The LabCyte CORNEA-MODEL24 EIT

February, 2017

ME-TOO VALIDATION REPORTVALIDATION STUDY FOR LabCyte CORNEA-MODEL24 EYE
IRRITATION TEST

Prepared by:
LabCyte Validation Management Team

Version 2.5
February, 2017

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February, 2017

TABLE OF CONTENTS
Page Number
MEMBERS OF LabCyte CORNEA-MODEL EIT VALIDATION MANAGEMENT TEAM .... 5
DATA ANALYSIS GROUP ................................................................................................... 5
OBSERVER ......................................................................................................................... 5
PATICIPATING LABORATORIES ....................................................................................... 5
LIST OF ACRONYMS AND ABBREVIATIONS................................................................... 6
APPENDICES ..................................................................................................................... 8
1. GOAL STATEMENT ...................................................................................................... 9
1-1.
1-2.

The Ultimate Goal ........................................................................................................... 9Primary Goal ................................................................................................................... 9

2. OBJECTIVE ................................................................................................................ 10
3. BACKGROUND .......................................................................................................... 11
4. RECONSTRUCTED HUMAN CORNEAL EPITHELIAL MODEL ............................... 12
4-1.
4-2.

LabCyte CORNEA-MODEL24 ....................................................................................... 12
Model Supplier .............................................................................................................. 12

5. COMPARISON OF ESSENTIAL TEST METHOD COMPONENTS BETWEENLabCyte24 EIT AND VRM OF THE PERFORMANCE STANDARD FOR THE OECD
TG492 ......................................................................................................................... 14
5-1. The Performance Standard for the OECD TG 492 .......................................................... 14
5-2. Essential Differences between Test Method Components of LabCyte24 EIT and OECD
TG492 VRM ................................................................................................................... 14
5-3. Comparison and Similarity Considerations between Each Test Component of
LabCyte24 EIT and the VRM in the Performance Standard ....................................... 16
5-3.1.
5-3.2.
5-3.3.
5-3.4.

Cell Source ..........................................................................................................................16
Number of RhCE Tissue Replicates ...................................................................................17
Chemical Application Amount ............................................................................................17
Negative Control and Positive Control...............................................................................175-3.5.
5-3.6.
5-3.7.
5-3.8.

Chemical Exposure, Post-exposure Immersion and Post-exposure Incubation Periods 18
Measurement of Tissue Viability ........................................................................................18
Detection and Correction of MTT/WST-8 Assay Interference ............................................20
Acceptance Criterion (SD) ..................................................................................................20

5-4. Similarity of LabCyte24 EIT to OCED TG 492 VRM ........................................................ 21

6. VALIDATION MANAGEMENT STRUCTURE ............................................................. 22
6-1.
6-2.
6-3.

Validation Management Team ...................................................................................... 22
Trial Coordinator (VMT Chairperson) .......................................................................... 23
Chemical Selection, Acquisition, Coding, and Distribution ....................................... 23

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6-4.
6-5.
6-6.
6-7.
6-8.
6-9.

February, 2017

Data Analysis Group .................................................................................................... 24
Record Management Group ......................................................................................... 24
Lead Laboratory ........................................................................................................... 24
VMT Observers ............................................................................................................. 24
Participating Laboratories ........................................................................................... 25
Sponsorship ................................................................................................................. 25

7. STUDY DESIGN.......................................................................................................... 26
7-1.
7-1.1
7-1.2

7-2.
7-2.1
7-2.2
7-2.3
7-2.4

7-3.
7-4.

Test Chemicals ............................................................................................................. 26
Chemical Selection .............................................................................................................26
Coding and Distribution......................................................................................................26

Defined Reliability and Accuracy Value ...................................................................... 28
Within-Laboratory Reproducibility .....................................................................................28
Between-Laboratory Reproducibility .................................................................................28
Predictive Capacity .............................................................................................................28
Study Quality Criteria..........................................................................................................29

Data Collection, Handling, and Analysis ..................................................................... 29
Quality Assurance and GLP ......................................................................................... 30

8. PROTOCOL ................................................................................................................ 31
8-1.
8-1.1
8-1.2

Protocol of the LabCyte24 EIT ..................................................................................... 31
LabCyte24 EIT Protocol for Liquid Chemicals ...................................................................31
LabCyte24 EIT Protocol for Solid Chemicals .....................................................................32

8-1.3
Detecting and Correcting Chemical Interference with WST-8 Endpoints .........................32
8-1.3.1 PRELIMINARY TEST ..............................................................................................................32
8-1.3.2 FUNCTIONAL CHECK ON VIABLE TISSUE...........................................................................33

8-2.
8-3.
8-3.1
8-3.2
8-3.3

8-4.

Prediction Model for the LabCyte24 EIT ..................................................................... 33
Acceptance Criteria ...................................................................................................... 34
Negative Control .................................................................................................................34
Positive Control ..................................................................................................................34
Standard Deviation .............................................................................................................35

Applicability Domain and Limitation ........................................................................... 35

9. RESULTS .................................................................................................................... 36
9-1.
9-2.
9-3.
9-4.
9-5.
9-6.
9-6.1
9-6.2

9-7.
9-8.

QC of the Tissue Models .............................................................................................. 36
Quality Assurance ........................................................................................................ 36
Negative Control ........................................................................................................... 37
Positive Control ............................................................................................................ 38
Predicting Eye Irritation Potential of the 30 Test Chemicals ..................................... 41
Reliability ...................................................................................................................... 43
Within-Laboratory Reproducibility .....................................................................................43
Between-Laboratory Reproducibility .................................................................................43

Predictive Capacity ...................................................................................................... 44
Study Quality Criteria ................................................................................................... 47

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February, 201710. DISCUSSION .............................................................................................................. 48
10-1.
10-2.
10-3.
10-4.

In Consideration of Invalid Runs ................................................................................. 48
Reliability ...................................................................................................................... 48
Predictive Capacity ...................................................................................................... 49
Similarity with the OECD TG 492 VRM ........................................................................ 49

11. CONCLUSION ............................................................................................................ 51
12. ACKNOWLEDGEMENT ............................................................................................. 52
13. REFERENCES ............................................................................................................ 53

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MEMBERS OF LabCyte CORNEA-MODEL EIT VALIDATION MANAGEMENT TEAM
Mr. Satoshi Nakahara

Maruishilabo Corp.

Mr. Hajime Kojima

JaCVAM: Japanese Centre for the Validation of Alternative Methods

Mr. Masakazu Katoh

Japan Tissue Engineering Co. Ltd.

DATA ANALYSIS GROUP
Mr. Takashi Omori

Kobe Univ.

Ms. Yuko Satake

Doshisha Univ.

OBSERVER
Mr. Jeong IK Lee

Konkuk Univ.

PATICIPANTING LABORATORIESDrug Safety Testing Center Co., Ltd.

Mr. Shinsuke Shinoda, Ms. Saori Hagiwara

Fujifilm Corp.

Mr. Toshihiko Kasahara, Mr. Yusuke Yamamoto

Nihon Kolmar Co., Ltd.

Mr. Hidefumi Ikeda, Mr. Hideki Nishiura

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February, 2017

LIST OF ACRONYMS AND ABBREVIATIONSBCOP:Bovine Corneal Opacity and Permeability

BLR:

Between-Laboratory Reproducibility

BRD

Background Review Document

CAS:

Chemical Abstracts Service

D-PBS:

Dulbecco’s Phosphate Buffered Saline

EBSS:

Earle’s Balanced Salt Solution

EIT:

Eye Irritation Test

FL:

Fluorescein Leakage

GHS:

Globally Harmonized System of Classification and Labeling of Chemicals

GLP:

Good Laboratory Practice

ICE:

Isolated Chicken Eye

JaCVAM:

Japanese Centre for the Validation of Alternative Methods

JSAAE:

Japanese Society for Alternative to Animal Experiments

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

QC:

Quality Control

RhCE:

Reconstructed human Corneal EpitheliumSD:Standard Deviation

SLS:

Sodium Lauryl Sulfate

SOP:

Standard Operating Procedure

STE

Short Time Exposure

TG:

Test GuidelineUN:

United Nations

VMT:

Validation Management Team

VRM:

Validated Reference Method

WLR:

Within-laboratory reproducibility

WNT:

Working Group of the National Coordinators of the Test Guideline Project

WST-1

Water soluble tetrazolium salt-12-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H

tetrazolium,monosodium salt
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WST-8:

February, 2017

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

XTT-1

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

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February, 2017

APPENDICES
Appendix 1:

Study Plan ver.2.0.

Appendix 2:

Datasheet_v2.5.2. (Japanese)

Appendix 3.

Material data sheet (Japanese)

Appendix 4.

Result of QC for each lot of LabCyte CORNEA-MODEL24

Appendix 5:

LabCyte CORNEA-MODEL24 validation study phase 1 and 2 Statistical analysis
report.

Appendix 6:

LabCyte CORNEA-MODEL24 validation study phase 1 and 2 Statistical analysis
report Supplement 1.

Appendix 7:

LabCyte CORNEA-MODEL24 EYE IRRITATION TEST OPERATION PROTOCOL
Ver. 2.5.6.

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1.

February, 2017

GOAL STATEMENT

1-1. The Ultimate Goal
The ultimate goal of this test method is partial replacement of the regulatory Draize eyeirritation test (OECD TG 405; OECD, 2012) in a bottom-up approach.

1-2. Primary Goal
The primary goal of this me-too validation study is to assess the within- andbetween-laboratory reproducibility, as well as the predictive capacity, of the LabCyte
CORNEA-MODEL24 Eye Irritation Test (LabCyte24 EIT) in accordance with PERFORMANCESTANDARD (OECD, 2015b) for OECD TG 492.

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2.

OBJECTIVE

The OECD Working group of the National Coordinators of the Test Guideline Project (WNT)
accepted OECD TG No. 492 in vitro eye irritation test guideline in July, 2015 (OECD, 2015a).
OECD TG 492 addresses the human health endpoint ―eye irritation.‖

One validated test

method that conforms to this TG and for which pre-validation, optimization, and validation
studies have been completed is an in vitro test method using a Reconstructed human Corneal
Epithelial tissue (RhCE) model. This method is commercially available as EpiOcular and has
been designated as Validated Reference Method (VRM).
The LabCyte24 EIT is another in vitro test method that employs a new RhCE model known as
the LabCyte CORNEA-MODEL24 and for which protocol optimization has been completed. The
objective of this me-too validation is to confirm that the LabCyte24 EIT conforms to OECD TG
492 by assessing its within- and between-laboratory reproducibility, as well as its predictive
capacity. The study was performed using a set of 30 test chemicals selected to meet
PERFORMANCE STANDARD for OECD TG 492. This validation study was undertaken inaccordance with the principles and criteria documented in the OECD Guidance Document on
the Validation and International Acceptance of New or Updated Test Methods for Hazard
Assessment (No. 34, OECD, 2005) and according to the Modular Approach to validation(Hartung et. al. 2004).

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3.

February, 2017

BACKGROUND

To date, a number of in vitro test methods have been developed as alternatives to the Draize
eye test (Draize et. al., 1944). In particular, the following tests have been found to predict ocular
irritancy adequately and were adopted as OECD TG.
Bovine Corneal Opacity and Permeability (BCOP) test (TG 437, OECD 2013a)
Isolated Chicken Eye (ICE) test (TG 438, OECD 2013b)
Fluorescein Leakage (FL) test (TG 460, OECD 2012)
Short Time Exposure (STE) test (TG491, OECD 2015c)
RhCE EIT (TG 492, OECD 2015a).
Since the use of a battery of in vitro tests is considered a viable means of improving overall
accuracy, there is a clear need for the development of additional in vitro test methods.
A newly developed RhCE tissue model known as the LabCyte CORNEA-MODEL24 wasrecently developed using human corneal epithelial cells. The histological structure of the
LabCyte CORNEA-MODEL24 is considered highly similar to that of a native human corneal
epithelium. The LabCyte CORNEA-MODEL24 EIT (LabCyte24 EIT) provides a promisingalternative to animal testing in assessing corneal irritation. Protocol optimization was studied inorder to establish a new alternative method for eye irritancy evaluation using the LabCyte
CORNEA-MODEL24 tissue. Through protocol optimization, the ring-study for the technicaltransferability and several pre-validation studies, an improved LabCyte24 EIT was finallyestablished as shown in the Background Review Document (BRD) for the LabCyte24 EIT.
Upon review, the LabCyte24 EIT Validation Management Team (VMT) determined that the
improved LabCyte24 EIT was similar to the EpiOcular EIT, which is a VRM of OECD TG 492.
Therefore, a me-too validation study of the LabCyte24 EIT was planned in accordance withperformance standard for OECD TG 492. On the advantages in terms of cost and animal
welfare, LabCyte24 EIT is considered comparably equal to the VRM of OECD TG 492.

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4.

February, 2017

RECONSTRUCTED HUMAN CORNEAL EPITHELIAL MODEL

4-1. LabCyte CORNEA-MODEL24
The LabCyte CORNEA-MODEL24 is a commercially available RhCE model produced by
Japan Tissue Engineering Co. Ltd. It comprises normal human corneal epithelial cells that arederived from a human cornea, which cells are neither cornified nor keratinized. The cells arecultured with 3T3-J2 cells as a feeder layer in order to expand them while maintaining theirphenotype (Rheinwald and Green, 1975; Green, 1978). Reconstruction of the human cultured
corneal epithelial tissue is achieved by cultivating and proliferating the corneal epithelial cells on
an inert filter substrate with a surface area of 0.3 cm2 at an air-liquid interface for 13 days using
an optimized medium containing 5% fetal bovine serum (FBS). This results in the formation of amultilayer and non-cornified structure comprising a fully differentiated corneal epithelium withfeatures mimicking those of a normal human corneal epithelium described in the section 2-1 of
the BRD. For delivery, LabCyte CORNEA-MODEL24 tissues are embedded in an agarose gel
containing a nutrient solution and shipped.
Even if the lots of some raw materials (e.g. FBS) change over time, quality and performanceof each batch of LabCyte CORNEA-MODEL24 is controlled to ensure a highly stable production.
Also the timing of FBS lot change was indicated in the QC information of each batch of LabCyte
CORNEA-MODEL24, and different FBS lots have not affect QC data of the LabCyte
CORNEA-MODEL24, as described in the section 2-2 of the BRD.

4-2. Model Supplier
According to OECD Good Laboratory Practice (GLP) Consensus Document No. 5―Compliance of Laboratory Suppliers with GLP Principles‖, responsibility for the quality andfitness for use of equipment and materials rests entirely with the management of the test facility(OECD, 1999).
The acceptability of equipment and materials in laboratories complying with GLP must
therefore be guaranteed to any regulatory authority to which studies are submitted. In somecountries where GLP has been implemented, suppliers belong to national regulatory or
voluntary accreditation schemes that can provide users with additional documentation provingthat they are using a test system of defined quality.
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Audits performed during the study focused on procedures established to guarantee a definedquality of the tissue models.

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5.

February, 2017

COMPARISON OF ESSENTIAL TEST METHOD COMPONENTS BETWEENLabCyte24 EIT AND VRM OF THE PERFORMANCE STANDARD FOR THE OECD
TG492
5-1. 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) Essential
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.
The (i) Essential Test Method Components of the PERFORMANCE STANDARD describes
the general and functional conditions of the RhCE model for the EIT method according theOECD TG492 and specifically explains the procedural conditions of the VRM EIT. The generaland functional conditions of LabCyte CORNEA-MODEL24 are described in detail in the BRD.
Essential test method components of RhCE EIT for the OECD TG492 and the corresponding
LabCyte24 EIT are shown as follows.

5-2. Essential Differences between Test Method Components of LabCyte24 EIT and
the OECD TG492 VRM
Difference between test components of LabCyte24 EIT and the OECD TG492 VRM are
shown in Table 1.

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Table 1February, 2017

Description of test components for the RhCE EIT (OECD TG 492)

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 )Phosphate

2(33 mg/cm )

50 mg
2

(83 μL/cm )(83 mg/cm2)

Does Not
Ultrapure H2O

Buffered Saline (D-PBS)

apply

Ethanol

Lauric acid

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)

Solid

50 μL

10 mg
2

Dulbecco’s

LiquidPositive control
Methyl acetate(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.)

Detection and correction of
WST-8/MTT interference

Using killed tissue

Colored: Using living tissue
MTT reducer: Using Killed tissue
2 tissue (difference ofAcceptance criteria SD≤ 20%

viability): ≤ 20%
If 3 tissue: ≤ 18%*1) Refer to previous report (Katoh, 2012)15 /54

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Difference between test components of LabCyte24 EIT and OECD TG492 VRM are as
follows:(1) Cell source(2) Number of RhCE tissue replicates(3) Chemical application(4) Negative control and positive control(5) Chemical application period(6) Measurement of tissue viability(7) Detection and correction of WST-8/MTT interference(8) Acceptance criterion (SD)

5-3. Comparison and Similarity Considerations between each Test Component
LabCyte24 EIT and the VRM of the Performance Standard
5-3.1. 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 in the
RhCE tissue, which should be composed of progressively stratified but not cornified cells.
LabCyte CORNEA-MODEL24 comprises normal human corneal epithelial cells that are derived
from a human cornea, which cells are neither cornified nor keratinized. This structure is amultilayered and non-cornified structure comprising a fully differentiated corneal epithelium withfeatures mimicking those of a normal human corneal epithelium (Fig. 1), as described in thesection 2-1 of the BRD.

Fig. 1.

Structure of LabCyte CORNEA-MODEL24

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Concerning the cell source of RhCE tissues, LabCyte CORNEA-MODEL24 tissue isconcordance with the PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD,
2015b).

5-3.2. Number of RhCE Tissue replicatesIn 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 are used for each chemical and control substance.
Concerning the number of RhCE tissue replicates, the LabCyte24 EIT protocol is inconcordance with the PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD,
2015b).

5-3.3. Chemical Application AmountIn 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, approximately 80 μL/cm2 of liquid
chemicals, and 80 μL/cm2 of solid chemicals are applied. On the other hand, in the LabCyte24
EIT, approximately 165 μL/cm2 of liquid chemicals, and 33 mg/cm2 of solid chemicals areapplied. Application amounts of the liquid and solid chemicals in the LabCyte24 EIT satisfy thecondition described in the PERFORMANCE STANDARD for the OECD TG 492 (applied touniformly cover the corneal epithelial surface while avoiding an infinite dose).
Concerning chemical application amounts, LabCyte24 EIT protocol is in concordance with the
PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD, 2015b).

5-3.4. Negative Control and Positive Control
In the PERFORMANCE STANDARD for the OECD TG 492 (OECD, 2015b), separate
negative and positive controls are needed for each protocol of the test method. In the VRM, thepositive control and the negative control substances used are neat methyl acetate andultra-pure H2O, respectively, for both liquid and solid chemicals. In the LabCyte24 EIT, thepositive control and the negative control substances used are ethanol and D-PBS for liquid

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February, 2017

chemicals, and lauric acid and no treatment for solid chemicals. Concurrently, the negative
control should be included in each run to demonstrate that the viability and the sensitivity
determined with the positive control of the tissues are within acceptance ranges defined basedon historical data, which is described in the section 3-2, 3-3, and 3-4 of the BRD. The concurrent
negative control provides the baseline to calculate the relative percent viability of the tissues
treated with the test chemicals.
Concerning selection and setting of negative and positive control, LabCyte24 EIT protocol is inconcordance with the PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD,
2015b).

5-3.5. Chemical

Exposure,

Post-exposure

ImmersionandPost-exposure

Incubation Periods
In the PERFORMANCE STANDARD for the OECD TG 492 (OECD, 2015b), it was describedthat two different treatment protocols may be used for different types of chemicals, for Liquid
test chemicals or for 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 andpost-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.
Concerning the selection and setting of the chemical exposure pattern, LabCyte24 EIT
protocol is in concordance with the PERFORMANCE STANDARD requirement.

5-3.6. Measurement of Tissue Viability
In the RhCE EIT test method for the OECD TG 492, the cell viability is measured as an
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February, 2017endpoint for the prediction of eye irritation. In the OECD TG 492, MTT assay is selected for themeasurement of cell viability. MTT assay is a tetrazolium reduction assay. MTT is reduced bycellular dehydrogenase and produces insoluble MTT formazan with blue color. A variety oftetrazolium salts which are reduced by cellular dehydrogenase in the same manner as the MTT
assay and change to water soluble formazan charging various colors have been developed.
Commonly used tetrazolium salts include MTT (which produces water-insoluble formazan),
MTS, XTT, WST-1 and WST-8 (which do not produce water-soluble formazan).
Since cellular hydrogenase is rapidily inactivated by damaged cells, the degree of coloring byformazan dye directly correlates to cell viability. Therefore, the cell vialibity can be quantified
through the determination of such coloring degree of formazan dye. Such tetrazolium reduction
assay is widely accepted as a simple test method for analysis of cell viability.
While many kinds of tetrazolium salts have been developed, MTT is the one that has been
widely used, and is the assay is selected for the EpiOcular EIT for the OECD TG492 VRM.
On the other hand, in the LabCyte24 EIT, WST-8 assay, which produces water soluble
formazan, was selected to determine cell viability. The principle of the WST-8 assay is the sameas the MTT assay. Furthermore, since the WST-8 assay does not require an extraction step, theprocedure is much simpler compared to the MTT assay.
WST-8 was demonstrated to be of value for use as an indicator for cell viability with higher
sensitivity than conventional tetrazolium salts, including MTT (Tominaga et. al., 1999).
The outline of MTT assay and WST-8 assay is described in BRD.
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 toobtain the number of viable cells. Unlike MTT, a dissolution step is not required in the WST-8
assay. This leads to shorter process periods, which in turn might contribute to more accurate
test results compared to the MTT assay.
The reaction principle of WST-8 assay is the same to that of MTT assay, as both aretetrazolium reduction assays that utilize dehydrogenase activity. Furthermore, from theknowledge and highly correlated results between WST-8 assay and MTT assay, described in
BRD, it is assumed that the measurement data from assays are almost equal.
Finally, the LabCyte24 EIT VMT determined that the measurement method of the cell viabilityof the LabCyte24 EIT was similar to that of the OECD TG 492 VRM, and its protocol was inconcordance with the PERFORMANCE STANDARD requirement for the OECD TG 492 (OECD,
2015b).

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February, 20175-3.7. Detection and Correction of MTT/WST-8 Assay Interference
A possible limitation of this EIT might be some test chemicals that directly affect the
WST-8/MTT endpoints. Colored chemicals and/or WST-8 reducers may interfere with the
MTT/WST-8 assay.
In the LabCyte24 EIT, both coloring chemicals and WST-8 reducers were detected using thesame protocol; coloring interference is corrected using killed tissues (see the Section 8.1.3).
However, the VRM in the PERFORMANCE STANDARD for the OECD TG 492 detects coloring
interference by spectral analysis and this interference is corrected using living tissues without
MTT reaction. The reason why LabCyte24 EIT adopted a common protocol for both types ofinterference is because the WST-8 detection system can sufficiently detect and correct coloringchemicals, as well as WTS-8 reducers. This was determined taking into consideration the factthat the adsorption and residual pathway of both types of chemicals are similar in both living andkilled 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.
Note
The VMT further requested that the protocol be updated to change detection and correction
protocols for coloring chemicals according to OECD T G492 VRM (Appendix 7).

5-3.8. Acceptance Criterion (SD)In the OECD TG 492 (OECD, 2015a) it is defined that the SD value between three tissues
should not exceed 18%. On the other hand, in the LabCyte24 EIT, the acceptance criterion of
SD has been set to ≤ 20%, which is the accepted limit of difference of viability between two
tissue replicates in the EpiOcular EIT. VMT considered that a SD of 20% was acceptablebecause the difference between ≤ 20% and ≤ 18% is little.
However, in this validation report, it is reported that the reliability and the predictive
performance of LabCyte24 EIT should be evaluated with the SD set to ≤ 18%, because the VMTdetermined that the data analysis of the validation study should be in accordance with theacceptance criteria of OECD TG 492.
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February, 20175-4. Similarity of LabCyte24 EIT to OCED TG 492 VRM
Based on the points described in the section 5-3, the VMT considers the LabCyte24 EIT to bea derivative of the RhCE EIT method described in OECD TG 492, and this validation study for
LabCyte24 EIT was planned in accordance with PERFORMANCE STANDARD for OECD TG
492 (OECD, 2015b).

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6.

February, 2017

VALIDATION MANAGEMENT STRUCTURE

This validation study was funded in part by the Japanese Society for the Alternative to Animal
Experiments (JSAAE).
The management structure is shown in Fig. 2.

Validation Management Team
Chair

Biostatistician
Kobe Univ.

Maruishilabo Corp.

Doshisha Univ.

Study Sponsor
JSAAE

Lead
laboratory&
Training
J-TEC

Chemical coding
and distribution
Record Management

ObserverKonkuk Univ.

JaCVAM

Nihon Kolmar Co., Ltd.

Fig. 2.

Fujifilm Corp.

Drug Safety Testing
Center Co., Ltd.

Management structure for me-too LabCyte24 EIT validation study

6-1. Validation Management TeamA Validation Management Team (VMT) was organized to ensure that the LabCyte24 EIT was
validated smoothly and in a scientifically pertinent manner.
The VMT comprised of a trial coordinator, a chemical management group, a data analysis
group, a record management group, and the lead laboratory (assay developer). The leadlaboratory provided support to the participating laboratories. The VMT was responsible forpreparing, reviewing, and finalizing a draft study plan and a study protocol. In addition, the VMT
was also responsible for managing the validation study in terms of monitoring its progress,
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The LabCyte CORNEA-MODEL24 EIT

February, 2017assuring the quality of study records, and communicating with the participating laboratories.
The VMT played a central role in overseeing the conduct of the validation study, including theplanning and implementation of the following:
1.

Goal statement

2.

Project plan including objective

3.

Study protocol / amendments

4.

Outcome of quality control (QC) audits

5.

Test chemicals

6.

Data management procedures

7.

Timeline / study progression

8.

Data collection and analysis

9.

Study interpretation and conclusions

10.

Reports and publications

6-2. Trial Coordinator (VMT Chairperson)
Trial coordinator was Satoshi Nakahara of Maruishilabo Corp. He declared no conflict ofinterest associated with this validation study. He was independently responsible for preparing a
draft study plan (Appendix 1), study protocol, and test chemical list, as well as for convening ad
hoc meetings of the VMT for reviewing and finalizing the content of these documents, and allother aspects of conducting the validation study.

6-3. Chemical Selection, Acquisition, Coding, and Distribution
The chemical management group was chaired by Hajime Kojima of the Japanese Center for
the Validation of Alternative Test Method (JaCVAM) and included other JaCVAM staff members.
The validation study was conducted in accordance with the PERFORMANCE STANDARD for
EpiOcular EIT, which is a VRM of the RhCE EIT VRM, and the chemical management group
was not involved in selecting the chemicals.
The test chemicals used for this validation study were encoded and distributed to theparticipating laboratories by JaCVAM. Thus, the participating laboratories performed the tests
without knowing the identity of the test chemicals. The coding procedure used in these studies
is not known, and the test chemicals thus coded were used only for this validation study.
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February, 2017

6-4. Data Analysis GroupThe Data Analysis Group was headed by Takashi Omori of Kobe University and included Mai
Endo and Mayuko Satake, both of Doshisha University. They declared no conflict of interest
associated with this validation study. The Data Analysis Group performed an independent
third-party review of the data obtained in this validation study. They were responsible forindependent statistical processing to ensure that measured values were recorded appropriately
in the data sheets (Appendix 2).

6-5. Record Management GroupThe Record Management Group was headed by Hajime Kojima and included JaCVAM staff
members.
The Record Management Group prepared material datasheets (Appendix 3) and distributed
them to the participating laboratories. They also collected filled out forms and data sheets after
completion of experiments, pointing out omissions or flaws in recording, if any, and requestingcorrection of such errors.

6-6. Lead Laboratory
J-TEC provides support to the participating laboratories as the lead laboratory.

6-7. VMT Observers
Each participating laboratory sent a representative to observe VMT proceedings. These
representatives were responsible for supervising implementation of the testing by laboratory
personnel and for creating and submitting all data records and other required documentation to
the Record Management after completion of all testing.
Additionally, Jeong Ik Lee of Konkuk University, Korea, was invited to observe VMTproceedings as part of an international validation per the PERFORMANCE STANDARD.

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February, 2017

6-8. Participating Laboratories
Validation of the LabCyte24 EIT with thirty test chemicals was performed at the following
participating laboratories.
Laboratory A (Lab A): Drug Safety Testing Center Co., Ltd.; Shinsuke Shinoda, Saori Hagiwara
Laboratory B (Lab B): Nihon Kolmar Co., Ltd.; Hidefumi Ikeda, Hideki Nishiura
Laboratory C (Lab C): Fujifilm Corp.; Toshihiko Kasahara, Yusuke Yamamoto
All participants underwent training for the LabCyte24 EIT in the ring-study for the technicaltransferability described in the section 3-2 of the BRD. Through several pre-validation studies by
their laboratories described in the section 3-3 and 3-4 of the BRD, participation of thelaboratories on the validation study were permitted by agreement of VMT.
J-TEC, which was lead laboratorie, is a company part of the Fujifilm Corp. group. Fujifilm Corp.
participated as an independent laboratory and declared no conflict of interest associated withthis validation study.

6-9. Sponsorship
The study was managed and funded jointly by JSAAE, Maruishilabo Corp., JaCVAM, and
J-TEC.
1) JSAAE provided funding.
2) Maruishilabo Corp. provided support for:- management of the validation study3) JaCVAM provided funding for:- management of the validation study- purchase, coding, and distribution of chemicals to the participating laboratories- independent QC audit of the data
- publication of the validation study results4) J-TEC provided support as lead laboratory for:- training the participating laboratories- independent QC audit of the LabCyte24 EIT- funding the participating laboratories

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7.

February, 2017

STUDY DESIGN

7-1. Test Chemicals
7-1.1

Chemical Selection

The VMT selected thirty reference chemicals from the PERFORMANCE STANDARD forOECD TG 492 (OECD, 2015b), as shown in Table 3. Reference Chemicals are used todetermine if the reproducibility and predictive capacity of a proposed test method are equal to or
better than the defined minimum values of an existing VRM, thus demonstrating that the
proposed test method is both structurally and functionally similar to an existing VRM.
This validation study was set in two phases (Table 2), because the VMT considered that thetesting process of all 30 reference chemicals at once using one batch would exceed theperforming capacity of each participating laboratory.
Table 2. Breakdown of substances used for the LabCyte24 EIT validation study
Phase

No. of the substances

No. of the repetitions

I

18

3

Examination
Within and between laboratory
reproducibility and predictivity
Within and between laboratory

II

12

3
reproducibility and predictivity

Division of thirty test chemicals for each phase was managed by the chemical management
group independently, with consideration to proportionally distribute between phases thesubstances classified by the United Nations (UN) Globally Harmonized System (GHS) as No
category, Category 1 and 2.

7-1.2

Coding and Distribution

The coding and distribution of test chemicals was subcontracted by JaCVAM to a reliableindependent laboratory. A list of the coded chemicals is shown in Table 2. A single coding was
given to each chemical, but not to each repeat chemical.

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February, 2017

Table 3. Reference Chemicals list in the OECD TG 492
No
Test chemical

CAS number.

PhysicalIn vivo

State

class

Chemical CodeSupplier

Phase
Lab A

Lab B

Lab C

1

(Ethylenediaminepropyl)-trimethoxysilane

1760-24-3

LiquidCat 1

Sigma-Aldrich

LaA101

LaB106

LaC127

II

2

Methylthioglycolate

2365-48-2

LiquidCat 1

Sigma-Aldrich

LaA112

LaB101

LaC130II

3Tetraethylene glycol diacrylate

17831-71-9

LiquidCat 1

Sigma-Aldrich

LaA113

LaB111

LaC101I

4

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

2634-33-5

Solid

Cat 1

WAKO*1

LaA124

LaB115

LaC104II

5

110-03-2

Solid

Cat 1

Sigma-Aldrich

LaA125

LaB120

LaC107

I27344-41-8

Solid

Cat 1

WAKO

LaA102

LaB124

LaC110I

62-76-0

Solid

Cat 1

Sigma-Aldrich

LaA111

LaB102

LaC113I

18472-51-0

LiquidCat 2A

Sigma-Aldrich

LaA114

LaB107

LaC116I

92,5-Dimethyl-2,5-hexanediol
Disodium
2,2'-([1,1'-biphenyl]-4,4'-diyldivinylene)bis- (benzenesulphonate)
Sodium oxalate
2,4,11,13-Tetraazatetradecane-diimidami
de,N,N''-bis(4-chlorophenyl)-3,12-diimino-,
di-D-gluconate (20%, aqueous)
Gamma-Butyrolactone

96-48-0LiquidCat 2A

Aldrich

LaA123

LaB112

LaC119I

101,5-Naphthalenediol

83-56-7

Solid

Cat 2ATCI*2

LaA126

LaB116

LaC122

II
I

6
7
8

11

Sodium benzoate

532-32-1

Solid

Cat 2A

Sigma-Aldrich

LaA103

LaB121

LaC125

12

2-Methyl-1-pentanol

105-30-6

LiquidCat 2B

Sigma-Aldrich

LaA110

LaB125

LaC128I

13Diethyl toluamide

134-62-3

LiquidCat 2B

Sigma-Aldrich

LaA115

LaB103

LaC102

II

14

104-36-9

Solid

Cat 2BTCI

LaA122

LaB108

LaC105

II

79-92-5

Solid

Cat 2B

Aldrich

LaA127

LaB117

LaC108I342573-75-5

LiquidNo CatSigma-Aldrich

LaA104

LaB122

LaC111I

17

1,4-Dibutoxy benezene
2,2-Dimethyl-3-methylenebicyclo [2.2.1]
heptane
1-Ethyl-3-methylimidazolium
ethylsulphate
2-Ethoxyethyl methacrylate

2370-63-0

LiquidNo CatSigma-Aldrich

LaA109

LaB126

LaC114I

18

3-Phenoxybenzyl alcohol

13826-35-2

LiquidNo CatSigma-Aldrich

LaA116

LaB130

LaC117II

19

4-(Methylthio)-benzaldehyde

3446-89-7

LiquidNo CatSigma-Aldrich

LaA121

LaB104

LaC120II

20

Dipropyl disulphide629-19-6

LiquidNo CatSigma-Aldrich

LaA128

LaB109LaC123I

21

Ethyl thioglycolate

623-51-8

LiquidNo CatSigma-Aldrich

LaA105

LaB113

LaC126II

22

Piperonyl butoxide
Polyethylene glycol (PEG-40)
hydrogenated castor oil
1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl) urea2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol
2,2'-Methylene-bis-(6-(2H-benzotriazol-2yl)-4-(1,1,3,3-tetramethylbutyl)-phenol)
4,4'-Methylenebis-(2,6-di-tert-butylphenol)
Cellulose, 2-(2-hydroxy-3-(trimethylammonium) propoxy) ethyl
ether chloride (91%)
Potassium tetrafluoroborate
Trisodiummono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo- 2,5-dihydro-furan-3-yl) phosphate51-03-6

LiquidNo CatSigma-Aldrich

LaA108

LaB118

LaC129I

61788-85-0

Viscous

No CatWAKO

LaA117

LaB127

LaC103

I

101-20-2

Solid

No CatSigma-Aldrich

LaA120

LaB129LaC106

II3179-89-3

Solid

No CatAK Scientific

LaA129

LaB105

LaC118II103597-45-1

Solid

No CatSigma-Aldrich

LaA106

LaB110

LaC112

I

118-82-1

Solid

No CatSigma-Aldrich

LaA107

LaB114

LaC115I

68610-92-4

Solid

No CatSigma-Aldrich

LaA118

LaB119LaC109I

14075-53-7

Solid

No CatSigma-Aldrich

LaA119

LaB123

LaC121

I

66170-10-3

Solid

No CatSigma-Aldrich

LaA130

LaB128

LaC124

II

15
16

23
24
25
26
27
28
29
30

*1) WAKO: Wako Pure Chemical Industries, Ltd.
*2) TCI: Tokyo Chemical Industry Co., Ltd.

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February, 20177-2. Defined Reliability and Accuracy ValueAll thirty of the reference chemicals listed in Table 2 were tested at each of the threeparticipating laboratories, in order to evaluate the reliability and relevance of the LabCyte24 EIT.
Each laboratory performed three independent runs for each test chemical with different tissue
batches and at sufficient time intervals. Each run comprised three concurrently tested replicatesfor each test chemical, negative control, and positive control.

7-2.1

Within-Laboratory Reproducibility

The VMT decided that the target value for within-laboratory reproducibility of the LabCyte24
EIT should be equal to or better than a value derived from a VRM of OECD TG 492. Since theresults for the EpiOcular EIT were 93%, 93% and 97% at three participating laboratories, thetarget for this validation study of the LabCyte24 EIT was set to 90% or higher.

7-2.2

Between-Laboratory Reproducibility

The VMT decided that the target value for between-laboratory reproducibility of the
LabCyte24 EIT should be equal to or better than a value derived from a VRM of OECD TG 492.
Since the results for the EpiOcular EIT was 90% concordance between the three participating
laboratories, the target for this validation study of the LabCyte24 EIT was set to 85% or higher.

7-2.3

Predictive Capacity

The VMT decided that the target value for accuracy (sensitivity, specificity, and overall
accuracy) of the LabCyte24 EIT should be equal to or better than a value derived from a VRM
of OECD TG 492, as well as suitable for the species of interest, as shown in Table 4. Thus, thetarget value for sensitivity was set to be equal to or higher than 90%, and that for specificity was
set to be equal to or higher than 60%. No restrictions to the sensitivity or specificity wereapplied; non-concordance with the in vivo classification of any test chemical was acceptablejust as long as the final sensitivity and specificity of the test method achieved the target value,
and the overall accuracy was equal to or higher than 75%.
The restriction was added, however, that none of the UN GHS Category 1 reference
chemicals were to be under-predicted as no category from valid test results from the

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February, 2017

participating laboratories.
Table 4.

7-2.4

Required predictive values for sensitivity, specificity and overall accuracy.
Sensitivity

Specificity

Overall Accuracy

≥ 90%

≥ 60%

≥ 75%

(EpiOcular EIT: 93%)

(EpiOcular EIT: 63.0%)(EpiOcular EIT: 78.2%)

Study Quality Criteria

In the event that a test result is determined to be invalid due to failure to meet acceptancecriteria for the test chemical, control chemicals, or any other reason, a maximum of two
additional retests per test chemical are permitted to augment the data set. Since retesting
requires concurrent testing with a positive control and negative control, a maximum number of
two additional runs are permitted for each test chemical.
It is conceivable that, even after retesting, one or more of the participating laboratories will failto obtain a minimum of three valid runs for each test chemical, which could result in an
incomplete data matrix. A dataset is considered valid, however, as long as the following threecriteria are all met:
1) There is one complete test sequence for each of the thirty test chemicals at any one of thethree participating laboratories.
2) Each of the three participating laboratories must achieve a minimum of 85% complete test
sequences. This means that, there are no more than four incomplete test sequences out of
30 test sequences at any one laboratory.
3) At least 90% of all the test sequences at all three laboratories must be complete. Thismeans that, for 30 test chemicals each at three participating laboratories, there are a total ofno more than nine incomplete test sequences out of 90 test sequences in total.
In this context, the term ―test sequence‖ refers to the total number of independent tests
performed for a single test chemical in a single laboratory, including retests, or between three tofive tests. A complete test sequence comprises three valid test results. A test sequencecomprising less than three valid test results is considered incomplete.

7-3. Data Collection, Handling, and Analysis

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Working in close collaboration with JaCVAM, the independent biostatistician collected andorganized the data using custom data collection software, which included decoding the codedchemicals and performing statistical analyses using statistical tools that were approved by theVMT.

7-4. Quality Assurance and GLP
All participating laboratories conducted testing in the spirit of GLP.
Quality assurance of all the data and records was performed by JaCVAM. After completion of
all testing, all study documents were submitted to the chairperson of VMT and only data sheets
were forwarded by e-mail to the biostatistician. The chairperson reviewed the contents of thestudy documents and clarified illegible or unclear content by contacting each group by e-mail or
telephone.

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February, 20178.

PROTOCOL

8-1. Protocol of the LabCyte24 EIT
Prediction of eye irritation potential of test chemicals using the LabCyte24 EIT was performed
according to the protocol described in LabCyte CORNEA-MODEL24 Eye Irritation Test
Operation Protocol Ver. 2.5.2. Test Operation Protocol Ver. 2.5.2 was also used to estimate thepredictive performance of the LabCyte24 EIT method using 136 test chemicals over a wide
range of chemical classes described in BRD.
The LabCyte CORNEA-MODEL24 tissues were shipped to the participating laboratories, andthe tissues were aseptically removed from the agarose medium, placed in wells containing 500
μL of assay medium, and then incubated overnight at 37°C in a humidified atmosphere of 5%
CO2 in air.

8-1.1 LabCyte24 EIT Protocol for Liquid Chemicals
After incubation, the tissue was topically exposed to 50 μL of a liquid test chemical, which wasapplied with a micropipette. Each test chemical was applied to three tissue replicates.
Additionally, three tissue replicates were treated with 50 μL of Dulbecco’s Phosphate Buffered
Saline (D-PBS, Invitrogen, CA, USA) as a negative control and three more with ethanol as apositive control. The exposed tissue replicates were then incubated for one minute. Next, eachtissue was carefully rinsed at least ten times with D-PBS applied from a washing bottle toremove any residual test chemical from the tissue surface. The tissues were then blotted andplaced in new wells containing 500 μL of fresh assay medium. The tissues were again
incubated for 24 hours at 37°C in a humidified atmosphere of 5% CO 2 in air. After incubation,
the tissues were blotted and placed in new wells containing 300 μL of freshly prepared WST-8
solution for a WST-8 assay (Ishiyama et. al., 1997). WST-8 solution comprised a 1:10 dilution of
Cell Counting Kit-8 (Dojindo Co., Japan) with Earle’s balanced salt solution (EBSS;
Sigma-Aldrich, MO, USA). The tissues were again incubated for 4 hours at 37°C in a humidified
atmosphere of 5% CO2 in air. Finally, 200 μL of culture supernatant was placed in a microtiterplate and the optical density (OD) value of the culture supernatant was measured at 450 nm andat 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 cell viability was calculated as the ratio of the OD of the test chemical to OD of negative
control. The mean of three replicate tissues was used to predict eye irritation potential of the test
chemical according to the prediction model.

8-1.2 LabCyte24 EIT Protocol for Solid Chemicals
After incubation, the tissue replicates were placed in 500 μL of fresh assay medium andtopically exposed to 10 mg of a solid test chemical from a microtube. Each test chemical wasapplied to three replicate tissues. Additionally, three untreated tissue replicates were set apartfor use as a negative control and three more were treated with Lauric acid as a positive control.
The exposed tissue replicates were incubated for 24 hours and then carefully rinsed with D-PBS
just like the liquid chemicals. Solid chemicals were not incubated after exposure to test
chemicals. After the rinsing, the tissue replicates were blotted and analyzed for cell viability
using the WST-8 assay, just like the liquid chemicals.

8-1.3

Detecting and Correcting Chemical Interference with WST-8 Endpoints

A possible limitation of this EIT might be some test chemicals that will affect the WST-8
endpoints directly. There are two kinds of test chemicals that can interfere with the WST-8
assay.
A.

Chemicals that stain epithelial tissues directly.

B.

Chemicals that reduce WST-8 directly.

Test chemicals that stain the corneal epithelial tissues could possibility transfer from thecorneal epithelial tissue to the WST-8 reaction buffer and affect OD measurements.
Test chemicals that reduce WST-8 directly could possibly affect OD measurements if the test
chemical is present in the corneal epithelial tissues when the WST-8 viability test is performed.
A procedure for detecting such test chemicals is described below.

8-1.3.1 PRELIMINARY TEST
WST-8 solution was prepared by making a 1:10 dilution of the Cell Counting Kit-8 with EBSS.
Either 50 μL of a liquid test chemical or 10 mg a solid test chemical are added to the wells of a

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February, 201724-well assay plate that contains 0.3 mL of WST-8 solution in each well. Untreated WST-8
solution is used as control. Close the lid of 24-well assay plate and incubate the mixture in CO2
for about 4 hours. After incubation, shake the mixture gently and examine the staining of thediluted WST-8 medium macroscopically.

8-1.3.2 FUNCTIONAL CHECK ON VIABLE TISSUE
For each test chemical that clearly changed the color of the diluted WST-8 solution in the
preliminary test above, apply either 50 μL of liquid test chemicals or 10 mg of solid test
chemicals to the surface of corneal epithelial tissues. Perform all steps of the LabCyte24 EIT
described in sections 7-1.1 to 7-1.2 using corneal epithelial tissues that have been freeze-killed
twice at -80°C or lower for 30 minutes instead of viable ones. Then calculate the corrected OD
as follows:
Corrected OD = A − (B − C),where:A is the OD of viable tissue exposed to a test chemical,
B is the mean OD of freeze-killed tissue exposed to a test chemical, andC is the mean OD of freeze-killed tissue exposed to the negative control.
If the corrected OD is below 0, the OD is considered to be 0.
When the cell viability is <40%, the test chemical is predicted to be GHS category 1 or 2, andthere is no need to calculate a corrected value.

8-2. Prediction Model for the LabCyte24 EIT
The in vivo eye irritation classification of test chemicals was based on the UN GHS
classification (United Nations, 2003).
According to the UN GHS , an irritant (Category 1 or 2) is predicted if the mean relative tissue
viability of three individual tissues exposed to the test chemical falls below 40% of the mean
viability of the negative control (Table 5).

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The LabCyte CORNEA-MODEL24 EIT

Table 5.

February, 2017

Prediction model for the LabCyte24 EIT
In vitro results

Prediction for UN GHS

Tissue viability is ≤ 40%

Irritant (Category 1 or 2)

Tissue viability is > 40%

Non Irritant (No Category)

8-3. Acceptance Criteria
8-3.1

Negative Control

The absolute OD of the negative control for liquid test chemicals on tissue treated with sterile
D-PBS or of the negative control for solid test chemicals on untreated tissue in the WST-8 assay
is an indicator of tissue viability obtained in the testing laboratory after shipping and storingprocedures and under specific conditions of use.
The negative control for both liquids and solids has to be tested for each run.
0.5 ≤ Mean OD (A450/650) measured value ≤ 1.3
This acceptance range of negative control OD was established from the historical QC data oftissue viability, described in the section 2-2 (Table 2-2) of BRD, and consideration reflecting
both the shipment stress of LabCyte CORNEA-MODEL24 and the stress by all test processesof LabCyte24 EIT.
It has been confirmed that acceptance lower limit (0.5 OD) could provide sufficiently reliableprediction for the LabCyte24 EIT, through the ring-study for the technical transferability andseveral pre-validation studies described in the section 3 (Fig 3-3, 3-4 and 3-6) of the BRD. Thisis supported by the assessment for reliability of LabCyte24 EIT in this validation study.

8-3.2

Positive Control

Ethanol is used as the positive control for liquid test chemicals and is tested concurrently withthe liquid test chemicals. Lauric acid is used as the positive control for solid test chemicals andis tested concurrently with the solid test chemicals.
Concurrent here means the positive control for both liquids and solids has to be tested foreach run.

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February, 2017

Mean tissue viability ≤ 40%

8-3.3

Standard Deviation

Since eye irritation potential is predicted from the mean viability of three individual tissues, thevariability of tissue replicates must be kept at an acceptably low level.
Standard Deviation (SD) of tissue viability of three identically treated replicatesfor negative control, positive control, and test chemicals ≤ 20%

Note
Results of the validation study were evaluated under the condition of SD ≤ 18%, as well as
SD ≤ 20% by the data analysis group, as reported in the statistical analysis reports (Appendices
5 and 6). In addition, the reliability and the predictive performance of LabCyte24 EIT that were
evaluated with the SD ≤ 18% acceptance criteria was reported in this validation report because
VMT had finally determined to adopt the OECD TG 492 acceptance criteria, although thepresent validation study has been performed according to the SD ≤ 20% acceptance criteria
defined as described above.

8-4. 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.
LabCyte24 EIT is applicable to substances and mixtures, and to solids, liquids, semi-solids
and waxes with the same applicability as the OECD TG 492 VRM (OECD, 2015a). The liquids
may be aqueous or non-aqueous; solids may be soluble or insoluble in water. However,
LabCyte24 EIT does not allow the testing of gases and aerosols for the same reasonsdescribed in the OECD TG492 VRM (OECD, 2015a).

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9.

RESULTS

9-1. QC of the Tissue Models
The QC data for the tissue models used in this validation study demonstrated that the tissue
viability as measured in the MTT/WST-8 assay, and barrier function as measured IC50 after
treatment for one hour with various concentrations of SLS solution, was stable among thedifferent batches provided to each laboratory, as shown in the results of QC for each lot of
LabCyte CORNEA-MODEL24 (Appendix 4). Also, all batches of LabCyte CORNEA-MODEL24
showed multilayered corneal epithelium-like tissue (at least three layers) of viable cells and a
non-keratinized surface (Appendix 4). Using this data, the VMT was able to confirm thecompleteness of the epithelial tissue layers used in this validation study. All the batches used forthe validation study had passed the manufacturer’s model supply criteria for LabCyte
CORNEA-MODEL24 (Appendix 4).

9-2. Quality Assurance
Assays and quality assurance were carried out in the spirit of GLP, although not all
participating laboratories were GLP certified. The participating laboratories conducted theexperiments according to the protocol described in ver. 2.5.2 of the SOP. All raw data and data
sheets were reviewed at each laboratory and then checked for errors and omissions by both the
VMT data analysis group and the record management group. The results accurately reflect theraw data (Appendices 5 and 6).

36 /54

The LabCyte CORNEA-MODEL24 EIT

February, 20179-3. Negative Control
Table 6 shows absorbance values for the negative control.
Table 6.

Absorbance and SD of cell viability in negative control
Phase 1Lab A

Liquid

Lab B

Lab C

1

2

3

0.79

0.54

0.58

(2.1%)(7.7%)(9.7%)

0.70

0.64

0.97

(4.6%)(5.8%)(4.0%)

0.82

0.75

0.61

Solid

Lab B

Lab C

R02

(9.3%) (11.0%) (1.1%)
0.65

Lab A

R01

Phase 2

0.51

SD

0.64

0.13

0.77

0.18

0.73

0.11

0.58

0.07

0.7

0.14

0.61

0.07

1

2

3

0.76

0.64

0.61

(9.3%)(5.4%) (10.8%)

0.72

0.61

(4.5%) (12.2%)

0.85

0.59

(8.7%)

0.60

0.71

0.61

0.61

1.08

0.70

(8.5%)(2.6%)(4.7%)(2.4%)

0.57

0.63

0.76

(4.4%) (12.3%) (11.8%)
0.54

0.78

0.62

(6.3%)(6.1%)(5.1%)

0.65

0.53

0.91

0.52

(15.6%) (8.5%) (10.9%) (11.8%) (13.5%)

Mean

SD

0.67

0.08

0.71

0.10

0.76

0.22

0.65

0.10

0.65

0.12

0.70

0.19

0.81

0.63

0.65

(11.8%) (8.0%)

R01(11.7%) (15.7%) (9.7%)

0.57(5.2%)

0.60

Mean(11.1%) (15.6%) (5.6%)

Upper row: OD (Absorbance 450nm/650nm); Lower row: SD of cell viability.

All data (19 test runs of liquid and 20 test runs for solid protocol) for the negative control met
acceptance criteria for both the OD range (0.5 ≤ Mean OD ≤ 1.3) and SD (≤ 18%). The
frequency of invalid test runs for the negative control was 0%.

37 /54

The LabCyte CORNEA-MODEL24 EIT

February, 20179-4. Positive Control
Table 7 shows absorbance values for the positive control. All data for the positive control met
all acceptance criteria.
Table 7.

Cell viability and its SD of positive control
Phase 1
1

2

3

7.0

19.3

17.9

R01

Phase 2
R02

Lab A

Liquid

(0.9)

(5.8)

(3.7)

25.9

30.3

10.5

Lab B

SD

14.7

6.7

22.2
(3.7)

(8.9)

(3.3)

14.8

16.1

21.0

Lab C

17.3
(11.2)

(1.2)

(6.5)

0.8

2.1

1.9

Lab A

Solid

Mean

1.6
(0.3)

(0.9)

(1.4)

7.7

4.5

5.5

Lab B

5.9
(2.5)

(1.4)

(1.9)

1.7

0.1

0.0

0.6

0.9
(0.3)

(0.2)

(0.0)

(0.4)

(0.5)

2

3

R01

6.4

6.9

11.6

(3.2)

(1.7)

(4.1)

22.0

34.3

24.3

(5.0)

(3.4)

(9.0)

13.7

9.6

10.3

9.0

(1.1)

(1.6)

(1.1)

(0.5)

0.9

0.7

0.9

(0.6)

(0.5)

(1.1)

9.1

4.9

4.1

(2.6)

(1.2)

(1.7)

1.0

1.8

0.3

(0.0)

(0.6)

(0.1)

10.4

3.3

0.7

1.6

1.9

Lab C

1

0.9

Mean

SD

8.3

2.9

26.9

6.5

10.7

2.1

0.8

0.1

6.0

2.7

1.0

0.6

Upper row: Cell viability (%); Lower row: SD of cell viability (%).
All data (19 test runs of liquid and 20 test runs for solid protocol) for the positive control met
acceptance criteria for both cell viability (≤ 40%) and SD (≤ 18%). The frequency of invalid test
runs for the positive control was 0%.

38 /54

The LabCyte CORNEA-MODEL24 EIT

Table 8.

February, 2017

Mean cell viability of each qualified test run for 30 reference chemicals andconcordance of prediction within-laboratory for LabCyte24 EIT

No.

UN GHS
in vivo Cat.

Lab A
WST-8
interference

12Cat. 1

Cat. 1

3

Cat. 1

4

Cat. 1

5

Cat. 1

6

Cat. 1

7

Cat. 1

8

Cat. 2A

9

Cat. 2A

10

Cat. 2A

11

Cat. 2A

12

Cat. 2B

13

Cat. 2B

14

Cat. 2B

15

Cat. 2B

1

2

Lab B
3

WST-8

6.6

5.8

15.6

interference

(1.4)

(1.8)

(8.7)

WST-8

0.8

3.9

1.2

interference

(0.0)

(0.0)

(0.0)

18.0

27.4

26.1

(1.6)

(3.7)

(3.2)

7.0

8.8

6.4

(1.0)

(3.9)

(0.5)

0.6

1.8

1.6

(0.3)

(0.8)

(0.3)

0.0

0.1

0.0

(0.0)

(0.2)

(0.0)

2.3

4.0

2.4

(0.7)

(0.6)

(0.6)

0.0

1.2

0.2

(0.0)

(1.0)

(0.2)

10.5

15.3

17.7

(4.9)

(4.8)

(11.2)

WST-8

1.9

0.0

4.8

interference

(3.3)

(0.0)

(8.4)

0.4

1.6

1.9

(0.5)

(0.5)

(0.1)

18.6

39.0

31.9

(4.5)

(5.3)

(5.9)

29.9

23.1

29.4

(11.9)

(3.6)

(6.0)

35.4

35.3

25.7

(7.4)

(5.5)

(3.8)

0.3

1.0

0.2

(0.3)

(0.2)

(0.2)

WLR

WST-8

Lab C

1

2

3

WLR

8.3
(5.2)

25.6
(2.0)

16.1
(2.9)

C

0.4
(0.7)

0.7
(1.3)

0.0
(0.0)

C

C

19.1
(3.9)

20.7
(0.8)

27.7
(11.2)

C

14.0
(3.9)

12.1
(0.3)

C

6.1
(2.1)

C

WST-8

1

2

3

0.0
(0.0)

4.9
(1.3)

5.1
(1.6)

8.0
(10.5)

30.5(45.4)

0.0
(0.0)

C

17.2
(0.9)

12.5
(2.1)

21.3
(3.4)

C

13.0
(1.1)

C

4.9
(0.7)

10.1
(1.7)

7.9
(1.0)

C

4.8
(1.4)

6.4
(1.6)

C

2.4
(0.8)

1.3
(0.5)

0.0
(0.0)

C

6.4
(2.1)

6.4
(1.0)

5.9
(3.7)

C

0.8
(0.3)

0.6
(0.3)

0.0
(0.0)

C

C

4.0
(0.8)

6.0
(0.9)

6.0
(2.0)

C

3.0
(1.0)

3.6
(0.3)

1.6
(0.5)

C

C

1.0
(0.7)

0.3
(0.3)

0.9
(0.7)

C

0.0
(0.0)

0.0
(0.0)

0.0
(0.0)

C

C

39.7
(10.0)

25.4
(7.2)

29.4
(4.9)

C

9.0
(6.0)

15.2
(2.5)

22.7
(3.0)

C

0.0
(0.0)

0.0
(0.0)

0.0
(0.0)

C

0.0
(0.0)

0.0
(0.0)

0.0
(0.0)

C

C

4.3
(1.2)

6.6
(2.5)

6.8
(3.0)

C

1.8
(0.7)

1.7
(0.6)

1.3
(0.2)

C

C

29.5
(8.4)

37.9
(1.4)

27.5
(2.1)

C

23.4
(3.7)

30.0
(2.0)

32.5
(6.9)

C

C

34.0
(6.9)

59.8
(7.4)

35.5
(7.4)

NC

29.1
(2.4)

25.2
(6.5)

25.5
(2.0)

C

C

47.8
(9.0)

27.8
(3.3)

46.9
(0.6)

NC

30.7
(8.6)

37.1
(6.8)

33.3
(2.4)

C

C

4.0
(0.8)

8.8
(9.0)

2.8
(0.2)

C

1.6
(0.3)

1.0
(0.3)

0.0
(0.0)

C

C

C

C

interference

WST-8
interference

WST-8
interference

WST-8
interference

interference

WST-8
interference

WST-8
interference

WST-8
interference

R1

R2

WLR
C

0.0
(0.0)

Upper row: viability in %, Lower row (in bracket): SD in %.
Yellow cells indicate an invalid test run.

39 /54CThe LabCyte CORNEA-MODEL24 EIT

February, 2017

(Continued)
No.

UN GHS
in vivo Cat.

Lab A
WST-8
interference

Lab B

1

2

3

WLR

16

No Cat.

40.5
(8.2)

55.2
(7.5)

51.9
(5.5)

C

17

No Cat.

37.9
(5.8)

33.0
(6.1)

25.9
(5.9)

C

18

No Cat.

52.3
(5.2)

57.0
(9.4)

55.9
(4.7)

C

19

No Cat.

63.8
(2.5)

69.4
(3.2)

81.4
(11.0)C

20

No Cat.

103.3(12.3)

78.7
(2.9)

141.0
(15.0)C

21

No Cat.

2.5
(4.3)

0.0
(0.0)

3.2
(5.6)

C

WST-8
interference

22

No Cat.

76.5
(4.5)

104.9
(14.1)

89.7
(6.6)

C

23

No Cat.

48.0
(4.8)

56.3
(8.6)

84.8
(13.2)C

24

No Cat.

68.9
(3.9)

76.3
(2.0)

73.6
(8.2)

C

25

No Cat.

0.5
(0.2)

3.5
(1.8)

1.3
(0.6)

C

WST-8
interference26

No Cat.

98.9
(6.6)

83.9
(6.4)

96.7
(18.4)C

27

No Cat.

102.9
(2.0)

80.5
(5.6)

88.8
(8.9)

C

28

No Cat.

9.1
(3.4)

23.8
(3.7)

9.2
(2.9)

C

29

No Cat.

60.7
(3.4)

56.5
(3.7)

82.3
(9.8)

C

30

No Cat.

0.0

0.0

0.0

WST-8
interference

(0.0)

(0.0)

(0.0)

C

WST-8
interference

1

2

Lab C
3

57.7

57.4

53.6

(4.4)

(8.8)

(3.4)

44.9

51.6

41.6

(9.8)

(7.9)

(3.3)

52.4

52.7

68.5

(7.5)

(9.1)

(10.8)

75.2

91.3

79.7

(7.6)

(2.2)

(13.0)

69.8

101.1

89.9

(4.6)

(10.6)

(7.3)

WST-8

12.4

0.0

0.0

interference

(7.2)

(0.0)

(0.0)

64.3

105.9

85.0

(10.6)

(7.8)

(3.7)

52.0

55.0

45.9

(10.9)

(1.9)

(5.9)

73.0

91.5

100.1

(1.9)

(8.1)

(2.9)

WST-8

0.0

5.8

2.9

interference

(0.0)

(0.9)

(0.6)

74.0

82.1

125.8

(2.0)

(12.2)

(8.8)

95.0

93.5

97.7

(7.3)

(9.9)

(7.6)

14.1

19.0

20.7

(2.0)

(3.5)

(1.5)

50.8

91.4

58.2

(4.7)

(11.3)

(5.4)

WST-8

0.0

13.5

20.6

interference

(0.0)

(1.7)

(8.5)

WLR

WST-8
interference

C

C

C

C

C

C

3

42.9

39.9

32.6

(5.4)

(6.9)

(8.1)

46.4

40.5

50.1

(11.8)(2.3)(19.7)

R1

35.8

48.8

54.4

74.6

(22.9)(7.3)(11.0)(12.8)

77.0

72.0

61.0

(12.5)(0.7)(10.0)

96.3

85.0

121.3

(5.6)

(7.5)

(3.6)

C

17.2

0.0

0.0

interference

(0.0)

(0.0)

121.0

101.4

112.1

(3.7)

(8.6)

(17.5)

91.3

57.5

72.9

(8.4)

(12.3)

59.6

82.6

74.5

(8.5)

(10.8)

(4.4)

WST-8

0.0

0.1

1.6

interference

(0.0)

(0.2)

(0.1)

C

C

C

C

C

C

(26.0)(16.4)

C

C

C

C

C

108.9

98.0

90.0

64.3

88.3

(20.0)(19.6)(22.8)(10.2)(12.3)

89.6

106.6

104.8

(11.2)

(6.9)

(5.3)

11.1

11.8

8.5

(3.7)

(0.3)

(3.8)

77.5

51.7

70.0

(11.0)

(8.1)

(5.1)

WST-8

0.0

0.0

0.0

interference

(0.0)

(0.0)

(0.0)

WLR

C

3.6

C

R2

NC

(6.2)

C

C

2

WST-8

C

C

1

C

C

C

C

C

Upper row: viability in %, Lower row: SD in %.
Yellow cells indicate an invalid test run.
Blue cells indicate non irritancy prediction (Mean cell viability >40%).
Red cells indicate irritancy prediction (Mean cell viability ≤40%).
WLR: Within-laboratory reproducibility; C: Concordance, NC: Non-concordance.

40 /54

The LabCyte CORNEA-MODEL24 EIT

February, 20179-5. Predicting Eye Irritation Potential of the 30 Test Chemicals
The results of chemical interference detection with WST-8 endpoints are shown in the Table 8.
(Ethylenediaminepropyl)-trimethoxysilane (No 1), Methylthioglycolate (No. 2), 1,5-Naphthalenediol(No. 10), Ethyl thioglycolate (No. 21), 2,2'-[[3-Methyl-4- [(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol(No. 25) and Trisodium mono-(5-(1,2 -dihydroxyethyl)-4-oxido-2-oxo- 2,5-dihydro-furan-3-yl)
phosphate (No. 30) were detected as interfering chemicals in all participating laboratories. WST-8
endpoint ODs of the chemicals were corrected using freeze-killed tissues in each participating
laboratories.

Table 8 also shows the mean viability of the test chemicals. All data from Lab B for the 30 test
chemicals met the acceptance criteria of SD (≤ 18%) and the frequency of invalid test run for test
chemicals was 0% (0/90).
On the other hand, one data point at Lab A and seven data points at Lab C showed a SD of >
18%,

which

fails

to

meet

the

acceptancecriteria.

One

test

run

of

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol (No. 26) at Lab A failed to meet
the acceptance criteria (Table 8).

Also one test run each of Methylthioglycolate (No. 2),

2-Ethoxyethyl methacrylate (No.17), 3-Phenoxybenzyl alcohol (No. 18), and Ethyl thioglycolate
(No.

21)

as

well

as

three

test

runs

of

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol (No. 26) at Lab C failed to meet
the acceptance criteria (Table 8). Three chemicals (No. 2, No.18 and No21) at Lab C were
submitted as complete data matrices by re-testing up to two times. On the other hand, chemical(No. 26) at Lab C was tested for five times and resulted three times in non-qualified results, andtherefore only two qualified tests were available for this chemical. Also, chemical (No. 26) at Lab
A and chemical (No. 17) at Lab C resulted in only two qualified test because re-testing was not
performed. The chemicals with only two qualified tests available produced three incomplete runsequences. The frequencies of invalid test runs at Lab A and Lab C were 1% (1/90) and 7%(7/95), respectively.

41 /54

The LabCyte CORNEA-MODEL24 EIT

Table 9.

February, 2017

Mean cell viability of 3 independent runs for 30 reference chemicals andconcordance of prediction between the laboratories.

No.

UN GHS
in vivo Cat.

Mean

Lab A
SD

Judge

Mean

Lab B
SD

Judge

Mean

Lab C
SD

Judge

1

Cat. 1

9.3

5.4

I

16.7

8.7

I

3.3

2.9

I

C

2

Cat. 1

2.0

1.7

I

0.4

0.4

I

2.7

4.6

I

C

3

Cat. 1

23.8

5.1

I

22.5

4.6

I

17.0

4.4

I

C

4

Cat. 1

7.4

1.2

I

13.0

1.0

I

7.6

2.6

I

C

5

Cat. 1

1.3

0.6

I

5.8

0.9

I

1.2

1.2

I

C

6

Cat. 1

0.0

0.1

I

6.2

0.3

I

0.5

0.4

I

C

7

Cat. 1

2.9

1.0

I

5.3

1.2

I

2.7

1.0

I

C

8

Cat. 2A

0.5

0.6

I

0.7

0.4

I

0.0

0.0

I

C

9

Cat. 2A

14.5

3.7

I

31.5

7.4

I

15.6

6.9

I

C

10

Cat. 2A

2.2

2.4

I

0.0

0.0

I

0.0

0.0

I

C

11

Cat. 2A

1.3

0.8

I

5.9

1.4

I

1.6

0.3

I

C

12

Cat. 2B

29.8

10.4

I

31.6

5.5

I

28.6

4.7

I

C

13

Cat. 2B

27.5

3.8

I

43.1

14.5

NI

26.6

2.2

I

NC

14

Cat. 2B

32.1

5.6

I

40.8

11.3

NI

33.7

3.2

I

NC

15

Cat. 2B

0.5

0.4

I

5.2

3.2

I

0.9

0.8

I

C

16

No Cat.

49.2

7.7

NI

56.2

2.3

NI

38.5

5.3

I

NC

17

No Cat.

32.3

6.0

I

46.0

5.1

NI

43.5*

NI

NC

18

No Cat.

55.1

2.5

NI

57.9

9.2

NI

59.3

13.6

NI

C

19

No Cat.

71.5

9.0

NI

82.1

8.3

NI

70.0

8.2

NI

C

20

No Cat.

107.7

31.4

NI

86.9

15.9

NI

100.9

18.6

NI

C

21

No Cat.

1.9

1.7

I

4.1

7.2

I

1.2

2.1

I

C

22

No Cat.

90.4

14.2

NI

85.1

20.8

NI

111.5

9.8

NI

C

23

No Cat.

63.0

19.3

NI

51.0

4.6

NI

73.9

16.9

NI

C

24

No Cat.

72.9

3.7

NI

88.2

13.8

NI

72.2

11.7

NI

C

25

No Cat.

1.8

1.5

I

2.9

2.9

I

0.6

0.9

I

C

26

No Cat.

91.4*

NI

94.0

27.9

NI

76.3*

NI

C

27

No Cat.

90.7

11.3

NI

95.4

2.1

NI

100.3

9.3

NI

C

28

No Cat.

14.0

8.5

I

17.9

3.4

I

10.5

1.7

I

C

29

No Cat.

66.5

13.8

NI

66.8

21.6

NI

66.4

13.3

NI

C

30

No Cat.

0.0

0.0

NI

11.4

10.5

NI

0.0

0.0

NI

C

BLR

I: Irritant; NI: Non irritant; BLR: Between-Laboratory Reproducibility,
C: Concordance, NC: Non-concordance.

*: Mean of two independent valid test run.

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9-6. Reliability
9-6.1
OECD

Within-Laboratory Reproducibility
TG

492

PERFORMANCESTANDARD(OECD,

2015b)

describes

thatwithin-laboratory reproducibility should be calculated based on concordance of classifications
using only qualified tests obtained with Reference Chemicals for which at least two qualified
tests are available. All participating laboratories produced two or three qualified tests from allreference chemicals in this validation study (Table 8).
An assessment of within-laboratory reproducibility was based on concordance of predictions(UN GHS Category 1/2 and No Category) obtained from replicate test runs of 30 test chemicals
at each of the participating laboratories. As shown in Table 8, Lab A consistently predicted the
30 test chemicals in three independent experiments and had a within-laboratory reproducibility
rate of 100%. Lab B had non-concordant predictions twice (No. 13 and No. 14) and had awithin-laboratory reproducibility of 93% (28/30, Table 8). Lab C had one non-concordant
prediction (No. 16) and a within-laboratory reproducibility of 97% (29/30, Table 8). Thus, the≥ 90% target for within-laboratory reproducibility was achieved at each laboratory.

9-6.2

Between-Laboratory Reproducibility

OECD TG 492 PERFORMANCE STANDARD (OECD, 2015b) describes that for the
calculation of between-laboratory reproducibility (BLR) the final classification for each
Reference Chemical in each participating laboratory should be obtained by using the arithmetic
mean value of viability over the different qualified tests performed. It also describes thatbetween-laboratory reproducibility should be calculated based on concordance of classifications
using only qualified tests from Reference Chemicals for which at least one qualified test perlaboratory is available. All participating laboratories produce two or three qualified tests from allreference chemicals in this validation study (Table 9).
An assessment of between-laboratory reproducibility was based on concordance ofpredictions (UN GHS Category 1/2 and No Category) obtained from replicate test runs of 30 test
chemicals at all three participating laboratories. As shown in Table 9, between-laboratory
reproducibility was 87% (26/30) because there were four non-concordant predictions fourth(No.13, No.14, No16 and No.17). Thus, the ≥ 85% target for between-laboratory reproducibility
was achieved.

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The LabCyte CORNEA-MODEL24 EIT

February, 20179-7. Predictive Capacity
An assessment of predictive capacity of the LabCyte24 EIT was based on the concordance
between the predictions made with data obtained during this validation study with the in vivo
categories specified in the PERFORMANCE STANDARD for OECD TG 492 (OECD, 2015b).
The 30 test chemicals chosen from the TG 492 Reference Chemicals included 15 UN GHS
Category 1/2 chemicals and 15 UN GHS No Category chemicals. OECD TG 492
PERFORMANCE STANDARD (OECD, 2015b) describes that calculation of predictive capacity(i.e., sensitivity, false negatives, specificity, false positives and accuracy) should be done using
all qualified tests obtained for each Reference Chemical from at least three laboratories andalso describes that the calculations should be based on the individual predictions of eachqualified test for each Reference Chemical in each laboratory and neither on the arithmetic
mean values of viability over the different qualified tests performed nor on the mode of all
predictions obtained (or any other procedure used to summarize the multiple test results
obtained into a single prediction per Reference Chemical). All participating laboratories
produced two or three qualified tests from all reference chemicals in this validation study (Table
8).
Lab A and Lab C predicted correctly 15 UN GHS Category 1/2 chemicals. On the other hand,
Lab B predicted correctly 13 of 15 UN GHS Category 1/2 chemicals, however one valid run ofDiethyl toluamide (No. 13) and two valid runs of 1,4-Dibutoxy benzene (No. 14) were predicted
as false-negatives in Lab B. (Table 8). Furthermore, all three participating laboratories
accurately predicted the seven UN GHS Category 1 test chemicals. Sensitivity was 100% at Lab
A, 93.3% at Lab B, and 100% at Lab C, while a cumulative two-by-two table for all threeparticipating laboratories gave a sensitivity of 97.8% (Table 10, 11).
Lab A and Lab C predicted 10 of 15 UN GHS No Category chemicals, while Lab B predicted
11 of 15 UN GHS No Category chemicals. False positives were obtained by all threeparticipating laboratories for four UN GHS No Category chemicals: Ethyl thioglycolate (No. 21),2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol

(No.

25),

Cellulose,

2-(2-hydroxy-3-(trimethylammonium)propoxy)ethyl ether chloride (91%) (No. 28), and Trisodiummono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl) phosphate (No. 30). Lab C
obtained a false positive for two valid runs of the UN GHS No Category chemical
1-Ethyl-3-methylimidazolium ethylsulphate (No. 16). Lab A obtained a false positive for UN GHS
No Category chemical 2-Ethoxyethyl methacrylate (No. 17). Thus, specificity was 65.9% at Lab
A, 73.3% at Lab B, and 67.4% at Lab C, while a cumulative two-by-two table for all threeparticipating laboratories gave a specificity of 68.9% (Table 10, 11).
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The LabCyte CORNEA-MODEL24 EIT

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Overall accuracy was 83.1% at Lab A, 83.3% at Lab B, and 84.1% at Lab C, while acumulative two-by-two table for all three participating laboratories gave an overall accuracy of
83.5% (Table 10, 11).

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The LabCyte CORNEA-MODEL24 EIT

Table 10.

February, 2017

2 × 2 tables for the each participating laboratory

Lab A

In vitro prediction

Cat 1/2
No CatTotal

Sensitivity (%)
Specificity (%)
Accuracy (%)

In vivo classification (GHS classification)
Cat 1/2
No Category
Total
45
15
60
0
29
29
45
44
89
100
65.9
83.1

Lab B

In vitro prediction

Cat 1/2
No CatTotal

Sensitivity (%)
Specificity (%)
Accuracy (%)

Cat 1/2
42
3
45
93.3
73.3
83.3

Lab C

In vitro prediction

Cat 1/2
No CatTotal

Sensitivity (%)
Specificity (%)
Accuracy (%)

Table 11.

Cat 1/2
45
0
45
100
67.4
84.1

In vivo classification
No Category
12
33
45

Total
54
36
90

In vivo classification
No Category
14
29
43

Total
59
29
88

2 × 2 tables for cumulative of the all participating laboratories

In vitro prediction
Sensitivity (%)
Specificity (%)
Accuracy (%)

Cat 1/2
No CatTotal

Cat 1/2
132
3
135
97.8
68.9
83.5

In vivo classification
No Category
41
91
132

Total
173
94
267

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The LabCyte CORNEA-MODEL24 EIT

February, 20179-8. Study Quality Criteria
In this validation study, the LabCyte24 EIT was performed on 30 test chemicals at threeparticipating laboratories. One invalid run at Lab A and seven invalid runs at Lab C occurred with SD>18%, however retesting augmented the incomplete run sequences for the acceptance criteria SD ≤
20% (Table 8), and then three incomplete run sequences (Chemical No17 at Lab C, Chemical No.
26 at Lab A and Chemical No. 26 at Lab C) were produced in the case of acceptance criteria SD ≤18% but not SD ≤ 20%.
The dataset had at least one or more than one complete test sequence for each of the thirty test
chemicals at all three participating laboratories. Thus, the target (one complete test sequence foreach of the thirty test chemicals at any one of the three participating laboratories) for the study
quality criteria was achieved.
Each Lab A, Lab B and Lab C achieved 97% (29/30), 100% (30/30) and 93% (28/30) complete
test sequence, respectively. Therefore the target (each ≥85%) for the study quality criteria wasachieved.

Also all three laboratories achieve 97% (87/90) complete test sequence. Therefore this study
quality criteria (≥90%) was achieved.

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The LabCyte CORNEA-MODEL24 EIT

10.
10-1.

February, 2017

DISCUSSIONIn Consideration of Invalid Runs

All test runs for negative and positive controls met the acceptance criteria. The VMTconcluded that the results of the positive and negative control were highly reproducible overtime (phase I and phase II) in this validation study (Table 6 and Table 7).
One test run each for Methylthioglycolate (No. 2), 2-Ethoxyethyl methacrylate (No.17),
3-Phenoxybenzyl alcohol (No. 18), and Ethyl thioglycolate (No. 21) at Lab C, as well as one test
run

at

Lab

A

and

three

test

run

at

Lab

C

for

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis -ethanol (No. 26) had a SD > 18%, which
failed to meet the acceptance criteria, so these test runs were invalid. Methylthioglycolate (No.
2) and Ethyl thioglycolate (No. 21) are strong WST-8 reducers and the VMT felt that residual
test chemicals after the washing process seem to have caused some variability in cell viability.
3-Phenoxybenzyl

alcohol

(No.

18)

and

2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol (No. 26) were difficult to remove completely during the washingprocess, because they adhere to the cell culture insert, and thus the VMT felt that residual test
chemicals in the cell culture inserts after the washing process might have caused somevariability in cell viability.
Finally the run sequences for three chemicals (No. 1, No.18 and No.21) were augmented byretesting with an improved washing procedure. The VMT proposed an improved andstandardized washing process as a means to decrease variability in cell viability andrecommended that a suitable video presentation of the washing procedure be prepared as aneffective means to train naïve laboratories planning to perform the LabCyte24 EIT. The VMTfurther requested that the protocol be updated to include precautions in the description of thewashing process (Appendix 7).
On the other hand, although three incomplete run sequences (chemical No. 17 at Lab C andChemical No 26 at Lab A and Lab C) were produced, the data quality of this validation study
was fully achieved according to the study quality criteria for OECD TG 492 PERFORMANCESTANDARD (OECD, 2015b).

10-2.

Reliability

Within-laboratory reproducibility was 100% at Lab A, 93% at Lab B, and 97% at Lab C, thus
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The LabCyte CORNEA-MODEL24 EIT

February, 2017

achieving the ≥ 90% requirement specified in the TG 492 PERFORMANCE STANDARD(OECD, 2015b).
Also, between-laboratory reproducibility for all three participating laboratories was 87%, thusachieving the ≥ 85% requirement specified in the TG 492 PERFORMANCE STANDARD.
This result demonstrates that the robustness and reliability of the LabCyte24 EIT method is
sufficient to meet the PERFORMANCE STANDARD for OECD TG 492 (OECD, 2015b).

10-3.

Predictive Capacity

The test results correctly predicted the eye irritation potential for almost all UN GHS Category
1 or 2 chemicals, with only false negative at Lab B: one valid run of Category 2B chemical
Diethyl toluamide (No. 13) and two valid runs of UN GHS Category 2B chemical 1,4-Dibutoxy
benzene (No. 14). Sensitivity was 93.3 to 100% at each of the participating, as well as 97.8% at
all three laboratories collectively, thus meeting the PERFORMANCE STANDARD for OECD TG
492 (OECD, 2015b).
In contrast, there were four false positives among the GHS No Category chemicals: Ethyl
thioglycolate (no. 21), 2,2'-[[3-Methyl-4-[(4-nitrophenyl)azo]-phenyl]imino]bis-ethanol (No. 25),
Cellulose, 2-(2-hydroxy-3-(trimethylammonium)propoxy)ethyl ether chloride (91%) (No. 28), andTrisodium mono-(5-(1,2-dihydroxyethyl)-4-oxido-2-oxo-2,5-dihydro-furan-3-yl) phosphate (No.
30). Of the No Category chemicals, Nos. 21, 25, 30 were also false positives in the EpiOcular
EIT. Moreover, although not a false positive per se in the EpiOcular EIT, the results for No. 28 inthat test were near the cut off line. Given that 26 of 30 test chemicals were predicted correctly,
the VMT feels that the LabCyte24 EIT has a predictive capacity similar to that of the EpiOcular
EIT.
Sensitivity at all participating laboratories individually, as well as collectively, was from 97.8%,
specificity was from 68.9%, and accuracy was from 83.5%.
PERFORMANCESTANDARD—which

specify 90%

Some deviation from the OECDsensitivity,

60% specificity, 75%

accuracy—were made for the LabCyte24 EIT. Predictive capacity at all laboratories was
sufficient to meet the acceptance criteria of the OECD PERFORMANCE STANDARD (OECD,
2015b).

10-4.

Similarity with the OECD TG 492 VRM

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The LabCyte CORNEA-MODEL24 EIT

February, 2017

As previously described the section 5, the VMT considers LabCyte24 EIT to be functionallysimilar to the EpiOcular EIT, which is a RhCE EIT VRM for OECD.
Using 30 Reference Chemicals from TG 492 as test chemicals, LabCyte24 EIT and the
EpiOcular EIT both gave four false positives for GHS No Category chemicals.
The LabCyte24 EIT yielded a false positive for Cellulose, 2-(2-hydroxy-3-(trimethylammonium)
propoxy) ethyl ether chloride (91%) (No. 28), even though the EpiOcular EIT predicted it correctly.
Also, the EpiOcular EIT yielded a false negative for 1,4-Dibutoxy benzene (No. 14), even though
the LabCyte24 EIT predicted it correctly. The EpiOcular EIT also yielded false positives for
2-Ethoxyethyl methacrylate (No. 17) and 3-Phenoxybenzyl alcohol (No. 18), even though the
LabCyte24 EIT predicted it correctly. Predictions of the other 26 chemicals were concordant
between the LabCyte24 EIT and the EpiOcular EIT.
These results suggest that LabCyte24 EIT has a predictive capacity similar to or higher thanthat of the EpiOcular EIT.

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The LabCyte CORNEA-MODEL24 EIT

11.

February, 2017

CONCLUSION

This validation study was intended to demonstrate that the LabCyte24 EIT is capable offulfilling the PERFORMANCE STANDARD stipulated in OECD TG 492 for similar or modified invitro RhCE EIT methods based on the EpiOcular EIT. The study was designed both to provide
the information necessary to validating the test method as well as to minimize the burden placedon the three participating laboratories. Assessment of reliability and accuracy of the method was
performed using 30 test chemicals selected from the Reference Chemicals listed in the
PERFORMANCE STANDARD of TG 492.
Having achieved within-laboratory reproducibility from 93% to 100% at each of the threeparticipating laboratories as well as a between-laboratory reproducibility of 87% for all threeparticipating laboratories combined, the LabCyte24 EIT clearly meets the PERFORMANCESTANDARD for TG 492.
The LabCyte24 EIT also demonstrated good predictive capacity with an overall accuracy from
83.5%, an overall sensitivity from 97.8%, and an overall specificity from 68.9%, thereby meetingthe acceptance criteria stipulated in the OECD PERFORMANCE STANDARD of 75% for
accuracy, 90% for sensitivity, and 60% for specificity.
These results suggest that the LabCyte24 EIT is a robust and reliable method for predicting
eye irritation potential. Most importantly, the test data provides information useful to proposing
the LabCyte24 EIT as me-too method for inclusion in OECD TG 492.

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The LabCyte CORNEA-MODEL24 EIT

12.

February, 2017

ACKNOWLEDGEMENTThe VMT would like to thank the JSAAE for its generosity in supporting this validation study
with a grant.

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The LabCyte CORNEA-MODEL24 EIT

13.

February, 2017

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