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October, 2018

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MODEL: LabCyte CORNEA-MODEL24

Japan Tissue Engineering
Co., Ltd.

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LabCyte CORNEA-MODEL24
EYE IRRITATION TESTOPERATION PROTOCOL
Ver. 2.7

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TABLE OF CONTENTS

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12
13
14
15
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18
19
20
21
22
23
24
25
26

1. RATIONALE AND BACKGROUND ..............................................................................4

27
28
29
30
31
32
33
34

2. MATERIALS ..................................................................................................................9

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36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56

3. TEST METHOD...........................................................................................................12

1.1 The LabCyte CORNEA-MODEL24 EYE IRRITATION TEST .....................................................4
1.2 BACKGROUND OF LabCyte24 EIT ..........................................................................................4
1.3 BASIS OF THE TEST METHOD ................................................................................................5
1.4 LIMITATION OF THE METHOD .................................................................................................5
1.5 BRIEF BASIC PROCEDURE.....................................................................................................5
1.5.1
1.5.1.1
1.5.1.2
1.5.2
1.5.2.1
1.5.2.2
1.5.2.3

LabCyte CORNEA-MODEL24 ....................................................................................................... 6
QUALITY CONTROL OF THE TEST SYSTEMS ............................................................................ 6
PRECAUTIONS............................................................................................................................ 7
ASSAY QUALITY CONTROL ........................................................................................................ 7
ASSAY ACCEPTANCE CRITERION 1: NEGATIVE CONTROL ...................................................... 7
ASSAY ACCEPTANCE CRITERION 2: POSITIVE CONTROL........................................................ 7
ASSAY ACCEPTANCE CRITERION 3: STANDARD DEVIATION (SD) ........................................... 8

1.6 DATA INTERPRETATION PROCEDURE (PREDICTION MODEL).............................................8
2.1 LabCyte CORNEA-MODEL24...................................................................................................9
2.1.1
2.1.2
2.1.3

LabCyte CORNEA-MODEL24 KIT COMPONENTS ....................................................................... 9
SHIPMENT OF LabCyte CORNEA-MODEL24 ............................................................................... 9
INSTRUCTIONS FOR USE OF LabCyte CORNEA-MODEL24 ..................................................... 10

2.2 CONSUMABLES..................................................................................................................... 10
2.3 OTHERS ................................................................................................................................. 11
2.3.1

EQUIPMENT/INSTRUMENTS ..................................................................................................... 11

3.1 PREPARATIONS ..................................................................................................................... 12
3.1.1
3.1.2
3.1.3

POSITIVE CONTROL ................................................................................................................. 12
NEGATIVE CONTROL................................................................................................................ 12
POLY WASH BOTTLE FOR PBS ................................................................................................ 12

3.2 TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH WST-8 ENDPOINT ............. 13
3.2.1
3.2.1.1
3.2.1.2
3.2.2
3.2.2.1
3.2.2.2

DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE ................................................... 13
STEP 1 (PRELIMINARY TEST) ................................................................................................... 13
STEP 2 (FUNCTIONAL CHECK ON VIABLE TISSUE) ................................................................. 13
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE WST-8 .............................................. 14
STEP 3 (PRELIMINARY TEST) ................................................................................................... 14
STEP 2 (FUNCTIONAL CHECK ON FREEZE-KILLED TISSUE) .................................................. 15

3.3 EXECUTION OF THE TEST .................................................................................................... 17
3.3.1
PREPARATION OF LabCyte CORNEA-MODEL24 (DAY -1)......................................................... 17
3.3.2
APPLICATION OF LIQUID TEST CHEMICALS AND RINSING (DAY 0~1) .................................... 18RD
3.3.2.1
PREPARATION OF WELLS FOR POST-INCUBATION (3 ROW) .............................................. 18
3.3.2.2
APPLICATION OF LIQUID TEST CHEMICALS AND RINSING..................................................... 19
3.3.2.3
POST-EXPOSURE INCUBATION ............................................................................................... 21
3.3.3
APPLICATION OF SOLID TEST CHEMICALS AND RINSING (DAY 0–1) ..................................... 22
ND
3.3.3.1
PREPARATION OF WELLS FOR SOLID TEST CHEMICAL APPLICATION (2 ROW) AND FOR
RD
POST-INCUBATION (3 ROW) ............................................................................................................. 22
3.3.3.2
APPLICATION OF SOLID TEST CHEMICALS ............................................................................. 22

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59
60
61
62

3.3.3.3
3.3.4
3.3.4.1
3.3.4.2
3.3.4.3
3.3.4.463
64
65

4. ASSESSMENT ............................................................................................................2966

5. References .................................................................................................................30

67

MDS 1-1:

68
69

MDS 1-2: TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH WST-8
ENDPOINT STEP-1 (3.2.1.1) ......................................................................................32

70
71

MDS 1-3: TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH WST-8
ENDPOINT STEP-1 (3.2.2.1) ......................................................................................33

72

MDS 1-4:

PREPARATION OF FREEZE KILLED TISSUE (3.2.2) ...................................3473

MDS 2:

PREPARATION OF LabCyte CORNEA-MODEL24 (3.3.1) ...............................35

74
75

MDS 3-1(LIQUID): APPLICATION OF LIQUID TEST CHEMICALS, RINSING AND
POST-INCUBATION (3.3.2) ........................................................................................36

76
77

MDS 3-2(SOLID): APPLICATION OF SOLID TEST CHEMICALS AND RINSING (3.3.3)....................................................................................................................................38

78

MDS 4-1:

79
80

MDS 4-2: SAMPLING OF REACTING WST-8 SOLUTION (3.3.4.3) AND
MEASUREMENT (3.3.4.4) ..........................................................................................41

81

REVISION HISTORY.........................................................................................................4282

RINSING OF SOLID TEST CHEMICALS ..................................................................................... 23
WST-8 ASSAY (DAY 1) ............................................................................................................... 25
PREPARATION OF WELLS FOR WST-8 ASSAY ......................................................................... 25
WST-8 REACTION ..................................................................................................................... 25
SAMPLING THE REACTED WST-8 MEDIUM.............................................................................. 26
OPTICAL DENSITY MEASUREMENTS OF THE REACTION MEDIUM ........................................ 27

4.1 CONDITIONS FOR A VALID TEST.......................................................................................... 29
4.2 ASSAY CRITERIA ................................................................................................................... 29

RECEIPT OF LabCyte CORNEA-MODEL24 (2.1.2) ......................................31

WST-8 ASSAY (3.3.4.1, 3.3.4.2) .....................................................................40

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1. RATIONALE AND BACKGROUND

84

1.1

85

The LabCyte CORNEA-MODEL24 EYE IRRITATION TESTThe LabCyte CORNEA-MODEL24 eye irritation test (LabCyte24 EIT) is designed to identify test

86

chemicals

that

cause

acute

eye

irritationbymeasuring

cytotoxic

effects

using

the

87

2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt

88

(WST-8) assay on the reconstructed human corneal epithelial (RhCE) model. The LabCyte24 EIT is not a89

kit, but LabCyte CORNEA-MODEL24 tissues are commercially available at a minimum of 24 LabCyte

90

CORNEA-MODEL24 tissues per order.

91
92

1.2

BACKGROUND OF LabCyte24 EIT

93

Assessment of ocular irritation is an essential part of early testing procedures for the evaluation and94

hazard classification of substances. Therefore, it plays an important role in the safety evaluation of95

general consumer products and materials. Novel substances used in consumer products must undergo

96

comprehensive toxicological evaluation for eye irritation and a variety of other adverse outcomes. To

97date, the in vivo Draize eye test has been the international standard assay for acute ocular toxicity

98

evaluation (irritation and corrosion) and is described (including optimizations and refinements) in OECD

99

Test Guideline 405. However, the use of this test has been questioned and strongly criticized for ethical100

concerns related to animal welfare, because it is painful to the rabbits. Thus, alternative strategies and101

tests are urgently required in order to evaluate the eye irritation potential of new chemicals. Corneal

102

epithelial cells on the surface of the eye are to first to be exposed to substances and have been widely

103

studied for links to the biological role of tissue and gene regulation. Three-dimensional RhCE models are104

useful as a multilayered, standardized tissue that mimics the human corneal epithelium.

105

The LabCyte24 EIT was developed as a replacement for the Draize eye irritation test. The Draize

106

scoring system is heavily weighted towards corneal damage (80 out of 110 total score), because

107

irreversible damage to the cornea can lead to blindness. Since damage to the cornea is so crucial for108

human health, corneal tissue can be considered a useful tool for the development of in vitro eye irritation

109

testing.

110
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1.3

BASIS OF THE TEST METHOD

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Damage induced by eye irritants generally progresses from the corneal epithelium through the stroma

113

and potentially to the endothelium, and the LabCyte24 EIT is able to provide information on the first114

stages of this progression. Irritants damage cells while penetrating the corneal epithelium layer, and the115

cytotoxic progression can be estimated by analyzing cell viability in the LabCyte CORNEA-MODEL24

116

tissue using standardized methods. Although the tissues represent only the corneal epithelium, (very mild

117

responses would also be reflective of some conjunctival irritation), it can be used to estimate deeper

118

damage as far as the stroma by the analyzing cell viability.

119

The relative viability of the tissue exposed to a test chemical is measured using the WST-8 assay

120

immediately after exposure and again after a post-exposure period. A viability of 40% of the negative

121

control value was used as the cutoff in identifying test chemicals as an irritant (GHS Category 1 or 2) or

122

an non-irritant (GHS No Category). Some culture environments might allow the detection of very small

123

quantities of cytokines secreted by the corneal epithelial tissue in response to topical application of test

124

chemicals.

125
126

1.4

LIMITATION OF THE METHOD127

One limitation of this assay method is potential interference of the test chemical with the WST-8

128

endpoint. A colored test chemical or one that directly reduces WST-8 (and thereby mimics dehydrogenase129

activity of the cellular mitochondria) could interfere with the WST-8 endpoint. This is only a problem,130

however, if there are significant residual levels of the test chemical on or in the tissue at the time of the131

WST-8. Although this is considered unlikely, if it did happen, both the actual metabolic WST-8 reduction

132

and the quasi direct WST-8 reduction by a colored test chemical can be quantified using the procedure133

described in Section 3.2 “TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH

134

WST-8 ENDPOINT”.

135
136

1.5

137
138

BRIEF BASIC PROCEDURE

On the day of receipt, LabCyte CORNEA-MODEL24 tissues are incubated overnight to releasetransport–stress-related compounds and debris.
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For liquid test chemicals, tissues are topically exposed for 1 minute. Preferably, three tissues are used

140

for each test chemical as well as for the positive and negative controls. After exposure, tissues are141

thoroughly rinsed and blotted to remove the test chemical, then transferred to fresh medium and142

post-incubated for 24 hours. For solid test chemicals, tissues are exposed for 24 hours but are not143

post-incubated.

144

After post-incubation of tissue exposed to liquid test chemicals or after exposure of tissue exposed to145solid test chemicals, the tissues are each transferred to a well containing the WST-8 medium in a 1:10

146

dilution with Earle balanced salt solution (EBSS). After a four-hour WST-8 incubation, the orange

147

water-soluble formazan salt is formed in the WST-8 medium by cellular mitochondria and optical density

148(OD) of the WST-8 medium is measured using a spectrophotometer at 450 nm and 650 nm as reference.

149

Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Test

150

chemicals that produce a relative cell viability below 40% of the negative control are predicted to be

151

irritants.

152
153

1.5.1

LabCyte CORNEA-MODEL24

154

The LabCyte CORNEA-MODEL24 is a commercially available RhCE model produced by Japan155

Tissue Engineering Co. Ltd. It comprises normal human corneal epithelial cells that are derived from a156

human cornea. The cells are cultured with 3T3-J2 cells as a feeder layer in order to expand them while157

maintaining their phenotype (Rheinwald and Green, 1975; Green, 1978). Reconstruction of the human158

cultured corneal epithelial tissue is achieved by cultivating and proliferating the corneal epithelial cells

159

on an inert filter substrate with a surface area of 0.3 cm2 at an air-liquid interface for 13 days using an

160

optimized medium containing 5% fetal bovine serum. This results in the formation of a multilayer

161

structure comprising a fully differentiated corneal epithelium with features mimicking those of a normal

162

human corneal epithelium. For delivery, LabCyte CORNEA-MODEL24 tissues are embedded in an

163

agarose gel containing a nutrient solution and shipped in 24-well plates.

164
165

1.5.1.1

166

QUALITY CONTROL OF THE TEST SYSTEMSLabCyte CORNEA-MODEL24 tissue is manufactured in accordance with well-defined quality
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assurance procedures. Each production batch was comes with quality control documentation that168

identifies storage conditions, RhCE instructions for use, lot number and origin, histology (demonstration

169

of human multilayered corneal epithelial-like structure), cell viability, and barrier function integrity

170(0.1 ≤ IC50 ≤ 0.4).

171
172
173

1.5.1.2

174

PRECAUTIONSCorneal epithelial cells are taken from healthy donors who are free of HIV or hepatitis. Nevertheless,175

always adhere to the following procedures for the handling of biological materials:

176(a) Always wear gloves during handling of the eye and kit components.

177(b) All corneal epithelial tissue as well as all material and media that came in contact with it should be178

decontaminated prior to disposal using special containers or autoclaving.

179
180

1.5.2

ASSAY QUALITY CONTROL

1.5.2.1

ASSAY ACCEPTANCE CRITERION 1: NEGATIVE CONTROL

181
182
183

The absolute OD of the negative control for either liquid or solid test chemicals (NC-L or NC-S)

184

tissues (treated with sterile PBS for liquid test chemicals or untreated for solid test chemicals) in the185

WST-8 assay is an indicator of tissue viability obtained in the testing laboratory after shipping and186

storing procedures and under specific conditions of use.

187

0.5 ≤ Mean OD (A450/650) measured value ≤ 1.6

188
189

1.5.2.2

ASSAY ACCEPTANCE CRITERION 2: POSITIVE CONTROL

190

Ethanol is used as the positive control (PC) for liquid test chemicals (PC-L) and is tested concurrently

191

with the liquid test chemicals. Lauric acid is used as the PC for solid test chemicals (PC-S) and is tested

192

concurrently with the solid test chemicals.

193

Concurrent here means that the PC-L and the PC-S are to be tested for each run.

194

Mean tissue viability ≤ 40%
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196

1.5.2.3

197
198

Since eye irritation potential is predicted from the mean viability of three individual tissues, thevariability of tissue replicates must kept at an acceptably low level.

199
200

ASSAY ACCEPTANCE CRITERION 3: STANDARD DEVIATION (SD)Standard Deviation (SD) of tissue viability of three identically treated replicates for the negative
control, positive control, and test chemicals ≤ 18%

201
202

1.6

203
204

The United Nations Globally Harmonized System (GHS) (United Nations, 2003) was used as areference for the in vivo eye irritation classification of test chemicals.

205
206

DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)For the purpose of this EIT, an eye irritant is defined as a substance that induces reversible ocularlesions after administration to rabbits.

207

According to GHS classifications, a substance is an irritant (Category 1 or 2) if the mean relative

208

viability of three individual tissues exposed to the test chemical is falls below 40% of the mean viability

209of the negative control. (Refer to Table 1.)

210

Table 1

211
212

8

Prediction model of LabCyte24 EIT

In vitro results

Prediction

Tissue viability is ≤ 40%

Category 1 or 2

Tissue viability is > 40%

No Category

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213

2. MATERIALS

214

2.1

215

2.1.1

216
217

LabCyte CORNEA-MODEL24
LabCyte CORNEA-MODEL24 KIT COMPONENTS

LabCyte CORNEA-MODEL24 kit components are shown in Table 2.
Table 2

LabCyte CORNEA-MODEL24 Kit Components

Component

Qty

LabCyteCORNEA-MODEL24 plate

Description
Contains 24 culture inserts with tissues fixed in

1 platenutritive agar medium for transport (usable area:
0.3 cm2).

Assay Medium

24-well plate

1 bottle

1 plateBasic medium for incubation (30 mL). Keeprefrigerated.
Blank plate for use in assay. Store at room
temperature.

218
219

2.1.2

SHIPMENT OF LabCyte CORNEA-MODEL24

220

LabCyte CORNEA-MODEL24 is packed in a special Icompo container available from and delivered

221

by NIPPON EXPRESS CO., LTD. After the kit is delivered, examine the contents and make sure that all

222

components (LabCyte CORNEA-MODEL24 plate, assay medium, and 24-well assay plate) are included

223

in the package. Also confirm lot numbers and expiration dates. Record details in Methods Documentation

224

Sheet (MDS) 1. (See MSD-1).

225
226

NIPPON EXPRESS will pick up the Icompo container at a later date (generally, the day after delivery),and it should be returned together with an shipping invoice and the insulating materials.

227
228

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230
231

2.1.3

INSTRUCTIONS FOR USE OF LabCyte CORNEA-MODEL24

Always incubate all of the culture inserts after opening the package. Do not store the culture inserts
after opening.

232

The human corneal epithelial tissue cells used in the LabCyte CORNEA-MODEL24 originate from a

233

normal human donor and are HIV-, HBV-, HCV-, and HPV-negative. They are to be handled,234

nevertheless, with due care and in accordance with laboratory biosafety guidelines for handling235

human-derived materials.

236
237

2.2

CONSUMABLES

238

The following consumables are required.

239*The following quantities are necessary to assay between one and six 6 test chemicals at a time.

240

・ Assay Medium, 100 mL (J-TEC: 402250)

1

241・ Cell Counting Kit-8, 500 test (Dojindo: CK04)

4242

・ 24-well assay plate (Becton, Dickinson and Company: 353047)

243

・ 96-well plate (Becton, Dickinson and Company: 353072 or equivalents)

244

・ PBS, 500 mL (Invitrogen: 14190-144 or equivalents)

245

・ Earle balanced salt solution (EBSS), 500 mL (SIGMA-ALDRICH: E3024)

1 bottle

246

・ Sterile cotton buds (JAPAN COTTON BUDS: 10A754D or equivalents)

1 box

247

・ Micro-pipette tips (sterile: 10~200µL, 200–1000µL)

248

・ Microtubes (1.5mL)

249

・ Dish (10cm)

250

・ Paper towel

251
252

Convenient consumable items are shown followings.

253

Also, it would be convenient to have the following.

254

・ Capillary & piston for positive-displacement-type pipette (10-100µL)

255
256
10

7-8
1
2 or 3 bottle

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2.3

OTHERS

258

2.3.1

259

・ Safety cabinet (or clean bench)

260

・ Water bath (37°C)

261

・ CO2 incubator (37°C, 5% CO2, capable of maintaining high humidity)

262

・ Autoclave

263

・ 96-well multi-plate reader (required filters: 450 nm, 650 nm)

264

・ Precision balance (0.1 mg)

265

・ Aspirator

266

・ Stop-watches

267

・ Adjustable micro-pipette (10–200 μL, 200–1000 μL)

268・ Sharp-edged forceps (sterile)269

・ Micro spatula (sterile)

270

・ Beaker (1–2 L: sterile)271

・ Sterilizable poly wash bottle (500–1000 mL: sterile) with wide mouth (mouth > 3-mm dia.)272

・ Mortar with pestle

EQUIPMENT/INSTRUMENTS

273
274

Also, it would be convenient to have the following.

275

・ Positive-displacement-type pipette (10–100 μL)

276

11

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277

3. TEST METHOD

278*Procedures described in Sections 3.1.1 to 3.1.3 and Sections 3.3.1 to 3.3.3 are to be performed

279

aseptically in a safety cabinet or clean bench. Procedures other than those mentions in the previous

280

sentence need not be performed aseptically. Refer to Section 2.1.3 “INSTRUCTIONS FOR USE

281

OF LabCyte CORNEA-MODEL24”.

282
283

3.1

PREPARATIONS

284

3.1.1

285

(1) Ethanol is used as a positive control for liquid test chemicals.

286(2) Lauric acid is used as a positive control for solid test chemicals.

POSITIVE CONTROL

287
288

3.1.2

NEGATIVE CONTROL

289(1) PBS is used as a negative control for liquid test chemicals.

290(2) Non-treatment is a negative control for solid test chemicals.

291
292

3.1.3

293

(1) Sterilize poly wash bottle by autoclave.

294(2) Fill the sterilized poly wash bottle with sterile PBS.

295
296
297

12

POLY WASH BOTTLE FOR PBS

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299

3.2

TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH WST-8 ENDPOINT

There are two kinds of test chemicals that interfere with the WST-8 assay.

300(a) Test chemicals that stain corneal epithelial tissues directly.

301(b) Test chemicals that react directly with WST-8.

302

The test chemicals that can dye the corneal epithelial tissues will be extracted from the colored tissue

303

during WST-8 reaction, affecting the OD measurement. Also, the test chemicals that can directly304

reduce the WST-8 medium will affect the OD measurement due to unexpected reduction reaction by305

residues of test chemicals in the culture inserts. These test chemicals are needed to perform

306

additional experiments. The procedure is described below.

307
308
309

3.2.1

DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE

310

3.2.1.1

STEP 1 (PRELIMINARY TEST)

311(1) Add 50μL (Liquid) or 10mg (Solid) of the test chemical into wells of 24-well assay plate

312

preliminarily filled with 0.5mL of distilled water. Untreated distilled water is used as control.

313(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 15 minutes.

314(3) After incubation, mix gently and evaluate the color change of the distilled water by visual check.

315(4) When the color of the solution changes significantly, the test chemical is presumed to have the316potential to stain the tissue. A functional check on viable tissues (Step2) should be performed. If the317

color of the solution does not change significantly, the test chemical is determined not to have a318

potential to stain the tissue.

319(5) Record the details of steps 1 to 4 above in MDS 1-2.

320
321

3.2.1.2

322(1) Add 50 μL (liquid) or 10 mg (solid) of the test chemical, which could clearly change the color of the

323

distilled water in step 1, onto the surface of the epidermis tissues. PBS are used as negative control.

324(2) Go to the Section 3.3 EXECUTION OF THE TEST and perform the experiments according to the325

procedures, expect for the WST-8 reaction. In the section of WST-8 assay, use the EBSS that does
13

STEP 2 (FUNCTIONAL CHECK ON VIABLE TISSUE)

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326

not contain WST stock solution, instead of diluted WST-8 medium to evaluate the affected OD

327

values by colored diluted water. (correcting tissue).

328(3) The corrected OD is calculated using the following formula.

329

Corrected OD = A − (B − C)

330
331

A : the OD of viable tissue exposed to a test chemical.

332

B : the mean OD of correcting tissue exposed to a test chemical.

333

C : the mean OD of correcting tissue exposed to the negative control.

334
335

(4) If a corrected OD is below 0, the OD is considered to be 0.

336(5) When a cell viability that is calculated according to the procedures described in Section 3.3.4.4 is337≤ 40%, the test chemical is predicted to be an irritant (GHS Category 1 or 2) and there is no need338

to calculate a corrected value.

339
340
341

3.2.2

DETECTION OF CHEMICALS THAT DIRECTLY REDUCE WST-8

342

3.2.2.1

STEP 3 (PRELIMINARY TEST)

343(1) Dilute the cell counting kit-8 (WST-8 stock solution) with EBSS (Cell Counting Kit-8:EBSS = 1:10),344

and then prepare the diluted WST-8 medium.

345*Prepare WST-8 medium before use.

346

Dispense 0.3 mL of diluted WST-8 medium into each well of the 24-well assay plate.

347(2) Add 50 μL of a liquid test chemical or 10 mg of a test chemical to the wells of 24-well assay plate.

348

The diluted WST-8 medium is used as control.

349(3) Put on the lid of 24-well assay plate and incubate in a CO2 incubator for about 4 hours.

350(4) After incubation, shake the mixture gently and evaluate the color change of the diluted WST-8

351

medium by visual check.

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Ethanol

352

Photo 1

5% Triton X100

Glycolic acidm-Phenylene
diamineMonoethanolamine

Calcium
thioglycollate

Example of test chemicals to interfere the WST-8 assay directly (STEP 1).

353

When test chemicals like m-Phenylene diamine, Monoethanolamine, or Calcium thioglycollate have354

colored the diluted WST-8 medium, Step 4 must be performed.

355(5) Significant coloring of the diluted WST-8 medium by the test chemical indicates the interference

356

between test chemicals and the WST-8 assay medium. The additional functional check is required for

357

these interference chemicals. Go to step 4 described below.

358(6) Record the details of steps 1 to 5 above in MDS 1-3.

359
360

3.2.2.2

361(1) Add 50 μL of a liquid test chemical or 10 mg of solid a test chemical that clearly changed the color362

of the diluted WST-8 medium (3.2.2.1. STEP 3) to the surface of the corneal epithelial tissues.

363(2) Go to Section 3.3 “EXECUTION OF THE TEST” and perform the experiments using364

freeze-killed corneal epithelial tissues, instead of using viable corneal epithelial tissues. . The

365

freeze-killed tissues are prepared by placing untreated Labcyte CRNEA-MODEL in-80°C or lower

366

for more than 30 min.

367

Record the details of freeze-killing the tissue in MDS 1-3.

368(3) The corrected OD is calculated using the following formula.

369

STEP 4 (FUNCTIONAL CHECK ON FREEZE-KILLED TISSUE)

Corrected OD = A − (B − C)

370
371

A : the OD of viable tissue exposed to a test chemical.

372

B : the mean OD of freeze-killed tissue (correcting tissue) exposed to a test chemical.

373

C : the mean OD of freeze-killed tissue (correcting tissue) exposed to the negative control.

374
375

(4) If a corrected OD is below 0, the OD is considered to be 0.
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376(5) Calculate a cell viability according to the procedures described in Section 3.3.4.4. In case that the377

cell viability is ≤ 40%, the test chemical is predicted to be an irritant (GHS Category 1 or 2) and378

there is no need to calculate a corrected value.

379
380

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381

3.3

EXECUTION OF THE TEST

382
383

3.3.1

384

(1) Warm the assay medium to 37°C for 30 minutes

385

PREPARATION OF LabCyte CORNEA-MODEL24 (DAY -1)

in a water bath.

Fig. 1386(2) Dispense 0.5 mL/well of the warm assay

387

medium into the six wells of the 1st row of each(pre-incubation)388

assay plate for LIQUID/SOLID. .

2nd row

389

See Fig. 1.

3rd row390(3) Open

391
392

the

LabCyteCORNEA-MODEL24

Assay Plate for LIQUID/SOLID

1st row

Test chemical 1

Test chemical 2(chemical application)

4th rowaluminum package.
(4) Open the LabCyte CORNEA-MODEL24 plate lid and pick up the culture inserts using sterile

393

forceps.

394*Do not touch the surface of the corneal epithelial tissue in the culture inserts.

395*Use forceps to remove any agar medium

396

sticking to the outside of the culture inserts.

397(5) Transfer the culture inserts to the assay medium

398

in the wells of the 1st row using sterile forceps.

399

See Fig. 2.

Fig. 2

Assay Plate for LIQUID/SOLID

st

1 row
(pre-incubation)(chemical application)

Test

nd

2 row

chemical1

Test chemical 2

rd

3 row
th

400

(6) * Avoid the formation of air bubbles under the401tissue inserts. Close the lid on the plate and402

place it in a CO2 incubator.

4 row

403

(7) Incubate overnight (15–30 hours) until ready to perform Section 3.3.2 “APPLICATION OF

404

LIQUID TEST CHEMICALS, RINSING AND POST-INCUBATION” or Section 3.3.3

405“APPLICATION OF SOLID TEST CHEMICALS AND RINSING”.

406(8) Record the details of steps 1 to 7 above in MDS 2.

407
408
409

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3.3.2

APPLICATION OF LIQUID TEST CHEMICALS AND RINSING (DAY 0~1)412

3.3.2.1

PREPARATION OF WELLS FOR POST-INCUBATION (3RD ROW)

413

(1) Warm the assay medium to 37°C for 30 minutes

410
411

414
415

using a water bath.
(2) Take out the assay plate for LIQUID from theFig. 3(pre-incubation)(chemical application)

416

CO2 incubator.

417(3) Open the lid of the assay plate for LIQUID, and418

use a micropipette to fill the six wells of the 3 rd419

row with 0.5 mL/well of the warm assay medium.

420

See Fig. 3.

421

chemical1

3rd row

Test chemical 2(post-incubation)
4th row

CHEMICALS AND RINSING” continuously.
(5) If the test chemicals are not applied immediately, store the Assay Plate for LIQUID in a CO2

424
425

Test

2nd row(4) Close the lid of the assay plate and perform Section 3.3.2.2 “APPLICATION OF LIQUID TEST

422
423

Assay Plate for LIQUID

1st row

incubator until ready apply but for no more than 12 hours.
(6) Record the details of steps 1 to 5 above in MDS 3-1.

426
427

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428

3.3.2.2

429(1) Take out the assay plate for LIQUID from the CO2 incubator.

430(2) Using a micropipette, apply 50 μL of a liquid test

431

chemical to the surface of the corneal epithelial

432

tissues in the 1st row of the assay plate. Each test

433

chemical is to be tested in three wells (N=3).

434

Carefully apply the test chemical to the central

435

part of each corneal epithelial tissue. After

436

application, close the lid of the assay plate and tap

437

the sides of the plate to spread the liquid test

438

chemicals to spread out over the entire corneal

439

epithelial surface. If necessary, use a micro spatula to spread the liquid test chemical over the entire

440

surface. Take care not to press down on the surface of the corneal epithelial with the spatula.

441*For viscous LIQUID test chemicals, use a wide orifice cell saver tip (See Photo 2.) or positive

442

APPLICATION OF LIQUID TEST CHEMICALS AND RINSING

Photo 2 Pipette tips for viscous liquid
test chemicals

displacement type pipette.

443*Use a pipette or other equipment to familiarize

444

yourself beforehand with the characteristics

445

the test chemicals.

Fig. 4

Assay Plate for LIQUID

Test chemical 1

Test chemical 2

1st row(pre-incubation)(chemical application)

446*Assay no more than two test chemicals on one

447

24-well assay plate.

448

See Fig. 4.

449

Each chemical is tested in three wells, using

450

three tissues (N = 3).

2nd row
3rd row(post-incubation)
4th row

451(3) Apply test chemicals to each well at an interval of one to three minutes.

452(4) Close the lid and incubate each well for 60 ±10 seconds in the cabinet at room temperature.

453*Keep the lid of the assay plate closed at all times except when applying test chemicals. Leaving the454

lid open could affect the quantity of the test chemical in the well due to air circulation in the455

cabinet.
19

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456(5) Wait for 60 ±10 seconds after applying a test chemical,

457

then open the assay plate for LIQUID and pick up a458

culture insert with sterile forceps.

459(6) Discard the test chemical on the tissue by decantation460

and tapping it on a beaker. Fill the culture insert to461

overflowing with PBS from a poly wash bottle.

462* Keep PBS flush during rinsing to wash away the test

463

Rinse 1

Photo 4

Rinse 2chemical from the tissue surface..

464

See Photo 3.

465*To avoid damaging the tissue with too forceful a466

stream, use a wide-mouth nozzle on the poly wash

467

bottle.

468(7) Decant the PBS into the beaker. Remove as much of the469

PBS inside the culture insert as possible by tapping it on470

the beaker.

471

See Photo 4.

472

Photo 3(8) Repeat steps 6 and 7 at least 10 times to remove as much as possible of the residual test chemical on473

the tissue surface.

474*Depending upon the physical properties of the test

475

chemical, bubbles might form in an insert during476

washing.

477

disappear.

Continue

washing

until

all

bubbles478(9) Using a sterile cotton bud, gently remove as much as

479

possible of the leftover PBS both inside and outside the480

culture insert.

481

See Photo 5.

482*Take care not to press down on the surface of the tissue483

with the sterile cotton bud.
20

Photo 5 Rinse 3

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484
485(10) Wipe the culture insert and then place it in the well486

of the same column in the 3rd row.

487

See Fig. 5.

488*Take care to prevent air bubbles from forming

489

under the culture inserts.

Fig.5

Assay Plate for LIQUID
Test chemical 1

Test chemical 2

1st row(pre-incubation)
2nd row(chemical application)490(11) Repeat steps 1 to 11 for all culture inserts at one- to

491
492

3rd row(rinse)
4th row

three-minute intervals.
(12) Record the details of steps 1 to 12 above in MDS 3-1.

493
494

3.3.2.3

POST-EXPOSURE INCUBATION

495(1) After performing Section 3.3.2.2 “APPLICATION OF LIQUID TEST CHEMICALS AND

496

RINSING”, close the lid of the assay plate for LIQUID and place it in a CO2 incubator as soon as

497

possible.

498(2) Incubate for 24 ±1 hours.

499(3) Record the details of steps 1 and 2 above in MDS 3-1.

500
501
502

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503

3.3.3

APPLICATION OF SOLID TEST CHEMICALS AND RINSING (DAY 0–1)

3.3.3.1

PREPARATION OF WELLS FOR SOLID TEST CHEMICAL APPLICATION

504
505

ROW) AND FOR POST-INCUBATION (3RD ROW)

506
507

(1) Warm the assay medium to 37°C for 30 minutes using a water bath.

508(2) Take out the assay plate for SOLID from the CO 2

509
510

(3) Open the lid of the assay plate for SOLID and use
a micropipette to fill the 12 wells in the 2nd and 3rd

512rows with 0.5 mL/well of warm assay medium .

513

See Fig. 6.

1st row(pre-incubation)
2nd row(chemical application)
3rd row(rinse)
4th row

3.3.3.2 “APPLICATION OF SOLID TEST CHEMICALS” continuously.
(5) If the test chemicals are not applied immediately, store the Assay Plate for LIQUID in a CO2

517
518

Assay Plate for SOLID(4) Close the lid of the assay plate and go to Section

515
516

Fig.6

incubator.

511

514

(2ND

incubator until ready apply but for no more than 12 hours.
(6) Record the details of steps 1 to 5 above in MDS 3-2.

519
520

3.3.3.2

APPLICATION OF SOLID TEST CHEMICALS521(1) Take out the assay plate for SOLID from the CO2
Photo 6

Application of SOLID

522

incubator.

523

Using a precision balance, weigh out 10 ±2 mg of524

the solid test chemicals. If necessary, crush and525

grind the solid test chemicals in a mortar with526

pestle. Apply the solid test chemical to the surface

527

of the corneal epithelial tissue.

528

See Photo 6.

529

If necessary, use a micro spatula to spread the test chemical gently over the entire surface. Use three530

wells per test chemical (N = 3).
22

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531

See Fig. 7.

532

Assay Plate for SOLID

Fig.7

Test chemical 1
1st row(pre-incubation)

533(2) Place the exposed culture insert in the well of534

the same column in the 2nd row (chemical

535

application).

536

See Fig. 8.

537*Take care to prevent air bubbles from forming538

2nd row(chemical application)
3rd row(rinse)
4th row

Assay Plate for SOLID

Fig.8

under the culture inserts.

Test chemical 1539(3) Close the lid of the assay plate for SOLID and540

place it in a CO2 incubator. Incubate for(pre-incubation)
2nd row541

24 ±1 hours.

(chemical application)
3rd row

542

Test chemical 2(4) Record the details of steps 1 to 3above in

Test chemical 2

1st row(rinse)
4th row

MDS 3-2.

543
544
545

3.3.3.3

RINSING OF SOLID TEST CHEMICALS

546(1) After incubation, take out the assay plate for SOLID from the CO 2 incubator.

547(2) Open the assay plate for SOLID and pick up a culture insert with sterile forceps.

548(3) Discard test chemicals on the tissue by tilting the insert and tapping if on a beaker. Fill the culture549

insert to overflowing with PBS from a poly wash bottle.

550* Keep PBS flush during rinsing to wash away the test

551

chemical from the tissue surface.

552

See Photo 7.

553*To avoid damaging the tissue with too forceful a554

stream, use a wide-mouth nozzle on the poly wash

555

bottle.

556(4) Tilt the insert to discard the PBS into the beaker.

557

Remove as much of the PBS inside the culture insert as558

possible by tapping it on the beaker.
23

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559

See Photo 8.

560(5) Repeat steps 3 and 4 at least 10 times to remove as

561

much as possible of the residual test chemical on the562tissue surface.

563(6) Using a sterile cotton bud, gently remove as much as564

possible of the leftover PBS both inside and outside565

the culture insert.

566

See Photo 9.

567(7) If it proves difficult to remove completely all the568

residual test chemical from the corneal epithelial tissue569

surface, remove as much as possible and continue to570

step 8.

571

Rinse 2

Photo 9Rinse 3(8) Place the rinsed culture insert in the well of the same572

column in the 3rd row (rinse).

573

See Fig. 9.

574*Take care to prevent air bubbles from forming

575
576

Photo 8under the culture inserts.
(9) Record the details of steps 1 to 8 above in MDS

577

3-2.

578

After rinsing step, perform Section 3.3.4

579“WST-8 assay” continuously.

580
581

24

Fig.9
1st row(pre-incubation)
2nd row(chemical application)
3rd row(rinse)
4th row

Assay Plate for SOLID

Test chemical 1

Test chemical 2

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3.3.4

WST-8 ASSAY (DAY 1)

584

3.3.4.1

PREPARATION OF WELLS FOR WST-8 ASSAY

585(1) Warm the EBSS to 37°C for 30 minutes using a water bath.

586(2) Dilute the cell counting kit-8 (WST-8 stock solution) with EBSS (Cell Counting Kit-8:EBSS = 1:10),587

and then warm the diluted WST-8 medium. Prepare the additional dilute WST-8 medium for blanks588

in the WST-8 assay.

589*Prepare WST-8 medium before performing the

582
583

590

WST-8 assay.

Fig.10

Assay Plate for LIQUID/SOLID
Test chemical 1

Test Chemical 2

1st row(pre-incubation)(chemical application)591(3) Take out the assay plate for LIQUID from the CO 2592

incubator or prepare the assay plate for SOLID.

593(4) Open the lid of the assay plate and use a594

micropipette to fill each well of the 4th row with595

0.3 mL/well of the warm diluted WST-8 medium.

596

See Fig. 10.

597

Close the lid of the assay plate and perform Section 3.3.4.2 “WST-8 REACTION” continuously.

598

2nd row(post-incubation)(chemical application)
3rd row(rinse)
4th row(WST-8 reaction)(5) Record the details of steps 1 to 4 above in MDS 4-1.

599
600

3.3.4.2

WST-8 REACTION

601(1) Add about 20 mL of PBS each to two dishes: PBS dish 1 and PBS dish 2.

602(2) Open an assay plate (for either LIQUID or SOLID) and pick up a culture insert with sterile forceps.

603(3) Remove residual culture medium on a cluture604

insert by washing in PBS dish 1(first time) and the605

in PBS dish 2 (second time). After washing, wipe606

the bottom with a paper towel.

607(4) After washing and wiping, place the culture insert

608

in the well of the same column in the 4th row.

609

See Fig. 11.
25

Fig.11

Assay Plate for LIQUID/SOLID

1st row(pre-incubation)(chemical application)
2nd row(post-incubation)(chemical application)
3rd row(rinse)
4th row(WST-8 reaction)

Test chemical 1

Test Chemical 2

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610* Avoid the formation of air bubbles under the culture inserts.

611(4) Close the lid of the assay plate and place it in the CO 2 incubator.

612

Incubate for 4 hours ± 20 minutes.

613

Record the details of steps 1 to 5 above in MDS 4-1.

614
615

3.3.4.3

SAMPLING THE REACTED WST-8 MEDIUM

616(1) After incubation, take out the assay plate from the CO2 incubator.

617(2) Open the lid of the assay plate and remove the culture inserts from the 4th row with forceps.

618

Transfer 200 μL of the reacted WST-8 dilution medium into the wells of a 96-well plate.

619*Figs. 12A and 12B show typical allocations on a 96-well plate for both living and correcting tissue

620(freeze-killed tissue or tissue reacted with only EBSS that does not contain WST-8 stock solution).
Fig.12A

Allocation on a 96-well plate for living tissue
1

A

26

4

5

6

NC forNC forPC forPC forPC for

7

8

9

10

11

12

Blank
NC forNC forNC forPC forPC forPC forSOLID-1

SOLID-2

SOLID-3

SOLID-1

SOLID-2

SOLID-3C

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

SOLID

SOLID

SOLID

SOLID

SOLID

SOLID

1-1

1-2

1-3

2-1

2-2

2-3

1-1

1-2

1-3

2-1

2-2

2-3

D

LIQUID
3-1

LIQUID
3-2

LIQUID
3-3

LIQUID
4-1

LIQUID
4-2

LIQUID
4-3

SOLID
3-1

SOLID
3-2

SOLID
3-3

SOLID
4-1

SOLID
4-2

SOLID
4-3

E

LIQUID
5-1

LIQUID
5-2

LIQUID
5-3

LIQUID
6-1

LIQUID
6-2

LIQUID
6-3

SOLID
5-1

SOLID
5-2

SOLID
5-3

SOLID
6-1

SOLID
6-2

SOLID
6-3

F

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

SOLID

SOLID

SOLID

SOLID

SOLID

SOLID

7-1

7-2

7-3

8-1

8-2

8-3

7-1

7-2

7-3

8-1

8-2

8-3

G

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

SOLID

SOLID

SOLID

SOLID

SOLID

SOLID

9-1

9-2

9-3

10-1

10-2

10-3

9-1

9-2

9-3

10-1

10-2

10-3

H

622

3

LIQUID-1 LIQUID-2 LIQUID-3 LIQUID-1 LIQUID-2 LIQUID-3

B

621

2

NC forVersion 2.7

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Fig.12B

Allocation on a 96-well plate for correcting tissue.
1

A
B

2

3

4

5

6

7

8

9

NC forNC forNC forNC forNC forLIQUID-1 LIQUID-2 LIQUID-3

SOLID-1

SOLID-2

SOLID-3

10

11

12

Blank
NC forC

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

SOLID

SOLID

SOLID

SOLID

SOLID

SOLID

1-1

1-2

1-3

2-1

2-2

2-3

1-1

1-2

1-3

2-1

2-2

2-3

D

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

SOLID

SOLID

SOLID

SOLID

SOLID

SOLID

3-1

3-2

3-3

4-1

4-2

4-3

3-1

3-2

3-3

4-1

4-2

4-3

E

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

SOLID

SOLID

SOLID

SOLID

SOLID

SOLID

5-1

5-2

5-3

6-1

6-2

6-3

5-1

5-2

5-3

6-1

6-2

6-3

F

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

SOLID

SOLID

SOLID

SOLID

SOLID

SOLID

7-1

7-2

7-3

8-1

8-2

8-3

7-1

7-2

7-3

8-1

8-2

8-3

G

LIQUID
9-1

LIQUID
9-2

LIQUID
9-3

LIQUID
10-1

LIQUID
10-2

LIQUID
10-3

SOLID
9-1

SOLID
9-2

SOLID
9-3

SOLID
10-1

SOLID
10-2

SOLID
10-3

H
Add reacted WST-8 medium to the same locations as living tissues (Fig.12A).
623
624(3) Record the details of steps 1 to 2 above in MDS 4-2.

625
626

3.3.4.4

627(1) Using a 96-well plate reader, measure OD at 450 nm and 650 nm and then used the following

628

OPTICAL DENSITY MEASUREMENTS OF THE REACTION MEDIUM

equation to determine a composite OD for each well.

629
630

Composite OD = (ODTC at 450 nm – ODblank at 450 nm) – (ODTC at 650 nm – ODblank at 650 nm)

631*If the plate reader can be programmed to perform this calculation automatically, then only the632

composite OD value need be recorded.

633
634

(2) Calculate the mean OD for the negative control, a cell viability for each individual tissue, and a635

mean cell viability (including SD) for each test chemical using the following equations.

636
27

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Mean ODNC ＝

Sum of the ODNC for three replicate tissues
3637
Tissue cell viability (%) ＝Each tissue ODTC
Mean ODNC

x 100

638
Mean cell viability(%) ＝

Sum total of cell viability (%) for three replicate tissues

639
640

(3) Record the details of steps 1 and 2 above in MDS 4-2.

641
642

28

3

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643

4. ASSESSMENT

644
645

4.1

646

CONDITIONS FOR A VALID TESTAn eye irritation test is considered valid if all three of the following criteria have been met.

647
648



Tissue viability: 0.5 ≤ mean OD (A450/650) measured value for negative control ≤ 1.6

649

Positive control: mean tissue viability for positive control ≤ 40%

650



SD: SD (negative control, positive control and each test chemicals) of tissue viability of 3

651

identically treated replicates ≤ 18 %

652
653

4.2

ASSAY CRITERIA

654

The criteria for in vitro prediction are shown below.

655

After exposure to a chemical, if cell viability is 40% or less, the chemical is predicted to be an

656

irritant (GHS Category 1 or 2), otherwise it is predicted to be a non-irritant (GHS No Category).

657

See Table 3.

658
659

Table 3

660
661

29

Prediction model of LabCyte24 EIT

Tissue Viability

Prediction≤ 40%

Category 1 or 2

> 40%

No Category

Version 2.7

Page 30 of 45

IN VITRO EYE IRRITATION TEST
October, 2018

USING

HUMAN CORNEAL TISSUE
MODEL: LabCyte CORNEA-MODEL24

Japan Tissue Engineering
Co., Ltd.

662

5. References

663

Green H. (1978) Cyclic AMP in relation to proliferation of the epidermal cell: New view. Cell, 15,664

801-811.

665

Rheinwald J.G. and Green H. (1975) Serial cultivation of strains of human epidermal keratinocytes:

666

The formation of keratinizing colonies from single cells. Cell, 6, 331-343.

667

30

Version 2.7

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IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

MODEL: LabCyte CORNEA-MODEL24

Japan Tissue Engineering
Co., Ltd.

668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686

MDS 1-1:
RECEIPT OF LabCyte CORNEA-MODEL24 (2.1.2)
Laboratory name:1.

Test name:

Test no. :LabCyte CORNEA-MODEL24
Date received:
Lot no.:Expiration date:
Accessories:

Assay medium, 30mL

□ (Lot no.:Expiration date:

24 well assay plate □
Note

687
688
689
690
691
692
693
694
695
696
697
698

2.

Assay medium
Date received:
Lot no.:Expiration date:

Note

699
700
701
702
703
704
705
706
707

Date:
Secretariat

31

Operator:
Check date:

Check date:
Name:

Study director:)

Version 2.7

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IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730

MDS 1-2:
TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH WST-8 ENDPOINT
STEP-1 (3.2.1.1)
Laboratory name:
Test name:
Test no. :1.

Add distilled water (0.5 mL) to the wells of the 24-well assay plate.
To add distilled water (0.5 mL)□

Execution date/time:2.

Apply test chemicals to the wells of the 24-well assay plate.

3.

Culture the 24-well assay plate in CO2 incubator for 4 hours.
Time of WST-8 reaction started:
Time of WST-8 reaction completed:4.

Check the color of water.

5.

Test chemical information and check list of coloring potential.
Test chemical

Physicalstate

PBS (NC)

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

731
732

AmountColoring

Test chemical.

Physicalstate

Amount

SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID

mg
mg
mg
mg
mg
mg
mg
mg
mg
mg

Non treatment
(NC)
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL

Note

733
734
735
736
737
738
739
740

Date:
Secretariat

32

Operator:
Check date:

Check date:
Name:

Study director:

Coloring

Version 2.7

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IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772

MDS 1-3:
TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH WST-8 ENDPOINT
STEP-3 (3.2.2.1)
Laboratory name:
Test name:
Test no. :1.

Preparation of WST-8 dilution medium
Warm EBSS for 30 minutes.
CCK-8:
EBSS:
Volume2.

□

Time/date:(Lot no.:(Lot no.:mL

Expiration date:
Expiration date:
Time/date completed:))

Add WST-8 dilution medium (0.3mL) to the wells of the 24-well assay plate.
To add WST-8 dilution medium (0.3mL)□

Time/date executed:

3.

Apply test chemicals to the wells of the 24-well assay plate.

4.

Culture the 24-well assay plate in CO2 incubator for 4 hours.
Time of WST-8 reaction started:
Time of WST-8 reaction completed:5.

Check the color of WST-8 medium.

6.

Test chemical information and checked list of WST-8 assay interfere.
Test chemical

Physicalstate

PBS (NC)

LIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUIDLIQUID

773
774

Amount

WST-8 assay
interfere

Test chemical.

Physicalstate

Amount

SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID
SOLID

mg
mg
mg
mg
mg
mg
mg
mg
mg
mg

Non treatment
(NC)
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL
50 µL

Note

775
776
777
778
779
780
781
782

Date:
Secretariat

33

Operator:
Check date:

Check date:
Name:

Study director:

WST-8 assay
interfere

Version 2.7

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IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
783
784
785

MDS 1-4:
PREPARATION OF FREEZE KILLED TISSUE (3.2.2)

786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816

Laboratory name:

Test name:

Test no. :1.

Transfer LabCyte CORNEA-MODEL24 tissues to 50 mL tube or appropriate sterile container.

2.

Freeze tissues in the -80℃ deep-freezer for 30 minutes (1st freezing).
Store for 30 minutes.

3.

□Time/date:

Thaw tissues in the 37℃ incubator for 15 minutes.
Store for 15 minutes.

□

Time/date:Freeze tissues in the -80℃ deep-freezer for more than 30 minutes (2nd freezing).

4.

Store for more 30minutes.

5.

□Time/date:

Just before using, thaw tissues in the 37℃ incubator for 15 minutes.
Store for 15 minutes.

□

Time/date:

Note

817
818
819
820
821
822
823
824

Date:
Secretariat

34

Operator:
Check date:

Check date:
Name:

Study director:

Version 2.7

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IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851

MDS 2:
PREPARATION OF LabCyte CORNEA-MODEL24 (3.3.1)
Laboratory name:1.

Test name:

Test no. :Warm up the assay medium and add 0.5mL of the assay medium to the wells of the 1st row on the
24-well assay plate.
Assay medium:(Lot no.:Warm for 30 minutes.
□
Add 0.5mL of assay medium to each well □2.

Expiration date:
Time/date:
Time/date:)
Number of plate:

Transfer culture inserts to wells in the 1st row on the 24-well assay plate.
LabCyte CORNEA-MODEL24(Lot no.:Expiration date:

Time/date executed:
Confirm that there are no bubbles under the cell culture insert.

3.
.

□

LabCyte CORNEA-MODEL24 is cultured in CO2 incubator overnight.
Time/date of culture started:

Note

852
853
854
855
856
857
858
859
860
861
862

)

Date:
Secretariat

35

Operator:
Check date:

Check date:
Name:

Study director:

Version 2.7

Page 36 of 45

IN VITRO EYE IRRITATION TEST
October, 2018

USING

HUMAN CORNEAL TISSUE
Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879

MDS 3-1(LIQUID):APPLICATION OF LIQUID TEST CHEMICALS, RINSING AND POST-INCUBATION (3.3.2)
Laboratory name:
Test name:
Test no. :rd1.

Warm up the assay medium and add 0.5mL of the assay medium to the wells of the 3 row on the
24-well assay plate for LIQUID.
Assay medium:

Expiration date:Warm for 30 minutes.
□
Add 0.5mL of assay medium to each well □2.

Time/date:
Time/date:)
Number of plate:

Apply test chemicals to the LabCyte CORNEA-MODEL24.
Time/date execution started:

3.

LIQUID test chemical information
Physical state

Test chemical
vol.

PBS
(Negative control)

LIQUID

50 µL

Ethanol(Positive control)

LIQUID

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

LIQUID, viscous

50 µL

Test chemical.

880
881
882
883
884
885
886
887
888
889
890
891
892

(Lot no.:4.

Lot no.

Time ofapplication
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:

Time ofrinsing
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:

Exposure period(1 minute)
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□
□

After exposure to test chemical for 1 minute, wash out the LabCyte CORNEA-MODEL24 andtransfer the culture inserts to the 3rd row on the 24-well assay plate.
PBS:(Lot no.:Hit PBS stream on the tissue surface directly.

Expiration date:□

Confirm that there are no bubbles under the cell culture insert.

5.

□

Time/date completed:

Culture LabCyte CORNEA-MODEL24 in CO2 incubator for 24 hours.
Time/date post-incubation started:
Time/date post-incubation completed:36)

Version 2.7

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IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

MODEL: LabCyte CORNEA-MODEL24

Japan Tissue Engineering
Co., Ltd.

893

Note

894
895
896
897
898
899
900
901
902
903
904
905
906

Date:
Secretariat

37

Operator:
Check date:

Check date:
Name:

Study director:

Version 2.7

Page 38 of 45

IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924

MDS 3-2(SOLID):
APPLICATION OF SOLID TEST CHEMICALS AND RINSING (3.3.3)
Laboratory name:
Test name:
Test no.
nd

1.

Assay medium:(Lot no.:Expiration date:Warm for 30 minutes.
□
Add 0.5mL of assay medium to each well □2.

Time/date:
Time/date:)
Number of plate:

Apply test chemicals to the LabCyte CORNEA-MODEL24 and transfer the culture inserts to the
2nd row on the 24-well assay plate.
Time/date started:

3.

Time/date completed:

SOLID test chemical information
Test chemical

Lot no.

Non treatment(Negative
control)
Lauric acid
(Positive
control)

925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942

rd

Warm up the assay medium and add 0.5mL of the assay medium to the wells of 2 and 3 row on
the 24-well assay plate for SOLID.

4.

Physical state

Crush andgrind

Test chemical amount.

Time ofapplication

Exposureperiod(24hours)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

SOLID

□

(

mg,

mg,

mg)

:

□

Culture LabCyte CORNEA-MODEL24 in CO2 incubator for 24 hours.
Time/date exposure started:
Time/date exposure completed:

5.

After exposure to test chemical, wash out the LabCyte CORNEA-MODEL24 and transfer theculture inserts to the 3rd row on the 24-well assay plate.
PBS:(Lot no.:Hit PBS stream on the tissue surface directly.

Time/date started:

38

Expiration date:□

Time/date completed:)Version 2.7

Page 39 of 45

IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

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MODEL: LabCyte CORNEA-MODEL24

Japan Tissue Engineering
Co., Ltd.

Note

943
944
945
946
947
948

Date:
Secretariat

39

Operator:
Check date:

Check date:
Name:

Study director:

Version 2.7

Page 40 of 45

IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
949
950
951

MDS 4-1:
WST-8 ASSAY (3.3.4.1, 3.3.4.2)

952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987

Laboratory name:1.

Test name:

Test no. :Preparation of WST-8 dilution medium
Warm EBSS for 30 minutes.
CCK-8:
EBSS:
Volume2.

□

Time/date:(Lot no.:(Lot no.:mL

Expiration date:
Expiration date:
Time/date completed:

Add WST-8 dilution medium (0.3mL) to the wells in the 4th row on the 24-well assay plate.
To add WST-8 dilution medium (0.3mL)3.

□Time/date executed:

After the operation, the blotted tissue transfer to wells of 4th row of 24-well assay plate.
Time/date started:

Time/date completed:

Confirm that there are no bubbles under the cell culture insert.
4.

□

Culture LabCyte CORNEA-MODEL24 in CO2 incubator for 4 hours.
Time of WST-8 reaction started:
Time of WST-8 reaction completed:

Note

988
989
990
991
992
993
994
995
996
997
998

)
)

Date:
Secretariat

40

Operator:
Check date:

Check date:
Name:

Study director:

Version 2.7

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IN VITRO EYE IRRITATION TESTUSINGHUMAN CORNEAL TISSUE

October, 2018

Japan Tissue Engineering

MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.
MDS 4-2:
SAMPLING OF REACTING WST-8 SOLUTION (3.3.4.3) AND MEASUREMENT (3.3.4.4)

999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011

Laboratory name:1.

Test name:

Test no. :Reacting WST-8 solution (200 µL) is transferred to each well on the 96-well plate.
Transfer to the 96-well plate.

□

Time/date executed:
Sample location on 96-well plate.
LIQUID
1
A

blank

B

PBS-1

2

3

PBS-2

PBS-3

SOLID
4

Ethanol-1

5Ethanol-2

6Ethanol-3

7
Non-treatment-18
Non-treatment-2

9
Non-treatment-31011

12Lauricacid-1

Lauricacid -2

Lauricacid -3

C
D
E
F
G

1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
1022
1023
1024
1025

H

2.

Analyze extract OD at 450nm and 650nm, and calculate the OD(450nm-650nm).
□
□
□
□
□
□

Analyze OD at 450nm and 650nm.
Calculate the OD (450nm-650nm).
Calculate cell viability and SD.
Cell viability and SD are recorded on a separate data sheet.
The data sheet is attached to the back of this sheet.
Check for input errors.
Time/date executed:

Note

1026
1027
1028
1029
1030
1031

Date:
Secretariat

41

Operator:
Check date:

Check date:
Name:

Study director:

Version 2.7

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MODEL: LabCyte CORNEA-MODEL24

Co., Ltd.

1032

REVISION HISTORY

1033

Rev.

CONTENT

Date Revised

Ver.1.1

1) First version

Aug., 2012

Ver.1.2

1) Revised clerical error.

Aug., 2012

Ver.1.3

1) Revised clerical error.

Sep., 2012

Ver.1.3PS

Added the following supplementary explanation.

Sep., 2012

1) Added more detail explanation about the conditions of WST-8
reaction.
2) Added the formula of SD.

Ver.2.1

1) Revised clerical error.

May, 2013

2) In the section 1.2 “BACKGROUND”, changed statement aboutanimal testing (Draize test).
3) In the section 3.3. “TEST METHOD”, explained the washingprotocol of the LIQUID and SOLID test chemical more briefly.
4) In the section 3.3. “TEST METHOD”, changed the WST-8
dilution rate with PBS from 1:10 to 1:5.
5) In the section 3.3. “TEST METHOD”, changed the reaction
period of WST-8 from 5 hours to 4 hours.
6) In the section 3.3. “TEST METHOD”, changed the condition of
WST-8 reaction from shaking to standing.
7) In

the

section

3.3.

“TEST

METHOD”,

changedtheapplication-amount of SOLID chemicals from 50mg to 10mg.
8) As assay acceptance criteria, added that SD (test chemicals) oftissue viability of 3 identically treated replicates ≤ 20 %.
9) In the prediction model of this EIT, changed the cut-off value ofthe mean viability from 50% to 40%.

42

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Co., Ltd.

Ver.2.2

1) Revised clerical error.

Sep., 2013

2) In the section 3.3. “TEST METHOD”, additionally explained thatthe solid test chemical is crush and grind in a mortar with pestle
if necessary.
3) In the section 3.3.2 “APPLICATION OF LIQUID TESTCHEMICALSAND

RINSING

temperature

condition

(room

(DAY

0~1)”,

temperature)atexplainedchemicalapplication.
Ver.2.3

1) Revised clerical error.

Feb., 2014

2) In the section 3.3. “TEST METHOD”, explained about a blank
preparation at WST-8 assay.
3) In the section 3.3. “TEST METHOD”, changed the condition of
WST-8 reaction from standing to shaking.
5) In the section 3.3. “TEST METHOD”, explained about a blank
preparation at WST-8 assay.

Ver.2.3.1

1) Revised clerical error.

Mar., 2014

2) About the prediction result of eye irritation, changed theclassification from irritation/no irritation to GHS classification.

Ver.2.3.2MTT

1) At the analysis of cell viability, changed the assay method fromJul., 2014

WST-8 assay to MTT assay.
Ver.2.4.1

1) At the analysis of cell viability, changed the assay method from
MTT assay to WST-8 assay.
2) In the section 3.3. “TEST METHOD”, changed the condition of
WST-8 reaction from shaking to standing.
3) In the section 3.3. “TEST METHOD”, changed the dilution
solution of WST-8 reaction from PBS to EBSS.
4) In the section 3.3. “TEST METHOD”, changed the reaction
period of WST-8 from 5 hours to 4 hours.
5) The standard of the additional testing was mentioned about a

43Jan., 2015

Version 2.7

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Japan Tissue Engineering
Co., Ltd.

borderline result.
9) In the prediction model of this EIT, changed the cut-off value ofthe mean viability from 40% to 50%.

Ver.2.4.2

1) Changed the assay acceptance criterion of negative control

Mar., 2015

from 0.5 < and <2.0 to 0.6 < and <1.5.

Ver.2.4.2m

1) The judgement of the chemical which is a result of theJul., 2015

borderline was added.

Ver.2.5.1

1) Revised clerical error.

Aug., 2015

2) In the prediction model of this EIT, changed the cut-off value ofthe mean viability from 50% to 40%.
3) Changed the judgement of equivocal results.
4) Changed the assay acceptance criterion of negative control
from 0.6 < and <1.5 to 0.5 < and <1.3.
5) In the section 3.2. “TEST FOR DETECTING CHEMICALSTHAT INTERFERE WITH WST-8

ENDPOINT”,

reviseddescription has clearly.
6) Added MDS1-2 and MDS 1-3.

Ver.2.5.1m

1) Revised clerical error.

Sep., 2015

2) Changed allocation for a 96-well plate of pattern which consistsof freeze killed tissue (Fig.12B).

Ver. 2.5.1mr

1) Revised Fig.2B

Sep.,2015

Ver. 2.5.2

1) In the section 3.3. “TEST METHOD”, explained the washingSep.,2015

protocol of the LIQUID and SOLID test chemical more briefly.
2) In the MDS 3-1 and the MDS 3-2, added the check box about
the washing procedure.
3) In the MDS 3-2, added the check box about crush and grind of44

Version 2.7

Page 45 of 45

IN VITRO EYE IRRITATION TEST
October, 2018

USING

HUMAN CORNEAL TISSUE
MODEL: LabCyte CORNEA-MODEL24

Japan Tissue Engineering
Co., Ltd.

test chemicals.

Ver. 2.5.4

1) In the section 3.3. “TEST METHOD”, added attention point ofJune, 2016

the washing procedure.
2) In the section 4.3 “ASSESSMENT FLOWCHART”, Change
numbers of test run from three independent run to single run.
Ver. 2.5.5

1) Revised clerical error.

October, 20162) As assay acceptance criteria, changed that SD (negative
control, positive control and test chemicals) of tissue viability of
3 identically treated replicates from ≤ 20 % to ≤ 18 %.
Ver.2.5.6

1) Revised clerical error.

February, 20172) Detection protocol of coloring interference is changed fromusing WST-8 medium to distilled water and correction ofcoloring interference is changed from using freeze-killed tissue
to using living tissue without WST-8 reaction.
Ver.2.6

1) Changed the assay acceptance criterion of negative control
from 0.5 ≤ and ≤1.3 to 0.5 ≤ and ≤ 1.6.

Ver.2.7

1034
1035

45

1) Revised clerical errors. There is no change of protocol.

June, 2018