BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017

BACKGROUND REVIEW DOCUMENTNew Established In Vitro Eye Irritation Test Method
using Reconstructed Human Corneal Epithelium,
LabCyte CORNEA-MODEL24

Prepared by:
1

R&D Department, Japan Tissue Engineering Co., Ltd.,
2

Faculty of Biomedical Sciences, Kobe University

3

Faculty of Culture and Information Science, Doshisha University,
4

Maruishilabo Corporation,

5

R&D, Nihon Kolmar Co., Ltd.,

6

Examination Room 3, Drug Safety Testing Center Co., Ltd.,
7

Safety Evaluation Center, Fujifilm Corporation,
8

Product Assurance, Mandom Corporation,

9

Research & Development Division, Oppen Cosmetics Co., Ltd.,

10

Institute of Biomedical Science & Technology, Konkuk University,

11

Japanese Center for the Validation of Alternative Methods (JaCVAM), National
Institute of Health Sciences.

Authors:
1

Masakazu Katoh, 2,3Takashi Omori, 3Aimi Yamashita, 3Mai Endo, 3Maiko Satake,

4,8

Satoshi Nakahara, 5Hideki Nishiura, 5,8Hidefumi Ikeda, 6Shinsuke Shinoda, 6Saori

Hagiwara, 7Toshihiko Kasahara, 7Haruna Tahara, 7Yusuke Yamamoto, 9Yuichiro
Yoshitake, 10Jeong Ik Lee, 10Yuna Han, 10Lee, Soo Jung, 1Katsura Sugawara,
11

Hajime Kojima
Version 2.2

February, 2017

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TABLE OF CONTENTS
Page Number
LIST OF TABLE .................................................................................................................. 7
LIST OF FIGURES .............................................................................................................. 9
LIST OF ACRONYMS AND ABBREVIATIONS................................................................. 10
PREFACE .......................................................................................................................... 12
EXECTIVE SUMMARY ...................................................................................................... 14
1. INTRODUCTION AND RATIONALE FOR THE PROPOSED USE OF IN VITRO TESTMETHODS TO IDENTIFY OCULAR IRRITANTS ....................................................... 17
1-1.

INTRODUCTION ............................................................................................................ 17

1-1.1.
1.1.2.

1-2.

Historical Background of Ocular Irritation Tests and Rationale Their Development .....17
The Proposed EIT Method using LabCyte CORNEA-MODEL24 ......................................19

Scientific Basis for the Proposed Test Method .......................................................... 20

1.2.1.

Propose and Mechanism Basis of LabCyte24 EIT ...........................................................20

1.2.2.

Similarities and Differences of Modes of Action of Between the LabCyte24 EIT and the
Draize Eye Test ..................................................................................................................20
The Draize Eye Test ...........................................................................................................20
Comparison of the LabCyte24 EIT with Draize Eye Test .................................................21
Applicability and Limitations of LabCyte24 EIT ...............................................................21

1-2.2.1.
1-2.2.2.
1.2.3.

1-3.

Regulatory Rationale and Applicability ....................................................................... 22

1-3.1.

Current Regulatory Testing Requirements ......................................................................22

1-3.2.
1-3.3.

Intended Regulatory Use(s) ..............................................................................................23
The Similarities and Differences in the Endpoint Measured in the Proposed Test andCurrently Used In Vivo Reference Test ............................................................................23

1-3.4.

How the proposed test fits into the overall strategy of hazard or safety ........................23

1-4.

Objection of This Background Review Document ..................................................... 24

2. DESCRIPTION OF LabCyte CORNEA-MODEL24 .................................................... 25
2-1.
2-2.

Condition of the Functional Reconstructed human Corneal Epithelial Tissue Model,
LabCyte CORNEA-MODEL24 ....................................................................................... 25
Quality Control for LabCyte CORNEA-MODEL24 ....................................................... 28

2-2.1.
Quality Control Procedures for LabCyte CORNEA-MODEL24 ........................................28
2-2.1.1. Tissue Viability ..................................................................................................................28
2-2.1.1a. MTT Assay .........................................................................................................................28
2-2.1.1b. WST-8 Assay (Collection as Reference Data not to Control Release Criteria) ...............28
2-2.1.2. Barrier Function: 50% Inhibitory Concentration (IC50) Assay..........................................29
2-2.1.3.
2-2.2.

Morphology .......................................................................................................................29
Batch Control Information for LabCyte CORNEA-MODEL24 ..........................................30

3. OPTIMIZATION AND IMPROVMENT STUDY OF THE LabCyte24 EIT .................... 34
3-1.

Optimization of Suitable Time Periods for Test Chemicals Combining Sample
Exposure and Post-exposure Incubation ................................................................... 34
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3-1.1.

Purpose of This Study ......................................................................................................34

3-1.2.

Optimization of Suitable Exposure and Post-exposure Incubation Periods for the Liquid
Test Chemicals ..................................................................................................................34

3-1.2.1.
3-1.2.2.

Study Conditions ..............................................................................................................34
Results of Optimization Study for Liquid Chemicals.......................................................35

3-1.3.

Optimization of Suitable Exposure and Post-exposure Incubation Periods for the Solid
Test Chemicals ..................................................................................................................38
Study Conditions ..............................................................................................................38
Results of Optimization Study for Solid Chemicals ........................................................39
Discussion and Conclusion ..............................................................................................39
Publication ........................................................................................................................40

3-1.3.1.
3-1.3.2.
3-1.4.
3-1.5.

3-2.

Ring-Study for the Technical Transferability of the LabCyte24 EIT .......................... 41

3-2.1.
Purpose of This Study ......................................................................................................41
3-2.2.
Study Plan .........................................................................................................................41
3-2.2.1. Participating Laboratories ................................................................................................41
3-2.2.2. Selected Test Chemicals ...................................................................................................423-2.2.3. Test Protocol .....................................................................................................................43
3-2.2.3a. Protocol for Liquid Chemical ............................................................................................43
3-2.2.3b. Protocol for Solid Chemical ..............................................................................................43
3-2.2.3c. Assay Criteria ....................................................................................................................43
3-2.2.3d. Prediction Model in This Study ........................................................................................443-2.3.
3-2.3.1.

Results...............................................................................................................................44
Technical Transferability ...................................................................................................443-2.3.2.
3-2.3.3.
3-2.3.4.
3-2.4.

Within-Laboratory Reproducibility ...................................................................................46
Between-Laboratory Reproducibility ...............................................................................47
Predictive Capacity ...........................................................................................................47
Discussion and Conclusion ..............................................................................................47

3-3.

Modification of LabCyte24 EIT

3-3.1.
3-3.2.
3-3.2.1.
3-3.2.2.
3-3.2.3.
3-3.2.3a.

(Pre-Validation Study Phase 01) ............................ 48

Purpose of This Study ......................................................................................................48
Study plan .........................................................................................................................48
Participating Laboratories ................................................................................................48
Selected Test Chemicals ...................................................................................................49
Test Protocol .....................................................................................................................49
Protocol for Liquid Chemical ............................................................................................49

3-3.2.3b. Protocol for Solid Chemical ..............................................................................................50
3-3.2.3c. Assay Criteria ....................................................................................................................50
3-3.2.3d. Prediction Model in This Study ........................................................................................50
3-3.3.
Results...............................................................................................................................51
3-3.3.1. Negative control and positive control ..............................................................................51
3-3.3.2. Within-Laboratory Reproducibility ...................................................................................53
3-3.3.3. Between-Laboratory Reproducibility ...............................................................................53
3-3.3.4. Predictive capacity ............................................................................................................53
3-3.3.5. Proficiency Level of Preset Criteria..................................................................................53
3-3.4.
Discussion and Conclusion ..............................................................................................54

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3-4.

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Comparison study of MTT assay for the LabCyte24 EIT (Pre-Validation Study
Phase-02, 03) ................................................................................................................ 55

3-4.1.

Purpose of This Study ......................................................................................................55

3-4.2.
3-4.2.1.

Study Plan .........................................................................................................................55
Participating Laboratories ................................................................................................55

3-4.2.2.
3-4.2.3.

Selected Test Chemicals ...................................................................................................55
Test Protocol .....................................................................................................................56

3-4.2.3a. Protocol for Liquid Chemical ............................................................................................56
3-4.2.3b. Protocol for Solid Chemical ..............................................................................................57
3-4.2.3c. Assay Criteria ....................................................................................................................57
3-4.2.3d. Prediction Model in This Study ........................................................................................57
3-4.3.
Results...............................................................................................................................57
3-4.3.1. Negative Control ...............................................................................................................57
3-4.3.2. Test chemicals...................................................................................................................59
3-4.4.
Discussion and Conclusion ..............................................................................................60

3-5.

Optimization Study for WST-8 Assay Condition ......................................................... 62

3-5.1.
3-5.2.
3-5.2.1.
3-5.2.2.
3-5.2.3.
3-5.3.
3-5.3.1.

Purpose of This Study ......................................................................................................62
Study plan .........................................................................................................................62
Study 1...............................................................................................................................62
Study 2...............................................................................................................................62
Study 3...............................................................................................................................62
Results...............................................................................................................................63
Study 1...............................................................................................................................63

3-5.3.2.
3-5.3.3.
3-5.4.

Study 2...............................................................................................................................64
Study 3...............................................................................................................................64
Discussion and Conclusion ..............................................................................................65

4. DISCRIPTION OF THE IMPROVED LabCyte24 EIT.................................................. 67
4-1.

Overview of LabCyte the Improved CORNEA-MODEL24 EIT Procedure .................. 67

4-1.1.

The Improved LabCyte24 EIT Procedure for Liquid Chemicals ......................................67

4-1.2.
4-1.3.

The Improved LabCyte24 EIT Procedure for Solid Chemicals ........................................68
Prediction Model of the Improved LabCyte24 EIT ...........................................................68

4-2.

Description and Rationale for the Test Components of the Improved LabCyte24 EIT....................................................................................................................................... 69

4-2.1.
4-2.2.
4-2.2.1.
4-2.2.2.
4-2.3.
4-2.4.

Pre-Exposure Incubation ..................................................................................................69
Duration of Chemical Exposure........................................................................................69
Amount of Chemical Exposure .........................................................................................69
Exposure Periods of Chemicals and Post-exposure incubation Period.........................69
RhCE Tissue Replication ..................................................................................................70
Endpoint(s) ........................................................................................................................70

4-2.5.
4-2.5.1.
4-2.5.2.
4-2.6.

Appropriate Positive Control and Negative Control ........................................................71
Negative Control ...............................................................................................................71
Positive Control.................................................................................................................71
Acceptance Criteria...........................................................................................................72

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4-2.6.1.
4-2.6.2.

Negative Control ...............................................................................................................72
Positive Control.................................................................................................................72

4-2.6.3.
4-2.7.

Cell Variability ...................................................................................................................72
Applicability Domain and Limitation ................................................................................73

5. EVALUATION OF THE PREDICTIVE PERFORMANCE OF THE IMPROVED
LabCyte24 EIT USING WIDE-RANGE OF TEST CHEMICALS ................................. 74
5-1.
5-2.

Purpose ......................................................................................................................... 74
Materials and Methods ................................................................................................. 74

5-2.1.
Chemical Selection of Wide-Range Classes ....................................................................74
5-2.2.
Protocol of the improved LabCyte24 EIT .........................................................................82
5-2.2.1. The Improved LabCyte24 EIT Procedure for Liquid Chemicals ......................................82
5-2.2.2. The Improved LabCyte24 EIT Procedure for Solid Chemicals ........................................83
5-2.2.3. Prediction Model of the Improved LabCyte24 EIT ...........................................................83
5-2.2.4. Detecting chemical interference with WST-8 endpoints and correction procedures .....84
5-2.2.4a. STEP 1 (PRELIMINARY TEST) ..........................................................................................84
5-2.2.4b. STEP 2 (FUNCTIONAL CHECK ON VIABLE TISSUE) .......................................................84

5-3.

Results .......................................................................................................................... 86

5-3.1.
5-3.2.

5-4.

Detecting chemical interference with WST-8 endpoints and correction.........................86
Predictivity of the Improved LabCyte24 EIT.....................................................................88

Discussion and Conclusion ......................................................................................... 94

6. THE AVAILABILITY OF LabCyte24 EIT OUTSIDE OF JAPAN: EXPORT OF LabCyte
CORNA-MODEL TO KOREA AND CONFIRMATION OF ITS PERFORMANCE ....... 96
6-1.
6-2.

Purpose ......................................................................................................................... 96
Export to Korea and Test Protocol .............................................................................. 96

6-2.1.
6-2.2.

Shipment Schedule ...........................................................................................................96
Test Facility .......................................................................................................................96

6-2.3.

Evaluation Procedure of LabCyte CORNEA-MODEL24 after Shipment ..........................97

6-3.

Results .......................................................................................................................... 97

6-3.1.
6-3.2.
6-3.3.
6-3.4.

6-4.

Export to Korean Laboratory ............................................................................................97
Results of Negative Control in the LabCyte24 EIT after Shipment .................................98
Results of Positive Control in LabCyte24 EIT after Shipment.........................................99
Results of Proficiency Chemicals in LabCyte24 EIT .......................................................99

Discussion and Conclusion ....................................................................................... 101

7. VALIDATION STUDY OF LabCyte24 EIT ACCORDING TO THE PERFORMANCESTANDARD FOR THE OECD TG492....................................................................... 102
7-1.
7-2.
7-3.

The Performance Standard for the OECD TG 492 .................................................... 102
Difference between LabCyte24 EIT and the OECD TG492 VRM Procedural
Conditions ................................................................................................................... 102
Comparison and Similarity Consideration about Each Test Component of Between
the VRM in the Performance Standard and LabCyte24 EIT ..................................... 104

7-3.1.
7-3.2.

Cell Source ......................................................................................................................104
Replication of RhCE Tissues ..........................................................................................104

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7-3.3.
7-3.4.

Amount of Chemical Application ....................................................................................105
Negative Control and Positive Control ...........................................................................105

7-3.5.

Chemical Exposure, Post-exposure Immersion and Post-exposure Incubation Periods.........................................................................................................................................105
Measurement of Tissue Viability ....................................................................................106

7-3.6.

7-3.6.1. Cell Viability Measurement with Tetrazolium-Salt..........................................................106
7-3.6.1a. MTT Assay .......................................................................................................................107
7-3.6.1b. WST-8 Assay....................................................................................................................1087-3.6.2. Similarity between MTT Assay and WST-8 Assay ..........................................................109
7-3.6.2a. Additional Data: Comparison of the LabCyte CORNEA-MODEL24 QC data ................109
7-3.6.2b. Additional Data: Time Course Experiment ..................................................................... 110
7-3.6.3. Advantage of WST-8 Assay to MTT Assay ..................................................................... 111
7-3.6.4. Similarity of LabCyte24 EIT to the OECD TG492 VRM ................................................... 112
7-3.7.
Detection and Correction of MTT/WST-8 Assay Interference ........................................ 112
7-3.8.
Acceptance Criterion SD................................................................................................. 113

8. CONCLUSION .......................................................................................................... 114
9. ACKNOWLEDGEMENT ........................................................................................... 115
10. REFERENCES .......................................................................................................... 116
11. APENDECS .............................................................................................................. 121

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LIST OF TABLE
Page Number
Table 2-1.

QC acceptable range of barrier function. .................................................................. 29

Table 2-2.

Batch information for LabCyte CORNEA-MODEL24 ................................................. 30

Table 3-1.

The test chemical set for initially optimization of the LabCyte24 EIT ......................... 34

Table 3-2.

The Solid test chemical set for initially optimization of the LabCyte24 EIT ................ 38

Table 3-3.

Optimal application period ........................................................................................ 40

Table 3-4.

Participating laboratories .......................................................................................... 41

Table 3-5.

Selected test chemicals. ........................................................................................... 42

Table 3-6.

Prediction of three independent run .......................................................................... 45

Table 3-7.

Participating laboratories. ......................................................................................... 49

Table 3-8.

Selected test chemicals. ........................................................................................... 49

Table 3-9.

Prediction of three independent run .......................................................................... 52

Table 3-10. Prediction of three independent run .......................................................................... 53
Table 3-11. Participating laboratories. ......................................................................................... 55
Table 3-12. Selected test chemicals. ........................................................................................... 56
Table 3-13. PH value of various buffers after the cultivation under 37°C in a humidified
atmosphere with or without 5% CO2 ......................................................................... 63
Table 3-14. Effect of various buffer on OD value after WST-8 reaction for 5 hours under 37°C in ahumidified atmosphere with 5% CO2......................................................................... 64
Table 3-15. Change of OD values with time-dependent manner in the WST-8 reaction under
37°C in a humidified atmosphere with 5% CO 2 ......................................................... 65
Table 4-1.

Prediction model of LabCyte24 EIT .......................................................................... 68

Table 4-2.

Amount of chemical application ................................................................................ 69

Table 4-3.

Exposure Periods of Chemicals and Post-exposure incubation Period ..................... 70

Table 4-4.

Negative control........................................................................................................ 71

Table 4-5.

Chemical of positive control ...................................................................................... 71

Table 4-6.

Acceptability ranges for negative control ................................................................... 72

Table 4-7.

Acceptability ranges for positive control .................................................................... 72
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Table 4-8.

Acceptability SD limit ................................................................................................ 72

Table 5-1.

Wide range of the test chemicals for evaluation of the LabCyte CORNEA-MODEL24
SIT ............................................................................................................................ 75

Table 5-2.

Category of the test chemicals for evaluation of the LabCyte24 EIT ......................... 81

Table 5-3.

Number by physical state and UN GHS classification of the wide range chemicals .. 81

Table 5-4.

Prediction model of the improved LabCyte24 EIT ..................................................... 83

Table 5-5.

Detection of WST-8 reducer in 139 chemicals .......................................................... 87

Table 5-6.

Wide range of the test chemicals for evaluation of the LabCyte24 EIT ...................... 89

Table 5-7.

Wide range of the test chemicals for evaluation of the LabCyte24 EIT ...................... 94

Table 6-1.

Shipments to Korean laboratory. ............................................................................... 97

Table 6-2.

OD and SD of negative controls in the EIT using LabCyte CORNEA-MODEL24 after
shipment to Korean laboratory. ................................................................................. 98

Table 6-3.

Cell viability and SD of positive control for EIT using LabCyte CORNEA-MODEL24
exported to Korean laboratory. .................................................................................. 99

Table 6-4.

Prediction results of proficiency chemicals in the OECD TG492 by EIT using
LabCyte CORNEA-MODEL24 exported to Korean laboratory. ............................. 100

Table 7-1.

Test component of LabCyte24 EIT .......................................................................... 103

Table 7-2.

Commonly used tetrazolium salts ........................................................................... 106

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LIST OF FIGURES
Page Number
Fig.2-1.

Histological cross-sectional views of the LabCyte CORNEA-MODEL24 with H&E
staining (original magnification:

Fig.2-2.

×40). ..................................................................... 25

Histological cross-sectional views of the LabCyte CORNEA-MODEL24 withimmuno-staining: ...................................................................................................... 26

Fig.2-3.

Transmission electron micrographs of the LabCyte CORNEA-MODEL24. ................ 27

Fig.3-1.

Optimization of exposure periods and post-exposure incubation periods for the
LabCyte24 EIT for Liquid chemicals.......................................................................... 37

Fig.3-2.

Optimization of exposure periods and post-exposure incubation periods for the
LabCyte24 EIT for Solid chemicals. .......................................................................... 39

Fig.3-3.

Results of negative control and positive control ........................................................ 44

Fig.3-4.

Results of negative control........................................................................................ 51

Fig.3-5.

Results of positive control. ........................................................................................ 51

Fig.3-6.

Results of negative control........................................................................................ 58

Fig.3-7.

Results of Cell viability of test chemicals ................................................................... 60

Fig.5-1.

Direct WST-8 reduction ability (STEP 1). .................................................................. 86

Fig.7-1.

MTT assay: Reaction mechanism. .......................................................................... 108

Fig.7-2.

WST-8 assay: Reaction mechanism. ...................................................................... 109

Fig.7-3.

WST-8 assay and MTT assay in LabCyte CORNEA-MODEL24 QC results ............ 110

Fig.7-4.

Time Courses of WST-8 reaction and MTT reaction in LabCyte24 EIT. .................... 111

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LIST OF ACRONYMS AND ABBREVIATIONSBCOPBovine Corneal Opacity and Permeability

BRD

Background Review Document

CAS

Chemical Abstracts Service

CV

Coefficient of variation

D-PBS

Dulbecco’s phosphate buffered saline

EBSS

Earle’s balanced salt solution

EC

European Commission

ECETOC

European Centre for Ecotoxicology and Toxicology of Chemicals

EIT

Eye irritation test

EU

European Union

FBS

Fetal bovine serumFL

Fluorescein Leakage

GHS

Globally Harmonized System (of Classification and Labeling of Chemicals)

GLP

Good Laboratory Practices

HPLC/UPLC Highperformanceliquidchromatography/Ultra

highperformanceliquidchromatography
ICE

Isolated Chicken Eye

JaCVAM

Japanese Center for the Validation of Alternative Methods

JSAAE

Japanese Society for Alternatives to Animal Experiments

LabCyte CORNEA-MODEL24 EIT

LabCyte24 EIT

MMAS

Modified maximum average score

MTS

3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium

MTT

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OD

Optical density

OECD

Organisation for Economic Co-operation and Development

RhCE

Reconstructed human corneal epithelium

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SD

Standard deviation

SLS

Sodium Lauryl Sulfate

SOP

Standard operation procedure

STE

Short time exposure assay

TG

Test GuidelineUN

United Nations

VMT

Validation management team

VRM

Validated reference method

WST-1

2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H

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tetrazolium,monosodium salt
WST-8

Water soluble tetrazolium salt-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliu
m, monosodium salt

XTT

2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide

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PREFACEThe location, physiological structure and sensitivity of the ocular surface predispose it toexposure from a variety of potentially hazardous environmental conditions and substances on adaily basis. Many different materials and chemicals can result in damage to the cornea that mayvary from irritation and inflammation causing mild discomfort to tissue corrosion resulting in
irreversible blindness. These include household, industrial, agricultural and military products,
cosmetics, toiletries and may even include certain ocular drugs. Eye toxicity tests are therefore
required to ensure that the risks associated with products meet suitable safety criteria and areclearly labeled.
For many years, the ocular irritation potential of these substances has mainly been evaluatedby the Draize eye test, which was developed in the 1940s (Draize et. al., 1944). The Draize eyetest is the eye toxicity test officially accepted in the Organization for Economic Co-operation andDevelopment (OECD) test guidelines (TG) 405 (OECD, 2012a) for regulatory purposes in theclassification of eye-irritancy substances. The Draize eye test is mainly based on the scoring ofobserved macroscopic changes in the rabbit cornea, conjunctiva, and iris. However, it is often
criticized for both ethical (animal welfare) and scientific reasons (subjective scoring, lowinter-laboratory reproducibility, or sensitivity differences with humans) (Christian et. al., 1996).
Therefore, there is a strong need for an in vitro evaluation method which can be used to assess
eye irritancy.
The public interests in animal alternative tests have increased recently and the developmentof these tests has become a critical task for the cosmetic industry globally. In addition, thedevelopment of alternative methods is accelerating in the world due to new regulations like thebanning of cosmetics in animal ocular irritation tests in the EU (Directive 2003/15/EC, 2003). To
date, a lot of ex vivo or in vitro ocular irritancy test methods have been developed as alternative
method to the Draize eye test. Regarding those ex vivo, the Bovine Corneal Opacity andPermeability (BCOP) test and the Isolated Chicken Eye (ICE) test were found to adequately
predict severe irritancy and non-irritancy although they could not identified materials for irritancy,
they were adopted as OECD TG 437 and 438 (OECD, 2013a; OECD, 2013b). In addition,
concerning those in vitro, the fluorescein leakage (FL) test method, short time exposure test
using a monolayer culture system, and the eye irritation test (EIT) method using reconstructed
human corneal epithelial tissue (RhCE) were adopted as OECD TG 460, 491 and 492 (OECD,
2012b; OECD, 2015c, OECD, 2015a).
The LabCyte CORNEA-MODEL24 EIT (LabCyte24 EIT) that is proposed in this backgroundreview document (BRD) is an alternative in vitro eye irritation test method using the RhCE tissue
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model. The LabCyte CORNEA-MODEL24 displays a high similarity to human corneal
epithelium in the histological morphology (Katoh et. al., 2012). A test protocol based on asimplified cytotoxic approach was set up in the same manner as an already the validated skinirritation test method using a reconstructed human epidermal tissue model for OECD TG 439(OECD, 2010), so as to promptly and easily predict the eye irritation potential of a test
substance.
Category classification of ocular irritation by LabCyte24 EIT is determined based on therelative cell viability assessed. A test substance that has a relative viability of 40% or less iscategorized as an irritant (the United Nations (UN) Globally Harmonized System (GHS)
Category 1, 2A and 2B) and a test substance that has a relative viability greater than 40% iscategorized as a non-irritant (UN GHS No Category).
The objective of this BRD is to describe the brief explanation of the feature of the LabCyte 24
EIT, that is the optimization study, several pre-validation studies and also that the current statusof the LabCyte24 EIT, including what is known about its accuracy and reliability.

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EXECTIVE SUMMARY
This Background Review Document (BRD) reviews available data and information regarding
the eye irritation test (EIT) method using LabCyte CORNEA-MODEL24 (LabCyte24 EIT) for
identifying ocular irritants. The test method was reviewed for its ability to predict ocular irritant as
defined by the United Nations (UN) Globally Harmonized System (GHS) of classification andlabeling of chemicals (UN 2003). The objective of this background review document is todescribe the brief explanation of the feature of the LabCyte24 EIT, that is the optimization study,
several pre-validation studies and also that the current validation status of the LabCyte24 EIT,
including what is known about its accuracy and reliability.
Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test,
which uses rabbits, is essential from the standpoint of animal welfare. It has been developed areconstructed human corneal epithelial (RhCE) tissue model, the LabCyte CORNEA-MODEL24,
which has a representative corneal epithelium like structure. Histological analysis of LabCyte
CORNEA-MODEL24 shows a complete corneal epithelium containing three major corneal
epithelial layers, including a superficial layer, a wing cell layer, and a basal layer. The corneal
epithelial marker, Cytokeratin 3, mucins (mucin-1 and mucin-16), cell adhesion molecules(E-cadherin, Claudin-1 and Desmoglein-3) and basement membrane consistent (Laminin 332)
were expressed in the appropriate regions, as seen in a human corneal epithelium. In analysis
of LabCyte CORNEA-MODEL24 tissue by electron microscope, well developing microvilli wasshown in the superficial layer of the LabCyte CORNEA-MODEL24. Therefore the histologicalstructure of the LabCyte CORNEA-MODEL24 is considered that is highly similar to that of anative human corneal epithelium. The LabCyte CORNEA-MODEL24 provides a promisingalternative to animal testing, a means to assess corneal irritation.
Protocol optimization was examined in order to establish a new alternative method for eye
irritancy evaluation with the LabCyte CORNEA-MODEL24 tissue. A test protocol based on asimplified cytotoxic approach was set up in the same manner as an already validated skinirritation testing method as the Organization for Economic Co-operation and Development(OECD) test guideline (TG) 439 (OECD, 2010), so as to promptly and easily predict theeye-irritation potential of a test chemical. Therefore, to optimize EIT protocol using the LabCyte
CORNEA-MODEL24, two important conditions were examined in order to designate exposure
periods and post-exposure incubation periods in this optimization study. From the results of the
optimization study, the application periods for chemicals were set at 1 minute for Liquid
chemicals or 24 hours for Solid chemicals, and the post-exposure incubation periods were set at
24 hours for Liquids or zero for Solids to see below the Section 3-1 in this BRD. Furthermore,
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through the ring study (to see below Section 3-2), the study of protocol modification (see below
theSection

3-3),

the

comparison

study

of

between3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Water soluble
tetrazolium

salt-8(WST-8;[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]) assay (see below
Section 3-4) and the optimization study of WST-8 assay condition (to see below Section 3-5),
the improved LabCyte24 EIT method was finally established.
For the evaluation of predictive performance of the improved LabCyte24 EIT by in-housestudy, 139 chemicals with wide-range were selected from some published database (ECETOC,
1998; ICCVAM, 2006; OECD, 2015b) and some previous reports (Doucet et. al, 2006;
Takahashi et. al., 2008; Takahashi et. al., 2009; Kaluzhny et. al., 2011; Kolle et. al., 2011), andthese chemicals were examined to assess the performance of the improved LabCyte
COLNEA-MODEL24 EIT. The predictions of the improved LabCyte CORNEA-MODEL24 eye
irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%,
specificity 73.8%, and accuracy 88.2%).
In order to confirm the availability of LabCyte CORNEA-MODEL24 after shipment to outside ofJapan, LabCyte CORNEA-MODEL24 is exported to the laboratory of Konkuk University (Korea)
laboratory and the improved LabCyte24 EIT was performed at the laboratory of Konkuk
University. From the results of the negative control and the positive control in the laboratory of
Konkuk University were adapted to the acceptance criteria in three batches were suggested thatthe performance of the LabCyte CORNEA-MODEL24 after shipping to outside of Japan isenough usefulness for the eye irritation testing. Furthermore, proficiency chemicals listed in theOECD TG 492 (OECD, 2015a) which was OECD TG for the RhCE EIT method were correctlypredicted by improved LabCyte24 EIT in the laboratory of Konkuk University, it was concluded
that the improved LabCyte24 EIT is able to perform outside of Japan.
In several developing RhCE EITs, the EIT test method using EpiOcular model has only
completed a formal validation study and the EpiOcular EIT was implemented as validated
reference method (VRM) into the OECD TG 492 in 2015 (OECD, 2015a). The LabCyte24 EIT
procedure is based on the measurement of viable cells and therefore its assay principle is sameto that of the EpiOcular EIT which is VRM of OECD TG 492 (OECD, 2015a). The PerformanceStandard (OECD, 2015b) for the OECD TG 492 is described that cell source, pre-exposure step,and each step of chemical application were appropriately set each condition if necessary. The
LabCyte24 EIT has been set each condition of such test component as shown below the section
4. On the other hands, method of cell viability measurement is used MTT assay in the OECD
TG 492 (OECD, 2015a), however the LabCyte24 EIT is used WST-8 assay. From MTT assay
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and WST-8 assay are commonly one of tetrazolium reduction assay method and therefore it
was thought that both assays were functionally similar.
Finally, the validation management team (VMT) of the LabCyte24 EIT judged the improved
LabCyte CORNEA-MODEL24 is similar assay method to the RhCE EIT for the OECD TG 492.
Therefore, the me-too validation study of LabCyte24 EIT is planned according to the
PERFORMANCE STANDARD for the OECD TG 492 in older to assess its reliability(reproducibility within and between laboratories) and its relevance (predictive capacity).

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INTRODUCTION AND RATIONALE FOR THE PROPOSED USE OF IN VITROTEST METHODS TO IDENTIFY OCULAR IRRITANTS

1-1. INTRODUCTION
1-1.1. Historical Background of Ocular Irritation Tests and Rationale Their
Development
Many chemicals and materials may result in damage to the cornea that may vary fromirritation and inflammation causing mild discomfort to tissue corrosion resulting in irreversible
blindness. These include household, industrial, agricultural, cosmetics, toiletries products andocular drugs if incorrectly administered. To reduce the risk of exposure to dangerous
substances, all manufactured consumer products and their ingredients should be evaluatedtheir eye irritation potential.
Evaluation methods of eye irritancy are therefore indispensable to ensure that the risks
associated with such materials. For many years, the eye irritation potential of these substances
has mainly been evaluated by the Draize eye test which has been established by Draize et al(Draize et. al., 1944). The procedure involves the application of 0.1 mL (or 0.1 g Solid) test
substance onto the cornea and conjunctival sac of one eye of a conscious rabbit for up to 72
hours while the other eye serves as an untreated control. After assessment of the eye irritation
potential according to the Organization for Economic Co-operation and Development (OECD)
test guidelines (TG) 405 (OECD, 2012a) which is the TG for the Draize eye test, test substances
are classified according to the United Nations (UN) Globally Harmonized System (GHS) (UN,2009), as Category 1 (severe irritants causing irreversible damage to the eye), Category 2A(moderate irritants having reversible effects on the eyes) or Category 2B (mild ocular irritants).
Non-irritating substances are not labelled according to the UN GHS system. The UN GHS isbased upon averaged single tissue observations which can account for the reversibility of theobserved chemical effects (Eskes et. al., 2005). However, it is often criticized for both ethical(animal welfare) and scientific reasons (subjective scoring, low inter-laboratory reproducibility,
or sensitivity differences with humans) (Christian et. al., 1996). Therefore, there is a strong needfor alternative method which can be used to assess eye irritancy.
To date, numbers of ex vivo or in vitro assay methods have been developed as alternative
methods to the Draize eye test.
Above all, the Bovine Corneal Opacity and Permeability (BCOP) test and the Isolated Chicken
Eye (ICE) test were found to adequately predict severe irritancy although they were not

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recommended for the identification of materials not classified for irritation, they were adopted as
OECD TG 437 and 438 (OECD, 2009a; OECD, 2009b). Furthermore, their OECD TGs
extending the applicability will be revised, which are accepted for the identification of non-irritantchemicals in the field of eye irritation (OECD, 2013a; OECD, 2013b). However, these test
methods still have many shortcomings such as use of non-human tissue and unsatisfactory
predictive capacity for human ocular irritation, reflecting a considerable demand for novel andmore human-like alternative tests.
On the other hands, as in vitro eye irritation test, the fluorescein leakage (FL) test method was
also adopted as an OECD test guideline (OECD, 2012).
Furthermore, a rabbit corneal epithelial cell line has been made available as a usefulalternative when evaluated by a cytotoxicity assay (Hagino et. al.. 2008; Takahashi et. al., 2008;
Takahashi et. al. 2009). In such proposed test methods, the short time exposure (STE) assay
using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay has completed
a formal validation study and the STE assay was implemented into the OECD TG 491 in 2015
(OECD, 2015c).
Although these assays are easy to perform economically, there are some disadvantages, inthat it is often not possible to assess accurately any insoluble or volatile test material because
they must be dissolved in the test solvent.
To solve their problems, there have been increasing expectations for the development of aneye irritation test (EIT) method using a reconstructed human corneal epithelial (RhCE) tissue
model, fabricated with tissue engineering techniques. Since eye irritants affect corneal
epithelium directly, the disruption of structural integrity, and decreased viability of the cornea areimportant indices of eye irritation. Especially, the RhCE models well recapitulate the stratified
and differentiated human corneal epithelium and are capable of evaluating neat test materials
directly without using any test solvent (Lotz et. al., 2016).
EITs using four RhCE models have been reported up to now; EpiOcular (MatTek, MA, USA)model to reconstruct using normal human epidermal keratinocyte (Stern et al., 1998; Kaluzhny
et. al., 2011; Pfannenbecker et. al., 2013), SkinEthic HCE (SkinEthic, France) model toreconstruct using immortalized human corneal epithelial cell line (Van Goethem et. al., 2006,
Cotovio et. al., 2010; Alépée et. al., 2013), LabCyte CORNEA-MODEL24 (Japan Tissue
Engineering Co., Ltd (J-TEC), Japan) model to reconstruct using normal human corneal
epithelial cells (Katoh et. al., 2012; Katoh et. al., 2013) and MCTT-HCE (Modern Cell & Tissue
Technologies, Korea) model to reconstruct using normal human corneal epithelial cells which issame to LabCyte CORNEA-MODEL24 tissue (Jung et. al., 2011; Jang et. al., 2015). All EITs
using four RhCE models are based on the cell viability measurement as endpoint of eye
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irritancy prediction.
In these EITs using RhCE models, the EIT test method using EpiOcular model has only
completed a formal validation study and the EpiOcular EIT was implemented as validated
reference method (VRM) into the OECD TG 492 in 2015 (OECD, 2015a).

1.1.2. The Proposed EIT Method using LabCyte CORNEA-MODEL24
The LabCyte CORNEA-MODEL24 which is proposed in this background review document(BRD) is generated from corneal epithelial cells originating from normal human corneal
epithelial tissue. The culture of normal human corneal epithelial cells on a membrane insert
results in three-dimensional RhCE tissue equivalent similar to the in vivo human corneal
epithelium with a verifiable expression of corneal epithelium marker (Katoh et. al., 2012). The
LabCyte CORNEA-MODEL24 tissue construct consists of at least three viable layers which iscomposed superficial cell layer, wing cell layer and basal cell layer and a no cornified surface,showing a corneal epithelium like structure analogous to that found in human eye (Katoh et. al.,2012; Poumay et. al., 2004).
In order to develop an ocular irritancy test method using an RhCE model, it was made to refer
the strategy of a validated skin irritation test method using a reconstructed human epidermal
tissue (OECD, 2010). In the LabCyte CORNEA-MODEL24 EIT (LabCyte24 EIT), the test
material is applied topically to the RhCE tissue surface and then tissue viability is measured
following exposure and a post-exposure incubation periods, that its strategy of its test procedure
is equal to that of skin irritation test method for the OECD TG 439 (OECD, 2010).
The RhCE tissue viability in the LabCyte24 EIT is measured by enzymatic conversion of the
vital

dye

Water

SolubleTetrazolium

Salt-8(WST-8;[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]) by theviable cells of the tissue into an orange-color WST-8 formazan salt that is quantitatively
measured assay solution directly. A cut-off value of 40% viability of the negative control value
was considered and used to classify test chemicals as eye irritancy (UN GHS Category 1, 2A
and 2B) or eye no irritancy (UN GHS No Category).
Also the assay principles of the LabCyte24 EIT is considered to be similar to the EpiOcular
ETI which is VRM of RhCE EIT for OECD TG492 (OECD, 2015a).

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1-2. Scientific Basis for the Proposed Test Method
1.2.1. Propose and Mechanism Basis of LabCyte24 EIT
Chemical-induced serious eye damage/eye irritation, manifested in vivo mainly by corneal
opacity, iritis, conjunctival redness and/or conjunctival chemosis, is the result of a cascade ofevents beginning with penetration of the chemical through the cornea and/or conjunctiva andproduction of damage to the cells. Cell damage can occur by several modes of action, including
cell membrane lysis, coagulation of macromolecules, saponification of lipids and alkylation orother covalent interactions with macromolecules (Scott et. al., 2010).
A recent report showed the general concept that slight eye irritants affect only the superficial
corneal epithelium, the mild and moderate irritants (UN GHS Category 2A and 2B) affectprincipally the epithelium and superficial stroma, and the severe irritants (UN GHS Category 1)
act through to deeper parts of the stroma, potentially as far as full stromal depth (Maurer et. al.,2002). Since the LabCyte CORNEA-MODEL24 reconstructs only the corneal epithelium, it isthought that the toxicity at all layers of the corneal epithelium can be distinguished from toxicity
only at the superficial layer of tissue in the LabCyte24 EIT. Therefore, it is suggested that the
LabCyte24 EIT might be able to distinguish between no or slight eye irritants (UN GHS No
Category) and mild to severe eye irritants (UN GHS Category 1, 2A and 2B).
The relative cell viability of the treated tissues at the end of test chemicals exposure and thenpost-exposure periods is measured using WST-8 assay. Chemicals not requiring classificationand labelling according to UN GHS Category are identified as those that do not decrease tissue
viability below a defined threshold (i.e., tissue viability > 40%, for UN GHS No Category).

1.2.2. Similarities and Differences of Modes of Action of Between the LabCyte24
EIT and the Draize Eye Test
1-2.2.1. The Draize Eye Test
For many years, the ocular irritation potential of chemicals mostly has been evaluated by the
Draize eye test. This test method involves instillation of the test substance into the lower
conjunctival sac of the rabbit eye, and evaluates the cornea, the iris, and the conjunctiva foradverse effects after exposure to the potential irritant.
The cornea is evaluated both for the degree of corneal opacity and the area of the cornea inwhich opacity is involved.

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The iris is assessed for inflammation, iridal folds, congestion, swelling, circumacorneal
injection, reaction to light, hemorrhage, and gross destruction.
The conjunctiva is evaluated for the degree of redness, chemosis (swelling), and discharge(Draize et. al. 1944).

1-2.2.2. Comparison of the LabCyte24 EIT with Draize Eye Test
In the LabCyte24 EIT, cytotoxicity is determined by the viability of the RhCE tissues. The
tissue viability is measured by WST-8 assay method. While these in vitro toxicity measurements
using the RhCE tissue are correlated with in vivo ocular irritation corneal effects, they represent
only one aspect of the overall complex response of the eye to irritants, which involves othertissues such as the iris and conjunctiva.
In contrast, the in vivo rabbit eye test involves a qualitative visual evaluation of the severity ofadverse effects on the cornea, the iris, and the conjunctiva, as well as the reversibility of any
ocular effects detected at selected intervals up to 21 days after exposure. In the LabCyte24 EIT,a test substance is exposed directly to cells for just 1 minute for Liquid chemicals and 24 hours
for Solid chemicals, and then rinsed off. And then blotted tissues were post-exposure incubatedfor 24 hours for only Liquid chemicals. In the in vivo rabbit eye test, test substances are applied
to the conjunctival sac. Because the rabbit eye can blink and/or tear, exposure of the test
substance to the cornea will be affected by these factors in terms of coverage or duration. The
production of tear film for the protection of eye tissue is also not present in the LabCyte
CORNEA-MODEL24. When compared with an in vivo rabbit eye test, application of a test
substance in the absence of this protective barrier might be expected to cause an increase infalse positive outcomes. On the other hands, in some test substances (e.g., Solids), blinking canalso induce mechanical damage in vivo, contributing to a higher degree of irritation. However,this protective mechanism for the eye are absent in the LabCyte24 EIT.

1.2.3. Applicability and Limitations of LabCyte24 EIT
One limitation of the RhCE EIT method is that it does not allow discrimination between eye
irritation/reversible

effects

on

the

eye

(UN

GHS

Category2)

andseriouseyedamage/irreversible effects on the eye (UN GHS Category 1), nor between eye irritants (UNGHS optional Category 2A) and mild eye irritants (UN GHS optional Category 2B), as definedby UN GHS (UN, 2003). For these purposes, further testing with other suitable test methods is
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required.
Other limitation of this assay method is a possible interference of the test chemical with the
WST-8 endpoint. A colored test chemical or one that directly reduces WST-8 (and thereby
mimics dehydrogenase activity of the cellular mitochondria) may interfere with the WST-8
endpoint. However, these test chemical are a problem only if at the time of the WST-8 test
sufficient amounts of the test chemical are still present on (or in) the tissues. In case of thisunlikely event, the (true) metabolic WST-8 reduction and the contribution by a colored test
material or (false) direct WST-8 reduction by the test material can be quantified using
freeze-killed RhCE tissues.
LabCyte24 EIT is applicable to substances and mixtures, and to solids, liquids, semi-solids
and waxes as same applicability to OECD TG 492 VRM (OECD, 2015a). The liquids may beaqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible,
solids should be ground to a fine powder before application; no other pre-treatment of thesample is required.

1-3. Regulatory Rationale and Applicability
1-3.1. Current Regulatory Testing Requirements
In recent years, several regulations and regulatory agencies have contributed to a greater
emphasis on alternative animal testing for ocular irritation (7th amendment to the Cosmetic
Directive [Directive 2003/15/EC, 2003], Registration Evaluation Authorization and Restriction ofChemicals [REACH]).
For the ocular irritation animal alternative test, it may be unlikely to completely replace the
Draize test by a single in vitro test because the Draize eye test evaluates a range of criteria for
injury and inflammation to the eye. The tiered approach of several in vitro assays combined was
proposed in order to estimate the irritation potential for a wide range of chemical classes;
Hayashi et. al., 2012a; Hayashi et. al, 2012b).
The LabCyte24 EIT is a cytotoxicity test involves exposing to the RhCE tissue. Furthermore,
to apply neat testing material on the RhCE tissue directly in spite of physical state of test
material, it's applicable at the wide ranges of test chemicals in the LabCyte24 EIT. For thesereasons, the LabCyte24 EIT method could be predict the eye irritancy of wide range chemical toconsider the bottom-up assay according to the tiered approach to establishing an ocular

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irritation animal alternative testing paradigm.

1-3.2. Intended Regulatory Use(s)The RhCE EIT test methods such as the LabCyte24 EIT have been proposed for identifying ofocular irritancy (e.g., Category 1 or Category 2 per the GHS classification system [UN 2003]) in
the accepted the OECD TG 492 (OECD, 2015a).

1-3.3. The Similarities and Differences in the Endpoint Measured in the Proposed
Test and Currently Used In Vivo Reference Test
As mentioned in Section 1.1.2, this test method was based on the cytotoxicity of the RhCE
tissue because the corneal tissue is one of the main targets during accidental eye exposures,and damage to the corneal tissues can result in visual impairment or loss.
In the LabCyte24 EIT, cytotoxicity is determined by the viability of the RhCE tissues. The
viability is measured by WST-8 assay method. While these in vitro toxicity measurements using
the cultured cell line are correlated with in vivo ocular irritation corneal effects, they represent
only one aspect of the overall complex response of the eye to irritants, which involves othertissues such as the iris and conjunctiva.

1-3.4. How the proposed test fits into the overall strategy of hazard or safety
As mentioned in Section 1.3.1, for the ocular irritation animal alternative test, it may be unlikely
to completely replace the Draize eye test by a single in vitro test because the Draize eye test
evaluates a range of criteria for injury and inflammation to the eye. The tiered approachcombined several in vitro assays was proposed in order to estimate the irritation potential for awide range of chemical classes (Hagino et. al., 2008; McNamee et. al., 2009; Scott et. al., 2010;
Hayashi et. al., 2012a; Hayashi et. al, 2012b).
The LabCyte24 EIT is being considered for use in identification ocular not irritancy (UN GHS
No category) from ocular irritancy (UN GHS category 1 or category 2). For these reasons, theLabCyte24 EIT could be considered a bottom up assay in the tiered approach to establishing anocular irritation animal alternative testing paradigm.

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1-4. Objection of This Background Review Document
The objective of this BRD is to describe the brief explanation of the feature of the LabCyte24
EIT, that is the optimization study, several pre-validation studies and also that the current statusof the LabCyte24 EIT, including what is known about its accuracy and reliability.

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2. DESCRIPTION OF LabCyte CORNEA-MODEL24
2-1. Condition of the Functional Reconstructed human Corneal Epithelial Tissue
Model, LabCyte CORNEA-MODEL24
LabCyte CORNEA-MODEL24 is a new, commercially available reconstructed human cultured
corneal epithelial model produced by Japan Tissue Engineering Co. Ltd.

It consists of normal

human corneal epithelial cells whose biological origin is human eye. In order to expand thehuman corneal epithelial cells while maintaining their phenotype, they are cultured with 3T3-J2
cells as a feeder layer (Green, 1978; Rheinwald et. al., 1975). Reconstruction of human cultured
corneal epithelial tissue is achieved by cultivating proliferating corneal epithelial cells on an inert
filter substrate (surface 0.3 cm2) at the air-liquid interface for 13 days, with an optimized medium
containing 5% fetal bovine serum. The result is a multilayer structure consisting of a fully
differentiated corneal epithelium with features of the normal human corneal epithelial tissues,consisting of superficial layer, wing cell layer and basal layer (Fig.2-1). The LabCyte
CORNEA-MODEL24 is embedded in an agarose gel containing a nutrient solution and shippedin 24-well plates at around 18°C.

Fig.2-1. Histological cross-sectional views of the LabCyte CORNEA-MODEL24 with H&E
staining (original magnification:×200).

Cytekeratin-3 is a specific marker of corneal epithelium, and it was expressed in all layers of
the LabCyte CORNEA-MODEL24 (Fig. 2-2(A)). Mucin-1 and mucin-16 that composed thetransmembrane glycocalyx in the surface of the corneal epithelium were well expressed in the
superficial layer of the LabCyte CORNEA-MODEL24 (Fig. 2-2(B)). Cells in the corneal
epithelium are connected by desmosomes, tight junctions and adherence junctions. Claudin-1
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which is a marker of a tight junction, desmograin-3 which is a marker of desmosome andE-cadherin which is a marker of an adherence junction are localized in the interface between
cells at all cell layers, including the superficial layers (Fig. 2-2(C)). Laminin is an important
constituent in the basement membrane at the basal corneal epithelium junction. It was
expressed continuously in basal cells of the LabCyte CORNEA-MODEL24 basal layer (Fig.
2-2(C)).
(A)(B)

Cytokeratin 3

Mucin 1

Mucin 16(C)
Claudin-1

Desmoglein-3E-cadherin

Laminin 332

Fig.2-2. Histological cross-sectional views of the LabCyte CORNEA-MODEL24 withimmuno-staining:(a) Immunohistochemical analysis for cytekeratin-3, which is an epithelial marker.
(b) Immunohistochemical analysis for mucin 1 and mucin 16, which are mucous
components of superficial cell layers.
(c)Immunohistochemical analysis for Claudin-1, Desmoglein-3, and E-cadherin,
which are cell adhesion molecules, and for laminin-332 which is a componentof the basement membrane.

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(A)

(B)

(C)

Superficial layer

Wing cell layer

Basal layer

Fig.2-3. Transmission electron micrographs of the LabCyte CORNEA-MODEL24.
(A) Superficial cell layer. Note microvilli-like structure (arrows).
(B) Wing cell layer. Note desmosome (dotted circle line).
(C) Basal cell layer. Note hemidesmosome (arrows).

The specific corneal differentiation markers, cell adhesion molecules and basement
membrane constituents are expressed in the appropriate regions as seen in a human corneal
epithelium.
The basement membrane is smooth like that of human corneal tissues. In the fully developed
basement membrane zone, hemidesmosomes are quite numerous along the basal cell layer(Fig. 2-3(A)). The wing cell layer and superficial cell layer of the LabCyte CORNEA-MODEL24
are composed of round and irregular cells connected by highly developed desmosomes (Fig
2-3(B)). Furthermore, the microvilli-like structure is well formed on the surface of the superficial
layer in the LabCyte CORNEA-MODEL24 (Fig 2-3(C)). In conclusion, the LabCyte
CORNEA-MODEL24, a reconstructed human corneal epithelial model, reproduces many of thecharacteristics of the native human corneal tissue. Therefore, it provides a morphologically
relevant means to assess eye irritation, percutaneous absorption, and other ocular-related
research as an alternative to animal testing.
Their histological feature of LabCyte CORNEA-MODEL24 adapts the general condition of the
new RhCE for EIT indicated in the OECD TG 492.
Their histological evaluation was already published in our scientific report (Katoh et. al., 2013).

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February, 20172-2. Quality Control for LabCyte CORNEA-MODEL24
2-2.1. Quality Control Procedures for LabCyte CORNEA-MODEL24
LabCyte CORNEA-MODEL24 is manufactured according to a defined standard operation
procedure (SOP). All batches of the LabCyte CORNEA-MODEL24 production are checked fortheir viability, barrier function and morphology.
The product is released following stringent quality control procedures.
The quality of the final product is assessed by the following protocol and decision criteria;
1) Tissue viability2) Barrier function3) Morphology

2-2.1.1. Tissue Viability
2-2.1.1a. MTT Assay
Three LabCyte CORNEA-MODEL24 tissues are subjected to an MTT assay as follows.
Tissues are put in the wells of 24-well plates containing 0.5 mL of MTT medium (0.5 mg/mL;
Dojindo Co., Kumamoto, Japan) and are incubated for 3 hours (37°C, 5% CO 2, humidified
atmosphere). Formazan produced in the tissues is extracted with isopropanol (300 μL) and theoptical density (OD) of the extract (200 μL) is measured at 570 nm and at 650 nm as a reference
absorbance, with isopropanol as a blank.
The mean of the OD values indicates tissue viability for each LabCyte CORNEA-MODEL24
batch.
QC acceptance criteria: 2.5 ≥ OD ≥ 0.8
Since the start of the commercial release of the product until December 2015, no batch has
been outside the QC acceptance criteria.

2-2.1.1b. WST-8 Assay (Collection as Reference Data not to Control Release
Criteria)
Three LabCyte CORNEA-MODEL24 tissues are subjected to a WST-8 assay as follows.
Tissues are put in the wells of 24-well plates containing 0.3 mL of WST-8 medium {1:10 dilution
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February, 2017of Cell counting kit-8 (Dojindo Co., Japan): Earle’s balanced salt solution (EBSS; Sigma-Aldrich,
MO, USA)} and are incubated for 4 hours (37°C, 5% CO 2, humidified atmosphere). The OD ofculture supernatant (200 μL) is measured at 450 nm and at 650 nm as a reference absorbance,with WST-8 medium as a blank.
The mean of the OD values indicates tissue viability for each LabCyte CORNEA-MODEL24
batch.

2-2.1.2. Barrier Function: 50% Inhibitory Concentration (IC50) Assay
To evaluate whether the LabCyte CORNEA-MODEL24 tissue resists the rapid penetration of
the cytotoxic marker chemical sodium lauryl sulphate (SLS), the viability of the corneal epithelial
tissue is estimated in terms of the half maximal inhibitory concentration of 50% (IC50). Various
concentrations (0.1, 0.2, 0.3, and 0.4 %(w/v)) of SLS (50 μL) are applied to the LabCyte
CORNEA-MODEL24, and cell viability was measured after 1 hour using an MTT assay.
experiments

areperformed

in

triplicate.

The

acceptable

range

for

the

All

LabCyteCORNEA-MODEL24 is shown in Table 2-1.
Table 2-1.

QC acceptable range of barrier function.

LabCyte CORNEA-MODEL24

Lower limit

Mean

Upper limit

0.10 (w/v)%

0.24 (w/v)%

0.40 (w/v)%

IC50(1 hour SLS)

Since the start of the commercial release of the product until December 2015, no batch has
been outside the QC acceptance criteria.

2-2.1.3. Morphology
A piece of LabCyte CORNEA-MODEL24 is fixed with 4% parafolmaldehyde and 2% sucrose
in Dulbecco’s Phosphate Buffered Saline (D-PBS, Invitrogen, CA, USA) for more than threehours and processed for embedding in paraffin. Five-micrometer vertical sections are cut andstained with hematoxylin and eosin for light-microscopic examination.

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QC acceptance criteria:

Confirmation

February, 2017

of

the

formationofmultilayered

cornealepithelium-like tissue.
Since the start of the commercial release of the product until October 2010, no batch has beenoutside the QC acceptance criteria.

2-2.2. Batch Control Information for LabCyte CORNEA-MODEL24
Quality control data for the tissue viability and barrier function of each LabCyte
CORNEA-MODEL24 batch is shown in Table 2-2.
Table 2-2.

Year

2010

2011

Batch information for LabCyte CORNEA-MODEL24

Month

Tissue viability(Mean OD)

Barrier function

MTT

WST-8

Mean (%)(A570/650)(A450/650)

February

1.17

0.28

March

1.44

0.25

April

1.23

0.28

May

1.24

0.28

June

1.60

0.29

July

1.48

0.24

September

1.74

0.25

October

1.63

0.28

November

1.52

0.21

December

1.47

0.25

January

1.40

0.20

February

1.46

0.28

March

1.48

0.28

April

1.44

0.20

May

1.40

0.22

June

1.29

0.19

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BRD of the LabCyte CORNEA-MODEL24 EIT

July

1.24

0.19

August1.34

0.19

September

1.23

0.26

October

1.20

0.21

1.28

0.20

December

1.25

0.19

January

1.22

0.19

February

1.17

0.21

March

1.23

0.25

April

1.16

0.22

May

1.18

0.19

June

1.04

0.22

July

1.11

0.19

August1.14

0.23

September

1.33

0.27

October

1.38

0.22

November

1.48

0.19

December

1.44

0.26

January

1.47

0.20

February

1.37

0.24

March

1.17

0.25

April

1.18

0.23

May

1.16

0.21

June

1.02

0.23

July

1.29

0.23

August1.27

0.18

September

1.29

0.20

October

1.23

0.24

November

1.29

0.19

December

1.20

0.20

January

1.30

0.25

February

1.14

0.26

March

1.35

0.25

1.35

0.22

November

2012

2013

2014

February, 2017

April

FBS lot changed

FBS lot changed

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2015

February, 2017

May

1.25

0.27

June

1.18

0.26

July

1.34

0.31

August1.22

0.27

September

1.25

0.25

October

1.13

0.29

November

1.11

0.24

December

1.14

0.25

January

1.12

1.02

0.26

February

1.18

1.06

0.24

March

1.15

1.13

0.22

April

1.07

1.02

0.24

May

1.16

1.00

0.26

June

1.06

0.92

0.24

July

1.06

1.13

0.26

August

0.99

1.03

0.28

September

1.02

1.03

0.22

October

1.23

0.87

0.25

November

0.92

0.84

0.25

December

1.04

0.99

0.24

Mean±SD

1.26±0.16

1.00±0.09

0.24±0.03

12.7%

9.0%

13.4%

Coefficient Variation

As shown in Table 2-2, the mean±SD of tissue viability (MTT assay / WST-8 assay) andbarrierfunction(IC50)

fromtheevaluationofcontinuous

batchesof

LabCyteCORNEA-MODEL24 were 1.26 ± 0.16% (MTT assay), 1.00 ± 0.09% (WST-8 assay) and 0.24 ±
0.03%, respectively.

The tissue viability and barrier function of LabCyte CORNEA-MODEL24

tissue have remained constant, indicating reproducibility [low coefficient variation (CV): 12.7%(MTT assay), 9.0% (WST-8 assay) and 13.4%, respectively] and monthly consistency is high.
Note
FBS lot used LabCyte CORNEA-MODEL24 cultivation had been changed twice (at November,2011 and at April, 2014).

Performance of FBS lot has been checked before changing lot using

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BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017and only passed FBS lot was accepted for production of LabCyte CONREA-MODEL24. Each
LabCyte CORNEA-MODEL24 batch between acceptable QC range have been consistently
produced using all lot of FBS.

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February, 2017

3. OPTIMIZATION AND IMPROVMENT STUDY OF THE LabCyte24 EIT
3-1. Optimization of Suitable Time Periods for Test Chemicals Combining Sample
Exposure and Post-exposure Incubation
3-1.1. Purpose of This Study
For the confirmation of suitable LabCyte24 EIT protocol, two important conditions were
examined in order to designate exposure periods and post-exposure incubation periods for both
Liquid chemicals and Solid chemicals, respectively.

3-1.2. Optimization of Suitable Exposure and Post-exposure Incubation Periods
for the Liquid Test Chemicals
3-1.2.1. Study Conditions
This study condition was summarized followings.
Table 3-1.

The test chemical set for initially optimization of the LabCyte24 EIT
CAS number1)

In vivo class2)

Physical state

3-Methoxy-1,2-propanediol

623-39-2

NC

LiquidPolyethylene glycol 400

25322-68-3

NCLiquidGlycerol

56-81-5NC

LiquidMethyl cyclopentane

96-37-7NC

LiquidTween 209005-63-5

NC

Liquid2-Methyl -1-pentanol

105-30-62LiquidTriton X-100 (5%)

9002-93-1

2

Liquid1-Octanol

111-87-5

2Liquid2-Ethyl-1-hexanol

103-76-7

2

Liquidn-Hexanol

111-27-3

2

LiquidName1) CAS No.: Chemical abstracts service registry number.
2) In vivo class was referred from the UN GHS classification.
NC: No category (no eye irritant); 2: Category 2 (eye irritant) 2A: 1: Category 1 (eye corrosion)

In order to determine optimal exposure periods for the Liquid chemicals, tissues that applied

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February, 2017

Liquid chemicals (50 μL) were incubated for varying time periods (1, 3, 5, 10 and 20 minutes).
After exposure, each tissue was rinsed with D-PBS, using a washing bottle to remove any
remaining test chemical from the tissue surface. The blotted tissues were then transferred to
new wells on 24-well plates containing 500 μL of fresh assay medium. In order to determine
optimal post-exposure incubation periods, the blotted tissues were post-exposure incubated forvarious time periods (0, 2 and 24 hours) under the standard cultivation conditions.
After the post-exposure incubation periods, blotted tissues were transferred to new wells on
24-well plates containing 300 μL of freshly prepared WST-8 solution (1:10 dilution of Cell
Counting Kit-8 with D-PBS) for a WST-8 assay. Tissues were incubated for 5 hours under the
standard cultivation conditions. Subsequently, 200 μL of culture supernatant were transferred toa 96-well microtiter plate. The OD value of culture supernatant was measured at 450 nm and at
650 nm as a reference absorbance, with WST-8 solution as a blank.
The cell viability was equal to or lower than 50%, the chemical was judged to be UN GHS
category 1 or 2, otherwise it was considered GHS No category.
Ten Liquid test chemicals (initially optimization set) as shown in Table 3-1 was used thisoptimization study.

3-1.2.2. Results of Optimization Study for Liquid Chemicals
Ten test chemicals (5 No Category: no irritancy, and 5 Category 2: irritant, in the GHS
classification) were selected for the optimization experiments.
When no post-exposure incubation period was set both false-positive predictions for threechemicals and false-negative predictions for four chemicals were observed in 5 minutes
exposure period (Fig. 3-1(A)). Even if exposure periods were gradually prolonged to 20 minutes,
the number of chemicals showing a false-positive increased while that of false negative
chemicals does not decrease (Fig 3-1(A)), so that, in the end, the correlation with the GHS classwas not improved.
When the post-exposure incubation periods were set at 2 hours, four chemicals showing
irritant in vivo had shown a false negative for a 1 minute exposure period and their sensitivity
result was low (Fig 3-1(B)). On the other hand, in the case of exposure periods that were 5
minute or longer, 3 of 5 in vivo non-irritant chemicals showed a false-positive, though there wasno chemical that showed a false negative (Fig. 3-1(B)), and therefore the correlation with the invivo class was low during exposure periods.

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February, 2017

(A)
Exposure periods (minute)
5

10

20

120

Viability (% of Control)

100

80

60

40

20

0
3-Methoxy-1,2propanediol

Polyethylene
glycol 400

Glycerol

Methylcycｌopentane

Tween 20

2-Methyl -1pentanol

5% Triton X-100

No Category

1-Octanol

2-Ethyl-1-hexanol

n-Hexanol

Category 2

GHS class(B)
Exposure periods (minute)
1

5

10

120

Viability (% of Control)

100

80

60

40

20

0
3-Methoxy-1,2propanediol

Polyethylene
glycol 400

Glycerol

Methylcycｌopentane

Tween 20

2-Methyl -1pentanol

No Category

5% Triton X-100

1-Octanol

2-Ethyl-1hexanol

n-Hexanol

Category 2

GHS class36 /121

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February, 2017

(Continue)(C)
Exposure periods (minute)
1

5

10

120

Viability (% of Control)

100

80

60

40

20

0
3-Methoxy-1,2propanediol

Polyethylene
glycol 400

Glycerol

Methylcycｌopentane

Tween 20

2-Methyl -1pentanol

No Category

5% Triton X-100

1-Octanol

2-Ethyl-1hexanol

n-Hexanol

Category 2

GHS classFig.3-1. Optimization of exposure periods and post-exposure incubation periods for the
LabCyte24 EIT for Liquid chemicals.
Post-exposure incubation period was set at 0 hours (A), 2 hours (B) and 24 hours (C),respectively.

When the post-exposure incubation periods were set at 24 hours, all 5 non-irritant chemicals
and 5 irritant chemicals were correctly predicted at 1 minute exposure periods, and theprediction results completely correlated to the UN GHS classification (Fig 3-1(C)). The numberof chemicals that showed a false-positive increased relatively as the exposure period was
extended, while the accuracy of prediction showed a tendency to decrease gradually.
Finally, it was decided that the optimal application conditions for exposure and post-exposure
incubation periods for Liquid chemicals should be set at 1 minute and 24 hours, respectively.

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BRD of the LabCyte CORNEA-MODEL24 EIT

February, 20173-1.3. Optimization of Suitable Exposure and Post-exposure Incubation Periods
for the Solid Test Chemicals
3-1.3.1. Study Conditions
This study condition was summarized followings.
In order to determine optimal exposure periods for the Solid chemicals, tissues that applied
Solid chemicals (50 mg) were incubated for varying time periods (1, 4 and 24 hours). After
exposure, each tissue was rinsed with D-PBS, using a washing bottle to remove any remaining
test chemical from the tissue surface. The blotted tissues were then transferred to new wells on
24-well plates containing 500 μL of fresh assay medium. In order to determine optimal
post-exposure incubation periods, the tissues were post-incubated under the same conditions
as that of the Liquid chemicals for respective periods (23 hours for an exposure of 1 hour, 20
hours for an exposure of 4 hours and not set for an exposure of 24 hours). After the exposure
and post-expose periods, blotted tissues were analyzed for their cell viability through means of a
WST-8 assay, in the same manner as for Liquid chemicals.
Table 3-2.

The Solid test chemical set for initially optimization of the LabCyte24 EIT
CAS number1)

In vivo class2)

Physical state

4,4'-Methylenebis(2,6-di-tert-butylphenol)

118-82-1

NC

Solid

Silicic acid7699-41-4

NC

Solid

Sucrose fatty acid ester

-

2

Solid

3,3-Dithiodipropionic Acid

1119-62-6

2

Solid

1-naphtalen acetic acid

86-87-3

2

SolidDiisopropanolamine

110-97-4

2

Solid

Lauric acid143-07-7

1

Solid

Name

1) CAS No.: Chemical abstracts service registry number.
2) In vivo class was referred from the GHS classification.
NC: No category (no eye irritant); 2: Category 2 (eye irritant); 1: Category 1 (eye corrosion)

The cell viability was equal to or lower than 50%, the chemical was judged to be UN GHS
category 1 or 2, otherwise it was considered UN GHS no category.
Seven Solid test chemicals (initially optimization set) as shown in Table 3-2 was used thisoptimization study.

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February, 20173-1.3.2. Results of Optimization Study for Solid Chemicals
Seven test chemicals (two UN GHS No Category: no irritancy, and five UN GHS Category 1 or
2 irritant, in the GHS classification) were selected for the optimization experiments.
When the chemical exposure period has been set to 1 or 4 hours, a sucrose fatty acid esterthat was classified in the No Category in the UN GHS classification was wrongly predicted as a
non-irritant (Fig. 3-2). On the other hand, all the irritant and non-irritant chemicals in the GHS
classification could apparently be predicted between irritating chemical and non-irritatingchemicals when the exposure time was set at 24 hours (Fig. 3-2).
Finally, it was decided that the optimal application conditions for exposure and post-exposure
incubation periods for Solid chemicals should be set at 24 hours and none, respectively.

120
Exposure periods (hour)
1

4

24

Viability (% of Control)

100

80

60

40

20

0
4,4'-Methylenebis(2,6di-tert-butylphenol)

Silicic acid

Sucrose fatty acid ester

No Category

3,3-DithiodipropionicAcid1-naphtalen acetic acid

Category 2

Diisopropanolamine

Lauric acid

Category 1

GHS classFig.3-2. Optimization of exposure periods and post-exposure incubation periods for the
LabCyte24 EIT for Solid chemicals.

3-1.4. Discussion and Conclusion
In this optimization study, it was decided the optimal application conditions for exposure andpost-exposure incubation periods for Liquid and Solid chemicals as shown in the Table 3-3.

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BRD of the LabCyte CORNEA-MODEL24 EIT

Table 3-3.

February, 2017

Optimal application period
Periods

Liquid Chemicals

Solid Chemicals

Chemical exposure periods

1 minute

24 hours

Post-exposure incubation periods

24 hours

Not set

It is thought that the optimized LabCyte24 EIT protocol for Liquid may reflect the Draize eyetest well, as it has very short exposure periods (1 minute) and long recovery periods (24 hours).
On the other hand, some Solid chemicals may often adhere to a corneal tissue because theirrejection from the eye is not completely accomplished by physiological mechanisms. In the
optimized LabCyte24 EIT for Solid chemicals, it was thought that the set-up for long exposure
periods (24 hours) could reflect the situation where Solid chemical might often remain in the eyeof a test animal in an in vivo Draize test, because of possibly low mobility.

3-1.5. Publication
Detailed results and conclusion of this optimization study were already published in ourprevious report (Katoh et. al, 2013).

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February, 2017

3-2. Ring-Study for the Technical Transferability of the LabCyte24 EIT
3-2.1. Purpose of This Study
In order to assess the technical transferability of the LabCyte24 EIT method, the technicaltransfer-course for twenty-four laboratories which participated by the general invitation of theplanning committee in the Japanese Society for Alternatives to Animal Experiments (JSAAE)
has been held and joint research was carried out.
After the technical transfer-course, participating laboratories were performed the LabCyte24
EIT according to technical transfer-course methods with 16 selected test chemicals in order toconfirm technical transferability, the reliability and the predictive performance.

3-2.2. Study Plan
3-2.2.1. Participating Laboratories
Twenty-four laboratories were participated in this study (Table 3-4).
Table 3-4.

Participating laboratories

Lab No.

Laboratory Name

1

Hatano Research Institute, Food and Drug Safety Center2

Nihon Kolmar Co.,Ltd

3

ISHIHARA SANGYO KAISHA,LTD

4

Drug Safety Testing Center Co., Ltd

5

FujiFilm Corp.

6

Chemicals Evaluation and Research Institute, Japan

7

TOYO BEAUTY CO.,LTD

8

Safety Research Institute for Chemical Compounds Co., Ltd.

9Japan Food Research Laboratories

10

ROHTO Pharmaceutical Co., Ltd.

11

OPPEN COSMETICS Co.

12Taisho Pharmaceutical Co. ,Ltd.

13

Nihon Nohyaku Co., Ltd.

14IVY COSMETICS Co.

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BRD of the LabCyte CORNEA-MODEL24 EIT

February, 201715

Kao Corp.

16

LION CORP.

17

Kobayashi Pharmaceutical Co., Ltd.

18

Mandom Corp.

19

NOEVIR Co.,Ltd.,20DRC CO.,LTD

21

BOZO Research Center Inc.

22

Kamakura Techno-Science

23

BioSafety Research Center (BSRC)

24

Japan Tissue Engineering Co., Ltd

3-2.2.2. Selected Test Chemicals
Sixteen test chemicals were selected in this study (Table 3-5).
Table 3-5.

Selected test chemicals.

Chemical No.

Test Chemicals

CAS no.

Category

Physical StateGHS class

L0101-L0601

Sucrose fatty acid ester-

Surfactants (nonionic)

Solid

2

L0102-L0602

4,4'-Methylenebis(2,6-di-tert-butylphenol)

118-82-1

Aromatics

Solid

NC

L0103-L0603

1-Butanol

71-36-3

Alcohols

Liquid1

L0104-L0604

Sodium hydroxide (1%)

1310-73-2

Inorganic bases

Liquid2B

L0701-L1201

1-Naphtalen acetic acid

86-87-3

Pesticides

Solid

2

L0702-L1202

Sodium salicylate

54-21-7

Organic salts

Solid

1

L0703-L1203

Tween 209005-64-5

Surfactants (nonionic)

LiquidNC

L0704-L1204

Isopropylalcohol

67-63-0

Alcohols

Liquid2A

L1301-L1801

2,5-dimethyl-2,5-hexanediol

110-03-2

Alcohols

Solid

1

L1302-L1802
L1303-L1803

105-30-6
57-09-0

Alcohols
Surfactants (nonionic)

LiquidLiquid2B
27699-41-4

Inorganics

Solid

NC

L1901-L2401

2-Methyl -1-pentanol
Triton X-100(5%)
Silic acid
(Silicon Dioxide n-Hydrate)
Glycolic acid79-14-1

2

3,3-Dithiodipropionic Acid

1119-62-6

Carboxilic acid
Sulphur-containing
compounds

Solid

L1902-L2402

Solid

2B

L1903-L2403

Benzalkonium chloride (10%)

8001-54-5

Surfactants (cationic)

Liquid1

L1904-L2404

3-Methoxy-1,2-propanediol

623-39-2

Alcohols

LiquidNC

Positive control
Positive control

Ethanol
Lauric acid64-17-5
143-07-7

Alcohols
Fatty acids

LiquidSolid

2A
1

L1304-L1804

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BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017

3-2.2.3. Test Protocol
3-2.2.3a. Protocol for Liquid Chemical
Liquid chemicals (50 μL) were applied to three tissues. In addition, three tissues were treatedwith 50 μL of D-PBS serving as negative controls and ethanol serving as positive controls. And
then tissues applied chemicals were incubated for 1 minute. After exposure, each tissue wasrinsed with D-PBS ten times or more, using a washing bottle to remove any remaining test
chemical from the tissue surface. The blotted tissues were then transferred to new wells on
24-well plates containing 500 μL of fresh assay medium. And then the tissues were
post-exposure incubated for 24 hours under the standard cultivation conditions (at 37°C in ahumidified atmosphere of 5% CO 2 in air). After the post-exposure incubation periods, blottedtissues were transferred to new wells on 24-well plates containing 300 μL of freshly prepared
WST-8 solution (1:10 dilution of Cell Counting Kit-8 with D-PBS) for a WST-8 assay. Tissues
were incubated for 5 hours under the standard cultivation conditions. Subsequently, 200 μL of
culture supernatant were transferred to a 96-well microtiter plate. The OD value of culture
supernatant was measured at 450 nm and at 650 nm as a reference absorbance, with WST-8
solution as a blank.
The tissue viability was calculated as a percentage relative to the viability of negative controls.
The mean of the three values from identically treated tissues was used to classify a chemicalaccording to the prediction model.

3-2.2.3b. Protocol for Solid Chemical
Solids (50 mg) in a microtube are applied by decantation on the tissue. Each test chemical
was applied to three tissues. In addition, three tissues serving as negative controls were set
apart as non-treated samples and Lauric acid (50 mg) served as positive control. And thentissues were incubated for 24 hours. After exposure, each tissue was rinsed with D-PBS in thesame manner as that of Liquid chemicals. In the Solid chemicals, post-exposure incubation was
not set. After the rinsing, blotted tissues were analyzed for their cell viability through means of a
WST-8 assay, in the same manner as for Liquid chemicals.

3-2.2.3c. Assay Criteria
Only when all three following conditions were satisfied, the test result was accepted. .
Negative control

0.5 ≤ Mean of OD (450nm/650nm) ≤ 2.0

Positive control

Mean of cell viability ≤ 50%
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SD

February, 2017

SD of viability of three RhCE tissues (negative control, positive
control and test chemical) ≤ 20 %3-2.2.3d. Prediction Model in This Study
If the mean cell viability was equal to or lower than 50%, the chemical was judged to be an
irritant, otherwise, it was considered a non-irritant.

3-2.3. Results
3-2.3.1. Technical Transferability
As shown the Fig.3-3 one run of negative control for Solid did not met the acceptance criteria
of OD range (0.5 ≤ Mean OD ≤ 2.0) and then the frequency of invalid test run for the negative
control was 0.7% (1/145 test).
On the other hands, all positive control for both Liquid and Solid chemicals met theacceptance criteria of both cell viability (≤ 50%) and SD (≤ 20 %) and then the frequency ofinvalid test run for the positive control was 0% (0/144 test).

Negative control (Solid)

2.0

2.0

1.5

1.5

OD (A450/650)

OD (A450/650)

Negative control (Liquid)

1.0

0.5

0.0

1.0

0.5

0.0
1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

1

2

3

4

5

6

7

8

Lab No.

Lab No.

Positive control (Solid)

120

120

100

100

80

80

Cell viability (%)

Cell viability (%)

Positive control (Liquid)

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

60
40
20

60
40
20

0

0
1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

1

2

3

4

Lab No.

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Lab No.

Fig.3-3. Results of negative control and positive control

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Table 3-6.

February, 2017

Prediction of three independent run
Cell viability (%)
1-Butanol

Lab No.
1
R1
R2
R3
2
R1
R2
R3
3 R1/R2
R2/R3
R3/R4

4

5

6

R1
R2
R3
R1
R2
R3
R1
R2
R3

Lab No.
7
R1
R2
R3
8
R1
R2
R3
9
R1
R2
R3
10
R1
R2
R3
11
R1
R2
R3
12
R1
R2
R3

Mean
SD
4.5
2.4
6.8
2.3
7.2
7.4
6.2
2.6
11.4
3.3
13.3
5.2
11.9
2.8
11.3
4.4
11.6
2.8
5.1
3.9
15.4
9.1
12.6
4.9
6.5
2.8
4.9
1.2
9.0
1.0
10.6
7.0
14.7
1.6
11.8
2.6
Tween 20
Mean
87.9
82.8
82.6
4.6
92.2
94.3
86.9
115.5
111.1
95.5
65.2
83.5
30.7
117.4
110.3
78.6
96.3
117.2

SD
0.8
0.9
2.3
1.1
2.8
4.0
6.3
14.7
1.9
18.8
2.7
18.0
10.6
10.2
13.3
62.8
22.0
9.7

LiquidSodium hydroxide (1%)
1
Judge
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
NC
Judge
NI
NI
NI
I
NI
NI
NI
NI
NI
NI
NI
NI
I
NI
NI
NI
NI
NI

Mean
SD
2.6
1.3
3.8
2.0
7.6
11.0
4.1
0.3
3.6
0.6
7.1
3.7
2.0
1.1
4.2
1.8
7.5
0.3
1.0
0.3
2.4
1.6
1.9
0.6
1.6
0.2
1.3
0.7
1.2
0.0
2.9
1.6
5.0
3.6
4.4
3.1
Isopropylalcohol
Mean
9.0
6.3
5.6
3.9
10.8
9.2
6.5
8.7
5.2
3.0
6.0
8.3
7.8
4.5
11.3
7.1
2.7
21.9

SD
0.8
0.4
0.4
4.5
4.1
1.8
0.9
3.0
2.7
2.2
0.8
4.3
3.9
1.1
1.4
3.7
1.5
9.1

Solid
2B Sucrose fatty acid ester
2
Judge
Mean
SD
Judge
I
13.9
2.4
I
I
11.3
3.6
I
I
16.0
1.2
I
I
13.7
1.0
I
I
15.8
1.7
I
I
24.1
1.9
I
I
13.6
3.8
I
I
16.3
1.1
I
I
9.0
3.1
I
I
17.2
2.1
I
I
18.3
9.0
I
I
19.2
2.9
I
I
20.4
5.7
I
I
15.0
4.9
I
I
13.1
4.1
I
I
14.3
4.6
I
I
21.6
1.8
I
I
10.6
1.6
I
2A 1-Naphtalen acetic acid
2
Judge
Mean
SD
Judge
I
6.6
0.2
I
I
7.1
0.8
I
I
8.0
0.5
I
I
7.1
9.3
I
I
10.7
5.7
I
I
11.1
6.1
I
I
1.8
0.5
I
I
2.6
0.3
I
I
2.4
1.0
I
I
6.2
5.1
I
I
4.2
2.0
I
I
2.7
0.7
I
I
1.5
0.7
I
I
1.3
0.3
I
I
2.7
0.8
I
I
5.4
2.4
I
I
1.4
0.1
I
I
4.7
1.0
I

4,4'-Methylenebis(2,6-di-tert-butylphenol)

Mean
SD
97.0
2.7
77.4
3.9
108.8
13.3
91.4
10.7
92.3
3.4
76.7
2.0
32.9
4.2
51.0
7.3
73.4
9.6
106.1
6.4
90.2
15.2
108.4
2.8
110.5
5.6
110.2
0.9
89.1
11.1
97.2
4.3
102.3
6.7
77.7
4.0
Sodium salicylate
Mean
4.3
5.6
6.2
0.7
0.8
0.7
1.3
1.5
1.0
1.4
2.0
1.3
0.9
0.7
1.5
0.7
0.8
0.7

SD
0.0
0.7
0.5
0.1
0.1
0.1
0.1
0.1
0.0
0.1
0.1
0.1
0.1
0.4
0.5
0.0
0.1
0.0

NC
Judge
NI
NI
NI
NI
NI
NI
I
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
1
Judge
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I

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(Continue)
Cell viability (%)
2-Methyl -1-pentanol

Lab No.
13
R1
R2
R3
14
R1
R2
R3
15
R1
R2
R3
16
R1
R2
R3
17
R1
R2
R3
18
R1
R2
R3

Mean
34.3
30.0
30.0
28.0
36.5
43.8
23.8
8.7
29.4
21.7
24.0
21.0
27.2
27.9
25.1
30.0
17.6
25.0

SD
3.9
5.9
7.9
8.9
6.9
6.3
3.7
9.3
5.4
4.4
10.0
2.1
9.0
1.5
4.2
2.3
0.9
3.3

Benzalkonium chloride (10%)

Lab No.
19
R1
R2
R3
20
R1
R2
R3
21
R1
R2
R3
22
R1
R2
R3
23
R1
R2
R3
24
R1
R2
R3

Mean
-0.8
1.4
0.8
0.7
1.1
1.2
2.2
2.5
1.8
1.6
1.5
1.5
1.6
0.7
1.5
0.7
1.3
0.6

SD
0.3
0.2
0.1
0.2
0.5
0.3
0.4
1.2
0.1
0.1
0.0
0.0
0.2
0.1
0.3
0.2
0.3
0.2

2B
Judge
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
1
Judge
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I

Triton X-100(5%)
Mean
4.5
4.3
4.2
23.5
21.3
21.8
3.3
7.4
2.4
1.6
1.1
1.5
1.7
2.1
1.8
5.0
5.0
11.0

SD
0.2
0.7
1.3
3.4
2.7
2.3
1.4
7.7
0.3
0.5
1.1
0.4
0.1
0.8
0.1
2.3
2.3
1.2

3-Methoxy-1,2-propanediol

Mean
47.9
40.0
48.9
54.5
62.1
51.6
70.6
95.2
93.4
76.3
81.6
62.6
75.4
50.1
47.7
85.7
73.0
82.0

SD
6.6
3.0
13.5
4.0
17.1
4.8
5.9
3.6
4.5
8.7
39.4
13.4
2.2
6.2
12.8
9.0
12.1
8.0

2
Judge
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
NC
Judge
I
I
I
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
I
NI
NI
NI

2,5-dimethyl-2,5-hexanediol

Mean
SD
4.7
0.4
3.2
0.2
2.7
0.3
1.4
0.5
1.0
0.4
0.7
0.0
4.0
0.2
7.2
6.3
3.5
0.3
1.4
0.1
1.7
0.1
1.4
0.1
2.4
0.7
2.1
0.2
1.5
0.0
1.6
0.2
2.9
0.5
2.4
0.3
Glycolic acid
Mean
-2.4
-0.1
0.3
-0.1
0.1
0.0
1.0
0.7
0.6
0.4
0.6
0.6
1.2
0.9
1.7
2.4
2.2
0.6

SD
0.0
0.4
0.4
0.5
0.2
0.2
0.1
0.1
0.1
0.1
0.2
0.2
0.7
0.1
0.2
0.8
1.6
0.2

Silic acid
1
NC
Judge
Mean
SD
Judge
I
67.2
1.4
NI
I
53.2
1.5
NI
I
63.4
13.4
NI
I
35.6
7.6
I
I
47.0
3.5
I
I
41.5
9.2
I
I
44.0
15.7
I
I
35.8
5.1
I
I
47.6
3.2
I
I
51.0
8.2
NI
I
45.4
8.3
I
I
61.4
7.6
NI
I
39.1
6.3
I
I
71.0
1.9
NI
I
42.0
2.7
I
I
37.6
2.4
I
I
42.5
1.4
I
I
43.4
4.1
I
3,3-Dithiodipropionic acid
2
2B
Judge
Mean
SD
Judge
I
-0.2
0.7
I
I
11.8
1.4
I
I
4.6
3.0
I
I
2.6
1.4
I
I
1.1
0.1
I
I
3.5
1.1
I
I
32.0
5.0
I
I
13.9
0.6
I
I
14.2
2.2
I
I
5.3
4.8
I
I
4.3
2.8
I
I
13.4
2.1
I
I
1.8
0.2
I
I
2.3
1.0
I
I
2.8
0.7
I
I
3.4
1.2
I
I
4.1
1.1
I
I
2.9
2.5
I

3-2.3.2. Within-Laboratory Reproducibility
In three independent runs within a single laboratory, the non-concordance of prediction of invitro eye irritation was occurred by chemicals of 6 test (Incidence rate: 6/96 test = 6.3%; Table
3-6). These chemicals belonged to the group classified no-category (no-eye irritant) in the GHS

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February, 2017classification (Incidence rate: 6/24 test= 25%).

3-2.3.3. Between-Laboratory Reproducibility
The results of ocular irritancy prediction of 4 data in 4 laboratories were not in agreement with
the GHS classification (Table 3-6).
These chemicals belonged to the group classified no-category (non-eye irritant) in the GHS
classification.

3-2.3.4. Predictive Capacity
Specificity of this protocol was not high (53/72 test=73.6%; Table 3-6).
Sensitivity of this protocol was 100% (216/216 test = 100%; Table 3-6).
Overall accuracy of this protocol was 93.4% (269/288 test = 93.4%; Table 3-6).

3-2.4. Discussion and Conclusion
It was thought that technical transferability of this EIT was easy from the results acceptancecriterion of positive control and negative control in this ring study.
From these results, it was found that both within- and between-laboratory reproducibility in the
LabCyte24 EIT were enough high.
It was also confirmed that the LabCyte24 EIT was the method which chemicals showing false
negative did not generate easily.
In spite of having been the test chemical selection which put weight on the negative detection
rate, some case showing false positive generated (19/72 test = 26.4%).
Because it was considered that the washing method for Solid or viscous chemicals (Tween20) caused these results, the opinion that modification of the LabCyte24 EIT protocol should beindispensable was offered from some participating laboratories.
From the above result, although it might be useful method to prediction of a non-eye irritantchemicals, it seemed that the intensity of eye irritation cannot be distinguishable with this EIT.

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3-3. Modification of LabCyte24 EIT(Pre-Validation Study Phase 01)
3-3.1. Purpose of This StudyIn the ring study (see the section 3.2), it was requested that protocol modification of
LabCyte24 EIT was distinguished for decrease variation and false-positive.
Study team was discussed about protocol modification. Variable widely of cell viability weresometime incidence at viscus chemicals or Solid chemicals because they might be remain on
the tissue after washing. On the other hands, because false-positive chemicals were often aborderline chemicals, it might be effective to change the cut-off value.
Finally, study team was accepted the modification of LabCyte24 EIT protocol as followings
points;1) The cut-off value of eye irritation:

From 50% to 40%.

2) Solid application amount:

From 50 mg to 10 mg.

3) Washing method:

From mild to more strong.

Then, in order to confirm the technical transferability, within- and between-laboratory
reproducibility of this modified EIT protocol, it was carried out the pre-validation study phase 01
by five laboratories. A purpose of this study is to examine whether the result of the modified
CORNEA-MODEL24 EIT method can achieve proficiency level of preset criteria. In this study, it
was set the assay criteria as follows;
1) SD of negative control, positive control and the test chemicals are ≤ 20%.
2) The dispersion of the cell viability by viscus chemical (Tween 20) is smaller than the ring
study (see the section 3.2).
3) The incidence rate of false positive is less than the ring study (see the section 3.2).

3-3.2. Study plan
3-3.2.1. Participating Laboratories
Five laboratories were participated in this study (Table 3-7).

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Table 3-7.

February, 2017

Participating laboratories.

Lab No.

Laboratory NameA

Mandom Corp.

B

Drug Safety Testing Center Co., LtdC

Fujifirm Corp.

D

Nihon Kolmar Co.,LtdE

OPPEN COSMETICS Co.

3-3.2.2. Selected Test Chemicals
Twelve test chemicals were selected in this study (Table 3-8).
Table 3-8.

Selected test chemicals.

Test chemical

CAS No.

Category

Physical state

GHS class

1-Butanol

71-36-3

Alcohols

Liquid13-Methoxy-1,2-propanediol

623-39-2

Alcohols

LiquidNC

2-Methyl-1-pentanol

105-30-6

Alcohols

Liquid2B

Glycerol

56-81-5

Polyols

LiquidNC

Tween 209005-64-5

Surfactants (nonionic)

LiquidNC

TritonX-100 (5%)

9002-93-1

Surfactants (nonionic)

Liquid24,4'-Methylenebis(2,6-di-tert-butylphenol)

118-82-1

Aromatics

Solid

NC

Sucrose Fatty Acid Ester

none

Surfactants (nonionic)

Solid

2

Silicic acid7699-41-4

Inorganics

Solid

NC

Phenothiazine

92-84-2

AminesSolidNC

3,3-Dithiodipropionic Acid

1119-62-6

Sulpha-containing compounds

Solid

2B

1-naphtalen acetic acid

86-87-3

Pesticides

Solid

2

3-3.2.3. Test Protocol
3-3.2.3a. Protocol for Liquid Chemical
Liquid chemicals (50 μL) were applied to three tissues. In addition, three tissues were treatedwith 50 μL of D-PBS serving as negative controls and ethanol serving as positive controls. And
then tissues applied chemicals were incubated for 1 minute. After exposure, each tissue wasstrongly washed with D-PBS ten times or more, using a washing bottle to remove any remaining
test chemical from the tissue surface. The blotted tissues were then transferred to new wells on
24-well plates containing 500 μL of fresh assay medium. And then the tissues were49 /121

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February, 2017

post-exposure incubated for 24 hours under the standard cultivation conditions (at 37°C in ahumidified atmosphere of 5% CO 2 in air). After the post-exposure incubation periods, blottedtissues were transferred to new wells on 24-well plates containing 300 μL of freshly prepared
WST-8 solution (1:10 dilution of Cell Counting Kit-8 with D-PBS) for a WST-8 assay. Tissues
were incubated for 5 hours under the standard cultivation conditions. Subsequently, 200 μL of
culture supernatant are transferred to a 96-well microtiter plate. The OD value of culture
supernatant was measured at 450 nm and at 650 nm as a reference absorbance, with WST-8
solution as a blank.
The tissue viability was calculated as a percentage relative to the viability of negative controls.
The mean of the three values from identically treated tissues was used to classify a chemicalaccording to the prediction model.

3-3.2.3b. Protocol for Solid Chemical
Solids (10 mg) in a microtube were applied by decantation on the tissue. Each test chemical
was applied to three tissues. In addition, three tissues serving as negative controls were set
apart as non-treated samples and Lauric acid (10mg) served as positive control. And thentissues are incubated for 24 hours. After exposure, each tissue was rinsed with D-PBS in thesame manner as that of Liquid chemicals. In the Solid chemicals, post-exposure incubation was
not set. After the rinsing, blotted tissues were analyzed for their cell viability through means of a
WST-8 assay, in the same manner as for Liquid chemicals.

3-3.2.3c. Assay Criteria
Only when all three following conditions were satisfied, the test result was accepted. .
Negative control

0.5 ≤ Mean of absorbance (450nm/650nm) ≤ 2.0

Positive control

Mean of cell viability ≤ 40%

SD

SD of viability of three RhCE tissues (negative control, positive
control and test chemical) ≤ 20 %3-3.2.3d. Prediction Model in This Study
If the mean cell viability was equal to or lower than 40%, the chemical was judged to be an
irritant, otherwise, it was considered a non-irritant.

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3-3.3. Results
3-3.3.1. Negative control and positive control
Fig. 3-4 shows the absorbance values for the negative control.

LiquidSolid

Fig.3-4. Results of negative control
All data for the negative control met the acceptance criteria of both OD (0.5 ≤ OD ≤ 2.0) andSD (≤ 20 %) and then the frequency of invalid test run for the negative control was 0%.
Table 3-5 shows the absorbance values for the positive control.
LiquidSolid

Fig.3-5. Results of positive control.

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February, 2017

All data for the positive control met the acceptance criteria of both cell viability (≤ 40%) and SD(≤ 20 %) and then the frequency of invalid test run for the positive control was 0%.
Table 3-9.

Prediction of three independent run

Test chemical

3-Methoxy1,2-propanediol

GlycerolTween 20

GHS

Lab

Class

run

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

Cell viability

57.4

48.5

55.3

88.6

84.5

120

47.9

41.5

95.4

65.8

62.5

104

40.7

41.2

70.3

SD

9.8

4.5

9.6

13.8

6.2

5.3

8.1

2.2

3

3.5

8.6

4.6

5.9

6.1

15.1

Judgement

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NC

NC

NC

4,4'-Methylenebis (2,6-di-tert-NCbutylphenol)
Silicic acid

Phenothiazine

TritonX-100(5%)
Sucrose Fatty
Acid Ester
1-naphtalen
acetic acid
2-Methyl-1pentanol

NC

NC

2

2

2

2B

3,3Dithiodipropionic

2B

Acid
1-Butanol

1

A

B

C

D

E

Cell viability

47

82

78.2

98.2

107

141

72.8

42.2

73.7

78.7

73.9

114

58.3

57.9

81.3

SD

2.5

1.5

11.2

5.3

2.8

18.8

16.1

13.4

18.3

12.1

14.9

6.1

13.7

6

5.6

Judgement

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

Cell viability

69.4

90.2

75

107

116

127

85.7

102

57.9

94.9

86.7

109

109

95.2

95.8
13

SD

3.8

5

0

5.9

3.6

10

19.6

16.6

15.8

1.9

5.7

2

5.1

8

Judgement

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

Cell viability

101

76.9

77.9

102

91.1

98.6

100

110

103

88.8

94.4

114

70

52.2

66.5

SD

2.6

7.3

6.8

8

3.1

9.9

9.6

5.5

6.6

1.8

2.1

6.6

4.6

1.7

3.7

Judgement

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

Cell viability

77

40.9

48.5

101

90.2

128

80.4

78.5

82.9

71

101

78.1

87.4

56.7

53.4
2.5

SD

2.6

10.4

5.6

13

4.9

7.8

7.6

7.2

6.6

2.9

9.8

17.3

6.9

7.5

Judgement

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

Cell viability

113

106

57

82

91.5

139

108

97.4

94.4

83

103

96.3

71.7

75

83.7
6.2

SD

9.5

6.7

9.4

8.8

5.3

8.6

6.3

11.7

6.8

6.4

3.5

16.4

4.7

8.9

Judgement

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

Cell viability

3.3

15.5

2.1

1.3

1.4

1.5

9.7

8.7

2

2.6

6.8

9.9

17.6

24.1

18.2
12.1

SD

0

1.5

0.9

0

0

0

7.7

3.4

0

0.6

5.9

3.4

6.4

6.5

Judgement

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Cell viability

20.7

26.3

9.4

16.9

16.7

23

14.4

18.3

14.7

15.8

25.1

38.1

10

11.2

13.9
1.4

SD

1.7

5.2

4.5

5.8

0.7

4.3

2.9

3.5

4.2

2.4

5

5.7

3.3

4

Judgement

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Cell viability

3.4

3.2

2.6

7.8

4.5

0

3.3

3.2

3.2

3.1

2.8

10

1.7

3

2.8

SD

0

0

0

3.6

1.4

0

0.8

0.6

0.7

0.7

0

1.1

0.8

0.6

1.4

Judgement

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Cell viability

27.3

21.1

14.9

22.8

33.6

7

27.6

24.7

23.7

14

18.9

21.9

26

19.3

30.2

SD

2.5

3.6

0.9

5.5

6.1

9.6

0

3.7

6.8

11

2.9

9.9

9.5

3

8.9

Judgement

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Cell viability

3.4

3.8

2.6

3.9

3.7

1.4

3.3

2.9

2.8

2.7

3.3

14.4

0.4

2.2

0.4
0.7

SD

0

0.9

0

4.8

3.2

0

0.8

1

0.7

0.7

0.8

1.1

0.8

1.1

Judgement

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Cell viability

4.9

2.6

4.3

15.4

16.8

10.6

5.1

1.7

7.2

5.9

18.2

17.9

4.4

4.4

11.5

SD

1.6

0.9

2.4

2

5.5

10.6

2.9

1.2

4.1

2.3

1.1

6.9

3.9

2

9

Judgement

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

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3-3.3.2. Within-Laboratory Reproducibility
In three independent runs within a single laboratory, the non-concordance of prediction of invitro eye irritation was not occurred (Table 3-9). Therefore, reproducibility of within-laboratory inthis study was 100% at the all participating laboratories.

3-3.3.3. Between-Laboratory Reproducibility
The rate of between-laboratory reproducibility in this study was 100% (Table 3-9).

Therefore,

all laboratories had high between-laboratory reproducibility.

3-3.3.4. Predictive capacity
In this study, prediction results at all laboratories were able to correctly identify eye irritation all
chemicals. Therefore, the sensitivity, specificity, and accuracy of this EIT protocol were all 100%
(Table 3-9).

3-3.3.5. Proficiency Level of Preset Criteria
This EIT method achieved the all criteria. Proficiency level was summarized in Table 3-10.
Table 3-10. Prediction of three independent run(A) Incidence of out of acceptance criteria (SD: > 20%)
Ring study (Section 4.2)

Pre-validation study 01

Negative control

0.7% (1/145 test)

0% (0/30 test)

Positive control

0% (0/145 test)

0% (0/30 test)

Test chemicals

0.5% (3/576 test)

0% (0/180 test)(B) The dispersion of the cell viability by Tween 20
Ring study (Section 4.2)

Pre-validation study 01

False positive

11.1% (2/18 test)

0% (0/30 test)

SD > 20%

11.1% (2/18 test)

0% (0/30 test)

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(Continue)(C) The dispersion of the cell viability by Tween 20
Ring study (Section 4.2)

Pre-validation study 01

6.3% (4/96 test)

0% (0/60 test)

26.4% (19/72 test)

0% (0/90 test)

Non concordance results in threedependen test
False positive

SD of negative control, positive control and the test chemicals were met assay criteria andincidence of invalid run was smaller than the ring study (Table 3-10),.
The dispersion of the cell viability by Tween 20 was smaller than the ring study results (Table
3-10).
The false positive was less than the ring study results (to see the Section 4.2).

3-3.4. Discussion and Conclusion
From these results, it was confirmed that both within- and between-laboratory reproducibilityby this EIT protocol were higher than the ring Study (see the Section 4.2).
Furthermore, it was also confirmed that the predictive performance in this EIT protocol werehigher than the ring study (see the Section 4.2).
However, it was confirmed that large dispersion of the cell viability sometimes occurred,especially it had varied widely the absorbance of negative control and then also the cell viabilityof the test chemicals of GHS no category. As the cause of such large dispersion, it wassuggested the possibility that the reaction of WST-8 assay was not stable among inter-test run.
Therefore it was assumed that reconsideration of WST-8 assay condition and comparative
examination between the WST-8 assay and the MTT assay might be necessary.

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3-4. Comparison study of MTT assay for the LabCyte24 EIT(Pre-Validation Study Phase-02, 03)
3-4.1. Purpose of This StudyIn the pre-validation study phase 01 (see the Section 3-3), large dispersion of the cell viability
was sometimes occurred, it was pointed out the possibility that WST-8 reaction is unstable as
their reason.
Therefore, it was requested that the comparison study of WST-8 assay and MTT assay for the
LabCyte24 EIT.
In this comparison study, it was confirmed as follow points;
1) The comparison of OD range of negative control between WST-8 assay and MTT assay.
2) The comparison of the prediction results of test chemicals between WST-8 assay and MTT
assay.

3-4.2. Study Plan
3-4.2.1. Participating Laboratories
Three laboratories were participated in this study (Table 3-11).
Table 3-11. Participating laboratories.

Lab No.

Laboratory NameB

Drug Safety Testing Center Co., LtdC

Fujifirm Corp.

D

Nihon Kolmar Co., Ltd

3-4.2.2. Selected Test Chemicals
As chemical shown bolder line or variable widely in the pre-validation study phase 01, four test
chemicals were selected in this comparison study (Table 3-12).

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Table 3-12. Selected test chemicals.

Test Chemical

CAS no.

Category

Physical state

GHS class

3-Methoxy-1,2-propanediol

623-39-2

Alcohols

LiquidNC

Glycerol

56-81-5

Polyols

LiquidNC

Sucrose Fatty Acid Ester-

Surfactants (nonionic)

Solid

2

Silicic acid7699-41-4

Inorganics

Solid

NC

3-4.2.3. Test Protocol
3-4.2.3a. Protocol for Liquid Chemical
Liquid chemicals (50 μL) were applied to three tissues. In addition, three tissues were treatedwith 50 μL of D-PBS serving as negative controls and ethanol serving as positive controls. And
then tissues applied chemicals were incubated for 1 minute. After exposure, each tissue wasstrongly washed with D-PBS ten times or more, using a washing bottle to remove any remaining
test chemical from the tissue surface. The blotted tissues were then transferred to new wells on
24-well plates containing 500 μL of fresh assay medium. And then the tissues were
post-exposure incubated for 24 hours under the standard cultivation conditions (at 37°C in ahumidified atmosphere of 5% CO 2 in air). After the post-exposure incubation periods, the cell
viability were determined by WST-8 assay or by MTT assay.
WST-8 assay
Blotted tissues were transferred to new wells on 24-well plates containing 300 μL of freshly
prepared WST-8 solution (1:10 dilution of Cell Counting Kit-8 with D-PBS) for a WST-8 assay.
Tissues were incubated for 5 hours under the standard cultivation conditions. Subsequently,
200 μL of culture supernatant were transferred to a 96-well microtiter plate. The OD value of
culture supernatant are measured at 450 nm and at 650 nm as a reference absorbance, with
WST-8 solution as a blank.
MTT assay
Tissues were put in the wells of 24-well plates containing 500 μL of MTT medium (0.5 mg/mL;
Dojindo Co., Japan) and were incubated for 3 hours (37°C, 5% CO2, humidified atmosphere).
MTT Formazan produced in the tissues was extracted with isopropanol (300 μL) and the extract(200 μL) was measured at 570 nm and at 650 nm as a reference absorbance, with isopropanol
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The tissue viability was calculated as a percentage relative to the viability of negative controls.
The mean of the three values from identically treated tissues was used to classify a chemicalaccording to the prediction model.

3-4.2.3b. Protocol for Solid Chemical
Solids (10 mg) in a microtube were applied by decantation on the tissue. Each test chemical
was applied to three tissues. In addition, three tissues serving as negative controls were set
apart as non-treated samples and Lauric acid (10 mg) served as positive control. And thentissues were incubated for 24 hours. After exposure, each tissue was rinsed with D-PBS in thesame manner as that of Liquid chemicals. In the Solid chemicals, post-exposure incubation was
not set. After the rinsing, blotted tissues were analyzed for their cell viability through means of a
WST-8 assay or MTT assay, in the same manner as for Liquid chemicals.

3-4.2.3c. Assay Criteria
Only when all three following conditions were satisfied, the test result was accepted. .
Negative control

0.5 ≤ Mean of absorbance (450nm/650nm) ≤ 2.0

Positive control

Mean of cell viability ≤ 40%

Standard Deviation (SD)SD of viability of three RhCE tissues (negative control, positive
control and test chemical) ≤ 20 %3-4.2.3d. Prediction Model in This Study
If the mean cell viability was equal to or lower than 40%, the chemical was judged to be an
irritant, otherwise, it was considered a non-irritant.

3-4.3. Results
3-4.3.1. Negative Control
Fig. 3-9 shows the OD for the negative control at three independent test run.

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Liquid negative control
WST-8 assay

MTT assay

Solid negative control
WST-8 assay

MTT assay

Fig.3-6. Results of negative control

All data for the negative control of both the WST-8 assay and the MTT assay met theacceptance criteria of OD (0.5 ≤ OD ≤ 2.0) and SD (OD ≤ 20%) (Fig. 3-6).
OD range of the negative control of WST-8 assay was between 0.6 and 1.4, on the other hand,that of MTT assay was between 0.5 and 1.2.
From these results, it was not apparent difference of OD value and variably between the
WST-8 assay and the MTT assay.

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3-4.3.2. Test chemicals
All data for the test chemicals of both the WST-8 assay and the MTT assay met theacceptance criteria of SD (OD ≤ 20%) (Fig. 3-7).
In the MTT assay, Sucrose Fatty Acid Ester which GHS Category 2 chemical was predicted as
false negative at all participating laboratories although results of two laboratories werenon-concordance between three independent run. Cell viability range of that chemical in the
MTT assay are between 28% and 100% and therefore it was resulted varied widely.
3-Methoxy-1,2-propanediol (GHS No category)
WST-8 assay

MTT assay

Glycerol (GHS No Category)
WST-8 assay

MTT assay

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(Continue)
Sucrose Fatty Acid Ester (GHS Category 2)
WST-8 assay

MTT assay

Silicic acid (GHS No category)
WST-8 assay

MTT assay

Fig.3-7. Results of Cell viability of test chemicals

Other three GHS No category chemicals (3-Methoxy-1,2-propanediol, Glycerol, and Silicicacid) was not apparent difference of their cell viability between the WST-8 assay and the MTT
assay (Fig.3-7).

3-4.4. Discussion and Conclusion
In this comparison study between MTT assay and WST-8 assay, the LabCyte24 EIT using
MTT assay was not decreased large dispersion of the cell viability to compare with WST-8

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assay.
On the other hands, the cell viability of Sucrose Fatty Acid Ester in the MTT assay were
resulted viability widely than WST-8 assay and produced false-negative prediction. Furthermore,
it was pointed out from participating laboratories that the operation of MTT assay was more
complex than WST-8 assay.
From these results, it was concluded that it was distinguished the optimization study of WST-8
assay condition but not changing MTT assay

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3-5. Optimization Study for WST-8 Assay Condition
3-5.1. Purpose of This StudyIn the WST-8 assay in the LabCyte24 EIT, the OD of negative control has been sometimes
occulated variable widely or low OD (see below the Sections 3-2, 3-3 and 3-4). In order to solve
above these problems, the optimization of WST-8 assay condition was indispensable.
In the study 1, pH of several buffers or assay medium as the candidate of WST-8 assay
solution was evaluated after incubation at the standard cultivation condition.
In the study 2, WST-8 assay were performed using several buffers or assay medium in order
to select of optimal buffer for the WST-8 assay.
In the Study 3, WST-8 assay periods was examined by time course experiments using
selected buffer in the Study 2.

3-5.2. Study plan
3-5.2.1. Study 1
In order to prefer optimizing WST-8 reaction condition, it was used D-PBS, Hanks' Balanced
Salt Solution (HBSS), HBSS without calcium and magnesium (HBSS(-)), EBSS, and EBSS
without calcium and magnesium (EBSS(-)) as the buffer of WST-8 reaction. All buffers were notcontained phenol red. Each buffer was incubated at 37°C in a humidified atmosphere with orwithout 5% CO2 in air for 1 hour. After the incubation periods, pH of each buffer was measured
after incubation.

3-5.2.2. Study 2
WST-8 reaction buffer was prepared using each buffer (D-PBS, HBSS, HBSS (-), EBSS, andEBSS (-)). Each WST-8 solution was incubated under the cultivation conditions at 37°C in ahumidified atmosphere and 5% CO2 in air for 5 hours. After the incubation, the OD of each
WST-8 reaction solution was measured. Levels and variabilities of OD of between buffers were
evaluated.

3-5.2.3. Study 3
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WST-8 reaction buffer was prepared using each buffer (HBSS and EBSS). Both WST-8
solutions were incubated under the cultivation conditions at 37°C in a humidified atmosphereand no or 5% CO2 in air for 1, 3 or 5 hours. After the incubation, the OD of each WST-8 reaction
solution was measured. Levels and variabilities of OD of between buffers were compared.

3-5.3. Results
3-5.3.1. Study 1
From various buffers, it was selected five buffers (D-PBS, HBSS, HBSS(-), EBSS andEBSS(-)) often used for the cell washing and short incubation, it was measured pH of buffers
made in Gibco, Sigma and Wako. In addition, pH of various buffers were measured after 0.5 or
1 hour incubation under 37°C in a humidified atmosphere with or without 5% CO 2 in air. In
HBSS and HBSS(-), pH of all makers were around 7.5 at room temperature and when they wereincubated for 1 hour under 37°C, pH of these rose to around 8.2. While, when they wereincubated for 1 hour under 37°C with 5% CO2, pH of these fell to around 7.0.
Table 3-13. PH value of various buffers after the cultivation under 37°C in a humidified
atmosphere with or without 5% CO2
Culture condition

D-PBS(-)

HBSS

HBSS(-)EBSS

EBSS(-)

Assay
mediumPeriod
Gibco

Sigma

Wako

Gibco

Sigma

Wako

Gibco

Sigma

Wako

Sigma

Gibco

J-TEC

(hour)
RT

0

7.11

2)

7.14

7.42

7.53

7.21

7.43

7.74

7.48

7.51

7.41

7.84

---

37℃
1)
(ha )

0.5

7.12

7.18

7.37

7.97

7.96

8.09

8.27

8.02

8.07

8.30

8.52

8.22

1

7.12

7.13

7.37

8.14

8.23

8.30

8.31

8.43

8.25

8.61

8.84

8.62

37℃
(ha)
5%CO2

0.5

6.79

6.83

6.96

7.08

6.97

7.09

7.14

7.12

7.16

7.66

7.86

7.80

1

6.70

6.71

6.80

6.96

6.90

7.00

7.11

7.01

7.04

7.69

7.85

7.84

1) ha: humidified atmosphere

2) pH

In D-PBS, pH of all makers were 7.1 to 7.4 at room temperature and when they wereincubated for 1 hour under 37°C, pH of these were almost no change. While, when they wereincubated for 1 hour under 37°C with 5% CO2, pH of these degreased about 0.4.
PH of EBSS and EBSS(-) were 7.4 and 7.8 at room temperature and when they wereincubated for 1 hour under 37°C, both rose to about 8.6 and 8.8. While, when they were

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incubated for 1 hour under 37°C with 5% CO2, pH of these hardly changed in 7.7 and 7.8. When
LabCyte assay medium was incubated for 1 hour under 37°C with 5% CO2, pH of this medium
was around 7.8 and was nearly the same as these of EBSS and EBSS(-) under 37°C with 5%
CO2. There was little maker difference in pH of various buffers, but we decided to use productsof Gibco with precedence.

3-5.3.2. Study 2
Each OD values of D-PBS (-) and HBSS were around 0.8 after WST-8 reaction for 5 hours
under 37°C in a humidified atmosphere with 5% CO 2. OD values of EBSS and EBSS(-) wereabout 1.3 and 1.2 respectively, and OD values of EBSS had a slightly higher absorbance thanthat of EBSS(-) in a humidified atmosphere. However, both buffers caused a large increase in
OD value as compared with D-PBS and HBSS. While, OD value of assay medium was about
1.3, and was almost same as EBSS.
Table 3-14. Effect of various buffer on OD value after WST-8 reaction for 5 hours under
37°C in a humidified atmosphere with 5% CO2
OD after blank correction (450nm-650nm)

Buffer or

Blank OD

medium(450-650)nm

well 1

well 2well 3Mean

SD (%)

CV(%)

1

D-PBS

0.03100.8176

0.89890.75980.8254

6.99

8.47

2

HBSS

0.03700.82900.8936

0.88130.8680

3.43

3.95

3

EBSS

0.0604

1.38591.23671.31301.3119

7.46

5.69

4

EBSS(-)

0.0774

1.2375

1.17201.2412

1.2169

3.89

3.20

5

Medium0.37631.30371.20351.40971.3056

10.31

7.90

No.

Medium: Assay medium

3-5.3.3. Study 3
Because the difference between EBSS and EBSS (-) in component was only the thingwhether or not calcium and magnesium were included, and the values of pH and OD of WST-8
in two buffers was almost same in Study1 and 2, we predicted that the similar value will go out
whichever buffer is selected in Study 3. So there is no decisive factor, we selected EBSS withhesitancy because EBSS had an absorbance a slightly higher that of EBSS(-).
In Study 3, we focused on two buffers of HBSS and EBSS and measured OD with64 /121

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February, 2017

time-dependent manner in reaction (1, 3, 5 hours). Moreover, we measured about LabCyte
assay medium in the same manner.
OD of HBSS increased with time-dependent manner in WST-8 reaction under 37°C in ahumidified atmosphere with 5% CO2 and was about 0.8 at 5 hours reaction. The results wererecognized reproducibility with Study 2. OD of EBSS increased with time-dependent manner in
WST-8 reaction under 37°C in a humidified atmosphere with 5% CO2 and OD was about 1.4 at
5 hours reaction, the results was recognized reproducibility with Study 2. OD of EBSS greatly
increased than that of HBSS. OD of the LabCyte assay medium was about 1.3.
Table 3-15. Change of OD values with time-dependent manner in the WST-8 reaction under
37°C in a humidified atmosphere with 5% CO2
OD after blank correction (450-650)nm

WST-8
Buffer orNo.

Blank OD
incubation time

medium(450-650)nm(hours)1-1SDwell 1

Well 2well 3

Mean

CV(%)
(%)

1

0.031

0.097

0.118

0.132

0.116

1.76

15.23

3

0.035

0.455

0.550

0.451

0.485

5.60

11.55

1-3

5

0.034

0.806

0.889

0.814

0.836

4.58

5.47

2-1

1

0.043

0.164

0.174

0.175

0.171

0.61

3.56

3

0.056

0.814

0.888

0.882

0.861

4.11

4.77

5

0.067

1.383

1.386

1.389

1.386

0.30

0.22

1

0.306

0.119

0.108

0.114

0.114

0.55

4.85

3

0.392

0.621

0.710

0.703

0.678

4.95

7.30

5

0.476

1.224

1.280

1.264

1.256

2.88

2.30

1-2

2-2

HBSS

EBSS

2-3
3-1
Assay
3-2
medium3-3

It was thought that Incubation period of WST-8 assay were suitable 4 hours which OD mightreach around 1.0, because OD of EBSS was 0.86 for 3 hours incubation and OD was 1.39 for 5
hours incubation.

3-5.4. Discussion and Conclusion
Reaction buffer of WST-8 assay were usually used suitable medium. However phenol-red or

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other factor contained in the assay medium might be affected blank OD value to rise, D-PBS
were selected as WST-8 reaction buffer initially.
After the incubation of assay medium at 37°C with 5% CO2 for 1 hour, pH of assay medium
was around 7.8, which was similar to that of EBSS at same incubation condition. NaHCO 3
concentration of EBSS is almost the same to assay medium and then it was contributed tostabilization of pH of EBSS under 37°C with 5% CO2. On the other hands, pH of HBSS after the
incubation at 37°C with 5% CO2 for 1 hour was around 7.0, which was lower than that of EBSSor assay medium.
It was thought that it was resulted to lower pH value by CO2 because concentration of
NaHCO3 of HBSS was low with about 1/8 of that of EBSS.
Also, under 37°C with 5% CO2, pH of D-PBS was around 6.7 to 6.8, which was very lower tocompare with that of the assay medium.

From the results, the lower OD level and its wide

variably were sometimes happened in cultivation with D-PBS. Therefore it was thought that
D-PBS might be not appropriate as a solvent of WST-8 assay, not the most suitable cultivation
condition from these results.
On the other hands, because pH of EBSS was stable around 7.7 to similar to that of assay
medium, WST-8 reaction were linearly progressed in the good condition for RhCE tissue andtherefore level of OD value were increased similar level for that of assay medium.
Finally it was confirmed that EBSS is suitable buffer solution of WST-8 assay for viablecondition of the RhCE tissue and reaction period of WST-8 assay was set 4 hours from resultsof study 3.

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DISCRIPTION OF THE IMPROVED LabCyte24 EIT

4-1. Overview of LabCyte the Improved CORNEA-MODEL24 EIT Procedure
Prediction of the eye irritation potential of test chemicals by the improved LabCyte24 EIT was
performed according to the improved protocol described in the SOP for ver.2.5.2 (Appendix 1).
The LabCyte CORNEA-MODEL24 tissues is shipped from the supplier on Mondays anddelivered to recipients on Tuesdays or Wednesdays. Upon receipt, the tissues are asepticallyremoved from the transport agarose medium, transfer into wells on 24-well plates (BDBiosciences, CA, USA) with the assay medium (0.5 mL) and pre-incubated overnight at 37°C in
a humidified atmosphere of 5 % CO 2 in air (standard cultivation conditions).

4-1.1. The Improved LabCyte24 EIT Procedure for Liquid Chemicals
After the pre-incubation, the tissue is topically exposed to the Liquid test chemicals. Liquids(50 μL) are applied with a micropipette. Viscous Liquids are applied using a positive
displacement-type tip with a micropipette, such as the MICROMAN® (Gilson inc., France). Eachtest chemical is applied to three tissues. In addition, three tissues are treated with 50 μL of
D-PBS serving as negative controls and ethanol serving as positive controls. And then tissues
applied chemicals are incubated for 1 minute. After exposure, each tissue is rinsed with D-PBS
ten times or more, using a washing bottle to remove any remaining test chemical from the tissue
surface.

The blotted tissues are then transferred to new wells on 24-well plates containing 500

μL of fresh assay medium. And then the tissues are post-incubated for 24 hours under the
standard cultivation conditions (at 37°C in a humidified atmosphere of 5% CO 2 in air). After the
post-exposure incubation periods, blotted tissues are transferred to new wells on 24-well plates
containing 300 μL of freshly prepared WST-8 solution for a WST-8 assay (Ishiyama et. al.,
1997; Tominaga et. al., 1999). WST-8 solution is prepared by 1:10 dilution of Cell Counting Kit-8(Dojindo Co., Japan) with EBSS (Sigma-Aldrich, MO, USA).

Tissues are incubated for 4 hours

under the standard cultivation conditions (at 37°C in a humidified atmosphere of 5% CO 2 in air).
Subsequently, 200 μL of culture supernatant are transferred to a 96-well microtiter plate. The
OD value of culture supernatant is measured at 450 nm and at 650 nm as a reference
absorbance, with WST-8 solution as a blank.

Cell Viability (%)

OD of the test chemical
OD of negative control

x 100

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The tissue viability is calculated as a percentage relative to the viability of negative controls.
The mean of the three values from identically treated tissues is used to classify a chemicalaccording to the prediction model.

4-1.2. The Improved LabCyte24 EIT Procedure for Solid Chemicals
After pre-incubation, the medium is changed to 500 μL of fresh assay medium and then thetissues are topically exposed to the Solid test chemicals. Solids (10 mg) in a microtube areapplied by decantation. Each test chemical is applied to three tissues. In addition, three tissues
serving as negative controls are set apart as non-treated samples and Lauric acid (10mg)
served as positive control. And then tissues are incubated for 24 hours. After exposure, eachtissue is rinsed with D-PBS in the same manner as that of Liquid chemicals. In the Solidchemicals, post-exposure incubation is not set. After the rinsing, blotted tissues are analyzed fortheir cell viability through means of a WST-8 assay, in the same manner as for liquid chemicals.

4-1.3. Prediction Model of the Improved LabCyte24 EIT
As a reference for the in vivo eye irritancy classification of each chemical, we used the UN
GHS classification (United Nations, 2003).
According to the UN GHS classification (No Category, Category 2 or Category 1), an irritant(Category 1 or 2) is predicted if the mean relative tissue viability of three individual tissues
exposed to the test chemical is reduced below 40% of the mean viability of the negative controls(Table 4-1).
Table 4-1.

Prediction model of LabCyte24 EIT

In vitro results

Prediction of eye irritation(UN GHS classification)

Tissue viability is ≤ 40%

Category 1 or 2

Tissue viability is > 40%

No category

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February, 20174-2. Description and Rationale for the Test Components of the Improved
LabCyte24 EIT
4-2.1. Pre-Exposure Incubation
In the LabCyte24 EIT, the tissue is incubated for 15-30 hours before test chemical exposure.
On the other hand, in the OECD TG 492 (OECD, 2015a), before exposure to test chemicals,
the tissue surface of the VRM is pretreated with 20 μL of D-PBS and incubated in the dark atstandard culture conditions for 30 minutes to mimic the wetting conditions of human eye(pre-wetting treatment). The LabCyte24 EIT is not set such as pre-wetting treatment for not tobe necessary, because the surface of LabCyte CORNEA-MODEL24 tissue has already beenwetting condition for represent of corneal surface as shown below to the Section 2.2.

4-2.2. Duration of Chemical Exposure
4-2.2.1. Amount of Chemical Exposure
Exposure volume for Liquid chemical is set 50 μL and on the other hand the amount of Solidchemical is set 10 mg (Table 4-2).
Table 4-2.

Amount of chemical application

Liquid chemical

Solid chemicals

50 μL

10 mg

LabCyte24 EIT, application amounts of both Liquid chemicals and Solid chemicals have been
set up as sufficient amounts to cover the entire surface of LabCyte CORNEA-MODEL24 tissue.

4-2.2.2. Exposure Periods of Chemicals and Post-exposure incubation Period
Exposure period of test chemicals and post-exposure incubation period for Liquid test
chemicals are set 1 minute and 24 hours, respectively (Table 4-3). On the other hands,
exposure period for Solid test chemicals is 24 hours and post-exposure incubation periods is not

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set (Table 4-3). These have been set throughout optimization study of LabCyte24 EIT as
described below in the Section 3-1 and also the previous publication report (Katoh et. al, 2013).
Table 4-3.

Exposure Periods of Chemicals and Post-exposure incubation Period
Physical state of chemicals

Process of exposure

LiquidSolid

Chemical exposure periods

1 minuets

24 hours

Post-exposure incubation periods

24 hours

Not set4-2.3. RhCE Tissue Replication
In the LabCyte24 EIT, the tissue is used each test chemical and each control substance ineach run, respectively.

4-2.4. Endpoint(s)As described below in Section 1, the LabCyte24 EIT was based on the cytotoxicity of RhCE
tissue because the corneal epithelial tissues is one of the main target during accidental eye
exposures, and damage to the corneal epithelial tissue can results in visual impairment or loss.
In the LabCyte24 EIT, cytotoxicity is determined by the viability of the RhCE tissue. The viabilityof tissues is conventionally measured by WST-8 assay that is one of the tetrazolium reduction
assay.

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4-2.5. Appropriate Positive Control and Negative Control
4-2.5.1. Negative Control
Negative control is set for Liquid chemicals and Solid chemicals, respectively, because they
are different assay condition between Liquid and Solid.
D-PBS is used as negative control for Liquid chemical. D-PBS is widely-used as negative
control for the many cytotoxicity assays (Table 4-4).
Table 4-4.

Negative control

Liquid

Solid

D-PBS

No application

For Solid chemicals, none treatment is set as negative control. None treatment is also set as
negative control for the many cytotoxicity assays (Table 4-4).

4-2.5.2. Positive Control
Positive control is set for Liquid chemicals and Solid chemicals, respectively, because theirassay conditions are different between Liquid and Solid.
Positive control for Liquid chemicals is used ethanol (Table 4-5).
Table 4-5.

Chemical of positive control
LiquidSolid

Ethanol

Lauric acid

(CAS no.: 64-17-5)(CAS no.: 143-07-7)

GHS: Cat 2A

GHS: Cat 1

Positive control for Solid chemicals is Lauric acid (Table 4-5).

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4-2.6. Acceptance Criteria
4-2.6.1. Negative Control
In the OECD TG 492, it is described that the acceptability range (upper and lower limit) for thenegative control OD values (in the test method conditions) should be established by the RhCE
tissue construct developer/supplier. As an example, the acceptability ranges for the negative
control OD values for the LabCyte24 EIT given Table 4-6.
Table 4-6.

Acceptability ranges for negative control

Lower acceptance limit

Upper acceptance limit

0.5 <

< 1.3

4-2.6.2. Positive Control
In the OECD TG 492, it is described that the acceptability range for the positive control cell
viability should be established by the RhCE tissue construct developer/supplier. As an example,
the acceptability ranges for the negative control OD values for the LabCyte24 EIT given Table
4-7
Table 4-7.

Acceptability ranges for positive control

Liquid chemical

Solid chemicals< 40%

4-2.6.3. Cell Variability
Cell variability in the LabCyte24 EIT has been evaluated by calculated the mean±SD of theviability values with triplicate tissue performed is less than 20% as shown Table 4-8. If the SD ofcell viabilities is more than 20%, an additional assay should be performed.
Table 4-8.

Acceptability SD limit

Negative control

Positive control

Test chemicals< 20%

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4-2.7. Applicability Domain and Limitation
One limitation of the RhCE EIT method is that it does not allow discrimination between eye
irritation/reversible

effects

on

the

eye

(UN

GHS

Category2)

andseriouseyedamage/irreversible effects on the eye (UN GHS Category 1), nor between eye irritants (UNGHS optional Category 2A) and mild eye irritants (UN GHS optional Category 2B), as definedby UN GHS (UN, 2003). For these purposes, further testing with other suitable test methods isrequired.
Other limitation of this assay method is a possible interference of the test chemical with the
WST-8 endpoint. A colored test chemical or one that directly reduces WST-8 (and thereby
mimics dehydrogenase activity of the cellular mitochondria) may interfere with the WST-8
endpoint. However, these test chemical are a problem only if at the time of the WST-8 test
sufficient amounts of the test chemical are still present on (or in) the tissues. In case of thisunlikely event, the (true) metabolic WST-8 reduction and the contribution by a colored test
material or (false) direct WST-8 reduction by the test material can be quantified using
freeze-killed RhCE tissues.
LabCyte24 EIT is applicable to substances and mixtures, and to solids, liquids, semi-solids
and waxes as same applicability to OECD TG 492 VRM (OECD, 2015a). The liquids may beaqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible,
solids should be ground to a fine powder before application; no other pre-treatment of thesample is required. Gases and aerosols have not been assessed in a validation study of VRM of
OECD TG 492. While it is conceivable that these can be tested using RhCE technology, theOECD TG 492 does not allow testing of gases and aerosols.

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5. EVALUATION OF THE PREDICTIVE PERFORMANCE OF THE IMPROVED
LabCyte24 EIT USING WIDE-RANGE OF TEST CHEMICALS
5-1. Purpose
In this study, we aimed to estimate the predictive performance of the improved LabCyte24 EIT
method using 139 test chemicals over a wide range of chemical classes.

5-2. Materials and Methods
5-2.1. Chemical Selection of Wide-Range Classes
139 test chemicals over a wide range of classes were used for evaluation of the improved
LabCyte24 EIT, as shown in Table 5-1. These chemicals were referred to in the following
reports.
ECETOC database. (ECETOC, 1998)
Publicly-available documents (ICCVAM, 2006; OECD, 2015b)
Published report (Doucet et. al, 2006; Takahashi et. al, 2008; Takahashi et. al, 2009; Kaluzhny
et. al, 2011; Kolle et. al, 2011)
Available information on irritation potential and classification according to the UN GHS
systems are provided in Table 5-1, Table 5-2 and Table 5-3.

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Table 5-1.

Wide

range

of

the

test

February, 2017

chemicalsforevaluation

of

the

LabCyteCORNEA-MODEL24 SIT
ChemicalNo

Name

CAS number

1)

Category

Physical
state

GHS
class

1

Hydroxyethyl acrylate

818-61-1

Acrylate; Alcohol

Liquid1

2

Tetraethylene glycol diacrylate

17831-71-9

Acrylate; Ether

Liquid1

31-Butanol

71-36-3

AlcoholLiquid1

4

Cyclohexanol

108-93-0

AlcoholLiquid1

5

Benzyl alcohol

100-51-6

AlcoholLiquid16(Ethylenediamine-propyl)-trimethoxysilane

1760-24-3

Aliphatic amine, primary; Aliphatic amine,secondary; Alkoxy silane

Liquid1

7

Monoethanolamine

141-43-5

AlkanolAmine

Liquid18Diethylethanolamine

100-37-8

Amine

Liquid19Lactic acid50-21-5

Carboxilic acid

Liquid1

10 Acetic acid64-19-7

Carboxilic acid

Liquid1

11 Methylthioglycolate

2365-48-2

Carboxy lic acid ester; Thioalcohol

Liquid1

12 Methoxyethyl acrylate

3121-61-7

Ester

Liquid1

13 Imidazole(20%)

288-32-4

Hydrocarbone

Liquid1

14 Pyridine

110-86-1

N-containing heterocycle

Liquid1

15 Di(2-ethylhexyl) sodium sulfosuccinate （10%）

577-11-7

Surfactant (anionic)

Liquid1

16 Potassium laurate（10%）

10124-65-9

Surfactant (anionic)

Liquid1

17 Cethylpyridinium bromide (6%)

140-72-7

Surfactant (cationic)

Liquid1

18 Benzalkonium chloride (10%)

8001-54-5

Surfactant (cationic)

Liquid1

19 Domiphen bromide（10%）

538-71-6

Surfactant (cationic)

Liquid1

20 Cethylpyridinium chloride（10%）

6004-24-6

Surfactant (cationic)

Liquid1

21 Stearyltrimethylammonium chloride（10%）

112-03-8

Surfactant (cationic)

Liquid1

22 Cethyltrimethylammonium bromide（10%）

57-09-0

Surfactant (cationic)

Liquid1

23 2,5-Dimethyl-2,5-hexanediol

110-03-2

AlcoholSolid127344-41-8

Alkene; Bipheny l; Sulf onic acid

Solid

1

25 m-Phenylene diamine

108-45-2

Amine

Solid

1

26 Quinacrine

69-05-6

Amine

Solid

1

27 Chlorhexidine

55-56-1

Amine

Solid

1

65558-69-2

Amine

Solid

1

24

Disodium 2,2'-([1,1'-bipheny l]-4,4'-diy ldiv iny lene)bis(benzenesulphonate)

28 1,3-Diiminobenz[f]isoindoline

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(Continue)
ChemicalCategory

Physical
state

GHS
class

2634-33-5

Benzo-thiazolinone; Benzo-isothiazolinone

Solid

1

30 Paraformaldehyde

30525-89-4

Ether

Solid

1

31 Lauric acid143-07-7

Fatty acid

Solid

1

32 Imidazole

288-32-4

Hydrocarbone

Solid

1

33 Tetraoctylammonium bromide

14866-33-2

Hydrocarbone

Solid

1

34 Captan 90-concentrate

133-06-2

Imide

Solid

1

35 Dibenzoyl-L-tartaric acid2743-38-6

Organic acid

Solid

1

36 2,4-Dihydroxybenzoic acid89-86-1

Organic acid

Solid

1

37 Sodium salicylate

54-21-7

Organic salt

Solid

1

38 Promethazine hydrochloride

58-33-3

Organic salt

Solid

1

39 Sodium oxalate

62-76-0Oxocarboxylic acid

Solid

1

40 1-naphtalen acetic acid

86-87-3

Pesticide

Solid

1

41 p-t-Butylphenol

98-54-4

PhenolSolid

1

42 Benzalkonium chloride (1%)

8001-54-5

Surfactant (cationic)

Liquid2

43 Diisopropanolamine

110-97-4

AlkanolAmine

Solid

2

44 Glycolic acid79-14-1

Carboxilic acid

Solid

2

45 Sodium Perborate Tetrahydrate

10486-00-7

Inorganic salt

Solid

2

46 Calcium thioglycollate

814-71-1

Organic salt

Solid

2

47 Ethanol

64-17-5

AlcoholLiquid2A

48 Isopropylalcohol

67-63-0

AlcoholLiquid2A

49 2-Benzyloxyethanol622-08-2

AlcoholLiquid2A

50 1-Octanol

111-87-5

AlcoholLiquid2A

51 2-Ethyl-1-hexanol

104-76-7

AlcoholLiquid2A

52 n-Hexanol

111-27-3

AlcoholLiquid2A

53 Isobutanol

78-83-1

AlcoholLiquid2A

18472-51-0

Aromatic heterocyclic halide; Aryl halide;
Dihydroxyl group; Guanidine

Liquid2A

55 Acetone

67-64-1

KetoneLiquid2A

56 Methyl ethyl ketone

78-93-3

KetoneLiquid

2A

No

Name

CAS number

29 1,2-Benzisothiazol-3(2H)-one

54

2,4,11,13-Tetraazatetradecane-diimidamide, N,N''-bis(4-chlorophenyl)3,12-diimino-, di-D-gluconate (20%, aqueous)1)

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(Continue)
ChemicalNo

Name

CAS number

1)

Category

Physical
state

GHS
class

57 gamma-Butyrolactone

21645-51-2

Lactone; Oxolane; Saturated heterocyclic fragment

Liquid2A

58 Sodium dodecyl sulfate(15%)

151-21-3

Surfactant (anionic)

Liquid2A

59 Cethylpyridinium bromide (1%)

140-72-7

Surfactant (cationic)

Liquid2A

60 Triton X-100 (5%)

9002-93-1

Surfactant (nonionic)

Liquid2A

61 Sodium benzoate

532-32-1Aryl; Carboxylic acid

Solid

2A

62 1,5-Naphthalenediol

83-56-7

Fused carbocyclic aromatic; Naphthalene;
PhenolSolid

2A

63 p-Formylbenzoic acid619-66-9

Organic acid

Solid

2A

64 2-Methyl-1-pentanol

105-30-6

Alcohol; Alkane, branched withtertiary carbon

Liquid2B

65 Diethyl toluamide

134-62-3

Benzamide

Liquid2B

66 Methyl acetate79-20-9

Ester

Liquid2B

67 Sodium hydroxide (1%)

1310-73-2

Inorganic baseLiquid2B

68 2,2-Dimethyl-3-methylenebicyclo [2.2.1] heptane

79-92-5

Bicycloheptane

Solid

2B

69 1,4-Dibutoxy benzene

104-36-9

Alkoxy; Aryl; Ether

Solid

2B

70 2,6-Dichloro-5-f luoro-beta-oxo-3-py ridinepropanoate

96568-04-6

Amine

Solid

2B

71 Maneb12427-38-2

Amine

Solid

2B

72 Ammonium Nitrate

6484-52-2

Inorganic salt

Solid

2B

73 m-Dinitrobenzene

99-65-0

Nitro compound

Solid

2B

74 p-Nitrobenzoic acid

62-23-7

Organic acid

Solid

2B

75 Sodium chloroacetate

3926-62-3

Organic salt

Solid

2B

76 3,3-Dithiodipropionic Acid

1119-62-6

Sulphur-containing compound

Solid

2B

77 Poly ethy lene gly col (PEG-40) hy drogenated castor oil

61788-85-0

Acy lal; Alcohol; Ally l; Ether

Liquid(Viscous)NC

78 Propylene glycol

57-55-6

AlcoholLiquidNC

79 3-Methoxy-1,2-propanediol

623-39-2

AlcoholLiquidNC

80 Ethanol (10%)

64-17-5

AlcoholLiquidNC

81 3-Phenoxybenzyl alcohol

13826-35-2

Alcohol; Benzyl; Ether

LiquidNC

82 4-(Methylthio)-benzaldehyde

3446-89-7

Aldehyde; Aryl; Sulfide

LiquidNC

83 3,3-Dimethylpentane

562-49-2

AlkaneLiquidNC

84 Triethanolamine

102-71-6

AlkanolAmine

LiquidNC

Alkane, branched with tertiary carbon; Alkene;

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(Continue)
ChemicalCategory

Physical
state

GHS
class

342573-75-5

Alkoxy; Ammonium salt; Aryl; Imidazole; Sulphate

LiquidNC

86 Piperonyl butoxide51-03-6

Alkoxy ; Benzodioxole; Benzy l;
Ether

LiquidNC

87 n-Octyl Ether

629-82-3

Alkoxy ; Ether

LiquidNC

88 2-Ethoxyethyl methacrylate

2370-63-0

Alkoxy ; Ether; Methacry lateLiquidNC

89 Xylene

1330-20-7

Aromatic

LiquidNC

90 1,6-Dibromohexane

629-03-8

Brominated Derivative

LiquidNC

91 1-bromo-4chlorobutane

6940-78-9

Brominated Derivative

LiquidNC

92 Ethyl thioglycolate

623-51-8

Carboxy lic acid ester; Thioalcohol

LiquidNC

93 Methyl cycropentane

96-37-7

Cycloalkane

LiquidNC

94 Dipropyl disulphide629-19-6

Disulfide

LiquidNC

95 Isopropyl myristate110-27-0

Ester

LiquidNC

96 Ethyl acetate141-78-6

Ester

LiquidNC

97 Toluene

108-88-3

Hydrocarbone

LiquidNC

98 2-Methylpentane

107-83-5

Hydrocarbone

LiquidNC

99 n-Octyl bromide

111-83-1

Hydrocarbone

LiquidNC

100 cis-Cyclooctene

931-87-3

Hydrocarbone

LiquidNC

101 Dodecane112-40-3

Hydrocarbone

LiquidNC

102 Styrene

100-42-5

Hydrocarbone

LiquidNC

103 Methyl isobutyl ketone108-10-1

KetoneLiquidNC

104 2-Heptanone(Methyl amylketone)

110-43-0

KetoneLiquidNC

105 2-Ethylhexyl p-dimethylamino benzoate

21245-02-3

PABA derivatives

LiquidNC56-81-5

PolyolLiquidNC

107 Polyethylene glycol 400

25322-68-3

PolyolLiquidNC

108 Sodium N-lauroyl sarcosinate (30%)

137-16-6

Surfactant (anionic)

LiquidNC

109 Sodium dodecyl sulfate(1%)

151-21-3

Surfactant (anionic)

LiquidNC

110 Benzalkonium chloride (0.01%)

8001-54-5

Surfactant (cationic)

LiquidNC

111 Cethylpyridinium bromide (0.1%)

140-72-7

Surfactant (cationic)

LiquidNC

112 Benzalkonium chloride (0.1%)

8001-54-5

Surfactant (cationic)

Liquid

NC

No

Name

85 1-Ethyl-3-methylimidazolium ethylsulphate

106 Glycerol

CAS number

1)

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ChemicalNo

Name

CAS number

1)

Category

Physical
state

GHS
class

113 Tw een20

9005-64-5

Surfactant (nonionic)

LiquidNC

114 Tw een80

9005-65-6

Surfactant (nonionic)

LiquidNC

115 Polyethylene glycol monolaurate (10E.O.)

31943-11-0

Surfactant (nonionic)

LiquidNC

116 Tw een80(10％)

9005-65-6

Surfactant (nonionic)

LiquidNC

117 3,4-dimethoxy benzaldehyde

120-14-9

Aldehy de; Ary l; Ether

Solid

NC

68610-92-4

Alcohol; Ammonium salt; Ether

Solid

NC

3179-89-3

Alcohol; Aromatic amine; Azo;
Nitrobenzene

Solid

NC

120 tetramethy lbuty l)-phenol)

103597-45-1

Alkane branched with quaternarycarbonSolidNC

121 2-Mercaptopyrimidine

1450-85-7

Amine

Solid

NC

122 Tetraaminopyrimidine sulfate

5392-28-9

Amine

Solid

NC

Ethy lenediamine-tetraacetic acid dipotassium salt
123 dihy drate

25102-12-9

Amine

Solid

NC

124 Phenothiazine

92-84-2

Amine

Solid

NC

125 Het Anhydride

115-27-5

Anhydryde

Solid

NC

126 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl) urea

101-20-2

Aromatic heterocyclic halide; Aryl halide;
Urea derivatives

Solid

NC

127 4,4'-Methylene bis-(2,6-di-tert-butylphenol)

118-82-1

Benzyl; Phenol; tert-Butyl

Solid

NC

Trisodium mono-(5-(1,2-dihy droxy ethy l)-4-oxido-2-oxo128 2,5-dihy dro-f uran-3-y l) phosphate

66170-10-3

Dihydroxyl group; Enol; Furanone;
Furanondione; Phosphate ester

Solid

NC

129 D(+)-Glucono-1,5-lactone

90-80-2

Ester

Solid

NC

130 Propyl p-hydroxybenzoate

94-13-3

Ester

Solid

NC

131 Anthracene120-12-7

Hydrocarbone

Solid

NC

132 2,2'-Iminodibenzyl

494-19-9

Hydrocarbone

Solid

NC

133 Silicic acid7699-41-4

Inorganic

Solid

NC

134 Aluminium hydroxide, 50-57%

21645-51-2

Inorganic base

Solid

NC

135 Potassium tetrafluoroborate

14075-53-7

Inorganic Salt

Solid

NC

136 Magnesium carbonate hydroxide pentahydrate

56378-72-4

Inorganic salt

Solid

NC

137 2,3-Dimethyl-2,3-dinitrobutane

3964-18-9

Nitro compound

Solid

NC

138 2,4-Dichloro-5-sulfamoylbenzoic Acid2736-23-4

Organic acid

Solid

NC

139 N, N-Dimethylguanidine sulfate598-65-2

Organic salt

Solid

NC

Cellulose, 2-(2-hydroxy-3-(trimethylammonium)propoxy)ethyl ether

118 chloride (91%)

2,2'-[[3-Methy l-4-[(4-nitropheny l)azo]-pheny l]imino]bis-

119 ethanol

2,2'-Methy lene-bis-(6-(2H-benzotriazol-2-y l)-4-(1,1,3,3-

1) CAS No.: Chemical abstracts service registry number.
2) In vivo class was referred from the GHS classification.

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NC: No category (non-eye irritant); 2: Category 2 (eye irritant); 1: Category 1 (eye corrosion)

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Table 5-2.

February, 2017

Category of the test chemicals for evaluation of the LabCyte24 EIT

Category

NumberCategory

NumberAcrylate

2 Fatty acids

1

Alcohols

19 Hydrocarbons

11

Aldehydes

2 Imide1

Aliphatic amine

1 Inorganic

1

Alkane3 Inorganic base2

Alkanol amine

3 Inorganic salts

4

Alkene1 Ketones/Lactones

5

Alkoxy5 N-containing heterocycle

1

Amines

11 Nitro compound2

Anhydryde

1 Organic acid5

Aromatic

3 Organic salts

5

Benzamide

1 Oxocarboxylic acid1

Benzo-thiazolinone

1 PAPA derivatives

1

Benzyl1 Pesticide1

Brominated derivative

2 Phenol1

Carboxylic acids

6 Polyol

2

Cycloalkane

1 Sulphur-containing compound

1

Dihydroxyl group

1 Surfactants (anionic)

5

Disulphides

1 Surfactants(cationic)

11

Esters

7 Surfactants(nonionic)

5

Fused carbocyclic aromatic

1

Table 5-3.

Number by physical state and UN GHS classification of the wide range
chemicals

UN GHS classification

Liquid Chemicals

Solid Chemicals

Total

Category 1 or 2

41

35

76

No category

40

23

63

Total

81

58

139

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Prediction of the eye irritation potential of test chemicals by the improved LabCyte24 EIT was
performed according to the improved protocol described in the SOP for ver.2.5.2 (Appendix 1)
and described as follows.
The LabCyte CORNEA-MODEL24 tissues is shipped from to recipients, the tissues are
aseptically removed from the transport agarose medium, transfer into wells on 24-well plateswith the assay medium (500 μL) and pre-incubated overnight at 37°C in a humidified
atmosphere of 5 % CO2 in air.

5-2.2.1. The Improved LabCyte24 EIT Procedure for Liquid Chemicals
After the pre-incubation, the tissue was topically exposed to the Liquid test chemicals.
Liquids (50 μL) were applied with a micropipette. Each test chemical was applied to threetissues. In addition, three tissues were treated with 50 μL of D-PBS serving as negative controls
and ethanol serving as positive controls. And then tissues applied chemicals were incubated for
1 minute. After exposure, each tissue was rinsed with D-PBS ten times or more, using awashing bottle to remove any remaining test chemical from the tissue surface. The blottedtissues were then transferred to new wells on 24-well plates containing 500 μL of fresh assay
medium. And then the tissues were post-incubated for 24 hours under the standard cultivation
conditions (at 37°C in a humidified atmosphere of 5% CO 2 in air). After the post-exposure
incubation periods, blotted tissues were transferred to new wells on 24-well plates containing
300 μL of freshly prepared WST-8 solution for a WST-8 assay (Ishiyama et. al., 1997; Tominaga
et. al., 1999). WST-8 solution was prepared by 1:10 dilution of Cell Counting Kit-8 (Dojindo Co.,
Japan) with EBSS (Sigma-Aldrich, MO, USA).

Tissues were incubated for 4 hours under the

standard cultivation conditions (at 37°C in a humidified atmosphere of 5% CO 2 in air).
Subsequently, 200 μL of culture supernatant were transferred to a 96-well microtiter plate. The
OD value of culture supernatant was measured at 450 nm and at 650 nm as a reference
absorbance, with WST-8 solution as a blank.

Cell Viability (%)

OD of the test chemical
OD of negative control

x 100

The tissue viability was calculated as a percentage relative to the viability of negative controls.
82 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017

The mean of the three values from identically treated tissues was used to classify a chemicalaccording to the prediction model.

5-2.2.2. The Improved LabCyte24 EIT Procedure for Solid Chemicals
After pre-incubation, the medium was changed to 500 μL of fresh assay medium and then thetissues were topically exposed to the Solid test chemicals. Solids (10 mg) in a microtube wereapplied by decantation. Each test chemical was applied to three tissues. In addition, threetissues serving as negative controls were set apart as non-treated samples and Lauric acid(10mg) served as positive control. And then tissues were incubated for 24 hours. After exposure,each tissue was rinsed with D-PBS in the same manner as that of Liquid chemicals. In the Solidchemicals, post-exposure incubation was not set. After the rinsing, blotted tissues wereanalyzed for their cell viability through means of a WST-8 assay, in the same manner as for
Liquid chemicals.

5-2.2.3. Prediction Model of the Improved LabCyte24 EIT
As a reference for the in vivo eye irritancy classification of each chemical, we used the United
Nations globally harmonized system (GHS) classification (United Nations, 2003).
According to the GHS classification (No Category, Category 2 or Category 1), an irritant(Category 1 or 2) was predicted if the mean relative tissue viability of three individual tissues
exposed to the test chemical is reduced below 40% of the mean viability of the negative controls(Table 5-4).
Table 5-4.

Prediction model of the improved LabCyte24 EIT

In vitro results

Prediction of eye irritation(UN-GHS classification)

Tissue viability is ≤ 40%

Category 1 or 2

Tissue viability is > 40%

No category

83 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

February, 20175-2.2.4. Detecting chemical interference with WST-8 endpoints and correction
procedures
A possible limitation of this eye irritation protocol describe in the Section 3-3 might be due tothe effect of a small amount of test chemicals on the WST-8 endpoints directly. The following
two types of test chemicals can interfere with the WST-8 assay.
A.

Chemicals that stain epithelial tissues directly.

B.

Chemicals that can directly reduce WST-8.

Test chemical that stains the corneal epithelial tissues has a possibility to transfer from thecorneal epithelial tissues to the WST-8 reaction buffer and to affect the OD value
measurements.
Test chemical that is able to directly reduce WST-8 can affect the OD value measurements, if
the test chemical is present in the corneal epithelial tissues when the WST-8 viability test is
performed.
Detection procedure of these test chemicals is described below.

5-2.2.4a. STEP 1 (PRELIMINARY TEST)
WST-8 solution was prepared by 1:10 dilution of Cell Counting Kit-8 with EBSS.
Fifty-μL (Liquid) or 10mg (Solid) of the test chemical was added into wells of 24-well assay
plate pre-filled with 0.3mL of WST-8 solution. Untreated the WST-8 solution was used as control.
Close the lid of 24-well assay plate was closed and the mixture is incubated in CO2 incubator for
about 4 hours. After incubation, the mixture was gently shaken and the staining of the diluted
WST-8 medium was evaluated macroscopically.

5-2.2.4b. STEP 2 (FUNCTIONAL CHECK ON VIABLE TISSUE)
Fifty-μL (Liquid) or 10mg (Solid) of the test chemical, which clearly changed the color of thediluted WST-8 solution at preliminary test, was added onto the surface of the corneal epithelial
tissues. All procedures of the LabCyte24 EIT described in section from 5-2.2.1 to 5-2.2.3 was
performed, although the corneal epithelial tissues that have been freeze-killed at -80 °C or lower
for 30 minutes twice instead of viable corneal epithelial tissues was used. The tissue corrected

84 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017

OD was calculated as following;
Each tissue OD (viable tissue) test chemical
Each tissue corrected OD =– [Mean OD (freeze-killed tissue) test chemical– Mean OD (freeze-killed tissue) negative control]

If each tissue corrected OD is below 0, its tissue OD was regarded as 0. When the cell viability(%), which was calculated according to the procedures described in Section 5-2.2.3, is <40%,
the test chemical is determined as GHS category 1 or 2 and therefore correction of the results is
not necessary.

85 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

February, 20175-3. Results
5-3.1. Detecting chemical interference with WST-8 endpoints and correction
First, 139 chemicals were examined to detect interference with WST-8 endpoints. Of the 139
tested chemicals, in the experiment for direct WST-8 reduction (5-2.2.4a, STEP 1).

(Ethylenediamine-propyl)

Monoethanolamine

Methyl thioglycolatem-Phenylene diamine

Calcium thioglycollate

1,5-Naphthalenediol

Ethyl thioglycolate-trimethoxysilane

Diisopropanolamine

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol

Trisodiummono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydroT-furan-3-yl) phosphate

Fig.5-1. Direct WST-8 reduction ability (STEP 1).

As shown in Fig.5-1, (Ethylenediamine-propyl)-trimethoxysilane (no.6), Monoethanolamine(no.7), Methyl thioglycolate (no.11), m-Phenylene diamine (no.25), Diisopropanolamine (no.43),
Calcium thioglycollate (no.46), 1,5-Naphthalenediol (no.62), Ethyl thioglycolate (no.92),
86 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol(no.119),

Trisodiummono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl) phosphate (no.128) weredetected as potential WST-8 interference.
Table 5-5.

Detection of WST-8 reducer in 139 chemicals

Positive chemicals in step 1

6

Cell viability

WST-8 interfere

Corrected(%: not corrected)(%)

viability (%)

9

3

4

(Ethylenediamine-propyl)-trimetho
xysilane

7

Monoethanolamine

6

3

1

11

Methyl thioglycolate

279

360

0

25

m-Phenylene diamine

24

16

1

43

Diisopropanolamine

6

4

1

46

Calcium thioglycollate

19

2

16

62

1,5-Naphthalenediol

14

183

0

92

Ethyl thioglycolate

177

188

14

0

0

0

1

2

0

2,2'-[[3-Methyl-4-[(4-nitrophenyl)a
119zo]-phenyl]imino]bis-ethanol
Trisodiummono-(5-(1,2-dihydroxyethyl)-4-ox
128
ido-2-oxo-2,5-dihydro-furan-3-yl)
phosphate

It was examined whether these chemical reduced WST-8 using freeze-killed tissue instead ofliving tissue. As a result (Table 5-5), Methyl thioglycolate (no.11) and Ethyl tioglycolate (no.92)
were strongly reduced WST-8 and therefore stained over 100%. Even if such chemicals withstrong interference WST-8, the cell viabilities of Methyl thioglycolate and Ethyl tioglycolate after
corrected showed 0% and 1.4%, respectively. Prediction results of both chemicals were eyeirritant and parallel with a prediction result of EpiOcular EIT (the OECD TG492 VRM).
Other

8

chemicals

shown

WST-8

assay

interference,(Ethylenediamine-propyl)-trimethoxysilane (no.6), Monoethanolamine (no.7), m-Phenylene
diamine(no.25),

Diisopropanolamine(no.43),

Calcium

thioglycollate(no.46),87 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

1,5-Naphthalenediol(no.119),(no.62),

Trisodium

February, 2017

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanolmono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl)

phosphate (no.128), were all shown lower than 40%, so these chemicals were finallydetermined as irritants (Table 5-4).

5-3.2. Predictivity of the Improved LabCyte24 EIT
The performance of the improved LabCyte24 EIT was evaluated with the 139 test chemicals.
Results obtained with each chemical are given in 5-5.
2-Ethoxyethyl methacrylate (no 88), 1-bromo-4chlorobutane (no. 91), Toluene (no. 97) and2-Heptanonee (no. 104) showed different predictions in three independent test runs.
Concordance with classifications obtained within-laboratory had a sufficient level of
reproducibility for the improved LabCyte24 EIT at 97% (135/139).
In the group of 63 test chemicals (no.77 to no.139) that were classified No Category (notclassified for irritation) in the GHS classification, positive predictions for 17 test chemicals,
2-Ethoxyethyl methacrylate (no.88), Ethyl thioglycolate (no.92), Ethyl acetate (no.96), Toluene(no.97),Styrene(no.102),

3,4-dimethoxy

benzaldehyde

propoxy)ethyl

ether

Methylisobutyl ketone(no.103),

2-Heptanone(no.104),(no.117),

Cellulose,

2-(2-hydroxy-3-(trimethylammonium)(91%;

no.118),2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-chloride

phenyl]imino]bis-ethanol (no.119), Ethylenediamine-tetraacetic acid dipotassium salt dehydrate(no.123), Het Anhydride (no.125), Trisodium mono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo2,5-dihydro-furan-3-yl)
p-hydroxybenzoate

phosphate(no.130),(no.128),

D(+)-Glucono-1,5-lactone

2,4-Dichloro-5-sulfamoylbenzoic

Acid(no.129),(no.138),

PropylandN,

N-Dimethylguanidine sulfate (no.139) were not concordant with the results of the in vivo
classification (Table 5-5).
In the group of 76 test chemicals (no.1 to no.75) that were classified Category 1 or 2 (irritant)in GHS classification, no chemicals were wrongly predicted as non-irritant by the LabCyte24 EIT(Table 5-6).

88 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

Table 5-6.

February, 2017

Wide range of the test chemicals for evaluation of the LabCyte24 EIT
LabCyte CORNEA-MODEL EIT
Chemical

No

Name

GHS
class

R1

R2

R3

Mean

Viability (%) Judge

Viability (%) Judge

Viability (%) Judge

Viability (%)

Judge

1

Hydroxyethyl acrylate

1

3.9± 2.0 Cat2/1

5.6± 1.9 Cat2/1

5.7± 2.4 Cat2/1

5.0

±

1.0

Cat2/1

2

Tetraethylene glycol diacrylate

1

28.6

± 8.6 Cat2/1

22.8± 4.5 Cat2/1

24.9± 4.4 Cat2/1

25.4

±

2.9

Cat2/1

3

1-Butanol

1

4.9± 0.9 Cat2/1

14.9± 2.5 Cat2/1

18.0± 2.0 Cat2/1

12.6

±

6.8

Cat2/1

4

Cyclohexanol

1

16.8

± 0.8 Cat2/1

14.2± 5.7 Cat2/1

16.3± 1.9 Cat2/1

15.8

±

1.4

Cat2/1

5

Benzyl alcohol

12.8± 0.5 Cat2/1

9.6± 1.3 Cat2/1

13.2± 1.1 Cat2/1

8.5

±

5.3

Cat2/1

6(Ethylenediamine-propyl)-trimethoxysilane

1

8.5± 3.8 Cat2/1

5.2± 1.2 Cat2/1

5.2± 2.5 Cat2/1

6.3

±

1.9

Cat2/1

7

Monoethanolamine1

2.9± 0.2 Cat2/1

2.5± 1.0 Cat2/1

3.0± 0.3 Cat2/1

2.8

±

0.2

Cat2/1

8

Diethylethanolamine1

2.7± 0.2 Cat2/1

5.8± 0.6 Cat2/1

2.6± 0.3 Cat2/1

3.7

±

1.9

Cat2/19Lactic acid1

2.9

± 0.4 Cat2/1

3.2± 0.5 Cat2/1

5.5± 0.1 Cat2/1

3.9

±

1.4

Cat2/110 Acetic acid1

4.2

± 0.3 Cat2/1

2.8± 0.4 Cat2/1

5.3± 0.1 Cat2/1

4.1

±

1.3

Cat2/1

11 Methylthioglycolate

10.0± 0.0 Cat2/1

3.0± 5.3 Cat2/1

0.0± 0.0 Cat2/1

1.0

±

1.8

Cat2/1

12 Methoxyethyl acrylate

12.5± 0.9 Cat2/1

8.6± 1.1 Cat2/1

6.7± 0.8 Cat2/1

6.0

±

3.1

Cat2/113 Imidazole(20%)

1

12.4

± 0.6 Cat2/1

23.1± 4.7 Cat2/1

24.7± 3.3 Cat2/1

20.0

±

6.7

Cat2/1

14 Pyridine

1

2.1± 0.3 Cat2/1

1.2± 0.7 Cat2/1

2.3± 0.2 Cat2/1

1.9

±

0.6

Cat2/1

15 Di(2-ethylhexyl) sodium sulfosuccinate （10%）

1

4.1± 0.7 Cat2/1

14.0± 4.8 Cat2/1

15.4± 0.8 Cat2/1

11.2

±

6.2

Cat2/1

16 Potassium laurate（10%）

1

32.7

± 1.5 Cat2/1

8.6± 6.7 Cat2/1

20.2± 12.1Cat2/1

20.5± 12.1Cat2/117 Cethylpyridinium bromide (6%)

1

15.6

± 8.0 Cat2/1

15.5± 1.2 Cat2/1

11.7± 4.0 Cat2/1

14.3

±

2.2

Cat2/118 Benzalkonium chloride (10%)

10.7± 0.8 Cat2/1

2.7± 1.0 Cat2/1

1.3± 1.1 Cat2/1

1.6

±

1.0

Cat2/119 Domiphen bromide（10%）

1

0.8± 0.1 Cat2/1

0.9± 0.0 Cat2/1

0.4± 0.4 Cat2/1

0.7

±

0.2

Cat2/120 Cethylpyridinium chloride（10%）

1

2.9± 0.4 Cat2/1

7.2± 1.3 Cat2/1

22.8± 0.4 Cat2/1

11.0± 10.5

Cat2/121 Stearyltrimethylammonium chloride（10%）

1

9.0± 0.7 Cat2/1

16.7± 1.0 Cat2/1

37.1± 0.4 Cat2/1

20.9± 14.5

Cat2/1

22 Cethyltrimethylammonium bromide（10%）

1

7.0± 2.3 Cat2/1

12.1± 10.8

Cat2/129.7± 0.4 Cat2/1

16.3± 11.9Cat2/1

23 2,5-Dimethyl-2,5-hexanediol

1

3.1± 1.5 Cat2/1

2.3± 0.6 Cat2/1

0.8± 0.4 Cat2/1

2.1

±

1.2

Cat2/11

3.8± 0.5 Cat2/1

4.1± 0.3 Cat2/1

1.7± 0.4 Cat2/1

3.2

±

1.3

Cat2/125 m-Phenylene diamine1

8.9± 0.4 Cat2/1

2.0± 0.9 Cat2/1

1.4± 0.5 Cat2/1

4.1

±

4.2

Cat2/126 Quinacrine1

3.7± 4.1 Cat2/1

10.1± 6.6 Cat2/1

6.0± 0.7 Cat2/1

6.6

±

3.3

Cat2/127 Chlorhexidine

1

6.9± 2.6 Cat2/1

2.6± 2.6 Cat2/1

0.7± 0.4 Cat2/1

3.4

±

3.2

Cat2/1

28 1,3-Diiminobenz[f]isoindoline1

1.5± 0.2 Cat2/1

3.2± 1.2 Cat2/1

0.7± 0.2 Cat2/1

1.8

±

1.3

Cat2/1

29 1,2-Benzisothiazol-3(2H)-one

1

19.6

± 1.9 Cat2/1

16.1± 9.7 Cat2/1

16.4

Cat2/1

17.4

±

1.9

Cat2/130 Paraformaldehyde

1

0.9± 0.3 Cat2/1

8.9± 1.1 Cat2/1

3.5± 4.7 Cat2/1

4.4

±

4.1

Cat2/1

24

Disodium 2,2'-([1,1'-bipheny l]-4,4'-diy ldiv iny lene)bis(benzenesulphonate)± 11.089 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017

(Continue)
LabCyte CORNEA-MODEL EIT
Chemical
No

Name

GHS
class

R1

R2

R3

Mean

Viability (%) Judge

Viability (%) Judge

Viability (%) Judge

Viability (%)

Cat2/1

17.4

±

1.9

Cat2/1Judge

29 1,2-Benzisothiazol-3(2H)-one

1

19.6

± 1.9 Cat2/1

16.1± 9.7 Cat2/1

16.4

30 Paraformaldehyde

1

0.9± 0.3 Cat2/1

8.9± 1.1 Cat2/1

3.5± 4.7 Cat2/1

4.4

±

4.1

Cat2/131 Lauric acid1

2.1

± 0.8 Cat2/1

9.3± 0.2 Cat2/1

4.4± 4.3 Cat2/1

5.3

±

3.7

Cat2/132 Imidazole1

1.1± 1.1 Cat2/1

8.5± 0.1 Cat2/1

3.5± 4.4 Cat2/1

4.4

±

3.8

Cat2/133 Tetraoctylammonium bromide

1

13.3

± 4.5 Cat2/1

16.7± 2.5 Cat2/1

14.4± 2.0 Cat2/1

14.8

±

1.7

Cat2/1

34 Captan 90-concentrate

1

12.8

± 0.8 Cat2/1

5.3± 2.7 Cat2/1

3.2± 0.7 Cat2/1

7.1

±

5.0

Cat2/135 Dibenzoyl-L-tartaric acid1

8.9

± 1.4 Cat2/1

0.6± 0.2 Cat2/1

0.2± 0.1 Cat2/1

3.2

±

4.9

Cat2/1

36 2,4-Dihydroxybenzoic acid1

8.5

± 0.5 Cat2/1

0.0± 0.1 Cat2/1

0.8± 0.6 Cat2/1

3.1

±

4.7

Cat2/137 Sodium salicylate

19.6± 0.9 Cat2/1

1.6± 0.3 Cat2/1

2.8± 0.4 Cat2/1

4.7

±

4.4

Cat2/138 Promethazine hydrochloride

1

11.8

± 2.2 Cat2/1

5.0± 0.6 Cat2/1

5.4± 2.3 Cat2/1

7.4

±

3.8

Cat2/139 Sodium oxalate

1

2.3± 1.9 Cat2/1

4.8± 0.7 Cat2/1

4.2± 2.5 Cat2/1

3.8

±

1.3

Cat2/140 1-naphtalen acetic acid1

0.1

± 0.1 Cat2/1

9.4± 1.6 Cat2/1

1.8± 0.1 Cat2/1

3.8

±

5.0

Cat2/1

41 p-t-Butylphenol

1

8.5± 0.6 Cat2/1

0.1± 0.1 Cat2/1

1.3± 0.1 Cat2/1

3.3

±

4.5

Cat2/142 Benzalkonium chloride (1%)

2

3.0

± 1.1 Cat2/1

7.3± 4.4 Cat2/1

6.3± 4.1 Cat2/1

5.5

±

2.3

Cat2/143 Diisopropanolamine2

1.4± 0.1 Cat2/1

0.8± 0.1 Cat2/1

0.5± 0.4 Cat2/1

0.9

±

0.5

Cat2/1

44 Glycolic acid2

1.1

± 0.6 Cat2/1

8.3± 0.3 Cat2/1

4.1± 3.7 Cat2/1

4.5

±

3.6

Cat2/145 Sodium Perborate Tetrahydrate

2

9.3± 0.6 Cat2/1

1.4± 0.0 Cat2/1

3.1± 0.2 Cat2/1

4.6

±

4.2

Cat2/1

46 Calcium thioglycollate

2

14.4

± 6.6 Cat2/1

17.9± 3.0 Cat2/1

27.8± 1.2 Cat2/1

20.1

±

7.0

Cat2/147 Ethanol

2A

13.8

± 2.3 Cat2/1

12.3± 2.5 Cat2/1

13.5± 5.0 Cat2/1

13.2

±

0.8

Cat2/148 Isopropylalcohol

2A

7.2

± 2.9 Cat2/1

6.6± 5.0 Cat2/1

8.0± 2.2 Cat2/1

7.3

±

0.7

Cat2/1

49 2-Benzyloxyethanol

2A

5.3

± 1.1 Cat2/1

33.1± 7.4 Cat2/1

12.6± 1.4 Cat2/1

17.0± 14.4

Cat2/150 1-Octanol

2A

3.2

± 2.1 Cat2/1

4.7± 1.1 Cat2/1

4.1± 0.4 Cat2/1

4.0

±

0.7

Cat2/151 2-Ethyl-1-hexanol

2A

2.2

± 0.8 Cat2/1

4.8± 0.7 Cat2/1

3.0± 0.9 Cat2/1

3.4

±

1.3

Cat2/1

52 n-Hexanol

2A

25.3

± 5.5 Cat2/1

38.0± 1.3 Cat2/1

19.0± 1.4 Cat2/1

27.4

±

9.7

Cat2/153 Isobutanol

2A

10.1

± 3.9 Cat2/1

23.4± 4.9 Cat2/1

15.3± 1.4 Cat2/1

16.3

±

6.7

Cat2/12A

1.1± 0.2 Cat2/1

18.0± 2.5 Cat2/1

20.2± 2.9 Cat2/1

13.1± 10.5

Cat2/155 Acetone

2A

23.9

± 7.8 Cat2/1

9.3± 0.2 Cat2/1

5.7± 2.5 Cat2/1

12.9

±

9.7

Cat2/156 Methyl ethyl ketone

2A

7.1

± 1.6 Cat2/1

14.3± 4.8 Cat2/1

13.9± 0.8 Cat2/1

11.8

±

4.1

Cat2/154

2,4,11,13-Tetraazatetradecane-diimidamide, N,N''-bis(4-chlorophenyl)3,12-diimino-, di-D-gluconate (20%, aqueous)± 11.090 /121

BRD of the LabCyte CORNEA-MODEL24 EIT

February, 2017

(Continue)
LabCyte CORNEA-MODEL EIT
Chemical

R1

R2

R3

GHS
class

Viability (%) Judge

Viability (%) Judge

Viability (%) Judge

Viability (%)

Judge

57 gamma-Butyrolactone

2A

7.2

± 2.3 Cat2/1

9.2

6.3

± 2.5 Cat2/1

7.5

±

1.5

Cat2/158 Sodium dodecyl sulfate(15%)

2A

34.9

± 5.6 Cat2/1

25.8± 10.5

Cat2/128.6± 7.9 Cat2/1

29.8

±

4.7

Cat2/159 Cethylpyridinium bromide (1%)

2A

11.6

± 0.6 Cat2/1

22.4± 0.4 Cat2/1

28.6± 0.9 Cat2/1

20.9

±

8.6

Cat2/1

60 Triton X-100 (5%)

2A

0.9

± 0.4 Cat2/1

17.8± 1.3 Cat2/1

19.3± 5.4 Cat2/1

12.7± 10.2Cat2/1

61 Sodium benzoate

2A

2.1

± 1.2 Cat2/1

3.5± 0.4 Cat2/1

1.7± 0.3 Cat2/1

2.4

±

0.9

Cat2/1

62 1,5-Naphthalenediol

2A

6.7

± 5.4 Cat2/1

19.8± 6.9 Cat2/1

9.7± 4.9 Cat2/1

12.1

±

6.9

Cat2/163 p-Formylbenzoic acid

2A

12.2

± 0.9 Cat2/1

3.3± 1.1 Cat2/1

7.1± 0.3 Cat2/1

7.5

±

4.4

Cat2/164 2-Methyl-1-pentanol

2B

14.7

± 4.7 Cat2/1

37.7± 7.6 Cat2/1

19.5± 3.5 Cat2/1

24.0± 12.1Cat2/165 Diethyl toluamide

2B

13.3

± 5.9 Cat2/1

39.5± 5.9 Cat2/1

28.6± 7.4 Cat2/1

27.1± 13.2Cat2/1

66 Methyl acetate

2B

13.1

± 1.5 Cat2/1

15.5± 6.1 Cat2/1

13.3± 1.7 Cat2/1

14.0

±

1.3

Cat2/167 Sodium hydroxide (1%)

2B

0.9

± 0.4 Cat2/1

17.5± 1.1 Cat2/1

9.9± 1.7 Cat2/1

9.4

±

8.3

Cat2/1

68 2,2-Dimethyl-3-methylenebicyclo [2.2.1] heptane

2B

2.2

± 0.6 Cat2/1

4.3± 3.4 Cat2/1

5.1± 6.5 Cat2/1

3.9

±

1.5

Cat2/1

69 1,4-Dibutoxy benzene

2B

29.3

Cat2/1

26.4± 4.6 Cat2/1

30.1

±

4.1

Cat2/1

70 2,6-Dichloro-5-f luoro-beta-oxo-3-py ridinepropanoate

2B

71 Maneb

NoName± 2.7 Cat2/1

Mean

Cat2/134.5

1.3± 0.2 Cat2/1

1.5± 0.3 Cat2/1

1.2± 0.3 Cat2/1

1.3

±

0.1

Cat2/1

2B

7.4± 0.6 Cat2/1

2.8± 0.3 Cat2/1

0.0± 0.0 Cat2/1

3.4

±

3.8

Cat2/172 Ammonium Nitrate

2B

9.8

± 0.2 Cat2/1

1.6± 0.2 Cat2/1

3.0± 0.2 Cat2/1

4.8

±

4.4

Cat2/173 m-Dinitrobenzene

2B

9.6

± 0.1 Cat2/1

3.2± 0.1 Cat2/1

2.6± 0.7 Cat2/1

5.1

±

3.9

Cat2/1

74 p-Nitrobenzoic acid

2B

9.0

± 0.3 Cat2/1

2.0± 2.1 Cat2/1

1.1± 0.3 Cat2/1

4.1

±

4.3

Cat2/175 Sodium chloroacetate

2B

9.1

± 0.8 Cat2/1

1.1± 0.1 Cat2/1

0.8± 0.1 Cat2/1

3.7

±

4.7

Cat2/1

76 3,3-Dithiodipropionic Acid

2B

11.0

± 0.2 Cat2/1

3.4± 0.3 Cat2/1

5.1± 0.6 Cat2/1

6.5

±

4.0

Cat2/177 Poly ethy lene gly col (PEG-40) hy drogenated castor oil

NC

52.7

± 8.3

NC

107.9 ± 3.9

NC

74.1

± 6.8

NC

78.2

± 27.8NC

78 Propylene glycol

NC

80.8

± 10.3

NC

77.6± 10.7

NC

87.6

± 6.5

NC

82.0

±

5.1

NC

79 3-Methoxy-1,2-propanediol

NC

68.9

± 11.5

NC

76.5

± 7.1

NC

70.9

± 6.3

NC

72.1

±

3.9

NC

80 Ethanol (10%)

NC

117.9 ± 1.9

NC115.1 ± 16.9

NC

89.1

± 18.7

NC

107.4 ±

1.9

NC

81 3-Phenoxybenzyl alcohol

NC

50.3

± 8.0

NC

60.4

± 16.0NC

57.8± 11.8

NC

56.2

±

5.3

NC

82 4-(Methylthio)-benzaldehyde

NC

49.3

± 8.2

NC

58.9

± 4.5

NC

43.4

± 5.4

NC

50.5

±

7.8

NC

83 3,3-Dimethylpentane

NC

90.4

± 18.3

NC

88.5

± 0.7

NC

104.4 ± 4.6

NC

94.4

±

8.7

NC

84 Triethanolamine

NC

80.9

± 16.2NC

75.0± 12.2

NC

82.9

NC

79.6

±

4.1

NC

± 11.8± 11.2± 6.2

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(Continue)
LabCyte CORNEA-MODEL EIT
Chemical

R1

R2

R3

GHS
class

Viability (%) Judge

Viability (%) Judge

Viability (%) Judge

Viability (%)

85 1-Ethyl-3-methylimidazolium ethylsulphate

NC

53.2

± 19.4

NC

42.6

± 3.7

NC

49.0

± 19.8

NC

48.3

±

5.3

NC

86 Piperonyl butoxide

NC

93.8

± 3.1

NC

92.5

± 10.6

NC

85.8

± 5.4

NC

90.7

±

4.3

NC

87 n-Octyl Ether

NC

98.5

± 6.1

NC

89.5

± 3.8

NC

90.1

± 9.2

NC

92.7

±

5.0

NC

88 2-Ethoxyethyl methacrylate

NC

34.9

± 9.8 Cat2/1

41.2

± 1.4

NC

37.4

± 7.8 Cat2/1

37.8

±

3.2

Cat2/1

89 Xylene

NC

87.0

± 2.6

NC

71.4

± 8.8

NC

60.0

± 13.4

NC

72.8

±

2.6

NC

90 1,6-Dibromohexane

NC

74.6

± 17.7

NC

60.6

± 3.3

NC

53.7

± 3.1

NC

63.0

± 10.6

NC

91 1-bromo-4chlorobutane

NC

37.9

± 6.5 Cat2/1

49.8± 10.8

NC

50.2

± 1.6

NC

46.0

±

7.0

NC

92 Ethyl thioglycolate

NC

9.9

± 11.4

Cat2/10.0± 0Cat2/10.6± 1.0 Cat2/1

3.5

±

5.6

Cat2/193 Methyl cycropentane

NC

94.5

± 4.3

NC

107.7 ± 17.2

NC

84.7

± 11.2

NC

95.6± 11.6

NC

94 Dipropyl disulphide

NC

79.8

± 7.0

NC

102.6 ± 7.6

NC100.0 ± 7.4

NC

94.1

± 12.5NC

95 Isopropyl myristate

NC

85.4

± 16.2

NC

85.4± 18.3

NC

106.5 ± 0.8

NC

92.4

± 12.2NC

96 Ethyl acetate

NC

16.5

± 3.0 Cat2/1

20.4± 3.2 Cat2/1

20.7± 4.4 Cat2/1

19.2

±

2.3

Cat2/197 Toluene

NC

31.6

± 1.1 Cat2/1

25.4± 1.0 Cat2/1

47.8

± 4.6

NC

35.0

± 11.6

Cat2/1

98 2-Methylpentane

NC

89.9

± 4.3

NC

128.8 ± 3.7

NC

90.6

± 7.1

NC

103.1 ±

4.3

NC

99 n-Octyl bromide

NC

84.1

± 5.3

NC

126.5 ± 11.2

NC

97.3

± 13.6

NC

102.6 ±

5.3

NC

100 cis-Cyclooctene

NC

70.3

± 2.0

NC

120.0 ± 1.8

NC

92.0

± 3.5

NC

94.1

±

2.0

NC

101 Dodecane

NC

84.0

± 1.2

NC

101.4 ± 2.5

NC108.5 ± 1.9

NC

84.0

± 12.6

NC

102 Styrene

NC

28.8

± 5.0 Cat2/1

27.8± 8.4 Cat2/1

25.8± 13.5

Cat2/1

27.5

±

1.5

Cat2/1103 Methyl isobutyl ketone

NC

32.6

± 5.5 Cat2/1

31.1± 6.1 Cat2/1

33.2± 3.8 Cat2/1

32.3

±

1.1

Cat2/1

104 2-Heptanone(Methyl amylketone)

NC

25.1

± 6.9 Cat2/1

34.7± 1.8 Cat2/1

47.9

± 6.5

NC

35.9

± 11.5

Cat2/1

105 2-Ethylhexyl p-dimethylamino benzoate

NC

91.9

± 10.4

NC

81.3

± 6.4

NC

100.8 ± 2.0

NC

91.3

±

9.8

NC

106 Glycerol

NC

87.3

± 5.8

NC

79.8

± 11.8

NC

66.3

± 4.9

NC

77.8

± 10.6

NC

107 Polyethylene glycol 400

NC

97.0

± 8.3

NC

100.2 ± 4.9

NC

76.6

± 4.4

NC

91.3

± 12.8NC

108 Sodium N-lauroyl sarcosinate (30%)

NC

85.1

± 15.2NC

93.9± 16.6NC

87.0± 13.1

NC

88.6

±

4.6

NC

109 Sodium dodecyl sulfate(1%)

NC

79.3

± 3.8

NC

96.4

± 7.5

NC

97.4

± 1.0

NC

91.0

± 10.1NC

110 Benzalkonium chloride (0.01%)

NC

69.7

± 15.8NC

111.9 ± 4.5

NC109.5 ± 5.3

NC

97.1

± 23.7

NC

111 Cethylpyridinium bromide (0.1%)

NC

52.5

± 6.8

NC

87.2

± 5.1

NC

84.0

± 5.5

NC

74.6

± 19.2NC

112 Benzalkonium chloride (0.1%)

NC

47.9

± 4.0

NC

67.9

± 7.2

NC

72.2

± 5.1

NC

62.7

± 13.0

NC

No

Name

Mean

92 /121Judge

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(Continue)
LabCyte CORNEA-MODEL EIT
Chemical

R1

R2

R3

GHS
class

Viability (%) Judge

Viability (%) Judge

Viability (%) Judge

Viability (%)

113 Tw een20

NC

94.5

± 5.5

NC

104.5 ± 7.7

NC

83.6

± 9.6

NC

94.2

± 10.5

NC

114 Tw een80

NC

81.4

± 14.5NC

80.0± 11.2

NC

104.5 ± 8.5

NC

88.6

± 13.8NC

115 Polyethylene glycol monolaurate (10E.O.)

NC

83.8

± 2.1

NC

77.0

± 6.1

NC

90.7

± 10.8

NC

83.8

±

6.9

NC

116 Tw een80(10％)

NC

71.4

± 13.0NC

106.9 ± 2.1

NC

88.2

± 12.8NC

88.8± 17.8NC

117 3,4-dimethoxy benzaldehyde

NC

3.1

± 0.0 Cat2/1

2.3± 0.1 Cat2/1

2.6± 0.0 Cat2/1

2.7

±

0.4

Cat2/1118 chloride (91%)

NC

14.3

± 7.0 Cat2/1

7.9± 5.5 Cat2/1

6.6± 3.1 Cat2/1

9.6

±

4.1

Cat2/12,2'-[[3-Methy l-4-[(4-nitropheny l)azo]-pheny l]imino]bis119 ethanol

NC

1.6

± 0.2 Cat2/1

1.0± 0.6 Cat2/1

1.6± 0.3 Cat2/1

1.4

±

0.4

Cat2/1120 tetramethy lbuty l)-phenol)

NC

91.7

± 8.4

NC

115.2 ± 8.7

NC

94.2

± 9.8

NC

100.4 ± 12.9

NC

121 2-MercaptopyrimidineNC

103.1 ± 4.4

NC107.1 ± 5.8

NC

77.5

± 4.9

NC

95.9

± 16.0NC

122 Tetraaminopyrimidine sulfate

NC

118.1 ± 24.7

NC

109.8 ± 5.4

NC

81.7

± 5.2

NC

103.2 ± 19.1

NC

123 dihy drate

NC

5.4

± 0.8 Cat2/1

3.4

4.8

1.2

Cat2/1

124 Phenothiazine

NC

64.6

± 14.2NC

125 Het Anhydride

NC

2.4

126 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl) urea

NC

61.8

± 5.7

NC

72.8

± 5.9

NC

73.4

127 4,4'-Methylene bis-(2,6-di-tert-butylphenol)

NC

102.8 ± 3.4

NC

72.5

± 14.3

NC

86.4

Trisodium mono-(5-(1,2-dihy droxy ethy l)-4-oxido-2-oxo128 2,5-dihy dro-f uran-3-y l) phosphate

NC

4.9

± 0.8 Cat2/1

9.1± 2.9 Cat2/1

129 D(+)-Glucono-1,5-lactone

NC

3.1

± 0.5 Cat2/1

1.9

130 Propyl p-hydroxybenzoate

NC

3.5

± 2.4 Cat2/1

0.8

131 Anthracene

NC

91.5

± 3.8

NC

114.8 ± 9.8

NC

71.8

± 6.9

NC

132 2,2'-Iminodibenzyl

NC

80.8

± 4.2

NC

81.9

± 11.3

NC

81.1

± 2.1

133 Silicic acid

NC

48.3

± 3.9

NC

96.4

± 3.0

NC

71.1

134 Aluminium hydroxide, 50-57%

NC

84.1

± 3.1

NC

105.1 ± 12.1

NC

77.5

135 Potassium tetrafluoroborate

NC

90.4

± 7.7

NC

111.1 ± 4.3

NC105.8 ±

136 Magnesium carbonate hydroxide pentahydrateNC

102.0 ± 7.3

NC104.8 ± 1.3

NC

137 2,3-Dimethyl-2,3-dinitrobutane

NC

74.0

± 1.1

NC

86.7

± 8.6

138 2,4-Dichloro-5-sulfamoylbenzoic Acid

NC

16.6

± 0.5 Cat2/1

139 N, N-Dimethylguanidine sulfate

NC

1.6

± 0.2 Cat2/1

No

Name

Cellulose, 2-(2-hydroxy-3-(trimethylammonium)propoxy)ethyl ether

2,2'-Methy lene-bis-(6-(2H-benzotriazol-2-y l)-4-(1,1,3,3-

Ethy lenediamine-tetraacetic acid dipotassium salt

± 0.5 Cat2/1
± 9.6

5.5

NC

89.8

± 0.5 Cat2/1

0.0

± 4.5

NC

66.0

± 0.1 Cat2/1

0.0

Mean

± 1.3 Cat2/1
± 3.9

±

Judge

NC

73.5

± 0.0 Cat2/1

0.8

±

1.4

Cat2/1± 10.6

NC

69.3

±

6.5

NC

± 11.3

NC

87.2± 15.1

NC

5.2± 1.4 Cat2/1

6.4

±

2.3

Cat2/1± 0.1 Cat2/1

1.8± 0.2 Cat2/1

2.3

±

0.7

Cat2/1± 0.8 Cat2/1

1.9± 1.8 Cat2/1

2.1

±

1.3

Cat2/192.7± 21.5

NC

NC

81.3

±

0.6

NC

± 5.3

NC

71.9

± 24.1

NC

± 8.0

NC

88.9

± 14.4

NC

12.1

NC

102.4 ± 10.7

NC

81.6

± 16.8NC

96.1± 12.7

NC

NC

70.9

± 1.6

NC

77.2

±

8.4

NC

13.5

± 3.3 Cat2/1

2.6± 1.7 Cat2/1

10.9

±

7.3

Cat2/1

1.3± 0.7 Cat2/1

0.6± 0.2 Cat2/1

1.2

±

0.5

Cat2/1

93 /121

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In summary, based on the mean of the three independent runs (Table 5-7), 45 out of 61
category and all out of 75 Category 1 or 2 in the GHS classification were classified correctly by
the improved LabCyte24 EIT. The statistical parameters describing assay performance aredisplayed in Table 5-7.
Table 5-7.

Wide range of the test chemicals for evaluation of the LabCyte24 EIT
In vivo classification

In vitro prediction

Cat 1 or 2

No category

Total

Irritant

76

17

93

Non-irritant

0

46

46

Total

76

63

139

Sensitivity (%)

100

Specificity (%)

73.0

Accuracy (%)

87.8

Sensitivity and specificity of predictions by the improved LabCyte24 EIT were 100% and73.0%, respectively. Overall accuracy was 87.8% (Table 5-6).

5-4. Discussion and Conclusion
In the OECD TG 492 description, the formazan solution may be quantified using either astandard absorbance OD measurement or a high performance liquid chromatography/an ultrahigh performance liquid chromatography (HPLC/UPLC) spectrophotometry procedure.
Especially, it is pointed out that the HPLC/UPLC may be useful to detection correct coloredformazan after the colored chemical application because it might be a possibility to distinguish apeak of formazan color from a peak of material color. In the improved LabCyte24 EIT, theformazan solution was quantified using OD measurement, because ocular irritancy of all
chemicals could be predicted from measured ODs. But, HPLC/UPLC measurement may be
useful about unknown strong-colored chemicals.
When compared with the UN GHS classification, the LabCyte24 EIT classified 17 No
Category chemicals (chemicals not classified for irritation), 2-Ethoxyethyl methacrylate, Ethyl
94 /121

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thioglycolate, Ethyl acetate, Toluene, Styrene, Methyl isobutyl ketone, 2-Heptanone,
3,4-dimethoxy benzaldehyde, Cellulose, 2-(2-hydroxy-3-(trimethylammonium) propoxy)ethyl
ether

chloride(91%),

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol,

Ethylenediamine-tetraacetic acid dipotassium salt dehydrate, Het Anhydride, Trisodiummono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl) phosphate, D(+)-Glucono1,5-lactone,

Propylp-hydroxybenzoate,

2,4-Dichloro-5-sulfamoylbenzoic Acid,

and

N,

N-Dimethylguanidine sulfate as irritants.
Interestingly, while 11 of 17 chemicals, 2-Ethoxyethyl methacrylate, Ethyl thioglycolate, Ethyl
acetate, Toluene, Methyl isobutyl ketone, 2-Heptanone, 3,4-dimethoxy benzaldehyde,
2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol,acid

dipotassium

saltEthylenediamine-tetraacetic

dehydrate,

Trisodiummono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl) phosphate, D(+)-Glucono1,5-lactone, and N, N-Dimethylguanidine sulfate as irritants were classified as No Category by
the UN GHS classification, alternative eye irritation models such as the OECD TG492 VRM
define them as irritants (Van Goethem et. al., 2006; Kaluzhny et. al., 2011; OECD, 2015b).
Human study data also supports that these chemicals might be eye-irritants (Van Goethem et.
al., 2006), indicating that the performance of the improved LabCyte CORNEA-MODEL24 mightsimulate the response of a human eye to chemical irritants very well.
Even if the 17 above-mentioned test chemicals were false positives, the specificity of the
improved LabCyte24 EIT showed 73.0 % and it was enough correlation with the UN GHS
classification. Furthermore, all chemicals classified in Category 1 or 2 (eye irritant) in the UN
GHS classification were correctly predicted as irritants in the improved LabCyte24 EIT.

The

sensitivity of this EIT showed 100% correlation with the GHS classification. Finally, an overall
accuracy of 87.8 % was obtained.

The present results strongly suggest that the improved

LabCyte24 EIT could be distinguished No Category from Category 1 or 2 in the UN GHS
classification according to Bottom-up strategy and therefore it could be a useful alternative
method to the rabbit Draize eye test.

95 /121

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6. THE AVAILABILITY OF LabCyte24 EIT OUTSIDE OF JAPAN:
EXPORT OF LabCyte CORNA-MODEL TO KOREA AND CONFIRMATION OF ITSPERFORMANCE
6-1. Purpose
For the LabCyte24 EIT to be included as one of the validated method in the OECD TG492(OECD, 2015a), it is necessary to demonstrate that this testing method is available
internationally.
In order to confirm the availability of LabCyte CORNEA-MODEL24 after shipment to outside ofJapan, LabCyte CORNEA-MODEL24 is exported to a Korean laboratory and the EIT using
LabCyte CORNEA-MODEL24 was performed at the Korean laboratory

6-2. Export to Korea and Test Protocol
6-2.1. Shipment Schedule
The LabCyte CORNEA-MODEL24 was exported to Korean laboratory as following schedule.
1st export:

February 15, 2016.

Lot no. LCC24-160215-A

2nd export:

February 29, 2016.

Lot no. LCC24-160229-A

3rd export:

April 18, 2016.

Lot no. LCC24-160418-A

6-2.2. Test Facility
The LabCyte CORNEA-MODEL24 after shipment was evaluated at following laboratory in
Korea.
Laboratory name
Konkuk University;
Center for Stem Cell Research, Dept. of Biomedical Science & Technology, Institute of
Biomedical Science & Technology (IBST)
120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea.

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Laboratory member
Dr. Jeong Ik Lee; Associate Professor.
Dr. Yuna Han; Research Professor
Dr. Lee, Soo Jung; Research Professor

6-2.3. Evaluation Procedure of LabCyte CORNEA-MODEL24 after Shipment
Using LabCyte CORNEA-MODEL24 kit after shipment, the LabCyte24 EIT was performed at
the Korean Laboratory according to the improved protocol, which described in the SOPver.2.5.2 (Appendix 1) for confirmed followings objection.
(1) To confirm that OD and SD of the negative control (Liquid and Solid chemical protocol) are
adapted in the acceptance criteria in all independent 3 batches after shipment.
(2) To confirm that the cell viability (%) and SD of the positive control (Liquid and Solidchemical protocol) are adapted in the acceptance criteria in all independent 3 batches after
shipment.
(3) To confirm that be able to perform LabCyte24 EIT in according to the improved protocol
which described in the SOP ver.2.5.2 (Appendix 1) using proficiency test chemicals as
listed in the OECD TG492 (OECD, 2015a).

6-3. Results
6-3.1. Export to Korean Laboratory
Shipment schedules to Korean laboratory are shown in Table 6-1.
Table 6-1.

Shipments to Korean laboratory.

Lot No.

Shipping date

Delivery date

Transportation period

LCC24-160215-A

Feb. 15, 2016

Feb. 17, 2016

2 days

LCC24-160229-A

Feb. 29, 2016

Mar. 4, 2016

4 days

LCC24-160418-A

Apr.18 , 2016

Apr. 20, 2016

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BRD of the LabCyte CORNEA-MODEL24 EIT

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Transportation periods of the LabCyte CORNEA-MODEL24 were taken between two and fourdays from shipping from the supplier (J-TEC, Japan) to arrive to Korean laboratory (KonKuk
University, Korea).

6-3.2. Results of Negative Control in the LabCyte24 EIT after Shipment
Using three batches of the LabCyte CORNEA-MODEL24 after shipment, the LabCyte24 EIT
was performed according to the improved protocol which was described in the SOP ver.2.5.2(Appendix 1) at the Korean laboratory (Konkuk University). Results of the negative control (bothLiquid and Solid chemical protocol) of three independent batches were shown in Table 6-2.
Table 6-2.

OD

and

SD

of

negative

controls

in

the

EIT

using

LabCyteCORNEA-MODEL24 after shipment to Korean laboratory.

Physical state of
Negative Control

Batch No.

Tissue Viability
OD(450nm/650nm)

Judge

SD (%)

Judge

Liquid (D-PBS)

0.811

Adapt

14.0

Adapt

Solid (Not treat)

0.810

Adapt

6.0

Adapt

Liquid (D-PBS)

0.779

Adapt

17.9

Adapt

Solid (Not treat)

0.629

Adapt

8.4

Adapt

Liquid (D-PBS)

1.070

Adapt

10.6

Adapt

Solid (Not treat)

1.022

Adapt

9.9

Adapt

LCC24-160215-A

LCC24-160229-A

LCC24-160418-A

OD of the cell viability of the negative control in three independent batches of LabCyte
CORNEA-MODEL24 after shipment are all adapted to the acceptance criteria (0.5 < OD < 1.3)
of LabCyte24 EIT (Table 6-2).
Also, SD (%) of the cell viability of negative control in three independent batches of LabCyte
CORNEA-MODEL24 after shipment are all adapted to the acceptance criteria (SD < 20%) of
LabCyte24 EIT (Table 6-2).
From

these

resultsonthenegative

control,itwassuggested

that

LabCyteCORNEA-MODEL24 after shipment to outside of Japan might be able to be useful for the
LabCyte24 EIT.

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6-3.3. Results of Positive Control in LabCyte24 EIT after Shipment
Using three batches of LabCyte CORNEA-MODEL24 after shipment, the LabCyte24 EIT was
performed according to the improved protocol which was described in the SOP ver.2.5.2(Appendix 1) at the Korean laboratory (Konkuk University). Results of positive control(both

Liquid and Solid chemical protocol) of 3 independent batches were shown in Table 6-3
Table 6-3.

Cell viability and SD of positive control for EIT using LabCyte
CORNEA-MODEL24 exported to Korean laboratory.

Lot No.

Cell viability

Physical state of
Positive Control

Cell Viability (%)

Judge

SD (%)

Judge

Liquid (Ethanol)

12.9

Adapt

4.6

Adapt

Solid (Lauric acid)

13.3

Adapt

10.2

Adapt

Liquid (Ethanol)

15.4

Adapt

6.8

Adapt

Solid (Lauric acid)

0.9

Adapt

0.5

Adapt

Liquid (Ethanol)

4.5

Adapt

4.6

Adapt

Solid (Lauric acid)

1.2

Adapt

0.2

Adapt

LCC24-160215-A

LCC24-160229-A

LCC24-160418-A

The cell viability (%) of positive control in three independent batches of LabCyte
CORNEA-MODEL24 after shipment are all adapted to the acceptance criteria (Cell Viability <40%) of LabCyte24 EIT (Table 6-3).
Also, SD (%) of the cell viability of positive Control in three independent batches of LabCyte
CORNEA-MODEL24 after shipment are all adapted to the acceptance criteria (SD < 20%) of
LabCyte24 EIT (Table 6-3).
From

these

resultsonthepositive

control,itwassuggested

that

LabCyteCORNEA-MODEL24 after shipment to outside of Japan might be able to be use for the
LabCyte24 EIT.

6-3.4. Results of Proficiency Chemicals in LabCyte24 EIT
In older to confirm whether it is able to use the EIT with LabCyte CORNEA-MODEL24 after
shipping, its test was performed in Korean laboratory according to the improved protocol which
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described in the SOP ver.2.5.2 (Appendix 1) using proficiency test chemicals (6 GHS Cat 1/2
chemicals and 7 GHS No Cat Chemicals) listed in the OECD TG492.
Table 6-4.

Prediction results of proficiency chemicals in the OECD TG492 by EIT using
LabCyte CORNEA-MODEL24 exported toProficiency chemical

Tetraethylene glycol diacrylate
2,5-Dimethyl-2,5-hexanediol
Sodium oxalate

Physicalstate

In vivo
GHS CatKorean laboratory.

LabCyte24 EIT
in Konkuk Univ.
Viability (%)*1

EpiOcular EIT(the OECD TG492 VRM)

Judge

Viability (%)

Judge

SD
1.9
0.3
1.2

Cat.1/2
Cat.1/2
Cat.1/2

34.9
2.3
29.0

Cat.1/2
Cat.1/2
Cat.1/2

Liquid
Solid
Solid

Cat.1
Cat.1
Cat.1

mean
17.9
5.3
7.1

LiquidCat 2A

4.4

0.3

Cat.1/2

4.0

Cat.1/2

LiquidCat 2B

31.3

0.4

Cat.1/2

15.6

Cat.1/2

Solid

Cat 2B

6.7

2.5

Cat.1/2

4.7

Cat.1/2

LiquidNo Cat51.4

11.0

No Cat79.9

No CatLiquidLiquidNo CatNo Cat

92.7
76.0

4.1
15.0

No CatNo Cat81.7
104.2

No CatNo CatViscusNo Cat61.8

3.6

No Cat77.6

No CatSolid

No Cat75.3

7.1

No Cat106.7

No CatSolid

No Cat83.4

5.1

No Cat102.7

No CatSolid

No Cat54.1

3.4

No Cat88.6

No Cat2,4,11,13-Tetraazatetradecanedi
imidamide,N,N''-bis(4-chlorophenyl)-3,12-diimino-,
di-Dgluconate (20%, aqueous)
Diethyl toluamide
2,2-Dimethyl-3-methylenebicycl
o [2.2.1] heptane
1-Ethyl-3-methylimidazolium
ethylsulphate
Dipropyl disulphide
Piperonyl butoxide
Polyethylene glycol (PEG-40)
hydrogenated castor oil
1-(4-Chlorophenyl)-3(3,4-dichlorophenyl) urea
2,2'-Methylene-bis-(6-(2H-benzo
triazol-2-yl)-4-(1,1,3,3-tetrameth
ylbutyl)-phenol)
Potassium tetrafluoroborate*1: To refer th description of the OECD TG492 (OECD, 2015a).

The prediction of the eye irritation of a proficiency test chemical in the LabCyte24 EIT wasparallel with in vivo GHS classification perfectly in the eye irritation test conducted in a Korean
laboratory.
From these results, it was concluded that LabCyte24 EIT was able to be performed outside ofJapan.

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6-4. Discussion and Conclusion
Using three dependent batch of the LabCyte CORNEA-MODEL24 after the shipment fromJapan to Korean laboratory, in vitro eye irritation test was performed in the Korean laboratory
and the results of the negative control and the positive control were adapted in the acceptancecriteria in three batches. From such results are suggested that the performance of the LabCyte
CORNEA-MODEL24 after shipping to outside of Japan is enough usefulness for the eyeirritation testing.
Furthermore, from that the prediction of eye irritancy of the proficiency chemical is correctlypredicted by LabCyte24 EIT, it is concluded that the LabCyte24 EIT is be able to performoutside of Japan.

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7. VALIDATION STUDY OF LabCyte24 EIT ACCORDING TO THE PERFORMANCESTANDARD FOR THE OECD TG492
7-1. The Performance Standard for the OECD TG 492
The OECD document, series on Testing & Assessment No 216 includes PerformanceStandard in vitro RhCE test methods for identifying chemicals not requiring classification andlabelling for eye irritation or serious eye damage, based on the validated reference method
EpiOcular EIT described in the OECD TG 492 (OECD, 2015a), has been declassified andpublished in 2015 (OECD, 2015b). This PERFORMANCE STANDARD consists of; (i) Essestial
Test Method Components; (ii) Minimum List of Reference Chemicals, and; (iii) Defined
Reliability and Accuracy Values that the proposed test method should meet or exceed.
In the (i) Essential Test Method Components of the PERFORMANCE STANDARD, it was
described about the general conditions and the functional conditions of RhCE model for the EIT
method according the OECD TG492 condition and it was also explained specifically procedural
conditions of the VRM EIT. In the general condition and functional conditions of LabCyte
CORNEA-MODEL24 are detail described in the section 2.2.
In the next section, needed essential assay component of RhCE EIT for the OECD TG492
and the corresponding LabCyte CORENA-MODEL EIT are shown.

7-2. Difference between LabCyte24 EIT and the OECD TG492 VRM Procedural
Conditions
In the Table 7-1, it was described about each needed assay component between the RhCE
EIT method in the OECD TG492 and LabCyte24 EIT.

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Table 7-1.

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Test component of LabCyte24 EIT

Test component(Requiredper

LabCyte24 EIT

EpiOcular EIT

Human corneal epithelial cells

Human keratinocytes

Overnight incubation

Pre-soak incubation

3 tissues

2 tissues

PERFORMANCESTANDARD for OECD TG 492.)

Cell source(Relevant human-derived cells)

Pre-exposure(To select if necessary)

Tissue replicates(Min. of 2 tissues)

Application of test chemical
Quantity(Uniformity)

Negative control

Liquid

Solid

50 μL
(167 μL/cm )

Solid

50 μL

10 mg
2

Dulbecco’s

Liquid2(33 mg/cm )

50 mg
2

(83 μL/cm )(83 mg/cm2)

Phosphate
Not apply

Ultrapure H2OEthanol

Lauric acid

Methyl acetate

Application period

1 minute

24 hours(Determine as appropriate.)(Optimized )*1(Optimized )Post-exposure soak

None

None

(Optimize as appropriate)(Not required)(Not required)

Post-application period

24 hours

0 hours(Optimize as appropriate)(Optimized )(Determine as appropriate.)

Buffered Saline (D-PBS)

Positive control(Determine as appropriate.)

Cell viability measurement

*1

*1

*1

30 minutes

6 hours

12 minutes

25 minutes

2 hours

18 hours(Optimized )WST-8 assay

MTT assay

40%

60%

(MTT assay)

Cell viability threshold value(Determine as appropriate.)

Colored: Using living tissue
Detection and correction of
WST-8/MTT interference

Using killed tissue

MTT reducer: Using Killedtisue
2 tissue (difference ofAcceptance criteria SD≤ 20%

viability): ≤ 20%
If 3 tissue: ≤ 18%*1) Refer to previous report (Katoh, 2012)

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Difference of test component of Between the OECD TG492 VRM and the LabCyte24 EIT are
followings;(1) Cell source(2) Replication of RhCE tissue(3) Chemical application(4) Negative control and positive control(5) Chemical exposure pattern(6) Measurement of tissue viability(7) Detection and correction of WST-8/MTT interference(8) Acceptance criterion (SD)

7-3. Comparison and Similarity Consideration about Each Test Component of
Between the VRM in the Performance Standard and LabCyte24 EIT
7-3.1. Cell Source
In the PERFORMANCE STANDARD for the OECD TG 492 (OECD, 2015b), relevant
human-derived cells (e.g., human corneal epithelial cells or keratinocytes) should be used to the
RhCE tissue, tissue, which should be composed of progressively stratified but not cornified cells.
LabCyte CORNEA-MODEL24 is used human corneal epithelial cells, which should becomposed of progressively stratified but not cornified cells.
About cell source of RhCE tissues, LabCyte CORNEA-MODEL24 tissue is concordance with
the PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD, 2015b).

7-3.2. Replication of RhCE Tissues
In the PERFORMANCE STANDARD for the OECD TG 492 (OECD, 2015b), at least two
tissue replicates should be used for each test chemical and each control substance in each run.
In the LabCyte24 EIT, three tissue replicates use each chemicals and controls substance.
About replication of RhCE tissues, the LabCyte24 EIT protocol is concordance with the
PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD, 2015b).

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7-3.3. Amount of Chemical Application
In the PERFORMANCE STANDARD for the OECD TG 492 (OECD, 2015b), a sufficientamount of the chemical or control substance should be applied to uniformly cover the corneal
epithelial surface while avoiding an infinite dose. In the VRM, Liquid chemical is applied about
80 μL/cm2 and Solid chemicals applied 80 mg/cm2. On the other hands, in the LabCyte24 EIT,
the Liquid chemical is applied about 165 μL/cm2 and the Solid chemical is applied 33 mg/cm2.
Application amounts of the Liquid chemicals and Solids chemical in the LabCyte24 EIT aresatisfied the condition described in the PERFORMANCE STANDARD for the OECD TG 492(applied to uniformly cover the corneal epithelial surface while avoiding an infinite dose).
About chemical application amounts, LabCyte24 EIT protocol was concordance with the
PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD, 2015b).

7-3.4. Negative Control and Positive Control
In the PERFORMANCE STANDARD for the OECD TG 492, separate the negative control andthe positive control are needed for each protocol of the test method. In the VRM, the positive
control and the negative control substances used neat methyl acetate and ultra-pure H2O for
both Liquid chemicals and Solid chemicals. In the LabCyte24 EIT, the positive control and thenegative control substances used ethanol and D-PBS for Liquid chemicals and lauric acid andno treatment for Solid chemicals. Concurrent the negative control and the positive controls
should be included in each run demonstrate that the viability and the sensitivity determined withthe positive control of the tissues are within acceptance ranges defined based on historical data.
The concurrent negative control provides the baseline to calculate the relative percent viabilityof the tissues treated with the test chemicals.
About selection and setting of negative and positive control, LabCyte24 EIT protocol wasconcordance with the PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD,
2015b).

7-3.5. Chemical

Exposure,

Post-exposure

ImmersionandPost-exposure

Incubation Periods
In the PERFORMANCE STANDARD for the OECD TG 492, it was described that two different
treatment protocols may be used for different types of chemicals, for Liquid test chemicals or for

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Solid test chemicals. If different protocols are used to an EIT, they may differ in terms of their
exposure periods, post-exposure incubation immersion periods and post-exposure incubation
periods.
In order to establish suitable exposure and post-exposure incubation periods for the new
RhCE EIT using the LabCyte CORNEA-MODEL24 tissue, a protocol optimization study was
performed as described in the section 3-1 of the BRD. In the study for liquid chemicals, theprediction of LabCyte24 EIT only resulted in high correlation to the in vivo classification whenthe exposure was set to a short period (1 minute), as well as post-exposure incubation was set
to a long period (24 hours). In contrast, in the study for solid chemicals, a long exposure period(24 hours) was required to eliminate false-negative predictions in the LabCyte24 EIT. Throughthe protocol optimization study, the chemical exposure and post-exposure incubation periodsfor LabCyte24 EIT were set to 1 minute and 24 hours for liquid test chemicals, and 24 hours
without post-exposure incubation for solid test chemicals.
About selection and setting of the chemical exposure pattern, LabCyte24 EIT protocol wasconcordance with the PERFORMANCE STANDARD requirement.

7-3.6. Measurement of Tissue Viability
7-3.6.1. Cell Viability Measurement with Tetrazolium-Salt
In the RhCE EIT test method for the OECD TG 492, the cell viability is measured as an
endpoint for the prediction of eye irritation. In the OECD TG 492, MTT assay is selected bymeasurement of cell viability. MTT assay is one of tetrazolium reduction assay. MTT is reducedby cellular dehydrogenase and produced insoluble MTT formazan with blue color. A variety oftetrazolium salts which are reduced by cellular dehydrogenase as same manner of MTT assay
and changed to water soluble formazan charging various colors have been developed.
Commonly used tetrazolium salts are included MTT, MTS, XTT, WST-1 and WST-8 (Table 7-2).
Table 7-2.

Commonly used tetrazolium salts
Water solubility ofAbbreviation

Chemical nameproducing formazan
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H

tetrazolium

MTT

Insoluble
bromideMTS

3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-ssoluble106/121

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ulfophenyl)-2H-tetrazolium
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)
XTT

Solublecarbonyl]-2H-tetrazolium hydroxide
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H

WST-1

Solubletetrazolium, monosodium salt
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfoph

WST-8

Solubleenyl)-2H tetrazolium, monosodium salt

If cellular dehydrogenase are rapidly inactivated by damaging cells, degree of coloring byformazan dye is correlate reduced because amount of cellular dehydrogenase is related the cell
viability. Therefore, it can be quantified the cell viability by the determination of such coloring
degree of formazan dye. Such tetrazolium reduction assay is widely accepted as simple test
method for analysis of cell viability.
While many kinds of tetrazolium salts have been developed, MTT has been widely used oneof the tetrazolium salt, which is also selected the EpiOcular EIT for the OECD TG492 VRM.
On the other hands, in the LabCyte24 EIT, WST-8 assay which produced water soluble
formazan is selected as the determination of the cell viability. WST-8 was demonstrated to be ofvalue for use as indicator for cell viability with higher sensitivity than those of conventional
tetrazolium salts including MTT (Tominaga et. al., 1999).
The outline of a MTT assay and WST-8 assay is described to next section and it is mentioned
about the similarity of the both method.

7-3.6.1a. MTT Assay
A MTT assay is widely selected a tetrazolium reduction assay in worldwide. MTT assay is
selected in skin irritation test method using the reconstructed human epidermal tissue for theOECD TG 439.

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Fig.7-1. MTT assay: Reaction mechanism.

MTT penetrates through cell membrane and concentrate into the mitochondria in the cytosol.
MTT is reduced by dehydrogenase through NADH and NADPH which are a coenzyme and thenproduced blue MTT formazan in the cytosol (Fig.7-1). For MTT formazan is water-insoluble, it isprecipitated as crystals of needle-like structure in the cytosol. MTT formazan is solubilized withappropriate organic solvent such as isopropanol and then it is determined the OD (absorbancearound 570nm) of solubilized solution.
On the other hand, because MTT formazan precipitate as an insoluble crystal in the viable cell,
it induces highly cell toxicity and then their phenomenon might cause a decline of the sensitivityof assay.

For the solubilizing process of MTT formazan is indispensable, assay variability

might increase by such process.
In order to solve these problem, several tetrazolium salts to produce water-soluble formazan,such as MTS, XTT, WST-8 and WST-8 has been developed (Table 7-2). Among them, WST-8
has developed as an useful tetrazolium salt produces WST-8 formazan with highly
water-soluble potency (Ishiyama et. al., 1997).

7-3.6.1b. WST-8 Assay
WST-8 assay is one of tetrazolium reduction assay as same manner of MTT assay and it
widely used recently.

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http://www.dojindo.com/store/p/456-Cell-Counting-Kit-8.html

Fig.7-2. WST-8 assay: Reaction mechanism.
WST-8 is reduced to an orange-colored WST-8 formazan through electron mediator,
1-Methoxy PMS by NADH and NADPH activities which are generated by cellulardehydrogenase, as indicated in the Fig. 7-2. The amount of WST-8 formazan is dependent on
the activity of cellular dehydrogenase, so WST-8/1-Methoxy PMS system can be used todetermine the number of living cells and cell viability. For WST-8 formazan is highly
water-soluble, it can be directly determined OD (absorbance around 450 nm) of its culture
medium without an extraction step. Cell Counting Kit-8, which is a one-bottle solution of the
combination both WST-8 and 1-methoxy PMS, is commercially available in worldwide.
Because WST-8 assay is not required both extraction step of MTT formazan and preliminary
preparation step by the use of Cell Counting Kit-8, its assay periods is reduced substantially

7-3.6.2. Similarity between MTT Assay and WST-8 Assay
The reaction principle of both WST-8 assay and MTT assay are same, because both test
methods are commonly one of the tetrazolium reduction assay which determine the cellulardehydrogenase activity.

7-3.6.2a. Additional Data: Comparison of the LabCyte CORNEA-MODEL24 QC
data

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WST-8 assay OD (A450/650)

1.500

1.000

0.500
y = 0.9557x
0.000
0.000

0.500

1.000

1.500

MTT assay (OD A570/650)

R=0.77

Fig.7-3. WST-8 assay and MTT assay in LabCyte CORNEA-MODEL24 QC results

In the quality control of LabCyte CORNEA-MODEL24, it is performing both WST-8 assay and
MTT assay as cell viability test against every batch.

The coefficient of correlation (R) between

WST-8 assay and MTT assay indicated about 0.77 (Fig. 7-3) and it is shown that the results ofboth tetrazolium assay are highly correlate.

7-3.6.2b.

Additional Data: Time Course Experiment

In order to confirm linearity about both MTT reaction and WST-8 reaction, the time course
experiment of WST-8 reaction and MTT reaction are examined in the LabCyte24 EIT at
Mandom Co. Ltd. The results showed in Fig. 7-4.

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1.600

1.400

1.200

OD

1.000

0.800

WST-8

MTT

0.600

0.400

0.200

0.000

0

1

2

3

4

5

6

Periods (hour)Fig.7-4. Time Courses of WST-8 reaction and MTT reaction in LabCyte24 EIT.

ODs of reaction medium of both WST-8 assay and MTT assay are lineally increased
according to reaction periods.
From above results and reaction principles of tetrazolium reduction assay, it was supposedthat both reactions reflect of the same cellular dehydrogenase activity.

7-3.6.3. Advantage of WST-8 Assay to MTT Assay
Since WST-8 formazan is water soluble, it does not form crystals like MTT. Therefore, after
the incubation with the WST-8 solution, OD of reaction medium can be directly analyzed OD thenumber of viable cells.
From dissolution step such as MTT formazan are not required in the WST-8 assay, theholding periods of WST-8 assay is shorter to compare with MTT assay (CCK-8 catalog;
Attachment 2). Less handling time for WST-8 assay might contribute to more accurate test
results.

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7-3.6.4. Similarity of LabCyte24 EIT to the OECD TG492 VRM
Reaction principle of WST-8 assay is same to that of MTT assay which is the tetrazolium
reduction assay to utilize dehydrogenase activity, and then it is concluded that special quality of
WST-8 assay can be mostly covered one of MTT assay.
Furthermore, from the knowledge and the additional data confirmed so far, it is judged themeasurement data from WST-8 assay is almost equal to a MTT assay.
Finally, LabCyte24 EIT VMT judged that measurement method of the cell viability of the
LabCyte24 EIT was similar to the OECD TG 492 VRM and its protocol was concordance with
the PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD, 2015b).

7-3.7. Detection and Correction of MTT/WST-8 Assay Interference
A possible limitation of this EIT might that there might be some test chemicals that will affect
the WST-8/MTT endpoints directly. Colored chemicals and/or WST-8 reducer may interfere with
the MTT/WST-8 assay.
In the LabCyte24 EIT, both coloring chemicals and WST-8 reducer were detected using same
protocol using WST-8 solution (see the Section 8.1.3) macroscopically and coloring interference
were corrected used killed tissue (see the Section 8.1.3) as same way of WST-8 reducer.
However, VRM in the PERFORMANCE STANDARD for the OECD TG 492 is detected coloring
interference by spectral analysis and coloring interference is corrected using living tissue
without MTT reaction. The reason why LabCyte24 EIT adopted a common protocol for bothtypes of interference is because the WST-8 detection system can sufficiently detect and correctcoloring chemicals, as well as WTS-8 reducers. This was determined taking into consideration
the fact that the adsorption and residual pathway of both types of chemicals are similar in bothliving and killed tissues.
Because the principle of detection and correction of coloring interfering chemicals adopted by
the LabCyte24 EIT and the VRM of OECD TG492 is considered similar, LabCyte24 EIT protocol
is in concordance with the PERFORMANCE STANDARD requirement for the OECD TG 492(OECD, 2015b) with regards to the detection and correction of WST-8/MTT assay interference
chemicals.

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7-3.8. Acceptance Criterion SD
In the OECD TG 492 (OECD, 2015a), it was defined that SD value between three tissues
should not exceed 18%. On the other hand, acceptance criterion of SD has been set in the
LabCyte24 EIT, that was referred accept limit of difference of viability between two tissue
replicates in the EpiOcular EIT was ≤ 20%. VMT has judged that 20% of SD acceptance criteria
was permitted because the difference between ≤ 20% and ≤ 18% might be little.

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8. CONCLUSION
Through the optimization study and several pre-validation studies described in below thesection 3, it has been developed the improved LabCyte24 EIT is a robust and reliable method toaddress the initiating event of eye irritation.
Using wide ranged chemicals (139 chemicals), the LabCyte24 EIT demonstrated highpredictive performance (88.2% overall accuracy, 100% overall sensitivity, and 73.8% overall
specificity).
From the results of LabCyte24 EIT in the Korean laboratory, the performance of the LabCyte
CORNEA-MODEL24 after shipping to outside of Japan is enough usefulness for the eye irritation
testing and the LabCyte24 EIT is able to perform outside Japan.
Finally, the VMT of the LabCyte24 EIT judged the improved LabCyte CORNEA-MODEL24 issimilar assay method to the RhCE EIT VRM for the OECD TG 492 (OECD, 2015a). Therefore,
the me-too validation study of LabCyte24 EIT is planned according to the PERFORMANCESTANDARD for the OECD TG 492 in older to assess its reliability (reproducibility within andbetween laboratories) and its relevance (predictive capacity).

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9. ACKNOWLEDGEMENT
We would like to thank for the planning committee in JSAAE, because the ring study (seebelow Section 3-2) has been realized based on technical transfer course let by the planningcommittee in JSAAE.

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10. REFERENCES
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11. APENDECS
Appendix 1.

Eye irritation test protocol using the reconstructed human model “LabCyte
CORNEA-MODEL24” Ver.2.5.2

Appendix 2.

Cell counting kit-8 catalog.

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