Interim Quantitative Method for Evaluating the Efficacy of Antimicrobial Test Substances on Porous Surfaces against Viruses (12/12/2022)

Interim Quantitative Method for Evaluating the Efficacy of Antimicrobial Test Substances

on Porous Surfaces against Viruses

(12/12/2022)

Scope

The Environmental Protection Agency (EPA) Office of Pesticide Programs (OPP) recommends
that applicants utilize this interim method to support the registration of disinfectant products with
claims for efficacy against viruses when used on soft-porous surfaces.

This method is quantitative and provides log reduction (virus inactivation) as the quantitative
measure of efficacy for disinfectants against viruses on soft-porous surfaces

Method Overview

In brief, the method uses 1 cm diameter discs (carriers) of a set of materials to represent soft-
porous surfaces. Each disc receives 10 |iL of microbial inoculum (with a three-part organic and
inorganic soil load) deposited in the center of each carrier. The inoculum is allowed to dry and is
then exposed to 50 |iL of the antimicrobial treatment; control carriers receive an equivalent
volume of an innocuous fluid (e.g., growth media). The exposure time is allowed to elapse; a
liquid neutralizer is then added to the vial to halt the antimicrobial action. Each vial with the
carrier is vortexed, serially diluted, and plated onto cells to recover viable virus. The presence of
viable virus particles is determined as applicable to the test system (e.g., cytopathic effect (CPE),
direct fluorescent antibody (DFA) stain, hemagglutination, etc.). Based on the difference
between the mean logio density values of the untreated control and treated carriers, a logio
reduction (LR) in viable virus is calculated The I R value is used as the measure of product
effectiveness.

Appropriate biosafety 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)" 6th
edition, from the subject matter 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 of Health (N1H).

-------
30

31

TABLE OF CONTENTS

1)	Special Apparatus and Materials		3

2)	Carriers	 5

3)	Carrier Inoculation		6

4)	Performance Assessment - Efficacy		8

5)	Data Requirements		9

Attachment 1: Neutralization Assay and Flowchart		11

Attachment 2: Cytotoxicity Determination		13

32

2

-------
33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

1) Special Apparatus and Materials

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

b.	Cell Line, appropriate for the virus tested.

c.	Media and reagents:

i.	Complete Growth Media (CGM). Consisting of Minimum Essential Media and
FBS or other medium specified for the virus being tested. Used for cell line
propagation, 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
Dulbeeeo's). Used to prepare complete growth media. Prepare per
manufacturer's guidelines.

2.	Heat Inactivated Fetal Bovine Serum (IBS). Compatible for use with cell
lines. Often used to prepare complete growth media.

ii.	Neutralizer. Used to inactivate and/or dilute the antimicrobial treatment to end the
contact time.

1. Note: The recommended neutralizer for the 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.

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

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

v. Soil load, 3-part. Use as the soiling agent. Add to the test suspension in the
following manner:

1.	BSA: Add 0.5 g bovine serum albumin (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 (e.g., a minimum of 50 |iL), and store at -20±2°C.

2.	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 (e.g., a minimum of 70 |iL), and store at -20±2°C.

3.	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 at -20±2°C.

3

-------
70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

4. The three stock solutions of the soil load are single use only. Do not
refreeze; store up to one year at 20±2°C.

vi.	Antimicrobial Test Substance. Ready-to-use, activated, or concentrated
antimicrobial. If the antimicrobial test substance is prepared by diluting a
concentrate, adequately mix antimicrobial test substance with the appropriate
diluent (e.g., hard water), then use prepared test substance within 3 hours of
preparation or as otherwise instructed by the manufacturer. Measuring error
increases as delivery volume decreases. To minimize variability due to measuring
error, a minimum of 1.0 mL or 1.0 g of concentrated antimicrobial test substance
should be used when preparing use-dilutions for testing. Use v/v dilutions for
liquid antimicrobial test substances and w/v dilutions for solid antimicrobial test
substances. The use of a positive displacement pipette is recommended for
viscous liquids.

vii.	Water. De-ionized (Dl), distilled water or water with equivalent quality for
making reagent solutions and culture media.

d. Apparatus

i.	Carriers: Discs (1 cm in diameter) cut from porous material. Carriers are single
use only. See section 2 for carrier specifications.

ii.	Hole punch: If necessary, for use in the preparation of 1 cm discs from material.
Model number: SKU# HP-ME1448R or equivalent.

iii.	Calibrated 10 uL positive displacement pipette with corresponding 10 |iL tips, for
carrier inoculation.

iv.	Filter paper. Whatman No. 2, to line glass Petri plates.

v Calibratedmicropipettes (e.g., 200 |iL, 1 mL) with appropriate corresponding
lips, for deposition of lest substance on carriers and preparing dilutions.

vi.	iorceps, straight or curved, non-magnetic, disposable with smooth flat tips to
pick up llie carriers for placement in vials.

vii.	Vials ivilli In/s (plastic or comparable). Sterile, flat-bottomed, wide-mouthed (at
least 25 mm diameter), approximately 20 mL capacity, for holding inoculated
carriers lo he exposed to the test chemical and for accommodating neutralizer.

1. Transparent vials are more desirable to facilitate application of 50 |iL test
substance or control substance to inoculated carrier.

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

ix. Desiccation unit (with gauge to measure vacuum) with fresh desiccant (e.g.,
anhydrous CaCh). For drying inoculated carriers.

4

-------
106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

x.	Vacuum source. In-house line or suitable vacuum pump capable of achieving
0.068 to 0.085 MPa, for drying inoculated carriers in desiccation unit and to
perform membrane filtration.

xi.	Titration kit (e.g. Hach digital titrator). For measuring water hardness.

xii.	Vortex-style mixer. For vortex-mixing of various solutions.

xiii.	15 mL conical centrifuge tubes. For serial dilutions.

xiv.	Water bath. To maintain cell culture media at 37±1°C.

xv.	Tissue/cell culture flasks (tissue culture treated). Flasks for cell propagation.

xvi.	Cell plates. 24-well plates used to assay virus from control and treated carriers.

xvii.	Centrifuge (with swinging bucket rotor). For preparing frozen virus stock.

xviii.	Ultracentrifuge (capable of spinning 100,000 x g). For concentrating virus stock
if needed.

xix.	Inverted microscope. For viewing cells.

xx.	Incubator with 5% CO2. For incubation of virus/cell line test system

2) Carriers

a. Carrier Materials (see Figure 1)

i.	Privacy Curtain Fabric (PCF-03): 54% Polyester, 46% Fire Resistant (FR)
Polyester. CF Stinson, LLC. Mambo MAM34 Nights.

ii.	Non-PVC Fabric (NVF-01): Polyurethane Face made with Polycarbonate and
Polyether Resins, Polyester Backing. CF Stinson, LLC. Kid BlueSky K1D17.

iii.	Vinyl Seating Fabric (VF-01): Vinyl Face, Polyester Backing. CF Stinson, LLC.
Hopsack - HOP24 Fjord.

Figure 1: Examples of carrier materials cut into 1 cm discs; materials 2.a.i, 2.a.ii,
and 2.a.iii (from left to right)

b. Carrier Preparation

i. Punch 1 cm round carriers or use comparable cutting procedure from fabric.

5

-------
133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

ii. Visually screen carriers to ensure consistent surface characteristics; trim any
jagged edges or loose fabric.

iii. No pre-cleaning of carriers is necessary. To sterilize carriers, sterilize using a
gravity cycle, 121°C for 20 minutes; ensure carriers are dry following
sterilization. Test sterility of carriers prior to testing.

1. Carriers may not be entirely flat after autoclaving; however, minor
distortion of carriers is acceptable for testing.

2. Prior to use in testing, document the condition of the screened and sterile
carriers (e.g., digital photographs).

c. Carrier Cytotoxicity Check

i. Each carrier type should be tested for any cytotoxic ell eels on the cell line. Place
a carrier into 10 mL of the proposed neutralizer and lei soak for ten minutes. Add
1 mL per well of this solution to a 24 well plate with a eonlluent monolayer of
cells. Incubate plate at the required conditions and lime. ohser\ e daily for
cytotoxicity. No cytotoxicity should be observed

3) Carrier Inoculation

a. Propagate the virus on the appropriate cell line.

Note: Concentration of the virus stock (~ 100,000 x g for 4 hours at 4° C) may be necessary to
achieve adequate control counts.

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).

c. Dilute the virus stock with CGM to achieve control counts in the range of 4.0 to 5.5 logs
virus particles/carrier

d. Use the diluted virus lo 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 microbial test suspension,
i. 340 |iL virus test suspension

f. It is advisable to briefly rescreen each sterilized carrier for abnormalities prior to
inoculation. Place carriers screened side up inside an empty, sterile plastic Petri dish (no
more than 20 carriers/dish).

-------
169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

i. Privacy curtain carriers have no backing material and may be inoculated on either
side

ii. Non-PVC and vinyl carriers are layered materials comprised of a smooth, colored
top surface and a white fabric bottom; only the top surface will be inoculated.

g. Vortex-mix the final test suspension for 10 s following the addition of the virus test
suspension and immediately prior to use. Inoculate carriers within 30 min of preparation.

i. If a smaller test suspension is prepared, pipetting to mix may be used.

ii. Inoculate the number of carriers required for the evaluation of the test substance
(3 controls and 5 treated) along with a few extra carriers.

h. Using a calibrated positive displacement pipette with a 10 |iL tip, w iihdraw 10 |iL of the
final test suspension and deposit it at the center of each carrier (clean, screened and
sterile); avoid contact of pipette tip with carrier and do not spread the final test
suspension with the pipette tip.

i. For consistency, vortex-mix the final test suspension frequently during
inoculation of the carrier set.

ii. The same pipette tip may be used lo inoculate all carriers (unless the tip is
compromised).

iii. Discard any inoculated carrier u here the final test suspension has run over the
edge.

i. Transfer the Petri dish(es) with the inoculated carriers into a desiccation unit (with
desiccant) and completely remove the lid of I lie Petri dish. Close the desiccation unit door
(or lid) and seal the unit. Apply vacuum lo e\ acuate the desiccation unit.

j. Maintain and monitor the vacuum level using a gauge. Achieve and maintain consistent
level of vacuum (at 20-25 in of mercury, 508-635 torr, 677-847 mbar, or 68000-85000
Pascal) throughout the carrier drying process.

k. Hold the inoculated carriers in the evacuated desiccation unit at 21±3°C for 45 to 60 min.
Inspect inoculated carriers to verify that they are not visibly wet and remove from
desiccation unit. Do not use carriers that are visibly wet for testing.

i. Record the time for all timed events.

ii. Depressurize the desiccator slowly to avoid the potential for carriers to move or
flip.

1. Use dried inoculated carriers for testing within 30 min following removal from

desiccation unit; hold carriers in closed Petri dish at room temperature (21±3°C) until
use.

4) Performance Assessment - Efficacy

a. Evaluate 3 untreated control carriers and 5 treated carriers per test substance.

-------
206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

i. One set of untreated control carriers may be used for evaluating multiple test
substances against the same virus on the same test day (assuming the carrier
material, neutralizer, and soil load are the same).

b. Using sterile forceps, transfer each dried carrier with the inoculated side up to a flat-
bottom vial and cap the vial. Repeat until all carriers are transferred.

c. Prepare the antimicrobial test substance. Use antimicrobial test substance within 3 hours
of preparation or as specified by the manufacturer.

d. In a timed fashion with appropriate intervals, sequentially deposit 50 |iL of the test
substance (equilibrated to 21±3°C) with a calibrated micropipette over the dried
inoculum on each test carrier, ensuring complete coverage.

i. Note: Gently apply the antimicrobial test substance at a perpendicular angle to the
inoculated carrier; do not forcefully deposit the disinfectant.

e. Use a new tip for each carrier; do not touch the carrier surface with a pipette tip during
the application of the test substance or the control substance; replace with new carrier(s)
and vial(s) if this occurs. Do not cap the vials.

i. For non-foaming aerosols and pump/trigger spray products, obtain the test
substance by dispensing the product into a sterile vessel for collection. Cap the
vessel and use dispensed product within 30 min.

ii. For foaming spray formulations, allow the foam to break down for at least 5-10
minutes for the generation of a l-2mL liquid sample. Cap the vessel and use
dispensed product within 3d min

f. Do not process carriers where the test substance runs off the carrier or does not
completely cover the inoculum spot; replace with new carrier(s) and vial(s) if this occurs.

g. Conduct the test at room temperature (21±3°C) for the selected contact time. Use a
certified timer to ensure that each carrier receives the required contact time.

h. Process control carriers last. Each control carrier receives 50 |iL CGM equilibrated to
21±3°C. instead of the test substance. Hold the control carriers for the same contact time
as used for the test substance.

i. Within ±5 s of the end of the contact period, add neutralizer equilibrated to 21±3°C to
each vial in the specified order according to the predetermined schedule. Briefly vortex-
mix (2-3 s) each vial following the addition of the neutralizer.

i. The vial containing the neutralized solution with carrier is considered to be the
10° dilution.

ii. Use the same neutralizer for the control carriers as that for the treated carriers.

iii. If deemed necessary, the final volume of neutralizer may be increased to 20 mL
(for all carriers).

-------
242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

j. Following the neutralization of the entire set of carriers, vortex-mix each vial for 30±5 s
at high speed to recover and disaggregate the inoculum. Do not remove the carrier from
the vial.

k. Initiate dilutions within 30 min after neutralization and vortex-mixing. Initiate
inoculation of cell line within 30 min of preparing the dilutions.

1. Titrate the samples for virus infectivity using the appropriate cell line using 8 wells per
dilution.

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

i. 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 lo least dilute.

n. The elution steps for control carriers are the same as for the test carriers; use 10-fold
dilutions to achieve 4.0 - 5.5 logs virus particles/carrier.

o. If cytotoxicity was observed in pre-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) and wash them with pre-warmed PBS, then replace the PBS
with fresh CGM.

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

5) Data Requirements

a. Record all observations (presence absence of viable virus particles) and use in
calculations to estimate the log reduction Ixised on the TCIDso or MPN (most probably
number) technique.

b. Use values with at least three significant figures when performing calculations (e.g., log
density, mean log density). Report the final mean log reduction value with two significant
figures (e.g., round up to the nearest tenth).

c. Calculate the TC ID so/carrier or MPN/carrier. Calculate the log density of each carrier by
taking the logic of the density (per carrier).

d. Calculate the mean logio density across treated carriers.

e. Calculate the mean logio density across control carriers.

Calculate the logio reduction (LR) for treated carriers: logio reduction = mean logio
control - mean logio treated.

-------
275

276

Til

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

Attachment 1

Neutralization Assay

The purpose of this section is to assess the effectiveness of the neutralization processes
associated with this method. Perform the neutralization assay prior to testing to demonstrate
the neutralizer's ability to inactivate the chemical and determine if there is interference from
the carrier itself.

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

1) Prepare Test Suspension A: Dilute the virus stock suspension in CGM lo 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).

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

3) Neutralization Treatments (Figure 2)

a. Treatment 1: Neutralizer Effectiveness. Add 50 |iL of the test substance to each
of three test tubes. At timed i nlei \ ill s. add 10 mL (up to 20 mL) neutralizer to
each vessel and briefly swirl (by hand). After 10±2 s, gently add 10 |iL of Test
Suspension B using a micropipette to each vessel. Use a new tip for each tube.
Vortex each tube for 3-5 s Proceed with step 3.

b. Treatment 2: Neutralizer Toxicity Control Add 10 mL neutralizer to each of three
reaction vessel s. At timed intervals, add 10 |j.L of Test Suspension B using a
micropipette to each vessel. Use a new tip for each tube. Vortex each tube for 3-5

s. Proceed with step 3.

c. Treatment 3: Titer Control Add 10 mL CGM to each of three reaction vessels.
At timed intervals, add 10 |j.L of Test Suspension B using a micropipette to each
vessel. Use a new tip for each tube. Vortex each tube for 3-5 s. Proceed with step
3.

d. Treatment 4: Carrier Interference Control. Add 10 mL CGM and one sterile
carrier to each of three reaction vessels. At timed intervals, add 10 |j.L of Test
Suspension B using a micropipette to each vessel. Use a new tip for each tube.
Vortex each tube for 3-5 s. Proceed with step 3.

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

4) Hold the neutralization treatments for 10±1 minutes at room temperature (21±3°C).

-------
315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

5) 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).

a. 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.

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

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

ii. 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.

c. 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) and wash them with pre-warmed PBS, then replace the PBS with fresh
CGM. Also wash with pre-warmed PBS and replace the CGM in the same
dilutions for the control carriers.

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

e. For the neutralizer to be considered effective:

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

ii. 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.

iii. 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.

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

f. All criteria in step e must be met. If the criteria are not met, another neutralizer or
mixture of neutralizers must be identified and verified.

-------
352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

Attachment 2

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% CGM).

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° n confluent monolayer
of cells and add 1 mL per well of the neutralizer plus CGM solution. Mule at least 4 wells
per dilution. Have at least one well as a negative control (e.g., CGM with 2% PBS 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.

2) Neutralizer Plus Test Chemical Effect on Cell Line.

a) Add 50 |iL of test chemical and 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 an 80-95% confluent monolayer
of cells and add 1 mL per well of the neutralizer plus test chemical solution and dilutions.
Plate at least 8 wells for the 100 dilution, 6 wells for the 10"1 dilution, and 4 wells is 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.

-------
389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

i) For the 10° dilution: On the day of the test, change 2 wells 1-2 hours (1-hour
minimum) after the neutralizer/test chemical mixture was added to the cells. Change
2 more wells 3-5 hours after the neutralizer/test chemical mixture was added to the
cells. The next day, change 1 each of the 1-2 hour and 3-5 hour wells, as well as
another, previously unchanged well.

ii) For the 10"1 dilution: On the day of the test, change 2 wells 3-5 hours after the
neutralizer/test chemical mixture was added to the cells. The next day, change 1 of
these wells as well as another, previously unchanged well.

iii) For the 10"2 dilution: On the day after the test, change 1 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 lhal allows the media to stay on
the cells for as long as possible.

i) Example: In the 10° dilution, if the unchanged wells are lo\ic, but both the 1 hour
and 4 hour media changes are non-lo\ic. change the media in the 100 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 ASTM
Method E1482-12. Standard Practice for Use of Gel Filtration Columns for Cytotoxicity
Reduction and Neutralization (Reapproved 2017) for further information on column
filtration.

3) 3-Part Soil Effect on Cell Line.

a) Make the 3-part soil (see section 14e but withhold the virus).

b) Add 10 |il. ol'lhe 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 an 80-95% 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.

-------
422

423

Attachment 3

Treatment 1

Add 50 nL of test
substance to each
vessel. At timed
intervals add 10 mL
neutralizer and swirl by
hand.

Figure 2: Neutralization Schematic

000

Neutralizer Effectiveness

—^ Wait 10 s ~
50 [jL test substance

Add 10 nL of Test
Suspension B to each
vessel containing 50 nL
test substance and 10
mL neutralizer.

Vortex and hold for
—10 min at 21±3°C.
Proceed to
vortexing/plating.

Treatment 2^,

Add 10 nL of Test
Suspension B to each
vessel containing 10 mL
neutralizer.

Treatment 3

Add 10 nL of Test
Suspension B to each
vessel containing 10 mL
PBS.

0^0

Neutralizer Toxicity
Control

Titer Control

Vortex and hold for
10 min at 21±3°C.
Proceed to
vortexing/plating.

Vortex and hold for
10 min at 21±3°C.
Proceed to
vortexing/plating.

Treatment 4

Add 1 sterile carrier to
each vessel. At timed
intervals, add 10 mL
neutralizer and swirl by
hand.

Wait 10 s

Titer Interference Control

Add 10 nL of Test
Suspension B to each
vessel containing the
carrier and 10 mL
neutralizer.

Vortex and hold for
10 min at 21±3°C.
Proceed to
vortexing/plating

14

-------