EPA 600/R-14/332 I October 2014 I www.epa.gov/research
United States
Environmental Protection
Agency
                Parametric Testing of
                Decontamination Chemistries to
                Guide Decontaminant Selection I:
                Peracetic Acid
                                                 
                                   *
Office of Research and Development
National Homeland Security Research Center

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                                                       EPA/600/R-14/332
                                                          October 2014
Parametric Testing of Decontamination Chemistries to Guide
          Decontaminant Selection I: Peracetic Acid
              National Homeland Security Research Center
                 Office of Research and Development
                 U.S. Environmental Protection Agency
                  Research Triangle Park, NC 27711

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Disclaimer

The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and directed this investigation through EP-C-09-
027 WAs 4-76 and 5-76 with ARCADIS U.S., Inc. This report has been peer and administratively
reviewed and has been approved for publication as an Environmental Protection Agency document. It
does not necessarily reflect the views of the Environmental Protection Agency. No official endorsement
should be inferred. This report includes photographs of commercially available products. The
photographs are included for purposes of illustration only and are not intended to imply that EPA
approves or endorses the product or its manufacturer. Environmental Protection Agency does not
endorse the purchase or sale of any commercial products or services.

Questions concerning this document or its application should be addressed to:

Sang Don Lee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (E-311-K)
Office of Research and Development
109. T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-2973
Fax:919-541-4531
E-mail:lee.sangdon@epa.gov

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Acknowledgments

Contributions of the following individuals and organization to this report are gratefully acknowledged:


EPA Project Team:

Lukas Oudejans, EPA Alternate Work Assignment Manager, EPA National Homeland Security Research
Center, Decontamination and Consequence Management Division, Research Triangle Park, NC.
Worth Calfee, EPA Biocontaminant Laboratory Manager. National Homeland Security Research Center,
Decontamination and Consequence Management Division, Research Triangle Park, NC.


EPA Peer reviewers:

Tim Dean, EPA National Risk Management Research Laboratory (NRMRL), Air Pollution Prevention and
Control Division, Research Triangle Park, NC.
Matthew Magnuson EPA National Homeland  Security Research Center Water Infrastructure Protection
Division, Cincinnati, OH

Tiffany Yelverton, EPA National Risk Management Research Laboratory (NRMRL), Air Pollution
Prevention and Control Division, Research Triangle Park, NC.
ARCADIS US, Inc.

Abderrahmane Touati, Project Manager. ARCADIS U.S., Inc.
Gayatri Snigdha, Engineer. ARCADIS U.S., Inc.

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Table of Contents
Disclaimer	1
Acknowledgments	2
Table of Contents	3
List of Figures	5
List of Tables	6
List of Acronyms and Abbreviations	7
Executive Summary	9
1   Introduction	11
  1.1     Process	12
  1.2     Project Objectives	12
  1.3     Experimental Approach	13
2   Materials and Methods	14
  2.1     Task 1: PAA Formulation Characterization	14
    2.1.1     In-house PAA Formulations with Sulfuric Acid	15
    2.1.2     In-house PAA Formulations without Sulfuric Acid	17
    2.1.3     In-house PAA Formulations with Sulfuric Acid Derived from Chemicals Available OTC	18
    2.1.4     Modified AHP Decontamination Formulation Developed by SNL	20
    2.1.5     Diluted Hydrogen Peroxide Solution	20
  2.2     Task 2: Method Development for Neutralization	20
    2.2.1     Standard Neutralization Test Method	21
      2.2.1.1     Test A: Neutralizer Effectiveness	21
      2.2.1.2     Test B: Neutralizer Toxicity	22
      2.2.1.3     TestC: Organism Viability	22
      2.2.1.4     Test D: Test Material Control	22
    2.2.2     Sampling Frequency for Neutralization Testing	22
  2.3     Task 3: Liquid-Liquid Decontamination Testing	23
    2.3.1     Efficacy Testing	23
    2.3.2     Sampling Frequency for Decontamination Testing	24
  2.4     Task 4: Surface Decontamination Testing	26
    2.4.1     Spray Down Technical Approach	26
    2.4.2     Test Coupon  Preparation and Inoculation - Spray Down Approach	27
    2.4.3     Spray Down Neutralization Method Evaluation	27

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    2.4.4     Spray Down Decontamination Procedure	28
    2.4.5     Spray Down Decontamination Test Matrix	29
  2.5    Task 5: Material Compatibility Evaluation	31
3   Sampling and Analytical Procedures	32
  3.1     Sampling Procedures	32
  3.2    Sample Extraction/Analysis	32
    3.2.1     Liquid-Liquid Decontamination	32
    3.2.2     Surface Decontamination	32
  3.3    Sample Analysis	32
  3.4    Data Analysis	33
  3.5    Efficacy Testing	33
  3.6    Decontamination Efficacy	33
  3.7    Sample Preservation	35
  3.8    Holding Times	35
  3.9    Sample Archival	35
  3.10   Steady State Conditions	35
  3.11   PAA and  HP Characterization Measurements	35
4   Results and Discussion	36
  4.1     Formulation Characterization Results	36
    4.1.1     "In-House" PAA Formulations	36
    4.1.2     Modified AHP Decontamination Formulation	38
    4.1.3     Over-The-Counter (OTC) In-house PAA Formulation	38
  4.2    Neutralization Testing Results	40
    4.2.1     Neutralization Effectiveness	40
    4.2.2     Neutralizer Toxicity	41
    4.2.3     Test Material Control	42
  4.3    Task 3: Liquid-Liquid Decontamination Testing	43
    4.3.1     In-House PAA Formulation with Sulfuric Acid	43
    4.3.2     In-House PAA Formulation without Sulfuric Acid	45
    4.3.3     Modified AHP Formulation	47
    4.3.4     Diluted Hydrogen Peroxide Formulation	48
    4.3.5     OTC  Formulation	49

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      4.3.5.1     Liquid-Liquid Decontamination Approach	49
      4.3.5.2     Surface Decontamination Approach	50
  4.4    Material Compatibility Tests	53
  4.5    Results Summary	56
5   Quality Assurance	57
  5.1     Sampling, Monitoring, and Analysis Equipment Calibration	57
  5.2    Data Quality Objectives	58
  5.3    Acceptance Criteria for Critical Measurements	58
  5.4    Quality Assurance  (QA)/Quality Control (QC) Checks	60
  5.5    Data Quality Audits	61
  5.6    QA/QC Reporting	61
6   References	62
List of Figures
Figure 2-1.    Temporal Product/Reactant Evolution (modeled) for PAA Formulation with Acetic
             Acid	16
Figure 2-2.    Temporal Product/Reactant Evolution (modeled) for PAA Formulation without
             Acetic Acid	18
Figure 2-3.    Temporal Product/Reactant Evolution (modeled) for the OTC PAA Formulation	19
Figure 2-4.    Efficacy Testing Sequence Flow Chart	25
Figure 2-5.    Front View of Spray Apparatus with Orifice Plate	26
Figure 4-1.    Temporal Product/Reactant Evolution for the In-House PAA Formulation with
             SulfuricAcid	37
Figure 4-2.    Temporal Product/Reactant Evolution - In-House PAA without SulfuricAcid	37
Figure 4-3.    PAA and HP Concentration for the Modified AHP Formulation	38
Figure 4-4.    Temporal Product/Reactant Evolution - OTC PAA	39
Figure 4-5.    Temporal PAA Degradation over Time for the OTC and PAA with Sulfuric Acid
             Formulations	40
Figure 4-6.    Temporal Efficacy of the In-House PAA Formulations with SulfuricAcid	45
Figure 4-7.    Temporal Efficacy of the In-House PAA Formulations wthout SulfuricAcid	47
Figure 4-8.    Corrosion on Carbon Steel with pH ~2 using Procedure 1 (Post-Rinse)	53
Figure 4-9.    Front Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse)	53

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Figure 4-10.   Back Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse)	54
Figure 4-11.   Front Surface of Carbon Steel with pH 6 using Procedure 2 (No Post-Rinse)	54
Figure 4-12.   Galvanized Metal with pH 2 using Procedure 1 (Post Rinse)	55
Figure 4-13.   Salt Deposition on Carbon Steel and Stainless Steel with pH 6 using Procedure 1	55
Figure 4-14.   Salt Deposition on Stainless Steel and Aluminum with pH 6 using Procedure 2	56

List of Tables
Table 2-1.     Description and Sources of Compounds used in the PAA Formulations	15
Table 2-2.     Determination of Recipe Formulation - PAA with Sulfuric Acid Formulation (SA)	16
Table 2-3.     Determination of Recipe Formulation - PAA without Sulfuric Acid Formulation (AD)	17
Table 2-4.     Determination of Recipe Formulation - OTC PAA Formulation	19
Table 2-5.     AHP Decontamination Solution  Recipe	20
Table 2-6.     Components of Tests Ato D	21
Table 2-7.     Sampling Measurements for Neutralization Testing	22
Table 2-8.     Sampling Measurements for Decontamination Testing	24
Table 2-9.     Coupon Material Specifications	27
Table 2-10.   OTC PAA Formulation -Spray  Duration and Frequency (Series 1)	29
Table 2-11.   OTC PAA Formulation -Spray  Duration and Frequency (Series 2)	30
Table 2-12.   Neutralizer Amounts Added To  Samples -Spray Down Approach (Series 1)	31
Table 4-2.     Neutralizer Toxicity (Test B)	42
Table 4-3.     Test Material Control (Test D)	42
Table 4-4.     Test Parameters Measurements Verification	43
Table 4-5.     In-house PAA Formulation with  Sulfuric Acid - Efficacy Testing	44
Table 4-6.     In-house PAA Formulation without Sulfuric Acid - Efficacy Testing	46
Table 4-7.     Modified AHP Formulation - Efficacy Testing	48
Table 4-8.     Diluted HP Formulation - Efficacy Testing	49
Table 4-9.     OTC PAA Formulation - Efficacy testing (Liquid-Liquid Decontamination)	49
Table 4-10.   Test Parameters Measurements Verification (Series 1)	50
Table 4-11.   Test Parameter Measurement Verification (Series 2)	50
Table 4-12.   OTC PAA Formulation - Efficacy testing (Surface Decontamination - Series 1)	51
Table 4-13.   OTC PAA Formulation - Efficacy testing (Surface Decontamination -Series 2)	52
Table 5-1.     Analysis Equipment Calibration  Frequency	57
                                              6

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Table 5-2.    Critical Measurement Acceptance Criteria	59
Table 5-3:    QA/QC Sample Acceptance Criteria	61
List of Acronyms and Abbreviations
AA
AD
AHP
APPCD
ATCC
B.
Ba
BSC
CPU
DCMD
D-Value

Dl
DQI
DQO
DTRL
EPA
EtO
HP
HP
HSRP
K2C03
NaHC03
OH-
OOH"
LR
MOP
N
NA
NE
NHSRC
NIST
NT
Acetic Acid
"In-house" PAA formulation without sulfuric acid
Activated Hydrogen Peroxide
Air Pollution Prevention and Control Division
American Type Culture Collection
Bacillus
Bacillus atrophaeus
Biosafety Cabinet
Colony Forming Unit(s)
Decontamination and Consequence Management Division
Refers to decimal reduction time and is the time required at a certain temperature
to kill 90% of the organisms being studied
Deionized
Data Quality Indicator
Data Quality Objective
Decontamination Technologies Research Laboratory
U. S. Environmental Protection Agency
Ethylene Oxide
Hydrogen Peroxide
Hydrogen Peroxide solution alone without PAA
Homeland Security Research Program
Potassium Carbonate
Sodium bicarbonate (baking soda)
hydroxyl free radicals
Peroxy anion
Log Reduction
Miscellaneous Operating Procedure
Normal (Equivalent per liter)
Not Applicable
Neutralizer Effectiveness
National Homeland Security Research Center
National Institute of Standards and Technology
Neutralizer Toxicity

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oc

OTC
PAA
pAB
PBST
PPE
QA
QAC
QAPP
QC
RH
RSD
RTP
SA
SD
SNL
STS
ISA
UV
WA
WACOR
X
"In-house" PAA formulation with sulfuric acid derived from chemicals
available over-the-counter
Over-the-Counter
Peracetic Acid
pH-Adjusted Bleach
Phosphate Buffered Saline with 0.05% TWEEN20
Personal Protective Equipment
Quality Assurance
Quaternary Ammonium Compound
Quality Assurance Project Plan
Quality Control
Relative Humidity
Relative Standard Deviation
Research Triangle Park
"In-house" PAA formulation with sulfuric acid
Modified AHP decontamination formulation developed by SNL
Sandia National Laboratories
Sodium Thiosulfate
Tryptic Soy Agar
Ultraviolet
Work Assignment
Work Assignment Contracting Officer Representative
Stoichiometric equivalent

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Executive Summary

The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program (HSRP)
helps protect human health and the environment from adverse impacts of terrorist acts by carrying out
performance tests on homeland security technologies. The primary objective of this effort is to develop a
low-tech Bacillus anthracis decontamination method that would be beneficial for numerous building
interiors for effective decontamination under various environmental conditions. This research effort
focuses on low-tech peracetic acid (PAA) formulations that can be used as a green alternative to low-tech
chlorine-based decontamination applications and the less potent antimicrobial activated hydrogen
peroxide (AHP). The low-tech PAA must be effective against microorganisms including bacterial spores
and can be prepared in situ from over-the-counter (OTC) ingredients prior to use for a decontamination
event.

This project evaluated key parameters to decontaminate a nonpathogenic bacterial species Bacillus
atrophaeus (Ba), a simulant of virulent and toxic B. anthracis, using "low-tech" expedient decontaminants
such as PAA and AHP. Four PAA formulations, as well as a hydrogen peroxide (HP) solution without
PAA, were tested to determine their respective decontamination effectiveness when tested against direct
inoculation of Bacillus spores. The PAA formulations included an in-house solution prepared with and
without sulfuric acid, a modified AHP solution developed by Sandia National Laboratories (SNL), and a
fourth solution derived from OTC.

The target stock solution concentration for the in-house and the modified AHP formulations were set at
1.5 % PAA, which was then diluted successively to encompass a full range of PAA test concentrations
from 0.001 % to 1  %. The in-house PAA formulations with sulfuric acid were found to reach  maximum
PAA concentration and equilibrium within six days, while the formulation without sulfuric acid reached the
maximum target PAA concentration within 31 days. The OTC PAA formulation was prepared in a single 1-
liter batch and reached a maximum PAA equilibrium concentration of 0.07 % PAA in just over one  day.
The modified AHP formulation reached its target PAA concentration of 1.5 % immediately after mixing.
However, the solution was highly unstable and the concentration of both PAA and HP dropped within four
hours. The diluted HP antimicrobial solution was tested at 4.5 % HP concentration. This value is the same
estimated concentration of HP in the 1 % PAA solutions. Once equilibrium was reached,  all diluted
solutions were observed to be stable overtime with minor shifts in PAA and HP concentrations  (less than
10 % change in concentration over a period of weeks); note that the AHP solution was not monitored
beyond the four-hour suggested use time).

The first phase of the project evaluated the liquid inoculation/liquid decontamination solution approach for
decontamination efficiency of all five formulations at different PAA concentrations. The results from the
first phase of the project demonstrated a near complete decontamination at a two-minute exposure time
with the 1 % PAA in-house formulation. The colony forming unit (CPU) recovery between the two-  and
eight-minute exposure time lies within the uncertainty of the measurement technique, which suggests a
calculated D-value(linear interpolation) for this formulation of less than 0.26 min.  The 0.1 % PAA also
showed promise with an average D-value calculated to be  1.8 min, which suggests a 6-Log  CPU
reduction (LR) within an exposure time of about 10 minutes. The 0.01 % and 0.001 % PAA formulation
were found to be ineffective with D-values greater than 18 minutes.

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The liquid-liquid decontamination approach using the modified AHP formulation resulted in greater than 6
LR of spores when the concentration of the formulation was at 1 % PAA. However, the 0.1 % dilution
proved to be less effective, even with extended exposure times. The diluted HP (4.5 % HP, no PAA)
formulation was found to be ineffective with an LR of less than 0.5, suggesting that the sporicidal
ingredient of the PAA solutions is the PAA itself. The averaged D-value for the diluted HP formulation was
18 min, which suggests that this product may not be a practical sporicide for field application. The liquid-
liquid decontamination approach using the OTC formulation (0.07 % PAA) provided greater than 6 LR for
exposure times of 8 minutes or longer. As this solution is made from OTC  common household products,
this formulation could be a promising decontaminant for use by the general population. However, the PAA
solution is highly acidic (pH 1.7), which could potentially corrode some surfaces and potentially be
hazardous to the users.

The OTC PAA (0.07 % PAA) surface decontamination efficacy was evaluated using  a spray-down
approach on coupons of carpet, wood, glass, vinyl flooring, and concrete. These tests demonstrated that
increasing exposure time combined with frequent spraying resulted in greater decontamination efficacy as
compared to multiple sprays at the same time. However, the overall surface decontamination efficacy for
the OTC formulation was not sufficient to achieve 6 LR. These lower surface decontamination efficiencies
were due to the high degradation of the OTC PAA concentration observed during the spraying. The
surface decontamination approach can be improved significantly if the OTC formulation is  further
stabilized by using laboratory grade ingredients or stabilizing the formulation by using other OTC
ingredients. Further studies will be necessary to determine the cause of the OTC degradation.

The in-house PAA formulations (PAA with and without sulfuric acid and OTC components) are acidic (pH
1.7). To analyze the acidity effect of these formulations,  a material compatibility test was designed to
assess the corrosion or changes of low PAA formulations at different pH levels. This study was performed
on coupons made of stainless  steel, galvanized metal, aluminum, and carbon steel. These coupons,
tested in duplicate, followed two test procedures: (1) Post-rinse procedure and (2) No post-rinse
procedure. In the post-rinse procedure, two coupons of each material were immersed in the original  OTC
PAA (0.07 % PAA) formulation. They were then rinsed with deionized (Dl)  water and left to dry after a
contact time of 30  minutes with the decontaminant. In the no post-rinse procedure, two coupons of each
material were immersed  in OTC formulation and then left to dry without a Dl water rinse after a contact
time of 30 minutes with the decontaminant. No post-rinse materials appeared to have salt  deposits
(arising from the food grade baking soda added as an acid neutralizerto the OTC PAA formulation to
bring the solution to a neutral pH). Materials that did follow a post-rinse procedure did not show any
significant except for the carbon steel which was observed to have reacted most vigorously to the
decontamination solution (with both procedures), with visible amounts of rust and corrosion on its  surface.
Little change was observed on the surfaces of stainless steel and aluminum coupons that were post-
rinsed after being treated with the decontamination solutions. Typical  indoor room conditions  (71 F and
37 % RH) were observed throughout the duration of this material compatibility test.
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1      Introduction

This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Office of
Research and Development's (ORD) Homeland Security Research Program (HSRP) by providing
information relevant to the decontamination of areas contaminated as a result of an act of terrorism.
Under Homeland Security Presidential Directives (HSPD)-5, 7, 8, and 10, the EPA, in a coordinated effort
with other federal agencies, is responsible for "developing strategies, guidelines, and plans for
decontamination of equipment, and facilities" to mitigate the risks of contamination following a biological
agent contamination incident.

EPA's National Homeland Security Research Center (NHSRC) aims to help EPA address the mission of
the HSRP by providing expertise and products that can be widely used to prevent, prepare for, and
recover from public health and environmental emergencies arising from terrorist threats and incidents.
One of NHSRC's missions is to provide expertise and guidance on the selection and implementation of
decontamination methods and provide the scientific basis for a significant reduction in the time,  cost, and
to address the complexity of decontamination activities. Quick, effective, and economical decontamination
methods that have the capacity to be employed over wide areas (outdoor and indoor) are one specific
focus of this  research program. This project's aim is to assess the effectiveness of in situ decontamination
methods using peracetic acid (PAA) solutions formulated from over-the-counter (OTC) products. The
detrimental effect these decontamination solutions have on various surfaces (e.g., corrosion) is  also being
assessed.

Decontamination can be defined as the process of inactivating or reducing contaminants in or on humans,
animals, plants, food, water, soil, air,  areas, or items through physical, chemical, or other methods to
meet a cleanup goal. In terms of the surface of a material, decontamination can be accomplished by
physical removal of the contaminant or via inactivation of the contaminant with  antimicrobial chemicals,
heat, ultraviolet (UV) light, etc. Physical removal could be accomplished via in situ removal of the
contaminants from the material or by physical removal of the material itself (i.e., disposal) [1~51. Similarly,
inactivation of the contaminant can be conducted in situ or after removal of the material for ultimate
disposal[1 2 4 5I. Following the 2001 anthrax incidents, a combination of removal and in situ
decontamination was used [1 2I. The balance between the two procedures was  facility-dependent and
factored in many issues (e.g., the physical state of the facility). Such remediation was unprecedented for
the United States Government, and no wide-area decontamination techniques  had been  proven effective
at the time against spores of Bacillus anthracis. The cost of disposal proved to  be significant, and finding
an ultimate disposal site was complicated by the nature of the waste. Since 2001, a primary focus for
facility remediation has been improving the effectiveness and practical application of in situ
decontamination methods and evaluating waste treatment options to be able to provide the information
necessary to optimize the decontamination/disposal paradigm. This optimization has a significant impact
on reducing the cost of and time for the remediation effort.

Low-tech PAA formulations can be used as a method alternative to low-tech pH Adjusted Bleach (pAB)
and other chlorine-based decontamination applications and to the less effective antimicrobial activated
hydrogen peroxide (AHP). Peroxides (compounds which contain two oxygen atoms bonded together) are
strong oxidants that offer an environmentally friendly alternative to the toxic and corrosive chlorine-based
decontaminants [6 7I. The sporicidal effectiveness of hydrogen peroxide (HP) solutions is  due to  the
peroxy anion (OOH~) and hydroxyl free radicals (OH-)[7  8I. Another peroxy compound that is commonly

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used is PAA (CH3CO(OOH))[61. PAA is often added as a supplemental oxidizing agent in mixtures with
HP. PAA is effective against all microorganisms, including bacterial spores, because of its high oxidizing
potential, and is often added as an oxidizing agent in mixtures with HP. The co-presence of HP and PAA
have a synergistic sporicidal efficacy [6]. Due to stringent shipping and handling requirements, PAA is
often generated in situ, prior to decontamination, using AHP solutions. This method has many variations,
but the foundation of the chemistry is the same: generation of PAA through the hydrolysis of an acetyl
donor, with a specifically formulated HP solution as an  oxygen source [7 8I. A significant variable affecting
practical application of PAA is that different formulations take different amounts of time for the PAA to be
formed, because formation of PAA is an equilibrium process.

1.1   Process
This project examined the ability of five PAA formulations to remove B. anthracis surrogate spores (8.
atrophaeus (Ba)) from samples: three in-house PAA formulations (with and without sulfuric acid, and with
sulfuric acid derived from chemicals available OTC); a  modified AHP decontamination formulation
developed by Sandia National Laboratories (SNL) under the Wide Area Recovery and Resiliency
Program  research efforts; and a hydrogen  peroxide (HP) solution alone without PAA (with the same
estimated HP concentration as the 1 % PAA solution).  In the first phase of the project, the spores were
directly inoculated into the decontaminant samples and exposed to the decontaminant for contact times
ranging from two minutes to 20 minutes. Because these formulations were generated in s/fujust prior to
decontamination testing, an understanding of the time to reach equilibrium for simple PAA and the
relationship between PAA concentration, exposure time, and sporicidal effectiveness was developed. In
addition to looking at the decontamination efficacy, this effort also qualitatively assessed the tendency of
the decontaminant to corrode different building materials by visual inspection  and gravimetric analysis.
The subsequent phase of the project evaluated the decontamination potential and physical effects of the
OTC decontamination formulation on designated test coupons prepared from materials  typical of surfaces
found in an indoor environment.

1.2   Project Objectives
The primary objectives were:

       To develop a simple PAA solution that could easily be generated in the field, within a realistic time
       (i.e., a few hours) to reach equilibrium;

       To develop an understanding of the time to reach equilibrium  for simple PAA and the relationship
       between PAA concentration, exposure time, and sporicidal effectiveness;

       To determine the fate of the spores  during the decontamination process -LR of B.  anthracis
       surrogate spores on contaminated liquid media; and

       To assess the corrosion  produced  by application of low PAA formulations derived from OTC
       products.

The operational parameters of decontamination (e.g., pH and temperature of decontamination solution
within the pot life of the decontamination solution, the concentration of the PAA formulations, and the
exposure times) were considered important for understanding the sporicidal activity of the PAA-based
decontamination process and were characterized in addition to sporicidal effectiveness.
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1.3   Experimental Approach
The general testing approach consisted of the following Tasks:

       Task 1: PAA Formulation Characterization

       The concentration of active ingredients and other characteristics were measured overtime for
       four formulations of PAA.

       Task 2: Method Development for Neutralization of Decontaminant Solutions

       Neutralization methods to inactivate the sporicides were tested for compatibility with spore
       enumeration methods.

       Task 3: Liquid-Liquid Decontamination Testing

       The direct liquid inoculation/liquid decontamination solution approach was evaluated forsporicidal
       effectiveness of the target decontamination solutions at different concentrations.

       Task 4: Surface Decontamination Testing

       Surface decontamination testing was conducted using an OTC formulation that was applied to
       coupons of carpet, wood, glass, vinyl flooring and  concrete in the form of spray.

       Task 5: Material Compatibility Evaluation

       Visual and gravimetric assessments of the PAA formulation material compatibility were
       undertaken to qualitatively investigate any changes/corrosion on the surfaces of different material
       types.
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2      Materials and Methods

This section describes the test facilities and equipment, general decontamination approach, and test
conditions, test materials and methods that were used to evaluate the data related to the project
objectives. Testing was conducted at the EPA RTP facility.

The research presented in this report characterized the decontamination efficacy of diluted PAA
formulations against Ba (American Type Culture Collection [ATCC] 9372) spores. The overall
effectiveness of the methods used in this study was determined by the ability of the method to reduce the
number of and/or to  inactivate the spores of Ba  in the PAA solution.

2.1   Task 1: PAA Formulation Characterization
Each stock solution PAA formulation was prepared in a single one-liter batch, and the PAA and HP
concentration measurements of the stock solutions were monitored overtime until each of the solutions
reached a maximum PAA equilibrium concentration for a set of initial mixture conditions. The target stock
solution concentration was set in excess of the highest target test PAA solution, which was then diluted
successively to encompass a full range of PAA concentrations from 0.001 % to 1 %. The stock solutions
and the corresponding diluted solutions were measured overtime to determine the kinetics and stability of
the mixture. Stability, in this case, is  defined as a pseudo-steady state of a target solution  in which
variation of the PAA concentration is less than 10 % over a period of one hour. The following five
formulations were characterized in this study:

    1)   In-house PAA Formulation with Sulfuric Acid (SA)

    2)   In-house PAA Formulation without Sulfuric Acid (AD)

    3)   In-house PAA Formulation with Sulfuric Acid Derived from Chemicals Available OTC (OTC)

    4)   Modified AHP Decontamination Formulation Developed by SNL (SD)

    5)   HP solution alone without PAA (same estimated HP concentration as the 1 % PAA solution)

Descriptions and sources of the ingredients used in the formulations are provided in Table 2-1. The
formulations are described further in the following subsections.
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Table 2-1.   Description and Sources of Compounds used in the PAA Formulations
Formulat
ion
1.5%
Peracetic
Acid (SA
and AD)
OTC
Modified
AHPby
SNL(SD)
Diluted
HP (HP)
Ingredients
Hydrogen Peroxide
Glacial Acetic Acid
Sulfuric Acid
Dl Water
Hydrogen Peroxide
Topical Solution
White Distilled Vinegar
Battery Acid (Sulfuric
Acid)
Hydrogen Peroxide
Maquat(MC1412-80%E)
Potassium Carbonate
(K2C03)
Ethanol
Triacetin
Hydrogen Peroxide
CAS
Number
7722-84-1
64-19-7
7664-93-9
7732-18-5
7722-84-1
64-19-7
7664-93-9
7722-84-1
648424-85-1
64-17-5
68439-90-3
7732-18-5
584-08-7
64-17-5
102-76-1
7722-84-1
Label
Concentration
50%
99.7+ %
1 Normal (N)
100%
3%
5%
30^3 % (by wt)
50%
80-82 % (by wt)
Quaternary
Ammonium
Compounds (QACs)
>99 %
99.5 %
99%
50%
Origin / Manufacturer
Fisher Scientific
Acros Organ ics
Fisher Scientific
Produced in-house using Dl
Water System from Purologix
Water Services Inc.
Walgreens
Heinz
East Penn Manufacturing
Company, Inc
Fisher Scientific
Mason Chemical
Fisher Scientific
Acros Organics
Acros Organics
Fisher Scientific
City/State
Pittsburgh, PA
Pittsburgh, PA
Pittsburgh, PA
Holly Springs, NC
Deerfield, IL
Pittsburgh, PA
Lyons, PA
Pittsburgh, PA
Arlington Heights, IL
Pittsburgh, PA
Pittsburgh, PA
Pittsburgh, PA
Pittsburgh, PA
2.1.1  In-house PAA Formulations with Sulfuric Acid
This in-house PAA formulation was prepared by reaction of acetic acid (AA) and HP [9 10 13]. The rate at
which equilibrium is achieved is accelerated by adding a strong acid catalyst (sulfuric acid). The target
decontamination formulation was prepared by adding the AA to a specially cleaned volumetric flask (triple
rinsed with Dl water followed by a one-hour autoclave sterilization cycle at 121 C) with concentrated
sulfuric acid, before adding HP to the flask contents. An initial stock solution batch of one liter of the PAA
formulation was prepared to achieve  a high PAA concentration of 1.5 %. This solution was then diluted to
achieve PAA concentrations of 1 %, 0.1 %, 0.01 % and 0.001 % forsporicidal testing.

A mathematical kinetic model based on rate constants determined by other researchers [13] was
developed in this study to estimate the time when the equilibrium between the reactants and products
could be established. The input parameters for this model were the HP concentration, the molar ratio of
AAto HP, and the loading of the sulfuric acid. The recipe formulation for the target decontamination
solution was also established through this model to  determine the amount of reactants needed to achieve
the target PAA at equilibrium, as illustrated in Table 2-2 for the PAA with sulfuric acid formulation. The
time to reach the target  equilibrium of 1.5 % (0.2 mol/L) PAA is shown in Figure 2-1.
                                               15

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Table 2-2.   Determination of Recipe Formulation - PAA with Sulfuric Acid Formulation (SA)
Recipe Formulation
Recipe
HP
Acetic acid
Water
Sulfuric Acid
Input Parameters
15
100

1
%wt/wt
%

<-N v/v %->
Volume Calculated (mL)
403
128.34
404.2
62.5
Initial Set Conditions
Recipe
PAA
HP
Acetic acid
Water
Sulfuric Acid
mol/L
0.00
1.82
2.18
45.24
0.13
wt(g)
0.00
61.74
130.79
814.26
12.26
wt%
0.00
6
13
80
1
Expected Equilibrium Concentration
Recipe
PAA
HP
Acetic acid
Water
Sulfuric Acid
mol/L
0.20
1.61
1.98
45.44
0.13
wt(g)
15.24
54.92
118.75
817.87
12.26
wt%
1.50
5.39
11.65
80.26
1.20
          1.75.
          1.50-
          1.25-
         LOO-
       IS
         0.75-
         0.50-
         0.25-
         0.00-
                    10
                          20     30     40    50     60


                              Reaction Time (Hours)
                                                          70
                                                                  6.2
                                                                  6.0
                                                                  5.8:5
                                                                  5.6
                                                                  5.4 '
                                                                     CL
                                                                  5.2
                                                                  5.0
                                                                80
Figure 2-1.    Temporal Product/Reactant Evolution (modeled) for PAA Formulation with Acetic Acid
                                                   16

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2.1.2  In-house PA A Formulations without Sulfuric Acid
The in-house PAA formulation without sulfuric acid (SD) was prepared in the same way as the in-house
PAA acid solution, with the exception that no sulfuric acid was added as a catalyst to speed up the
reaction. As a result, the solution required approximately a month to reach the target maximum PAA
concentration (1.5 %) and equilibrium. A 15 % HP concentrated solution was used to prepare this
decontamination solution. The volumetric ratio of AA initial solution to HP initial solution was kept at 1.2:1
as well. This solution was then diluted to achieve PAA concentrations of 1 %, 0.1 %, 0.01 % and 0.001 %
for sporicidal testing. The  recipe for the target SD formulation is shown in Table 2-3. The time to reach
target equilibrium of 1.5 % (0.2 mol/L) PAA is shown in Figure 2-2.

Table 2-3.   Determination of Recipe Formulation - PAA without Sulfuric Acid  Formulation (AD)
Recipe Formulation
Recipe
HP
Acetic acid
Water
Input Parameters
15
100

% wt/wt
%

Volume Calculated (ml_)
403
129.05
461.0
Initial Set Conditions
Recipe
PAA
HP
Acetic acid
Water
mol/L
0.00
1.82
2.18
45.61
wt(g)
0.00
61.74
130.79
820.93
wt%
0.00
6.10
12.90
81.00
Expected Equilibrium Concentration
Recipe
PAA
HP
Acetic acid
Water
mol/L
0.20
1.62
1.98
45.81
wt(g)
15.14
54.96
118.83
824.51
wt%
1.49
5.42
11.73
81.36
                                              17

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              200    400    600    800    1000   1200   1400
                        Reaction Time (Hours)
                                                         6.2
                                                         6.0
                                                         5.8
                                                         5.6
                                                         5.4 '
                                                           Q_
                                                         5.2
                                                         5.0
Figure 2-2.    Temporal Product/Reactant Evolution (modeled) for PAA Formulation without Acetic Acid

2.1.3  In-house PAA Formulations with Sulfuric Acid Derived from Chemicals Available
       OTC
The OTC PAA formulation was prepared in a single one-liter batch. The PAA and HP concentrations,
along with the pH values, of the stock solution were monitored overtime until the solution reached a
maximum PAA equilibrium concentration (0.07 %) for a set of initial conditions. This solution was derived
from products that are available OTC or off-the-shelf so that they are easily accessible to the general
population. Battery acid (sulfuric acid) was used as the catalyst in this recipe to enable equilibrium to be
reached in hours. The volumetric ratio of the AA (from vinegar) initial solution to HP initial solution was
kept at 1.2:1 as well. A material compatibility study using the OTC solution was performed on metal
coupons and is discussed in Section 2.4. The  recipe for the target OTC decontamination solution is
shown  in Table 2-4. The time to reach target equilibrium of 0.07 % PAA is shown in Figure 2-3.
                                               18

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Table 2-4.   Determination of Recipe Formulation - OTC PAA Formulation
Recipe Formulation
Recipe
HP
Acetic acid
Water
Sulfuric Acid
Input Parameters
3
5

9.4
%wt/wt
%

<-N v/v
%->
Volume Added (mL)
439
559
0
3.5
Initial Set Conditions
Recipe
PAA
HP
Acetic acid
Water
Battery acid
mol/L
0.00
0.39
0.47
53.23
0.066
wt(g)
0.00
13.22
28.00
958.14
6.47
wt%
0.00
1.30
2.80
95.30
0.60
Expected Equilibrium Concentration
Recipe mol/L
PAA 0.009
HP 0.38
Acetic acid 0.46
Water 53.23
Sulfuric Acid 0.066
wt(g)
0.71
12.90
27.44
958.31
6.47
wt%
0.07
1.28
2.73
95.28
0.64
      0.075-
      0.060-
    S 0.045-
    o
      0.030-
      0.015-
      0.000
                                                              1.280
                 10    20    30     40     50     60    70    80
                           Reaction Time (Hours)
Figure 2-3.  Temporal Product/Reactant Evolution (modeled) for the OTC PAA Formulation
                                                  19

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2.1.4  Modified AHP Decontamination Formulation Developed by SNL
The AHP decontamination formulation was developed by SNL. The original recipe for preparation of AHP
provided by SNL is given in Table 2-5. For preparation of large batches of AHP, the original recipe was
modified as shown. Variquat was not available at the commencement of this study and was replaced by
Maquat. Variquat and Maquat are both quaternary ammonium compounds (QACs) that have microbicidal
properties.

Table 2-5.   AHP Decontamination Solution Recipe

Part A
PartB
PartC
Original Recipe
Dl water [mL]
K2C03 [g]
Variquat [mL]
Ethanol [mL]
50 % HP [mL]
Triacetin [mL]
76.9
3
0.1
5
12
3
Modified Recipe
Dl water [mL]
K2C03 [g]
Maquat [mL]
Ethanol [mL]
15%HP[mL]
Triacetin [mL]
489
30
1
50
400
30
The stock AHP solution was then diluted to achieve PAA concentrations of 1 %, 0.1 %, 0.01 % and 0.001
% for sporicidal testing. The target maximum PAA concentration (1.5 %) was achieved almost
immediately post-mixing. The solution has a shelf life of only four hours and thus had to be used in testing
within that time frame. A new solution was prepared fresh for each test sequence.

2.1.5  Diluted Hydrogen Peroxide Solution
The diluted  HP antimicrobial solution was tested at 4.5 % HP concentration. This value is the same
concentration of HP estimated to be in the 1 % PAA solutions.

2.2   Task 2: Method  Development for Neutralization
The presence of decontamination solution components in the test solution after the contact time could
negatively bias colony forming unit (CPU) quantification. Sporicide neutralization tests of a Ba spore
solution were carried out in advance of the decontamination solution evaluation to determine the most
suitable neutralization solutions  required for complete inactivation of the microbicidal properties for two
PAA decontamination solutions  (0.001 % and 1 %). Sodium thiosulfate (STS) was evaluated as a
neutralizing agent for PAA and HP in an aqueous solution containing  0.1 g  potassium carbonate (K2CO3),
phosphate buffered saline  with 0.05% TWEEN80 (PBST), and a specific decontamination solution.
Because  the PAA formulations are acidic, K2CO3 was added to the neutralizer solution to raise the
neutralizer-decontamination solution mixture to a pH between 5.5 - 8.5. If 0.1 g of K2CO3 was insufficient
to raise the  pH within the test parameter range, more K2CO3 was added as needed to satisfy the pH
criteria. The STS solution was evaluated at 1X, 2X, and 5X molar excess for the  1 % PAA, and  at 1000X
stoichiometric excess for the 0.001 % PAA solution (where X represents stoichiometric neutralizing
conditions of PAA and  HP). In addition to STS, a 2-mL lecithin/Tween solution was added to neutralize
the QACs present in the AHP formulation. The following recipe was used to prepare 30 mL of the lecithin/
TWEEN 80 solution:
                                             20

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       5 g Soybean Lecithin (Fisher Scientific, CAS No. 8002-43-5)
       5 ml TWEEN 80 (Fisher Scientific, CAS No. 9005-65-6)

       25 ml Dl Water (In-house Dl Water System, by Purologix Water Services Inc)

Contents were thoroughly mixed in a sterile 125 ml media bottle and autoclaved for 20 minutes at 121C.
The neutralization solution with STS was prepared once the lecithin/ TWEEN 80 solution cooled to room
temperature.

2. 2. 1  Standard Neutralization Test Method

Due to the specific interest in the exposure period of Bacillus spores to the test formulation, the
neutralization solution was evaluated according to the "Standard Test Methods for Evaluation of
Inactivators of Antimicrobial Agents," ASTM E1 054-8mi with recovery on semi-solid media. Table 2-6
shows that the test components for Tests A through D were kept the same, with an emphasis on
maintaining the same total solution volumes and ratios throughout all tests.

Table 2-6.   Components of Tests A to D
Components
Microbial Suspension
Decontaminant
Neutralizer
PBST
Test A
X
X
X

TestB
X

X
X
TestC
X


X
TestD
X
X

X
The microbial suspension was comprised of Ba ATCC 9372 and was manipulated so that the microbial
suspension used in Tests A through D resulted in the total components containing 25 to 250 CFU/ ml,
with the target concentration being 100 CFU/mL.

2.2.1.1   Test A: Neutralizer Effectiveness
This test determined the NE to quench the activity of the decontamination solution by simulating the
conditions of the efficacy test (i.e., neutralizer volume, decontamination solution volume, challenge
microbial solution volume, decontamination solution to neutralizer ratio, contact time). After the
concentrations of PAA and HP for the target decontamination solution were measured, the stoichiometric
equivalent (X) for STS to neutralize the decontaminant was calculated. Five ml of the target neutralizer
(an aqueous solution containing PBST and 0.1  g baking soda with 1X, 2X, and 5X STS for the 1 %  PAA,
and 1000X for the 0.001 % PAA solution STS) were added to 5 ml of the decontamination  solution
(0.001 % PAA or 1 % PAA).. Once the pH of the neutralizer/decontamination solution had been
aseptically checked, the solution was  inoculated with 0.1 ml of 1xio4 CFU/mL challenge microbial
suspension to result in a final suspension that contains approximately 100 CFU/mL (1000 total CFU) of
the microorganism. The decontamination/neutralizer/microbial suspension was allowed to stand for a total
holding time of 30 minutes. This test was conducted in triplicate

A 1 mL aliquot was removed from each of the decontamination/neutralizer/microbial suspension samples
and transferred, in triplicate, to a tube with 10 mL PBST. Once  homogenized, each 11  mL sample was
                                              21

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filtered using a Nalgene filter unit (cat no. 130-4020) and the filters were aseptically removed and placed
onto a pre-labeled Tryptic Soy Agar (ISA) media plate and incubated at 35  2 C overnight.

2.2.1.2   Test B: Neutralizer Toxicity
Test B was designed to determine whether the presence of neutralizer in excess was biostatic or biocidal.
This test sequence was a repeat of Test A, with the exception that the decontamination solution was
replaced with a sterile PBST solution that resulted in the same total sample volume as that used in Test
A.

2.2.1.3   Test C: Organism Viability
The organism viability test determined the recovery of the microorganisms exposed to the sterile buffer
(PBST) only. This test sequence was a repeat of Test A, with the exception that the decontamination/
neutralizer solution mixture was replaced with a sterile PBST solution that resulted in the same dilution
ratio as that used in Tests A and B. Samples from this test also serve as positive controls to calculate the
recoveries from Test A, B, and D.

2.2.1.4   Test D: Test Material Control
This test sequence was a repeat of Test A, with the exception that no STS was added to the neutralizer
solution, resulting in the same dilution  ratio as Tests A and B.

2.2.2  Sampling Frequency for Neutralization Testing
The neutralization testing sequence for each formulation is listed in Table 2-7.

Table 2-7.   Sampling Measurements for Neutralization Testing
Testing Sequence
For Each
Decontamination
Formulation
Measurement
Titration
(PAA concentration)
Titration
(HP concentration)
pH Probe
Decontamination Solution
Negative Controls CFU
Laboratory Blank Solution
CFU
Neutralization Test
Solution CFU
Measurement Range
0.001-1 %
0-5 %
0-9.5
0-1 00 CFU per filter
30-1 00 CFU per filter
Frequency
Once before and after completion
of the neutralization testing
One sample per decontamination
solution
(PAA 1 % and PAA 0.001 %)
One sample per neutralization
essay
Three samples per
decontamination solution per
neutralizer concentration per
neutralization assay type
                                              22

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2.3   Task 3: Liquid-Liquid Decontamination Testing
The testing sequence included both the decontamination formulation evaluation prior to testing (checks
for stability, concentration of active ingredients, and solution neutralization,) and the sporicidal efficacy
based on direct inoculation as a function of reaction time. The microbial suspension was tested for
sporicidal effectiveness, in quintuplicate, at four decontamination exposure times (2, 4, 6, and 8 minutes)
for the 1 % PAA formulations and at six decontamination exposure times (2, 4, 6, 8, 16 and 20 minutes)
for further dilutions of the PAA formulations. For each microbial suspension test, there was one negative
test control using the same approach as the efficacy testing, three positive test control tests using a
neutralizer/PBST solution, and one inoculum control test using a PBST solution alone.

2.3.1   Efficacy Testing
This test was designed  to determine the sporicidal effectiveness of each decontamination formulation at a
prescribed reaction time. After the concentrations of PAA and HP for the target decontamination solution
were measured, the target neutralizer containing appropriate amounts of STS (determined during the
neutralizer effectiveness test), PBST with baking soda was prepared. Lecithin was also added to the
neutralizer solution when testing the AHP formulation. Five ml samples of the decontamination solution
were inoculated with 0.1 ml of 5.0 x 108 CFU/mL challenge microbial suspension to result in a final
suspension (decontamination/neutralizer/microbial suspension) that contained approximately 5.0 xio7
CFU of the microorganism. After the required exposure time had passed, 5 ml of the neutralizer solution
was added to each sample to stop the decontamination reaction.

The decontamination/neutralizer/microbial suspension was held at room conditions for 30 minutes and
the mixture was then immediately homogenized for 15 seconds using a vortex mixer prior to a tenfold
serial dilution and plating onto TSA. In addition, a 1-mL aliquot was removed  and aseptically transferred
to a sterile, pre-labeled  tube containing 10 ml of PBST. The tube containing the 1  ml aliquot and the 10
ml of PBST was homogenized by vortex mixer for 15 seconds and then aseptically transferred to an
individual Nalgene filter unit, where the sample was filtered. Immediately following filtration, the filter unit
was rinsed  using another 10 ml of PBST, which was also  filtered. The filters were aseptically removed
and placed onto a pre-labeled TSA media plate that was incubated at 35  2 C overnight. Following
incubation, CFU were enumerated.

The pH  of the decontamination/neutralizer solution mixture was required to be within a pH range of 5.5 -
8.5 to provide an optimum medium for the spores post-neutralization. A separate test vial containing 5 ml
of decontamination solution  and  5 ml of neutralization solution was used to test the pH of the  solution
mixture  to maintain sterility of the solutions and to avoid contaminating the pH meter probe with the
spores.  The pH of all other samples used in efficacy testing was assumed to be the same as this test vial.
                                               23

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2.3.2  Sampling Frequency for Decontamination Testing
The decontamination testing sequence for each formulation is listed in Table 2-8. Figure 2-4 presents a
flowchart of the efficacy testing sequence.

Table 2-8.  Sampling Measurements for Decontamination Testing
Testing
Sequence
For Each
Decontamination
Formulation
Measurement
Titration (PAA concentration)
Titration (HP concentration)
pH Probe
Decontamination Solution Negative
Controls CFU
Laboratory Blank Solution CFU
Positive Controls CFU
Inoculum Controls CFU
Test Samples CFU
Concentration Range
0.001 - 1 %
0-5 %
0-9.5
0-1 00 CFU per filter
~5.0xlo7CFUper
ml_, 30-300 per plate
30-300 per plate for
dilution plating; 0-100
CFU per filter
Frequency
Once before and after
completion of the efficacy testing
One sample per
decontamination solution
One sample per neutralization
assay
Three samples per
decontamination solution
One sample in PBST per
decontamination solution
Five samples per
decontamination solution per
solution concentration per
reaction time
                                            24

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                                            Make the Diluted Decon Solution
                          Assess concentration of active ingredients using titrations (eerie sulfate,
                          followed by iodiometric determination of PAA)
                          After 1 hour, reassess concentration of active ingredients using
                          titrations (eerie sulfate, followed by iodiometric determination of PAA)
                                            Is solution stable ( 10% drift
                                            in PAA)?
                                         Is solution within 50% of desired PAA
                                                       target
No
                      Determine STS equivalents and use to prepare neutralization solution from Test
                                                        A-D
                               Remove 5 ml aliquot of decon solution. Spike with inoculum
                                             Allow required reaction time
                                             Add 5 ml neutralizer solution
                         Add 10 ml extraction buffer solution, vortex, and plate within 30 minutes
Figure 2-4.     Efficacy Testing Sequence Flow Chart
                                                        25

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2.4   Task 4: Surface Decontamination Testing
The study conditions for decontaminant solutions generated in-house during the first phase of the project
do not reflect the way in which decontaminants are applied. While laboratory studies are largely
conducted in solution, technical decontamination requires more readily available procedures such as
spraying for surface decontamination. The spraying approach using readily available equipment may
increase readiness to respond to a wide-area contaminant release.

2.4.1   Spray Down Technical Approach
The OTC PAA decontamination formulation was applied to coupons of carpet, wood, glass, vinyl flooring,
and concrete in the form of spray. Two series of tests were designed under this approach. The first series
of tests  required a 10 minute coupon-decontaminant exposure time. The second series of tests required a
30 minute coupon-decontaminant exposure time and  more frequent spraying to determine if these factors
had an effect on the surface decontamination efficacy. The spray application was conducted using a
custom-built testing apparatus shown in Figure 2-5. The spray testing apparatus was previously
employed in EPA decontamination testing, and a test method was developed for the use of this
apparatus. Decontamination solution was sprayed onto coupons using a 0.5-gallon chemical- and break-
resistant adjustable commercial hand sprayer (RL FloMaster Model # 1985VI, Lowell, Ml) made of high-
density  polyethylene with a Viton seal. The adjustable sprayer included a pump trigger that can  provide a
controlled delivery and was set on fine mist to minimize runoff and drips of the decontaminant. The
sprayer is also fitted with a pressure gauge that reads up to 30 psi.
Figure 2-5.    Front View of Spray Apparatus with Orifice Plate
                                              26

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2.4.2  Test Coupon Preparation and Inoculation - Spray Down Approach
Test coupons were made from five different materials that represent typical indoor materials consisting of
porous materials (carpet, wood and concrete) and nonporous materials (glass and vinyl flooring). The
size of the test coupons was approximately 1.7 cm x 5 cm to fit into the 50 ml conical tubes used during
the spore extraction process. Concrete coupons were coated with a waterproof epoxy coating on five
sides leaving only  one 1.7 cm x 5 cm surface of the coupon absorbent to the decontaminant solution
(MOP 3202). Similarly, wood coupons were coated with a polyurethane coating on five sides. Carpet,
vinyl and wood coupons were sterilized prior to use with ethylene oxide (EtO) from an Andersen EOGas
333 sterilization system  (Andersen Products, Haw River, N.C), while concrete and glass coupons were
sterilized in an autoclave. Supplier and specification for all five coupon materials are shown in Table 2-9.

Table 2-9.   Coupon Material Specifications
Material
Glass
Wood
Concrete
Carpet
Vinyl Flooring
Origin
Prism Research Glass
Maple Dowels, Cindoco
Wood Products
Made in-house
Beaulieu Solutions
Peel and Stick Vinyl Tile,
Home Depot
Location
Raleigh, N.C
Mt. Orab, OH
RTP, N.C
Dalton, GA
Gary, N.C
Specification
Dimensions: 1 .7 cm x 5 cm x 1 cm
Dimensions: 3/4" x 36"
Dimensions: 1 .7 cm x 5 cm x 1 cm
Model #657985861200AB; Dimensions:
18"x18"
Model #26295061;
Dimensions: 12"x12"
Final Coupon Size
1 .7 cm x 5 cm
The test coupons were inoculated with 100 uL of a Ba ATCC 9372 spore stock suspension. Coupons
were placed flat in a sterile Petri dish inside the biosafety cabinet (BSC) and inoculated with
approximately 1 x 104 viable spores per coupon for method development for neutralization, and 1 x 108
viable spores per coupon for the decontamination testing. Ten (10) droplets (each 10 uL in volume) of the
spore stock suspension were dispensed uniformly across the surface of the test coupon using a
micropipette. After inoculation, the coupons were allowed to dry overnight in the BSC.

2.4.3  Spray Down Neutralization Method Evaluation
A series of method development tests was performed to determine the required volume and strength of
the neutralizerto be preloaded in the 50 ml extraction conical tubes (BD Falcon cat. no. 352098, Franklin
Lakes, NJ) following the decontamination spray. This testing was conducted as follows:

   a.  The spray apparatus, with coupons and rinsate collection vials, was aseptically assembled. The
       vials were pre-weighed before spraying.

   b.  The decontamination solution was sprayed onto the coupons (spray frequency for each coupon
       and series of tests is detailed in Tables 2-10 and 2-11).

   c.  For Series 1 Tests, coupons were to remain  wetted for the entire duration of the exposure time
       (10 min). If the coupons appeared to dry  before the 10 minutes were up, they were sprayed
       again.  For Series 2 Tests, a 30-minute exposure time was required. The coupons were not
       required to remain wet for the entire duration of the exposure time.
                                              27

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    d.  After the specified exposure time (10 minutes or 30 minutes), the coupons were extracted
       aseptically using sterile thumb forceps and transferred to the coupon collection vial.

    e.  Final weights of the collection vials were recorded. The net volume of runoff collected was
       calculated from the recorded weights.

A stoichiometric equivalent (X) portion of neutralization solution was determined for the highest sprayed
volume of runoff collected independent of material type. The neutralization solution volume (containing
the required mass of baking soda to achieve a pH between 5 and 8.5) was adjusted  to achieve a  10X
STS strength. The funnels were rinsed with sterile Dl until a total of 20 ml was reached.

Four additional preliminary tests (Tests A-D) were conducted to determine the proper neutralization
procedures:

      Test A: Neutralization Effectiveness

      Test B: Neutralizer Toxicity

      Test C: Organism Viability

      Test D: Material Control

Tests (A-D) were similar to the method development tests that were employed in the first phase of this
project.

2.4.4  Spray Down Decontamination Procedure
The general decontamination procedure consisted of spraying  the coupons set up in the spray apparatus
and was conducted as follows:

    a.  The concentrations of PAA and HP for the decontamination solution were measured.

    b.  The neutralizer solution (an aqueous solution  containing baking soda and the appropriate amount
       of STS solution) was pre-loaded onto the coupon and rinsate collection vials. The neutralization
       solution volume (containing the required amount of baking soda to achieve a pH range of 5.5-8.5)
       was manipulated so that the STS was at 10X  strength, and the coupons were completely
       immersed in the decontamination/neutralizer solution mix without risk of overflow in the conical
       tubes.

    c.  Coupons were then sprayed with the decontamination  solution.

    d.  After the required contact time with the decontaminant solution, the coupons were aseptically
       transferred into each of their respective coupon collection vials (pre-loaded with PBST and the
       required amount of neutralizer totaling a volume of 26  ml).

    e.  The funnels were rinsed with sterile Dl until a  total of 20 ml was reached. After an  overall holding
       time of 30 minutes post extraction, the samples were subjected to a ten-minute sonication step,
       followed immediately by two minutes of continuous vortexing on setting 10.
                                              28

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    f.  All samples were then homogenized for one 15-second burst using a vortex mixer on setting 10
       prior to a tenfold serial dilution with spread-plating. In addition, a 1ml_ aliquot was removed for
       filter-plating.

    g.  The plates were placed into an incubator for 18 to 24 hours at 35  2 C, following which the CPU
       were enumerated.

2.4.5  Spray Down Decontamination Test Matrix
Two series of spray down decontamination tests were performed as part of this study.  In both series of
tests, OTC PAA decontamination formulation was sprayed onto coupons of carpet, wood, glass, vinyl
flooring and concrete. The duration and frequency of each decontaminant spray in both series of tests is
listed in Table 2-10 and 2-11. The adjustable spray wash bottle used in these approaches was
maintained at 20 psi to ensure a consistent spray each time. Because the coupons were to remain wetted
with the decontaminant solution for the entire exposure time of 10 minutes (for Series 1 tests only),
concrete (a porous, fast absorbing material) required more sprays as compared to the  other materials.
Tables 2-12 and 2-13 list the neutralizer amounts added to each of the five material sample types for
Series Tests 1 and 2, respectively.

Table 2-10.  OTC PAA Formulation - Spray Duration and Frequency (Series 1)
Material
Glass
Wood
Concrete
Carpet
Vinyl Flooring
Duration of Spray (sec)
2
2
2
2
2
Frequency
1 -Once every 10 minutes
1 -Once every 10 minutes
3 - Once every 3 minutes
1 -Once every 10 minutes
1 -Once every 10 minutes
                                              29

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Table 2-11.  OTC PAA Formulation - Spray Duration and Frequency (Series 2)
Material type
concrete
wood
carpet
glass
vinyl flooring
Number of Sprays (#)
1
2
2
4
3
4
1
2
2
4
3
4
1
2
2
4
3
4
1
2
2
4
3
4
1
2
2
4
3
4
Spray Frequency (min)
1 at time 0
1 at time 0 and 1 at 15 min
2 at time 0
2 at time 0, and 2 at 15 min
3 at time 0
4 at time 0
1 at time 0
1 at time 0 and 1 at 15 min
2 at time 0
2 at time 0, and 2 at 15 min
3 at time 0
4 at time 0
1 at time 0
1 at time 0 and 1 at 15 min
2 at time 0
2 at time 0, and 2 at 15 min
3 at time 0
2 at time 0, and 2 at 15 min
1 at time 0
1 at time 0 and 1 at 15 min
2 at time 0
2 at time 0, and 2 at 15 min
3 at time 0
4 at time 0
1 at time 0
1 at time 0 and 1 at 15 min
2 at time 0
2 at time 0, and 2 at 15 min
3 at time 0
4 at time 0
                                              30

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Table 2-12.  Neutralizer Amounts Added To Samples -Spray Down Approach (Series 1)
Material
Glass
Wood
Concrete
Carpet
Vinyl Flooring
STS
Strength
10X
10X
10X
10X
10X
Coupon (ml)
STS (0.1 N)
0.3
0.8
1.75
2.0
0.3
PBST
25.7
25.2
23.25
24.0
25.7
Total Volume
(ml)
26
26
26
26
26
Rinsate (ml)
STS(1.5N)
0.8
1.2
1.9
0.8
0.9
Baking Soda (g)
0.1
0.2
0.35
0.1
0.1
Total Volume
(ml)
20
20
20
20
20
2.5   Task 5: Material Compatibility Evaluation
The in-house PAA formulations are very acidic (pH <2). To analyze the effects of the acidity of these
formulations, a material compatibility test was designed to assess the corrosion or changes of the OTC
PAA formulation at different pH levels. This study was performed on 10 cm x 10 cm coupons made of
stainless steel, galvanized metal, aluminum and carbon steel. These coupons, tested in duplicate,
followed two test procedures: (1) post-rinse procedure, and (2) no post-rinse procedure. In the post-rinse
procedure, two coupons of each material were immersed in the original OTC PAA  formulation. The
coupons were rinsed with Dl water and left to dry after a contact time of 30 minutes with the
decontaminant. In the no post-rinse procedure, two coupons of each material were immersed in OTC
solution and left to dry without a Dl water rinse after a contact time of 30 minutes with the decontaminant.
The original  OTC solution has a pH of 1.7 -1.8 (~2).  Further assessment on the coupons was conducted
using the OTC solution at a higher pH of 4 and 6 to determine the extent of the acidity of the solution on
material compatibility. These higher pH values were achieved by adding baking soda (sodium
bicarbonate) to the OC solution. Visual and gravimetric analyses were performed on the coupons on a
weekly basis for a total assessment period of eight weeks. Temperature and relative humidity (RH) inside
the laboratory were monitored using a HOBO Data Logger U12 Series (Onset Computer Corporation,
Bourne, MA) throughout the eight-week assessment period.
                                              31

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3    Sampling and Analytical Procedures

3.1   Sampling Procedures
Within a single test, sampling was completed first for all procedural blank samples before sampling of any
test material. Prior to the sampling event, all materials needed for sampling were prepared using aseptic
techniques. The order of sampling was performed as follows: (1) all blank samples, (2) all
decontaminated samples, and (3) all positive control samples. This order ensures that coupons are
handled in an order from the least level of contamination to the most.. The general sampling supplies
were purchased sterile or were sterilized/disinfected prior to each sampling event. After sample collection
for a single test was complete, all biological samples were extracted typically within ten minutes.

3.2   Sample Extraction/Analysis
3.2.1  Liquid-Liquid Decontamination
For the neutralizer evaluation testing, the microbial decontamination/sterile PBST/neutralizer microbial
suspension solution was immediately homogenized for 15 seconds using a vortex mixer. A 1-mL
undiluted aliquot was  removed and aseptically transferred in triplicate to three tubes with 10 ml of PBST.
These three 11-mL tubes were homogenized using a vortex mixer for 15 seconds and then each solution
was aseptically transferred to an individual Nalgene filter unit, cat no.  130-4020 (Thermo Fisher Scientific,
Waltham, MA) where  the samples were filtered. Immediately following filtration, the filter unit was rinsed
using another 10 ml of PBST, which was also filtered. Each filter was aseptically removed using sterile
thumb forceps and placed onto a pre-labeled TSA media plate.

For the decontamination effectiveness testing (starting with a high microbial concentration), the solution
was immediately homogenized for 15 seconds using a vortex mixer. A 0.1-ml  aliquot was removed and
aseptically dilution-plated onto a pre-labeled TSA media plate. Further, three 1-mL undiluted aliquots
were removed in parallel with the 0.1-ml aliquot and aseptically transferred in triplicate to three tubes with
10 ml of PBST. These three 11 -ml tubes were homogenized for 15-seconds using a vortex mixer and
then aseptically transferred to an individual Nalgene filter unit for the filter plating sequence. Samples
from the neutralization Tests A through D were filtered in triplicate and the plates were incubated
overnight at 35 2 C.

3.2.2  Surface Decontamination
After the required contact time of 10 minutes with the decontaminant solution, the coupons were
aseptically transferred into each of their respective coupon collection vials (pre-loaded with PBST and the
required amount of neutralizer totaling a volume of 26 ml). After an overall holding time of 30 minutes
post extraction, the samples were subjected to a 10-minute sonication step, followed immediately by two
minutes of continuous vortexing at the maximum rotational speed. Prior to plating, all samples were then
homogenized for one  15-second burst using a vortex mixer at the maximum rotational speed.

3.3   Sample Analysis
Decontamination samples were  subjected to up to five-stage tenfold serial dilutions (10~1 to 10~5). The
resulting samples were plated in triplicate and incubated overnight at 35  2 C. Following incubation,
CFU were manually enumerated. The samples resulting in fewer than the reportable limit of 30 CFU/plate
on the undiluted sample underwent further analysis. When no detectable spores were detected from the

                                              32

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filter-plating, a value of 0.5 CFU/sample was assigned as the detection limit for efficacy determinations for
purposes of calculating LR.

3.4   Data Analysis

The total spore recovery for each test method was calculated by multiplying the mean CPU counts from
triplicate plates by the inverse of the volume plated (e.g., 1/0.1 ml or 10), by the dilution factor, and finally
by the volume of the sample extract as illustrated in Equation 3-1.

               average CPU from
  CPU    replicate dilution plates or on filter           1         ,   .     .    .        r x         , ,. N
	r =    ;	;.   ,.,	-H-: x       :	 x(extract vo lume, ml)         (3-1)
sample      volume plated or filtered,mL      (tube dilution factor)
3.5   Efficacy Testing
D-value, defined here as the time required to reduce the population of the target organism by 1 log (90 %)
and temporal CPU LR were used to determine the efficacy of each decontamination solution.

3.6   Decontamination Efficacy
The sporicidal effectiveness (efficacy) of a decontamination technique is a measure of the ability of the
method to inactivate the spores. The sporicidal effectiveness is evaluated by measuring the difference in
the logarithm of the measured CPU before decontamination  (determined from sampling positive control
coupons) and after decontamination (determined from the sampling of the test samples) for the same
type of material (represented by the neutralizer/decontamination suspension). The number of viable
spores recovered was measured as CPU and reported as a  LR for each specific decontamination solution
as defined in the Equation 3-2.
    n =^ -- -^ -                                               (3-2)
     ''         Nc               Ns

where:

               j,    _    Decontamination effectiveness; the average LR of spores for a
                ''        specific solution (designated by ;)
 NC
 V loe,(CFU   }         ~*~he avera9e f tne logarithm (or geometric mean) of the number of
 _i=i _ _    =    viable spores (determined by CPU) recovered on the positive controls
      Nc                (C indicates control and Nc is the number of positive controls)
 N,                      The average of the logarithm (or geometric mean) of the number of
 2_\og(CFU sk}    ^    viable spores (determined by CPU) remaining in the decontamination
  -         solution (S indicates a decontaminated solution and Ns is the number
       N
         s                of test samples).
                                              33

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The standard deviation of the average log reduction of spores corresponding to a specific
decontamination solution is calculated by Equation 3-3.
                 Ns-l
                                                                                      (3-3)
where:

  SZ>
         _
       ''

              Standard deviation of r|j, the average LR of spores for a
              specific solution

              The average LR of spores for a specific solution type
              designated by ;

              The average of the LR for a specific test sample (Equation 3-
              3)

              Number of test samples for a specific decontamination
              solution
and,
where:
log(CFUc) = 
             log(CTC7Cjt)
                   N
                     c
                       CFUS k

                           Ns
                                   Represents the "mean of the logs" (geometric mean),
                                   the average of the logarithm-transformed number of
                                   viable spores (determined by CPU) recovered on the
                                   positive controls (C = control samples, Nc = number of
                                   control samples, k = test sample number and Ns is the
                                   number of test samples)

                               =   Number of CPU in the kth decontaminated sample

                               =   Total number (1 ,k) of decontaminated samples.
                                                                                      (3-4)
                                             34

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In this report, decontamination efficacy is generally reported in terms of LR for a particular
decontamination formulation. We also occasionally report results by noting whether the average LR for a
particular test is > 6.0, since a decontaminant that achieves > 6 LR is considered effective ^

3.7  Sample Preservation
The presence of decontamination solution components in the test solution could negatively bias spore
recovery tests. Therefore, the coupon solutions were neutralized  immediately after the exposure time (ten
minutes) and plated within 30 minutes, which is the longest holding time set for the neutralization tests.

The prepared decontamination samples were stored in sealed 1-L jars and aseptically opened only for
sample retrieval for chemical analysis or testing. All samples were stored in the refrigerator at the  EPA
RTP facility Microbiology Laboratory at 4 C  2 C until further analysis was required.

3.8  Holding Times
The sample collection and analysis occurred in the NHSRC RTP Microbiology Laboratory. After sample
collection for a single test was complete, all biological samples were transferred to the microbiology
analyst for immediate extraction with typical holding times not exceeding ten minutes.

3.9  Sample Archival
Samples were archived by maintaining the primary extract at 4 C  2 C in a sealed extraction tube  until
the data set had been quality controlled and the sample released for disposal. Any deviations from
sampling protocols were documented in the laboratory notebook. Sampling duration, time of day, and
observations were also recorded in the laboratory notebook.

3.10 Steady State  Conditions
Decontamination solutions are not expected to be stable overtime. Solutions were prepared and
monitored overtime  until they reached a pseudo-steady state with a drift in PAA concentration not
exceeding 10 % of the  maximum concentration within one hour of testing.  The prepared decontamination
samples were stored in sealed 1 -L jars and opened only for aseptic samples retrieval for chemical
analysis or testing.

3.11 PAA  and HP Characterization  Measurements
The concentration of PAA and HP in the PAA formulations was determined using an iodometric
titration [12].
                                              35

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4      Results and Discussion

This section discusses results of the decontamination solution characterization tests (Section 4.1),
neutralization characterization testing (Section 4.2), liquid-liquid decontamination testing (Section 4.3),
and material compatibility testing (Section 4.4).

4.1   Formulation Characterization Results
The pH and concentration of HP and PAA in the working decontamination solutions were considered
critical to the understanding of the dynamic changes of key decontamination agents (HP, PAA) in the
formulations. These parameters were monitored throughout the entire testing (immediately after mixing
and before each application of decontamination solution).

4.1.1   "In-House" PAA Formulations
The target maximum concentration for the in-house PAA formulations (with and without sulfuric acid) was
set at 1.5 % PAA (0.2 mol/L). The diluted HP formulation reached its target concentration of 4.5 % HP
immediately post-mixing. The pH of this decontamination formulation was 4.7.

The in-house PAA with sulfuric  acid formulation reached its target maximum concentration and
equilibrium in approximately six days, while the in-house PAA without sulfuric acid formulation reached its
target maximum concentration and equilibrium in 31 days.  Once equilibrium was reached, the solutions
were observed to  be stable over the duration of the tests (over two months) with minor shifts in PAA and
HP concentrations (less than 10 % deviation). The average pH of the PAA with sulfuric acid formulation
was 1.05, while the average pH of the  PAA without sulfuric acid formulation was 1.9 during testing. The
temporal product/reactant evolution for the in-house PAA formulations with and without sulfuric acid is
presented in Figures 4-1  and 4-2, respectively.
                                              36

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                 Model HP results
                 Model PAA results
             ^   Actual HP solution results
             o   Actual PAA solution results
         0      100      200      300      400     500     600      700

             Time since start of the formulation preparation (hrs)


Figure 4-1.    Temporal Product/Reactant Evolution for the In-House PAA Formulation with Sulfuric Acid


       7-
       6-
    ^  s-\
    KM* U 
1 4^


8 :
    re
    a-  i-\
       0-
                        Model HP results
                        Model PAA results
                        Actual HP solution results
                        Actual PAA solution results
         0       100      200     300     400     500     600     700

             Time since start of the formulation preparartion (hrs)


Figure 4-2.    Temporal Product/Reactant Evolution - In-House PAA without Sulfuric Acid
                                              37

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4.1.2  Modified AHP Decontamination Formulation
The modified AHP decontamination formulation developed by SNL reached its target PAA concentration
of 1.5 % immediately after mixing. However, the solution was highly unstable, and the concentration of
both  PAA and HP dropped continuously, as illustrated in Figure 4-3. This drop is operationally important,
as low PAA concentrations affect the decontamination efficacy of the modified AHP solution against 8.
atrophaeus (Ba) spores (Section 4.3.3). PAA concentrations below 0.1 % produced inadequate spore
reductions (< 6LR). These results are discussed in further detail in Section 4.3.
      6-
      5-
    T3


    I
      3-
      2-
      1-
      0-
-- HP for target PAA of 1.5%
-- HP for target PAA of 1.0%
-o- PAA target of 1.5%
-- PAA target of 1.0%
                          PH = 7.4|
                    5       10       15       20
                 Time since start of the formulation (hrs)
                                                    25
Figure 4-3.   PAA and HP Concentration for the Modified AHP Formulation
4.1.3  Over-The-Counter (OTC) In-house PAA Formulation
PAA, pH, and HP concentrations of the OTC formulation were measured overtime to determine the
kinetics and stability of the mixture (potential loss of active ingredients by decomposition and
evaporation). The developed kinetic model reasonably predicted the  PAA formation rate from solution
prepared from the OTC products (Figure 4-4). The target maximum concentration (0.1 %) and equilibrium
were achieved in 25 hours. The OTC solution had a pH of 1.7 during  decontamination testing.
                                             38

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       0.12-
       0.10-
       0.08-
       o.oe-
       0.04-
       0.02-1
       0.00-
                                   Actual PAA results
                                   Model PAA results
           0      20     40     60     80     100     120     140
               Time since start of the formulation preparation (hrs)

Figure 4-4.   Temporal Product/Reactant Evolution - OTC PAA

The stability of the PAA formulations made, respectively, from laboratory grade ingredients (in-house PAA
with sulfuric acid) and from OTC ingredients was evaluated during the spraying down procedure. Initial
HP and PAA concentrations of both formulations were measured before proceeding to the spray
application. Each formulation was then sprayed using an adjustable spray wash bottle fitted with a
pressure gauge to help ensure uniform pressure during each spray. The sprayer pressure was kept
constant at 20 psi. The sprayer-dispersed liquid was collected in conical tubes, which were also used for
runoff/rinsate collection during decontamination testing. To determine the residual concentration of the
active ingredients in the two formulations, the collected solutions were titrated immediately post-spraying
(0 minutes) and subsequently at 15 minutes, 30 minutes and 60 minutes. The spraying is shown to have
a remarkable effect on the degradation of the PAA concentration from the OTC formulation compared
with negligible to no degradation of the PAA from the formulation made from laboratory grade ingredients
(Figure 4-5). This OTC PAA degradation during the spraying will be shown later to result in dramatic
reduction in the sporicidal efficacy of the OTC formulation during surface decontamination.
                                               39

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        0.11
        0.10-
        0.09-
        0.08-
        0.07-
     O 0.06-
        0.05-
        0.04-
        0.03.
                                            SA Formulation
                                            OTC Formulation
                     Time delay after the spraying process
Figure 4-5.    Temporal PAA Degradation over Time for the OTC and PAA with Sulfuric Acid Formulations

4.2   Neutralization Testing Results
Tables 4-1 through 4-3 in the following sections show the results from method development for
neutralization using STS as the neutralizing agent for the decontamination formulations. Neutralization
tests (Tests A-D, as described in Section 2.2) were conducted with three of the five decontamination
formulations used in this project: in-house formulation with sulfuric acid, modified AHP formulation, and
the diluted HP formulation. Neutralization testing was not performed on the in-house PAA formulation
without sulfuric acid or on the OTC PAA formulation since it was assumed that these solutions would
follow the same decontamination trend as the PAA without sulfuric acid formulation. The samples tested
were allowed to stand for a holding time of 30 minutes before being filter plated.

For neutralization tests, all PAA stock solutions tested were diluted to two concentrations,  1 % PAA and
0.001 %  PAA. The values represent the highest and the lowest PAA  concentrations used in efficacy
testing for this work. Diluted HP stock solution was tested as is and with 4.5 % HP.

4.2.1  Neutralization Effectiveness
Neutralization Test A determined the NE to stop the activity of the decontamination solution. The results
for these tests are presented in Table 4-1 for the three formulations cited above.

Initial Neutralizer Effectiveness (Test A) tests for the AHP formulation also showed that the 2X neutralizer
with STS and PBST alone was insufficient. In addition to PAA and  HP, the modified AHP formulation
contains  QACs that have microbicidal properties. To neutralize these QACs, a solution of lecithin in
TWEEN80 was used. Analysis of the data from neutralization tests  determined that 2X neutralizer
                                               40

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worked effectively, inactivating 1 % PAA concentrations in all formulations evaluated. For the 0.001 %
dilution, STS neutralizer of strength 1000X was also found to be an effective neutralizing agent.

Table 4-1.      Neutralization Effectiveness (Test A, In-house PAA Formulation with Sulfuric Acid
(SA), Modified AHP Decontamination Formulation Developed by SNL (SD), HP solution alone
without PAA (HP))
Formulation
Type
SA
SD+
HP
Concentration
1.0%
0.001 %
1.0%
0.001 %
4.5 %
Positive Controls
(three replicates)
Average
7.41 x102
8.01 x102
9.02 x102
Stdev
6.09 x101
5.92 x101
1.74x102
Stoichiometric
Ratio
1X
2X*
5X
1000X
1X
2X
5X
1000X
1X
2X
5X
Recovery (CFU)
(five replicates)
Average
2.76 x102
7.53 x102
3.13x102
5.39 x102
4.92 x102
9.60 x102
8.28 x102
7.71 x102
7.10x102
8.65 x102
5.96 x102
Stdev
1.76x102
1.38x102
3.03 x101
2.10x101
1.63 x102
1.31 x102
3.64 x101
4.77 x101
6.41 x102
1.08x102
3.64 x101
%
37
100
42
73
61
120
100
96
79
96
66
*Represent CFU counts from Test A repeat for 1 %
+Test A for SD was repeated with lecithin added to
 PAA (SA) evaluated at 2X.
neutralize QACs present in decontaminant
4.2.2  Neutralizer Toxicity
The neutralizer toxicity (Test B) determined the growth inhibition caused by the neutralizer on the test
organisms. The neutralizer toxicity (NT) was determined as the ratio between the recoveries of a
population exposed to the neutralizer to a population that was not exposed to the neutralizer. The results
of the neutralizer toxicity are presented in Table 4-2. The results confirm that the neutralizer used in these
tests did not have a sporicidal or growth-inhibiting effect on the spores.
                                              41

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Table 4-2.   Neutralizer Toxicity (Test B)
Formulation
Type
SA
SD
HP
Positive Controls
(three replicates)
Avg CPU
6.50 x102
7.91 x102
9.02 x102
Stdev
5.74 x101
5.74 x101
1.74x102
Stoichiometric
Ratio
1X
2X
5X
1X
2X
5X
1X
2X
5X
Recovery (CPU)
(five replicates)
Avg CPU
5.66 x102
5.79 x102
6.03 x102
7.64 x102
7.44 x102
8.28 x102
9.29 x102
8.25 x102
7.98 x102
Stdev
8.80 x101
6.72 x101
1.20x102
2.54 x101
1.11x102
7.28 x101
5.25 x102
1.08x102
7.07 x101
%
87
89
93
97
94
110
100
92
89
4.2.3   Test Material Control
Test D determined the efficacy of the decontamination solution and demonstrated that the neutralizer was
effective in deactivating the decontamination solution. Analysis of the data from neutralization tests (Table
4-3) demonstrated that the 0.001  % PAA solution was an ineffective decontaminant, while the 1 % PAA
solutions and the diluted HP solution proved to be effective when no neutralizer was added. The
combined results from Test A and Test D demonstrate that STS effectively quenched  the decontaminant
solution.

Table 4-3.   Test Material Control (Test D)
Formulation
Type
SA
SD
HP
Concentration
1.0%
0.001 %
1.0%
0.001 %
4.5 %
Positive Controls
(three replicates)
Avg CFY
6.50 x102
7.91 x102
9.02 x102
Stdev
5.74 x101
5.74 x101
1.74x102
Stoichiometric
Ratio
OX
OX
ox
ox
ox
Recovery (CPU)
(five replicates)
Avg CPU
ND
6.30 x102
ND
7.24 x102
ND
Stdev
-
1.26x102
-
7.65 x101
-
%
0.00
97.0
0.00
92.0
0.00
ND = non-detect
                                               42

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4.3   Task 3: Liquid-Liquid Decontamination Testing
Prior to each liquid-liquid decontamination efficacy test, the formulations were checked for stability and
compared to the target set conditions. The results of these measurements are presented in Table 4-4.

Table 4-4.   Test Parameters Measurements Verification
Decontamination
PAAwith sulfuric
acid
PAAwith sulfuric
acid
PAAwith sulfuric
acid
PAAwith sulfuric
acid
HP
Modified AHP
Modified AHP
PAA without
sulfuric acid
PAA without
sulfuric acid
PAA without
sulfuric acid
OTC
Test
ID
1
2
3
4
0
1
2
1
2
3
0
PAA Concentration
Target
Value
(%)
1
0.1
0.01
0.001
4.5
1
0.1
1
0.1
0.01
0.1
Test
Value
(%)
1.04
0.11
0.012
0.0014
4.52
1.08
0.11
1.04
0.11
0.012
0.09
Frequency
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
PH
Test
Value
1.42
2.38
3.09
3.62
4.68
8.7
8.9
2.15
2.6
3.4
1.7
Temp
C
25
25
25
25
25
21.3
25
25
25
25
25
Frequency
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
Once before
testing
4.3.1   In-House PAA Formulation with Sulfuric Acid
Decontamination efficacy is dependent upon the concentration of active ingredients in the sporicidal
solution (PAA and HP, in this case), pH, and exposure times. The results of the liquid-liquid
decontamination approach are presented in Table 4-5 and illustrated in Figure 4-6. An 8 LR was achieved
with the high PAA concentration (1  %) even for an exposure time of two minutes. The pH of the 1 % PAA
with sulfuric acid formulation was 1. For the 0.1% PAA with sulfuric acid dilution, longer exposure times
helped achieve a greater than 6 LR. The pH of the 0.1 % PAA with sulfuric acid solution was 2.4 during
efficacy testing. The 0.01 % dilutions (pH 3.1) and 0.001 % dilutions (pH 3.6) were less compelling as
decontaminants, with LR values less than 1.
                                              43

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Table 4-5.   In-house PAA Formulation with Sulfuric Acid - Efficacy Testing
In House SA Formulation Decontamination Results
Concentration

0.001 %
0.01 %
0.1 %
1.0%
Positive
Controls(CFU)
(three replicates)
Average
4.00 x107
5.17 x107
3.87 x107
4.24 x107
Stdev
5.74x1 0s
1.40 x 10s
6.73x1 0s
2.63x1 0s
Time
(min)
2
4
6
8
2
4
6
8
2
4
6
8
16
20
2
4
6
8
Avg. Recovery (CFU)
(five replicates)
Average
1.74x107
1.67 x107
1.72x107
1.51 x107
4.30 x107
3.04 x107
2.76 x107
2.61 x107
4.88 x 10s
3.04 x107
1.09x102
1.83x102
<1. 01x10
<0.67x10
<1. 40x10
<0.60x10
<2.10x10
<3.70x10
Stdev
2.76x1 0s
1.10x106
2.97 x 10s
2.08 x 10s
2.85 x107
3.18x106
3.05 x 10s
1.49x106
4.29 x105
3.18x106
8.02 x101
3.25 x102
<0.73x10
<0.25x10
<2.01x10
<0.22x10
<1. 24x10
<6.88x10
LR
Average
0.37
0.38
0.37
0.43
0.14
0.23
0.28
0.30
0.90
2.3
5.7
6.4
7.7
7.8
7.7
7.9
7.4
7.6
Stdev
0.07
0.03
0.08
0.07
0.22
0.04
0.05
0.02
0.04
0.15
0.54
1.4
0.27
0.13
0.45
0.13
0.33
0.65
D-Value
(min)
5.5
11
16
19
15
17
22
27
2.2
1.7
1.1
1.2
2.1
2.5
0.26
0.51
0.81
1.1
                                                44

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       8-
    2  7
    "i   "
    3
    "S
    (  6-

    |,

    
    S4-
    12^
    8
       o-
                              10         15
                        Exposure Time (minutes)
20
Figure 4-6.   Temporal Efficacy of the In-House PAA Formulations with Sulfuric Acid

When 0.1 % and 0.01 % PAA solutions (in-house PAA with and without sulfuric acid) were mixed with
their neutralizer solutions (10X and 100X, respectively), the pH of the combined decontaminant/
neutralizer mix was significantly higher (pH > 9) than the individual components, even with no baking
soda added. The pH rise suggested that the PBST buffer in the neutralizer solution could have been
overwhelmed when mixed with the decontamination solution, causing the pH to rise drastically. Adding an
additional 10 ml of PBST to the neutralizer/decontamination solution mix solved this problem, and a
stable neutral pH was achieved.

Based on the decontamination efficacy trend, a near complete decontamination was observed at the two-
minute exposure time with the 1 % PAA formulation. The CPU recovery between the two and eight minute
exposure time lies within the uncertainty of the measurement technique, which suggests a calculated D-
value for this formulation of less than 0.26 min. The 0.1 % PAA also showed promise with an average D-
value calculated to be around 1.8 min, which suggests a 6-Log CPU reduction within an exposure time of
approximately 10 minutes. The 0.01 % and 0.001 % PAA formulations were found to be too dilute to have
an effective sporicidal activity, with D-values calculated at the maximum exposure time of eight minutes
being greater than 18 minutes.

4.3.2  In-House PAA Formulation  without Sulfuric Acid
The decontamination efficacy of the AD solution (Table 4-6, and Figure 4-7) was found to be very similar
to that observed for PAA with sulfuric acid formulation. Complete kill was achieved with the 1 % PAA
dilution (pH 1.9), while longer exposure times resulted in greater than 6 LR for the 0.1 % PAA dilution (pH
2.6). Because the 0.01 % PAA dilution (pH 3.4) did not have very effective sporicidal activity, the  lowest
PAA dilution (0.001 %) was not tested for efficacy.
                                              45

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Table 4-6.  In-house PAA Formulation without Sulfuric Acid - Efficacy Testing
"In House" AD Formulation Decontamination Results
Concentration

0.01 %
0.1 %
1.0%
Positive Controls(CFU)
(three replicates)
Average
4.48 x107
1.30x107
4.39 x107
Stdev
5.23x1 0s
1.31 x106
5.10x106
Time
(min)
2
4
6
8
16
20
2
4
6
8
16
20
2
4
6
8
Avg. Recovery (CFU)
(five replicates)
Average
6.21 x106
6.81 x 10s
5.45x1 0s
4.82x1 0s
4.67x1 0s
4.61 x 10s
2.14 x105
6.81 x 10s
5.03x10
1.21 x101
0.50x10
1.20x10
0.50x10
0.50x10
0.50x10
0.50x10
Stdev
7.52 x105
6.55 x105
5.86 x105
5.23 x105
3.94 x105
8.67 x105
2.99 x104
6.55 x105
0.00x10
1.57x101
0.00x10
1.04x10
0.00x10
0.00x10
0.00x10
0.00x10
LR
Average
0.86
0.82
0.92
0.97
0.98
0.99
1.7
6.7
6.4
6.2
7.4
7.1
7.9
7.9
7.9
7.9
Stdev
0.05
0.04
0.05
0.05
0.04
0.08
0.06
0.97
0.00
0.40
0.00
0.32
0.00
0.00
0.00
0.00
D-Value
(min)
2.3
4.9
6.5
8.3
16
20
1.1
0.59
0.94
1.3
2.2
2.8
0.25
0.50
0.75
1.0
                                                46

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      7-
      3-
      2-
                        I
                                0.01 %PAA
                            I	I0.1%PAA
                                1.0% PAA
                            10         15
                      Exposure Time (minutes)
Figure 4-7.   Temporal Efficacy of the In-House PAA Formulations wthout Sulfuric Acid

4.3.3  Modified AHP Formulation
Table 4-7 summarizes LR values for the SNL-modified AHP formulation. These data suggest that AHP-
based liquid sporicide provides greater than 6 LR of spores when the concentration of the formulation is
at 1 % PAA. However, the 0.1 % dilution proved to be ineffective, even with extended exposure times.
Because the 0.1 % solution was  ineffective at complete decontamination, lower dilutions of 0.01 % PAA
and 0.001  % PAA were not tested for decontamination efficacy. The pH of the 1  % PAA dilution and 0.1
% PAA dilution during efficacy testing were 8.7  and 9.1, respectively.
                                              47

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Table 4-7.   Modified AHP Formulation - Efficacy Testing
Modified AHP Formulation Decontamination Results
Concentration

0.1 %
1 %
Positive Controls(CFU)
(three replicates)
Average
1.64x107
5.70 x107
Stdev
1.17x105
3.05 x 10s
Time
(min)
2
4
6
8
16
20
2
4
6
8
Avg. Recovery (CFU)
(five replicates)
Average
2.27 x107
1.86x107
1.68 x107
1.52x107
1.61 x107
1.57x107
1.91x101
3.77x10
1.91x10
4.35 x101
Stdev
7.57x1 0s
2.88x1 0s
1.09x106
1.24x106
1.20x106
2.39x1 0s
3.34 x101
5.07x10
1.33x10
7.54 x101
LR
Average
-0.13
-0.05
-0.01
0.03
0.01
0.02
6.9
7.5
7.6
6.7
Stdev
0.13
0.06
0.03
0.03
0.03
0.06
0.66
0.58
0.38
0.81
D-Value
(min)
-
-
-
240
2000
870
0.29
0.53
0.79
1.2
4.3.4  Diluted Hydrogen Peroxide Formulation
Table 4-8 summarizes LR values for the diluted HP formulation. The diluted HP formulation is not
effective at decontaminating the spores. The pH of this formulation during decontamination testing was
4.7, suggesting that the sporicidal activity of the PAA solutions is due largely to the individual or
synergistic effects of the PAA. The D-value averaged approximately 18 min/log CFU, so longer contact
times will be required compared to other PAA formulations to achieve effective decontamination (>6 LR).
                                              48

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Table 4-8.   Diluted HP Formulation - Efficacy Testing
Diluted HP Formulation Decontamination Results
Concentration

4.510%
Positive Controls(CFU)
(three replicates)
Average
4.79 x107
Stdev
4.16x106
Time
(min)
2
4
6
8
Avg. Recovery (CFU)
(five replicates)
Average
4.23 x107
2.27 x107
1.88x107
1.78x107
Stdev
5.01 x 10s
2.75 x 10s
2.62x1 0s
1.01x106
LR
Average
0.06
0.33
0.41
0.43
Stdev
0.05
0.05
0.06
0.02
D-Value
(min)
25
12
15
19
4.3.5  OTC Formulation
4.3.5.1   Liquid-Liquid Decontamination Approach
The OTC formulation provided nearly complete kill for exposure times of eight minutes or longer (Table 4-
9). Further extending the contact time of spores with the decontamination solution should result in
complete kill based on the efficacy trend. As this solution is made from OTC and off-the-shelf common
household products, this formulation could be a promising decontaminantthat can be readily and widely
available sporicide.  . However, the pH of this solution is low (1.7), indicating that the solution could
potentially corrode some surfaces and be hazardous to the users. The 0.1 % OTC solution showed
promise with greater than 6-Log CFU reduction found with  eight minutes exposure time.

Table 4-9.   OTC PAA Formulation - Efficacy testing (Liquid-Liquid Decontamination)
In-house OTC Liquid-Liquid Decontamination Results
Concentration

0.100%
Positive Controls(CFU)
(three replicates)
Average
5.17x107
Stdev
4.84x1 0s
Time
(min)
2
4
8
16
20
Avg. Recovery (CFU)
(five replicates)
Average
8.69 x105
1.76x104
0.57x10
0.56x10
0.56x10
Stdev
1.01 x105
9.15x103
0.70x10'2
0.00x10
0.00x10
LR
Average
0.18
3.5
8.0
8.0
8.0
Stdev
3.5
0.21
0.00
0.00
0.00
D-Value
(min)
1.1
1.1
1.0
2.0
2.5
                                              49

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4.3.5.2   Surface Decontamination Approach
Prior to each surface decontamination efficacy test, the formulations were checked for stability and
compared to the target set conditions. The results of these measurements are presented in Tables 4-10
and 4-11. As the results in these tables establish, there was no significant difference in how the
decontamination solution changed with respect to test parameters (PAA concentration and pH).

Table 4-10. Test Parameters Measurements Verification (Series 1)
Decon
OTC
OTC
OTC
OTC
OTC
Test
Material
WO
VF
GL
CO
CA
PAA Concentration
Target
Value (%)
0.07
0.07
0.07
0.07
0.07
Test
Value (%)
0.08
0.08
0.076
0.076
0.069
Frequency
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
PH
Test
Value
1.82
1.82
1.9
1.9
1.79
Temp
C
21.9
21.9
21.9
21.9
21.7
Frequency
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Table 4-11.  Test Parameter Measurement Verification (Series 2)
Decontamination
OTC
OTC
OTC
OTC
OTC
OTC
OTC
OTC
OTC
OTC
OTC
OTC
Test
Material
WO
WO
wo
VF
VF
VF
GL
GL
CO
CO
CA
CA
Test ID
11, 12
21,22,31
41
22,31
11, 12
21,41
11, 12,21
22,31,41
11, 12,21
22,31,41
11, 12,21
22,31,41
PAA Concentration
Target
Value (%)
0.07
0.07
0.07
0.07
0.07
0.07
0.07
0.07
0.07
0.07
0.07
0.07
Test
Value (%)
0.076
0.076
0.072
0.065
0.071
0.072
0.64
0.067
0.070
0.068
0.066
0.068
Frequency
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
PH
Test
Value
1.89
1.82
1.91
1.84
1.90
1.91
1.84
1.87
1.82
1.79
1.85
1.81
Temp
C
21.9
22.1
22.2
20.9
21.7
22.2
22.0
21.9
21.4
21.9
22.3
22.1
Frequency
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
Once before testing
The efficacy test results for Series 1 Tests, as outlined in Table 4-12, demonstrated that a ten-minute
exposure time was not sufficient to achieve full decontamination using the spray-down approach. Thus a
second series of tests (Series 2) was conducted to determine the effect of increased exposure time and
frequency of spraying. The efficacy test results for Series 2 Tests are outlined in Table 4-13. These tests
demonstrated that the increased exposure time (30 minutes) also contributed to the overall surface
                                               50

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decontamination against the spores. Further, frequent spraying was observed to result in greater
decontamination efficacy as compared to multiple sprays at the same time.

Both series of tests using the spray-down approach didn't prove sufficiently efficacious in achieving the
target 6 LR. These lower surface decontamination efficiencies were due to the high degradation of the
OTC PAA concentration observed during the spraying. The surface decontamination approach can be
improved significantly if the OTC formulation is further stabilized by using laboratory grade ingredients or
stabilizing the formulation by using other OTC ingredients. Further studies would have to be conducted to
determine the cause of the OTC degradation.

Table 4-12. OTC PAA Formulation - Efficacy testing (Surface Decontamination - Series  1)
In-house OTC Surface Decontamination Results
Material
Type
Carpet
Concrete
Wood
Vinyl
Glass
Exposure
Time (min)
10
Positive Controls
(CFU)
Average
3.94 x107
5.82 x107
2.39 x107
3.51 x107
2.91 x107
Stdev
6.34x1 0s
6.31 x 10s
1.77x106
1.63x107
1.57x107
Avg. Recovery (CFU)
Average
1.56x107
2.57x1 0s
3.36 x105
3.60x1 0s
1.37x106
Stdev
6.56x1 0s
1.41 x 10s
1.66 x105
6.15x106
1.01x106
LR
Average
0.33
1.4
1.9
1.5
1.4
Stdev
0.27
0.21
0.18
0.75
0.33
                                               51

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Table 4-13.  OTC PAA Formulation - Efficacy testing (Surface Decontamination - Series 2)
In House OTC Surface Decontamination Results -Amendment 3
Material
Carpet
Concrete
Glass
Vinyl
Flooring
Wood
Exposure
Time
(min)
30
Number and
Frequency of Sprays
1 at 0 min
1 at 0 min, 1 at 15 min
2 at 0 min
2 at 0 min, 2 at 15 min
3 at 0 min
4 at 0 min
2 at 0 min, 2 at 15 min
3 at 0 min
4 at 0 min
1 at 0 min
1 at 0 min, 1 at 15 min
2 at 0 min*
2 at 0 min, 2 at 15 min
3 at 0 min
4 at 0 min
1 at 0 min
1 atO min, 1 at 15
min**
2 at 0 min
4 at 0 min
3 at 0 min
2 at 0 min, 2 at 15 min
1 at 0 min
1 at 0 min, 1 at 15 min
2 at 0 min
2 at 0 min, 2 at 15 min
3 at 0 min
4 at 0 min
Positive controls (CFU)
avg
2.75 x107
3.21 x107
4.68 x107
2.09 x107
1.18x108
3.45 x107
1.81 x107
3.26 x107
5.48 x107
3.43 x107
stdev
4.97x1 0s
3.44x1 0s
1.11 x106
3.00x1 0s
1.21 x107
5.16 x 10s
4.84x1 0s
1.04x107
7.52x1 0s
4.39x1 0s
Avg CFU Recovered
avg
1.35x106
1.41 x105
1.57x106
2.68 x105
6.54 x105
8.11 x105
1.40 x107
2.29 x107
2.29 x107
4.39 x104
1.18x104
9.62 x104
1.62 x104
4.66 x104
1.02x106
3.30 x105
3.06 x103
3.41 x104
5.04 x104
1.44 x104
8.29 x104
1.21 x106
3.11 x104
2.35 x105
4.32 x104
4.04 x105
7.27 x104
stdev
1.40x106
4.78 x104
1.41x106
6.34 x104
3.71 x105
7.53 x105
1.12x107
6.34x1 0s
3.01 x 10s
7.98 x104
1.61x104
1.89x105
3.27 x104
2.35 x105
1.82x106
5.97 x105
2.68 x103
3.44 x104
4.01 x104
1.58x104
9.77 x104
1.27x106
3.80 x104
1.21x105
3.44 x104
4.32 x105
4.46 x104
LR
avg
1.5
2.3
1.4
2.1
1.8
1.7
0.7
0.3
0.3
3.3
4.9
3.5
5.3
3.5
2.6
2.6
4.2
2.9
2.6
3.7
3.0
2.0
3.7
2.2
3.0
2.1
2.7
stdev
0.49
0.14
0.45
0.09
0.26
0.32
0.47
0.12
0.06
0.80
2.0
1.2
1.8
0.61
0.73
0.72
0.33
0.38
0.30
0.80
0.73
0.86
0.89
0.30
0.32
0.48
0.25
'Average calculated from only four samples instead of five (possible contamination of one of the samples).
**28 and 29 CFU counted as valid results on one of the five samples.
                                                  52

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4.4   Material Compatibility Tests
Figures 4-8 through 4-14 show the results of the material compatibility tests. None of the test coupons
(subjected to either Post-Rinse or No Post-Rinse procedures) showed significant changes in mass. Any
changes in mass recorded were within 0.02 % of the initial coupon masses. Materials that followed No
Post-Rinse procedure appeared to have salt deposits (possibly arising from baking soda) on them.
Carbon steel was observed to have reacted most to the decontamination solution (with both procedures),
with a visible amount of rust and corrosion on its surface (Figures 4-8 and 4-10). Little change was
observed on the surfaces of stainless steel and aluminum coupons that were post rinsed after being
treated with the decontamination solutions. Typical indoor room conditions (70.8 F and 37.1 % RH) were
observed throughout the duration of the  Material Compatibility test.
Figure 4-8.    Corrosion on Carbon Steel with pH ~2 using Procedure 1 (Post-Rinse)
                                               t>
Figure 4-9.    Front Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse)
                                               53

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Figure 4-10.   Back Surface of Carbon Steel with pH 4 using Procedure 2 (No Post-Rinse)
Figure 4-11.   Front Surface of Carbon Steel with pH 6 using Procedure 2 (No Post-Rinse)
                                                54

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Figure 4-12.   Galvanized Metal with pH 2 using Procedure 1 (Post Rinse)
Figure 4-13.   Salt Deposition on Carbon Steel and Stainless Steel with pH 6 using Procedure 1
                                                55

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Figure 4-14.   Salt Deposition on Stainless Steel and Aluminum with pH 6 using Procedure 2

4.5   Results Summary
Based on the liquid-liquid decontamination testing approach evaluated under this study, PAA (at 0.1 % or
higher concentrations) shows strong efficacy against Ba spores. However, the techniques and
procedures used to decontaminate materials contaminated with Ba spores using this approach do not
effectively reflect the way in which decontaminants are applied. The spraying approach using readily
available equipment may increase readiness to respond to a wide-area containment release.

However, a concern regarding PAA usage, in the form of a spray, is its stability. When prepared using
chemicals available off-the-shelf (OTC PAA formulation), the PAA breaks down quickly (within an hour).
This deterioration has a direct effect on the potency of the solution overtime. Future study and research
is needed to  improve upon the spraying technique and stability of the OTC PAA formulation.

Although the PAA spray prepared using laboratory grade chemicals was observed to be stable overtime,
the practical use of this formulation is limited. Access to these laboratory grade chemicals is restricted.
Production and wide consumer access to supplies such as sulfuric acid would have to be enhanced.
Furthermore, PAA is a powerful oxidizer, and users will have to be thoroughly briefed on the safety
aspects of handling these chemicals.

The materials compatibility test performed under this study also suggests that users need to sufficiently
neutralize the decontaminant to prevent potential damage to the exposed surfaces. Environmental
precautions such as diluting the PAA with large amounts of water before allowing it to enter drains and
sewers will have to be addressed to reduce the risk of decomposition.
                                              56

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5      Quality Assurance

This project was performed under an approved Category III Quality Assurance Project Plan (QAPP)
entitled, "Parametric Testing of Decontamination Chemistries to Guide Decontamination Selection I:
Peracetic Acid" (July 2013).

All test activities were documented via narratives in laboratory notebooks and the use of digital
photography. The documentation included, but was not limited to, a record for each decontamination
procedure, any deviations from the Quality Assurance Project Plan (QAPP), and physical impacts on
materials.

5.1   Sampling, Monitoring, and Analysis Equipment Calibration
There were standard operating procedures for the maintenance and calibration of all laboratory and
microbiology laboratory equipment. All equipment was verified as being certified calibrated or having the
calibration validated by EPA's on-site (Research Triangle Park [RTP], NC) Metrology Laboratory at the
time of use. Standard laboratory equipment such as balances, pH meters, BSCs, and incubators were
routinely monitored for proper performance. Calibration of instruments was done at the frequency shown
in Table 5-1. If deficiencies were noted, the instrument was adjusted to meet calibration tolerances and
recalibrated within 24 hours. If tolerances were not met after recalibration, additional corrective action was
taken, possibly including, recalibration or/and replacement of the equipment.

Table 5-1.  Analysis Equipment Calibration Frequency
Equipment
pH meter
HOBO RH Sensor
Stopwatch
Micropipettes
Clock
Scale
Buret
Temperature of Incubation
Chamber
Calibration/Certification
Perform a two-point calibration with standard
buffers that bracket the target pH before each
use
Compare to calibrated RH sensor prior to use.
Compare against NIST* Official U.S. time at
http://nist.time.gov/timezone.cgi?Eastern/d/-
5/java once every 30 days
All micropipettes will be certified as calibrated
at least once per year. Pipettes are recalibrated
by gravimetric evaluation of pipette
performance to manufacturer's specifications
every six months by supplier (Rainin
Instruments)
Compare to office U.S. Time @ time.gov every
30 days
Check calibration with Class 2 weights
Manufacturer certified
Once a year against an NIST-traceable
thermometer
Expected Tolerance
 0.1 pH units
5%
1 min/30 days
5%
1 min/30 days
+ 0.1 % weight
0.1 ml_
+2C
*NIST = National Institute of Standards and Technology
                                              57

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5.2   Data Quality Objectives
The primary objective of this project was to develop an effective sporicidal low tech PAA formulation that
could easily be generated in the field, using over-the-counter ingredients. This formulation needs to reach
equilibrium within realistic (i.e., a few hours) time. The Data Quality Objectives (DQOs) assigned to this
study are summarized below:
    1.   Characterizing the decontamination solutions (four PAA formulations and one Hydrogen Peroxide
        formulation) by developing an  understanding of the time to reach equilibrium.
    2.   Assessing the relationship between PAA concentration, hydrogen peroxide concentration,
        exposure time, and sporicidal effectiveness.
    3.   Determining the fate of the spores during the decontamination processes (liquid-liquid and surface
        decontamination) and providing a 6-LR of 8. anthracis surrogate spores.
    4.   Assessing the corrosion of low PAA formulations derived from OTC products.
The operational parameters of decontamination (e.g., pH and temperature of decontamination solution
within the pot life  of the decontamination solution, the concentration of the PAA formulations, the exposure
times) were considered important in understanding the sporicidal activity of the PAA-based decontamination
process and were characterized in addition to sporicidal effectiveness.
5.3   Acceptance Criteria for Critical Measurements
The DQOs are used to determine the critical measurements needed to address the stated objectives and
specify tolerable levels of potential errors associated with simulating the prescribed decontamination
environments. The following measurements were deemed to be critical to accomplish part or all of the
project objectives:
        PAA concentration in the decontamination solutions
        HP concentration in the decontamination solutions
        pH
        Volume of the decontamination solution
        Volume of HP in the decontamination solution
        Volume of AA in the decontamination solution
        Volume of sulfuric acid in the decontamination solution
        Volumetric ratio of AA initial solution to HP initial solution
        Volume of the neutralizer solution
        CPU abundance per plate
        Decontamination time
        Decontamination solution stability overtime (drift)

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       Weight during titration
       Weight of the coupons from the material compatibility testing
Data Quality Indicators (DQIs) for the critical measurements were used to determine if the collected data
met the QAPP objectives. The critical measurement acceptance criteria is shown in Table 5-2.

Table 5-2.   Critical Measurement Acceptance Criteria
Measurement
Parameter
Time
Plated Volume
Dilution Ratio
Volumes
Weight
Counts of
CFU/plate
PH
PAA
HP
Analysis Method
NIST-calibrated
stopwatch
Pipette
Volumetric
Serological pipette
tips
Burettes
Scale
Manual counting
NIST-traceable
buffer solutions
lodometric Titration
per MOP 3200
lodometric Titration
per MOP 3200
Accuracy
 1 minute per hour
2%
0.2 %
0.1 ml_
0.1 ml_
NA
10% of all CPU
per plate between
first and second
count
0.1 pH units
NA
NA
Precision/
Repeatability
NA
1 %
NA
NA
NA
0.0001 g
50 % RSD between
triplicates for each
plate
NA
10%
10%
Detection
Limit
NA
NA
NA
NA
NA
NA
1 CPU
NA
0.001 %
NA
Completeness
(%)
100
100
100
100
100
100
100
100
100
100
NA = not applicable
RSD = relative standard deviation
Plated volume critical measurement goals were met. All pipettes are calibrated yearly by an outside
contractor (Calibrate, Inc, Carrboro, N.C).

Plates were quantitatively analyzed (CFU/plate) using a manual counting method. For each set of results
(per test), a second count was performed on 25 percent of the plates within the quantification range
(plates with 30-300 CFU). All second counts were found to be within 10 percent of the original count.

Many QA/QC checks were used to validate spore recovery measurements. These QA/QC checks include
samples that demonstrate the ability of the EPAS's onsite microbiology laboratory to culture and grow
spores as well as to demonstrate that materials used in this effort do not themselves contain spores. The
checks included:

       Laboratory material coupons: includes all materials, individually, used by the on-site microbiology
       laboratory in sample analysis; materials were confirmed sterile.
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       Positive control coupons: coupons inoculated but not decontaminated.

       Inoculation control coupons: PBST samples inoculated but not decontaminated to assess the
        variability of the inoculation operation.

       Procedural blank coupons: Spores were detected on some blank coupons. The potential impact
        on the decontamination efficacy was considered negligible.

5.4   Quality Assurance (QA)/Quality Control (QC) Checks
Uniformity of the  material sections was a critical attribute to assure reliable test results. Uniformity was
maintained by obtaining a large enough quantity of material that multiple material sections and coupons
could be constructed with  presumably uniform characteristics. Samples and test chemicals were
maintained to ensure their integrity. Samples were stored away from standards or other samples that
could cross-contaminate them.

Supplies and consumables were acquired from reputable sources and were NIST-traceable when
possible. Supplies and consumables were examined for evidence of tampering or damage upon receipt
and prior to use,  as appropriate. Supplies and consumables showing evidence of tampering or damage
were not used. All examinations were documented, and supplies were appropriately labeled. Project
personnel checked supplies and consumables prior to use to verify that they met specified task quality
objectives and did not exceed expiration dates.

Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did  not involve the use of analytical measurement devices. Rather, the CPU were
enumerated manually and recorded. QC checks for critical measurements/parameters are shown in Table
5-2. The acceptance criteria were set at the most stringent level that can routinely be achieved. Positive
controls and procedural blanks were included along with the test samples in the experiments so that well-
controlled quantitative values could be obtained. Background checks were also included as part of the
standard protocol. Replicate coupons were included for each set of test conditions. Standard operating
procedures using qualified, trained and experienced personnel were used to ensure data collection
consistency. The confirmation procedure, controls, blanks, and method validation efforts were the basis of
support for biological investigation results. If necessary, training sessions were conducted by
knowledgeable parties, and  in-house practice runs were used to gain expertise and proficiency prior to
initiating the research.
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Table 5-3:  QA/QC Sample Acceptance Criteria
Coupon or Sample Type
Decontamination Solution PAA
Stability (drift)
Decontamination Solution Negative
Controls (solution without biological
agent)
Positive Control Coupons
Laboratory Blank
Blank ISA, Sterility Control (plate
incubated, but not inoculated)
Acceptance Criteria
+ 10 % Drift over an hour of testing
No observed CPU
Target recovery of 1 *1 o7 CPU per
coupon (sample) with a standard
deviation of < 0.5. (5x1 06 through
5x1 07 CPU/coupon); 90 % recovery of
inoculum; Grubbs outlier test (or
equivalent)
No observed CPU
No observed growth following
incubation
Corrective Action
After 1 hour, reassess concentration of
active ingredients using titrations (eerie
sulfate, followed by iodiometric
determination of PAA)
If detected, discuss possible implications
with the EPA WACOR*to improve the
source of cross-contamination
Outside target range: discuss potential
impact on results with EPA WACOR;
check inoculum and prepare new
inoculum if necessary
Trace and remove source of
contamination. Reject results of the
same order of magnitude as the
laboratory blank recovery
Discard plate. Review plate making
procedures to identify source of
contamination
*WACOR = Work Assignment Contracting Officer Representative
Potential confounding organisms were excluded or controlled by sterilization of the materials and use of
aseptic technique, procedural blank controls, and a pure initial culture. Aseptic technique was used to
ensure that the culture remains pure. Procedural blank controls were run in parallel with the contaminated
materials. Assuming that the procedural blank controls showed no CPU, the observed colonies from
inoculated coupons indicated surviving spores from the inoculated organisms provided they were
consistent with the expected colony morphology (i.e., orange color, round form, flat elevation, rough
texture, and undulate margin).

5.5   Data Quality Audits
This project was assigned QA Category III and did not require technical systems, performance evaluation
or data quality audits by EPA or contractor QA personnel.

5.6   QA/QC Reporting
QA/QC procedures were performed in accordance with the QAPP for this investigation.
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14.  U.S.EPA, U. (2010). "Determining the Efficacy of Liquids and Fumigants in Systematic
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