirmed that the growth observed indicated only 

B. anthracis ames or B. subtilis, respectively, from 

the spiked coupons. 

■		 The only materials damage observed from 

decontamination with CASCADTM SDF was that the 

top coat of paint peeled away from the primer coat on 

painted cinder block coupons. 

12.5 Oxonia Active® Results 

■		 The quantitative efficacy of Oxonia Active® was 7.0 log 

reduction or greater on six of the seven test materials for 

B. anthracis and on five of the seven test materials for 

B. subtilis. Lower efficacy values were found only on 

bare pine wood and painted wallboard paper. Efficacy 

results for B. anthracis and B. subtilis on bare pine 

wood were 4.64 and 5.15 log reduction, respectively; on 

painted wallboard paper the efficacy for B. subtilis was 

≥ 6.69 log reduction. 

■		 Significant differences between efficacy values 

for B. subtilis and B. anthracis were found on four 

materials. However, no viable spores of either organism 

were found on coupons of any of these materials 

after decontamination (i.e., the efficacy values were 

all reported as “≥ ” values). Thus the differences in 

reported efficacy values are due to differing efficiencies 

of recovery of the two spore types from these materials, 

and cannot be attributed to actual differences in the 

efficacy of Oxonia Active®. 

■ In the qualitative tests, no viable spores were found 

on any decontaminated coupon after either one or 

seven days incubation, consistent with the quantitative 

efficacy results. 

■		 No visible damage was observed on any of the test 

materials after 60 minutes contact time with Oxonia 

Active®, or seven days later after completion of the 

qualitative assessment of residual spores.
	

12.6 Minncare® Cold Sterilant Results 

■		 The quantitative efficacy of Minncare® Cold Sterilant 

was 7.5 log reduction or greater on six of the seven test 

materials for both B. anthracis and B. subtilis. lower 

efficacy values were found only on bare pine wood, for 

which efficacy results for B. anthracis and B. subtilis 

were 5.40 and 6.00 log reduction, respectively. 

■		 Significant differences between efficacy values 

for B. subtilis and B. anthracis were found on four 

materials. However, no viable spores of either organism 

were found on coupons of any of these materials after 

decontamination (i.e., the efficacy values were all 

reported as “≥” values). Thus the differences in reported 

efficacy values are likely due to differing efficiencies of 

recovery of the two spore types from these materials, 

and cannot be attributed to actual differences in the 

efficacy of Minncare® Cold Sterilant. 

■ In the qualitative tests, no viable spores were found 

on any decontaminated coupon after either one or 

seven day’s incubation, consistent with the quantitative 

efficacy results. 

■ No visible damage was observed on any of the test 

materials after either 10 or 30 minutes contact time 

with Minncare® Cold Sterilant, or seven days later 

after completion of the qualitative assessment of 

residual spores. 

12.7 SanDes Results 

■		 The quantitative efficacy of SanDes was less than 

1.0 log reduction for six of the seven test materials for 

both B. anthracis and B. subtilis. Higher efficacy values 

were found only on glass for B. anthracis (4.65 log 

reduction) and on decorative laminate for B. subtilis 

(1.37 log reduction). 

64 
■		 Significant differences between efficacy values 

for B. subtilis and B. anthracis were found on four 

materials. In two such cases, both efficacy results were 

less than 1.0 log reduction. The largest differences were 

for glass (4.65 log reduction with B. anthracis and 

0.22 log reduction with B. subtilis), and for decorative 

laminate (0.18 log reduction with B. anthracis and 

1.37 log reduction with B. subtilis). As noted above 

these respective materials exhibited the highest efficacy 

results for each organism. 

■		 In the qualitative tests, viable spores were found on all 

of the decontaminated coupons after one day and after 

seven days of incubation, consistent with the relatively 

low quantitative efficacy results. 

■		 No visible damage was observed on any of the test 

materials after 70 minutes contact time with SanDes, 

or seven days later after completion of the qualitative 

assessment of residual spores. 

65 
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66 
13.0 

References
 

1.	 Technology Testing and Evaluation Program Test/QA Plan for Evaluating Liquid and Foam Technologies for the 

Decontamination of B. anthracis Spores on Building and Outdoor Materials, version 1, Battelle, Columbus, ohio, 

December 2007. 

2.	 Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP), version 2, Battelle, 

Columbus, ohio, 2006. 

3.	 Battelle, FSP Annex 5 to Appendix B, Guidelines for Safe Handling and Storage of Etiologic Agents at the MREF: West 

Jefferson, ohio. 

4.	 Battelle, Battelle MREF Facility Safety Plan (FSP) Annex 6 to Appendix B of the FSP, Guidelines for the Control of 

Etiologic Agents in the MREF Biofacility: West Jefferson, ohio. 

5.	 Battelle, FSP Annex 7 to Appendix B, Guidelines for Disinfection/Decontamination of Etiological Agents at the MREF 

Biofacilities: West Jefferson, ohio. 

6.		Rogers, J.V., et al., “Decontamination Assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus

stearothermophilus Spores on Indoor Surfaces Using a Hydrogen Peroxide Gas Generator”, J. Appl. Microbiol., 2005, 

99: p. 739–748. 

7.	 Battelle, Battelle MREF Facility Safety Plan Annex 12 to Appendix B, Guidelines for the Use of Class II and Class III 

Biological Safety Cabinets in the MREF Biofacility: West Jefferson, ohio. 

8.	 Battelle, Facility Safety Plan (FSP) for the Medical Research and Evaluation Facility (MREF) Biofacility: West Jefferson, 

ohio. 

9. Rogers, J.V., Sabourin, C.L., Taylor, M.L., Riggs , K., Choi, Y.W., Richter, W.R., Rudnicki, D.C., BIOQUELL, Inc. 

Clarus CTM Hydrogen Peroxide Gas Generator. U.S. EPA Environmental Technology Verification Program, ETV Building 

Decontamination Technology Center, March 2004. (http://www.epa.gov/NHSRC/tte_fumdecontech.html). 

10. Rogers, J.V., Sabourin, C.L., Taylor, M.L., Riggs, K., Choi, Y.W., Joseph, D.W., Richter, W.R., Rudnicki, D.C., CERTEK® 

Inc. 1414RH Formaldehyde Generator/Neutralizer. U.S. EPA Environmental Technology Verification Program, ETV 

Building Decontamination Technology Center, August 2004. (http://www.epa.gov/NHSRC/tte_fumdecontech.html). 

11. Rogers, J.V., Sabourin, C.L., Taylor, M.L., Riggs, K., Choi, Y.W., Richter ,W.R., Rudnicki, D.C., Stone, H.J., CDG Research 

Corp. Bench-Scale Chlorine Dioxide Gas:Solid Generator. U.S. EPA Environmental Technology Verification Program, ETV 

Building Decontamination Technology Center, September 2004. (http://www.epa.gov/NHSRC/tte_fumdecontech.html). 

12. Rogers, J.V., Richter, W.R., Choi, Y.W., Waugh, J.D., Taylor, M.L., Riggs, K.B., Stone, H.J., Willenberg, Z.J., Krile, 

R.T., Wood, J.P., Evaluation of Sporicidal Decontamination Technology: Sabre Technical Services Chlorine Dioxide Gas 

Generator. U.S. EPA Technology Testing and Evaluation Program Report, EPA/600/R-06/048, April 2006, (http://www.epa. 

gov/NHSRC/tte_fumdecontech.html). 

13. Rogers, J.v., Richter, W.R, Choi, y.W., Fleming, E.J., Shesky, a.M., Cui, J., Taylor, M.l., Riggs, K.B., Willenberg, Z.J., 

Stone, H.J., Wood, J.P.. Evaluation of Spray-Applied Sporicidal Decontamination Technologies. U.S. EPA Technology 

Testing and Evaluation Program Report, EPA/600/R-06/146, September 2006, (http://www.epa.gov/NHSRC/tte_ 

liquiddecontech.html). 

14. Standard Methods for the Examination of Water and Wastewater, 21st Edition, published jointly by the American Public 

Health Association (APHA), the American Water Works Association, and the Water Environment Federation, ISBN 

0875530478, APHA, Washington, D.C., September, 2005. 

15. Chlorine and Chlorine Dioxide in Workplace Atmospheres, Method ID-126SGX, Occupational Safety and Health 

Administration, Methods Development Team, Industrial Hygiene Chemistry Division, Salt Lake City, Utah, 2007. 

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68 
Appendix A 

DioxiguardTM Description
 

and Application Procedure
 

General Description 

DioxiGuardTM is a chlorine dioxide (ClO2) disinfectant 

solution. Clo2 is released when sodium chlorite (NaClO2) 

solution is mixed with an acid, in this case producing 

a concentration of about 190 ppm Clo2 in the resulting 

mixture.
	

DioxiGuardTM is supplied commercially in a dual chambered 

plastic spray bottle, with each chamber containing one of the 

two reagent solutions. Sodium chlorite solution is combined 

with acid solution at the time of use by squeezing the trigger 

of the spray bottle. The bottle holds 22 oz (650 mL) (total) 

and is easily hand held. The two solutions are mixed and 

dispensed in one operation, with Clo2 immediately produced 

in the mixed solution. The separate ingredients in the twin 

bottle are designed to be stable for two years or more. 

DioxiGuardTM is designed to suppress both ClO2 odor in 

the spray and the corrosion normally expected with ClO2 

preparations. The toxicity of DioxiGuardTM to humans is 

minimal, and many toxicity reports show the disinfectant 

liquid can be safely sprayed on the body. As is well known, 

Clo2 may bleach clothing and carpets, but not as readily as 

chlorine bleach. 

The DioxiGuardTM system of microorganism deactivation 

does not depend on Clo2 alone. Chlorous acid (HClO2) 

has a much higher oxidation potential than ClO2, and 

the product design attempts to maximize concentrations 

of this transitory molecule. This maximization can be 

done by adjusting the ph; if the ph is too high, too 

little hClo2 is formed, if the ph is too low, too much 

is formed at once. a high concentration of hClo2 

immediately disappears and disproportionates to Clo2:

 h+

 + NaClO2 → HClO2 + Na+ 

5hClo2 → 4ClO2 + HCl + 2H2o 

In addition, when hClo2 disproportionates as in the 

second equation above, several free radicals are formed 

instantaneously in the process. These radicals are shortlived, highly reactive, and strong oxidants. Accordingly, the 

production of Clo2 by way of an acid, rather than production 

directly from the chlorite, offers the advantage of these 

transient oxidizing species. On the other hand, forming ClO2 

and letting it remain in solution for a time before use will 

reduce the system’s oxidizing capability. In DioxiGuardTM, 

the chlorite is activated by organic acids and the solution also 

contains alcohol. 

Application Procedure for Testing 

For evaluation of DioxiGuardTM’s efficacy on diverse 

test surfaces in this evaluation, the following application 

procedure was used: 

•		 Spray the test coupons with DioxiGuardTM from a 

distance of about one foot (12 inches) using the vendorsupplied dual spray bottle. 

•		 Squeeze the trigger on the dual spray bottle repeatedly 

over a period of 10 seconds, or until the coupon surface 

is fully saturated with solution. 

•		 Let the test coupons remain in place, with the 

DioxiGuardTM solution on each coupon, for 10 minutes. 

No reapplication of DioxiGuardTM is needed. 

at the conclusion of the 10 minute contact time, each test 

coupon was placed into a separate aliquot of extraction 

solution along with any captured runoff of DioxiGuardTM 

from that coupon, and the DioxiGuardTM was neutralized 

with sodium thiosulfate (STS). The use of STS to neutralize 

DioxiGuardTM was established in a previous test program; the 

STS concentration used in this evaluation was based upon the 

concentration used previously. 

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70 
Appendix B 

pH-Amended Bleach Description 

and Application Procedure 

General Description 

ph-amended bleach consists of diluted normal household 

bleach (e.g., Clorox®) with its pH adjusted by addition of 

a small amount of acetic acid. Specifically, pH-amended 

bleach contains a total of about 5 to 6% by weight of 

sodium hypochlorite (NaOCl) in aqueous solution, with pH 

adjustment achieved by addition of a small amount of 5% 

acetic acid. The recipe for preparation of ph-amended bleach 

for use as a decontaminant is as follows: 

•		 Prepare 5% acetic acid solution by diluting 50 mL of 

glacial acetic acid up to 1 L with SFW in a volumetric 

flask. 

•		 Mix 9.4 parts SFW, 1 part commercial household 

bleach, and 1 part 5% acetic acid. The resulting solution 

will have a mean pH of about 6.8 and a mean total 

chlorine content of about 6,200 ppm. 

The active decontaminating agents in this solution are 

hypochlorite (OCl-) and hypochlorous acid. The effectiveness 

of this reagent as a biological decontaminant is widely 

known and well demonstrated through the common use 

of bleach as a sterilant and decontaminant. In testing of 

pH-amended bleach as a decontaminant under a previous 

TTEP Task order,(1) neutralization of the bleach solution 

was achieved using sodium thiosulfate (STS). Based on 

the chemical composition of the ph-amended bleach, the 

amount of that solution (0.325 mL) retained or run off from 

a test coupon with a specified 10-second application period, 

and the use of 10 mL of an extraction solution containing 

phosphate-buffered saline (PBS) + 0.1% Triton X-100, an 

STS concentration of 0.086% in the extraction solution was 

determined to be optimal for neutralizing the ph-amended 

bleach. The application equipment and procedures used in 

this evaluation differ from those used in previous testing,(1) 

so the determination of the neutralization procedure 

was repeated to establish conditions appropriate for this 

evaluation. 

Application Procedure for Testing 

Based on information available from previous use of pHamended bleach,(1) an application procedure for use in 

testing has been developed. The intent of this procedure is 

to employ conventional and readily available equipment 

in a relatively simple application process. Trial runs were 

conducted to establish the appropriate concentration of STS 

for neutralization of the ph-amended bleach. 

The test coupon materials used with ph-amended bleach 

were soil, bare wood, bare concrete, and painted cinder 

block. Good decontaminant efficacy has been demonstrated 

previously with pH-amended bleach on glass,(1) so that 

surface was replaced by painted cinder block as an outdoor 

surface in this test. 

The ph-amended bleach was prepared fresh shortly before 

use on each day of testing, as described above. The pH of the 

solution was measured and recorded as part of the test data. 

a non-corroding garden pump sprayer was used to apply the 

solution of ph-amended bleach to the test coupon surfaces. 

An identical sprayer was used to apply SFW to positive 

control test coupons. Each sprayer was fitted with a pressure 

gauge to indicate the internal delivery pressure of the sprayer. 

The internal pressure of each sprayer was maintained 

in a normal range for use (i.e., 4 to 6 psi) throughout all 

applications. Based on laboratory tests, such a range of 

pressures produces a stable spray suitable for application 

on the scale of coupon testing. The step-by-step application 

procedure was: 

•		 Apply the pH-amended bleach solution to the test 

coupons (or SFW to the positive control coupons) from 

a distance of about 30 cm (one foot or 12 inches) using 

the sprayer at a delivery pressure within the specified 

range, until the test coupon surfaces are fully wetted by 

the solution. 

•		 Reapply the solution if test coupon surfaces become dry, 

but no more frequently than at ten minute intervals. 

•		 If necessary, pump up the pressure in the sprayer 

before application to maintain pressure within the 

specified range. 

When 60 minutes had elapsed since the start of the first 

application, the coupons were placed into the extraction 

solution (containing the neutralization agent) along with any 

collected runoff of ph-amended bleach. 

Reference: 

1.	 Rogers, J.v., Richter, W.R, Choi, y.W., Fleming, E.J., 

Shesky, a.M., Cui, J., Taylor, M.l., Riggs, K.B., 

Willenberg, Z.J., Stone, H.J., Wood, J.P.. Evaluation of 

Spray-Applied Sporicidal Decontamination Technologies. 

U.S. EPA Technology Testing and Evaluation Program 

Report, EPa/600/R-06/146, September 2006. 

(http://www.epa.gov/NHSRC/tte_liquiddecontech.html). 

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72 
Appendix C 

Calcium Polysulfide Description 

and Application Procedure 

General Description 

Calcium polysulfide (CaSx), also known as “Lime Sulfur,” 

has been in use as an agricultural fungicide and insecticide 

since the early 1900s. Calcium polysulfide is also used in 

veterinary medicine as an effective treatment for various 

pet and livestock infections. Calcium polysulfide can be 

purchased from many manufacturers and vendors, with the 

typical concentration of 29% by weight in water. Calcium 

polysulfide is a yellow to orange aqueous solution with a 

density of 1.28 g/ml. 

Diluted calcium polysulfide solutions ranging from 

0.7 to 2.9% are commonly applied to treat fungus and 

insect infestation of agricultural crops, by spraying with 

non-corroding crop and garden sprayers to the point of 

solution runoff. 

Application Procedure for Testing 

Based on the information available on calcium polysulfide, an 

application procedure for use in testing has been developed. 

The intent of this procedure is to use a concentration of 

calcium polysulfide that is likely to be effective when 

applied with conventional and readily available equipment 

in a relatively simple application process. Trial runs were 

conducted to establish the appropriate concentration of 

D-E Neutralizing Agar for neutralization of the calcium 

polysulfide. Test surfaces used were glass, soil, bare wood, 

and bare concrete.
 

For testing, a concentration of 5.8% by weight calcium 

polysulfide (i.e., a 1:5 dilution with water of the commercial 

29% product) was used. The specific product used is AquaClear®, manufactured by VGS, St. Joseph, Missouri (www. 

calciumpolysulfide.com; site currently under construction). 

a non-corroding garden pump sprayer was used to apply the 

solution of calcium polysulfide to the test coupon surfaces. 

An identical sprayer was used to apply SFW to positive 

control test coupons. Each sprayer was fitted with a pressure 

gauge to indicate the internal delivery pressure of the sprayer. 

The internal pressure of each sprayer was maintained in 

a normal range for use (i.e., 4 to 6 psi) in all applications. 

Based on laboratory tests, such a range of pressures produces 

a stable spray, suitable for application on the scale of coupon 

testing. The step-by-step application procedure was: 

•		 Apply the calcium polysulfide solution to the test 

coupons (or SFW to the positive control coupons) 

from a distance of about one foot (12 inches) using 

the sprayer at a delivery pressure within the specified 

range. Spray the solution onto the coupons until the test 

coupons are fully wetted, and with no less than a fivesecond spray duration on any surface. 

•		 Reapply the solution if test coupon surfaces become dry, 

but no more frequently than at ten minute intervals. 

•		 Regardless of the wetness of the coupons, reapply the 

calcium polysulfide solution to all coupons 30 minutes 

after the initial application, again with at least a fivesecond spray duration on each surface. 

•		 If necessary, pump up the pressure in the sprayer 

before application to maintain pressure within the 

specified range. 

•		 When 60 minutes have elapsed since the start of the 

first application, place the coupons into the extraction 

solution (containing the neutralization agent) along with 

any collected runoff of calcium polysulfide solution. 

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74 
 

 

 

Appendix D 

CASCADTM SDF Description
 

and Application Procedure
 

General Description 

CASCADTM Surface Decontamination Foam (SDF) uses two 

liquid solutions (A and B) which react to form a foam as they 

are mixed upon release from the application device. These 

two solutions are made from three separate reagents, having 

chemical composition as follows: 

• GPA-2100 (decontaminant) – solid reagent in powder 

form consisting of dichloroisocyanuric acid sodium salt, 

70 to 100% by weight; 

•		 GPB-2100 (buffer) – solid reagent in powder form 

consisting of sodium tetraborate 10 to 30%, sodium 

hydroxide 1 to 5 %, and sodium carbonate 40 to 65% 

by weight; 

•		 GCE-2000 (surfactant) – liquid reagent consisting of 

sodium myristyl sulfate 10 to 30%, sodium (C14-16) 

olefin sulfonate 10 to 30%, ethanol denatured 3 to 9%, 

alcohols (C10-16) 5-10%, sodium sulfate 3 to 7%, 

sodium xylene sulfonate 1 to 5%, and a proprietary 

mixture of sodium and ammonium salts along with 

water and co-solvent >9% by weight. 

The a and B solutions are prepared from these reagents by 

the following procedure: 

1. Make solution A by adding 31.2 grams (four 7.8 gram 

packets) of GPA-2100 to 250 mL of SFW in a graduated 

cylinder, and then dilute with water to 300 ml. 

2. Mix with a micro stir bar until dissolved 

3. Make solution B by adding 7.2 grams (four 1.8 gram 

packets) of GPB-2100 to 250 mL of SFW in a graduated 

cylinder. 

4. Mix with a micro stir bar until dissolved. 

5. Add 18 mL (four 4.5 mL packets) of GCE-2000 to the 

solution from step 4, mix, and then dilute with SFW to a 

final volume of 300 mL 

For use on the small scale needed for testing, a manual spray 

application bottle developed by Allen-Vanguard (the 600 mL 

Hand Held Decontamination System) draws solutions A and 

B from separate compartments and delivers them as a foam 

through a single spray head. To fill and operate the Hand 

Held Decontamination System, follow these steps: 

1. Pull the Locking Lever on the front of the bottle housing 

forward and lift to open the housing and expose the 

solution bottles, which are labeled “A” and “B”. 

2. With the housing opened remove the caps (turn counter 

clockwise) and pull out the solution suction lines from 

the solution bottles. 

3. With the caps and suction lines removed from both the 

“A” and “B” solution bottles: 

a. Pour solution A into the bottle labeled “A”, and 

pour solution B into the bottle labeled “B”. 

b. assure that both bottles are seated in the housing 

with the “B” bottle at the front. 

c.		Place the suction lines back into the “A” and “B” 

bottles and tighten both the “A” and “B” caps by 

turning them in a clockwise direction. 

4. hold the suction line up with one hand while closing 

the top of the housing with the other hand. Make certain 

that the Locking Lever snaps into its recess when the 

housing top closes. The suction line may be pinched 

closed if this procedure is not followed correctly. Check 

for closure of the line by looking through the housing 

and checking the suction line. 

5. To use the 600 mL Hand Held Decontamination System, 

grasp the neck of the housing with your dominant hand 

and place the finger of this hand on the trigger of the 

foam nozzle. aim the tip of the foam nozzle in the 

direction of the area to be decontaminated and pump the 

trigger. The trigger may have to be squeezed three or 

four times to evacuate the air in the suction line before 

foam is discharged. 

Application Procedure for Testing 

CASCADTM SDF was applied to test coupons using the 

vendor-developed dual spray applicator. In previous testing,(1) 

neutralization of the CASCADTM SDF was achieved by 

addition of 0.5% sodium thiosulfate (STS) to the extraction 

solution. Trial runs were conducted before testing to establish 

the appropriate STS concentration for neutralization of the 

applied CASCADTM SDF. 

The step-by-step application procedure for testing was: 

• Follow the instructions provided above for preparation 

of the reagent solutions and loading of the manual spray 

applicator. 

• Squeeze the trigger of the applicator head a few times 

while pointing the applicator into a laboratory sink or 

other waste container, until any air is cleared from the 

applicator and CASCADTM SDF is delivered from the 

applicator as a foam. 

• Apply the CASCADTM SDF to the test coupons using 

the manual applicator from a distance of about 30 cm 

(12 inches) while moving the nozzle, until the test 

coupons are entirely covered with no less than one 

(1) centimeter (3/8") deep foam. 

75 
•		 Allow the foam to remain on the coupons for 30 

minutes. Do not reapply.
	

•		 When 30 minutes have elapsed since the application, 

place each coupon into the extraction solution 

(containing the STS neutralization agent) along with 

any associated collected runoff of CASCADTM SDF. 

•		 Empty and clean the manual spray applicator after use 

according to the instructions below. 

Cleaning the Hand Held Decontamination System 

Clean the CASCADTM SDF system after use by the following 

procedure. 

1. Dump any remaining decontamination solution from 

both the “A” and “B” bottles and dispose of the 

solutions following appropriate waste procedures. 

2. Thoroughly rinse both bottles with SFW, then fill each 

bottle with SFW. 

3. Place the filled bottles back into the housing, insert the 

suction lines, and close the housing. 

4. Pump the trigger until the suction lines and foam nozzle 

are free from the decontamination solution. 

5. Flush the interior and the exterior of the housing, and 

the caps used while mixing the solution, thoroughly 

with SFW. 

Reference: 

1.	 Rogers, J.v., Richter, W.R, Choi, y.W., Fleming, E.J., 

Shesky, a.M., Cui, J., Taylor, M.l., Riggs, K.B., 

Willenberg, Z.J., Stone, H.J., Wood, J.P.. Evaluation of 

Spray-Applied Sporicidal Decontamination Technologies. 

U.S. EPA Technology Testing and Evaluation Program 

Report, EPa/600/R-06/146, September 2006. 

(http://www.epa.gov/NHSRC/tte_liquiddecontech.html). 

76 
Appendix E 

Oxonia Active® Description 

and Application Procedure 

General Description 

Oxonia Active® is a liquid sanitizer made by Ecolab Inc., 

that consists of 27.5 % hydrogen peroxide (H2o2) and 

5.8% peroxyacetic acid (CH3CO(O2)H) by weight in 

water (density = 1.13 g/mL). According to the vendor, 

Oxonia Active is used for sterilizing a variety of surfaces 

and containers in food, packaging, and other industries, 

and can be applied as a liquid or foam, or as droplets 

by fogging the target area. A temporary approval (crisis 

exemption) of Oxonia Active® was granted by the u.S. 

Environmental Protection Agency for decontamination of 

Bacillus anthracis spores on non-porous surfaces, at defined 

temperatures, contact times, and dilution of the product. 

Application Procedure for Testing 

An application procedure for use of Oxonia Active® 

in testing has been developed, based on information 

provided by the vendor. The aim is to use a relatively 

simple application process that is likely to be effective 

when carried out with conventional and readily available 

equipment. Trial runs were conducted to establish the 

appropriate concentration of sodium thiosulfate (STS) 

for neutralization of Oxonia Active®. Test surfaces used 

include glass, decorative laminate, industrial-grade 

carpet, galvanized metal ductwork, painted wallboard 

paper, painted cinder block, and bare pine wood. 

For testing, a decontaminant solution containing 5,000 ppm 

peroxyacetic acid was prepared fresh daily by diluting 76 mL 

of Oxonia Active® to 1 L with SFW. The Ecolab High Oxonia 

Active® Test Kit 322 was used for periodic verification 

of the peroxyacetic acid concentration in the undiluted 

Oxonia Active® from which the decontaminant solution was 

prepared. 

a non-corroding garden pump sprayer was used to apply the 

diluted Oxonia Active® solution to the test coupon surfaces. 

An identical sprayer was used to apply SFW to positive 

control test coupons. Each sprayer was fitted with a pressure 

gauge to indicate the internal delivery pressure of the sprayer, 

which was maintained in a normal range for use (i.e., 4 to 

6 psi) in all applications. Based on laboratory tests, such 

a range of pressures produces a stable spray, suitable for 

application on the scale of coupon testing. all applications 

were done at normal room temperature (approximately 20 °C 

(68 °F)). 

The step-by-step application procedure for 

Oxonia Active® was: 

•		 Apply the decontaminant solution to the test coupons 

(or SFW to the positive control coupons) from a 

distance of about one foot (12 inches) using the sprayer 

at a delivery pressure within the specified range. Spray 

the solution onto the coupons until the test coupons are 

visibly wet and excess liquid drips from the coupons. 

•		 Reapply the decontaminant solution if coupon surfaces 

become visibly dry, and regardless of the wetness of 

the coupons reapply the decontaminant solution every 

10 minutes. 

•		 If necessary, pump up the pressure in the sprayer 

before application to maintain pressure within the 

specified range. 

•		 When 60 minutes have elapsed since the start of the 

first application, place the coupons into the extraction 

solution (containing the neutralization agent) along with 

any collected runoff of decontaminant solution. 

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78 
Appendix F 

Minncare® Cold Sterilant Description 

and Application Procedure 

General Description 

Minncare® Cold Sterilant is a liquid decontaminant consisting 

of 22.0% by weight hydrogen peroxide (H2o2) and 4.5% by 

weight peroxyacetic acid (CH3C(O)O2H) in aqueous solution. 

Minncare® Cold Sterilant is a clear liquid with a density of 

1.1 g/ml and a ph of 0.5 to 1.1. Minncare® Cold Sterilant is 

thus both an oxidizing agent and a strongly acid solution. 

at the direction of Minntech Corp., a solution of peptone, 

sodium thiosulfate (STS), and potassium dihydrogen 

phosphate (KH2Po4) with a small amount of catalase was 

used as the neutralizing agent for Minncare® Cold Sterilant. 

Trial runs were conducted to establish the appropriate 

chemical quantities and procedures for neutralization in this 

evaluation. 

Preparation of Minncare® Cold Sterilant: a 10% solution of 

Minncare® Cold Sterilant was prepared fresh shortly before 

use on each day of testing, by diluting 1 part of the Cold 

Sterilant with 9 parts of SFW. 

Preparation of Neutralization Solution: The neutralization 

solution was prepared fresh shortly before use on each day 

of testing, by diluting 10 g of peptone, 1 g of STS, and 14 g 

Kh2Po4 to 1 l in SFW, and adjusting the ph to 7 ± 0.5. That 

solution was then autoclaved for 20 minutes at 121 °C, and 

then allowed to cool to room temperature. Catalase (Sigma 

C-9322) was filter sterilized and added to the cooled solution 

just before neutralization to achieve a catalase concentration 

of 0.005%. 

Application Procedure for Testing 

Based on the vendor’s instructions, an application procedure 

for use of Minncare® Cold Sterilant in testing was developed. 

The intent of this procedure was to employ conventional 

and readily available equipment in a relatively simple 

application process. The test coupon materials used in testing 

of Minncare® Cold Sterilant included decorative laminate, 

galvanized metal ductwork, painted wallboard paper, glass, 

industrial-grade carpet, painted cinder block, and bare pine 

wood. 

The 10% Cold Sterilant solution was applied to test coupons 

using a hand-held plastic spray bottle. a similar bottle was 

used to apply SFW to positive control test coupons. The stepby-step application procedure was as follows: 

• Apply the Minncare® Cold Sterilant 10% solution to the 

test coupons (or SFW to the positive control coupons) 

from a distance of about 30 cm (one foot or 12 inches) 

using the handheld spray bottle, until the test coupon 

surfaces are fully wetted by the solution. 

•		 No reapplication of the Cold Sterilant solution is 

required.
	

•		 Allow the Cold Sterilant solution to remain in contact 

with the test coupon surfaces for the following contact 

times, which differ for different coupon materials: 

Decorative laminate 10 minutes
	

Galvanized metal ductwork 10 minutes
	

Painted wallboard paper 10 minutes 

glass 10 minutes 

Industrial-grade carpet 30 minutes 

Painted cinder block 30 minutes 

Bare pine wood 30 minutes

 • When the allotted contact time has elapsed since the 

application of the Cold Sterilant solution, place the 

coupons into the extraction solution (containing the 

pre-determined amount of neutralization solution) along 

with any collected runoff of the Cold Sterilant solution. 

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80 
Appendix G 

SanDes Description 

and Application Procedure 

General Description 

SanDes, a liquid decontaminant made by DTI-Sweden AB, 

consists of 1,500 ppm chlorine dioxide (ClO2) in aqueous 

solution. SanDes is a light yellow solution with a density of 

1.0 g/mL and a pH of less than 1.5. SanDes is thus both an 

oxidizer and a relatively strong acid solution. At the vendor’s 

direction SanDes was used without dilution for application to 

test coupons. This application procedure is based on technical 

information provided by the vendor; that information was not 

verified as part of the test program. 

Based on previous experience with ClO2 decontaminants,(1) 

sodium thiosulfate (STS) was used as the neutralizing 

agent for SanDes. Trial runs were conducted to establish 

the appropriate chemical quantities and procedures for 

neutralization in this evaluation. 

Application Procedure for Testing 

Based on the vendor’s instructions, an application procedure 

for use of SanDes in testing was developed. The intent of this 

procedure was to employ conventional and readily available 

equipment in a relatively simple application process. The 

test coupon materials used in testing of SanDes included 

decorative laminate, galvanized metal ductwork, painted 

wallboard paper, glass, industrial-grade carpet, painted cinder 

block, and bare pine wood. 

The undiluted SanDes solution was applied to test coupons 

using a push-button spray nozzle that replaced the cap on a 

30 mL bottle of SanDes. An identical spray nozzle was used 

to apply SFW to positive control test coupons. The step-bystep application procedure was as follows: 

•		 Apply SanDes to the test coupons (or SFW to the 

positive control coupons) from a distance of about 

30 cm (one foot or 12 inches) using the spray nozzle, 

until the test coupon surfaces are fully wetted by the 

solution. 

•		 Reapply SanDes at 10-minute intervals after the original 

application, or more often if surfaces become dry, until 

three reapplications have been made. 

• Make a final application of the SanDes at 60 minutes 

after the original application. 

•		 Allow the final application of SanDes to remain 

in contact with the test coupon surfaces for 

10 minutes, resulting in a total contact time of 

70 minutes since the original application. 

•		 When the 70 minutes total contact time has 

elapsed, place the coupons into the extraction 

solution (containing the pre-determined amount 

of neutralization solution) along with any 

collected runoff of the SanDes solution.
	

Reference: 

1.	 Rogers, J.v., Richter, W.R, Choi, y.W., Fleming, E.J., 

Shesky, a.M., Cui, J., Taylor, M.l., Riggs, K.B., 

Willenberg, Z.J., Stone, H.J., Wood, J.P.. Evaluation of 

Spray-Applied Sporicidal Decontamination Technologies. 

U.S. EPA Technology Testing and Evaluation Program 

Report, EPa/600/R-06/146, September 2006. 

(http://www.epa.gov/NHSRC/tte_liquiddecontech.html). 

81 
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