ESAC Opinion on the SkinEthic™
Human Corneal Epithelium (HCE)
Eye Irritation Test (EIT)ESAC Opinion No. 2016-02
of 24 June 2016

2016

EUR 28175 EN

This publication is an Opinion by ESAC, the EURL ECVAM Scientific Advisory Committee, for the Joint ResearchCentre (JRC), the European Commission’s science and knowledge service. The Opinion does not necessarilyreflect the views of the European Commission. Neither the European Commission nor any person acting on behalfof the Commission is responsible for the use which might be made of this publication.
Contact information
Name:João BARROSO
Address: Joint Research Centre, Via Enrico Fermi 2749, 21027 Ispra (VA), Italy
E-mail: Joao.BARROSO@ec.europa.eu
Tel.:+39 0332 78 5329
JRC Science Hub
https://ec.europa.eu/jrc

JRC103704
EUR 28175 EN

PDF

ISBN 978-92-79-63197-9

ISSN 1831-9424

doi:10.2787/390390

Luxembourg: Publications Office of the European Union, 2016
© European Union, 2016
Reproduction is authorised provided the source is acknowledged.
How to cite: EURL ECVAM Scientific Advisory Committee (2016). ESAC Opinion on the SkinEthic™ Human Corneal
Epithelium (HCE) Eye Irritation Test (EIT). ESAC Opinion No. 2016-02 of 24 June 2016; EUR 28175 EN;
doi:10.2787/390390. Available at: http://publications.jrc.ec.europa.eu/repository/handle/JRC103704.
All images © European Union 2016

EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)EURL ECVAMSCIENTIFIC
ADVISORYCOMMITTEE(ESAC)ESAC OPINION
on theSkinEthic™ Human Corneal Epithelium (HCE)
Eye Irritation Test (EIT)ESAC Opinion No.
Relevant ESAC Request No.
Date of Opinion

2016-02
2016-02
24/06/2016

Table of contents
Abstract ............................................................................................................... 1
ESAC Opinion ........................................................................................................ 2
References ........................................................................................................... 5
Annex 1: Composition of ESAC and ESAC Working Group ........................................... 7
Annex 2: EURL ECVAM Request for ESAC Advice ....................................................... 9Annex 3: ESAC Working Group Peer Review Consensus Report ..................................27

Abstract
ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientificissues. Its main role is to conduct independent peer review of validation studies ofalternative test methods and to assess their scientific validity for a given purpose. The
committee reviews the appropriateness of study design and management, the quality of
results obtained and the plausibility of the conclusions drawn. ESAC peer reviews areformally initiated with a EURL ECVAM Request for ESAC Advice, which provides thenecessary background for the peer-review and establishes its objectives, timelines andthe questions to be addressed. The peer review is normally prepared by specialised ESAC
Working Groups. These are typically composed of ESAC members and other external
experts relevant to the test method under review. These experts may be nominated by
ESAC, EURL ECVAM and partner organisations within the International Cooperation on
Alternative Test Methods (ICATM). ESAC ultimately decides on the composition of these
Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions'and 'Working Group Reports' at the end of the peer review. ESAC may also issue
Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM
but not directly related to a specific alternative test method.
The ESAC Opinion expressed in this report relates to the peer-review of the SkinEthic™
Human Corneal Epithelium (HCE) Eye Irritation Test (EIT).

Page | 1

EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

Ispra, 24 June 2016

ESAC Opinion
In April 2016, the EURL ECVAM Scientific Advisory Committee (ESAC) (Annex 1) received
from EURL ECVAM a request for scientific advice on the L'Oréal-coordinated validation of
the SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT) for serious eye
damage/eye irritation testing (Annex 2). ESAC established a working group (WG) (Annex
1) which delivered an ESAC WG report dated 6 June 2016 (Annex 3).
The ESAC WG Eye Irritation was established to conduct a peer review of, and provide
scientific advice on:
1. A multi-laboratory trial involving 3 laboratories of the SkinEthic™ HCE Eye
Irritation Test (EIT), a test method with a wide applicability domain for
liquids (EITL: Eye Irritation Testing of Liquids) and solids (EITS: Eye
Irritation Testing of Solids) and in particular to consider the relevance(biological/mechanistic relevance and predictive capacity; the latter in thecontext of an Integrated Approach to Testing and Assessment (IATA)(OECD, 2008)), and reliability (transferability; within and between
laboratory reproducibility) of the test method.
2. The usefulness of HPLC/UPLC-spectrophotometry as an alternative
endpoint detection system to measure MTT-formazan, in particular for
highly coloured chemicals interfering with the conventional endpoint
measurement by OD-photometry when used specifically with SkinEthic™
HCE EIT.
The analysis and conclusions of the ESAC WG were based primarily on the EURL ECVAM
Test Submission Template (TST) and supporting documents supplied by L'Oréal, andsupplementary information made available by L'Oréal during and after an 11 May 2016teleconference.
Details of the validation study were previously published by Alépée et al. (2016a, b).
The pre-defined study objectives were:

To formally validate the SkinEthic™ HCE EIT by a three laboratory ring trial study
using an appropriate number and range of test chemicals and to obtain data toassess the relevance (predictive capacity) and reliability (reproducibility within
and between laboratories) of the assay.



To produce data to facilitate its international acceptance in regulatory schemes forhazard assessment of liquid and solid chemicals to discriminate chemicals(substances and chemical mixtures) not requiring classification for serious eye
damage/eye irritancy (No Category) from chemicals requiring classification andlabelling (Category 1 and Category 2) according to the UN GHS Classification andLabelling of Chemicals (UN GHS. UN, 2015) and as implemented by the EU CLP
regulation (EU CLP. EC, 2008a).
o

N.B. The test method was not designed, intended, or evaluated todifferentiate between GHS Category 1 (irreversible effects) and 2A-B
(reversible effects); or to test gases or aerosols.

To produce evidence and analysis to support the test method being incorporated
into a tiered testing strategy (so-called Bottom-Up/Top-Down testing strategy,
Page | 2

Scott L. et al., 2010). The ultimate purpose of such a tiered testing strategy being
to replace the traditional in vivo Draize eye test [Method B.5 of EC Regulation
440/2008 (EC, 2008b) or OECD TG 405 (OECD, 2002)].
At its 42nd meeting, held on the 9th and 10th June 2016 at EURL ECVAM, Ispra, Italy, thenon-Commission members of ESAC unanimously endorsed the following statement which
was based on the ESAC WG report:
ESAC concludes that, subject to the specific qualifications set out below, the study design
was generally appropriate and robust, the acceptance criteria appropriate at the time,
and that the study findings have provided the evidence and analysis required to satisfy
the study objectives.
While the SkinEthic™ HCE EIT was not developed to account for all mechanisms
associated with eye irritation, the validation study demonstrated that this RhCE test
method is able to correctly predict chemicals not requiring classification for serious eye
damage/eye irritation independently of the types of ocular effects observed in vivo (i.e.,
corneal, iridal and conjunctival injuries). However, the available documentation does notallow an assessment of the test method’s performance with mixtures.
The protocols for the testing of liquid and solid chemicals were found to be highly
reproducible and to meet the predetermined acceptable performance values set by the
EURL ECVAM/Cosmetics Europe Eye Irritation Validation Study (EIVS) Validation
Management Group (VMG) for sensitivity, specificity and accuracy (Barroso et al., 2015;
EURL ECVAM, 2016).
ESAC believes the available documentation provides sufficient evidence and reasonedarguments in favour of this RhCE test method having the potential to be at least as
relevant and reliable as the currently validated in vitro methods for the identification of
non-irritants [OECD TGs 437 (OECD, 2013a), 438 (OECD, 2013b), 491 (OECD, 2015a),and 492 (OECD, 2015b)], when combined with other alternative methods within future(tiered) testing strategies to replace the Draize eye test (Scott et al., 2010).
The SkinEthic™ HCE EIT validation study was generally well designed and conducted,
allowing ESAC to offer an informed opinion about the performance of the test method.
ESAC believes that the overall relevance and reliability of the test method has beensatisfactorily demonstrated with a view to it being considered for regulatory use in atiered assessment strategy. The test method could be applied as a first step in BottomUp discrimination of 'non-irritants' (GHS No Category) or as a confirmatory last step in a
Top-Down approach, where the priority is to first discriminate chemicals inducing serious
eye damage (GHS Category 1). However, the method is not intended to differentiate
Category 1 from Category 2 on its own.

HPLC/UPLC-spectrophotometry
The study results tend to confirm the findings of the EIVS study that suitably qualified
HPLC/UPLC-spectrophotometry analytical systems can be used to quantify precipitated
MTT-formazan as an alternative endpoint detection system in particular for highly
coloured chemicals interfering with the conventional endpoint measurement of MTTformazan by OD-photometry.

Page | 3

Table 1. Eye Irritation Validation Study (EIVS) target values and values obtained by thethree laboratories participating in the ring test for within laboratory reproducibility (WLR),
between laboratory reproducibility (BLR), sensitivity, specificity and accuracy. Two-sided
95 %-Confidence Intervals (CIs) are also provided.

EIVS Target
ValuesEITL

EITS

WLR (Lab1)≥ 85 %WLR (Lab2)≥ 85 %

95.0 %
95 %-CI: 86.3 % - 98.3 %88.3 %
95 %-CI: 77.8 % - 94.2 %96.7 %
95 %-CI: 88.6 % - 99.1 %95.0 %
95 %-CI: 86.3 % - 98.3 %WLR (Lab 3)≥ 85 %

93.3 %
95 %-CI: 84.1 % - 97.4 %95.0 %
95 %-CI: 86.3 % - 98.3 %93.3 %
95.0 %
98.3 %
93.3 %
95 %-CI: 84.1 % - 97.4 %96.7 %
96.7 %
100 %96.7 %
95 %-CI: 88.6 % - 99.1 %BLR (Lab 1 v Lab 2)
BLR (Lab 1 v Lab 3)
BLR (Lab 2 v Lab 3)
BLR (Cumulative)≥ 80 %

Sensitivity (Lab 1)
Sensitivity (Lab 2)
Sensitivity (Lab 3)
Sensitivity (Cumulative)
Sensitivity(Bootstrap Resampling)≥ 90 %≥ 90 %≥ 90 %≥ 90 %≥ 90 %

100 %97.9 %
96.9 %
98.3 %
98.2 %
95 %-CI 93.8 % - 100 %92.2 %
92.2 %
92.2 %
92.2 %
91.9 %
95 %-CI 90.0 % - 93.3 %Specificity (Lab 1)
Specificity (Lab 2)
Specificity (Lab 3)
Specificity (Cumulative)
Specificity(Bootstrap Resampling)≥ 60 %≥ 60 %≥ 60 %≥ 60 %≥ 60 %

65.5 %
72.6 %
70.2 %
69.4 %
69.4 %
95 %-CI 60.7 % - 75.0 %75.6 %
78.9 %
75.3 %
76.6 %
76.6 %
95 %-CI 73.3 % - 80.0 %Accuracy (Lab 1)
Accuracy (Lab 2)
Accuracy (Lab 3)
Accuracy (Cumulative)
Accuracy(Bootstrap Resampling)≥ 75 %≥ 75 %≥ 75 %≥ 75 %≥ 75 %

83.9 %
86.1 %
84.4 %
84.8 %
84.8 %
95 %-CI 80.8 %- 88.3 %83.9 %
85.6 %
83.3 %
84.4 %
84.3 %
95 %-CI 81.7 % - 86.7 %Page | 4

References

Alépée N, Leblanc V, Adriaens E, Grandidier MH, Lelièvre D, Meloni M, Nardelli L,
Roper CS, Santirocco E, Toner F, Van Rompay A, Vinall J, Cotovio J (2016a).
Multi-laboratory validation of SkinEthic HCE test method for testing serious eye
damage/eye irritation using liquid chemicals. Toxicol In Vitro 31: 43-53.

Alépée N, Adriaens E, Grandidier MH, Meloni M, Nardelli L, Vinall CJ, Toner F,
Roper CS, Van Rompay AR, Leblanc V, Cotovio J (2016b). Multi-laboratory
evaluation of SkinEthic HCE test method for testing serious eye damage/eye
irritation using solid chemicals and overall performance of the test method withregard to solid and liquid chemicals testing. Toxicol In Vitro 34: 55-70.

Barroso J, Alépée N, Cole T, Eskes C, Freeman SJ, Liška R, McNamee P,
Pfannenbecker U, Reus AA, Rubingh CM, Schaeffer MW, Zuang V (2015). EURLECVAM — Cosmetics Europe prospective validation study of Reconstructed human
Tissue-based test methods for serious eye damage/eye irritation testing. Poster
Presented at the Congress of the European Societies of Toxicology (EUROTOX),
Porto, Portugal, September, 13–16, 2015. Available from:http://dx.doi.org/10.1016/j.toxlet.2015.08.507
https://eurl-ecvam.jrc.ec.europa.eu/posters-1/eurl-ecvam-cosmetics-europeprospective-validation-study-of-reconstructed-human-tissue-based-test-methodsfor-serious-eye-damage-eye-irritation-testing-poster/view

EC (2008a). Regulation (EC) No 1272/2008 on classification, labelling andpackaging of substances and mixtures (the 'CLP Regulation').

EC (2008b). COUNCIL REGULATION (EC) No 440/2008 of 30 May 2008 laying
down test methods pursuant to Regulation (EC) No 1907/2006 of the European
Parliament and of the Council on the Registration, Evaluation, Authorisation andRestriction of Chemicals (REACH).

EURL ECVAM (2016). The EURL ECVAM - Cosmetics Europe prospective validation
study of Reconstructed human Cornea-like Epithelium (RhCE)-based test methods
for identifying chemicals not requiring classification and labelling for serious eye
damage/eye
irritation:
Validation
Study
Report.
EUR
28125
EN;
doi:10.2787/41680. Available from:http://publications.jrc.ec.europa.eu/repository/handle/JRC100280

OECD (2002). Acute Eye Irritation/Corrosion. OECD Guideline for the Testing ofChemicals No. 405, OECD, Paris.

OECD (2008). OECD Environment Health and Safety Publications Series on
Testing and Assessment No. 88 Workshop on Integrated Approaches to Testingand Assessment. OECD, Paris.

OECD (2013a). Bovine Corneal Opacity and Permeability Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not
Requiring Classification for Eye Irritation or Serious Eye Damage. OECD Guideline
for the Testing of Chemicals No. 437, OECD, Paris.

OECD (2013b). Isolated Chicken Eye Test Method for Identifying i) Chemicals
Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for
Eye Irritation or Serious Eye Damage. OECD Guideline for the Testing ofChemicals No. 438, OECD, Paris.

OECD (2015a). Short Time Exposure In Vitro Test Method for Identifying i)
Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage. OECD Guideline for the
Testing of Chemicals No. 491, OECD, Paris.
Page | 5OECD (2015b). Reconstructed human Cornea-like Epithelium (RhCE) test method
for identifying chemicals not requiring classification and labelling for eye irritationor serious eye damage. OECD Guideline for the Testing of Chemicals No. 492,
OECD, Paris.



Scott L, Eskes C, Hoffmann S, Adriaens E, Alépée N, Bufo M, et al. (2010). A
proposed eye irritation testing strategy to reduce and replace in vivo studies using
Bottom-Up and Top-Down approaches. Toxicol In Vitro 24: 1-9.

UN (2015). United Nations Globally Harmonized System of Classification andLabelling of Chemicals (GHS), Sixth revised edition, UN New York and Geneva.

Page | 6

EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

Annex 1COMPOSITION OF ESAC AND
ESAC WORKING GROUP

Page | 7

EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

Composition of ESAC and ESAC Working GroupEURL ECVAM Scientific Advisory Committee (ESAC)















Dr. Neil CARMICHAEL (ESAC Chair)Prof. Jürgen BORLAK
Dr. Harvey CLEWELL
Prof. Lucio G. COSTA
Dr. Kristina KEJLOVÁ
Prof. David John KIRKLAND
Prof. Annette KOPP-SCHNEIDER
Dr. Renate KRÄTKE
Prof. Claus-Michael LEHR
Dr. José Maria NAVAS
Prof. Aldert PIERSMA
Dr. Jonathan RICHMOND
Dr. Erwin L. ROGGEN
Dr. Dorothea SESARDIC

ESAC Working Group (WG)








Dr. José Maria NAVAS (ESAC Member, WG Chair)
Dr. Kristina KEJLOVÁ (ESAC Member)Prof. Annette KOPP-SCHNEIDER (ESAC Member)Dr. Renate KRÄTKE (ESAC Member)Dr. Jon RICHMOND (ESAC Member)Dr. Dave ALLEN (NICEATM; ICATM nomination by NICEATM/ICCVAM)
Prof. Kyung-Min LIM (College of Pharmacy, Ewha Womans University; ICATM
nomination by KoCVAM)EURL ECVAM (Secretariat)

Dr. João BARROSO (ESAC Coordinator)
Dr. Thomas COLE
Prof. Maurice WHELAN (Head of Unit)

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EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

Annex 2EURL ECVAM REQUEST FOR
ESAC ADVICE

Page | 9

EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)ESAC Request 2016-02
EURL ECVAM Scientific Advisory Committee(ESAC)EURL ECVAM REQUEST FOR ESAC ADVICEon theSkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT)

Title page information
Abbreviated title of ESAC request
ESAC REQUEST No.
Template used for preparing request
Date of finalising request
Date of submitting request to ESAC
Request discussed throughOpinion expected at (date)
File name of this request

EURL ECVAM REQUEST FOR ESAC ADVICESkinEthic™ HCE EIT validation
2016-02
EP 3.02
27/05/2016
27/05/2016
Written procedure previous to ESAC 42
ESAC 42 (June 2016)
ER2016-02_ESAC_REQUEST_SKINETHIC_HCE_EIT.doc

Page | 10

TABLE OF CONTENTS
1.

TYPE OF REQUEST ........................................................................................................................................122.

TITLE OF STUDY OR PROJECT FOR WHICH SCIENTIFIC ADVICE OF THE ESAC IS REQUESTED.....................13

3.

BRIEF DESCRIPTION OF THE STUDY OR PROJECT ........................................................................................13

4.

OBJECTIVES, QUESTIONS, TIMELINES ..........................................................................................................19
4.1
4.2
4.3

5.

OBJECTIVE ..........................................................................................................................................19
QUESTION(S) TO BE ADDRESSED .......................................................................................................19
TIMELINES ..........................................................................................................................................20

EURL ECVAM PROPOSALS ON HOW TO ADDRESS THE REQUEST WITHIN ESAC ........................................21
5.1
5.2

EURL ECVAM PROPOSAL REGARDING REQUEST-RELATED STRUCTURES REQUIRED .......................21
DELIVERABLES AS PROPOSED BY EURL ECVAM .................................................................................21

6.

LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESAC ....................................................................22

7.

TERMS OF REFERENCE OF THE ESAC WORKING GROUP.............................................................................24
7.1
ESTABLISHMENT OF THE ESAC WORKING GROUP ............................................................................24
7.2
TITLE OF THE ESAC WORKING GROUP ...............................................................................................24
7.3
MANDATE OF THE ESAC WORKING GROUP.......................................................................................24
7.4
DELIVERABLES OF THE ESAC WORKING GROUP ................................................................................24
7.5
PROPOSED TIMELINES OF THE ESAC WORKING GROUP ...................................................................24
7.6
QUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WORKING GROUP ................................25
APPENDIX 1REPORTING TEMPLATE ........................................................................................................25

EURL ECVAM REQUEST FOR ESAC ADVICE

Page | 11

1.

TYPE OF REQUEST

Request Type

Identify request ("YES")

R1 ESAC Peer Review

YES, external validation studyof a Prevalidation Study or Validation Study(i.e. not coordinated by EURL ECVAM)

If R1)applies please specify further:►Prevalidation StudyNO►Prospective Validation Study

YES
Background
In December 2008, two reconstructed human eye tissue
models for in vitro assay of eye irritation potential,
EpiOcular™ Eye Irritation Test (EIT) and SkinEthic™ Human
Corneal Epithelium (HCE), were sponsored for validation asalternatives to the traditional in vivo standard practice withrabbits (Draize test). The eye irritation validation study(EIVS) was conceived as a ring trial of comparative
performance among six participant laboratories (three foreach test method), testing selected chemicals to evaluatereliability (reproducibility within and between laboratories
of results obtained in vitro) and relevance (predictive
capacity of effects documented in vivo).
Neither test method was able to comply fully with theacceptance criteria set by the validation management group(VMG). Therefore, further optimisation was recommended.
With minor refinement to the EpiOcular™ EIT protocol, themethod was successfully validated in 2013.
The SkinEthic™ HCE protocol was subject to morecomprehensive revision, followed by another validation ringtrial (three laboratories) completed in 2015.
In November 2015 the revised SkinEthic™ HCE test method
was submitted for assessment by EURL ECVAM and formal
peer-review by ESAC.

►Retrospective Validation StudyNO►Validation Study based on PerformanceStandards

NO

R2 Scientific Advice on a test method submitted to
EURL ECVAM for validation

NO(e.g. the test method's biological relevance etc.)

R3 Other Scientific AdviceNO(e.g. on test methods, their use; on technical issues such as cell
culturing, stem cells, definition of performance standards etc.)

EURL ECVAM REQUEST FOR ESAC ADVICE

Page | 12

2.

TITLE OF STUDY OR PROJECT FOR WHICH SCIENTIFIC ADVICE OF THE
ESAC IS REQUESTED

L’Oréal-coordinated validation of the in vitro SkinEthic™ Human Corneal Epithelium (HCE) Eye
Irritation Test (EIT)3.

BRIEF DESCRIPTION OF THE STUDY OR PROJECT

1) Serious eye damage/eye irritation and regulatory tests
Eye irritation is the result of reversible anterior surface tissue trauma, causing degeneration of vision.
Serious eye damage is not fully reversible within 21 days of exposure (UN, 2013). Traditionally, eyeirritation has been determined by the in vivo rabbit Draize eye test (OECD Test Guideline 405, 2012).
Validated in vitro alternative methods include:- organotypic assays: Bovine Corneal Opacity and Permeability test (OECD updated Test
Guideline 437, 2013) and Isolated Chicken Eye test (OECD updated Test Guideline 438, 2013).
- cell-based methods: Fluorescein Leakage assay (OECD Test Guideline 460, 2012), Cytosensor
Microphysiometer assay (OECD draft Test Guideline, 2012) and Short Time Exposure assay(OECD Test Guideline 491, 2015).
- reconstructed human cornea-like epithelium (RhCE) test: EpiOcular™ EIT (OECD Test Guideline 492,
2015).

2) SkinEthic™ HCE EIT purpose
At present, no single in vitro method can fully replace the in vivo Draize eye test for assessment ofserious eye damage/eye irritation. However, tiered combination of alternatives (so-called TopDown/Bottom-Up approach) can reduce/replace reliance on in vivo procedures (Scott et al., 2010;
OECD draft Guidance, 2015). Top-Down differentiates chemicals inducing serious eye damage (GHS
category 1) as priority, while Bottom-Up first discriminates 'non-irritants' (GHS no category).
The SkinEthic™ HCE EIT method is intended for inclusion in a Top-Down/Bottom-Up assessment
strategy, particularly relevant for industrial chemicals or chemicals used in human exposure products,
such as cosmetics ingredients which are banned from animal testing. The method is therefore
required as an alternative, also effectively reducing the need for animal studies by their partial
replacement. The SkinEthic™ HCE is the second RhCE test method that is validated following
EpiOcular™ EIT. It is however important to have at least two of these methods validated andaccepted by regulatory authorities in order to guarantee the widespread availability of thistechnology and avoid potential market monopolies.
In a tiered assessment strategy, the SkinEthic™ HCE EIT method is applicable as a first step in BottomUp discrimination of 'non-irritants' or as a confirmatory last step in a Top Down approach. However,
the method is not intended to differentiate category 1 from 2 on its own.
3) SkinEthic™ HCE EIT principle
The test method addresses eye irritation caused by topical exposure to chemicals, manifested in vivo
as local inflammation and/or opacity, resulting mechanistically from cell damage (cytotoxicity).
The in vitro test system uses immortalized human corneal epithelial cells, cultured to form a
Reconstructed human Cornea-like Epithelium (RhCE), i.e. a three-dimensional tissue similar to thehuman corneal epithelium. The test method was developed to model in vivo topical exposure, withEURL ECVAM REQUEST FOR ESAC ADVICE

Page | 13

prediction of positive or negative irritation response from cell viability assay. Tissue viability isdetermined quantitatively as a percentage, relative to a negative control (100% viable) bystandardised MTT assay (photometric measurement of purple formazan production from enzymatic
reduction of the vital dye MTT). Tissues treated with eye irritants show a decrease in viability relative
to the negative control, with discrimination of positive or negative GHS classification defined by an
optimised viability threshold percentage (prediction model).
The revised SkinEthic™ HCE test submission is complete with comprehensive protocols (SOPs) for eye
irritation testing of liquids (EITL) and solids (EITS) as used in the validation study (Attachments 1a &1b, respectively) and as intended for test method users (Attachments 1c & 1d, respectively).
Critical elements of the SOPs include:- test system description (Human Corneal Epithelium tissue model, with quality control).
- TT: test treatment (application, exposure, incubation, MTT-formazan extraction).
- viability determination (MTT formazan assay: OD measurement, HPLC).
- prediction model: EITL (60% threshold) and EITS (50% threshold).
Acceptance criteria (for qualified test, qualified run, and complete test):- NgC: negative control (PBS): 1.4 ≤ OD ≤ 2.5 (mean of 2 replicate tissues).
- PC: positive control (methyl acetate): viability ≤ 30% (mean of 2 tissue replicates).
- viability difference between run replicates ≤ 20 (NgC, PC, TT).
4) SkinEthic™ HCE EIT optimisation
The test method was developed by L’Oréal, with prediction model optimization using 125 chemicals,
including 71 liquids and 54 solids (Attachment 2).
A principal criterion for selection of test chemicals was availability of supporting complete and quality
assured in vivo Draize eye irritation data. The selection was limited to commercially available
chemicals.
The chemicals, incorporating 44/125 (35%) previously selected for the original ring trial eye irritation
validation study (EIVS) provided a range of properties, including:- Chemical class (functional group): soap/surfactant, organics (neutral, acid and base) andinorganic base.
- Several colour interfering chemicals, MTT reducers and MTT reducing coloured chemicals.
- GHS classification: 49% not classified (NC) and 51% classified (C) (divided as 53% Category 1
and 47% Category 2.
As distribution of physical state and GHS classification category, the 125 chemicals covered: 34
Category 1 (19 liquids and 15 solids), 21 Category 2A (16 liquids and 5 solids), 9 Category 2B (4 liquids
and 5 solids) and 61 No Category (32 liquids and 29 solids).
The complement of chemicals used for development and optimization represents a significant andbalanced set.
5) SkinEthic™ HCE EIT training and transfer
Transferability of the method was demonstrated using 18 chemicals (9 solids / 9 liquids) including
strong colorants and MTT reducers known to cause interference, aiming to cover all experimental
eventualities.
Two training days are required for a naïve laboratory, including practical application and data
evaluation. Actual transfer of the method was arranged over two weeks, testing the 18 chemicals in
replicate independent series to allow evaluation of:- adherence to acceptance criteria.
EURL ECVAM REQUEST FOR ESAC ADVICE

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- single and dual operator comparison.
- predictive concordance.
Results demonstrated accurate and reproducible implementation.
The training exercise has been described in full, with detailed method SOPs (Attachments 4a and 4b)
and assessment reports (Attachments 5a, 5b, 5c, and 5d).
SOP implementation (transfer) by the naïve laboratories has also been reported in full (Attachments
6a and 6b) indicating the method is both robust and transferable.
6) SkinEthic™ HCE EIT validation
The ring trial validation study (EITL and EITS) for evaluation of within/between laboratory
reproducibility (WLR/BLR) and predictive capacity (PC) included 120 chemicals (60 liquids, 60 solids)
tested in three laboratories (L'Oréal, Charles River, Vito) with an additional 80 chemicals (45 liquids,
35 solids) tested by L'Oréal (lead laboratory).
The chemical selection (Attachment 2) again covered a range of properties:- the full range of in vivo eye irritation GHS Categories (1, 2A, 2B, or No Category).
- the in vivo determinants of classification (cornea opacity, iritis, conjunctiva redness, chemosis,
reversibility/persistence).
- wide representation of organic functional groups.
- known chemical structures.
- coloured and/or direct MTT reducers.
- availability through laboratory retail supply, at reasonable cost.
The processing and analysis of all data from the three laboratories in the ring trial was contracted to
an independent consultant statistician who has compiled 2 comprehensive reports, respective of theliquid and solid protocols (Attachments 8a and 8b). The reports are clear and concise, uniformly
applying the acceptance criteria and prediction model to determine within laboratory reproducibility(WLR) between laboratory reproducibility (BLR) and predictive capacity (PC).
The test submitter (L'Oréal) has also compiled all data used for method evaluation, provided asattachments in pdf and xls formats:- Attachment 3a: Data used for relevance and reliability assessment (EITL and EITS).
- Attachment 3b: EITL: WLR assessment: 60 ring trial chemicals, 3 labs (L'Oreal, VITO, CRL).
- Attachment 3c: EITL: WLR assessment: 45 additional chemicals, 1 lab (L'Oreal).
- Attachment 3d: EITS: WLR assessment: 60 ring trial chemicals, 3 labs (L'Oreal, VITO, CRL).
- Attachment 3e: EITS: WLR assessment: 35 additional chemicals, 1 lab (L'Oreal).
- Attachment 7a: EITL: BLR assessment: 60 ring trial chemicals, 3 labs (L'Oreal, VITO, CRL).
- Attachment 7b: EITS: BLR assessment: 60 ring trial chemicals, 3 labs (L'Oreal, VITO, CRL).
- Attachment 9: EIT: Predictive capacity (PC) assessment.
7) SkinEthic™ HCE EIT results
Within Laboratory Reproducibility (WLR)
WLR (concordance of predicted classification) based on the set of 120 chemicals, was reported asfollows:- CRL:
91.7% (EITL 88.3% and EITS 95.0%).
- VITO:
94.2% (EITL 93.3% and EITS 95.0%).
- L'Oreal: 95.8% (EITL 95.0% and EITS 96.7%).
WLR for the extended set of 200 chemicals (tested by L’Oreal only) was:EURL ECVAM REQUEST FOR ESAC ADVICE

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- 95.0% (EITL 93.3% and EITS 96.8%).
The test submission report concluded that the SkinEthic™ HCE EIT method (liquids/solids) has beenshown to exceed the minimum requirement for WLR of 85% set by the validation management group(VMG) of EIVS. The WLR is also comparable to that obtained previously for a similar method,
EpiOcular™ EIT.
Between Laboratory Reproducibility (BLR)
Fifty six of the 60 liquid chemicals were consistently classified (NC/C) by the three laboratories
resulting in a BLR (concordance of predicted classification) of 93.3% (95% CI: 84.1% - 97.4%). BLR
based on pair-wise comparison, was reported as follows:- L'Oreal versus CRL: 93.3% (56/60 chemicals).
- L'Oreal versus VITO: 95.0% (57/60 chemicals).
- CRL versus VITO:
98.3% (59/60 chemicals).
Fifty eight of the 60 solid chemicals were consistently classified (NC/C) by the three laboratories
resulting in a BLR (concordance of predicted classification) of 96.7% (95% CI: 88.6% - 99.1%). BLR
based on pair-wise comparison, was reported as follows:- L'Oreal versus CRL and L'Oreal versus VITO: 96.7% (58/60 chemicals).
- CRL versus VITO:100%.
The test submission report concluded overall BLR for the SkinEthic™ HCE EIT method, based on theset of 120 chemicals, was 95.0% (EITL 93.3% and EITS 96.7%) exceeding the defined minimum
requirement of 80% set by the VMG of EIVS.
For comparison, the test submission reported BLR from the previous ring trial validation of the similar
test method EpiOcular™ EIT was 94.4% for liquids and 92.0% for solids.
Predictive Capacity (PC)PC (ring trial) was evaluated by comparing in vitro viability with respect to prediction model (all runs,
per laboratory and cumulatively) with documented in vivo classifications according to GHS.
The statistics report summarises the frequency distribution of true versus false predictions,
respective of irritant classification (C) and non-irritant classification (NC). From these frequencies arecalculated the sensitivity (rate of correct prediction for C, with false negatives), the specificity (rate ofcorrect prediction for NC, with false positives) and overall accuracy (rate of correct prediction, C orNC) expressed as percentages:
Liquids protocol (EITL) predictive capacity (ring trial):
in vivo
Classified
No Category
Total
Sensitivity (%)
False Negatives (%)
Specificity (%)
False Positives (%)
Accuracy (%)

Cumulative
C
NC
283 5
77
175
540

L’Oréal
C
NC
96
0
29
55
180

CRL
C
NC
94
2
23
61
180

VITO
C
NC
93
3
25
59
180

98.3
1.7
69.4
30.6
84.8

100
0
65.5
34.5
83.9

97.9
2.1
72.6
27.4
86.1

96.9
3.1
70.2
29.8
84.4

EURL ECVAM REQUEST FOR ESAC ADVICE

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From statistical bootstrap resampling (which estimates uncertainty in predictive capacity, as 95% CI)(10,000 re-samples at n=1 for the 60 chemicals) the statistics report indicates overall predictive
capacity for the liquids protocol (EITL):
Parameter
Sensitivity (%)
Specificity (%)
Accuracy (%)

Estimate
98.2
69.4
84.8

95% CI
93.8; 100
60.7; 75.0
80.0; 88.3

Solids protocol (EITS) predictive capacity (ring trial):
in vivo
Classified
No Category
Total
Sensitivity (%)
False Negatives (%)
Specificity (%)
False Positives (%)
Accuracy (%)

CumulativeCNC
249 21
63
206
539

L’Oréal
C
NC
83
7
22
68
180

CRL
C
NC
83
7
19
71
180

VITO
C
NC
83
7
22
67
179

92.2
7.8
76.6
23.4
84.4

92.2
7.8
75.6
24.4
83.9

92.2
7.8
78.9
21.1
85.6

92.2
7.8
75.3
24.7
83.3

From statistical bootstrap resampling (which estimates uncertainty in predictive capacity, as 95% CI)(10,000 re-samples at n=1 for the 60 chemicals) the statistics report indicates predictive capacity forthe solids protocol (EITS):Parameter
Sensitivity (%)
Specificity (%)
Accuracy (%)

Estimate
91.9
76.6
84.3

95% CI
90.0; 93.3
73.3; 80.0
81.7; 86.7

The test submission also reports sensitivity, specificity and accuracy for the extended set of chemicals(including 45 additional liquids and 35 additional solids tested by the lead laboratory only) quoting
similar figures.
8) HPLC spectrophotometry
The MTT-reduction assay for tissue viability, relevant to all in vitro test methods based on
Reconstructed human Tissues (RhT) is limited by interference with coloured chemicals.
The test method R&D has overcome this limitation using High/Ultra High Performance Liquid
Chromatography Performance (HPLC-UPLC)-spectrophotometry for endpoint detection of formazan.
The HPLC-UPLC method has been shown to be highly reproducible (BLR) between different
laboratories.
Based on this, the test submission report concludes that HPLC/UPLC is relevant to all in vitro RhT test
methods irrespective of the test system and test method and can be applied to any of the other RhT
test systems within the relevant OECD Test Guidelines. Indeed, the HPLC/UPLC-spectrophotometry
technique has already been implemented in OECD TGs 431 (in vitro skin corrosion based on RhE), 439(in vitro skin irritation based on RhE) and 492 (in vitro serious eye damage/eye irritation based on
RhCE).

EURL ECVAM REQUEST FOR ESAC ADVICE

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References
Hartung, T., Bremer, S., Casati, S., Coecke, S., Corvi, R., Fortaner, S., Gribaldo, L., Halder, M., Hoffmann, S., Roi
A.J., Prieto, P., Sabbioni, E., Scott, L., Worth, A. and Zuang. V. (2004): A modular approach to the
ECVAM principles on test validity. ATLA 32, 467-472.
OECD (2012), Test No. 405: Acute Eye Irritation/Corrosion, OECD Guidelines for the Testing of Chemicals,
Section 4, OECD Publishing, Paris.
DOI: http://dx.doi.org/10.1787/9789264185333-en
OECD (2012), Test No. 460: Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe
Irritants, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
DOI: http://dx.doi.org/10.1787/9789264185401-en
OECD (2012, draft) The Cytosensor Microphysiometer Test Method: An in vitro Method for Identifying Ocular 3
Corrosive and Severe Irritant Chemicals as well as Chemicals not Classified as Ocular Irritants.
http://www.oecd.org/env/ehs/testing/DRAFT%20Cytosensor%20TG%20(V9)%2021%20Dec%2012_cle
an.pdf
OECD (2013, updated), Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i)
Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation
or Serious Eye Damage, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing,Paris.
DOI: http://dx.doi.org/10.1787/9789264203846-en
OECD (2013, updated), Test No. 438: Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing
Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye
Damage, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
DOI: http://dx.doi.org/10.1787/9789264203860-en
OECD (2015), Test No. 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing
Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye
Damage, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
DOI: http://dx.doi.org/10.1787/9789264242432-en
OECD. (2015), Test No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying
chemicals not requiring classification and labelling for eye irritation or serious eye damage, OECD
Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
DOI: http://dx.doi.org/10.1787/9789264242548-en
OECD (2015, draft), Draft Guidance Document on an Integrated Approach to Testing and Assessment (IATA) for
Serious Eye Damage and Eye Irritation: http://www.oecd.org/env/ehs/testing/draft-guidance-reviewdocuments-monographs.htm
Scott, L., Eskes, C., Hoffmann, S., Adriaens, E., Alepée, N., Bufo, M., Clothier, R., Facchini, D., Faller, C., Guest, R.,
Harbell, J., Hartung, T., Kamp, H., Varlet, B.L., Meloni, M., McNamee, P., Osborne, R., Pape, W.,
Pfannenbecker, U., Prinsen, M., Seaman, C., Spielmann, H., Stokes, W., Trouba, K., Berghe, C.V.,
Goethem, F.V., Vassallo, M., Vinardell, P., Zuang, V. (2010): A proposed eye irritation testing strategy
to reduce and replace in vivo studies using Bottom-Up and Top-Down approaches. Toxicol In Vitro 24,
1-9.
th

United Nations (UN) Globally Harmonized System (GHS) for Classification and Labelling of Chemicals (2013): 5(revised) edition.

EURL ECVAM REQUEST FOR ESAC ADVICE

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4.

OBJECTIVES, QUESTIONS, TIMELINES

4.1

OBJECTIVE

Objective
Why does EURLECVAM require
advice on thecurrent issue?

EURL ECVAM requests an ESAC opinion on the reliability (reproducibility within andbetween laboratories of results obtained in vitro) and relevance (predictive capacity ofeffects documented in vivo) of the SkinEthic™ Human Corneal Epithelium (HCE) Eye

Irritation Test (EIT) for prediction of eye irritation potential of chemicals. The
opinion of ESAC should support EURL ECVAM with respect to the development of
an EURL ECVAM recommendation on the Reconstructed human Cornea-like
Epithelium (RhCE) assays for serious eye damage/eye irritation testing outlining (1)the scientific basis of the assays, (2) their overall performance (transferability,
reproducibility and predictive capacity) as assessed during the validation studies
and based on other (e.g. published) information, (3) their applicability andlimitations, and 4) their proposed use.
ESAC's advice should enable EURL ECVAM to conclude, within its EURL ECVAM
Recommendation, on the potential adequacy of the SkinEthic™ HCE EIT for routine
testing of serious eye damage/eye irritation for regulatory purposes.

4.2

QUESTION(S) TO BE ADDRESSEDQuestions
What are thequestions andissues that should
be addressed inview of achieving
the objective of
the advice?

The ESAC peer review of the SkinEthic™ HCE EIT should address the following
aspects:(1) Scientific basis in relation to serious eye damage/eye irritation.
(2) Clarity of the test definition, including:- purpose and need of the test method.
- biological/mechanistic relevance in relation to the test system used and theendpoint measured.
- protocol clarity and completeness.
- clarity and adequacy of the prediction model and its development.
(3) Clarity of the definition of the study objective(s).
(4) Appropriateness of the study design and execution considering the study
objective(s), including:- number and selection criteria for test chemicals (e.g., range of documented
effects in vivo, etc.).
- quality assurance of reference data (in vivo) for predictive capacity
assessment.
- number of participating laboratories.
- number of replicates, number of repetitions, rules for retesting and handlingof deviations.
(5) Study management and conduct.
(6) Results compilation and statistical analyses reporting.
- appropriateness of calculation of WLR and BLR on the basis of the generated
data.

EURL ECVAM REQUEST FOR ESAC ADVICE

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- appropriateness of calculation of Predictive Capacity on the basis of thegenerated data.
- appropriateness of identification of limitations/applicability domain on thebasis of the generated data.
(7) Transferability and reproducibility (WLR/BLR).
(8) Predictive capacity for distinguishing chemicals not requiring classificationfrom chemicals requiring classification as Category 1 (serious eye damage) or
Category 2 (eye irritation) and relevance to a tiered (Top-Down/Bottom-Up)
testing strategy.
(9) Applicability and any known limitations, assessed from the selection of thetest chemicals (range of molecular class and physical properties) and analysesof possible reasons for misclassifications.
(10) Possible gaps, if any, between study design and study conclusions.
(11) Whether the information provided in the submission is sufficient tosubstantiate the proposed use of the test method within a Bottom-Up/TopDown testing strategy.
(12) Usefulness and applicability of HPLC/UPLC-spectrophotometry as analternative endpoint detection system to standard photometry in SkinEthic™
HCE EIT.
(13) What additional work, if necessary, should be undertaken in future tofurther characterise the test method and its proposed use.
ESAC's advice should conclude on the regulatory applicability of the SkinEthic™
HCE EIT (i.e., for implementation as an EU test method and OECD Test Guideline).

4.3

TIMELINES

Timelines
concerning thisrequestTimeline

Indication

Finalised ESAC Opinion required by:June 2016

When does EURLECVAM require
the advice?

Request to be presented to ESAC bywritten procedure (e.g. due tourgency) prior to the next ESAC

YES

Request to be presented to ESAC at
ESAC plenary meetingNO

EURL ECVAM REQUEST FOR ESAC ADVICE

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5.

EURL ECVAM PROPOSALS ON HOW TO ADDRESS THE REQUEST WITHIN
ESAC

5.1

EURL ECVAM PROPOSAL REGARDING REQUEST-RELATED STRUCTURES REQUIREDSpecific
structures
required within
ESAC to address
the request

Structure(s) required

Required according(YES/NO)S1 ESAC RapporteurNO

S2 ESAC Working Group

Does the advice
require an ESAC
working group, an
ESAC rapporteuretc.?

S3 Invited Experts

to

EURL

ECVAM?

ESAC members- José M. Navas (Chair)- Kristina Kejlová- Annete Kopp-Schneider- Renate Kraetke- Jon Richmond
ICATM nominations- Dave Allen (NICEATM/ICCVAM)- Kyung-Min Lim (College of Pharmacy,
Ewha Womans University; nominated by
KoCVAM)NO

Ad S3: If yes – list names andaffiliations of suggested
experts to be invited andspecify whether these aremember of the EEP
If other than above (S1-S3):

5.2

DELIVERABLES AS PROPOSED BY EURL ECVAM

Deliverables
What deliverables(other than the
ESAC opinion) are
required for
addressing therequest?

Title of deliverable otherthan ESAC opinion

Required? (YES/NO)

D1 ESAC Rapporteur Report NO
and draft opinion

D2 ESAC Peer Review Report YES
and draft opinion
If other than above (D1-D2):

EURL ECVAM REQUEST FOR ESAC ADVICE

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6.

LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESACCount Description of document

Already
File nameavailable?
(YES/NO)1YES

TST SkinEthic HCE EIT_Amended.pdf

YES

SkinEthic_HCE_assessment_report_201605-09_final.pdf

YES

Attachment 1a.pdf

YES

Attachment 1b.pdf

YES

Attachment 1c.pdf

YES

Attachment 1d.pdf

YES

Attachment 2.pdf

YES

Attachment 3a.pdf

YES

Attachment 3b.pdf

YES

Attachment 3c.pdf

YES

Attachment 3d.pdf

YES

Attachment 3e.pdf

YES

Attachment 4a.pdf

YES

Attachment 4b.pdf

YES

Attachment 5a.pdf

YES

Attachment 5b.pdf

YES

Attachment 5c.pdf

YES

Attachment 5d.pdf

YES

Attachment 6a.pdf

YES

Attachment 6b.pdf

YES

Attachment 7a.pdf

YES

Attachment 7b.pdf

2
3
4
5
6

7

8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

SkinEthic™ HCE test submission (TST)EURL ECVAM Assessment Report on the
SkinEthic™ HCE test submission
Protocol of the SkinEthic™ HCE EITL
(Liquid)
Protocol of the SkinEthic™ HCE EITS (Solid)
SkinEthic™ HCE EITL (Liquid) DB-ALM
protocolSkinEthic™ HCE EITS (Solid) DB-ALM
protocolSkinEthic™ HCE EIT - List of test items
including their CAS number and basic
physical/chemical properties for
optimisation/transfer/WLR/BLR/Predictive
capacity
SkinEthic™ HCE EIT - Data used forrelevance and reliability assessment (EITL
and EITS)SkinEthic™ HCE EITL - WLR assessment(60 chemicals – 3 labs)
SkinEthic™ HCE EITL - WLR assessment(45 additional chemicals – 1 lab)
SkinEthic™ HCE EITS - WLR assessment(60 chemicals – 3 labs)
SkinEthic™ HCE EITS - WLR assessment(35 additional chemicals – 1 lab)
Training protocol of the SkinEthic™ HCEEITL (Liquids)
Training protocol of the SkinEthic™ HCEEITS (Solids)
Training report of the SkinEthic™ HCE EITL
(Liquids) - VITO
Training report of the SkinEthic™ HCE EITL
(Liquids) - CRL
Training report of the SkinEthic™ HCE EITS
(Solids) - VITO
Training report of the SkinEthic™ HCE EITS
(Solids) – CRL
Transfer report of the SkinEthic™ HCE EITL
(Liquids) – VITO & CRL
Transfer report of the SkinEthic™ HCE EITS
(Solids) – VITO & CRL
SkinEthic™ HCE EITL - BLR assessment(60 chemicals – 3 labs)
SkinEthic™ HCE EITS - BLR assessment(60 chemicals – 3 labs)

EURL ECVAM REQUEST FOR ESAC ADVICE

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23
24
25
26
27
28

Statistical analysis and reporting of the
SkinEthic™ HCE EITL (Liquids)
Statistical analysis and reporting of the
SkinEthic™ HCE EITS (Solids)
SkinEthic™ HCE EIT – Predictive capacity
assessment
Project plan of the SkinEthic™ HCE EITL
(Liquids)
Project plan of the SkinEthic™ HCE EITS
(Solids)
SkinEthic™ HCE EIT - HPLC/UPLCspectrophotometry (24 chemicals – 1 lab)

YES

Attachment 8a_Revised.pdf

YES

Attachment 8b.pdf

YES

Attachment 9.pdf

YES

Attachment 10a.pdf

YES

Attachment 10b.pdf

YES

Attachment 11.pdf

29

Publication on the validation of EITL

YES

30

Publication on the validation of EITSYESEURL ECVAM REQUEST FOR ESAC ADVICE

Alépée et al. 2016 - SkinEthic HCE
liquids.pdf
Alépée et al. 2016 - SkinEthic HCE
solids.pdf

Page | 23

7.

TERMS OF REFERENCE OF THE ESAC WORKING GROUP

7.1

ESTABLISHMENT OF THE ESAC WORKING GROUP

The ESAC unanimously agreed by written procedure on the 18th of February 2016 on the compositionof a new ESAC Working Group for the review of test methods in the area of serious eye damage/eye
irritation.

7.2

TITLE OF THE ESAC WORKING GROUP

Full title:
ESAC Working Group on Eye Irritation Test Methods
Abbreviated title:
ESAC WG Eye Irritation

7.3

MANDATE OF THE ESAC WORKING GROUP

The ESAC WG is requested to conduct a scientific review of the l'Oréal-coordinated validation study
concerning the SkinEthic™ HCE EIT. The review needs to address the questions put forward to ESAC
by EURL ECVAM under section 4.2 of the current request.
The review should focus on the appropriateness of design and conduct of the study in view of thestudy objective and should provide an appraisal to which extent the conclusions of the test submitterare substantiated by the information generated during the study and how the information generated
relates to the scientific background available.

7.4

DELIVERABLES OF THE ESAC WORKING GROUP

The ESAC WG is requested to deliver to the chair of the ESAC and the ESAC Coordinator a detailed
ESAC Working Group Report outlining its analyses and conclusions and a draft ESAC Opinion. A
template has been appended (Appendix 1) intended to facilitate the drafting of the WG report.
The conclusions drawn in the report should be based preferably on consensus. If no consensus canbe achieved, the report should clearly outline the differences in the appraisals and provide
appropriate scientific justifications.

7.5

PROPOSED TIMELINES OF THE ESAC WORKING GROUP

Item
1

Proposed date/time
6 May 20162

11-13 May 2016

3

27 May 20164

9-10 June 2016

ActionTeleconference of the Working
Group
Working Group meeting
Circulation of final WG reportand draft ESAC opinion to ESAC
Endorsement of WG report and
ESAC opinion at ESAC42 meeting

EURL ECVAM REQUEST FOR ESAC ADVICE

Deliverable
Agree procedure
Draft ESAC WG report and draft
ESAC opinion
Final draft ESAC WG report anddraft ESAC opinion
Final ESAC WG report and ESAC
opinion

Page | 24

7.6

QUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WORKING GROUP

The review should address the questions put forward to ESAC by EURL ECVAM (see section 4.2) andthe information requirements of the ESAC Working Group Template, where applicable. The ESAC
Coordinator will provide guidance if needed.
When preparing the final ESAC WG report to address these questions, the ESAC WG is requested touse a pre-defined reporting template. This template (see appendix 1) follows EURL ECVAM's modular
approach and addresses to which extent the standard information requirements have beenaddressed by the study. The template allows moreover for addressing the issues specific studies
outlined in section 4.2. The Secretariat will provide guidance if necessary.

APPENDIX 1REPORTING TEMPLATE

The appended ESAC WG template suggests a structure that is in close agreement with the EURLECVAM information requirements ("modules") for scientific review following validation and allows at
the same time for the description of the analysis and conclusions concerning more specific questions.
The template can be used for various types of validation studies (e.g. prospective full studies,retrospective studies, performance-based studies and prevalidation studies). Depending on the study
type and the objective of the study, not all sections may be applicable.
However, for reasons of consistency and to clearly identify which information requirements have notbeen sufficiently addressed by a specific study, this template is uniformly used for the evaluation ofvalidation studies.
The current template is
TEMPLATE_ESAC-WG_REPORT-v6.doc

EURL ECVAM REQUEST FOR ESAC ADVICE

Page | 25

EURL ECVAM REQUEST FOR ESAC ADVICE

Page | 26

EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

Annex 3

ESAC WORKING GROUP PEER REVIEWCONSENSUS REPORTPage | 27

EUROPEAN COMMISSION
DIRECTORATE-GENERALJOINT RESEARCH CENTRE
Directorate F - Health, Consumers and Reference Materials
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)EURL ECVAMSCIENTIFIC
ADVISORYCOMMITTEE(ESAC)ESAC Working Group Peer Review Consensus Reporton theSkinEthic™ Human Corneal Epithelium (HCE)
Eye Irritation Test (EIT)
Title page information
File name

ESAC_WG_Report_SkinEthic_HCE_EIT.doc

Abbreviated title of ESAC
requestSkinEthic™ HCE EIT validation

Relating to ESAC REQUEST No.

2016-02

Request discussed throughWritten procedure previous to ESAC 42

Report to be handed over to
ESAC Chair and EURL ECVAM
Coordinator by

José M. Navas

Version tracking
DateVersionAuthor(s)Description

13 May 2016

V1.0

ESAC WG

First ESAC WG agreed draft

17 May 2016

V2.0

ESAC WG

Second revised draft after commenting

27 May 2016

V3.0

ESAC WG

Third revised draft after commenting

01 June 2016

V4.0

ESAC WG

Fourth revised draft after commenting

06 June 2016

V5.0

ESAC WG

Final ESAC WG approved draft sent to ESAC for
endorsement

ESAC WORKING GROUP REPORTPage | 28

Table of Contents
ESAC WORKING GROUP ................................................................................................................................. 31
ABBREVIATIONS USED IN THE DOCUMENT .................................................................................................... 32
1. STUDY OBJECTIVE AND DESIGN .................................................................................................................. 33
1.1 ANALYSIS OF THE CLARITY OF THE STUDY OBJECTIVE'S DEFINITION ............................................................................. 33(a) ESAC WG summary of the study objective as outlined in the Test Submission .................................... 33(b) Appraisal of clarity of study objective as outlined in the Test Submission ........................................... 33
1.2 QUALITY OF THE BACKGROUND PROVIDED CONCERNING THE PURPOSE OF THE TEST METHOD ........................................ 33(a) Analysis of the scientific rationale provided in the Test Submission .................................................... 33(b) Analysis of the regulatory rationale provided in the Test Submission ................................................. 34
1.3 APPRAISAL OF THE APPROPRIATENESS OF THE STUDY DESIGN ................................................................................... 35
1.4 APPROPRIATENESS OF THE STATISTICAL EVALUATION .............................................................................................. 36
2. COLLECTION OF EXISTING DATA ................................................................................................................. 36
2.1 EXISTING DATA USED AS REFERENCE DATA ............................................................................................................ 36
2.2 EXISTING DATA USED AS TESTING DATA ................................................................................................................ 37
2.3 SEARCH STRATEGY FOR RETRIEVING EXISTING DATA ................................................................................................ 37
2.4 SELECTION CRITERIA APPLIED TO EXISTING DATA .................................................................................................... 38
3. QUALITY ASPECTS RELATING TO DATA GENERATED DURING THE STUDY ................................................... 38
3.1 QUALITY ASSURANCE SYSTEMS USED WHEN GENERATING THE DATA .......................................................................... 38
3.2 QUALITY CHECK OF THE GENERATED DATA PRIOR TO ANALYSIS ................................................................................. 38
4. QUALITY OF DATA USED FOR THE PURPOSE OF THE STUDY (EXISTING AND NEWLY GENERATED) ............. 38
4.1 OVERALL QUALITY OF THE EVALUATED TESTING DATA (NEWLY GENERATED OR EXISTING) .............................................. 38
4.2 QUALITY OF THE REFERENCE DATA FOR EVALUATING RELEVANCE............................................................................... 39
4.3 SUFFICIENCY OF THE EVALUATED DATA IN VIEW OF THE STUDY OBJECTIVE ................................................................... 39
5. TEST DEFINITION (MODULE 1) .................................................................................................................... 39
5.1 QUALITY AND COMPLETENESS OF THE OVERALL TEST DEFINITION .............................................................................. 39
5.2 QUALITY AND COMPLETENESS OF THE DOCUMENTATION CONCERNING SOPS AND PREDICTION MODELS.......................... 40
6. TEST MATERIALS ........................................................................................................................................ 40
6.1 SUFFICIENCY OF THE NUMBER OF EVALUATED TEST ITEMS IN VIEW OF THE STUDY OBJECTIVE .......................................... 40
6.2 REPRESENTATIVENESS OF THE TEST ITEMS WITH RESPECT TO APPLICABILITY................................................................. 40
7. WITHIN-LABORATORY REPRODUCIBILITY (WLR) (MODULE 2) .................................................................... 41
7.1 ASSESSMENT OF REPEATABILITY AND REPRODUCIBILITY IN THE SAME LABORATORY ...................................................... 41
7.2 CONCLUSION ON WITHIN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY .................................................. 41
8. TRANSFERABILITY (MODULE 3) .................................................................................................................. 41
8.1 QUALITY OF DESIGN AND ANALYSIS OF THE TRANSFER PHASE ................................................................................... 41
8.2 CONCLUSION ON TRANSFERABILITY TO A NAÏVE LABORATORY / NAÏVE LABORATORIES AS ASSESSED BY THE STUDY ............. 42
9. BETWEEN-LABORATORY REPRODUCIBILITY (BLR) (MODULE 4) .................................................................. 42
9.1 ASSESSMENT OF REPRODUCIBILITY IN DIFFERENT LABORATORIES............................................................................... 42
9.2 CONCLUSION ON BETWEEN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY ............................................... 42
10. PREDICTIVE CAPACITY AND OVERALL RELEVANCE (MODULE 5) ................................................................ 43
10.1 ADEQUACY OF THE ASSESSMENT OF THE PREDICTIVE CAPACITY IN VIEW OF THE PURPOSE ............................................ 43
10.2 OVERALL RELEVANCE (BIOLOGICAL RELEVANCE AND ACCURACY) OF THE TEST METHOD IN VIEW OF THE PURPOSE ............ 45

ESAC WORKING GROUP REPORTPage | 29

11. APPLICABILITY DOMAIN (MODULE 6) ....................................................................................................... 45
11.1 APPROPRIATENESS OF STUDY DESIGN TO CONCLUDE ON APPLICABILITY DOMAIN, LIMITATIONS AND EXCLUSIONS ............ 45
11.2 QUALITY OF THE DESCRIPTION OF APPLICABILITY DOMAIN, LIMITATIONS, EXCLUSIONS ................................................ 45
12. PERFORMANCE STANDARDS (MODULE 7) ................................................................................................ 45
12.1 ADEQUACY OF THE PROPOSED ESSENTIAL TEST METHOD COMPONENTS .................................................................. 45
12.2 ADEQUACY OF THE REFERENCE CHEMICALS......................................................................................................... 45
12.3 ADEQUACY OF PROPOSED PERFORMANCE TARGET VALUES..................................................................................... 45
13. READINESS FOR STANDARDISED USE........................................................................................................ 46
13.1 ASSESSMENT OF THE READINESS FOR REGULATORY PURPOSES ................................................................................ 46
13.2 ASSESSMENT OF THE READINESS FOR OTHER USES ................................................................................................ 46
13.3 CRITICAL ASPECTS IMPACTING ON STANDARDISED USE .......................................................................................... 46
13.4 GAP ANALYSIS ............................................................................................................................................... 46
14. OTHER CONSIDERATIONS ......................................................................................................................... 47
15. CONCLUSIONS ON THE STUDY .................................................................................................................. 47
15.1 ESAC WG SUMMARY OF THE RESULTS AND CONCLUSIONS OF THE STUDY ................................................................ 47
15.2 EXTENT TO WHICH STUDY CONCLUSIONS ARE JUSTIFIED BY THE STUDY RESULTS ALONE................................................ 48
15.3 EXTENT TO WHICH CONCLUSIONS ARE PLAUSIBLE IN THE CONTEXT OF EXISTING INFORMATION ..................................... 48
16. RECOMMENDATIONS ............................................................................................................................... 48
16.1 GENERAL RECOMMENDATIONS ......................................................................................................................... 48
16.2 SPECIFIC RECOMMENDATIONS (E.G. CONCERNING IMPROVEMENT OF SOPS) ............................................................ 48
17. REFERENCES ............................................................................................................................................. 49

ESAC WORKING GROUP REPORTPage | 30

ESAC Working Group
Full title: ESAC Working Group on Eye Irritation Test Methods
Abbreviated title: ESAC WG Eye irritation

The ESAC WG was established in March 2016 by written procedure to assist in the production of an
ESAC Opinion by undertaking a peer review of a three laboratory ring trial of the SkinEthic™ Human
Corneal Epithelium (HCE) Eye Irritation Test (EIT), a test method with a wide applicability domain for
liquids (EITL: Eye Irritation Testing of Liquids) and solids (EITS: Eye Irritation Testing of Solids),
developed for the prediction of the eye irritation potential of liquid and solid chemicals, specificallyto distinguish chemicals requiring official classification for eye irritation or serious eye damage
according to the United Nations Globally Harmonized System of Classification and Labelling ofChemicals (UN GHS. UN, 2015) from chemicals not requiring classification (no classification category,'non-irritants').
Following a teleconference on 6 May 2016 the ESAC WG met at EURL-ECVAM 11-13 May 2016 to
conduct its peer review.

The ESAC WG members appointed by ESAC were:

Dr. José Maria Navas (ESAC Member, WG Chair)

Dr. Kristina Kejlová (ESAC Member)Prof. Annette Kopp-Schneider (ESAC Member)Dr. Renate Krätke (ESAC Member)Dr. Jon Richmond (ESAC Member)

Dr. Dave Allen (Nomination by NICEATM/ICCVAM)

Prof. Kyung-Min Lim (Nomination by KoCVAM)ESAC Coordination:
Dr. João Barroso (ESAC Coordinator)



Dr. Thomas Cole

ESAC WORKING GROUP REPORTPage | 31

Abbreviations used in the document


BLR

Between-laboratory reproducibility

CIConfidence Interval

EIT

Eye Irritation Test



EITL

Eye Irritation Testing of Liquids



EITS

Eye Irritation Testing of Solids



EIVS

EURL ECVAM/Cosmetics Europe Eye Irritation Validation Study



ESAC

EURL ECVAM Scientific Advisory Committee



ESAC WG

ESAC Working GroupEU CLP

European Union Regulation on Classification, Labelling and Packaging
of Substances and Mixtures

EURL ECVAM

European Union Reference Laboratory for Alternatives toAnimal TestingFDA(US) Food and Drug Administration



GLP

Good Laboratory Practice

HCE

Human Corneal EpitheliumHPLC/UPLCHigh Performance Liquid Chromatography/Ultra Performance Liquid
Chromatography



IATA

Integrated Approach to Testing and Assessment

MTT

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

OD

Optical density



OECD

Organisation for Economic Co-operation and Development

PC

Positive Control



RhCE

Reconstructed Human Cornea-like Epithelium

RhT

Reconstructed Human Tissue

SOP

Standard Operating Procedure

TST

Test Submission TemplateUN GHS

United Nations Globally Harmonized System for the Classificationand Labelling of Chemicals.



VMG

Validation Management Group

WLR

Within-laboratory reproducibility

ESAC WORKING GROUP REPORTPage | 32

1. Study objective and design
1.1

Analysis of the clarity of the study objective's definition(a) ESAC WG summary of the study objective as outlined in the Test Submission
The TST and supporting documents provide an adequate summary of the main study objectives:

To formally validate the SkinEthicTM HCE test method by a three laboratory ring trial study
using an appropriate number and range of test chemicals and to obtain data to assess therelevance (predictive capacity) and reliability (reproducibility within and between
laboratories) of the assay.



To facilitate its international acceptance in regulatory schemes for hazard assessment ofliquid and solid chemicals to discriminate chemicals not requiring classification for serious
eye damage/eye irritancy (No Category) from chemicals requiring classification and labelling(Category 1 and Category 2) according to the UN GHS Classification and Labelling ofChemicals (UN GHS. UN, 2015) and as implemented by the EU CLP regulation (EU CLP. EC,
2008a).
o N.B. The test method was not designed, intended, or evaluated to differentiate
between GHS Category 1 (irreversible effects) and 2A-B (reversible effects); andthere is no protocol for the testing of gases and aerosols.



To produce evidence to support the test method being incorporated into a tiered testing
strategy (so-called Bottom-Up/Top-Down testing strategy, Scott L. et al., 2010). The ultimate
purpose of such a tiered testing strategy being to replace the traditional in vivo Draize eyetest [Method B.5 of EC Regulation 440/2008 (EC, 2008b) or OECD TG 405 (OECD, 2002)].

(b) Appraisal of clarity of study objective as outlined in the Test Submission
The ESAC WG believes that the study objectives were sufficiently clear, and were reflected in the way
the study was designed, conducted, analysed, and subsequently reported in the TST.

1.2 Quality of the background provided concerning the purpose of the test method
The TST clearly describes the intended application of the SkinEthicTM HCE test method and suitably
qualified HPLC/UPLC-spectrophotometry analytical measurement systems as being for regulatory
testing.
(a) Analysis of the scientific rationale provided in the Test Submission
The SkinEthicTM HCE test method validation study was designed and conducted to provide evidence of
whether the test method and EITL and EITS protocols are sufficiently relevant and reliable to identify
chemicals not requiring classification, i.e. chemicals that are not UN GHS/EU CLP Category 1 (serious
eye damage) or Category 2 (eye irritant). The results of the study (i.e. prediction of non-classified
chemicals) are to be considered for use within a testing strategy (Scott et al., 2010).
The SkinEthicTM HCE test method TST describes the relevance and scientific rationale of the test
method partly in terms of other validated and accepted RhT models’ proven ability to measurecytotoxicity by the OD-photometric quantification of MTT reduction products (Mosmann, 1983) as areliable surrogate measurement of a range of toxicological endpoints after the controlled exposureof an RhT air-tissue interface to solid or liquid test materials. The ESAC WG accepts that in othercontexts (for example skin irritation) cytotoxicity/cell viability following chemical exposure is arecognised surrogate measure for chemically induced in vivo toxicity: with the in vivo cytotoxicity

ESAC WORKING GROUP REPORTPage | 33

then stimulating an inflammatory response involving the innate immune system in proportion to thedegree of cytotoxicity produced by the chemical.
The TST also provides a scientific rationale and justification in the specific context of eye irritation,
reasoning that RhCE test system cytotoxicity/tissue viability is a potentially plausible surrogate
measure of ocular hazards capable of discriminating between UN GHS/EU CLP classified chemicals(Category 1 and Category 2) and non-irritants produced by a variety of relevant, known, in vivo
mechanisms of ocular toxicity.
While the SkinEthic™ HCE EIT was not developed to account for all mechanisms associated with eyeirritation, the validation study demonstrated that this RhCE test method is able to correctly predict
chemicals not requiring classification for serious eye damage/eye irritation independently of thetypes of ocular effects observed in vivo (i.e., corneal, iridal and conjunctival injuries).
The evidence and arguments advanced in the TST in support of the test method’s performance takeaccount of the findings of previous pre-validation studies and other peer reviewed publications. The
available documentation does not allow an assessment of the test method’s performance with
mixtures.
The TST acknowledges that, for the foreseeable future, the in vivo Draize rabbit eye test is unlikely tobe replaced by a single in vitro test (Eskes et al., 2005), and argues that the intended use of this RhCE
test method would be within a larger testing strategy designed to replace or reduce animal testingfor determining chemical-induced eye damage/irritation potential for regulatory purposes (Scott etal., 2010).
The ESAC WG believes the available documentation provides sufficient evidence and reasonedarguments in favour of this RhCE test method having the potential to be at least as relevant andreliable as the currently validated in vitro methods for the identification of non-irritants [OECD TGs
437 (OECD, 2013a), 438 (OECD, 2013b), 491 (OECD, 2015a), and 492 (OECD, 2015b)], when combinedwith other alternative methods within future (tiered) testing strategies to replace the Draize eye test
(Scott et al., 2010).
It is proposed by Scott et al. (2010) that within such future testing strategies, based on the expected
ocular toxicity profile of the test chemical, and using one of two tiered testing approaches (BottomUp or Top-Down), the systematic and sequential application of one or more in vitro test methods
would be used to determine chemically induced eye damage/irritation potential. The full details of
the appropriate integrated testing strategies and test methods have yet to be defined.
The TST discusses a known limitation of the currently used OD-photometric endpoint detection
system - its inability to reliably assess the cytotoxic effects of chemicals, which interfere with the ODphotometric endpoint measurement of MTT-formazan - and provides a rationale for the use of asuitably qualified HPLC/UPLC-spectrophotometry endpoint detection system as an alternative MTTformazan endpoint measure.
(b) Analysis of the regulatory rationale provided in the Test Submission
The TST identifies relevant regulatory requirements. In the view of the ESAC WG, the relevant
legislation and regulations are appropriately referenced; and the regulatory requirements and therole of the RhCE-based methods in the context of the regulatory requirements are adequately
specified.
RhCE test methods for eye damage/irritation testing such as the SkinEthicTM HCE test method could
contribute to a reduction in animal testing by reliably identifying chemicals not requiringclassification when used within an appropriate non-animal testing strategy (Scott et al., 2010).
With respect to ocular toxicity, the ESAC WG considers that in view of the high prevalence of nonclassified chemicals (Adriaens et al., 2014) RhCE test methods validated for this purpose and used
ESAC WORKING GROUP REPORTPage | 34

within a “bottom-up approach” could significantly reduce animal testing by identifying the muchlarger number of chemicals not requiring classification.

1.3 Appraisal of the appropriateness of the study design
The study and data reported in the TST complies with the principles and criteria set out in the OECD
Guidance Document on the Validation and International Acceptance of New or Updated Test
Methods for Hazard Assessment (No. 34, OECD, 2005), and described in the generally accepted
Modular Approach to validation (Hartung et al., 2004).
Subject to the comments below (and section 1.4 of this report), the TST and supporting
documentation tends to confirm compliance with the expected standards for validation study
organisation, management, and conduct, including:a reasoned scientific basis for the mechanistic relevance of the test method;

suitable quality assurance systems for tissue model production and batch release;comprehensive SOPs for method implementation with separate protocols for the testing of
liquid and solid chemicals;representative and balanced chemical (test item) selection;comprehensive training of and test method transfer to naïve laboratories;



clear coordination and suitably defined responsibilities for the ring trial (project management,chemicals management, data management);

appropriate use of positive and negative controls, criteria and limits for retesting, definitionof qualified and non-qualified runs, data recording, calculation of WLR, BLR, specificity,
sensitivity, accuracy;

independence of ring trial testing and data statistics analysis; andcomplete documentation and transparency.

The SkinEthicTM HCE test method (with separate protocols for testing liquids and solids) was assessed
for its potential to identify chemicals not requiring classification for serious eye damage/eye irritation(UN GHS / EU CLP No Category; “non-irritants”) and their reliable discrimination from all classes ofclassified (categories 1 and 2) chemicals with a view to it forming an initial step of a Bottom-Up
approach or a later stage of a Top-Down approach (Scott et al., 2010).

The relevance and reliability of the test method were assessed by a three laboratory ring trial using
coded test chemicals (N=120): a pre-study statistical power analysis (sample size calculation) having
calculated within the EIVS study as a minimum requirement N=104 (i.e. 26 classified chemicals and
26 non-classified chemicals for each of the two protocols).
The analysis of the test method performance in the TST takes account of the study test results withthese 120 chemicals, plus data from 125 chemicals used for test method development within L'Oréal,
and an additional 80 chemicals tested prospectively by L'Oréal.
The performance of the SkinEthicTM HCE test method (with separate protocols for testing liquids andsolids) as a potential stand-alone test method for the identification of chemicals not classified forserious eye damage/eye irritation in the framework of Bottom-Up test strategies was judged against
the test method performance acceptance criteria previously derived to evaluate the EIVS study.
In setting minimum acceptable values for test method performance, the EIVS VMG took into accountof:
1. The background and specific objectives of the EIVS validation study;
2. The requirements of regulatory authorities and industry when testing and classifying
chemicals for serious eye damage/eye irritation;
ESAC WORKING GROUP REPORTPage | 35

3. The within test variability inherent in the in vivo Draize eye test data and the manner inwhich these data are currently used for classifying ocular effects according to UN GHS / EU
CLP;
4. The performance of other test methods already considered validated for this purpose;
5. The way in which the in vitro tests are to be used (as one test within a tiered testing
strategy); and6. The desired statistical power of the EIVS validation study.
The ESAC WG concludes that, subject to specific qualifications set out below (see 1.4), the study
design was generally appropriate and robust, the acceptance criteria appropriate at the time, andthat the study findings have provided the evidence and analysis required to satisfy the study
objectives.
The ESAC WG believes the TST provides a detailed and reasoned rationale for use of the suitably
qualified HPLC/UPLC-spectrophotometry analytical method (FDA, 2001; Alépée et al., 2015).

1.4 Appropriateness of the statistical evaluation
The statistical and data reports annexed to the SkinEthicTM HCE test method TST are detailed andinformative. The method used for the calculation of the WLR is appropriate.
The method used for the calculation of predictive capacity using resampling methods (bootstrappingof single runs) is also appropriate.
However, in the view of the WG the most appropriate way of calculating the BLR would have been to
apply resampling methods that better reflected how test materials would be categorised forregulatory purposes – as in practice categorization would be based on a single run not the average ofthree. Nevertheless, in the context of this study it would appear that had this approach been
followed the reported difference in the performance of the test method would be marginal.
Non-qualifying test runs were not included in the data used to calculate final BLR or Predictive
Capacity values.
No test chemical used for the ring trial was considered incompatible with the method by any of thethree laboratories, with either the EITS or the EITL protocol. All chemicals were thus included in all of
the statistical analyses of this test method.
The results obtained with the EITS and EITL protocols were independently assessed with respect totheir reproducibility and predictive capacity.

2. Collection of existing data
2.1 Existing data used as reference data
The TST and annexes provide detailed information on this point. The TST references and relies on an
extensive collection of information generated before and during the development and pre-validationof the test method.
The essential requirements for chemical selection for the ring trial were toxicological andphysicochemical properties, and the availability of complete and quality assured supporting in vivo
data to allow comparative evaluation of the predictive capacity of the RhCE test method as measured
against in vivo (Draize eye test) reference method.

ESAC WORKING GROUP REPORTPage | 36

The chemicals selected are all commercially available chemicals and included chemicals:of different physical states;

known to induce the full range of in vivo serious eye damage/eye irritation responses basedon high quality results obtained in the reference in vivo rabbit eye test (Draize, 1944) and the
UN GHS classification system (i.e., Categories 1, 2A, 2B, or No Category) (UN GHS. UN, 2015);



representing the various known in vivo drivers of classification as reported by Adriaens et al.
(2014) and Barroso et al. (2016);

covering a good and wide representation of organic functional groups;having well-defined chemical structures;

known to be coloured chemicals and/or direct MTT reducers; and

not associated with prohibitive acquisition and/or disposal costs.

In total, 120 coded chemicals (60 liquids and 60 solids) were evaluated in three laboratories, with thetest chemical set having the following qualities:

The 120 chemicals were distributed as follows according to the UN GHS classification: 32
Category 1 (16 liquids and 16 solids), 17 Category 2A (8 liquids and 9 solids), 13 Category 2B(8 liquids and 5 solids) and 58 No Category (28 liquids and 30 solids) chemicals;

16 different functional groups: including organic bases, organic acids, neutral organic,
inorganic bases, soap/surfactant;

direct MTT reduction: 11 chemicals identified as MTT reducers and 43 as non MTT reducers
during Eye Irritation Validation Study (2010);



coloured chemicals (with 2 identified as coloured chemicals during EIVS) and non-coloured
chemicals;previously used in related validation studies: 55 % (66/120) tested in EIVS.

75 of the 120 test materials had NOT been in the development or optimisation of the test method.
The Draize eye test Reference Database (DRD) published in Barroso et al. (2016) was consulted to
identify the test chemicals used in the validation study. Such database includes data from thefollowing sources:
1. ECETOC database of eye irritation reference chemicals (ECETOC, 1998).
2. Database from Laboratoire National de la Santé (LNS) (Gautheron et al., 1992).
3. ZEBET database of eye irritation reference chemicals (Spielmann et al., 1996).
4. ICCVAM (NICEATM) database of eye irritation reference chemicals.
5. EURL ECVAM database of chemicals considered for selection in EIVS, including amongst other(i) chemicals notified in the EC New Chemicals Database (NCD), (i) chemicals in the EC (DGSANCO) Cosmetics Ingredients (CosIng) database, (iii) pesticide actives in the US EPA
database

2.2 Existing data used as testing data
Not applicable.

2.3 Search strategy for retrieving existing data
See Section 2.1 above.

ESAC WORKING GROUP REPORTPage | 37

2.4 Selection criteria applied to existing data
See Section 2.1 above.

3. Quality aspects relating to data generated during the study
3.1 Quality assurance systems used when generating the data
L’Oreal’s R&I laboratory developed the SkinEthic™ HCE EIT test method and was the lead laboratory
for the ring trial. It is not Good Laboratory Practices (GLP) certified. However, for the purposes of thering trial the following safeguards (Balls et al., 1995) were applied:Qualified personnel, and appropriate facilities, equipment and materials were available



Records of the qualifications, training and experience, and a job description for eachprofessional and technical individual, were maintained.

For each study, an individual with appropriate qualifications, training and experience wasappointed to be responsible for its overall conduct and for any report issued.

Instruments used for the generation of experimental data were inspected regularly, cleaned,maintained and calibrated according to manufacturers' instructions. Records of theseprocesses were kept, and made available for inspection on request.

Reagents were labelled, as appropriate, to indicate their source, identity, concentration andstability. The labelling included the preparation and expiry dates, and specific storage
conditions.

All data generated during a study were recorded by the individual(s) responsible. These
entries were attributable and dated.

The two additional laboratories involved in the ring trial were involved in the validation study
namely CHARLES RIVER LABORATORIES (Edinburgh, United Kingdom) and VITO NV (Flemish Institutefor Technological Research, Mol, Belgium) are GLP certified and performed the studies in accordance
and in compliance with the GLP standards (OECD, 1999).

3.2 Quality check of the generated data prior to analysis
The study documents state that:"...for the statistical analysis, a summary template was designed by the statistician, and the results
were transferred to this template by the statistician. This summary template was compared with thesummary templates received from the participating laboratories in order to check that no mistakes
were made in the transfer of the results. The final conclusions for each chemical were then comparedto the conclusions of the reports send by the laboratories as an additional check".

4. Quality of data used for the purpose of the study (existing andnewly generated)
4.1 Overall quality of the evaluated testing data (newly generated or existing)
The data generated and supplied are of sufficient quality to apply the predetermined acceptancecriteria and prediction model.

ESAC WORKING GROUP REPORTPage | 38

4.2 Quality of the reference data for evaluating relevance1
The quality of data used is considered to be the best available Draize eye test reference data and isequivalent to that of data used in previous RhCE validation studies.

4.3 Sufficiency of the evaluated data in view of the study objective
The sample size was sufficient to produce appropriately narrow confidence intervals (CIs).
There is sufficient data for the WG to offer a reasoned opinion about the performance of the test
method.

5. Test definition (Module 1)
5.1 Quality and completeness of the overall test definition
The SkinEthic™ HCE EIT test method is based on RhCE technology; the test material (solid or liquid
chemicals) is applied directly to the upper surface of the air-tissue interface, and the eyedamage/irritation potential of the chemical is derived from a prediction model based on percentage
tissue viability (adjusted against a negative control) as estimated by OD-photometry or HPLC/UPLCspectrophotometry of the MTT reduction product formazan.
The tissue component of the test kit is manufactured by controlled culture of immortalised humancorneal epithelial cells in a chemically defined medium to produce a multi-layered epithelium similar
to in vivo corneal epithelium with columnar basal cells, wing cells and squamous cells. The model isalso characterised by the presence of relevant ultra-structural features (such as intermediatefilaments, mature hemi-desmosomes and desmosomes) that characterise the corneal epithelium insitu. Specific cytokeratins 64kD (K.3) have also been described (Nguyen et al., 2003).
The test method SOP provides separate protocols for testing liquids and solids. There are four
essential differences between the two protocols:the length of time that the tissue interface is in contact with the test material (30 minutes
exposure for liquids and 4 hours exposure for solids);

a requirement for an 18 hours post-exposure incubation phase for solid chemicals;the extraction of the MTT formazan product from both the top and bottom surfaces of thetissue interface in the case of liquids, but only from the bottom surface for solids.



the prediction models are different (see immediately below).

Following treatment with a test chemical the relative tissue viability is determined against thenegative control value by the reduction of the vital dye MTT to formazan as measured by ODphotometry and/or HPLC/UPLC-spectrophotometry. For both Optical Density and HPLC/UPLCspectrophotometry endpoints, the result is accepted if:

The mean Optical Density (ODNgC) at 570 nm (± 30nm) of the two replicate tissues treatedwith negative control is ≥ 1.4 with an upper acceptance limit of ≤2.5.

The Mean Viability of the two replicate tissues (2 values from each of the two tissues)
treated with positive control, expressed as percentage of the negative control, is ≤ 30 %,1OECD guidance document No. 34 on validation defines relevance as follows: "Description of relationship of thetest to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent towhich the test correctly measures or predicts the biological effect of interest. Relevance incorporates
consideration of accuracy (concordance) of a test method."
ESAC WORKING GROUP REPORTPage | 39

what is much higher than the reported PC mean values (generally < 5 %), so that theestablished threshold (30 %) could be reduced.


The TST indicates that when OD is chosen as endpoint the difference of viability between thetwo replicate tissues of a single test chemical should be ≤ 20 in the same run whatever thetest item (for positive control, negative control, test substances, and all adapted controls).
The WG notes that this is probably also applicable to the HPLC/UPLC-spectrophotometry
measurements.

For the purposes of the Prediction Model tissues treated with chemicals inducing eye irritation orserious eye damage (UN GHS/EU CLP Category 2 or Category 1, respectively) were expected to show
a decrease in tissue viability below a specified threshold in comparison to the negative control. See
table below.
Table 5.1.1. Cell viability values used as threshold for the assessment of the eye irritation or serious
eye damage (UN GHS/EU CLP) caused by chemicals.
Cell viability (liquids)

Cell viability (solids)

UN GHS / EU CLP
classification> 60 %> 50 %No Category≤ 60 %≤ 50 %

Categories 1 and 2

Results are expressed as mean OD, mean percentage viability and difference of viability between thetwo replicate tissues.
The histological features of the test system, and the use of similar endpoints and endpoint detection
systems as used within other RhT systems already validated for other toxicological endpoints, make it
plausible that this test RhCE system may provide insights into the likely eye damage/irritation
potential of chemicals.
Although derived from cultured human corneal cells, ESAC WG notes that the cells used have beentransformed and immortalised. The ESAC WG offers no opinion on whether these cells are inherently
more biologically relevant in this context than other epithelial cells (e.g. eye irritation RhCE models
derived from normal untransformed human keratinocytes).

5.2 Quality and completeness of the documentation concerning SOPs and prediction
modelsThe SOPs, including the prediction model, were sufficiently detailed. See section 5.1 above.

6. Test materials
6.1 Sufficiency of the number of evaluated test items in view of the study objective
The WG believes that the number of test items was sufficient to draw meaningful conclusions withrespect to the objectives of the study. See 2.1 above.

6.2 Representativeness of the test items with respect to applicability
The WG believes that the number and nature of the test items selected were adequate to provide
insights into the applicability domain and the scope and limitations of the test method. See 2.1 above.
ESAC WORKING GROUP REPORTPage | 40

7. Within-laboratory reproducibility (WLR) (Module 2)
7.1 Assessment of repeatability and reproducibility in the same laboratory
For liquid test items
The reliability of the SkinEthic™ HCE EITL protocol was assessed in terms of concordance inpredictions for three independent experiments.
The resulting WLR was 95 % (95 %-CI: 86.3 % - 98.3 %) for L’Oréal, 93.3 % (95 %-CI: 84.1 % - 97.4 %)for VITO, and 88.3 % (95 %-CI: 77.8 % - 94.2 %) for CRL. In summary, WLR ≥ 88 % between the
independent runs was observed within the laboratories, exceeding the minimum acceptable value of≥ 85 % set by the VMG.
In addition, the lead laboratory (L’Oréal) prospectively tested 45 additional liquid chemicals in three
independent experiments. Twenty-two chemicals did not require classification in vivo and 23
chemicals were classified. Concordant prediction was obtained for 41 of the 45 chemicals, resulting
in a WLR of 91.1 %, exceeding the minimum values set by the VMG.
For solid test items
The reliability of the SkinEthic™ HCE EITS protocol was assessed in terms of concordance inpredictions for the independent valid runs. The WLR was 96.7 % (95 %-CI: 88.6 % - 99.1 %) for L’Oréal
and 95.0 % (95 %-CI: 86.3 % - 98.3 %) for VITO and CRL.
In conclusion, low variation (WLR ≥ 95 %) between the independent runs was observed within thelaboratories, indicating that the SkinEthic™ HCE EITS protocol is robust. This means that the WLR ishigher than 95 %, exceeding the minimum value (85 %) set by the EIVS VMG (Barroso et al., 2015;
EURL ECVAM, 2016).
The lead laboratory (L’Oréal) prospectively tested 35 additional solid chemicals in three independentruns. Twenty-three chemicals did not require classification in vivo and 12 chemicals were classified. A
concordant prediction was obtained for 34 of the 35 chemicals, resulting in a WLR of 97.1 %exceeding the minimum values set by the VMG.

7.2 Conclusion on within-laboratory reproducibility as assessed by the study
Section 7.1 confirms that the target value set by the EIVS VMG was exceeded by all laboratories in
the ring trial and further prospective testing undertaken by L'Oréal.

8. Transferability (Module 3)
8.1 Quality of design and analysis of the transfer phase
The two naïve laboratories participating in the validation of SkinEthic™ HCE received two days
training by the lead laboratory (L'Oréal).
Training and proficiency testing was done with coded test chemicals (9 solids, and 9 liquids), theseincluded colourants and direct MTT reducers.
The transfer phase included demonstrating proficiency with the SOP, and completion of the Excel
spreadsheets. As confirmed by the data, both laboratories demonstrated their proficiency inperforming the SkinEthic™ HCE EIT method for the Eye Irritation Testing of Liquids (EITL protocol) and
Solids (EITS protocol). Only one test chemical did not produce concordant results in all cases.
ESAC WORKING GROUP REPORTPage | 41

Accordingly, the ESAC WG considers that the technicians from the participating laboratories
demonstrated adequate proficiency in performing the SkinEthic™ HCE and readiness to enter theformal validation study.

8.2 Conclusion on transferability to a naïve laboratory / naïve laboratories as assessed by
the study
The ESAC WG considers that the technicians from the participating laboratories demonstrated
adequate proficiency in performing the SkinEthic™ HCE and readiness to enter the formal validation
study.

9. Between-laboratory reproducibility (BLR) (Module 4)
9.1 Assessment of reproducibility in different laboratories
For each laboratory, the mean tissue viability and standard deviation over the three independentvalid runs were calculated to obtain a final classification for each chemical. The evaluation of BLR wason the concordance of the final predictions Classified (C) or Not Classified (NC). BLR was reportedwith the Wilson 95 %-CI.
The minimum acceptable BLR value set by the EIVS VMG was ≥ 80 %.
Assessment of BLR for Liquids:
Fifty-six of the 60 liquid chemicals were consistently classified (NC/C) by the three laboratories
resulting in a BLR of 93.3 % (95 %-CI: 84.1 % - 97.4 %).
The BLR for the pair-wise comparisons was 93.3 % (56/60 chemicals) for L'Oréal and CRL, 95.0 %(57/60 chemicals) for L'Oréal and VITO, and 98.3 % (59/60 chemicals) for CRL and VITO.
Assessment of BLR for Solids:
Fifty-eight of the 60 solid chemicals were consistently classified (NC/C) by the three laboratories
resulting in a BLR of 96.7 % (95 %-CI: 88.6 % - 99.1 %).
The BLR for the pair-wise comparisons was 96.7 % (58/60 chemicals) for L’Oréal and CRL and for
L’Oréal and VITO, a 100 % concordance was obtained between CRL and VITO.

9.2 Conclusion on between-laboratory reproducibility as assessed by the study
The overall BLR for the SkinEthic™ HCE EIT method, based on the result of the ring trial with abalanced and representative set of 120 chemicals, was 95.0 % (EITL 93.3 % and EITS 96.7 %). The BLRof the SkinEthic™ HCE EIT test method was higher than the defined minimum value of 80 % set by the
EIVS VMG (Barroso et al., 2015; EURL ECVAM, 2016).

ESAC WORKING GROUP REPORTPage | 42

10. Predictive capacity and overall relevance (Module 5)10.1 Adequacy of the assessment of the predictive capacity in view of the purpose
The predictive capacity of the assay was evaluated by comparing the prediction results based on theindividual laboratory results with the existing proposed classification. Two-by-Two contingency tables(C versus NC) were constructed and sensitivity, specificity, and accuracy were calculated.
Bootstrap resampling (10,000 times with sample size = 1) was used to obtain 95 %-CIs for accuracy,
sensitivity and specificity. The rationale for performing bootstrap resampling with size n=1 was thefact that in reality a chemical will be tested and classified only once.
Sensitivity, specificity, and accuracy were calculated on 10,000 simulated sets of 120 chemicals (60liquids and 60 solids), based on observed predictions (9 predictions per chemical). Random samplingwith sample size n=1 was performed per chemical (with a pool of 9 predictions, based on 3
independent runs for each chemical at each of the 3 laboratories) for the set of 60 liquids or 60 solid
chemicals.
Accuracy, sensitivity and specificity were calculated for each of the 10,000 resampling sets. The meanof the bootstrap sample and 95 %-CI applying the percentile method was calculated for the three
performance parameters.
Reference data were use appropriately and the resulting data sets were adequate to assess test
method performance.
Assessment of predictive capacity for liquid chemicals:
Predictive capacity was calculated for each laboratory and for the cumulative results of the threelaboratories using the cut-off of 60 % viability to distinguish between chemicals not requiringclassification for serious eye damage/eye irritancy (No Cat) from chemicals requiring classificationand labelling (Cat 1 and Cat 2) according to UN GHS (UN, 2015) (Table 10.1.1 below).
Table 10.1.1. Predictive capacity for the set of 60 liquid chemicals based on individual laboratory
predictions: overall and for each laboratory.
In vivo UN GHS

Cumulative
I

Charles Riverlaboratories

L'Oréal

NI

I

NI

I

VITO

NI

I

NI

Classified (n)

283

5

96

0

94

2

93

3

No Category (n)

77

175

29

55

23

61

25

59

Total (n)

540

180

180

180

Sensitivity (%)
Specificity (%)

98.3

100

97.9

96.9

69.4

65.5

72.6

Accuracy (%)

70.2

84.8

83.9

86.1

84.4

The predictive capacity was also determined for the extended dataset (60 liquid chemicals of themulticenter study and 45 additional liquid chemicals) tested by the lead laboratory. This resulted inan overall accuracy of 83.5 % with 100 % sensitivity and 65.3 % specificity for L’Oréal only.
The bootstrap resampling yielded a sensitivity of 98.2 % (95 %-CI: 93.8 % - 100 %), a specificity of
69.4 % (95 %-CI: 60.7 % - 75.0 %), and an accuracy of 84.8 % (95 %-CI: 80.8 % - 88.3 %).

ESAC WORKING GROUP REPORTPage | 43

Assessment of predictive capacity for solid chemicals:
The predictive capacity was calculated for each laboratory and for the cumulative results of the threelaboratories using the cut-off of 50 % viability to distinguish NC (No Cat) from chemicals requiringclassification and labelling (Cat 1 and Cat 2) according to UN GHS (Table 10.1.2). The calculations
were based on the individual predictions derived from the qualified tests for each chemical in eachlaboratory. The three laboratories obtained a sensitivity of 92.2 %. The specificity varied between
75.3 % (VITO), 75.6 % (L’Oréal), and 78.9 % (CRL). An accuracy of 83.9 %, 85.6 %, and 83.3 % was
obtained by L’Oréal, CRL, and VITO, respectively (See table 10.1.2 below)
Table 10.1.2. Predictive capacity for the set of 60 solid chemicals based on individual laboratory
predictions: overall and for each laboratory
In vivo UN GHS

aCumulative

L'Oréal

CRL

VITO

C

NC

C

NC

C

NC

C

Classified (n)

249

21

83

7

83

7

83

NC
7

No Category (n)

63

206

22

68

19

71

22

67

Total (n)

539

180

180

179 a

Sensitivity (%)

92.2

92.2

92.2

92.2

Specificity (%)

76.6

75.6

78.9

75.3

Accuracy (%)

84.4

83.9

85.6

83.3

For chemical No. 2 only two valid runs were obtained over the five runs

The predictive capacity was also determined for the extended dataset (60 solid chemicals of themulticenter study and 35 additional solid chemicals) tested by the lead laboratory. This resulted in an
overall accuracy of 80.7 % with 89.7 % sensitivity and 73.6 % specificity for L’Oréal only.
The bootstrap resampling yielded a sensitivity of 91.9 % (95 %-CI: 90.0 % - 93.3 %), a specificity of
76.6 % (95 %-CI: 73.3 % - 80.0 %), and an accuracy of 84.3 % (95 %-CI: 81.7 % - 86.7 %).
All of the reported values exceeded the minimum acceptable values as set by the VMG which were asensitivity of at least 90 %, a specificity of at least 60 %, and an accuracy of at least 75 %. None of theCategory 1 chemicals were under-predicted in the majority of the runs across all laboratories.
However the working group notes that false negative results were obtained in a total of 2
independent runs at the same laboratory for 1 chemical ( [3-(2-Aminoethylamino)propyl]trimethoxysilane, CASRN 1760-24-3, classified Category 1 in the Draize eye test based on persistentconjunctival and corneal effects on day 21 in the majority of the animals). Therefore this chemical
was classified by this laboratory as a No Category. Having investigated this anomaly the testdeveloper reported that due to storage problems at this laboratory there was crystal formation forthis chemical during storage with the first run (viability: 25.1 %) giving the correct classification. This
first run was performed in the beginning of the experimental phase whereas the second and thirdruns, which gave the misclassification, were performed at the end of the experimental phase (morethan 60 days later). The effect of storage conditions on the stability of this chemical ([3-(2Aminoethylamino)propyl]trimethoxysilane) was evaluated after the validation study. Indeed,
confirmatory work has shown that the tissue viability increased when the container of the chemical
was not closed properly. After 14 and 30 days of storage with half open or open lid, mean viability
increased above 50 % (51.5 % to 66.3 %). In the two other laboratories, the independent runs for the[3-(2-Aminoethylamino)propyl]-trimethoxysilane were performed within a period of less than 30
days. The WG believes that had this chemical been properly stored then an appropriate classification
would have been achieved.
In addition, another chemical (Tetraethylene glycol diacrylate, CASRN 17831-71-9, classified as
Category 1 based on iritis that resulted in severe but delayed corneal opacity in the Draize eye test)

ESAC WORKING GROUP REPORTPage | 44was misclassified in 1 run at 1 laboratory – but in this case the overall classification by that laboratoryon the basis of three qualified run sequences was irritant in the in vitro assay.

10.2 Overall relevance (biological relevance and accuracy) of the test method in view ofthe purpose
See section 10.1. The WG believes that the overall relevance of the test method has beensatisfactorily demonstrated with a view to it being considered for regulatory use within an integrated
approach to testing and assessment (IATA) for the prediction of eye damage/irritation potential ofchemicals within its applicability domain.

11. Applicability domain (Module 6)
11.1 Appropriateness of study design to conclude on applicability domain, limitations andexclusions
The validation study was undertaken with a large number of liquid and solid test materials
representing a broad range of chemical classes (see 2.1) and a full range of ocular reactivity. However,there is limited information on test method performance with mixtures, and chemicals in the form ofgases and aerosols were not assessed in a validation study.

11.2 Quality of the description of applicability domain, limitations, exclusions
As indicated in section 11.1, gases and aerosols were not considered in the validation study.
SkinEthic™ HCE EIT test method is not intended to differentiate between UN GHS Category 1 (serious
eye damage) and UN GHS Category 2 (eye irritation). This differentiation will need to be addressed byanother tier of a testing strategy (Scott et al., 2010). A chemical that is identified as requiringclassification for eye irritation/serious eye damage with SkinEthic™ HCE EIT will thus require
additional testing (in vitro and/or in vivo) to establish a definitive classification, using e.g., OECD TG
437 (OECD, 2013a), 438 (OECD, 2013b), 460 (OECD, 2012) or 491 (OECD, 2015a).

12. Performance standards (Module 7)
Performance standards were not addressed by TST but are already available at OECD based on thesimilar method EpiOcularTM EIT (OECD, 2015c).

12.1 Adequacy of the proposed Essential Test Method Components
Not applicable – see immediately above.

12.2 Adequacy of the Reference Chemicals
Not applicable – see immediately above.

12.3 Adequacy of proposed performance target values
Not applicable – see immediately above.
ESAC WORKING GROUP REPORTPage | 45

13. Readiness for standardised use
13.1 Assessment of the readiness for regulatory purposes
The WG believes that the overall relevance and reliability of the test method has been satisfactorily
demonstrated with a view to it being considered for regulatory use in a tiered assessment strategy.
The test could be applied as a first step in Bottom-Up discrimination of 'non-irritants' (GHS No
Category) or as a confirmatory last step in a Top-Down approach, where the priority is to first
discriminate chemicals inducing serious eye damage (GHS Category 1). However, the method is notintended to differentiate Category 1 from Category 2 on its own.

13.2 Assessment of the readiness for other uses
The study findings justify the SkinEthic™ HCE EIT method being considered as a means of identifying
chemicals do not cause eye damage/irritation in non-regulatory contexts.

13.3 Critical aspects impacting on standardised use
The TST indicates that two training days are required for a naïve laboratory.
All HPLC/UPLC-spectrophotometry systems have to be suitably qualified (FDA, 2001; Alépée et al.,2015).

13.4 Gap analysis
1. The SkinEthic™ HCE EIT test method liquid and solid chemical protocols could form
components of future integrated approaches to testing and assessment (IATA) for
determining the eye damage/irritation potential of chemicals (Scott et al., 2010). The othercomponents of such a testing strategy and the precise role of these RhCE test methods have
yet to be formally defined.
2. As mentioned above the ESAC WG notes that there is limited information on theperformance of this test method with chemical mixtures and no information about gases andaerosols.
3. The WG acknowledges that including a wide range of chemical mixtures in validation studies
currently raises several problems, e.g. availability of in vivo data, selection of test mixtures,and continuity of supply. However, most of the chemicals which have to be classified are
mixtures and there is a need to confirm that in vitro methods can be used for theclassification of chemical mixtures. The ESAC WG recommends the inclusion of a broader
range of chemical mixtures in future validation studies, and proposes consideration of theuse of reference data available for the classification of mixtures, using the additivity
approach recommended by the UN GHS (UN, 2015) as well as the CLP (EC, 2008a), and/or theuse of mixtures already assessed and identified as Cat.1, Cat.2, or No Cat.
4. While the SkinEthic™ HCE EIT was not developed to account for all mechanisms associatedwith eye irritation, the validation study demonstrated that this RhCE test method is able tocorrectly predict chemicals not requiring classification for serious eye damage/eye irritation
independently of the types of ocular effects observed in vivo (i.e., corneal, iridal andconjunctival injuries).
5. The available documentation does not allow an assessment of the test method’s
performance with mixtures.
ESAC WORKING GROUP REPORTPage | 46

6. For RhCE test methods that use cytotoxicity as a surrogate measure of eye damage/irritation
potential it is plausible that related test methods could be developed to allow histologicaland other evaluation of the tissue damage to provide additional insights into the mechanismof injury.
7. There is a lack of information in the TST about inter-laboratory reproducibility of HPLC/UPLCspectrophotometry analytical method. However, this has been demonstrated for the
HPLC/UPLC-spectrophotometry measurement of formazan in isopropanol extracts obtained
from other RhTs (ESAC, 2014; Alépée et al., 2015).

14. Other considerations
The ESAC WG notes that due to the variability of individual animal responses within the in vivo
Draize eye test there is ≥ 12 % probability, if chemicals are retested, of chemicals currently
classified as UN GHS Category 2 by the in vivo test being identified as UN GHS No Category(Adriaens et al., 2014). As in vivo Draize eye test data served as reference data for chemical
selection and Predictive Capacity within validation studies, the reported performance of the invivo test should be borne in mind when evaluating the reported performance, and validity, of
alternative methods and testing strategies for detecting chemically induced eye
damage/irritation.
The reported PC mean values (generally < 5 %) are significantly lower than the 30 % acceptancethreshold set out in the SOP. The PC was also generally reproducible with maximum viabilities
unequivocally acceptable. The WG suggests that consideration be given to reducing the PC
acceptance criterion threshold.

15. Conclusions on the study
15.1 ESAC WG summary of the results and conclusions of the study
1. The SkinEthic™ HCE EIT test method study was generally well designed and conducted, andthe TST provides analysis and insights that allows the WG to offer an informed opinion about
the performance of the test method.
2. The ESAC WG considers that the SkinEthic™ HCE EIT test method liquid and solid chemical
protocols are scientifically valid (reproducible and accurate) in the context of identifying
chemicals not requiring classification for serious eye damage/eye irritation under UN GHS.
3. The ESAC WG considers that although a wide range of chemical types, chemical classes,
molecular weights, LogP, chemical structures, etc., were tested, no clear additional
limitations regarding applicability could be identified beyond those identified in the TST.
4. The ESAC WG has noted that currently there is only a limited range of chemical mixtures
available for use as test chemicals within eye damage/irritation validation studies, and would
like to see more data presented with respect to the test method performance in the case ofchemical mixtures requiring the classification for eye damage/irritation potential.
5. For coloured chemicals interfering too strongly with the MTT-reduction assay an alternative
endpoint detection system should be used (e.g., suitably qualified HPLC/UPLCspectrophotometry). The ESAC WG agrees, and believes that qualified HPLC/UPLCspectrophotometry endpoint measurement system described in the TST is suitable for thispurpose.

ESAC WORKING GROUP REPORTPage | 47

15.2 Extent to which study conclusions are justified by the study results alone
All of the ESAC WG conclusions given above can be derived and argued or justified from the TST andsupporting documents.

15.3 Extent to which conclusions are plausible in the context of existing information
In reaching its conclusions the ESAC WG has also taken account of the larger body of information andknowledge set out in the technical annexes supplied and the references cited in the study documents.
It is on consideration of both the study findings, and that larger body of knowledge of information,that the ESAC WG established and confirmed the plausibility of the conclusions set out above.

16. Recommendations
16.1 General recommendations
The ESAC WG believes that the overall relevance and reliability of the SkinEthic™ HCE EIT has beensatisfactorily demonstrated with a view to it being considered for regulatory use in a tiered
assessment strategy. The test method could be applied as a first step in Bottom-Up discrimination of'non-irritants' (GHS No Category) or as a confirmatory last step in a Top-Down approach, where thepriority is to first discriminate chemicals inducing serious eye damage (GHS Category 1). However,
the method is not intended to differentiate Category 1 from Category 2 on its own.
The validation study results tend to confirm the findings of the EIVS study that suitably qualified
HPLC/UPLC-spectrophotometry analytical systems can be used to quantify precipitated MTTformazan as an alternative endpoint detection system in particular for highly coloured chemicals
interfering with the conventional endpoint measurement of MTT-formazan by OD-photometry.

16.2 Specific recommendations (e.g. concerning improvement of SOPs)
The ESAC WG offers the following additional recommendations for consideration.
While the SkinEthic™ HCE EIT was not developed to account for all mechanisms associated with eyeirritation, the validation study demonstrated that this RhCE test method is able to correctly predict
chemicals not requiring classification for serious eye damage/eye irritation independently of thetypes of ocular effects observed in vivo (i.e., corneal, iridal and conjunctival injuries).
There may be an option to harmonise the protocols for liquid and solid chemicals in relation to theextraction procedures of formazan, if categorisation of chemicals based on extraction from only
under-surface of the tissue interface meets the performance of the different extraction procedures
given in the SOP for liquid and solid chemicals.
The ESAC WG recommends insights into test performance with chemical mixtures be obtained by
testing mixtures based on their current classification, or based on the known properties of theircomponents.
The ESAC WG believes that potential problems with the reliability of historical Draize eye test data
must also be taken into account when evaluating the predictive capacity of alternative test methods
and testing strategies.

ESAC WORKING GROUP REPORTPage | 48

17. References

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Wever B, Pfannenbecker U, Tailhardat M, Zuang V (2014). Retrospective analysis of the Draize test forserious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN
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Alépée N, Barroso J, De Smedt A, De Wever B, Hibatallah J, Klaric M, Mewes KR, Millet M,
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Barroso J, Alépée N, Cole T, Eskes C, Freeman SJ, Liška R, McNamee P, Pfannenbecker U, Reus AA,
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ESAC (2014). ESAC Opinion No. 2014-03 from 17 November 2014, based on the ESAC Scientific Peer
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