1

Validation Study of the Vitrigel-EIT method2

as an alternative to in vivo eye irritation testing

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Study Report, Version 2.0

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March 25, 2017

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VITRIGEL-EIT Validation Management Team (VMT)

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Table of Contents

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Abbreviations

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1

Abstract

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2

Introduction

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3

Methods

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3.1
Study Plan
3.1.1
Purpose
3.1.2
Organization
3.1.2.1
Trial coordinator
3.1.2.2
Chemical management group
3.1.2 3
Data analysis group
3.1.2.4
Record management group
3.1.2.5
Lead laboratory
3.1.2.6
Participating laboratories
3.1.3
Study design
3.1.3.1
Training of personnel at the participating laboratories
3.1.3.2
Phase 03.1.3.2
Phase I
3.1.3.3
Phase II
3.1.3.4
Phase III
3.1.4
Success criteria
3.2
Summary of protocol
3.2.1
Culturing HCE-T cells
3.2.2
Preparation of collagen vitrigel membrane chambers
3.2.3
Reconstruction of a human corneal epithelium model
3.2.4
Mode of action in vivo
3.2.5
Calculation of TEER values for HCE models
3.2.6
Exposure to test chemicals
3.2.7
Calculating eye irritancy of test chemicals
3.2.8
Correlation with the UN GHS classification
3.2.9
Commercial availability and/or intellectual property rights to the test method and its
components
3.3
Test chemicals
3.3.1
Selection and distribution of test chemicals
3.3.2
Test chemicals for Phases 0, I, II, and III
3.3.2.1
Test chemicals for Phase 0
3.3.2.2
Test chemicals for Phase I

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3.3.2.3
3.3.2.4

3.4
3.5

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Quality assurance
Record collection and analysis

Results
4.1
Study duration
4.1.1
Phase 04.1.2
Phase I
4.1.3
Phase II
4.1.4
Phase III
4.2
Quality assuarance

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Test chemicals for Phase II
Test chemicals for Phase III

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Discussion
5.1
5.2
5.3
5.4
5.5
5.6
5.7

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Purpose of the Validation
Transferability
Within- and between-laboratory reproducibility
Predictive capacity and relevance
Applicability domain
Other analysis
Comparison with other alternative to ocular irritation assay

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6Conclusion

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References

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Appendix

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8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
8.10
8.11
8.12
8.13
8.14

Study Plan ver.4
Protocol ver.1.80e
Data sheet format
Selection of test chemicals
Statistical analysis report for Vitrigel-EIT validation study
Report on the Quality assurance for Vitrigel-EIT validation study
Minutes of VMT meetings
Result of the Vitrigel-EIT at the lead laboratory for 57 chemicals
The draft list of proficiency chemicals
Yamaguchi H, et al., 2016. J. Applied Toxicology accepted
The raw data for the 118 in-house chemicals
Background by biostatistician
Predictive capacity of the Vitrigel-EIT for 114 chemicals considering DRD
Result of the Vitrigel-EIT at the lead laboratory for 114 chemicals

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Abbreviations
CVM

Collagen Vitrigel Membrane

EIT

Eye Irritancy Test

EURL ECVAM

European Union Reference Laboratory for Alternatives to Animal Testing

ECETOC

European Centre for Ecotoxicology and Toxicology of Chemicals

GHS

Globally Harmonized Systems of Classification and Labeling

GLP

Good Laboratory Practice

HCE

Human Corneal Epithelium

ICCVAM

Interagency Coordinating Committee on the Validation of Alternative
Methods

JaCVAM

Japanese Centre for the Validation of Alternative Methods

NI

Non-irritant

NICEATM

National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods

OECD

Organization for Economic Co-operation and Development

PET

Polyethylene terephthalate

SOP

Standard operating procedure

STE

Short time exposure

TEER

Transepithelial electrical resistanceUN

United Nations

VMT

Validation management team

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1

Abstract

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Collagen vitrigel membrane (CVM) comprises high-density collagen fibrils that are equivalent to in

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vivo connective tissues and is easily handled with tweezers. Takezawa et al. developed a human105

corneal epithelium (HCE) model by three-dimensional culturing of HCE-T cells on a CVM scaffold

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in a chamber that provided an air–liquid interface culture system. They further used their HCE model107

to establish a new test method, known as the Vitrigel-eye irritancy test (Vitrigel-EIT) method, which

108

can be used to estimate the ocular irritation potential of test chemicals by analyzing relative changes

109in transepithelial electrical resistance (TEER) over time.

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This trial was conducted to validate the reliability and relevance of the Vitrigel-EIT method at three111

participating laboratories in the spirit of GLP by verifying the within- and between-laboratory112

reproducibility for 42 test chemicals as well as the capacity for distinguishing non-irritants from113

irritants in a bottom-up approach.

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The results showed 80–100% within-laboratory reproducibility at all three laboratories and an

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excellent between-laboratory reproducibility of 92%. Unfortunately, the predictive capacity for

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distinguishing non-irritants from irritants per UN GHS categories in a bottom-up approach was not

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favorable because of false negative rates as high as 17%. After considerable review of the data,

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however, it was determined that excluding test chemicals with a pH level of 5 or less as well as solid

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test chemicals with a logP value of 2.5 or more and a density of less than 0.95 g/ cm3 or greater than120

1.10 g/cm3 improved the false negative rate to as low as 7%.

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These results suggest that, with a carefully defined applicability domain, the Vitrigel-EIT method is a122

useful alternative to the Draize test for distinguishing test chemicals that are ocular non-irritants from123

those that are irritants.

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2

Introduction

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Collagen vitrigel membrane (CVM) comprises high-density collagen fibrils that are equivalent to in

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vivo connective tissues and is easily handled with tweezers. In addition, it has excellent transparency

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and permeability of high molecular weight proteins and is now used as a cell culture scaffold in a

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number of advanced studies (Takezawa et al., 2004, 2007a–c). Takezawa et al. developed a corneal

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epithelium model utilizing a CVM scaffold that facilitates the maintenance of corneal epithelial

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phenotype in a monolayer of rabbit corneal epithelial cells (Takezawa et al., 2008). Still, there are

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significant differences in sensitivity to exogenous chemicals between humans and rabbits, so they also132

developed a human corneal epithelium (HCE) model by three-dimensional culturing of HCE-T cells

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on the CVM scaffold in a chamber that provided an air–liquid interface culture system (Takezawa et134al., 2011a). Here, HCE-T cells are a SV40-immortalized cell strain established by Araki-Sasaki et al

135(Araki-Sasaki et al., 1995). The HCE-T cell line is one of the most favored human cornea epithelium-

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derived cells and frequently used for various cornea epithelium-related studies because it is easy to137

maintain the stable characteristics of cornea epithelial cells in culture (Kim et al., 2016, Yamasaki et138al., 2009). The scaffold was fabricated on a polyethylene terephthalate (PET) membrane of a Millicell

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chamber suitable for assaying the transepithelial electrical resistance (TEER) of epithelial cells. The

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TEER assay is considered a suitable method for in vivo evaluation of the integrity of the tight junction

141of the corneal epithelium (Uematsu et al., 2007). Takezawa et al. then used the HCE model to verify

142that relative change over time in TEER is a useful indicator for assessing the ocular irritancy of four143

test chemicals, including mild irritants (Takezawa et al., 2011a). The HCE model, however, is not144

considered suitable for immuno-histological analyses due to difficulties in preparing frozen sections

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with a PET membrane. To overcome this inconvenience, they developed a novel chamber that merely

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accompanies a CVM without the PET membrane as well as established a process for its mass

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production (Takezawa et al., 2011b, 2012). More recently, they established a new test method for148

estimating the ocular irritancy of test chemicals by analyzing the relative changes over time in TEER

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after exposing HCE models reconstructed in CVM chambers to test chemicals. This new test method

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is called the Vitrigel eye irritancy test (Vitrigel-EIT) method. Thus far, thirty chemicals have been

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classified successfully as irritants or non-irritants without false negatives using the Vitrigel-EIT

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method (Yamaguchi et al., 2013).

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In association with the International Collaboration on Alternative Test Methods (ICATM), an

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international validation management team (VMT) was organized to validate the reliability and155

relevance of this test method, and a validation study was performed with the cooperation of three156

Japanese laboratories. Testing was conducted using a protocol developed by Yamaguchi and

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Takezawa using test chemicals distributed via the Japanese Center for the Validation of Alternative

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methods (JaCVAM). Descriptive statistics are used to summarize the data obtained from the testing.

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The aim of this trial is to validate the capability of the Vitrigel-EIT method as well as to assess

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transferability and between-laboratory reproducibility in preparation for incorporating this test into161

the screening of test chemicals for the eye irritation potential in accordance with the United Nations’162

Globally Harmonized System of Classification and Labelling of Chemicals (GHS) categories (United

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Nations, 2013). This multi-phase validation study of the Vitrigel-EIT method was undertaken in164

accordance with:

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i)

the principles and criteria documented in the Organization for Economic Co-operation and

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Development (OECD) No. 34 Guidance Document on the Validation and International

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Acceptance of New or Updated Test Methods for Hazard Assessment (OECD, 2005),

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ii)

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iii) the concepts discussed in The Principles of Good Laboratory Practice: Application to In Vitro

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the Modular Approach to Validation (Hartung et al., 2004), andToxicological Studies (Cooper-Hannan et al., 1999).

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Testing performed as part of a validation study should ideally be performed in accordance with GLP

172(OECD, 1998) and necessarily include, without being limited to, the use of standard operating

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procedures (SOP) and adequate recording of data as well as suitable reporting of results and archival

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record keeping.

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The “modular approach to validation” is a general conceptual framework for documenting the176

validation of a test method (Hartung et al., 2004; OECD, 2005). In this approach, the information

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needed to support the validity of the method is organized into modules, as follows.

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Module 1: Test Definition

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Module 2: Within-laboratory repeatability and reproducibility

180Module 3: Between-laboratory transferability

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Module 4: Between-laboratory reproducibility

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Module 5: Predictive capacity

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Module 6: Applicability domain

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Module 7: Performance standards

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The modular approach introduced by Hartung et al. (2004) allows the use of datasets from a variety

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of sources, and this principle was applied in our assessment of the scientific validity of the Vitrigel-

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EIT method. As a specific goal, this validation study was designed to clarify whether or not the188

Vitrigel-EIT test method is a useful alternative to the Draize test method in a bottom-up approach for

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distinguishing chemical substance.

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3

Methods

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3.1

Study Plan

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3.1.1

Purpose194

This validation study is designed to assess the reliability (within- and between-laboratory

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reproducibility) and relevance (predictive capacity) of the Vitrigel-EIT method using a challenging

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set of test chemicals for which high quality in vitro and in vivo data are available. The test chemicals

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are to include each type of UN GHS category as classified by in vivo data and predictive capacity is

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to be assessed primarily in accordance with UN GHS classification in a bottom-up approach (Scott,199

2010).

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3.1.2

Organization

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Members of the VMT contribute their collective expertise in the underlying science and scientific203

design, management, and evaluation of validation studies. The management structure for this204

validation study of the Vitrigel-EIT method is shown in Fig. 1.

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The VMT is responsible for overseeing the conduct of the validation study, including signing and206

dating the approval of all protocols, study plans, reports, and amendments.

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The members of the VMT as well as their respective roles and expertise for this validation study of208

the Vitrigel-EIT method are shown in Table 1 and Fig. 1.

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Table 1. The Vitrigel-EIT Validation Management Team
Name

Hajime Kojima

Toshiaki Takezawa
Hiroyuki Yamaguchi
Takashi Sozu

Role and expertise

Affiliation

Trial coordinator, Chemical
management and Quality assurance

Japanese Center for the Validation of
Alternative Methods (JaCVAM),
National Institute of Health Sciences(NIHS)Developer of this assay andexpertise underlying science as thelead laboratory

Institute of Agrobiological Sciences(NIAS),
National Agriculture and Food Research
Organization (NARO)

Data analysis and biostatistics
dossierTokyo Univ. of Science

Liaison members
National Toxicology Program
Interagency Center for the Evaluation of
Alternative Toxicological Methods
Nicole Kleinstreuer

Validation study expertise(NICEATM)/ Interagency Coordinating
Committee on the Validation of
Alternative Methods (ICCVAM), USA
European Union Reference Laboratory

Michael-Wilhelm
SCHAEFFER

Validation study expertisefor Alternatives to Animal Testing(EIRL ECVAM), Italy
Korean Center for the Validation ofLim, Chae-Hyung

Validation study expertise
Alternative Methods (KoCVAM), Korea

9Wannhsin Chen

Validation study expertise

Industrial Technology Research Institute,(ITRI), Taiwan

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Fig.1. Management Structure for the Vitrigel-EIT validation study

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3.1.2.1

Trial coordinator

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A trial coordinator was appointed by the VMT to be responsible for preparing draft study plans, a216

study protocol, and a list of test chemicals as well as to convene ad hoc VMT meetings for review and217

finalization of the study plan, the study protocol, and the test chemical list. The trial coordinator was

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also responsible for other administrative duties related to the validation study.

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3.1.2.2

Chemical management group

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The chemical management group comprised at least one member selected from the VMT and was

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responsible for preparing a list of test chemicals as well as conferring with the trial coordinator to

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finalize the list test chemicals to be used in the validation study. It also prepared and distributed non-224

coded or coded lists of test chemicals to chemical distributors.

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3.1.2 3

Data analysis group

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The data analysis group comprised at least one member selected from the VMT and was responsible

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for providing an objective analysis of data obtained in this validation study as well as for performing

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statistical processing of the data.

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3.1.2.4

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The record management group comprised at least one member selected from the VMT as well as a

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representative of the lead laboratory was responsible for preparing the test protocol, the test chemical

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preparation sheets, blank data sheets, and any other necessary materials as well as for distributing

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these materials to the participating laboratories. It also collected the completed forms and data sheets

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after testing, reviewed the records for errors and omissions, and requested correction as necessary.

Record management group

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3.1.2.5

Lead laboratory

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The lead laboratory represents the test developers and was responsible for providing the test method239

protocol as well as test chemical preparation record forms, blank data sheets, and all other necessary

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documentation. The lead laboratory was also responsible for providing revised versions of the241

protocol as necessary throughout the entire validation study. The VMT consulted with both the lead242

laboratory and the other participating laboratories on technical issues.

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3.1.2.6

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The following three laboratories in Japan participated in the testing of substances using the Vitrigel-246

EIT method. The name of the on-site study director is given in parenthesis.

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Participating laboratories

Lab A: Hatano Research Institute, Food and Drug Safety Center (FDSC), Hatano, Kanagawa(Mika Watanabe)249

Lab B: Bozo Research Center (BRC), Tokyo (Takayuki Fukuda)

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Lab C: Daicel Corporation (Daicel), Himeji, Hyogo (Kunihiko Yamashita)

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All three of these laboratories were naïve and were selected for participation by the VMT after252

practical training that provided a good indication of the robustness of the test method.

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A coordinator from each of these three laboratories participated in VMT activities as observers and254

was responsible for ensuring that the tests were performed in accordance with the study protocol as

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well as for filling out and submitting all necessary records and forms upon completion of testing.

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3.1.3

Study design

257This validation study of the Vitrigel-EIT method was carried out in four phases in accordance with258

the study plan as described in Appendix 8.1 and summarized in Table 2.

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Table 2. Overview of the Vitrigel-EIT validation study
The number of

The number of

the test chemicals

the repetitions

0

5

3

I

10

3

II

10

1

III

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1

Phase

Examination
Within- laboratory transferability
Between- laboratory transferability & Within- andbetween- laboratory reproducibility
Between- laboratory reproducibility
Between- laboratory reproducibility andpredictability

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3.1.3.1

Training of personnel at the participating laboratories

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A technical transfer workshop to explain the principles of and protocol for validation of the Vitrigel-

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EIT method was held May 22 and 23, 2013, with personnel from all three laboratories in attendance.

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Instructors from the lead laboratory explained the test method while demonstrating the protocol. All

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personnel in attendance performed the assay themselves, using saline, ethanol and silicic acid267

anhydrate. After the workshop, the coordinators from each participating laboratory agreed to purchase268

the cell line from RIKEN BioResource Center (Tsukuba, Japan) and to sign a memorandum pertaining

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to borrowing the TEER recorder.

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3.1.3.2

Phase 0272

Phase 0 was designed to assess between-laboratory transferability by testing five non-coded test

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chemicals using protocol ver. 1.30e. Each test chemical was determined to be either positive or274

negative by obtaining consistent results from each of three runs.

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3.1.3.2

Phase I

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Phase I was designed to assess within and between-laboratory reproducibility by testing ten coded test

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chemicals using protocol ver. 1.51e. Each test chemical was determined to be either positive or279

negative by obtaining consistent results from each of three runs in three different sets.

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3.1.3.3

Phase II

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The original plan was split into two parts: A and B. Phase IIA was designed to assess the between-

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laboratory reproducibility of ten coded test chemicals using protocol ver. 1.61e, after which Phase IIB

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was to validate an additional thirty coded test chemicals using the same protocol. Phase IIB was

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canceled when the results of Phase IIA led to a decision to undertake a major revision of protocol ver.

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1.61e. Consequently, Phase IIA was renamed Phase II, and the planned Phase IIB was incorporated

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into a newly designed Phase III using the protocol ver. 1.71e.

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3.1.3.4

Phase III

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Phase III was designed to assess the between-laboratory reproducibility and predictive capacity of the291

Vitrigel-EIT method for thirty-six coded test chemicals using protocol ver. 1.71e. Each test chemical292

was determined to be either positive or negative based on obtaining consistent results from each of293

three runs in one set.

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3.1.4

Success criteria

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Success criteria for within and between-laboratory reproducibility was 80%. The predictive capacity297

was assessed using thirty-six coded test chemicals. The results of statistical analysis were used to298

determine the preliminary design for validation study as well as automatization of the test leading to299

an increased dataset.

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Issues related to the applicability domain were discussed by the VMT decision during assessment of301

between-laboratory reproducibility.

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3.2

Summary of protocol

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The current test protocol is ver. 1.80e, which was designed per Yamaguchi et al., 2013, 2015 and is

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shown in Appendix 8.2. The data sheet format is shown in Appendix 8.3.

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3.2.1

Culturing HCE-T cells

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An SV40-immortalized HCE cell strain (HCE-T cells, RCB no. 2280) was obtained from RIKEN

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BioResource Center (Tsukuba, Japan). The cells were maintained in a culture medium comprising a

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1:1 mixture of Dulbecco’s modified eagle medium and nutrient mixture F-12 supplemented with 5%

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heat-inactivated fetal bovine serum, 5 μg/mL recombinant human insulin, 10 ng/mL recombinant312

human epidermal growth factor, 0.5% dimethyl sulfoxide, 100 units/mL penicillin and 100 μg/mL

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streptomycin (Araki-Sasaki et al., 1995; Yamasaki et al., 2009). Cells were grown at 37°C in a314

humidified atmosphere of 5% CO₂.

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3.2.2

Preparation of collagen vitrigel membrane chambers

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A collagen xerogel membrane chamber (ad-MED VitrigelTM) was purchased from Kanto Chemical

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Co., Inc. (Tokyo, Japan). The collagen xerogel membrane chamber was set in the well of a 12-well319

plate. Then, the collagen xerogel membrane was immersed in the culture medium by pouring 1.5 mL

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outside and 0.5 mL inside the chamber in the well for 10 min to convert the xerogel into vitrigel

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immediately before use.

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3.2.3

Reconstruction of a human corneal epithelium model

324

The culture medium outside the chamber in the well of a 12-well plate was replaced with 1.5 mL of

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fresh medium. The medium inside the chamber was removed and 0.5 mL of a cell suspension in a

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culture medium at a density of 1.2 × 105 cells/mL was poured onto the CVM in the chamber and327

cultured for 2 days at 37°C. Subsequently, the cells were cultured for 4 days at the air–liquid interface

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to fabricate a HCE model after removing the inside medium and changing the outside medium outside329

of the chamber. The medium outside the chamber was changed on the third day of culturing at the330

air–liquid interface.

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3.2.4

Mode of action in vivo

333

Time-dependent relative changes of TEER values after exposing chemicals to in vitro human corneal

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epithelial models are considered to be an excellent indicator for extrapolating the destructive activity

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of the chemicals against the barrier function of human corneal epithelium in vivo. For this reason, the336

TEER assay is a simple and suitable method for evaluating corneal irritancy and permeability

337

quantitatively and continuously (Uematsu et al., 2007). Therefore, it is important to develop an assay

338

system that can facilitate not only the reconstruction of human corneal epithelial model but also the339

TEER measurement and the chemical exposure.

340

Our preliminary results based on the testing of four chemicals demonstrated a correlation between

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irritancy potential and changes in TEER. We found that non-irritants caused virtually no change in

342

TEER, moderate irritants caused only a gradual decrease of limited magnitude in TEER, and strong

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irritants caused a rapid decrease of significant magnitude in TEER (Takezawa et al. 2011a). During344

further testing of 30 chemicals, we consistently observed these three patterns, which we were able to345

express mathematically using three parameters, namely, time lag, intensity, and plateau (Yamaguchi346

et al. 2013).

347

In this study, we aimed to develop such an ideal assay method utilizing HCE-T cells and the collagen348

vitrigel membrane chamber useful for TEER measurement.

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350

3.2.5

Calculation of TEER values for HCE models

351

The electrical resistance of a HCE model in a CVM chamber (Rmodel) and of a blank CVM chamber

352(Rblank) were measured using the TEER recorder shown in Fig. 2. The TEER value was calculated as353

follows:

354

TEER = (Rmodel – Rblank) × effective surface area (1.0 cm2)

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356

Fig.2. Schematic illustrations on the TEER measurement electrodes for HCE model and gross357

observation of TEER recorder system.

358

The electrode unit (A), the electrode unit applied for the culture media via HCE model (B) and359

the TEER recorder system (C).

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361

3.2.6

Exposure to test chemicals

362

A solution of test chemical was prepared in a culture medium at a concentration of 2.5% (weight/

363

volume), which is considered appropriate for measuring TEER values without undue influence from364

the electrical resistance of the test chemical itself. Test chemicals were manually mixed in the medium

365

until the test chemical dissolves or for a maximum of one minute. If the test chemical does not

366

dissolve readily, try using the following techniques in the following order to dissolve it: a) mix

367

mechanically for a maximum of one minute using a vortex mixer, b) sonication for a maximum of 20

368

minutes, or c) heating to a maximum temperature of 70°C.

369

temperature of each test chemical solution was checked.

370

dissolved or homogeneously dispersed, was moved to the next step. For test chemicals that proved to371

be insoluble or immiscible using the above technique, a test chemical solution was prepared as a372

homogeneous suspension by mixing the test chemical in the medium by vortex for up to 1 minute373

immediately before use (Fig.3). The pH level of each 2.5% test chemical solution was measured using

374

universal pH test paper from ADVANTEC (Tokyo, Japan).

375

The HCE models were exposed to a test chemical on day 6, as follows: First, 500 μL of culture

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After trying each technique, theTest chemical solution that is well

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medium was poured in the chamber and the TEER recorder was used to obtain a pre-exposure Rmodel

377

value for each model. Next, the medium inside the chamber was replaced with 500 μL of test chemical

378

solution and Rmodel values were measured at intervals of 10 seconds for a period of 3 min after exposure379

to the test solution. Here, it is essential to obtain the reproducible data that the measurement is started

380

within 2 to 5 seconds after adding the test chemicals.

381

electrode is often unstable within 2 seconds after exposing the test chemical solution. Also, the HCE

382

model has already been influenced with the test chemicals over 5 seconds. Three runs were made383

for each test chemical and a new HCE model was used in each. Test chemical exposure was conducted384

at an ambient temperature of 28±2°C. The ambient temperature of 28±2°C for the HCE model was

385

achieved by regulating the temperature of the 12-well plate using a hot plate, a water bath or an air

386

conditioner. Here, it is important to confirm that the actual temperature of culture medium is 28±2°C.

Because the liquid condition around the

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389

Fig.3. Preparation of test chemical solution per the revised protocol

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3.2.7

Calculating eye irritancy of test chemicals

392

The TEER values for each test chemical were measured during the three runs and then copied to a393

data sheet, where eye irritancy was calculated automatically. The mean TEER values for all three tests

394

were plotted on a time line and a profile of TEER values (dP/dT) was automatically analyzed for three395

parameters: time lag (t1), intensity (−[P2 − P1]/[t2 − t1]), and plateau level (100 − P2). Time lag (t1) is396

defined as the maximum time at which a profile was maintained at 0 ≥ dP/dT > −0.03%/second. The

397

starting time of plateau level (t2) after the profile was maintained at dP/dT ≤ −0.03%/second for a398

particular period of time was defined as the initial time at which the profile was maintained at399

0 ≥ dP (P3 − P2)/dT (t3 − t2) > −0.03%/s.

400(t3 = t2 + 30 seconds) because the plateau level was evaluated by the profile for 30 seconds. P1, P2,

401

and P3 are the percentages against the initial TEER value at t1, t2, and t3 after exposure to the test402

chemical, as shown in Fig. 4. A score for each index was calculated using the above formula.

403

Subsequently, the eye irritation potential of test chemicals was determined to be either irritant or non-

404

irritant, in accordance with the criteria shown in Table 3.

The

time

(t3)

is

represented

in

the

equation

405
406

407

408

Fig. 4. Schematic illustration showing an analysis of a TEER profile after exposure of a model to a409

test chemical.

410

t1 represents time lag, and t2 represents the start of the plateau level. t3 is defined as t2 + 30 s.

411

P1, P2, and P3 indicated a percentage relative to the initial TEER value at t1, t2, and t3, respectively.

412
413
414

18

415

Table 3. Eye irritancy criteria.
Criteria

Prediction

Time lag ≤ 180 or Intensity ≥ 0.05 or Plateau level > 5.0

Irritant (I)Time lag > 180 and Intensity < 0.05 and Plateau level ≤ 5.0

Non-irritant (NI)

416
417

3.2.8

Correlation with the UN GHS classification

418

The correlation with the UN GHS classification of test chemicals was estimated by calculating419

sensitivity, specificity, and accuracy, as follows.

420

Sensitivity (%) = A/(A + B) × 100

421

Specificity (%) = D/(C + D) × 100

422

Accuracy (%) = (A + D)/(A + B + C + D) × 100

423

A is the number of test chemicals classified as irritants by both the traditional UN GHS classification

424

and the Vitrigel-EIT method. B is the number of test chemicals classified as irritants by the traditional

425

UN GHS classification and as non-irritants by the Vitrigel-EIT method. C is the number of test

426

chemicals classified as non-irritants by the traditional UN GHS classification and as irritants by the427

Vitrigel-EIT method. D is the number of test chemicals classified as non-irritants by both the428

traditional UN GHS classification and the Vitrigel-EIT method.

429
430
431

3.2.9
Commercial availability and/or intellectual property rights to the test method and its
components

432

All components and reagents using in the test method are commercially available. HCE-T cells can433

be globally distributed from RIKEN BioResource Center. The Vitrigel-EIT method is available

434

without any restriction by its intellectual property rights. Vitrigel is registered trade mark of National

435

Agriculture and Food Research Organization (Tsukuba, Japan).

436
437

3.3

Test chemicals

438

3.3.1

Selection and distribution of test chemicals

439

The test chemicals were selected to ensure that a diverse range of substances were represented, and

19

440

aspects such as eye-irritant level per UN GHS categories, physical state, chemical class, and incidence441

of eye lesions were considered. Preference was given to test chemicals for which high-quality in vivo

442

data is available, especially when the data included results from individual animals. The list includes

443

test chemicals that were previously used in the 3-dimensional corneal model (such as EpiOcular)444

validation studies by EURL-ECVAM (ECETOC, 1998), the Short Time Exposure test validation

445

study by JaCVAM and independent peer review (ICCVAM, 2010, 2013), and the OptiSafe™

446

evaluation study by NICEATM.

447

All the test chemicals selected for this validation study are available commercially, were selected by448

the chemical management group, and approved by the VMT. All the test chemicals used in Phases I,

449

II, and III were coded, and their names were provided only after completion of the study. A total of 42

450

substances were tested by all three laboratories.

451
452

3.3.2

Test chemicals for Phases 0, I, II, and III

453

3.3.2.1

Test chemicals for Phase 0454

Five test chemicals were selected by the VMT for use in validating between-laboratory transferability

455

during Phase 0, as shown in Table 4. The five non-coded test chemicals were delivered to each456

participating laboratory by the VMT.

457
458

Table 4. List of test chemicals selected for Phase 0No.

Test chemical

Positive
Ethanol
control
0-1
Benzalkonium chloride

CASRN

State

64-17-5

Liquid8001-54-5

Density

logP

pH

GHS

-0.31

7

2A

Category 1

Solid

0.99

1.68

7

Category 1(g/cm3)0-2

2-Propanol

67-63-0Liquid

0.78

0.05

7

Category 2A

0-3

Glycerol

56-81-5

Liquid

1.26

-1.76

7

No Category

0-4n-Hexanol

111-27-3

Liquid

0.82

2.03

7

Category 2A

0-5

Silicon dioxide n-hydrate

7699-41-4

Solid

1.58

-

7

No Category

459
460

3.3.2.2

Test chemicals for Phase I

461

Ten test chemicals were selected by the VMT for use in validating within- and between-laboratory
20

462

reproducibility during Phase I, as shown in Table 5. The ten test chemicals comprised five irritants

463

and five non-irritants, five of which were solid and five of which were liquid, as shown in Table 5.

464

To assess the within-laboratory reproducibility, the VMT selected ten test chemicals in Phase I. The

465

VMT decided this scale based on our biostatistician’s opinion about the statistical validity of the466

number of test chemicals used for the ECVAM validation study for skin sensitization. A detailed

467

background is addressed at appendix 8-12. The ten test chemicals were coded and delivered in three468

sets to each participating laboratory by the VMT. Refer to the chemical selection report in Appendix

469

8.4 for code numbers.

470
Table 5. List of test chemicals selected for Phase I
No.

Test chemical

CASRN

State

Density(g/cm3)

logP

pH

GHS

1-1

Imidazole288-32-4

Solid

1.03

-0.08

9

Category

1-2

Cyclohexanol

108-93-0

Liquid

0.96

1.23

7

1

1-3

3,3-Dithiodipropionic acid1119-62-6

Solid

1.45

-0.15

4

1-4

Acetone67-64-1

Liquid

0.79

-0.24

7

Category

1-5

3-Chloropropionitrile542-76-7

Liquid

1.16

0.18

5

2A or 2B

1-6

Ammonium nitrate

6484-52-2

Solid

1.72

-

8

598-65-2

Solid

-

-

7

108-88-3

Liquid

0.87

2.73

7

623-39-2

Liquid

1.11

-1.13

7

90-80-2

Solid

1.61

-2.48

6

1-7
1-8
1-9
1-10

n,n-Dimethylguanidine
sulfateToluene3-Methoxy-1,2propanediol
Gluconolactone

No
Category

471
472

3.3.2.3

Test chemicals for Phase II473

Ten test chemicals were selected by the VMT for use in validating between-

474

laboratory reproducibility during Phase II, as shown in Table 6. The ten test

475

chemicals comprised four classified UN GHS Category 1, three classified UN GHS

476

Category 2A or 2B, and three classified UN GHS No Category, five of which were

21

477

solids and five of which were liquid, as listed in Table 6. The ten test chemicals were478

coded and delivered in one set to each participating laboratory by the VMT. Refer to479

the chemical selection report in Appendix 8.4 for code numbers.

480
481

Table 6. List of test chemicals selected for Phase II
No.

Test chemical

CASRN

State

Density(g/cm3)

logP

pH

GHS

2-1

Imidazole288-32-4

Solid

1.03

-0.08

9

2-2

Cyclohexanol

108-93-0

Liquid

0.96

1.23

7

Category

2-3

Sodium dodecyl sulfate

151-21-3

Solid

0.40

1.60

7

1

2-4

Sodium salicylate

54-21-7

Solid

0.32

0.42

7

2-5

Cyclopentanol

96-41-3

Liquid

0.95

2.41

7

2-6

2-Methyl-1-pentanol

105-30-6

Liquid

0.83

1.76

7

2-7

α-Hexylcinnamaldehyde

101-86-0

Liquid

0.95

5.12

7

598-65-2

Solid

-

-

7

2-8

n,n-Dimethylguanidine
sulfateCategory
2A or 2BNo

2-9Toluene108-88-3

Liquid

0.87

2.73

7

2-10

Gluconolactone

90-80-2

Solid

1.61

-2.48

6

Category

482
483

3.3.2.4

Test chemicals for Phase III

484

Thirty-six test chemicals were selected by the VMT for use in validating between-laboratory

485

reproducibility and predictive capacity during Phase III, as shown in Table 7. The number of chemicals,

486

total 36 chemicals, was decided in consideration of Kanto Chemical’s ability to supply the CVM

487

chambers as well as the participating laboratories’ testing capacity. All test chemicals were selected to488

ensure that a diverse range of substances were represented, and aspects such as eye-irritant level per489

UN GHS categories, physical state, chemical class, and incidence of eye lesions were considered.

490

Preference was given to test chemicals for which high-quality in vivo data is available, especially491

when the data included results from individual animals. The number of test chemicals in each GHS

492

classification is shown in Table 8. The number of solid and liquid test chemicals is show in Table 9.

493

The thirty-six test chemicals were coded and delivered in one set to each participating laboratory by

22

494

the VMT. Refer to the chemical selection report in Appendix 8.4 for code numbers.

495

The chemical master at Lab C revealed the name of test chemical No. 3-16, sodium chloroacetate,496

which was subsequently eliminated from the list and cyclopentanol was delivered by the VMT as an

497

alternative.

498
Table 7. List of test chemicals selected for Phase III
No.

Test chemical

CASRN

State

3-1

2,5-Dimethyl-2,5-hexanediol

110-03-2

Solid

Density(g/cm3)0.90

3-2

2-Benzyl-4-chlorophenol

120-32-1

Solid

3-3

2,2-Dimethyl butanoic acid595-379

3-4

Captan3-5Tetra-n-octylammonium bromide

3-6

logP

pH

1.19

7

1.19

3.60

7

Liquid

0.93

1.90

4

133-06-2

Solid

1.74

2.80

7

14866-33-2

Solid

0.94

3.45

7

Butanol

71-36-3

Liquid

0.81

0.88

8

Categ

3-7

3-(2-Aminoethylamino)
propyl]trimethoxysilane

1760-24-3

Liquid

1.01

-1.00

10

ory 1

3-8

Sodium dodecyl sulfate

151-21-3

Solid

0.40

1.60

7

3-9

m-Phenylenediamine

108-45-2

Solid

1.14

-0.33

8

3-10

Tetraethylene glycol

17831-71-9

Liquid

1.13

1.26

7

3-30

Imidazole288-32-4

Solid

1.03

-0.08

9

3-32

Sodium salicylate

54-21-7

Solid

0.32

0.42

7

3-11

gamma-Butyrolactone

96-48-0Liquid

1.13

-0.64

7

3-12

Methyl acetate79-20-9Liquid

0.93

0.18

7

3-13

Myristyl alcohol

112-72-1

Solid

0.82

6.03

7

3-14

2,6-Dichlorobenzoyl chloride

4659-45-4

Liquid

1.47

2.54

3

3-15

1623-08-1

Solid

1.46

1.71

3

Categ

29911-27-1

Liquid

0.94

1.14

7

ory

3-18

Dibenzyl phosphate
1-(2-Propoxy-1-methylethoxy)-2propanol
Camphene

79-92-5

Solid

0.84

1.94

7

2A or

3-19

Ethyl-2-methylacetoacetate609-14-3

Liquid

1.00

0.78

7

3-20

Propylene glycol propyl ether

1569-01-3

Liquid

0.89

0.56

8

3-31

2-Methyl-1-pentanol

105-30-6

Liquid

0.83

1.76

7

3-33

α-Hexylcinnamaldehyde

101-86-0

Liquid

0.95

5.12

7

3-37

Cyclopentanol

96-41-3

Liquid

0.95

2.41

7

3-21

Methyl amyl ketone

110-43-0

Liquid

0.82

1.98

7

3-22

2-(n-Dodecylthio)ethanol

1462-55-1

Liquid

0.91

-

7

3-23

iso-Octylthioglycolate

25103-09-7

Liquid

0.97

4.36

7

3-24

2,4-Difluoronitrobenzene

446-35-5

Liquid

1.46

-1.18

7

3-17

23

GHS

2B

No
Categ
ory

499
500

3-25

tetra-Aminopyrimidine sulfate

5392-28-9

Solid

1.65

0.27

3

3-26

2,4-Pentanediol

625-69-4

Liquid

0.96

0.35

8

3-27

iso-Octyl acrylate

29590-42-9

Liquid

0.88

4.61

7

3-28

Silicon dioxide n-hydrate

7699-41-4

Solid

1.58

-

7

3-29

Potassium tetrafluoroborate

14075-53-7

Solid

2.51

-

7

3-34

n,n-Dimethylguanidine sulfate598-65-2

Solid

-

-

7

3-35

Toluene108-88-3

Liquid

0.87

2.73

7

3-36

Gluconolactone

90-80-2

Solid

1.61

-2.48

6

Table 8. Breakdown of test chemicals used in Phase III
GHS

Total

Category 1

Category 2A/2B

Category 2B

No Category

12

8

4

12

36

501
502

Table 9. Breakdown of test chemicals used in Phase III per physical state
Solid
Liquid
Total
16

20

36

503
504

3.4

Quality assurance

505

All testing at the participating laboratories was conducted in accordance with the principles of Good

506

Laboratory Practice (GLP, OECD 1998), and were well documented, including a discussion of any

507

impact on study results. Records were kept of the maintenance of measuring instruments, the508

production of HCE models, and the preparation and application of test chemicals using a format

509

prepared by the lead laboratory. The data was input using a format developed for this validation study

510

by the lead laboratory and the biostatistician. Personnel at the participating laboratories recorded the511

necessary information, including the code names of each test chemical, names and date of preparation

512of solvents, degree of solubility or suspensibility, and concentration of the test solution. These records

513

were sent from the participating laboratories to JaCVAM, where they were checked for validity and514

accuracy as well as archived.

515
516

3.5

Record collection and analysis

517

Data collection and analysis were performed in close collaboration with biostatisticians and the quality

24

518

assurance group. Independent biostatisticians collected and organized data as shown in Appendix 8.5519

using custom data collection software, and all records were checked by the quality assurance group.

520

Any concerns at the participating laboratories over record keeping were resolved by the on-site study

521

director and reported at VMT meetings.

522

At the final VMT meeting, all data was finalized and decoded by the trial coordinator, after which the523

biostatisticians performed a statistical analysis. Data management procedures and statistical tools were524

approved by the trial coordinator and the data analysis group. Any deviation found in the analysis was

525

well documented, including a discussion of any impact on study results. Test results were evaluated526

for correlation with UN GHS classification based on predetermined criteria.

527

Predictive capacity of the Vitrigel-EIT method was evaluated using data from Phase III. First, an528

analysis was performed to assess predictive capacity in accordance with UN GHS classification per

529

either a bottom-up or a top-down approach (Scott, 2010). Further analysis was then performed to530

reduce false negatives by limiting the scope of the applicability domain.

531
532

4

Results

533

All data were analyzed by biostatisticians as shown in Appendix 8.5. The quality assurance group534

checked all records, following the quality assurance protocol, as summarized in Appendix 8.6.

535

4.1

536

Phase 0 was conducted from June to December 2013, using protocol ver. 1.30e.

537

Phase I was conducted from March to April 2014, using protocol ver. 1.51e.

538

Phase II was conducted from June to September 2014, using protocol ver. 1.61e.

539

Phase III was conducted from November 2014 to January 2015, using protocol ver. 1.71e.

540

VMT meetings were held during the intervals between these phases. The minutes of the VMT

541

meetings are show in Appendix 8.7.

Study duration

542
543

4.1.1

Phase 0544

Phase 0 was designed to assess between-laboratory transferability by testing five non-coded test

25

545

chemicals using protocol ver. 1.30e.

546

Although the results were generally good, two issues were identified: the results for glycerol obtained547

at BRC were inconsistent, and those for ethanol (positive control) obtained at Daicel did not meet the548

success criteria for between-laboratory reproducibility shown in Tables 10 and 11. With the exception549

of the results for glycerol obtained at BRC, the data was overall highly consistent. The results for two

550of the three runs of ethanol at Daicel fell below the acceptance criteria for positive control (plateau

551

level: 20 to 30%) in Fig.5. At the 1st VMT meeting, members discussed a proposal to use

552

benzalkonium chloride as the positive control instead of ethanol, in order to ensure clear and consistent553

results. Ultimately, ethanol was used as a reference control, and its range was modified to 15–30% at

554plateau level. This exact range was to be finalized based on the results of Phase I.

555

The VMT requested additional testing at BRC and Daicel using a revised protocol, ver. 1.40e. After

556

confirming the results of the additional testing (data not shown), all VMT members agreed to proceed557

with Phase I. The following key issues were addressed by revising the protocol to ver. 1.51e prior to558

the start of Phase I.

559



Success criteria for the reference control: Range at plateau level of 10–30%

560

Ambient temperature during TEER measurement: 18–30ºC

561

Time from start of exposure to start of measurement: within 2 seconds

26

562

Table 10-1. Data for Phase 0, Trial 1
No.

Test chemical

BRC

FDSC

DaicelTime lag

Intensity

190 (NI)-0.03 (NI)

0 (NI)

NI

190 (NI)-0.01 (NI)

0 (NI)

NI

Positive control (ethanol)

20 (I)

0.13 (I)

23 (I)I

0 (I)0.11 (I)

21 (I)

0-1 Benzalkonium chloride

0 (I)0.32 (I)58 (I)I

0 (I)0.30 (I)

0-2 2-Propanol

10 (I)

0.17 (I)

32 (I)I

0 (I)

0-3 Glycerol

0 (I)0.31 (I)

22 (I)

I

0-4 n-Hexanol

0 (I)0.21 (I)38 (I)

190 (NI)-0.02 (NI)

0 (NI)

Negative control

0-5 Silicon dioxide n-hydrate

Plateau level Result Time lag Intensity Plateau level Result Time lag

Intensity

Plateau level

Result190 (NI)-0.01 (NI)

0 (NI)NII

10 (I)

0.09 (I)18 (NI)N

54 (I)

I

0 (I)0.21 (I)

37 (I)

I

0.16 (I)

29 (I)

I

10 (I)

0.13 (I)

24 (I)

I

10 (I)

0.12 (I)

4 (NI)I

0 (I)0.25 (I)

13 (I)I

I

30 (I)

0.14 (I)

23 (I)

I

10 (I)

0.15 (I)

27 (I)

INI

190 (NI)-0.01 (NI)

0 (NI)

NI

190 (NI)-0.01 (NI)

0 (NI)

NI

Intensity

Plateau level

Result

563
564
565

Table 10-2. Data for Phase 0, Trial 2
No.

BRC

FDSC

Test chemical

DaicelTime lag

Intensity

Plateau level Result Time lag Intensity Plateau level Result Time lag

Negative control

190 (NI)-0.01 (NI)

0 (NI)

NI

190 (NI)-0.02 (NI)

0 (NI)

NI

190 (NI)-0.01 (NI)

0 (NI)

NI

Positive control (ethanol)

10 (I)

0.12 (I)

22 (I)

I

0 (I)0.12 (I)

21 (I)

I

10 (I)

0.13 (I)

24 (I)

I

0-1 Benzalkonium chloride

0 (I)0.32 (I)

57 (I)I

0 (I)0.30 (I)

54 (I)I

0 (I)0.33 (I)60 (I)

I

0-2 2-Propanol

0 (I)0.13 (I)

24 (I)

I

0 (I)0.18 (I)

32 (I)

I

10 (I)

0.13 (I)

24 (I)

I

0-3 Glycerol

0 (I)0.31 (I)

12 (I)

I

190 (NI)-0.10 (NI)

2 (NI)

NI

0 (I)0.21 (I)

12 (I)

I

0-4 n-Hexanol

10 (I)

0.15 (I)

28 (I)

I

0 (I)0.21 (I)

37 (I)

I

40 (I)

0.11 (I)

19 (I)I

0-5 Silicon dioxide n-hydrate

190 (NI)-0.01 (NI)

0 (NI)

NI

190 (NI)-0.01 (NI)

0 (NI)

NI

190 (NI)

0.00 (NI)

0 (NI)

NI

566

27

567

Table 10-3. Data for Phase 0, Trial 3
BRC

FDSC
No.

Test chemical

DaicelTime lag

Intensity

Plateau level

ResultTime lag

Intensity

Plateau level

ResultTime lag

Intensity

Plateau level

ResultNegative control

190 (NI)-0.01 (NI)

0 (NI)

NI

190 (NI)-0.10 (NI)

0 (NI)

NI

190 (NI)-0.01 (NI)

0 (NI)

NI

Positive control (ethanol)

20 (I)

0.12 (I)

22 (I)

I

0 (I)0.14 (I)

21 (I)

I

10 (I)

0.10 (I)

19 (NI)NI

0-1 Benzalkonium chloride

0 (I)0.34 (I)

62 (I)I

0 (I)0.29 (I)

52 (I)I

0 (I)0.30 (I)

55 (I)

I

0-2 2-Propanol

10 (I)

0.15 (I)

29 (I)

I

0 (I)0.16 (I)

29 (I)

I

10 (I)

0.13 (I)

24 (I)

I

0-3 Glycerol

0 (I)0.30 (I)

18 (I)

I

0 (I)0.41 (I)

16 (I)I

0 (I)0.19 (I)

13 (I)

I

0-4 n-Hexanol

0 (I)0.22 (I)

39 (I)

I

0 (I)0.16 (I)

28 (I)

I

20 (I)

0.15 (I)

27 (I)

I

190 (NI)-0.01 (NI)

0 (NI)

NI

190 (NI)-0.04 (NI)

0 (NI)

NI

190 (NI)-0.01 (NI)

0 (NI)

NI

0-5 Silicon dioxide n-hydrate

568
569
570

Table 11. Combined results for Phase 0
FDSC
No.

BRC

1

2

3

1

2

3

1

2

3

Negative control

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Positive control (ethanol)

Pass

Pass

Pass

Pass

Pass

Pass

NG

Pass

NG

0-1

Benzalkonium chloride

I

I

I

I

I

I

I

I

I

0-2

2-Propanol

I

I

I

I

I

I

I

I

I

0-3

Glycerol

I

I

I

I

NI

I

I

I

I

0-4

n-Hexanol

I

I

I

I

I

I

I

I

I

0-5

Silicon dioxide n-hydrate

NI

NI

NI

NI

NI

NI

NI

NI

NI

571
572

DaicelTest chemical

28

Negative control

40

20

1 23

1 23

1 23

FDSC

BOZO

DaicelLaboratory

573
574

60

40

20

1 23

1 23

Benzalkonium chloride

80

2
1 3

Plateau level (%)

60

0Positive control

80

Plateau level (%)

Plateau level (%)

80

60

12

3

2

1 23

40

3

1

20

0

0
FDSC

BOZO
Laboratory

DaicelFDSC

BOZO

DaicelLaboratory

Fig. 5. Distribution of the three trials of Phase 0

575
576

4.1.2

Phase I

577

Phase I was designed to assess within and between-laboratory reproducibility by testing ten coded test

578

chemicals using protocol ver. 1.51e.

579

The results for two of nine runs of the reference control (ethanol) at FDSC did not initially meet the580

success criteria, but were successfully retested, as shown in Tables 12 and 13. Analysis of Phase 0 and

581

Phase I results as well as concerns for quality assurance of the HCE models led the VMT to include

582

success criteria for the reference control in the next version of the test protocol. Consequently, the583

VMT recommended that the range for the reference control should be revised, so expanded success584

criteria for the positive and reference controls were developed by the lead laboratory. Furthermore,585

the results for test chemical No. 1-7, n,n-dimethyl guanidine sulfate, and No. 1-10, gluconolactone at

586

FDSC as well as for test chemical No. 1-8, toluene, at Daicel failed to satisfy the success criteria for587

the within-laboratory reproducibility, as shown in Tables 12 and 14. All results at BRC met the success588

criteria. Thus, the within-laboratory reproducibility was 80% at FDSC, 90% at Daicel, and 100% at

589

BRC, which was sufficient to satisfy the success criteria of 80% as stated in the study plan. Although590

the results for No. 1-1, imidazole, and No. 1-8, toluene, were somewhat inconsistent, the data showed591

a between-laboratory reproducibility of 80%, which met the acceptance criteria of 80% as stated in592

the study plan. The following key issues were addressed by revising the protocol to ver. 1.61e prior to593

the start of Phase II.

29

594



595
596

because control of ambient temperature is necessary.


597
598

Included success criteria for the reference control and changed the phrase “Plateau level isbetween 10% and 30%, inclusive” to “Plateau level is between 10% and 40%, inclusive”.



599
600

Revised the term “room temperature” to read “ambient temperature for the experiment,”

Change the ambient temperature for TEER measurement from “between 18 and 30°C” to“between 22 and 30°C,” because temperature of the HCE model can affect TEER.



Changed the description of the procedure for preparing test chemical solutions

601

Old: If the test chemical has not been dissolved, try to dissolve it by the mechanical mixture

602

for a maximum 1-minute period using a vortex, by the sonication for a maximum 20-minute603

period, or by the heating to70°.

604

New: If the test chemical does not dissolve readily, try one of the following techniques: a) mix

605

mechanically for a maximum of one minute using a vortex mixer, b) sonication for a

606

maximum of 20 minutes, or c) heating to a maximum temperature of 70°C.

607This was done, because some personnel at the participating laboratories misunderstood the608

procedure during Phase 1 and thought that all three of these techniques should be performed.

609

Also, the term “vortex” was corrected to “vortex mixer.”

610



Added a precaution to seal the 15-mL tube tightly during testing to prevent volatilization of611

the test chemical solutions, as follows: “To prevent volatilization of test chemical solutions,612

the 15-mL tube should be sealed tightly after weighing test chemicals, except when adding

613

culture medium and sampling the 2.5% test chemical solution.”614



Added instructions to reject and retest any result in which there is a significant discrepancy

615

between the initial TEER value and the TEER value measured at 0 seconds, which would

616

indicate some technical issue, such as electrical noise or improper use of electrode, as follows:

617“If there is a discrepancy of 40 Ω·cm2 or more between the initial TEER value and the TEER

618

value measured at 0 seconds, reject the test results and retest using another HCE model.”

30

619

Table 12-1. Data for Phase I, Trial 1
FDSC
No.

Test chemicalTime lag

Intensity

Negative control

190 (NI)-0.02 (NI)

Positive control

0 (I)Reference control

BRC

Plateau

ResultTime lag Intensity

0 (NI)

NI

190 (NI) -0.04 (NI)0.35 (I)

64 (I)I

0 (I)

20 (I)

0.09 (I)

16 (I)

I

1-1 Imidazole

190 (NI)

0.00 (NI)

0 (NI)

1-2 Cyclohexanol

10 (I)

0.23 (I)

1-3 3,3-Dithiodipropionic acidlevel

DaicelPlateaulevel

Result Time lag

0 (NI)

NI

0.39 (I)

51 (I)

I

0 (I)

0 (I)

0.18 (I)

16 (I)

INI

120 (I)

0.15 (I)

11 (I)

42 (I)I

0 (I)

021 (I)

190 (NI) -0.12 (NI)

0 (NI)

NI

190 (NI)

1-4 Acetone

30 (I)

0.08 (I)

15 (I)

I

10 (I)

1-5 3-Chloropropionitrile

10 (I)

0.18 (I)

32 (I)I

1-6 Ammonium nitrate

0 (I)0.77 (I)

54 (I)

1-7 n,n-Dimethylguanidine sulfate

0 (I)0.40 (I)

1-8 Toluene

190 (NI)1-9 3-Methoxy-1,2-propanediol
1-10 Gluconolactone

Intensity

level

Result0 (NI)

NI

0.35 (I)

64 (I)

I

10 (I)

0.14 (I)

26 (I)

II

130 (I)

0.12 (I)

9 (I)I

37 (I)I

0 (I)0.29 (I)

51 (I)

I

0 (NI)

NI

190 (NI) -0.06 (NI)

0 (NI)

NI

0.07 (I)

6 (I)

I

0 (I)

0.16 (I)

28 (I)

I

10 (I)

0.12 (I)

22 (I)

I

20 (I)

0.22 (I)38 (I)

I

I

0 (I)

1.36 (I)

27 (I)

I

0 (I)0.79 (I)

48 (I)

I

32 (I)I

0 (I)0.37 (I)

7 (I)

I

0 (I)

0.44 (I)

26 (I)

I

0.01 (NI)

0 (NI)

NI

170 (I)

0.02 (NI)1 (NI)

I

190 (NI)

0.02 (NI)1 (NI)

NI

190 (NI) -0.07 (NI)

0 (NI)

NI

190 (NI)

0 (NI)

NI

190 (NI) -0.12 (NI)

0 (NI)

NI

11 (I)I

0 (I)

3 (NI)

I

9 (I)

I

0 (I)

0.21 (I)

620

31

-0.07
(NI)

-0.08
(NI)
0.34 (I)190 (NI) -0.02 (NI)

Plateau

0 (I)0.22 (I)621

Table 12-2. Data for Phase I, Trial 2
FDSC
No.

Test chemical

BRC

Plateau

Time lag

Intensity

Negative control

190 (NI)

0.00 (NI)

0 (NI)

NI

Positive control

0 (I)0.33 (I)59 (I)I

0 (I)

Reference control

20 (I)-

3 (NI)NG

level

ResultTime lag

Intensity

190 (NI) -0.03 (NI)

DaicelPlateaulevel

ResultTime lag

0 (NI)

NI

0.23 (I)

42 (I)

I

0 (I)

0 (I)

0.12 (I)

21 (I)I

Intensity

190 (NI) -0.04 (NI)

Plateaulevel

Result0 (NI)

NI

0.42 (I)75 (I)

I

0 (I)0.17 (I)30 (I)

I

1-1 Imidazole

190 (NI) -0.03 (NI)

0 (NI)

NI

160 (I)

0.13 (I)

4 (NI)

I

140 (I)

0.11 (I)

7 (I)I

1-2 Cyclohexanol

30 (I)

0.16 (I)

25 (I)I

0 (I)0.40 (I)

48 (I)

I

0 (I)0.34 (I)

51 (I)

I

1-3 3,3-Dithiodipropionic acid

190 (NI) -0.06 (NI)

0 (NI)

NI

190 (NI) -0.07 (NI)

0 (NI)

NI

190 (NI) -0.05 (NI)

0 (NI)

NI

1-4 Acetone

10 (I)

0.04 (NI)10 (I)

I

0 (I)0.12 (I)

21 (I)

I

0 (I)0.17 (I)30 (I)

I

1-5 3-Chloropropionitrile

90 (I)

0.19 (I)

20 (I)

I

10 (I)

0.20 (I)

36 (I)

I

10 (I)

0.21 (I)

39 (I)I

1-6 Ammonium nitrate

0 (I)0.69 (I)

21 (I)

I

0 (I)0.67 (I)

27 (I)I

0 (I)1.05 (I)

52 (I)

I

1-7 n,n-Dimethylguanidine sulfate

190 (NI) -0.05 (NI)

2 (NI)

NI

0 (I)0.53 (I)

21 (I)I

0 (I)0.54 (I)

27 (I)I

1-8 Toluene

190 (NI) -0.01 (NI)

0 (NI)

NI

150 (I)

0.02 (NI)1 (NI)

I

190 (NI)

0.00 (NI)

0 (NI)

NI

1-9 3-Methoxy-1,2-propanediol

190 (NI) -0.05 (NI)

0 (NI)

NI

190 (NI) -0.12 (NI)

0 (NI)

NI

190 (NI)-0.11 (NI)

0 (NI)

NI

1-10 Gluconolactone

190 (NI) -0.04 (NI)

0 (NI)

NI

8 (I)

I

10 (I)

0.18 (I)

10 (I)

I

0 (I)

32

0.28 (I)622

Table 12-3. Data for Phase I, Trial 3
FDSC
No.

Test chemicalTime lag

Intensity

Negative control

190 (NI)-0.02 (NI)

Positive control

0 (I)Reference control

BRC
Plateau

DaicelPlateau

ResultTime lag

Intensity

0 (NI)

NI

190 (NI)-0.02 (NI)

0 (NI)

NI

0.36 (I)65 (I)I

0 (I)0.31 (I)

55 (I)

I

0 (I)

0 (I)

0.18 (I)

33 (I)NG

0 (I)0.12 (I)

22 (I)

I

190 (NI)-0.01 (NI)

0 (NI)

NI

110 (I)

0.17 (I)

13 (I)

0 (I)0.26 (I)

46 (I)I

0 (I)0.27 (I)

1-3 3,3-Dithiodipropionic acid

190 (NI)-0.05 (NI)

0 (NI)

NI

190 (NI)

1-4 Acetone

10 (I)

0.10 (I)

19 (I)

I

1-5 3-Chloropropionitrile

10 (I)

0.22 (I)

39 (I)

1-6 Ammonium nitrate

0 (I)0.62 (I)

1-7 n,n-Dimethylguanidine sulfate

0 (I)1-8 Toluene

level

level

Result Time lag Intensity

level

Result0 (NI)

NI

0.37 (I)

66 (I)

I

10 (I)

0.15 (I)

27 (I)I

I

60 (I)

0.14 (I)

20 (I)

I

48 (I)I

0 (I)0.33 (I)59 (I)

I-0.08 (NI)

0 (NI)

NI

190 (NI) -0.0 (NI)

0 (NI)

NI

0 (I)0.20 (I)

36 (I)

I

0 (I)0.18 (I)

32 (I)I

I

20 (I)

0.12 (I)

22 (I)

I

10 (I)

0.25 (I)

44 (I)

I

37 (I)I

0 (I)0.25 (I)50 (I)I

0 (I)0.94 (I)

47 (I)

I

0.36 (I)

25 (I)I

0 (I)0.45 (I)

23 (I)

I

0 (I)0.46 (I)

27 (I)

I

190 (NI)-0.03 (NI)

0 (NI)

NI

80 (I)

0.05 (I)8 (I)I

130 (I)

0.03 (NI)

2 (I)

I

1-9 3-Methoxy-1,2-propanediol

190 (NI)-0.09 (NI)

0 (NI)

NI

190 (NI)-0.12 (NI)

0 (NI)

NI

190 (NI)

0 (NI)

NI

1-10 Gluconolactone

10 (I)

0.10 (I)

5 (I)

I

0 (I)

0.30 (I)

9 (I)I

10 (I)

0.18 (I)

10 (I)

I

10 (I)

0.12 (I)

22 (I)

I

-

-

-

-

-

-

-

-

1-1 Imidazole
1-2 Cyclohexanol

Reference control (2)62333

190 (NI) -0.04 (NI)

Plateau

-0.11
(NI)

624

Table 13. Combined results for Phase I control chemicals
FDSC

Daicel

1

2

3

1

2

3

1

2

3

Negative control

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Positive control

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Reference

Pass

NG

NG

Pass

Pass

Pass

Pass

Pass

Pass

Reference (2)
625
626
627

BRC

Pass

Table 14. Combined results for Phase I test chemicals
GHS

No.

Test chemical

FDSC

BRC

Daicel

1

2

3

1

2

3

1

2

3

1-1

Imidazole

NI

NI

NI

I

I

I

I

I

I

1-2

Cyclohexanol

I

I

I

I

I

I

I

I

I

1-3

3,3-Dithiodipropionic acid

NI

NI

NI

NI

NI

NI

NI

NI

NI

1-4

Acetone

I

I

I

I

I

I

I

I

I

1-5

3-Chloropropionitrile

I

I

I

I

I

I

I

I

I

1-6

Ammonium nitrate

I

I

I

I

I

I

I

I

I

1-7

n,n-Dimethylguanidine sulfate

I

NI

I

I

I

I

I

I

I

No Category

1-8

Toluene

NI

NI

NI

I

I

I

NI

NI

I

(NC)

1-9

3-Methoxy-1,2-propanediol

NI

NI

NI

NI

NI

NI

NI

NI

NI

1-10

Gluconolactone

I

NI

I

I

I

I

I

I

I

Cat.1

Cat. 2A & 2B

628
629
630

34

631

4.1.3

Phase II

632

Phase II was designed to assess the between-laboratory reproducibility of ten coded test chemicals

633

using protocol ver. 1.61e.

634

Results for two of the ten test chemicals failed to satisfy the success criteria for between-laboratory635

reproducibility: No. 2-1, imidazole, and No. 2-9, toluene, as shown in Tables 15, 16, and 17. Although636

the concordance was 80% between the three laboratories, which was sufficient to satisfy the success637

criteria, the VMT was concerned over the failure to properly identify No. 2-1, imidazole, which is a638

UN GHS category 1 irritant. Therefore, the VMT was unanimous in recognizing the need to clarify639

the reason for this failure.

640

During a VMT teleconference to discuss the results of Phase II, the lead laboratory suggested that it

641

might be necessary to control the ambient temperature at which tests were conducted. The lead642

laboratory had obtained data at the relatively high ambient temperature of 28°C. In addition, the time643

dependent TEER profile after exposing imidazole was affected by the temperature. In case the644temperature below 22°C, imidazole was classified as non-irritant. All laboratories performed

645

additional testing of No. 2-1, imidazole, under the modified parameters given in Fig.6 and as shown646

in Table 18. All laboratories correctly identified No. 2-1, imidazole, as an irritant, which suggested the647need for rigorous control of the ambient temperature, and led to a major revision of the protocol prior

648

to Phase III.

649

Due to this revision, the VMT recognized that Phase II data should not be combined with Phase III

650

data to assess predictive capacity and decided to undertake validation of between-laboratory651

reproducibility and predictive capacity in Phase III using revised protocol ver. 1.71e. In consideration652

of the capacity of the participating laboratories, the number of test chemicals for Phase III was reduced

653

from 40 in Phases IIA and IIB of the original study plan to just 36. Thus, a total of four chemicals (two654from UN GHS category 1, 1 from UN GHS category 2, and 1 No Category) were removed from the655

original list of test chemicals.

656

The following key issues were addressed by revising the protocol to ver. 1.71e prior to the start of657

Phase III.

35

658



Having recognized the need to control ambient temperature, we replaced the instruction “Let659

stand for 10 minutes (within 2 hours) at the ambient temperature for the experiment” to660“Adjust the temperature of the model to 28±2°C.”

661



662
663

Replaced all instances of the phrase “ambient temperature for the experiment” to “between
22 and 30°C.”



Changed the success criteria for the reference control from “Plateau level is between 10%664

and 30%, inclusive” to “Plateau level is 10% or more.” The upper limit for this success665

criterion will be determined after reviewing the results of Phase III.

666

36

667

Table 15. Data for Phase II
FDSC

BRC

DaicelTest chemical
No.

Time lag

Intensity

Plateau level

ResultTime lag

Intensity

Plateau level

ResultTime lag

Intensity

Plateau level

ResultNegative control

190 (NI)-0.04 (NI)

0 (NI)

NI

190 (NI)-0.08 (NI)

0 (NI)

NI

190 (NI)-0.02 (NI)

0 (NI)

NI

Positive control

0 (I)0.41 (I)74 (I)I

0 (I)0.33 (I)59 (I)I

0 (I)0.30 (I)

54 (I)

I

Reference control

0 (I)0.24 (I)

31 (I)

I

0 (I)0.14 (I)

25 (I)

I

0 (I)0.16 (I)

24 (I)

I

190 (NI)

0.00 (NI)

3 (NI)

NI

140 (I)

0.15 (I)8 (I)I

190 (NI)

0.00 (NI)

0 (NI)NI

2-1

Imidazole

2-2

Cyclohexanol

0 (I)0.51 (I)

51 (I)I

0 (I)0.32 (I)

48 (I)I

0 (I)0.25 (I)38 (I)

I

2-3

Sodium dodecyl sulfate

0 (I)0.41 (I)74 (I)I

0 (I)0.32 (I)58 (I)I

0 (I)0.31 (I)

56 (I)

I

2-4

Sodium salicylate

0 (I)0.80 (I)

48 (I)

I

0 (I)0.41 (I)

33 (I)I

0 (I)0.54 (I)

33 (I)

I

2-5

Cyclopentanol

0 (I)0.28 (I)

39 (I)

I

0 (I)0.22 (I)

40 (I)

I

0 (I)0.17 (I)30 (I)

I

2-6

2-Methyl-1-pentanol

0 (I)0.30 (I)

54 (I)I

0 (I)0.23 (I)

35 (I)

I

10 (I)

0.14 (I)

26 (I)I

2-7

α-Hexylcinnamaldehyde

190 (NI)-0.03 (I)

0 (NI)

NI

190 (NI)-0.03 (NI)

0 (NI)

NI

190 (NI)-0.01 (NI)

0 (NI)

NI

2-8

n,n-Dimethylguanidine sulfate

0 (I)0.83 (I)

42 (NI)I

0 (I)0.37 (I)

26 (I)I

0 (I)0.54 (I)

27 (I)

I

2-9

Toluene60 (I)

0.06 (I)9 (I)I

190 (NI)

0.00 (NI)

0 (NI)

NI

190 (NI)-0.02 (NI)

0 (NI)

NI

2-10

Gluconolactone

0 (I)0.48 (I)

19 (I)I

0 (I)0.30 (I)

12 (I)I

0 (I)0.23 (I)

9 (I)

I

668
37

669

670
671
672

Table 16. Results for Phase II control chemicals
FDSC

BRC

DaicelNegative control

Pass

Pass

Pass

Positive control

Pass

Pass

Pass

Reference

Pass

Pass

Pass

Table 17. Results for Phase II test chemicals
GHS

No.

Cat. 1

Cat. 2A & 2B

No Category
673
674
675

Test chemical

FDSC

BRC

Daicel

2-1 Imidazole

NI

I

NI

2-2 Cyclohexanol

I

I

I

2-3 Sodium dodecyl sulfate

I

I

I

2-4 Sodium salicylate

I

I

I

2-5 Cyclopentanol

I

I

I

2-6 2-Methyl-1-pentanol

I

I

I

2-7 α-Hexylcinnamaldehyde

NI

NI

NI

2-8 n,n-Dimethylguanidine sulfate

I

I

I

2-9 Toluene

I

NI

NI

2-10 Gluconolactone

I

I

I

Table 18. List of test conditions at each lab.
Phase II study

Additional

CircumstancesPartmeasured

Temp.
(℃)

CircumstancesPartmeasured

Temp.
(℃)

FDSC

Room temp.

Room
temp.

24-26

On hot plate

Mediumat a well

27-28

BRC

Room temp.

Room
temp.

22-25

In Water bath

Mediumat a well27.4-28.6

DaicelRoom temp.

Room
temp.

22

Room temp.

Room
temp.

28±2

Lead
Lab

Room temp.

Mediumat a well

28

676

38

677

678

679
680

Fig.6. Additional data of Imidazole on Vitrigel-EIT phase II study

681

39

682

4.1.4

Phase III683

During Phase III, the VMT received a question about test chemical No. 3-16, sodium chloroacetate,684

from the on-site study director at Daicel, who opened the MSDS due to concerns over legal

685

compliance in handling deleterious substances. After considering the possibility of using this chemical,

686

the VMT decided instead to delete it from the list of test chemicals, and in its place, distributed to all

687

laboratories a new test chemical: No. 3-37, cyclopentanol. This test chemical is a UN GHS category688

2B substance, just like No. 3-16, sodium chloroacetate.

689

There were some discrepancies in the Phase III results that can be attributed to differences the690

techniques used to dissolve the test chemicals. To resolve this issue, the protocol was revised to691ver. 1.80e by limiting the techniques to be used to dissolve test chemicals.

692

Also, an additional procedure was included, which calls for the pH level of each 2.5% test chemical693

solution to be measured using universal pH test paper to ensure that the test chemical falls within the694

applicability domain.

695
696

Other procedural inconsistencies that require further study to determine whether or not standardization697

is necessary include the following.

698
699
700
701

a. The time interval from the start of exposure to a test chemical until the start of TEER
measurement: 4 s at FDSC, 3 s at BRC, and 2 s at Daicel
b. Temperature of the models: 27.0–28.7°C in culture medium at FDSC, 26.4–28.0°C in a water
bath at BRC, and 26.9–28.4°C in culture medium at Daicel

702

c. Number of insoluble test chemicals: Of the 21 test chemical solutions prepared at FDSC, four703

exhibited sediment and two exhibited supernatants (Nos. 212, 216); of the 19 test chemicals

704

prepared at BRC, 10 exhibited sediment and seven exhibited supernatants (Nos. 213, 221,

705

222, 223, 232, 234, and 236); and of the 17 test chemicals prepared at Daicel, seven exhibited

706

sediment and 10 exhibited supernatant (Nos. 202, 210, 218, 219, 220, 224, 230, 231, 233,707

and 235).

708

d. Other issues:

40

709

At Daicel, different batches of the frozen cell lines were used.

710

At FDSC, test chemical No. 216 was tested twice, but the data was not approved due to and711

inappropriate procedure.

712
713

All of the aforementioned issues were reported to the VMT, which unanimously agreed that these714

were minor issues that did not impact data analysis.

715

In Tables 19, 20, and 21, the between-laboratory reproducibility was 92% (33/36), which met the716

acceptance criteria of 80%. The results of a few insoluble test chemicals were inconsistent between

717

the laboratories, including No. 3-5, tetra-N-octylammonium bromide, No. 3-14, 2,6-dichlorobenzyl

718

chloride, and No. 3-18, camphene, and the VMT discussed the difficulties inherent in assessing these719

substances due to low solubility in the culture medium.

720

The following key issues were addressed by revising the protocol to ver. 1.80e after completion of

721

Phase III.

722



Added the term “Universal pH test paper (ADVANTEC, 07011030)” to section 3.

723



Added a description of the applicability domain, which was determined per the results for 93

724
725

test chemicals.
Changed the description of the procedure for preparing test chemical solution as follows.

726

Old: If the test chemical has not been dissolved, try to dissolve it by selecting an appropriate

727

technique(s) from the following; mechanical mixture for a maximum 1-minute period using728

a vortex mixer, sonication for a maximum 20-minute period, or heating to maximum 70°C.

729

New: If the test chemical does not dissolve readily, try using the following techniques in the730

following order to dissolve it: a) mix mechanically for a maximum of one minute using a731

vortex mixer, b) sonication for a maximum of 20 minutes, or c) heating to a maximum

732

temperature of 70°C. After trying each technique, adjust the temperature of each test chemical733

solution to 28±2°C and check solubility. Move to the next step of the procedure once the test

734

chemical solution is well dissolved or homogeneously dispersed.

735



Added a precaution that techniques for dissolving test chemicals are to be set according to

41

736

the physiochemical properties of the test chemicals.

737
738

The Vitrigel-EIT method was developed primarily to identify ocular non-irritants in a bottom-up

739

approach. As shown in Tables 22, the Vitrigel-EIT method demonstrated an accuracy of between 64740

and 69% (23 to 25/36), a sensitivity of between 75 and 83% (18 to 20/24), and a specificity of 42%741(5/12). These figures are lower than those of in house data obtained by the lead lab and there are too

742

many false negatives for this test method to be useful in a bottom-up approach. Substances that yielded

743

either false negative or false positive results are listed in Table 23.

744

42

745

Table 19-1. Results for Phase III control chemicals
FDSC
Set

Test chemical

BRC

Temp.

Temp.
Time lag

Intensity

Plateau level

Result

(°C)*

1

2

3

746
747
748

*

DaicelTemp.

Time lag

Intensity

Plateau level

Result(°C)*

Time lag

Intensity

Plateau level

Result(°C)*

Negative control

28.0

190 (NI)-0.03 (NI)

0 (NI)

NI

26.4

190 (NI)

0.00 (NI)

0 (NI)

NI

28.4

190 (NI)-0.02 (NI)

0 (NI)

NI

Positive control

28.2

0 (I)

0.45 (I)82 (I)

I

27.5

0 (I)

0.41 (I)75 (I)

I

28.4

0 (I)

0.52 (I)

67 (I)

I

Reference control

27.4

0 (I)

0.20 (I)

28 (I)

I

27.5

0 (I)

0.19 (I)34 (I)

I

28.4

0 (I)

0.24 (I)

29 (I)

I

Negative control

27.2

190 (NI)-0.03 (NI)

0 (NI)

NI

27.1

190 (NI)-0.04 (NI)

0 (NI)

NI

27.9

190 (NI)-0.03 (NI)

0 (NI)

NI

Positive control

27.3

0 (I)

0.44 (I)

79 (I)

I

27.2

0 (I)

0.40 (I)73 (I)

I

27.9

0 (I)

0.40 (I)

72 (I)

I

Reference control

27.2

0 (I)

0.19 (I)

29 (I)

I

27.3

0 (I)

0.19 (I)

35 (I)

I

27.9

0 (I)

0.14 (I)

26 (I)

I

Negative control

27.6

190 (NI)-0.05 (NI)

0 (NI)

NI

27.6

190 (NI)-0.04 (NI)

0 (NI)

NI

Positive control

27.5

0 (I)

0.36 (I)65 (I)

I

27.7

0 (I)

050 (I)

90 (I)

I

Reference control

27.2

0 (I)

0.17 (I)

31 (I)

I

27.7

0 (I)

0.27 (I)

48 (I)

I

Temperature of the model at the time of exposure to the test chemical solution

Table 19-2. Results for Phase III test chemicals
FDSC
No.

Test chemical

Temp.
(°C)*

BRC
Plateau

Time lag

Intensity

Temp.
Result

level

(°C)*

DaicelPlateau

Time lag

Intensity

Temp.
Result

level

(°C)*

Plateau
Time lag

Intensity

Resultlevel

3-1 2,5-Dimethyl-2,5-hexanediol

27.1

10 (I)

0.14 (I)

26 (I)

I

27.6

10 (I)

0.14 (I)

26 (I)

I

27.7

40 (I)

0.18 (I)

27 (I)

I

3-2 2-Benzyl-4-chlorophenol

28.5

0 (I)

0.40 (I)

72 (I)

I

28.0

0 (I)

0.40 (I)

72 (I)

I

27.7

0 (I)

0.40 (I)73 (I)

I

3-3 2,2-Dimethyl butanoic acid

27.6

0 (I)

0.50 (I)60 (I)

I

27.5

0 (I)

0.50 (I)60 (I)

I

27.7

50 (I)

0.23 (I)

31 (I)

I

3-4 Captan

27.0

190 (NI) -0.05 (NI)

0 (NI)

NI

27.5

190 (NI) -0.05 (NI)

0 (NI)

NI

27.9

190 (NI) -0.01 (NI)

0 (NI)

NI

43

Tetra-n-octylammonium
3-5

27.5

60 (I)

0.17 (I)

23 (I)

I

27.5

60 (I)

0.12 (I)

17 (I)

I

27.6

190 (NI)

0.01 (NI)

0 (NI)

NI

27.6

0 (I)

0.27 (I)

49 (I)

I

27.6

0 (I)

0.27 (I)

49 (I)

I

27.6

0 (I)

0.33 (I)59 (I)

I

28.1

0 (I)

0.33 (I)60 (I)

I

27.6

0 (I)

0.33 (I)60 (I)

I

27.6

0 (I)

0.41 (I)73 (I)I

3-8 Sodium dodecyl sulfate

28.4

0 (I)

0.45 (I)

81 (I)

I

27.6

0 (I)

0.45 (I)

81 (I)

I

27.6

0 (I)

0.46 (I)82 (I)

I

3-9 m-Phenylenediamine

27.0

0 (I)

0.39 (I)70 (I)

I

27.6

0 (I)

0.39 (I)70 (I)I

26.9

10 (I)

0.42 (I)74 (I)

I

3-10 Tetraethylene glycol

27.8

0 (I)

0.24 (I)

43 (I)

I

27.7

0 (I)

0.24 (I)

43 (I)

I

26.9

20 (I)

0.20 (I)

35 (I)

I

3-30 Imidazole

28.1

90 (I)

0.24 (I)

23 (I)I

27.8

90 (I)

0.24 (I)

23 (I)I

27.7

80 (I)

0.31 (I)

33 (I)

I

3-32 Sodium salicylate

27.7

0 (I)

0.54 (I)

43 (I)

I

27.5

0 (I)

0.54 (I)

43 (I)

I

28.0

0 (I)

0.35 (I)38 (I)

I

3-11 gamma-Butyrolactone

27.5

0 (I)

0.22 (I)

40 (I)I

27.7

10 (I)

0.23 (I)

42 (I)

I

26.9

0 (I)

0.21 (I)

37 (I)

I

3-12 Methyl acetate

28,4

0 (I)

0.20 (I)

36 (I)

I

27.6

0 (I)

0.18 (I)

32 (I)

I

26.9

0 (I)

0.18 (I)

32 (I)

I

3-13 Myristyl alcohol

27.1

190 (NI) -0.02 (NI)

0 (NI)

NI

27.3

190 (NI) -0.05 (NI)

0 (NI)

NI

28.2

190 (NI) -0.03 (NI)

0 (NI)

NI

3-14 2,6-Dichlorobenzoyl chloride

28.0

190 (NI)

0.00 (NI)

21 (NI)NI

27.3

110 (I)

0.46 (I)

33 (I)

I

28.2

190 (NI) -0.07 (NI)

0 (NI)

NI

3-15 Dibenzyl phosphate

28.1

0 (I)

0.51 (I)71 (I)

I

27.3

0 (I)

0.39 (I)59 (I)

I

28.2

0 (I)

0.32 (I)

57 (I)

I

27.8

0 (I)

1.65 (I)

37 (I)

I

27.4

0 (I)

151 (I)

30 (I)

I

28.1

0 (I)

1.57 (I)

31 (I)

I

3-18 Camphene

27.7

160 (I)

0.08 (I)

4 (NI)

I

27.3

190 (NI) -0.03 (NI)

0 (NI)

NI

28.4

190 (NI) -0.01 (NI)

0 (NI)

NI

3-19 Ethyl-2-methylacetoacetate

27.2

0 (I)

0.25 (I)

46 (I)I

27.5

10 (I)

0.19 (I)34 (I)

I

28.3

0 (I)

0.23 (I)

42 (I)

I

3-20 Propylene glycol propyl ether

28.1

0 (I)

0.23 (I)

42 (I)

I

27.5

0 (I)

0.23 (I)

42 (I)

I

28.3

10 (I)

0.20 (I)

36 (I)

I

3-31 2-Methyl-1-pentanol

27.9

0 (I)

0.42 (I)75 (I)

I

27.5

0 (I)

0.42 (I)75 (I)

I

28.0

0 (I)

0.26 (I)

48 (I)

I

3-33 α-Hexylcinnamaldehyde

27.4

190 (NI) -0.04 (NI)

0 (NI)

NI

28.0

190 (NI) -0.04 (NI)

0 (NI)

NI

27.7

190 (NI) -0.03 (NI)

0 (NI)

NI

3-37 Cyclopentanol

27.4

0 (I)

0.28 (I)

51 (I)

I

28.0

0 (I)

0.28 (I)

51 (I)

I

28.0

0 (I)

0.30 (I)

55 (I)

I

3-21 Methyl amyl ketone

27.4

10 (I)

0.10 (I)

20 (I)I

27.2

10 (I)

0.10 (I)

20 (I)

I

28.3

30 (I)

0.16 (I)

26 (I)

I

bromide3-6 Butanol
3- (2-Aminoethylamino)propyl]
3-7trimethoxysilane

1- (2-Propoxy-1-methylethoxy)3-17
2-propanol

44

749

3-22 2- (n-Dodecylthio)ethanol

28.7

190 (NI) -0.05 (NI)

0 (NI)

NI

27.4

190 (NI) -0.05 (NI)

0 (NI)

NI

28.3

190 (NI)

0.00 (NI)

0 (NI)

NI

3-23 iso-Octylthioglycolate

27.3

190 (NI)

0.00 (NI)

2 (NI)

NI

27.4

190 (NI)

0.00 (NI)

2 (NI)

NI

27.4

190 (NI) -0.02 (NI)

0 (NI)

NI

3-24 2,4-Difluoronitrobenzene

27.8

30 (I)

0.13 (I)

20 (I)

I

27.4

30 (I)

0.13 (I)

20 (I)

I

27.4

40 (I)

0.11 (I)

18 (I)

I

3-25 tetra-Aminopyrimidine sulfate

28.7

190 (NI) -0.09 (NI)

0 (NI)

NI

27.2

190 (NI) -0.09 (NI)

0 (NI)

NI

27.4

190 (NI) -0.09 (NI)

0 (NI)

NI

3-26 2,4-Pentanediol

27.4

120 (I)

0.08 (I)

7 (I)

I

27.6

120 (I)

0.08 (I)

7 (I)

I

27.4

130 (I)

0.12 (I)

8 (I)I

3-27 iso-Octyl acrylate

27.9

190 (NI)

0.00 (NI)

0 (NI)

NI

27.7

190 (NI)

0.00 (NI)

0 (NI)

NI

27.7

190 (NI) -0.01 (NI)

0 (NI)

NI

3-28 Silicon dioxide n-hydrate

27.3

190 (NI) -0.04 (NI)

0 (NI)

NI

27.8

190 (NI) -0.04 (NI)

0 (NI)

NI

27.7

190 (NI) -0.04 (NI)

0 (NI)

NI

3-29 Potassium tetrafluoroborate

27.8

0 (I)

0.45 (I)

13 (I)

I

27.7

0 (I)

0.45 (I)

13 (I)

I

27.7

0 (I)

0.47 (I)

14 (I)

I

3-34 n,n-Dimethylguanidine sulfate

27.8

0 (I)

0.40 (I)

32 (I)

I

27.7

0 (I)

0.40 (I)

32 (I)

I

28.0

0 (I)

0.84 (I)

25 (I)

I

3-35 Toluene

28.0

80 (I)

0.16 (I)

19 (I)I

27.7

80 (I)

0.16 (I)

19 (I)I

28.0

30 (I)

0.12 (I)

20 (I)

I

3-36 Gluconolactone

27.2

0 (I)

0.26 (I)

10 (I)

I

27.5

0 (I)

0.26 (I)

10 (I)

I

28.0

0 (I)

0.31 (I)

9 (I)

I

*

Temperature of the model at the time of exposure to the test chemical solution

750

45

751

Table 20. Results for Phase III control chemicals
FDSC

BRC

DaicelChemical
1

2

3

1

2

3

1

2

3

Negative control

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

-

Positive control

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

-

Reference

Pass

Pass

Pass

Pass

Pass

Pass

Pass

Pass

-

FDSC

BRC

DaicelLead Lab

752
753

Table 21. Results for Phase III test chemicals
GHS

Cat. 1No.

Test chemical

3-1

2,5-Dimethyl-2,5-hexanediol

I

I

I

I

3-2

2-Benzyl-4-chlorophenol

I

I

I

I

3-3

2,2-Dimethyl butanoic acid

I

I

I

I

3-4

Captan

NI

NI

NI

NI

3-5

Tetra-n-octylammonium bromide

I

I

NI

I

3-6

Butanol

I

I

I

I

I

I

I

I

3- (2-Aminoethylamino)propyl]
3-7trimethoxysilane
3-8Sodium dodecyl sulfate

I

I

I

I

3-9

m-Phenylenediamine

I

I

I

I

3-10

Tetraethylene glycol

I

I

I

I

3-30

Imidazole

I

I

I

I

3-32

Sodium salicylate

I

I

I

I

3-11

gamma-Butyrolactone

I

I

I

I

3-12

Methyl acetate

I

I

I

I

3-13

Myristyl alcohol

NI

NI

NI

NI

3-14

2,6-Dichlorobenzoyl chloride

NI

I

NI

I

3-15

Dibenzyl phosphate

I

I

I

I

1-(2-Propoxy-1-methylethoxy)-2-propanol

I

I

I

I

3-18

Camphene

I

NI

NI

I

3-19

Ethyl-2-methylacetoacetate

I

I

I

I

3-20

Propylene glycol propyl ether

I

I

I

I

3-31

2-Methyl-1-pentanol

I

I

I

I

3-33

α-Hexylcinnamaldehyde

NI

NI

NI

I

3-37

Cyclopentanol

I

I

I

I

Cat. 2A & 2B 3-1746

No Category

754

3-21

Methyl amyl ketone

I

I

I

I

3-22

2-(n-Dodecylthio)ethanol

NI

NI

NI

NI

3-23

iso-Octylthioglycolate

NI

NI

NI

NI

3-24

2,4-Difluoronitrobenzene

I

I

I

I

3-25

tetra-Aminopyrimidine sulfate

NI

NI

NI

NI

3-26

2,4-Pentanediol

I

I

I

I

3-27

iso-Octyl acrylate

NI

NI

NI

NI

3-28

Silicon dioxide n-hydrate

NI

NI

NI

NI

3-29

Potassium tetrafluorobroate

I

I

I

I

3-34

n,n-Dimethylguanidine sulfate

I

I

I

I

3-35

Toluene

I

I

I

I

3-36

Gluconolactone

I

I

I

NI

*In-house data from the lead lab was obtained from non-coded chemicals.

755
756

Table 22-1. Phase III contingency table used at FDSC and BRC in a bottom-up approach

Vitrigel-EIT

UN GHS

Total

I

NI

Cat.1, 2A, 2B

20

4

24

No Category

7

5

12

27

9

36

Total
Sensitivity: 83% (20/24)
Specificity: 42% (5/12)
Accuracy: 69% (25/36)
757
758

Table 22-2. Phase III contingency table used at Daicel in a bottom-up approach

Vitrigel-EIT
Cat.1, 2A, 2B
No Category

UN GHS
Total

Sensitivity: 75% (18/24)
Specificity: 42% (5/12)
Accuracy: 64% (23/36)

47

I
18
7

NI
6
5

25

11

Total
24
12
36

759
760

Table 22-3. Phase III contingency tables used at the lead lab in bottom-up approach

Vitrigel-EIT
Cat.1, 2A, 2B
No Category

UN GHS

Total
Sensitivity: 92% (22/24)
Specificity: 50% (6/12)
Accuracy: 78% (28/36)

I
22
6
28

Total

NI
2
6
8

24
12
36

761
762

Table 23. Limitations on applicability at a bottom-up approach in phase III
No.

Test chemicals

Rank

Applicability limitation

3-4

CaptanInsoluble after 5 m.

3-5

Tetra-n-octylammonium bromide

Insoluble after 5 m.

3-13

Myristyl alcohol

3-14

2,6-Dichlorobenzoyl chloride

3-18

Camphene

3-33

α-Hexylcinnamaldehyde

3-21

Methyl amyl ketone

3-24

2,4-Difluoronitrobenzene

3-26

2,4-Pentanediol

3-29

Potassium tetrafluoroborate

3-34

n,n-Dimethylguanidine sulfate

3-35

Toluene3-36

Gluconolactone

False negatives

Insoluble after 5 m.
pH of 2.5% solution < 5.0
Protocol revised

False positive

pH of 2.5% solution < 5.0 after 10 m.

763
764

4.2 Quality assurance

765

All the records (data sheets and record sheets) from the participating laboratories were checked by

766

JaCVAM, As a result, six record sheets were uncompleted. They were the record sheets on the767

maintenance of measuring instruments, the culture of HCE models, and the preparation and768

application of test chemicals at phase I and the preparation and application of test chemicals at phase769

II in BRC, and application of test chemicals at phase I and phase III in Daicel. Although there are770

these defectiveness records, JaCVAM considered these records had less effects on quality of data in the771

validation study.

48

772

5

Discussion

773

5.1

Purpose of the Validation

774

The validation study was conducted to assess the reliability (within- and between-laboratory775

reproducibility) and relevance (predictive capacity) of the Vitrigel-EIT method with a challenging set

776

of test chemicals for which high quality in vitro and in vivo data are available. Preference should be

777

given the selection of test chemicals that were classified under UN GHS using individual animal.

778

Unfortunately, the VMT is unable to establish a correlation between results obtained using the779

Vitrigel-EIT method and EPA categories due to a lack of individual animal data. Therefore, results

780

obtained using the Vitrigel-EIT method are correlated with UN GHS categories only. The Vitrigel-

781

EIT method was developed primarily to identify ocular non-irritants in a bottom-up approach. The

782

VMT also undertook an analysis of a top-down approach to identifying UN GHS Category 1 ocular

783

irritants for comparison with the results from a bottom-up approach.

784
785

5.2

Transferability

786

All test chemicals were successfully identified during Phase 0 in conformance with the results from787

the lead laboratory, and the protocol was then revised from ver. 1.30e to ver. 1.51e. Further revisions

788

were made to eliminate inconsistencies that were identified during Phase I and Phase II testing. The

789

VMT confirmed that these inconsistencies had been resolved, thereby validating transferability of the790

test method. A history of revisions made to the Vitrigel-EIT protocol during this process is shown in

791

Fig.7. Significant milestones during this process include:792

Changed the positive control from ethanol to benzalkonium chloride793



Adopted ethanol as reference control for checking the quality of the HCE models

794

Defined a procedure for dissolving test chemicals in the culture medium (Fig.3)

795



Defined a standard ambient temperature for the experiment796



Revised other minor points in the protocol

797

In order to check of transferability for regulatory use, a representative set of proficiency chemicals

798

address for regulatory acceptance in appendix 8.9.

49

799

800
801

Fig. 7 Vitrigel-EIT protocol revision history

802

5.3

803

The results of Phase I showed that within-laboratory reproducibility was 80% at FDSC, 90% at Daicel,

804

and 100% at BRC, which was sufficient to satisfy the success criteria of 80% as stated in the study

805plan. The results of Phase II, however, were problematic and not accepted by the VMT, because

806

irrespective of the fact that the results satisfied success criteria for between-laboratory reproducibility,807

all three participating laboratories obtained a false-negative result for imidazole, a GHS Category 1

808

irritant. The results of Phase III showed that imidazole was identified correctly by all laboratories and809

that overall between-laboratory reproducibility was 90%, which was sufficient to satisfy the success810

criteria of 80% as stated in the study plan. Thus, the VMT concluded that through the process of811

revising the test protocol, the Vitrigel-EIT method attained an elevated level of between-laboratory812

reproducibility.

813

On the other hand, there were nine test chemicals that were used in both Phases II and III. Although814

there was a significant difference between Phases II and III in the temperature at which measurements

815

were made, results of 7 of these 9 test substances were concordant. Only imidazol and toluene were816

not concordant between Phases II and III. In order to predict imidazole correctly as an irritant, theWithin- and between-laboratory reproducibility

50

817

temperature at which measurements were made was revised in the protocol prior to Phase III.

818

Regarding the inconsistencies for toluene in Phase II, Daicel and BRC performed the test at 22 to819

25°C and predicted it to be a non-irritant, although FDSC performed the test at a relatively high 24 to

820

26°C and predicted it to be an irritant (Table 18). However, in Phase III, all three laboratories tested821

at 28±2°C and predicted toluene to be an irritant. These results suggest that the temperature at which822

measurements are made is important for achieving reproducible results. Therefore, this data also

823

indicates a high between-laboratory reproducibility for this test method.

824
825

5.4

Predictive capacity and relevance826

The results obtained from thirty-six test chemicals during Phase III were analyzed to assess their827

correlation with both existing in vitro and in vivo data and thereby evaluate predictive capacity. The

828

Vitrigel-EIT method was developed primarily to identify ocular non-irritants in a bottom-up approach.

829

Therefore, the test chemicals included UN GHS category 1, 2, 2A and 2B ocular irritants for which in830

vivo data was available. The Vitrigel-EIT method demonstrated an accuracy of between 64 and 69%

831(23 to 25/36), a sensitivity of between 75 and 83% (18 to 20/24), and a specificity of 42% (5/12).

832

Sensitivity was low due to six false negatives and specificity (predictive capacity for identifying non-

833

irritants) was low due to seven false positives, as shown in Table 23. The VMT requested the further

834

analysis to determine whether or not predictive capacity could be improved by defining the835

applicability domain. Ultimately, it was determined that although the results of the validation

836

confirmed an elevated level of reproducibility for this assay, the sample size was insufficient either to837

evaluate predictive capacity or define a proper applicability domain. Therefore, the VMT

838

recommended that data obtained at the lead laboratory should be used to define an applicability

839

domain suitable for use in a regulatory context.

840

Total 132 test chemicals were tested at the lead laboratory and were composed of 118 test chemicals

841(Appendix 8.10 and Appendix 8.11) including 22 used during Phase III and additional 14 chemicals

842

during Phase III. According to the latest version of the protocol, however, the available data limited

843

at lead laboratory were 57 chemicals tested at 28±2°C in 96 chemicals subtracted 36 chemicals for

51

844

Phase III from the total 132 chemicals. Hence, the predictive capacity was evaluated by the 93

845

results comprise the data for 36 chemicals during Phase III shown in Table 21 and for 57 chemicals

846

obtained at the lead laboratory shown in Appendix 8.8. The test chemicals were selected to ensure that847

a diverse range of substances were represented, and aspects such as eye-irritant level per UN GHS

848

categories, physical state, chemical class. The 93 test chemicals are composed of 56 liquids and 37

849

solids. Also, their contents are 28 Category 1 chemicals, 32 Category 2, 2A, 2B chemicals, and 33 No

850

Category chemicals by UN GHS classification. There were 36 coded chemicals tested for Phase III851

and 57 non-coded chemicals were tested at the lead laboratory. These 93 test chemicals were examined852

by the Vitrigel-EIT method in accordance with the protocol versions described in Chapter 3.1.3.4 and853

Appendix 8.8. However, the temperature at which all measurements were made during the chemical

854

exposure experiments was strictly controlled at 28±2°C (Table 19 and Appendix 8.8). Thus we855

consider this data sufficient for assessing the suitability of the Vitrigel-EIT method for use in a bottom-

856

up approach for identifying ocular non-irritants and in a top-down approach for identifying UN GHS

857

Category 1 ocular irritants. In a bottom-up approach, 60 of the test chemicals were classified as irritant858

and the other 33 as non-irritant, with results for 73 of the 93 test chemicals matching their UN GHS

859

categories. In contrast, 10 of the 60 test chemicals classified as irritants by in vivo data were identified860

as non-irritants, a false-negative rate of 17%. Additionally, 10 of the 33 test chemicals classified as

861

non-irritants under UN GHS were identified as irritants, a false-positive rate of 30%. Thus, the862

Vitrigel-EIT method achieved a sensitivity of 83%, a specificity of 70%, and an accuracy of 78%, as863

shown in Table 24-1. Data from the lead laboratory also demonstrated that predictive capacity could864

be improved by expanding the sample size. For example, the specificity achieved in Phase III of this865

validation study was lower than that obtained from the data of 33 non-irritants resulted in a higher

866

specificity. The list of test chemicals that were either false negative or false positives is shown in Table

867

25.

868

On the other hand, analysis per a top-down approach for identifying UN GHS Category 1 ocular869

irritants was also performed as a part of this validation study, as shown in Tables 24-2. Regarding

870

identifying test chemicals classified as UN GHS Category 1 in a top-down approach, the Vitrigel-EIT

52

871

method demonstrated a sensitivity of 89% (25/28), a specificity of 46% (30/65), and an accuracy of872

59% (55/93). Specificity is an important criterion in a top-down approach, which means that Vitrigel-873

EIT method is not well suited for use in a top-down approach to identifying UN GHS Category 1

874

ocular irritants.

875

Table 24-1. Contingency table used for 93 test chemicals in a bottom-up approach

Vitrigel-EIT

UN GHS

Total

I

NI

Cat.1, 2A, 2B

50

10

60

No Category

10

23

33

60

33

93

Total
Sensitivity: 83% (50/60)
Specificity: 70% (23/33)
Accuracy: 78% (73/93)
876

Table 24-2. Contingency table used for 93 test chemicals in a top-down approach

Vitrigel-EIT

UN GHS

Total

I

NI

Cat.1

25

3

28

Cat.2A, 2B, No Category

35

30

65

Total

60

33

93

Sensitivity: 89% (25/28)
Specificity: 46% (30/65)
Accuracy: 59% (55/93)
877
878
879
880
881
882
883
884
885
886
887
888

53

889

Table 25. False test chemicals in a bottom-up approach for 93 test chemicals
No.*

Rank

Applicability limitation

3-4

CaptanInsoluble after 5 min.

3-13

Myristyl alcohol

Insoluble after 5 min.

3-14

2,6-Dichlorobenzoyl chloride

3-18

Camphene

3-33

α-Hexylcinnamaldehyde

192-Methylbutanoic acid24

3,3'-Dithiodipropionic acid

pH of 2.5% solution ≤ 5.0

Ethyl 2,6-dichloro-5-fluoro-beta-oxo-

pH of 2.5% solution ≤ 5.0

26

890

Test chemicals

pH of 2.5% solution ≤ 5.0
Protocol revised
False negatives

3-pyridinepropanoate

39

6-Methylpurine40

Lactic acid

3-21

Methyl amyl ketone

3-24

2,4-Difuroronitrobenzene

3-26

2,4-Pentanediol

3-29

Potassium tetrafluoroborate

3-34

n,n-Dimethylguanidine sulfate

3-35

Toluene3-36

Gluconolactone

8

Methyl isobutyl ketone

28

Triethanolamine

37

Cyclohexanone

pH of 2.5% solution ≤ 5.0

pH of 2.5% solution ≤ 5.0

False positive
pH of 2.5% solution < 5.0 after 10 min.

*Each number corresponds to the number in Table 21 and Appendix 8.8.

54

891

5.7

892

Analysis of the false-negative reactions shows that five of the ten false-negative chemicals were acidic,893

and the 2.5% solutions used for exposure had a pH level lower than 5, as shown in Table 25. The

894

TEER values of the HCE models after exposures to each of these five acidic test chemicals that yielded895

false-negatives increased from their initial values. Interestingly, it was reported that isolated rabbit

896

esophageal mucosal epithelium and normal human bronchial epithelial cell layers in culture displayed897

increased TEER values when exposed to weak acidic solutions (Farré et al., 2008; Oshima et al., 2012).

898

On the other hand, two of the five non-acidic false-negative chemicals were water-insoluble solids

899

that were easily separated from the culture medium at room temperature, as shown in Table 25.

900

Therefore, the lead laboratory added two restrictions to the applicability domain in consideration of901

above scientific rationales:

902

Applicability domain



903
904
905

Exclude all test chemicals that have a pH level of 5 or less in solution (affected 11 tested
chemicals).



Exclude all solids that have both a logP value of 2.5 or more and a density of either less than
0.95 g/cm3 or over 1.10 g/cm3 (affected 6 test chemicals).

906

Under this applicability domain, 17 of the original 93 test chemicals were excluded, as shown in

907

Tables 26, which improve sensitivity from 83 to 93%, specificity from 70 to 69%, and accuracy from908

78 to 83%, as shown in Table 27.

909

Of the 44 irritants, one other that yielded a false-negative was 6-methylpurine, a non-acidic, water-

910

soluble powder. The reason for the false-negative judgment is currently under investigation. The

911

classification of the test chemical in vivo was identified as “Study Criteria Not Met” because the study912

was terminated before 21 days without full reversibility (scores equal to 0) of all endpoints in all

913

animals, in the absence of any other effects driving a Cat 1 classification (Barroso et al, 2017).

914

Eight of the 36 test chemicals in Phase III are excluded under the new applicability domain:915

No. 3-2

2-Benzyl-4-chlorophenol (insoluble)

916

No. 3-3

2,2-Dimethyl butanoic acid (pH ≤ 5)917

No. 3-4

Captan (insoluble)

55

918

No. 3-5

tetra-n-Octylammonium bromide (insoluble)

919

No. 3-13

Myristyl alcohol (insoluble)

920

No. 3-14

2,6-Dichlorobenzoyl chloride (pH ≤ 5)921

No. 3-15

Dibenzyl phosphate (pH ≤ 5)922

No. 3-25

tetra-aminopyrimidine sulfate (pH ≤ 5)

923

After excluding these eight test chemicals, sensitivity improved from between 75 and 83% to between

924

88 and 94% (15 to 16/17), specificity changed from 42% to 36% (4/11), and accuracy improved from925

between 64 and 69% to between 68 and 71% (19 to 20/28).

926

Of the 17 irritants, two others that yielded false-negatives were No. 3-18, camphene, and No. 3-33,

927

alpha-hexylcinnamaldehyde. Camphene is a waxy, water-insoluble solid, and the false-negative was928

due to the technique used for dissolving, as described in section 4.1.4 Phase III. Alpha-929

hexylcinnamaldehyde is a water-immiscible liquid and was identified as an irritant by the lead930

laboratory (Yamaguchi, 2016). The reason for the discordance of the judgment is currently under931

investigation, although the classification of alpha-hexylcinnamaldehyde in several studies in vivo was932reported as NC and 2A or higher (Barroso et al, 2017). In consideration of the Draize eye test

933

Reference Database (DRD; Barroso et al, 2017), additional testing was performed in the lead934

laboratory using 114 test chemicals selected from the list of DRD (Appendix 8.13, 8.14).

935
936

Table 26-1. Limitations on applicability (pH level 5 or less in 2.5% solution) in a bottom-up approachNo.*

Test chemical

3-3

2,2-Dimethyl butanoic acid

1

3-14

2,6-Dichlorobenzoyl chloride

2A

3-15

Dibenzyl phosphate

2A

3

3-25

tetra-Aminopyrimidine sulfate

NC

3

19

2-Methylbutanoic acid1

False negative

4

24

3,3’-Dithiodipropionic acid

2B

False negative

426

Ethyl 2,6-dichoro-5-fluoro-beta-oxo3-pyridinepropanoate

2B

False negative

5

GHS category

56

Vitrigel-EIT results

pH
4

False negative

3

937
938
939
940

27

3-Chloropropionitrile

2B

40

Lactic acid1

49

Citric acid

2A

3

52

Glycolic acid2

4

5
False negative

3*

Each number corresponds to the number in Table 21 and Appendix 8.8.

Table 26-2. Limitations on applicability (solid chemicals with a logP value of 2.5 or more and a density
under 0.95 g/cm3 or over 1.10 g/cm3 in a bottom-up approach.
GHS
category

No.*

Test chemical

3-2

2-Benzyl-4-chlorophenol

1

3-4

Captan1

3-5

Tetra-n-octylammonium bromide

1Vitrigel-EIT
results

False negative

LogP

Density(g/cm3)

3.60

1.19

2.80

1.74

3.45

0.94

6.03

0.82

3-13 Myristyl alcohol

2A

22

Acid red 92

2

7.13

2.16

35

Potassium laurate

1

4.57

1.12

False negative

941

*

942
943

Table 27. Contingency tables used for 76 test chemicals within the applicability domain in bottom-up
approach.

Each number corresponds to the number in Table 21 and Appendix 8.8

Vitrigel-EIT
UN GHS

I
41
10
51

Cat.1, 2A, 2B
Not Classified

Total
Sensitivity: 93% (41/44)
Specificity: 69% (22/32)
Accuracy: 83% (63/76)

NI
3
22
25

Total
44
32
76

944
945

5.6

Other analysis

946

The VMT discussed the use of an area over the curve (or weighted area under the curve: wAUC) of

947

the TEER measurement to obtain high predictive capacity and requested that the biostatisticians

948

develop new prediction algorithm. As a result, a new statistical algorithm was designed and proposed949

to improve the predictive capacity, particularly in the area of specificity.

57

950

The proposed algorithm involved evaluating the eye irritancy of a test chemical using two parameters:

951(1) the TEER value measured at the final time point (180 seconds) and (2) the decrease in TEER value952

across the 180-second measurement period. A suitable cut-off value was determined for these two953

parameters based on the results of Phase III and in reference to the Youden index. The sensitivity,954

specificity, and accuracy obtained with the proposed algorithm were then compared with those955

obtained with the original algorithm. Finally, the validity of the proposed algorithm was confirmed

956

using the results obtained from 118 test chemicals by the lead laboratory (Yamaguchi et al., 2016).

957

Using a cut-off value of 0.15 for the decrease in TEER value across the measurement period yielded958

a sensitivity of 67%, a specificity of 92%, and an accuracy of 75%. Based on these results, the VMT

959

decided not to accept the new prediction algorithm to analyze data from this validation study.

960
961

5.7

Comparison with other alternative to ocular irritation assay

962

The Vitrigel-EIT method was developed by measuring relative changes in TEER for a period of 180

963

second after exposure to 30 test chemicals as previously reported (Yamaguchi et al., 2013). It is964

generally accepted that at least 100 substances should be tested to assess the predictive capacity of a965

new test method, and to this end, the developers tested a total of 118 test chemicals of various physical966

and chemical properties (Yamaguchi et al., 2016). The results of this testing showed that the Vitrigel-

967

EIT test method had a predictive capacity that was comparable to other test methods for which OECD

968

test guidelines are currently being developed. For example, the EpiOcular-EIT method demonstrated969

a sensitivity of 98%, a specificity of 73%, and an accuracy of 85% (Kaluzhny et al., 2011). Used in a970

bottom–up approach, the short time exposure (STE) test demonstrated a sensitivity of 88%, a971

specificity of 80%, and an accuracy of 85% (ICCVAM, 2013) and the predictive capacity of the972

Vitrigel-EIT method is similar with ones of the other methods (the sensitivity of 93%, a specificity of

973

69%, and an accuracy of 83%) under the applicability domain.

974

In addition, the vitrigel-EIT method has some advantages in required time, practicality and cost shown975

in Table 28. Each of these test methods, however, yields some false-negatives or false-positives. Thus,976

it is important to clarify the mechanism that results in these false-negatives and false-positives,

58

977

particularly when developing an in vitro test method suitable for use as an alternative to in vivo testing.

978

The VMT has confirmed the applicability domain proposed by the lead laboratory. Meanwhile,979

scientists at the lead laboratory consider immuno-histology to be a powerful tool for clarifying the980

mechanism of false-positive reactions, because the culture model can be easily utilized as frozen

981

sections after completing the Vitrigel-EIT.

982
983

Table 28. Comparative table between the Vitrigel-EIT method and other test methods.
Test method

Required time(for 24 test)

STE (TG491)

EpiOcular-EIT(TG492)

4days for preparing
SIRC cell monolayer
3hours for chemical
exposure experiment

1day for preparing HCEmodels9hours(liquid)or
30hours (solid) for
chemical
exposureexperiment

Vitrigel-EIT

6days for preparing
HCE models
2hours for chemicals
exposure experiment

Practicality

Easy

Easy

Difficult to remove test
chemicals from HCE
modelsCost¥84,000 for ad-MED
Vitrigel

Relatively low¥144,000modelsMechanistic
relevance

Epithelial
function

Cell viability

Cell viability

Limitation oftest chemicals

Excludeacidicchemicals and easily
separable
waterinsoluble solids

Exclude highly volatile
substances and all solid
chemicals other thanSurfactants

Colored sample(needadditional
procedure)

barrier

984
985
986

59

for

HCE

987

6

Conclusion

988

This study was performed in the spirit of GLP at three participating laboratories using a total of 42989

test chemicals to validate the Vitrigel-EIT method for within- and between-laboratory reproducibility990

as well as for the capacity to distinguish non-irritants from irritants in a bottom up approach.

991

The results showed good within-laboratory reproducibility between 80 and 100% as well as an

992

excellent between-laboratory reproducibility of 92% (33/36). Unfortunately, predictive capacity for

993

distinguishing non-irritants from irritants per UN GHS categories in a bottom-up approach was not

994

favorable because of a high incidence of false negatives as high as 17% (10/60). After considerable

995

review of the data, the applicability domain was revised to exclude test chemicals that have a pH level996

of 5 or less in solution as well as those that are solids and have both a logP value 2.5 or more and a997

density of either less than 0.95 g/cm3 or a density of over 1.10 g/cm3, which improved the false

998

negative rate to 7% (3/44).

999

From the above described results, the VMT concluded that the Vitrigel-EIT method demonstrated

1000

excellent within- and between-laboratory replicability and that, with a carefully defined applicability

1001

domain, it is a useful alternative to the Draize test for distinguishing test chemicals that are ocular non-1002

irritants from those that are irritants.

1003
1004

Acknowledgments

1005

This work was supported by Agri-Health Translational Research Project (no. 6320) from the Ministry1006

of Agriculture, Forestry and Fisheries of Japan.

1007
1008

7

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