Standard Operating Procedure for Method for the Evaluation of Antimicrobial Activity of Hard, Non-porous Copper-Containing Surface Products SOP Number: MB-41-00 Date Revised: 09-26-22


S Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Standard Operating Procedure for

Method for the Evaluation of Antimicrobial Activity of Hard, Non-porous Copper-
Containing Surface Products

SOP Number: MB-41-00
Date Revised: 09-26-22

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SOP No. MEM 1-00
Date Revised 09-26-22
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SOP Number

MEM 1-00

Title

Method for the Evaluation of Antimicrobial Activity of Hard, Non-
porous Copper-Containing Surface Products

Revisions Made

• N/A, new SOP.

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SOP No. MB-41-00

Date Revised 09-26-22
Page 1 of 30

SOP Number

MB-41 -00

Title

Method for the Evaluation of Antimicrobial Activity of Hard, Non-
porous Copper-Containing Surface Products

Scope

Describes the methodology used for evaluating the durability and
efficacy of copper-containing surfaces against Staphylococcus
aureus, Pseudomonas aeruginosa, and viruses.

Application

This methodology described in this SOP is limited to copper-
containing surfaces against the prescribed test microbes.





Approval Date

SOP Developer:

(2<€i09/26/22

tr

Print Name: Marc Carpenter

SOP Reviewer

09/26/22

Print Name: Amanda Strauch

Quality Assurance Unit

KLtmv VfrrmA/ 09/26/22

Print Name: Kiran Venn a

Branch Chief

xm> 09/26/22

Print Name: Rebecca Pines



Data SOP issued:

09/26/22

Controlled copy number:

0

Date SOP withdrawn:

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SOP No. MEM 1-00
Date Revised 09-26-22
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Method for the Evaluation of Antimicrobial Activity of Hard, Non-
porous Copper-Containing Surface Products

Scope

The Environmental Protection Agency (EPA) Office of Pesticide Programs (OPP) recommends
that applicants utilize this test method to support efficacy requirements for the registration of
hard, non-porous copper-containing surface products with non-food contact surface antimicrobial
claims and are designed to be supplements to standard disinfection practices. The claim is
intended for indoor use only including claims for "continuous reduction of bacteria and viruses."
The test method applies to solid copper products, impregnated copper products, and copper
coated products (sold as coated pre-market). The test method provides guidance for the
evaluation of durability and efficacy of the copper surfaces against Staphylococcus aureus,
Pseudomonas aeruginosa, and viruses; the test method can be adapted for additional organisms.
A minimum 3 log reduction of test organisms within 1-2 hours is the required level of
performance. This test method is a revised version of the interim method posted on 10/26/2020
(EPA-HQ-OPP-2020-0529).

Method Overview

In brief, the test method is comprised of two parts: 1) abrasion and chemical treatment, and 2)
product efficacy. The method specifies the use of 1" x 1" copper (product) carriers and stainless
steel control carriers. Carriers are exposed to abrasion and chemical treatment five times a day,
five days a week for six consecutive weeks. The abrasion and chemical exposure processes are
intended to represent a degree of normal and relevant physical wear, as well as reproduce
potential effects resulting from repeated exposure of copper-containing surfaces to three different
biocidal materials (chemical solutions). The impact of the abrasion and chemical exposure on the
integrity of product carriers is documented - these carriers are considered "exposed" carriers.
Following completion of abrasion and chemical exposure, each product and control carrier
receives a 20 |iL mixture of the test organism and soil load. Following a 1-2 hour contact time,
the carriers are neutralized, and the number of viable microorganisms is determined
quantitatively. The log reduction (LR) in the viable test organisms on exposed carriers is
calculated in relation to the viable test organisms on the unexposed control carriers. The impact
of the abrasion and chemical exposure on product efficacy is also determined by comparing
carriers not exposed to abrasion and chemical treatment.

Appropriate safety procedures should always be used when working with laboratory test systems
which include human pathogenic microorganisms. Laboratory safety is discussed in the current
edition of "Biosafety in Microbiological and Biomedical Laboratories (BMBL)" from the subject
matters experts within the U.S. Department of Health and Human Services (HHS), including
experts from the Centers for Disease Control and Prevention (CDC) and National Institutes for
Health (NIH).

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Table of Contents

1)	Terminology		4

2)	Apparatus		4

3)	B acteri al Reagents		5

4)	Viral Reagents		6

5)	Common Reagents		7

6)	Carriers		8

7)	Chemical Exposure and Abrasion Treatment Process		9

8)	Preparation of Test Cultures: P. aeruginosa and S. aureus		12

9)	Preparation of Test Culture: Viruses		13

10)	Efficacy Assessment		13

11)	Bacterial Recovery		15

12)	Virus Recovery		16

13)	Calculations/Data Analysis		17

14)	References		19

Appendix A: Preparation of Bacterial Frozen Stock Cultures		20

Appendix B: Examples of Failed Physically Screened Carriers		22

Appendix C: Example of Dry Inoculated Stainless Steel Carrier		23

Appendix D: Bacterial Neutralization Assay		24

Appendix E: Cytotoxicity Determination		26

Appendix F: Viral Neutralization Assay		28

Appendix G: Synthetic Broth Recipe		30

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1. Terminology

a. Control carriers, n—stainless steel carriers

b. Cycle (abrasion cycle), n—a series of 12 sequential, single passes across the surface of
carriers.

c. Exposed carriers, n—carriers that are exposed to chemical exposure and abrasion cycles
via a non-scratch scour pad on the surface of the abrasion tester tray.

d. Min, n—minutes.

e. Pass, n—within an abrasion cycle, one movement across the surface of the carriers.

f. S, n—seconds.

g. Product carriers, n—copper or copper coated carriers

h. Unexposed carriers, n—carriers that are not exposed to chemical exposure and abrasion
cycles via a non-scratch scour pad on the surface of the abrasion tester tray.

2. Apparatus

a. -20°C Freezer. Used for storage of soil aliquots.

b. -80°C Freezer. Used for storage of frozen stock cultures.

c. 0.2 pim pore diameter poly ether sulfone (PES) membrane filters, 47 mm diameter. Used
for recovery of test microbe (bacteria). Filtration units (reusable or disposable) may be
used.

d. 3M Scotch Brite Non-scratch scour pads. UPC 051141373195

e. Abrasion Boat. Used to abrade carriers with non-scratch scour pad attached.

f. Carriers, Copper (product) carriers. Die/machine cut 1" x 1" square from copper test
product, single use.

g. Carriers, Stainless steel (control) carriers. Die/machine cut 1" x 1" square made from
sheet stainless steel (AISI #304). The carriers should physically match the product
carriers as closely as possible with respect to thickness, degree of polish and/or brushed
surface machining, etc. Carriers are single use.

h. Centrifuge (with rotor capable of achieving 5,000 g). Used for test culture preparation.

i. Certified timer. Readable in minutes and seconds, for tracking of timed events and
intervals.

j. Conical tubes (e.g., 15 mL, 50 mL). Capable of being centrifuged at 5,000 g. Used for
culture preparation and neutralization.

k. Cryovials. Used for storage of frozen stock culture.

1. Dilution tubes (glass/plastic tubes). Used for preparing serial dilutions.

m. Environmental chamber. Used for efficacy testing, to hold carriers during microbe
contact time at 21±3°C and 30-40% relative humidity.

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SOP No. MEM 1-00
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n. Filter paper (Whatman No. 2). Used to line Petri dishes associated with carriers.

o. Forceps, sterile. Used to handle carriers and membrane filters.

p. Gardco Model D10Vor comparable abrasion instrument. Used to simulate wear on
carriers.

q. Identification system (optional). Used for appropriate identification of test microbes
(e.g., VITEK identification system).

r. Incubator. Used to incubate test cultures and growth medium plates at 36±1°C.

s. Incubator with 5% CO2. Used for growing cell lines and viruses (when applicable).

t. Liquid nitrogen dewar or comparable freezer capable of long-term storage of coll lines.
Used for storing viral cell lines.

u. Kimwipes. Lint free cloth; used for drying and cleaning carriers.

v. Microcentrifuge tubes. Used for storage of soil single use aliquots.

w. Microscope (e.g., lOOx optics and lOx ocular). Used for microbial observation.

x. Micropipettes, calibrated. Used with corresponding tips for preparing dilutions.

y. Petri dishes. Glass/plastic used as a flat surface for inoculating and incubating carriers.
Also used with filter paper for carrier drying, storage, and chemical treatment.

z. Positive displacement pipette, 20 piL calibrated. Used with corresponding tips for carrier
inoculation.

aa. Refrigerator (2-8°C). Used to store media and post-incubated plates.

bb. Serological Pipettes. Used for removing/adding larger volumes of liquid (e.g., 10 mL, 25
mL).

cc. Sonicator (capable of producing 45 Hz). Used for removal of organism from carriers. If
necessary, verify the sonicator to determine the impact of sonication on the culture by
placing the standardized broth culture into sonicator for 5 min, serially dilute, and
recover. Compare sonicated counts to a non-sonicated control. The sonicated and non-
sonicated counts should be comparable.

dd. Spray Bottle. Used to apply chemical treatments (solutions A, B, and C) to carriers.

ee. Titration kit (e.g., Hach digital titrator). Used for measuring total chlorine.

ff Vacuum source (in-house line or suitable vacuum pump). Used to facilitate rapid
membrane filtration.

gg. Vortex. Used for vortex mixing of various solutions including carriers.

3. Bacterial Reagents

a. Test microbes: Pseudomonas aeruginosa (ATCC #15442) and Staphylococcus aureus
(ATCC #6538)

b. Culture media for P. aeruginosa and S. aureus.

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i.	10% (w/v) dextrose solution. Used as a supplement to synthetic broth. Add 5.0 g
dextrose to 50 mL de-ionized water (to bring to volume in a volumetric flask) and
mix by stirring. Filter sterilize the solution using a 0.2 |im filter. Store the sterile
solution at 2-8°C for up to 30 days.

ii.	Phosphate buffered saline stock solution (e.g., 10X). Used for preparing IX
phosphate buffered saline. The stock solution has a pH of approximately 7.2±0.2.

iii.	Phosphate buffered saline (PBS), IX. Used for dilution blanks and filtration. PBS
has a pH of approximately 7.0±0.5.

iv.	Selective media (optional). Cetrimide agar (P. aeruginosa) and Mannitol salt agar
(S. aureus). See Table 5 in Appendix A for use. Purchase from a reputable source
or prepare according to manufacturer's instructions.

v.	Synthetic broth (SB). Used as the growth medium for test cultures. Commercial
media (HIMEDIA, Synthetic Broth, AOAC, #M334-500G). Store prepared SB at
2-8°C.

1. Alternatively, SB made in-house per the recipe provided in Appendix G
and AOAC Methods 955.15, 964.02, and 955.14 may be substituted.

vi.	Trypticase Soy Agar (TSA). Used as a recovery medium for bacterial enumeration
and purity checks. Prepare TSA according to manufacturer's instructions.

1. Equivalent commercially prepared agar culture medium may be
purchased.

vii.	Trypticase Soy Agar with 5% sheep blood (BAP). Used for performing streak
isolation of microbial cultures as a purity check (quality control purposes).

viii.	Trypticase Soy Broth (TSB), (30g/L). Used for rehydrating lyophilized/frozen
vegetative culture of test microorganism. Prepare TSB according to
manufacturer's instructions.

ix. TSB with 15% (v/v) glycerol. Used as a cryoprotectant solution. Suspend 7.5 g
TSB in 212.5 mL de-ionized water. Using a positive displacement pipette,
dispense 37.5 mL glycerol and stir, warm slightly to dissolve. Dispense into
bottles and steam sterilize for 15 min at 121°C.

1. Alternatively, purchase broth from a reputable source or prepare according
to manufacturer's instructions.

c. Gram stain kit. Used for diagnostic staining.

4. Viral reagents

a.	Test virus: use appropriate virus to be claimed on the label

b.	Cell line: use an appropriate cell line for virus selected for efficacy testing.

c.	Viral media

i. Complete Growth Media (CGM). Consisting of Minimum Essential Media and
FBS or other medium specified for the test virus. Used for cell line propagation,

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SOP No. MEM 1-00
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viral propagation, and serial dilution. Antibiotics and/or antifungals may be added
to reduce potential contamination.

1. Minimum Essential Media (MEM). Liquid or powder form (e.g., Eagle's
or Dulbecco's). Used to prepare complete growth media. Prepare per
manufacturer's guidelines.

2. Heat Inactivated Fetal Bovine Serum (FBS), compatible for use with cell
lines. Often used to prepare CGM.

ii. Dulbecco's Phosphate buffered saline (DPBS), or other equivalent buffer (e.g.,
PBS, Earle's Balanced Salt Solution). Prepare per manufacturer's guidelines.

d. Antibiotic/antifungal. lOOx Amphotericin B/Penicillin/Streptomycin solution or other
equivalent antibiotic/antimycotic solution. May be used to prevent contamination of cell
culture.

5. Common Reagents

a. 95-98% ethanol. Used to decontaminate carriers prior to efficacy testing.

b. De-ionized (DI) water. Used for preparation of reagents, media, and rinsing test solutions
off carriers.

c. Liquinox or equivalent non-ionic solution. To clean carriers.

d. Neutralizer. Various confirmed neutralizers may be used, including letheen broth. If
necessary, other ingredients may be added to letheen broth. Purchase letheen broth from a
reputable source or prepare according to manufacturer's instructions.

i. The recommended neutralizer for the viral test system is the same medium used to
grow the virus (e.g., CGM). If the neutralization confirmation assay demonstrates
that CGM is ineffective, other neutralizers may be used.

e. Soil Load, 3-part. The standard soil load to be incorporated in the final test suspension.

i. Bovine Serum Albumin (BSA). Add 0.5 g BSA (radio immunoassay (RIA) grade
or equivalent, CAS# 9048-46-8) to 10 mL of PBS, mix and pass through a 0.2 |im
pore diameter polyethersulfone membrane filter, aliquot, and store frozen at
-20±2°C for up to one year. Aliquots are single use only; do not refreeze once
thawed.

ii. Yeast Extract. Add 0.5 g yeast extract to 10 mL of PBS, mix, and pass through a
0.2 |im pore diameter polyethersulfone membrane filter, aliquot, and store frozen
at -20±2°C for up to one year. Aliquots are single use only; do not refreeze once
thawed.

iii. Mucin. Add 0.04 g mucin (from bovine submaxillary gland, CAS# 84195-52-8) to
10 mL of PBS, stir or vortex-mix until thoroughly dissolved, and pass through a
0.2 |im pore diameter polyethersulfone membrane filter, aliquot, and store frozen
at -20±2 °C for up to one year. Note: mucin may require vigorous stirring or
vortex-mixing to fully dissolve. Aliquots are single use only; do not refreeze once
thawed.

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SOP No. MEM 1-00
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iv. See section 8j and 9e for addition of soil load to bacterial and viral inoculum
respectively.

f. Treatments used in chemical exposure of carriers

i. Treatment A. A 3000±150 ppm sodium hypochlorite (NaOCl) solution (e.g.,
Sigma-Aldrich reagent grade sodium hypochlorite) prepared in deionized water.
Verify the final concentration of the treatment using a suitable titration method
(e.g., Hach digital titrator). Identify concentration in the study report.

ii. Treatment B. Use an EPA-registered hospital disinfectant product containing
hydrogen peroxide (between 3.0% and 6.0%) and peracetic acid as active
ingredients that allows spray application to hard, non-porous surfaces. The
treatment concentration for the peracetic acid component is not limited to a
defined range. Identify product in the study report.

iii. Treatment C. Use an EPA-registered hospital disinfectant product with quaternary
ammonium compound as the active ingredient labeled as a one-step
cleaner/disinfectant that allows spray application to hard, non-porous surfaces.
Identify product in the study report.

6. Carriers

a. The following section provides guidelines for preparation of both stainless steel control
carriers and product carriers. Two production lots of the product should be used to
evaluate efficacy. Lot 1 is used for both abrasion/chemical treatments and efficacy. Lot 2
is used for efficacy testing only. See Table 1 for a summary of carrier distribution.
"Exposed" refers to carriers subjected to the physical abrasion/chemical treatment, while
"unexposed" refers to those carriers not subjected to the physical abrasion/chemical
treatment.

Table 1. Carrier Distribution for Testing Copper-Containing Surface Products

Carrier Type

# of carriers for S. aureus

# of carriers for P. aeruginosa

Exposed Product

5 per exposure*
(15 total)

Lot 1

Exposed Stainless Steel

3 per exposure*
(9 total)

Unexposed Product

Unexposed Stainless Steel

->
J)

Lot 2

Unexposed Product

Unexposed Stainless Steel

->
J)

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SOP No. MB-41-0Q
Date Revised 09-26-22
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*3 separate treatments (A, B, and C) with abrasion

b.	Screen and clean carriers prior to abrasion/chemical exposure and maintain production lot
identity throughout the testing process.

c.	Inspect each carrier to ensure uniformity. Discard carriers with visible surface or edge
abnormalities (e.g., corrosion/rust, chipping, gouges, or deep striations, etc.); refer to
examples in Appendix B.

d.	Soak screened carriers in a non-ionic detergent solution (e.g., Liquinox) for 2-4 hours to
degrease and then rinse thoroughly in deionized water. Gently wipe with a clean lint-free
cloth and allow to completely dry.

e.	Prepare at least one additional product carrier and one additional stainless steel control
carrier for sterility assessment.

7. Chemical Exposure and Abrasion Treatment Process

Figure 1. Abrasion and Chemical Exposure Treatment Diagram - for exposed carriers of
a single production lot for 1 day

Solution A

5

V

3 Exposed



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Solution B

CO

L-

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1—

03

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===== Abrasion Cyc&c^—Disinfectant |—| Rinse with PI ^ Minute*0 ~|

Solution C

Kr-jh -jil SX

5 E |-|



-| Abrasion Cycte^

3 Expo-sed U

H

H

Rinif with Dl

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V

Lei dry 30

a. Preparation for physical abrasion.

i. Select and prepare carriers as described in Section 6 of this document prior to the
abrasion and chemical exposure process.

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SOP No. MEM 1-00
Date Revised 09-26-22
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ii.	Orient individual carriers with the exposed copper surface side-up (i.e., the
bactericidal copper-containing surface). Maintain this orientation throughout the
exposure treatment. For the stainless steel control carriers, either side of the
carrier may be selected but maintain the orientation throughout the exposure
treatment.

iii.	A single abrasion and chemical exposure treatment is twelve passes of an abrasive
material (i.e., a non-scratch scour pad) against the product surface followed by a
20-minute exposure of the product surface to a specific treatment.

iv.	Perform exposure treatments on Lot 1 five times per day and five days a week for
six consecutive weeks, resulting in a total of 150 abrasion and chemical
exposures.

v.	Maintain all product and stainless steel control carriers under comparable
conditions during each abrasion and chemical treatment exposure treatment.
Perform the exposure treatment of the carriers at room temperature (21±3°C).

b. Conducting the Abrasion Treatment

Figure 2. Recommended Abrasion Process - Carrier Configuration

Stainless Steel

Copper

i.	Perform the abrasion exposure with the Gardco, Model D10V abrasion tester or
comparable equipment. Consult the owner's manual to ensure proper set up and
maintenance and calibration.

ii.	Calibrate instrument to achieve 2-2.5 seconds for one pass and 24-30 seconds per
abrasion cycle.

iii.	Attach the pad to the abrasion tester as specified in the manual or using double
sided tape. Each exposed treatment must have its own abrasion pad. Do not use
the same abrasion pad for product and stainless steel control carriers. Replace
abrasion pads daily.

iv.	The total weight of the abrasion boat plus the abrasion material must be 415 to
435 g.

v.	Load carriers onto abrasion instrument and conduct abrasion cycle.

vi.	Product and stainless steel control carriers should be situated in parallel with one
another (see Figure 2) or abraded separately.

vii.	Do not adhere carriers to the abrasion instrument; use a template with cut-outs or
similar device to hold the carriers in place during abrasion process.

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SOP No. MEM 1-00
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viii. One abrasion cycle consists of twelve passes of the abrasive pad against the
carrier surface (the pad to contact the carrier surface twelve times).

ix. Following the abrasion cycle, wipe the carriers with a clean, dry cloth (Kimwipe),
and subject the carriers to the chemical treatment.

c. Chemical Treatment

Table 2. Carrier Exposure to Chemical Treatments

Treatment A

Treatment B

Treatment C

Sodium Hypochlorite
(NaOCl)

Hydrogen peroxide

Quaternary ammonium

5 exposed product carriers

3 exposed stainless steel
carriers

i. Place the carriers, product and stainless steel control carriers, exposed surface up
on a flat surface (e.g., inside a Petri dish) with absorbent material (e.g., filter

paper) to absorb excess chemical treatment. Apply each chemical treatment to the
exposed carriers by spraying two to three pumps of the appropriate treatment at 6-
8 inches from the carrier surface.

ii. Allow each carrier to be in contact with the chemical treatment for 20±1 min at
room temperature.

iii. After the contact period, rinse each carrier thoroughly with DI water, air dry, and
store uncovered at room temperature until the next exposure cycle.

1. Wait a minimum of 30 min between each exposure cycle.

d. Following the 150 abrasion/chemical exposures, rinse all carriers thoroughly with DI
water, air dry, and store at room temperature in individual covered Petri dishes lined with
filter paper. Visually inspect carrier surfaces (for the designated production lot). Describe
and document any physical disruption or degradation of the exposed product surface
(e.g., decolorization, cracking, peeling, and chipping).

e. After completing the abrasion/chemical exposure (for Lot 1 only) immerse all product
and stainless steel control carriers in 95-98% ethanol for approximately 10 min. Using
sterile forceps, remove individual carriers and place face up in pre-sterilized Petri dishes
(one carrier per dish) lined with filter paper. Allow carriers to dry with lid open. Orient
individual carriers with the treated exposed side (i.e., copper surface) up; maintain this
orientation. Handle carriers aseptically.

f. Include all carrier storage conditions (temperature and humidity range) in the study
report.

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SOP No. MEM 1-00
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g. Initiate efficacy testing within 7 days of completion of the final abrasion/chemical
exposure process.

8. Preparation of Test Cultures: P. aeruginosa and S. aureus

a. Refer to Appendix A for preparation of the frozen stock cultures.

b. Defrost a cryovial rapidly to avoid loss in the viability of the preserved cells. Each
cryovial is single use only.

c. No more than 15 min prior to inoculation, use a calibrated micropipette to aseptically add
0.1 mL of 10% sterile dextrose (w/v) solution to each 10 mL tube of SB in a 20x 150 mm
glass culture tube with a Morton closure; mix well.

d. Using a calibrated micropipette, add 100 |iL of defrosted stock culture to the same tube
containing 10 mL SB + 0.1 mL dextrose, briefly vortex-mix and incubate for 24±2 h at
36±1°C.

i. In addition, inoculate TSA or TSA with 5% sheep blood plate from the inoculated
tube and streak for isolation. Incubate plate with the test culture.

ii. Incubate without disrupting the culture (i.e., do not move culture while
incubating).

e. Following incubation, use the SB cultures to prepare a test suspension for each organism.
Record results from the streak isolation plate.

f. The 24±2 h culture should exhibit a titer of at least 108 CFU/mL. Record time of culture
harvest.

i. For P. aeruginosa, inspect culture prior to harvest; visible pellicle on the surface
of the culture is expected to form during incubation (record its presence). Discard
the culture if pellicle has been disrupted (fragments in culture).

1. Remove visible pellicle on surface of medium and around associated
interior edges of the tube with vacuum suction.

2. Using a serological pipette, withdraw the remaining broth culture (approx.
7-8 mL) avoiding any sediment on the bottom of the tube and transfer it
into a 15 mL conical centrifuge tube.

3. Record approximate volume harvested and transferred to 15 mL conical
tube.

ii. For S. aureus, briefly vortex the 24±2 h culture and transfer to a 15 mL centrifuge
tube.

1. Record approximate volume harvested and transfer to 15 mL conical tube

g. Within 15 minutes, centrifuge the 24±2 h harvested broth cultures at 5,000xg for 20 min.
Record time of centrifugation.

h. Remove the supernatant without disrupting the pellet. Once supernatant is removed, re-
suspend the pellet in 5-10 mL PBS. Record resuspension volume and time of
resuspension.

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SOP No. MEM 1-00
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i. If necessary, disrupt the pellet using vortex-mixing or repetitive tapping/striking
against a hard surface to disaggregate the pellet completely. If necessary, add 1
mL of PBS to the pellet first to aid in disaggregation.

i. For efficacy testing, further dilute the 5-10 mL of resuspended culture in PBS as

necessary to achieve a mean control carrier count level of 4.0-5.0 logs CFU/carrier fori5.
aeruginosa and S. aureus. Each inoculated carrier must be within this range following the
2-hour exposure time for a valid test. Record time of test suspension dilution.

i. Optical density/absorbance (e.g., 650 nm) may be used as a tool to monitor/adjust
the diluted test suspension; record if measured.

j. Use the diluted culture within 30 min to prepare the final test suspension with the
addition of the soil load.

i. Vortex-mix the test suspension for 10-30 s.

ii. To obtain 500 |iL of the final test suspension with the 3-part soil load, vortex-mix
each component and combine in the following order using a calibrated
micropipette:

1. 25 |iL BSA stock

2. 35 |iL yeast extract stock

3. 100 |iL mucin stock

4. Vortex soil suspension for 10 s prior to adding microbial test suspension.

5. 340 |iL microbial test suspension.

k. Briefly vortex the final test suspension with 3-part soil load (at room temperature,

21±3°C) and use to inoculate carriers within 30 min of preparation. Record time of final
test suspension preparation.

i. Within the 35 carriers, there are a total of 9 independent "sets" of carriers (5 sets
of 3 carriers, 4 sets of 5 carriers). A 90-minute use period for the culture with the
3-part soil load provides the opportunity to stagger inoculations, 10 min per
carrier set.

ii. Inoculate 1 carrier from Control Set #1 first. Continue inoculations with Treated
Set #1, Treated Set #2, Control Set #2; then inoculate the remaining 2 carriers
from Control Set #2.

1. Streak inoculate an agar plate (TSA or TSA with 5% sheep blood) with a loopful of the
final test suspension. Incubate at 36±1°C for 48±4 h and visually examine for purity.

9. Preparation of the Test Culture: Viruses

a. Propagate the test virus on the appropriate cell line.

i. Note: Concentration of the test virus stock (~100,000xg for 4 h at 4°C) may be
necessary to achieve adequate control counts.

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SOP No. MEM 1-00
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b. Defrost a cryovial rapidly to avoid loss in the viability of the preserved virus (e.g., place
in a 37°C water bath and use within 15 min after thawing). Record time placed in water
bath.

c. Dilute the virus stock with CGM to achieve control counts in the range of 4.0 to 5.0 logs
virus particles/carrier. Record time of virus stock dilution.

d. Use the diluted virus within 30 min to prepare the final test suspension with the addition
of the soil load.

e. To obtain 500 |iL of the final test suspension with the 3-part soil load, vortex-mix each
component and combine in the following order using a calibrated micropipette (smaller
volumes may be used proportionally):

i. 25 |iL BSA stock

ii. 35 |iL yeast extract stock

iii. 100 |iL mucin stock

iv. Vortex soil suspension for 10 s prior to adding viral test suspension.

v. 340 |iL virus test suspension

f. Briefly vortex the final test suspension with 3-part soil load (at room temperature,
21±3°C) and use to inoculate carriers within 30 min of preparation. Record time of final
test suspension preparation.

ii. Inoculate 1 carrier from Control Set #1 first. Continue inoculations with Treated
Set #1, Treated Set #2, Control Set #2; then inoculate the remaining 2 carriers
from Control Set #2.

10. Efficacy Assessment

a. Conduct efficacy testing on all product and stainless steel control carriers within 7 days
of completing the abrasion/chemical exposure cycles.

b. Perform the neutralization assay for all organisms prior to testing to demonstrate the
neutralizer's ability to inactivate the copper and copper ions; see Appendix D (bacteria)
or Appendix F (viruses).

c. In preparation for efficacy testing, it is advisable to determine the appropriate dilution of
the test suspension that will ensure control counts in the appropriate range after drying by
inoculating three stainless steel control carriers, placing them in the environmental
chamber for 1-2 hours, and determining the counts per carrier.

d. Prepare test cultures per Section 8 (bacteria) or Section 9 (viruses) to achieve a final
target control count on stainless steel control carriers of 4.0-5.0 logs CFU or viral
particles per carrier after the 1-2 hour contact time.

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e. Set environmental chamber to achieve 21±2°C and 30-40% relative humidity during the
1-2 h contact period; record temperature and humidity over the contact period.

f. Record the time for all timed events.

g. Process product carriers first and stainless steel control carriers last.

h. Inoculate each carrier with 20 |iL of final test culture using a calibrated pipette. Spread
the inoculum to within 1/8 inch of the edge of each carrier using a sterile transfer loop or
the pipette tip. Place in environmental chamber within 10 minutes of inoculation.

i. Allow carriers to remain in a flat, horizontal position with the lid on the Petri dish in the
environmental chamber for 1-2 hours. Refer to Appendix C for picture of dried inoculum
on carrier.

11. Bacterial Recovery

a. Following the contact time, sequentially and aseptically transfer each carrier to a 50 mL
conical tube containing 20 mL of the appropriate neutralizer solution. Remove and
neutralize all carriers within 10 minutes

i. The tube with the neutralizer and the carrier represents the 10° dilution.

b. After all the carriers have been transferred into the neutralizer, vortex-mix for 30 s then
sonicate for 5 min±30 s at 45 Hz to suspend any surviving organism in the neutralizer. If
necessary, refer to section 2cc for sonicator verification.

c. Initiate serial dilutions of the neutralizer tubes in PBS within 30 min.

d. Initiate filtration within 30 min of preparing dilutions.

e. Prior to filtration, pre-wet each membrane filter with -10 mL PBS. Apply vacuum to
filter contents; leave the vacuum on for the duration of the filtration process regardless of
filtration apparatus used (e.g., filter manifold, single filter unit).

f. Use separate PES membrane filters for each eluate; however, the same filtration unit may
be used for processing eluates from a given carrier set starting with the most dilute
sample first.

g. Pour the eluate into the filter unit. Rinse tubes (conical tube and/or dilution blank) once
with -10 mL PBS, briefly vortex-mix, and pour into filter unit.

h. Swirl the contents of the filter unit and quickly filter with limited pooling of liquid in the
filter apparatus.

i. Rinse the inside of the surface of the funnel unit with -20 mL PBS and filter contents.

j. Aseptically remove the membrane filter and place onto TSA. Avoid trapping any air
bubbles between the filter and agar surface.

k. Filter appropriate dilutions which yield countable numbers (up to 200 CFU per plate).

1. Carrier Sterility Control: add one sterile product carrier and one sterile stainless steel
carrier to separate tubes containing 20 mL of TSB. Incubate at 36±1°C for 48±4 h and
examine for growth. The acceptance criterion is lack of turbidity/growth in the tube.

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m. Neutralizer Sterility Control: add 1 mL of neutralizer into 9 mL of TSB. Incubate at
36±1°C for 48±4 h and examine for growth. The acceptance criterion is lack of
turbidity/growth in the tube.

n. Incubate plates from Unexposed Stainless Steel carriers at 36±1°C for 48±4 h. Incubate
plates from Exposed Product, Exposed Stainless Steel, and Unexposed Product carriers at
36±1°C for 72±4 h.

i. Monitor all plates (filters) after 24 h of incubation to facilitate appropriate timing
for counting colonies.

ii. For colony counts on filters in excess of 200 CFU, record as Too Numerous to
Count (TNTC).

o. If isolated colonies are present, perform a Gram stain to assess one representative colony
per carrier set (product exposed, product unexposed, stainless steel exposed, stainless
steel unexposed).

p. If confluent growth is present, perform a streak isolation on the appropriate agar on
growth taken from at least 1 representative filter per carrier set.

q. The results of the streak isolation plates should be consistent with characteristics in Table
5.

r. If additional verification of the test organism is required, perform further confirmatory
analyses (e.g., VITEK or biochemical analyses) and isolation streaks on selective media.

12. Virus Recovery

i. The tube with the neutralizer and the carrier represents the 10° dilution.

b. After all the carriers have been transferred into the neutralizer, vortex-mix for 30 s to
suspend any surviving organism in the neutralizer. If necessary, to increase viral
recovery, sonicate for 5 min±30 s at 45 Hz; refer to section 2cc for sonicator verification.

c. Initiate dilutions within 30 min after neutralization and vortex-mixing.

d. Initiate inoculation of cell line within 30 min of preparing the dilutions.

e. Titrate the samples for virus infectivity.

f. Plate a minimum of 80% of the volume (8 mL for 10 mL volumes, 16 mL for 20 mL
volumes) of the 10° vial and of each dilution tube.

g. Remove the growth medium from each well of the plate with a confluent monolayer of
cells and replace with the maximum volume of the dilution tube (i.e., add 1 mL per well
for a 24 well plate) working from most dilute to least dilute.

h. The elution steps for stainless steel control carriers are the same as for the product
carriers; use 10-fold dilutions to achieve 4.0-5.0 logs virus particles/carrier.

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i. For each test, use at least one well as a negative control (CGM alone) and one well as a
positive growth control (e.g., one of the dilutions from a stainless steel control carrier).

j. If cytotoxicity was observed in the neutralization testing and/or on the cytotoxicity

control, CGM may be removed from all wells in the affected dilutions at the appropriate
time (one hour minimum). Wash the wells with pre-warmed PBS, then replace the PBS
with fresh CGM.

k. Incubate test and control plates as appropriate for the test system.

1. Record all observations (presence/absence of viable virus particles) and use in

calculations to estimate the log reduction based on the TCIDso or MPN (most probable
number) technique.

13. Calculations/Data Analysis

a.	Use values with at least three significant figures when performing calculations (e.g., log
density, mean log density). Report the final log reduction and difference in log reduction
values with two significant figures.

b.	For bacteria, calculate the Colony Forming Units (CFU)/carrier using the following
equation:

Y	= CFU per filter,

C	= volume filtered,

V	= total volume of neutralize^

D	= 10k,

k	= dilution,

n	= number of dilutions, and

i	= lower limit of summation (the fewest number of dilutions).

c.	For viruses, calculate the TCIDso/carrier or MPN/carrier. Calculate the log density of
each carrier by taking the logio of the density (per carrier).

d.	Calculate the mean log density (LD) of viable cells or virus particles for each microbe for
the carrier sets in Lot 1 (Exposed Product (per chemical exposure/abrasion treatment, 3
total), Exposed Stainless Steel (per chemical exposure/abrasion treatment, 3 total),
Unexposed Product, Unexposed Stainless Steel) as follows:

number of carriers assayed.

e. Calculate the mean LD of viable cells or virus particles for each microbe for the carrier
sets in Lot 2 (Unexposed Product, Unexposed Stainless Steel) using the above equation.

where:

Mean LD = £

Log10(Carrier 1 + Carrier 2 + ... + Carrier X)
X

¦, where "X" refers to the total

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Date Revised 09-26-22
Page 18 of 30

f.	For bacteria, when TNTC (Too Numerous to Count) values are observed for each dilution
filtered, substitute 200 for the TNTC at the highest (most dilute) dilution and account for
the dilution factor in the calculation.

g.	Conduct additional calculations for Lot #1 and Lot #2; include in study report. See Tables
3 and 4 for calculations and outcome requirements.

Table 3. Additional calculations for Lot #1

LOG DIFFERENCE BETWEEN UNEXPOSED AND EXPOSED STAINLESS STEEL

Outcome
(Difference)

Difference
between
Unexposed
Stainless Steel
and Exposed
Stainless Steel

Mean LD Unexposed Stainless Steel - Mean LD Exposed Stainless Steel:

Solution A

<0.5

Mean LD Unexposed Stainless Steel - Mean LD Exposed Stainless Steel:

Solution B

<0.5

Mean LD Unexposed Stainless Steel - Mean LD Exposed Stainless Steel:

Solution C

<0.5

LOG REDUCTION CALCULATIONS

Outcome
(LR)

LR Unexposed
Product

Mean LD Unexposed Stainless Steel - Mean LD Unexposed Product

>3.0

LR Exposed
Product

Mean LD Unexposed Stainless Steel - Mean LD Exposed Product: Solution A

>3.0

Mean LD Unexposed Stainless Steel - Mean LD Exposed Product: Solution B

>3.0

Mean LD Unexposed Stainless Steel - Mean LD Exposed Product: Solution C

>3.0

LOG DIFFERENCE BETWEEN UNEXPOSED AND EXPOSED PRODUCT

Outcome
(Difference)

Difference

between

Unexposed

Product and

Exposed

Product

Mean LR Unexposed Product - Mean LR Exposed Product: Solution A

< i.o

Mean LR Unexposed Product - Mean LR Exposed Product: Solution B

< i.o

Mean LR Unexposed Product - Mean LR Exposed Product: Solution C

< 1.0

Table 4. Additional calculations for Lot #2

LOG REDUCTION CALCULATIONS

Outcome
(LR)

LR Unexposed
Product

Mean LD Unexposed Stainless Steel - Mean LD Unexposed Product

>3.0

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Page 19 of 30

14. References

a.	Krieg, Noel R. and Holt, John G. 1984. Bergey's Manual of Systematic Bacteriology
Volume 1. Williams & Wilkins, Baltimore, MD. P. aeruginosa p. 164.

b.	Sneath, P., Mair, N., Sharpe, M.E., and Holt, J. eds. 1986. Bergey's Manual of Systematic
Bacteriology Volume 2. Williams & Wilkins, Baltimore, MD. S. aureus p. 1015.

c.	ASTM Method E1482-12. Standard Practice for Use of Gel Filtration Columns for
Cytotoxicity Reduction and Neutralization (Reapproved 2017)

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Appendix A

Preparation of Bacterial Frozen Stock Cultures

1. Initiate new stock cultures from lyophilized cultures of Pseudomonas aeruginosa and
Staphylococcus aureus from ATCC (or other reputable vendor) at least every 18 months.

a. New frozen stock culture may be initiated one time using an existing, unexpired frozen
stock culture as the source. Begin process at step 3 below, by streaking a loopful of the
frozen stock culture onto 2 TSA plates.

2. Open ampule of freeze-dried organism per manufacturer's instructions. Using a tube
containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and rehydrate the lyophilized
culture. Aseptically transfer the entire rehydrated pellet back into the original tube of broth.
Mix thoroughly. Incubate broth culture at 36±1°C for 24±2 h.

3. At the end of the incubation timeframe, streak a loopful of the broth culture onto 2 TSA
plates to obtain isolated colonies. Perform a streak isolation of the broth culture onto BAP as
a purity check and streak the broth culture onto the appropriate selective media. Refer to
appropriate selective media in Table 5. Incubate all plates for 24±2 h at 36±1°C.

a. Record results at the end of the incubation timeframe. Refer to Table 5 for results on
selective media and diagnostic characteristics of the test microbes.

4. From the TSA plates, select 3-5 isolated colonies of the test organism and re-suspend in 1
mL of TSB. For S. aureus, select only golden yellow colonies. Fori5, aeruginosa, select
colonies from each of the two possible phenotypes present. Spread plate 0.1 mL of the
suspension onto each of 6-10 TSA plates. Incubate the plates for 24±2 h at 36±1°C. If
necessary, to obtain more frozen stock cultures, a larger suspension (e.g., 2 mL) may be
prepared using the same ratio of TSB (1 mL) to number of colonies (3-5 colonies).

a. Using the TSB suspension, perform a streak isolation of the suspension onto a BAP as a
purity check, and streak on the appropriate selective media (refer to Table 5).

b. Incubate all plates for 24±2 h at 36±1°C. Record results. Refer to Table 5 for results on
selective media and diagnostic characteristics of the test microbes.

5. After the incubation period, harvest growth from TSA plates by adding approximately 5 mL
sterile cryoprotectant solution (TSB with 15% (v/v) glycerol) on the surface of each plate.
Re-suspend the growth in the cryoprotectant solution using a sterile spreader without
damaging the agar surface. Aspirate the suspension from the plate with a pipette and place it
in a sterile vessel large enough to hold about 30 mL.

6. Repeat the growth harvesting procedure with the remaining plates and continue adding the
suspension to the vessel (more than 1 vessel may be used if necessary). Mix the contents of
the vessel(s) thoroughly; if more than 1 vessel is used, pool the vessels prior to aliquoting
culture.

7. Immediately after mixing, dispense 0.5-1.0 mL aliquots of the harvested suspension into
cryovials; these represent the frozen stock cultures.

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a. For QC purposes, perform a streak isolation of the pooled culture onto a BAP as a
purity check and streak on appropriate selective media (refer to Table 5).

b. Incubate all plates for 24±2 h at 36±1°C.

c. Record results.

d. After incubation, perform a Gram stain on growth from the BAP; observe the Gram
reaction by using brightfield microscopy at 1000X magnification (oil immersion).

e. Conduct Vitek confirmation from growth taken from the BAP. Conduct VITEK
according to the manufacturer's instructions.

f. Record all confirmation results.

8. Store the cryovials at approximately -80°C for a maximum of 18 months. These cultures are
single-use only.

9. If the characteristics of the organism are not consistent with the information in Table 5 at any
step in the process, or the Vitek profile is inconsistent with the organism, discard the cultures
and re-initiate the process.

Table 5. Selective media and diagnostic characteristics fori5, aeruginosa and S. aureus.

Aspect

P. UlTHxillO.SU'"

S. aureus

(ii'am slum reaction

\egali\ e

Posim e

Mamiilol Sail \uar
Selecli\ e medium

N/A

Circular, small, yellow colonies, agar
turning fluorescent yellow

Ceiriniide \uar
Selecli\ e medium

Circular, small, initially opaque,
lu ruing fluorescent green over time;
auar fluorescent yellowish green

N/A

13ku»d anai" (15 \l'i

1 lat, opaque to off-white, round
spreading (1), metallic sheen, slightly
beta hemolytic

Small, circular, yellow or white,
glistening, beta hemolytic

lApiciil Microscopic Characteristics

Cell appearance

Si might or slightly curved rods, single
polar flagella, rods formed in chains;
ii 5-1.0 nm in diameter x 1.5-5.0 ^m
in length

Spherical, occurring singly, in pairs
and tetrads, sometimes forming
irregular clusters; 0.5-1.0 |im in
diameter

*After 24±2 h (1) P. aeruginosa may display two phenotypes.

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Appendix B

Examples of Failed Physically Screened Carriers

A and B fail due to discoloration of surface.
C and D fail due to surface scratches.

E and F fail due to deep gouge in surface.

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Appendix C

Example of a Dry Inoculated Stainless Steel Carrier

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Appendix D

Bacterial Neutralization Assay

The neutralization of the product carriers is confirmed in triplicate by using product carriers
in neutralizer, neutralizer only (without carriers), and PBS only (used to compare counts
from the neutralizer and product carriers).

Preparation of the Test Organism

a. Refer to the preparation of test cultures section (8a-h) of for preparation of the test
cultures. Conduct preliminary tests as necessary to determine appropriate dilution(s)
of Test Suspension A (used to prepare Test Suspension B) to achieve the target
challenge of 20-200 CFU per 20 |iL.

b. Prepare Test Suspension A (without soil load). Serially dilute the microbial test
suspension with PBS (e.g., through 10"4 or 10"5). Select appropriate dilutions of Test
Suspension A so that after the addition of the soil load, Test Suspension B will achieve
an average challenge of 20-200 CFU per 20 |iL. Use Test Suspension A within 30 min
of preparation.

i. Two separate serial dilutions of Test Suspension A may be used to prepare two
different concentrations of Test Suspension B to ensure at least one dilution
with an average challenge of 20-200 CFU per 20 |iL.

ii. A calibration curve (OD @ 650nm) may be used to estimate the number of
viable organisms in Test Suspension A.

c. Prepare Test Suspension B (with soil load). Prepare the 3-part soil load: using a
vortex, mix each component and combine 25 |iL bovine serum albumin (BSA), 35 |iL
yeast extract, and 100 |iL of mucin; then vortex-mix the solution. Add 340 |iL of
diluted Test Suspension A to the 160 |iL of the soil load (SL) and vortex-mix for 10 s.

i. Ensure at least one preparation of Test Suspension B provides an average
challenge of 20-200 CFU per 20 |iL.

Neutralization Assay Components

a. Treatment 1: Neutralizer Effectiveness. Add a product carrier (one per market
relevant lot) to each of three 50 mL conical tubes. At timed intervals, add 20 mL of
neutralizer to each 50 mL conical tube and vortex-mix for 30 s on highest vortex
setting. Immediately add 20 |iL Test Suspension B to each vessel using a micropipette
and briefly vortex-mix. Proceed with Appendix D, section 4.

b. Treatment 2: Neutralizer Toxicity Control. Add 20 mL of neutralizer to each of three
50 mL conical tubes. At timed intervals, add 20 |iL of Test Suspension B to each
vessel using a micropipette and briefly vortex-mix. Proceed with Appendix D, section
4.

c. Treatment 3: Titer Control. Add 20 mL of PBS to each of three 50 mL conical tubes.
At timed intervals, add 20 |iL of Test Suspension B to each vessel using a
micropipette and briefly vortex-mix. Proceed with Appendix D, section 4.

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4.	Processing and Recovery

a.	Hold the mixtures from Appendix D, section 3 for 10±1 min at room temperature
(21±3°C).

b.	At the conclusion of the holding period, vortex-mix each tube.

c.	Initiate filtration as soon as possible (e.g., within 30 min).

d.	Prior to filtration, pre-wet each membrane filter with -10 mL PBS. Filter each
mixture through a separate, pre-wetted 0.2 |im PES membrane filter. Apply vacuum
to filter contents; leave the vacuum on for the duration of the filtration process
regardless of filtration apparatus used (e.g., filter manifold, single filter unit).

e.	Wash each tube with -20 mL PBS and vortex-mix; filter the wash through the same
filter membrane. Finish the filtering process by rinsing the inside of the funnel unit
with -20 mL PBS and filter the rinsing liquid through the same filter membrane.

f.	Remove the membrane aseptically with sterile forceps and place it carefully over the
surface of the TSA. Avoid trapping air bubbles between the filter and the agar
surface.

g.	Incubate plates at 36±1°C for 48±4 h and count the colonies.

h.	Incubate plates with no growth or few colonies an additional 24±4 h and count the
number of colonies.

5.	Data Analysis/Calculations

a.	Compare the average CFU of the Titer Control with the average CFU of the
Neutralizer Toxicity Control and Neutralizer Effectiveness treatment. Determine
the percent difference in CFU.

b.	For determining the suitability of the neutralizer, ensure that the average CFU in the
Neutralizer Toxicity Control is at least 50% of the Titer Control. A count lower
than 50% indicates that the neutralizer is harmful to the test organism.

i. Average CFU for the Neutralizer Toxicity Control that are higher than the
Titer Control (e.g., 120% of the Titer Control) are also deemed valid.

c.	To verify effectiveness of the neutralizer, the average number of CFU in the
Neutralizer Effectiveness treatment is at least 50% of the Titer Control.

i. Average CFU for the Neutralizer Effectiveness treatment that are higher than
the Titer Control (e.g., 120% of the Titer Control) are also deemed valid.

d.	If the criteria are not met, verify another neutralizer or mixture of neutralizers.

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Appendix E
Cytotoxicity Determination

Prior to performing the neutralization assay, ensure the proposed neutralizer, neutralizer and test

chemical, and the soil used do not impact the quality of the cell line by performing the following:

1. Neutralizer Effect on Cell Line (for neutralizers other than CGM with 2% FBS).

a. Add 0.5 mL of the proposed neutralizer to 4.5 mL CGM with 2% (v/v) FBS, equilibrated
to 37±1°C (this is the 10"1 dilution). It is suggested to do further dilutions out to 10"2 or
10"3 depending on the expected cytotoxicity of the neutralizer.

b. Remove the CGM from the wells of a 24 well plate with an 80-95% confluent monolayer
of cells and add 1 mL per well of the neutralizer plus CGM solution. Plate at least 4 wells
per dilution. Have at least one well as a negative control (e.g., CGM with 2% FBS alone).

c. Incubate plate as appropriate and observe closely for cytotoxicity.

d. If cytotoxicity is observed after one hour, remove the media in a single well of the
affected dilution, rinse once with pre-warmed DPBS (the DPBS wash step may be
omitted if the cytotoxicity is mild), and replace media.

e. If cell death occurs in under one hour, the neutralizer cannot be tested.

f. The effect of the media change in the single well can be compared to the other wells in
the dilution and the negative control. If cytotoxicity cannot be overcome with washing
and replacing of media, column filtration (e.g., Sephadex) may be used in future testing.
See section 14c for further information on column filtration.

2. Neutralizer Plus Test Carrier Effect on Cell Line.

a. Add one test carrier to 20 mL of neutralizer, equilibrated to 21±3°C, and vortex 2-3
seconds. Let this solution sit at room temperature for 10 minutes.

b. Add 1.0 mL of this solution to 9 mL CGM with 2% (v/v) FBS, equilibrated to 37±1°C
(this is the 10"1 dilution). It is suggested to do further dilutions out to 10"2 depending on
the expected cytotoxicity.

c. Remove the CGM from the wells of a 24 well plate with a confluent monolayer of cells
and add 1 mL per well of the neutralizer plus test carrier and dilutions. Plate at least 8
wells for the 10° dilution, 6 wells for the 10"1 dilution, and 4 wells for the 10"2 dilution.
Extra wells will be needed to observe the effect of no media changes or for further media
changes as needed.

d. For highly toxic test chemicals, washing the cells with pre-warmed DPBS before the
addition of CGM with 2% FBS will help remove cytotoxicity.

e. Have at least one well on each plate as a negative control (e.g., CGM with 2% (v/v) FBS
alone).

f. At a minimum, change the media in the wells as outlined below. Change the media at the
lower time interval if they look more toxic. Other media changes can be made at other
times if necessary.

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i. For the 10° dilution:

i. On the day of the test, change two wells 1 -2 hours (1 -hour minimum)
after the neutralized test chemical mixture was added to the cells.

ii. Change two more additional wells 3-5 hours after the neutralized test
chemical mixture was added to the cells.

iii. The following day, change one 1-2 hour well, one 3-5 hour well, and one
previously unchanged well, plus one of the two previously changed well.

ii. For the 10"1 dilution:

i. On the day of the test, change tow wells 3-5 hours after the neutralized
test chemical mixture was added to the cells.

ii. The following day, change one 3-5 hour well and one previous
unchanged well.

iii. For the 10"2 dilution:

i. On the day after the test, change one well.

g. Incubate the plate as appropriate and observe the cells for cytotoxicity. The test cells
should be compared to the negative control cells to determine toxicity.

h. Score the cells as toxic or non-toxic in each in each test conditions.

i. Identify the test condition that removed the cytotoxicity and use that condition for further
neutralization and efficacy testing. Use the test condition that allows the media to stay on
the cells for as long as possible.

i. Example: In the 10° dilution, if the unchanged wells are toxic, but both the 1 hour
and 4 hour media changes are non-toxic, change the media in the 10° dilutions
after 4 hours in all future testing.

j. If cell death occurs in under one hour, that test condition cannot be used.

k. Cytotoxicity past the 10"1 dilution is unacceptable for testing. Alternative neutralizers or
column filtration (e.g., Sephadex) may be used to mitigate cytotoxicity. See section 14c
for further information on column filtration.

3. 3-Part Soil Effect on Cell Line.

a. Make the 3-part soil (see section 9e but withhold the virus).

b. Add 10 |iL of the soil to 20 mL of CGM, equilibrated to 37±1°C.

c. Remove the CGM from the cells and add 1 mL of this solution to 4 wells on a 24 well
plate with a confluent monolayer of cells. Have at least one well as a negative control
(e.g., CGM alone).

d. Incubate plate as appropriate and observe daily for cytotoxicity. No cytotoxicity should
be observed.

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Appendix F
Viral Neutralization Assay

1. Perform the neutralization assay prior to testing to demonstrate the neutralizer's ability to
inactivate the product carrier.

2. Select a neutralizing medium that is not inhibitory to the virus and is not cytotoxic to the
cells. The acceptance criteria for acceptable neutralization are 0.5 log differences between the
neutralization effectiveness, neutralization toxicity control, and titer control. Interaction
between the neutralizer and product and its effect on the cell line must be determined prior to
testing.

3. Prepare Test Suspension A. Dilute the virus stock suspension in CGM to achieve an average
recovered concentration of approximately 2-3 logs (i.e., 100-1000 virus particles) per vessel
for the Titer Control sample. To achieve this, dilute the virus stock suspension through 10"4
(or as necessary).

4. Prepare Test Suspension B. Prepare the soil load: vortex each component and combine 25
|iL bovine serum albumin (BSA), 35 |iL yeast extract, 100 |iL of mucin, and add 340 |iL of
Test Suspension A (0.5 mL total volume) and mix well. Use Test Suspension B within 30
minutes of preparation.

5. Neutralization Treatments

c. Treatment 3: Titer Control. Add 20 mL CGM to each of three 50 mL conical tubes.
At timed intervals, add 20 |iL of Test Suspension B to each vessel using a
micropipette and briefly vortex-mix. Proceed with Appendix F, section 6.

i. Note: Steps should be conducted at timed intervals (e.g., 30 s) to ensure
consistent time of contact.

6. Processing and Recovery

a. Hold the mixtures from Appendix F, section 5 for 10±1 min at room temperature
(21±3°C).

b. At the conclusion of the holding period, vortex each tube for 3-5 s. Serially dilute the
sample as needed (e.g., remove 1 mL of sample and dilute in 9 mL of CGM).

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i. Initiate dilution and plating as soon as possible (e.g., within 5 minutes). Two
analysts are recommended to perform vortexing and dilution steps to reduce
holding time after vortexing.

ii. Titrate the samples for virus infectivity using the appropriate cell line - plate a
minimum of 80% of the 10° vessel and all dilutions.

iii. For each well plated, add the maximum volume of the well (i.e., add 1 mL per
well for a 24 well plate).

iv. Note: If any 10° (vessel) dilution is used that does not contain CGM (e.g.,
Treatment 2 with proposed neutralizer), allow it to adsorb on the cells for 1 hr,
then remove and replace with fresh CGM.

7. If cytotoxicity was observed in pre-neutralization testing, CGM may be removed from all
wells in the affected dilutions at the appropriate time (one hour minimum), the wells washed
with pre-warmed PBS, and then the PBS replaced with fresh CGM. Follow the same
procedure for dilutions from the control carriers.

8. Incubate test and control plates as appropriate for the test system.

9. For the neutralizer to be considered effective:

a. Ensure that the recovered virus in the Titer Control using Test Suspension B is
between approximately 2-3 logs per vessel.

b. The recovered virus in the Neutralizer Effectiveness treatment is within 0.5 logs of
the Titer Control; this verifies effective neutralization. A log reduction greater than
0.5 logs indicates that the neutralizer was not effective. Note: a value higher than the
Titer Control is also deemed valid.

c. The recovered virus in the Neutralizer Toxicity Control is within 0.5 logs of the
Titer Control. A log reduction greater than 0.5 logs indicates that the neutralizer is
harmful to the test system. Note: a value higher than the Titer Control is also
deemed valid.

10. All criteria in Appendix F, section 9 must be met. If the criteria are not met, another neutralizer
or mixture of neutralizers must be identified and verified.

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SOP No. MEM 1-00
Date Revised 09-26-22
Page 30 of 30

Appendix G
Recipe for Synthetic broth

Solution A: Dissolve the following in 500 mL H2O containing 18 mL 1 N NaOH:

•	0.05 g L-cystine

•	0.37 g DL-methionine

•	0.4 g L-argininexHCl

•	0.3 g DL-histidine

•	0.85 g L-lysinexHCl

•	0.21 g L-tyrosine

•	0.5 g DL-threonine

•	1.0 g DL-valine

•	0.8 g L-leucine

•	0.44 g DL-isoleucine

•	0.06 g glycine

•	0.61 g DL-serine

•	0.43 g DL-alanine

•	1.3 g L-glutami c aci d x HC1

•	0.45 g L-aspartic acid

•	0.26 g DL-phenylalanine

•	0.05 g DL-tryptophan

•	0.05 g L-proline

Solution B: Dissolve the following in 500 mL H2O:

•	3.0 g NaCl

•	0.2 g KC1

•	0.1 gMgS04x7H20

•	1.5 g KH2PO4

•	4.0gNa2HPO4

•	0.01 g thiaminexHCl

•	0.01 niacinamide

Mix Solutions A and B, and steam sterilize 20 min at 121°C.

Final pH should be 7.1±0.1.

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