ESAC Opinion on the L'Oréalcoordinated
study
on
the
transferability and reliability of the
U-SENS™ test method for skinsensitisation testing

ESAC Opinion No. 2016-03
of 24 June 2016

2016

EUR 28178 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

JRC103705
EUR 28178 EN

PDF

ISBN 978-92-79-63200-6

ISSN 1831-9424

doi:10.2787/815737

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 L'Oréal-coordinated study
on the transferability and reliability of the U-SENS™ test method for skin sensitisation testing. ESAC Opinion No.
2016-03
of
24
June
2016;
EUR
28178
EN;
doi:10.2787/815737.
Availableat:
http://publications.jrc.ec.europa.eu/repository/handle/JRC103705.
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 theL'Oréal-coordinated study on the transferability
and reliability of the U-SENS™ test method
for skin sensitisation testing

ESAC Opinion No.
Relevant ESAC Request No.
Date of Opinion

2016-03
2016-03
24/06/2016

Table of contents
Abstract ............................................................................................................... 1
ESAC Opinion ........................................................................................................ 2
References ........................................................................................................... 4
Annex 1: Composition of ESAC and ESAC Working Group ........................................... 5
Annex 2: EURL ECVAM Request for ESAC Advice ....................................................... 7
Annex 3: ESAC Working Group Peer Review Consensus Report ..................................23

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 L'Oréalcoordinated study on the transferability and reliability of the U-SENS™ test method forskin sensitisation testing.

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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)

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 U-SENS™ test method for skin sensitisation testing (Annex 2). ESAC established aworking group (WG) (Annex 1) which delivered an ESAC WG report dated 6 June 2016
(Annex 3).
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:
The replacement of traditional animal-based test methods by alternative ones should
ideally be one-to-one replacements. However, the replacement of traditional animalbased test methods for skin sensitisation by animal-free approaches is likely to requirean Integrated Approach to Testing and Assessment (IATA). This combines reliable andmechanistically relevant test methods addressing the currently recognised key events inthe adverse outcome pathway for skin sensitisation induction (OECD, 2012; OECD,
2016a, b).
Currently, three animal-free test methods have been validated becoming OECD Test
Guidelines (TG). The DPRA (TG 442C) provides information about the capacity of thechemical to react with cysteine and/or lysine groups (key event 1). The KeratinoSens™
test method (TG 442D) addresses skin inflammation by assessing the impact of a
substance on ARE mediated Nrf2 activation (key event 2). The h-CLAT (TG 442E)
addresses key event 3 by measuring the upregulation of cell membrane markers CD86
and CD54 to predict a skin sensitisation potential.
The test method under evaluation (U-SENS™) addresses key event 3 by measuring CD86
upregulation as a surrogate measure for dendritic cell migration/maturation. This test
method shows similarities with the h-CLAT test method, which measures the impact of
substances on CD86 and CD54 expression levels. A comparison with the similar h-CLAT
method in terms of its benefit would be helpful to end users enabling them to make adecision regarding which method would be most appropriate for their purpose.
The study design was reasonable. The within- and between-laboratory reproducibility arecomparable, arguably better, than the validated methods listed above. However, a lower
reproducibility was observed with one of the naïve laboratories. This may highlight theimportance of proper training. Since proper training opportunities were provided by thetest method developer, a lower reproducibility may also indicate additional challenges fora naïve laboratory trying to apply the method. The predictive capacity is good as far as
the overall binary classification (Y/N) is concerned and the assay seems to have a goodsensitivity, perhaps at the expense of specificity. Overall, the test method's accuracy iscomparable with accepted in vitro methods. Where directly compared, performanceagainst human data and LLNA data was equivalent.
Notwithstanding the above, the prediction model was found to be complex and the
documentation was insufficiently clear to enable proper understanding of the conclusions
drawn by the test submitter from "inconclusive" results. In particular, the application of
the 'six rules' developed to solve inconclusive results was difficult to understand. Thismay jeopardise the application of this method by the end users. For these reasons, ESAC
has the concern that the 'six rules' used to resolve inconclusive outcomes were fitting thePage | 2

outliers in the absence of a reasonable biological and/or chemical rational. The presented
data suggest that the 'six rules' have limited accuracy (sensitivity: 90-100 %; specificity:
33 %) and add no value to simply considering all inconclusive results to be positive by
default. Indeed, considering all inconclusive results as sensitiser by default instead of
applying the 'six rules' would slightly decrease the number of false negatives with only anequally small increase of false positives.
The explanations given for false positive and false negative results were not always
consistent, as they appeared to be laboratory specific. Taking all the data together, abroad range of potency and chemical reactivity classes was covered. Analysis of thesedata revealed that outliers did not correspond with a particular potency or chemical
reactivity class. The data also showed that pre-haptens were correctly identified. Overall,
the presented evidence seems to indicate that volatile substances or poorly soluble
substances may require additional attention in the technology transfer and training
phase, as improper handling of such substances during exposure could affect the
reproducibility of the results rather than their accuracy.
ESAC concludes that in terms of test performance, the U-SENS™ test method appears to
be comparable to the currently available validated animal-free test methods in general,and to the h-CLAT method specifically. ESAC therefore considers that the U-SENS™ test
method is ready to be considered for regulatory use in the context of an IATA for skinsensitisation and remains a useful tool for screening and early decision making during
product development, within the applicability domain defined in the validation study.
However, ESAC has concerns with the complexity of the prediction model and theuncertainty of the applicability domain and the predictivity of non-sensitisers (specificityof 33 %) of the 'six rules' for resolving inconclusive results. Given that the 'six rules'
appear to change 'inconclusive' to 'sensitisers' in the majority of cases, the testdevelopers are recommended to consider removing these 'six rules' from the prediction
model and to consider any inconclusive result as positive by default. This would
significantly simplify the prediction model and would allow standardising the maximum
number of runs per experiment to three. Considering the validation dataset, modifying
the prediction model and the maximum number of runs as suggested above would havelittle impact on the predictive capacity of the U-SENS™ test method and could lead to adecrease in testing requirements of about 8 %.

Page | 3

ReferencesOECD (2012). The adverse outcome pathway for skin sensitisation initiated bycovalent binding to proteins. Part 1: Scientific evidence. Series on Testing andAssessment No. 168, OECD, Paris.

OECD (2016a). Guidance Document on the reporting of Defined Approaches to be
usedwithinIntegrated
Approaches
toTesting
andAssessment.
ENV/JM/MONO(2016)28. Series on Testing and Assessment No. 255, OECD, Paris.

OECD (2016b). Guidance Document on the reporting of Defined Approaches andindividual information sources to be used within Integrated Approaches to Testingand Assessment (IATA) for skin sensitisation. ENV/JM/MONO(2016)29. Series on
Testing and Assessment No. 256, OECD, Paris.

Page | 4

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 | 5

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. Erwin L. ROGGEN (ESAC Member, WG Chair)Prof. Annette KOPP-SCHNEIDER (ESAC Member)Dr. David BASKETTER (EURL ECVAM nomination)
Dr. Steve ENOCH (EURL ECVAM nomination)
Prof. Yong HEO (Catholic University of Daegu; ICATM nomination by KoCVAM)
Dr. Reiko ADACHI (NIHS; ICATM nomination by JaCVAM)EURL ECVAM (Secretariat)

Dr. João BARROSO (ESAC Coordinator)
Dr. Silvia CASATI
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 | 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)ESAC Request 2016-03
EURL ECVAM Scientific Advisory Committee(ESAC)EURL ECVAM REQUEST FOR ESAC ADVICEon theL'Oréal-coordinated study on the transferability and reliability of the U-SENS™
test method for skin sensitisation testing

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 ADVICEU-SENS™ validation
2016-03
EP 3.02
23/03/2016
01/04/2016
Written procedure previous to ESAC 42
ESAC 42 (June 2016)
ER2016-03_ESAC_REQUEST_U-SENS.doc

Page | 8

TABLE OF CONTENTS
1.

TYPE OF REQUEST ........................................................................................................................................10

2.

TITLE OF STUDY OR PROJECT FOR WHICH SCIENTIFIC ADVICE OF THE ESAC IS REQUESTED.....................11

3.

BRIEF DESCRIPTION OF THE STUDY OR PROJECT ........................................................................................11

4.

OBJECTIVES, QUESTIONS, TIMELINES ..........................................................................................................14
4.1
4.2
4.3

5.

OBJECTIVE ..........................................................................................................................................14
QUESTION(S) TO BE ADDRESSED .......................................................................................................14
TIMELINES ..........................................................................................................................................15

EURL ECVAM PROPOSALS ON HOW TO ADDRESS THE REQUEST WITHIN ESAC ........................................16
5.1
5.2

EURL ECVAM PROPOSAL REGARDING REQUEST-RELATED STRUCTURES REQUIRED .......................16
DELIVERABLES AS PROPOSED BY EURL ECVAM .................................................................................17

6.

LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESAC ....................................................................18

7.

TERMS OF REFERENCE OF THE ESAC WORKING GROUP.............................................................................20
7.1
ESTABLISHMENT OF THE ESAC WORKING GROUP ............................................................................20
7.2
TITLE OF THE ESAC WORKING GROUP ...............................................................................................20
7.3
MANDATE OF THE ESAC WORKING GROUP.......................................................................................20
7.4
DELIVERABLES OF THE ESAC WORKING GROUP ................................................................................20
7.5
PROPOSED TIMELINES OF THE ESAC WORKING GROUP ...................................................................20
7.6
QUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WORKING GROUP ................................21
APPENDIX 1REPORTING TEMPLATE ........................................................................................................21

EURL ECVAM REQUEST FOR ESAC ADVICE

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1.

TYPE OF REQUEST

Request Type

Identify request ("YES")

R1 ESAC Peer Review

YES, external validation study (i.e. not coordinatedby EURL ECVAM)

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

The U-SENS™ assay for skin sensitization testing(formerly known as Myeloid U937 Skin Sensitisation
Test –MUSST) has been evaluated in a two-phasestudy coordinated by l'Oréal. The study has been
designed to generate information on the test
method's transferability and reproducibility to allow
recommendations to be made on these two aspects ofthe method. In addition, data generated with thevalidation study's chemicals and a larger set of
l'Oréal's historical data has been used to estimate thepredictive capacity of the method compared toresponses in the LLNA and in humans. Data generated
with the U-SENS™ will be used in defined approaches
to testing and assessment or in the context of a WoE
assessment (i.e. within Integrated Approaches to
Testing and Assessment – IATA) for determining theskin sensitization potential of chemicals.
NO►Retrospective Validation Study►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 | 10

2.

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

L'Oréal validation study on the U-SENS™ assay for skin sensitisation testing3.

BRIEF DESCRIPTION OF THE STUDY OR PROJECT

1) Background to skin sensitization and current regulatory adopted tests
Skin sensitisation is the toxicological endpoint associated with substances that have the intrinsic
ability to cause allergic contact dermatitis (ACD) in humans.
The identification of the skin sensitisation potential represents an important component of the safetyassessment of new and existing chemicals including cosmetic ingredients. Traditional regulatory
predictive tests for skin sensitisation rely on the use of animals. These include: the guinea-pig tests(Buehler Test and Guinea-pig Maximisation Test) (OECD TG 406), the murine Local Lymph Node Assay(LLNA, OECD TG 429) and its non-radio-isotopic variants (OECD TG 422a and 422b).
In recent years progress has been made in the development and regulatory adoption of alternative
methods for skin sensitization hazard identification. Two methods validated and/or peer-reviewed by
EURL ECVAM, the direct peptide reactivity assay (DPRA) and the KeratinoSens™ have been adopted
by the OECD as Test Guidelines 442C and 442D respectively. A third method, the human Cell Line
Activation Test is in the final stages of the OECD adoption process at the time of writing of thisrequest.
Methods developed in the area are addressing key chemical/biological mechanisms leading to theacquisition of skin sensitization. Such mechanisms have been described in the form of an Adverse
Outcome Pathway (AOP; OECD 2010a, 2010b). Within the skin sensitisation AOP the molecular
initiating event (i.e. the first key event - KE) is the covalent binding of electrophilic substances tonucleophilic centres in skin proteins. The second key event takes place in the keratinocytes andincludes inflammatory responses as well as changes in gene expression associated with specific cellsignalling pathways such as the antioxidant/electrophile response element (ARE)-dependent
pathways. The third key event is the activation of dendritic cells (DC), typically assessed by expressionof specific cell surface markers, chemokines and cytokines. The fourth key event is T-cell proliferation,
which is indirectly assessed in the murine Local Lymph Node Assay (LLNA).
The U-SENS™ is proposed to address KE3 of the AOP. As for the other skin sensitization test methods
evaluated by EURL ECVAM and adopted by the OECD, also the U-SENS™ does not have the potential
to function as a full-replacement stand-alone method. Instead, it is proposed that a combination of inin silico, in chemico and in vitro tests, addressing the key biological events of skin sensitisation, will beneeded to achieve this goal. A defined approach to testing and assessment combining U-SENS datawith other information has been developed.
2) The U-SENS™ test method
The U-SENS™ addresses the role that Langerhans cell (LC) and dermal dendritic cells (DC) play in the
induction of skin sensitization. These cells are important mediators in the skin sensitization processsince they are capable of presenting the hapten-protein conjugate to responsive T lymphocytes in the
EURL ECVAM REQUEST FOR ESAC ADVICE

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lymph nodes draining the site of exposure (Kimber and Cumberbatch, 1992). The maturation processof LC and DC from antigen processing cells to antigen presenting cells is considered a key event in theacquisition of skin sensitisation. This maturation process involves the modulation of the expression ofcell surface phenotypic markers, those most commonly reported being CD54, CD80, CD86 and major
histocompatibility complex (MHC) class II (Galvao dos Santos et al., 2009). This knowledge has been
exploited in the development of in vitro tests based on the use of human monocytic immortalized
cell-lines to screen the skin sensitization potential of chemicals.
The U-SENS™ measures the modulation of the CD86 protein markers on the surface of U-937 cells(human monocyte cell line) by flow cytometric analysis, following 45 hours of exposure to at least 4
concentrations of a test chemical. The concentrations used in the test are selected among
concentrations predefined in the SOP. The test method is designed to discriminate between
sensitising and non-sensitising chemicals whereby chemicals are classified as sensitisers if the CD86IgG1 percent of positive cells exceeds a defined threshold (i.e. Stimulation Index ≥150) compared to
the vehicle control at least to consecutive tested concentration, in at least two independentmeasurements (i.e. run repetitions). Cell viability is measured concurrently by Propidium Iodide (PI)staining and CD86 values are considered for the prediction only if cell viability is above 70%.
3) History of development of the test method and background to the U-SENS™ validation study
The U-SENS ™ (former MUSST) protocol and prediction model were developed within L'Oréal'slaboratories using about 100 reference chemicals with animal and/or human clinical data. The
protocol and the prediction model were then assessed within the Colipa's (now Cosmetics Europe)
Skin Tolerance Project Team ring study with a small set of chemicals (N=10). In 2009 the test method
entered the EURL ECVAM-coordinated validation study together with the DPRA and the h-CLAT. At
the time of the EURL ECVAM validation study, the U-SENS was transferred to three naïve laboratories
but was withdrawn at the end of the first phase of the study because of reproducibility issues derived
by the fact that the prediction model allowed three final calls to be made, Sensitiser, Non-sensitiser
and Inconclusive. The prediction model was then optimized within L’Oréal by the addition of a scorederived from six new rules allowing only 2 final calls, Sensitiser vs Non Sensitiser to be made for thechemicals that were considered Inconclusive by using the original prediction model only.
Between 2013 and 2014 the U-SENS™ underwent a two-phase evaluation study coordinated byl'Oréal and both submitted to EURL ECVAM.
The first L'Oréal-coordinated study (study 2013) involved the participation of three laboratories (inaddition to L’Oréal in-house facility), two of which (i.e. Bioassay and WIL Research (former Ricerca))
had been involved in the evaluation of the method at the time of the EURL ECVAM-coordinatedstudy. Thus the method was newly transferred only to one of the laboratories (CiToxLab), although allof them received training on the new rules of the prediction model and on the software developed
for the automated application of the prediction model.
The within-laboratory reproducibility (WLR) was evaluated in L’Oréal laboratory only by testing 21
chemicals (14 coded + 7 not coded) in two independent experiments. The between-laboratory
reproducibility (BLR) was assessed on the basis of 14 coded chemicals tested once in each laboratory.
Following the evaluation by EURL ECVAM of the information submitted, l'Orèal was advised togenerate additional information in order to progress the method into peer review.
A second study was coordinated by l'Oréal in 2014. In addition to l'Oréal's in-house facility the 2014
study involved the participation of the three laboratories, mentioned above, that took part also in the2013 study. The 2014 study experimental designed foresaw the testing of 24 coded test chemicals forthe evaluation of the between-laboratory reproducibility and the testing of a subset of these
EURL ECVAM REQUEST FOR ESAC ADVICE

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chemicals (15) for the evaluation of the within-laboratory reproducibility.
The predictive capacity of the U-SENS™ method was firstly evaluated on the basis of the results
obtained in the two studies of 2013 and 2014 for a total of 38 chemicals. It was further assessed usinga larger number of chemicals (175) covering the entire range of skin sensitisation potency based onhuman and LLNA data.
Conclusions of the Test Submitter on the information submitted to EURL ECVAM
According to l'Oréal the information submitted to EURL ECVAM for entering the peer-review process
indicates the potential of the U-SENS™ test method to contribute to the discrimination between
sensitisers and non-sensitisers as defined by the Globally Harmonised System (GHS) of classificationand labelling of substances (category 1; no category) (UN, 2011) and as implemented in the European
Commission Regulation on classification, labelling and packaging (CLP) of substances and mixtures(EC, 2008), although the use of U-SENS™ as a standalone method for this purpose will not berecommended as stated as well for all preceded validated test methods addressing the skinsensitization endpoint. The U-SENS™ is foreseen to be combined with complementary informationand evaluated in the context of Integrated Approaches to Testing and Assessment (IATA). In suchcontext, the U-SENS™ test method is part of a decision strategy for skin sensitization hazard
identification based on in silico, in chemico, and in vitro data. The decision strategy has been alreadysubmitted to the OECD as a case study in the context of the draft guidance document on thereporting of defined approaches to testing and assessment.
The U-SENS™ test method is also foreseen to be a part of an integrated test battery for risk and safetyassessment which will be able to fully replace the in vivo test methods In such context, the U-SENS™
test method is part of The European Cosmetics Industry Trade Association current (Cosmetics Europe)
program.
References
1. Anon (2003) Globally Harmonised System of Classification and Labelling of Chemicals (GHS).
Part 3: Health and Environmental Hazards, pp. 151-158. New York, NY, USA, and Geneva,
Switzerland: United Nations Organisation.
2. EC (2008) Regulation (EC) No 1272/2008 (16 December 2008) of the European Parliamentand of the Council on classification, labelling and packaging of substances and mixtures,
amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation(EC) No 1907/2006. Official Journal of the European Union L 353, (31/12/2008) p. 1-1355.
3. Galvao dos Santos G, Reinders J, Ouwehand K, Rustemeyer T, Schepper RJ & Gibbs S (2009)
Progress on the development of human in vitro dendritic cell based assays for assessment of
the sensitizing potential of a compound. Toxicology and Applied Toxicology 236(3); 372-382.
4. Kimber I & Cumberbatch M (1992) Dendritic cells and cutaneous immune responses tochemical allergens. Toxicology and Applied Pharmacology 117; 137-46.
5.

OECD 2012a. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding toProteins. Part 1: Scientific Evidence. Series on Testing and Assessment No.168.
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2012)10/par
t1&doclanguage=en

6.

OECD 2012b. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding toProteins. Part 2: Use of the AOP to Develop Chemical Categories and Integrated Assessment andTesting
Approaches.
Serieson
Testing
andAssessment
No.
168
http://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-molecular-screening-andtoxicogenomics.htm

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?

4.2

The opinion of ESAC should support EURL ECVAM with respect to the developmentof an EURL ECVAM recommendation on the U-SENS™ assay outlining (1) the
scientific basis of the assay, (2) its overall performance as assessed during thestudy and based on other (e.g. published) information, (3) its applicability andlimitations, 4) proposed use of the method 5) additional work to be undertaken in
future to further characterise the test method.

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

1) DESIGN & CONDUCT OF STUDY: The ESAC is requested to review whether the
study was conducted appropriately in view of the objective of the study. The study
objective was to assess(1) the reproducibility of the method within one laboratory (WLR)(2) its transferability to other laboratories(3) its reproducibility between laboratories (BLR)(4) An indication of the predictive capacity of the test method for distinguishing
between sensitisers and non-sensitisers and, where possible, to appraise its
potential to contribute to a further sub-categorisation of sensitisers into two
subcategories (1A and 1B).
When reviewing the design and conduct of the study, the following issues should
be addressed in particular:(a) Clarity of the test definition (module 1)(b) Clarity of the definition of the study objective and study management(c) Appropriateness of the study design & execution in view of the study
objectives, inter alia:- Is the number of tested chemicals sufficient for the purposes of thestudy?
- Are the reference data used for assessing in particular the predictive
capacity appropriate and of good quality?
- Was the identification of chemicals conducted in an appropriatemanner (i.e. presence or absence of selection criteria, justification etc.)?
- Is the adverse effect range of the selected chemicals appropriate for the
purpose of the study- In case of gaps (chemical class etc.) – are these justified?
- Is the number of laboratories sufficient?
(d)Appropriateness of the study execution (e.g. was the number ofrepetitions sufficient? How were exceptions / deviations handled? Wereprovisions specified for retesting? etc.)

EURL ECVAM REQUEST FOR ESAC ADVICE

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(e)Appropriateness of the statistical analysis used for analysing WLR,
transferability, BLR and for providing an indication of the predictive capacity.
2) CONCLUSIONS OF STUDY: The ESAC is requested to assess whether theconclusions, as presented in the Validation Study Report, are substantiated by the
information generated in the study and are plausible with respect to existing
information and current views (e.g. literature).
In particular:(a)
Are the conclusions on reproducibility (WLR and BLR) as well astransferability justified and plausible?
(b)
Are the conclusions on the predictive capacity justified and plausible withrespect to existing information(c)
Are there possible gaps between study design and study conclusions which
remain to be addressed in view of the suggested conclusions / use (see also point
3)?
3) APPLICABILITY AND LIMITATIONS OF THE TEST METHOD: The ESAC is requested
to evaluate, whether the information provided in the submission, provides
sufficient information on the applicability and possible limitations of the test
method.
4) PROPOSED USE OF THE TEST METHOD: The ESAC is requested to evaluate,
whether the information provided in the submission are sufficient to substantiate
the proposed use of the method
5) FUTURE ACTIVITIES ON THE METHOD: The ESAC is requested to recommend
what additional work, if necessary, should be undertaken in future to further
characterise the test method and its proposed use.

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

Page | 15

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- Erwin Roggen (Chair)- Annette Kopp-Schneider
EURL ECVAM nominations- David Basketter (DABMEB Consultancy)- Steven Enoch (Liverpool John Moores
University)
ICATM nominations- Joanna Matheson (CPSC; nominated by
ICCVAM)- Yong Heo (Catholic University of Daegu;
nominated by KoCVAM)- Reiko Adachi (NIHS; nominated by
JaCVAM)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):

EURL ECVAM REQUEST FOR ESAC ADVICE

Page | 16

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

Page | 17

6.

LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESACCount

Description of document

Already
available?
(YES/NO)

File name1

Test Submission on the U-SENS™

YESTST U-SENS revision_11 11 2015.pdf

2

EURL ECVAM assessment reporton the U-SENS™ and l'Oréal
responses addressed also in the
Test Submission

YES

TM2013_02_U-SENS_assessment report
responses.pdf

3

Protocol(s) of the test method

YES

Attachment 1a_MUSST SOP.pdf
Attachment 1c_6 rules examples.pdf
Attachment 1d_SOP MUSST
automation.pdf
Attachment 1b_DB-ALM U-SENS.pdf

4

Test items used to assess WLR

YES

Attachment 3_WLR test items.pdf

5

Data used for WLR assessment

YES

Attachment 4_WLR assessment.pdf

6

Test items used for transferability

YES

Attachment 5_TF test items.pdf

YES

Attachment 6 & 7_ Training and Transfer
protocol.pdf

YES

Attachment 8a _ Transfer report
Bioassay.pdf

78

Training and transfer protocols

Transfer reports
Ricerca, CiToxlab

toBioassay,

Attachment 8b _ Transfer report
Ricerca .pdf
Attachment 8c_Transfer report
CiToxLab.pdf
910
11

Test items used to assess BLR
Data used for BLR assessment(Tables and/or Figures)
List of test items used to assess PC

EURL ECVAM REQUEST FOR ESAC ADVICEYES

Attachment 9_BLR test items.pdf

YES

Attachment 10_BLR assessment.pdf

YES

Attachment 11_PC test items v2.pdf

Page | 18

12

Data used for PC assessment

YES

Attachment 12a_PC assessment.pdf
Attachment 12b_PC 175 test items v2.pdf
Attachment 12c_PC assessment 175 test
items.pdf
Attachment 12d_PC assessment protein
binding classes.pdf

13
14

15

16

Project plan
List of test items used in thevalidation study
Coding and distribution of chemicals

Statistical reports

YES

Attachment 13_Project plan.pdf

YES

Attachment 14_Validation study tests
items.pdf

YES

Attachment 18a_2014 Study productcoordination report.pdf

YES

Attachment 18b_2014 Statisticalreport.pdf
Attachment 18m_2014 Amendment 1 tostatistical report.pdf
Attachment 18n_2015 Amendment 2 tostatistical report.pdf

17

Results from individual laboratories

YES

Attachment 18c_Bioassay 2014 results.pdf
Attachment 18d_CiToxLAB 2014
results.pdf
Attachment 18e_L'Oreal 2014 results.pdf
Attachment 18f_WIL Research 2014
results.pdf
Attachment 18g_L'OrÃ©al manual results2013.pdf
Attachment 18h_L'OrÃ©al auto results2013.pdf

18

Test presubmission form to EURL
ECVAM

YES

Attachment 18i_ TM203-02.pdf

19

IATA case study reported in the draft
OECD GD

YES

Attachment 18j_ OECD IATA submittedcase study v2.pdf

YES

Attachment 18k_ Piroird et al 2015.pdf

20

Relevant publications

Attachment 18l_AlÚpÚe et al 2015.pdf

EURL ECVAM REQUEST FOR ESAC ADVICE

Page | 19

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 2nd of March 2016 on the composition ofa new ESAC Working Group for the review of test methods in the area of skin sensitisation.

7.2

TITLE OF THE ESAC WORKING GROUP

Full title:
ESAC Working Group on Skin Sensitisation Test Methods
Abbreviated title:
ESAC WG Sensitisation

7.3

MANDATE OF THE ESAC WORKING GROUP

The EWG is requested to conduct a scientific review of the L'Oréal-coordinated validation study
concerning the U-SENS. The review needs to address the questions put forward to ESAC by EURLECVAM.
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
reporting template has been appended (Appendix 1) intended to facilitate the drafting of the 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
17-19 May 2016

ActionWorking group meeting2

27 May 2016

Circulation of final WG report and draft
ESAC opinion to ESAC

3

9-10 June 2016

Endorsement of WG report and ESAC
opinion at ESAC42 meeting

EURL ECVAM REQUEST FOR ESAC ADVICE

Deliverable
Draft ESAC WG reportand draft ESAC opinion
Final draft ESAC WG
report and draft ESAC
opinionFinal ESAC WG reportand ESAC opinion

Page | 20

7.6

QUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WORKING GROUP

The ESAC WG is requested to address the questions posed to the ESAC which have been broken
down further in more specific questions by the ESAC chair, the chair of the ESAC WG and theSecretariat (see section 4.2).
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 | 21

EURL ECVAM REQUEST FOR ESAC ADVICE

Page | 22

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 | 23

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 theL'Oréal-coordinated study on the transferability
and reliability of the U-SENS™ test method
for skin sensitisation testing
Title page information
File name

ESAC_WG_Report_U-SENS.doc

Abbreviated title of ESAC
requestU-SENS™ validation

Relating to ESAC REQUEST No.

2016-03

Request discussed throughWritten procedure previous to ESAC 42

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

Erwin L. Roggen

Version tracking
DateVersionAuthor(s)Description

19 May 2016

V1.0

ESAC WG

First draft agreed by the ESAC WG

27 May 2016

V2.0

ESAC WG

Second revised draft after commenting

01 June 2016

V3.0

ESAC WG

Third revised draft after commenting

06 June 2016

V4.0

ESAC WG

Final ESAC WG draft sent to ESAC for
endorsement

ESAC WORKING GROUP REPORTPage | 24

Table of Contents
ESAC WORKING GROUP ........................................................................................................................................27
ABBREVIATIONS USED IN THE DOCUMENT ..........................................................................................................28
1. STUDY OBJECTIVE AND DESIGN ........................................................................................................................29
1.1 ANALYSIS OF THE CLARITY OF THE STUDY OBJECTIVE'S DEFINITION .............................................................................29(a) ESAC WG summary of the study objective as outlined in the Test Submission ....................................29(b) Appraisal of the clarity of study objective as outlined in the Test Submission ....................................29
1.2 QUALITY OF THE BACKGROUND PROVIDED CONCERNING THE PURPOSE OF THE TEST METHOD ........................................29(a) Analysis of the scientific rationale provided in the Test Submission ....................................................29(b) Analysis of the regulatory rationale provided in the Test Submission .................................................29
1.3 APPRAISAL OF THE APPROPRIATENESS OF THE STUDY DESIGN ...................................................................................30
1.4 APPROPRIATENESS OF THE STATISTICAL EVALUATION ..............................................................................................30
2. COLLECTION OF EXISTING DATA .......................................................................................................................31
2.1 EXISTING DATA USED AS REFERENCE DATA ............................................................................................................31
2.2 EXISTING DATA USED AS TESTING DATA ................................................................................................................31
2.3 SEARCH STRATEGY FOR RETRIEVING EXISTING DATA ................................................................................................31
2.4 SELECTION CRITERIA APPLIED TO EXISTING DATA ....................................................................................................31
3. QUALITY ASPECTS RELATING TO DATA GENERATED DURING THE STUDY ......................................................31
3.1 QUALITY ASSURANCE SYSTEMS USED WHEN GENERATING THE DATA ..........................................................................31
3.2 QUALITY CHECK OF THE GENERATED DATA PRIOR TO ANALYSIS .................................................................................32
4. QUALITY OF DATA USED FOR THE PURPOSE OF THE STUDY (EXISTING AND NEWLY GENERATED) ...............32
4.1 OVERALL QUALITY OF THE EVALUATED TESTING DATA (NEWLY GENERATED OR EXISTING) ..............................................32
4.2 QUALITY OF THE REFERENCE DATA FOR EVALUATING RELEVANCE...............................................................................32
4.3 SUFFICIENCY OF THE EVALUATED DATA IN VIEW OF THE STUDY OBJECTIVE ...................................................................32
5. TEST DEFINITION (MODULE 1) ..........................................................................................................................32
6. TEST MATERIALS ................................................................................................................................................33
6.1 SUFFICIENCY OF THE NUMBER OF EVALUATED TEST ITEMS IN VIEW OF THE STUDY OBJECTIVE ..........................................33
6.2 REPRESENTATIVENESS OF THE TEST ITEMS WITH RESPECT TO APPLICABILITY.................................................................33
7. WITHIN-LABORATORY REPRODUCIBILITY (WLR) (MODULE 2) ........................................................................34
7.1 ASSESSMENT OF REPEATABILITY AND REPRODUCIBILITY IN THE SAME LABORATORY ......................................................34
7.2 CONCLUSION ON WITHIN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY ..................................................34
8. TRANSFERABILITY (MODULE 3) .........................................................................................................................34
8.1 QUALITY OF DESIGN AND ANALYSIS OF THE TRANSFER PHASE ...................................................................................34
8.2 CONCLUSION ON TRANSFERABILITY TO A NAÏVE LABORATORY / NAÏVE LABORATORIES AS ASSESSED BY THE STUDY .............35
9. BETWEEN-LABORATORY REPRODUCIBILITY (BLR) (MODULE 4) ......................................................................35
9.1 ASSESSMENT OF REPRODUCIBILITY IN DIFFERENT LABORATORIES...............................................................................35
9.2 CONCLUSION ON BETWEEN-LABORATORY REPRODUCIBILITY AS ASSESSED BY THE STUDY ...............................................35
10. PREDICTIVE CAPACITY AND OVERALL RELEVANCE (MODULE 5) ....................................................................35
10.1 ADEQUACY OF THE ASSESSMENT OF THE PREDICTIVE CAPACITY IN VIEW OF THE PURPOSE ............................................35
10.2 OVERALL RELEVANCE (BIOLOGICAL RELEVANCE AND ACCURACY) OF THE TEST METHOD IN VIEW OF THE PURPOSE ............37

ESAC WORKING GROUP REPORTPage | 25

11. APPLICABILITY DOMAIN (MODULE 6) .............................................................................................................37
11.1 APPROPRIATENESS OF STUDY DESIGN TO CONCLUDE ON APPLICABILITY DOMAIN, LIMITATIONS AND EXCLUSIONS ............37
11.2 QUALITY OF THE DESCRIPTION OF APPLICABILITY DOMAIN, LIMITATIONS, EXCLUSIONS ................................................38
12. PERFORMANCE STANDARDS (MODULE 7) .....................................................................................................38
12.1 ADEQUACY OF THE PROPOSED ESSENTIAL TEST METHOD COMPONENTS ..................................................................38
12.2 ADEQUACY OF PROPOSED REFERENCE CHEMICALS ...............................................................................................38
12.3 ADEQUACY OF PROPOSED PERFORMANCE TARGET VALUES.....................................................................................38
13. READINESS FOR STANDARDISED USE .............................................................................................................38
13.1 ASSESSMENT OF THE READINESS FOR REGULATORY PURPOSES ................................................................................38
13.2 ASSESSMENT OF THE READINESS FOR OTHER USES ................................................................................................39
13.3 CRITICAL ASPECTS IMPACTING ON STANDARDISED USE ..........................................................................................39
13.4 GAP ANALYSIS ...............................................................................................................................................39
14. OTHER CONSIDERATIONS................................................................................................................................39
15. CONCLUSIONS ON THE STUDY ........................................................................................................................39
15.1 ESAC WG SUMMARY OF THE RESULTS AND CONCLUSIONS OF THE STUDY ................................................................39
15.2 EXTENT TO WHICH STUDY CONCLUSIONS ARE JUSTIFIED BY THE STUDY RESULTS ALONE................................................39
15.3 EXTENT TO WHICH CONCLUSIONS ARE PLAUSIBLE IN THE CONTEXT OF EXISTING INFORMATION .....................................40
16. RECOMMENDATIONS ......................................................................................................................................40
16.1 GENERAL RECOMMENDATIONS .........................................................................................................................40
16.2 SPECIFIC RECOMMENDATIONS (E.G. CONCERNING IMPROVEMENT OF SOPS) ............................................................40
17. REFERENCES .....................................................................................................................................................41

ESAC WORKING GROUP REPORTPage | 26

ESAC Working Group
This report was prepared by the "ESAC Working Group for Skin Sensitisation" (ESAC WG SS), charged
with conducting a detailed scientific peer review of on the L'Oréal-coordinated study on thetransferability and reliability of the U-SENS™ assay for skin sensitisation testing.
The ESAC WG had the following members:
ESAC members
 Erwin Roggen (Chair) Annette Kopp-Schneider
EURL ECVAM nominations
 David Basketter (DABMEB Consultancy)
 Steven Enoch (Liverpool John Moores University)
ICATM nominations
 Yong Heo (Catholic University of Daegu; ICATM nomination by KoCVAM) Reiko Adachi (NIHS; ICATM nomination by JaCVAM)ESAC Coordination:João Barroso (ESAC Coordinator)
Silvia Casati

ESAC WORKING GROUP REPORTPage | 27

Abbreviations used in the document


BLR

Between-laboratory reproducibility



EURL ECVAM

European Union Reference Laboratory for Alternatives to Animal
Testing

ESAC

EURL ECVAM Scientific Advisory Committee



ESAC WG

ESAC Working GroupGLP

Good Laboratory Practice



IATA

Integrated Approach to Testing and Assessment



INC

Inconclusive

SOP

Standard Operating Procedure (used here as equivalent to 'protocol')

VMT

Validation Management Team

WLR

Within-laboratory reproducibility

ESAC WORKING GROUP REPORTPage | 28

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
From the project plan:
Formal validation of the U-SENS™ (improved MUSST) test, focusing on its transferability andreproducibility. This validation includes an earlier ‘ring trial’ evaluation of the transferability andreproducibility of the test.
The above is formulated as a study goal (goal 1), as is a preliminary assessment of the accuracy of themethod in discriminating sensitisers and non-sensitisers (according to UN GHS for classification andlabelling of chemicals for skin sensitisation (cat. 1; no cat.) (UN, 2015) and ECR No. 1272/2008 on
classification, labelling and packing of substances and mixtures (EC, 2008)) (goal 2).
(b) Appraisal of the clarity of study objective as outlined in the Test Submission
The study objective (and goals 1 and 2) is sufficiently clear to allow persons skilled in the process ofvalidation of cell-based assays to understand.

1.2 Quality of the background provided concerning the purpose of the test method
The U-SENS™ test method is considered to be a potentially important component of the safety
evaluation of chemicals. Skin sensitisation assessment represents a standard requirement ofchemical legislation (UN GHS for classification and labelling of chemicals for skin sensitisation (cat. 1;
no cat.) (UN, 2015) and ECR No. 1272/2008 on classification, labelling and packing of substances andmixtures (EC, 2008)).
The U-SENS™ test method is foreseen to be part of a battery or Integrated Approach to Testing andAssessment (IATA) for replacement of the existing in vivo assays for hazard identification.
(a) Analysis of the scientific rationale provided in the Test Submission
The scientific background of the U-SENS™ is identical to the background of the h-CLAT, which
recently became an OECD test guideline (OECD, 2016). Several membrane markers, including CD86,
are generally accepted to reflect dendritic cell (DC) migration/maturation. DC maturation results inmigration of the DC to the lymphoid tissue and an increasing capacity of the DC to present antigen to
T-cells. This sequence of events is known to result in an immune response. Sufficient reference to therelevant body of scientific literature was made.
It is well known that also skin sensitisers induce DC migration/maturation, and thus CD86
upregulation, leading to an (adverse) immune response. This sequence of events was outlined by thetest submitters in a concise but precise way.
The U-SENS™ was compared with the very similar h-CLAT (measuring CD86 and CD54). The added
value of the U-SENS™ (as compared to the h-CLAT) was not provided.
The provided scientific background correctly profiles the U-SENS™ as a test method covering key
event 3 of the OECD Adverse Outcome Pathway for Skin Sensitisation (OECD, 2012).
(b) Analysis of the regulatory rationale provided in the Test Submission
The provided regulatory rationale builds largely on the Cosmetics Regulation, and the emergence andapplication of OECD test guidelines in the context of REACH.
ESAC WORKING GROUP REPORTPage | 29

Skin sensitisation assessment represents a standard requirement of chemical legislation (UN GHS forclassification and labelling of chemicals for skin sensitisation (cat. 1; no cat.) (UN, 2015) and ECR No.
1272/2008 on classification, labelling and packing of substances and mixtures (EC, 2008)).

1.3 Appraisal of the appropriateness of the study design
The validation of the U-SENS™ comprises 2 independent studies.
1. A ring trial conducted in 2013 for a preliminary assessment of the reliability of the test (2013)(14 substances);
2. A formal validation study following EURL ECVAM recommendations (2014) (24 substances).
The results of the 2013 ring trial were submitted to EURL ECVAM. EURL ECVAM recognised that the
study was performed properly, but with an insufficient number of substances, leading to a decision
to initiate the 2014 validation study.
All the measures required for proper formal validation (2014 study) of a test method were taken:
independent Study Product Coordinator and Study Data Coordinator; substances were blinded andstatistical analysis of the reproducibility was conducted blind.
Four laboratories were involved in the study, instead of the required minimum of three. The
laboratories were involved in both the 2013 and 2014 studies.
All laboratories tested the 24 substances once. Fifteen of them were tested in three independent
experiments by all laboratories with three differently coded samples provided. A rationale for thenumber of test substances (other than adaption of the EURL ECVAM study design) was not provided
in the study plan.
Three laboratories worked under GLP compliance. Also for the non/GLP laboratory, measures forassuring Quality were defined (pp. 16/17 of the TST 112015). In case of non/GLP, these measures
were specified.

1.4 Appropriateness of the statistical evaluation
The Prediction Model is complex. First, two runs are performed. If they lead to concordantclassification as S or NS, no more runs are performed. In case of discordance, a third run isperformed. If this run leads to a classification as S or NS, the classification is based on the majority
vote. U-SENS™ may also lead to inconclusive (INC) results. Concordant INC results after two, or, if it
was required, three runs, trigger a decision requiring the application of a system of six decision ruleson the averaged dose-response data from all runs. The prediction model is shown in a decision tree.
However, when applied on the data shown in Annex A of attachment 18n (second amendment to thestatistical report) of the submission to EURL ECVAM, the ESAC WG observed a number of apparent
deviations from the decision tree. It was suggested that a more detailed flow chart was incorporated
into the SOP.
Analysis of the performance of this complex prediction model using the Bootstrap resampling
approach, although ideal, would be challenging and very time consuming. Consequently, the second
amendment to the statistical report (attachment 18n of the submission to EURL ECVAM) shows amodel-based Monte-Carlo simulation for the evaluation of BLR, WLR and predictive capacity. The
proposed method seems to rely on the evaluation of single runs for which INC results are mapped to
S or NS based on the system of six decision rules for the individual run. Each experiment was thensimulated to consist of 2 or 3 runs after which a majority voting rule is applied. Hence the assessment

ESAC WORKING GROUP REPORTPage | 30

of BLR, WLR and predictive capacity offered in the second amendment to the statistical report does
not match the prediction model shown in the study report.

2. Collection of existing data
2.1 Existing data used as reference data
The chemicals selected for the formal validation were selected from the list of reference chemicals(animal and/or human clinical data) of L’Oréal’s laboratories. Both liquids and solids were evaluated.
In order to analyse the predictivity of the U-SENS™ test method a set of 175 substances has been
evaluated by L’Oréal. The human data was the main criterion considered for the prediction.
According to human, LLNA and EU-CLP classifications, all potency classes, from extreme sensitiser tonon-sensitiser were represented.

2.2 Existing data used as testing data
See section 2.1.

2.3 Search strategy for retrieving existing data
Selection criteria for substances to be used for transferability and reproducibility assessment were:-

Availability of reliable in vivo LLNA reference data: sensitisers (S) and non-sensitisers (NS),-Commercial availability,-

Diversity in terms of chemical properties with different reactivity toward proteins (asdescribed by Aptula and Roberts (2006) and determined with the Toxtree software) and withingredients used in Cosmetics and non-cosmetic chemicals

Selection criteria for substances to be used for predictive capacity evaluation were:-

Availability of robust in vivo data to allow a proper comparative evaluation of in vitro results.
As such, availability of human (Basketter et al., 2014) and/or LLNA (OECD, 2010) in vivo skinsensitisation classification data was considered.

2.4 Selection criteria applied to existing data
Not specified, beyond the availability of animal and/or human clinical data.

3. Quality aspects relating to data generated during the study
3.1 Quality assurance systems used when generating the data
Three laboratories worked under GLP compliance. Also for the non/GLP laboratory, measures forassuring Quality were defined (e.g. pp. 16/17 of the Study report 112015). In case of non/GLP, thesemeasures were specified.
Good cell culture practice was not mentioned.

ESAC WORKING GROUP REPORTPage | 31

3.2 Quality check of the generated data prior to analysis
No additional measures were taken beyond the quality assurance mentioned in 3.1.

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)Overall, application of the quality criteria to the test system seems to assure good quality data, but
no independent data analysis before transfer to the biostatistician seems to exist.
However, the unexpected increase in non-qualified tests during the formal validation study at two of
the participating laboratories does raise concerns.

4.2 Quality of the reference data for evaluating relevance1
The quality of the reference data was sufficient for the purpose of the study.

4.3 Sufficiency of the evaluated data in view of the study objective
The overall quality of the data is sufficient to draw conclusions on the transferability andreproducibility (within as well as between laboratories).

5. Test definition (Module 1)
The test method is adequately described, and covers the parameters that are relevant for good test
performance.
1. The test description specifies the cell line used for the test and addresses the IPR issues
related to commercial use of this cell line.
2. The relevance of the endpoints measured is explained on the background of the available
scientific knowledge.
3. Quality and acceptance criteria for untreated cells, as well as positive and negative controls,
are described.
4. Acceptance criteria for the results are outlined. It is not clear though why there is no referral
to the quality and acceptance criteria for unexposed viability and CD86 levels nor the positive
and negative control values.
5. The prediction model was not found to be described in sufficient detail to repeat theconclusions made by the VMT as presented in Annex A of attachment 18n (second
amendment to the statistical report) of the submission to EURL ECVAM.

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 | 32

6. The ESAC WG has concerns about the 'six rules', which were not alleviated by receiving
details of the 60 chemicals used to develop the rules. According to the submitter, 60
chemicals were used as training set to develop the 'six rules', of which 33 were sensitisersand 27 were non-sensitisers.
Thirteen chemicals of the training set (11 sensitisers and 2 non-sensitisers) were included in
the 175 chemicals tested at L'Oréal to assess the predictive capacity of U-SENS™, whereas a
total of 35 out of these 175 chemicals required the application of the 'six rules'. Therefore,
only 37 % of the chemicals requiring the application of the rules in the list of 175 were part of
the training set (13/35). However, of these 35 chemicals only three were non-sensitisers invivo and only one of these three was correctly predicted by the 'six rules'. Indeed, for the 35
out of 175 chemicals for which the 'six rules' were applied, the sensitivity obtained was
100 % (32/32), while the specificity was only 33 % (1/3).
Similarly, of the 38 chemicals tested in the multi-laboratory validation trial (300 experiments),
only two (12 experiments) were part of the training set, whereas 17 of the 38 chemicals (38experiments) had at least one experiment requiring application of the 'six rules'. Again, of
these 17 chemicals only six were non-sensitisers in vivo, accounting for a total of nine
experiments. Considering the 38 experiments from the multi-laboratory validation trial forwhich the 'six rules' were applied (17 out of 38 chemicals), only 3 out of 29 experiments withsensitisers (10 %) were misclassified by the 'six rules' as compared to 6 out of 9 experimentswith non-sensitisers (67 %).
There is therefore no evidence of any predictive value of the 'six rules'. In fact, it appears thatthe rules have no added value to a simple decision that all INC results should be considered
positive: Of the 38 experiments in the multi-laboratory validation trial that required
application of the 'six rules', 26 were true positives, 6 were false positives, 3 were true
negatives and 3 were false negatives. Applying a positive outcome to all INC results in thisdataset by default would lead to three extra false positive experiments but also to three lessfalse negative experiments.
7. The expected frequency of non-qualified tests based on the experience of the test developer
was defined.

6. Test materials
6.1 Sufficiency of the number of evaluated test items in view of the study objective
The number of test items is considered sufficient to assess the transferability, and within andbetween laboratory reproducibility of the test method.

6.2 Representativeness of the test items with respect to applicability
The 2013 ring trial included eleven sensitisers (1 extreme, 3 strong, 5 moderate, 2 weak) and three
non-sensitisers. The underrepresentation of the non-sensitisers was corrected by the 2014 study.
The test substances selected (for the 2014 validation study) include strong (N=3), moderate (N=3)
and weak (N=2) sensitisers, one false positive substance and fifteen clear negative substances.
While the distribution of the selected test substances may be sufficient to indicate major issues
related to applicability domain and limitations, it may not be sufficient to identify minor (e.g.
chemical reactivity class related) issues.

ESAC WORKING GROUP REPORTPage | 33

7. Within-laboratory reproducibility (WLR) (Module 2)
7.1 Assessment of repeatability and reproducibility in the same laboratory
The WLR was assessed based on data from the 2013 ring trial and the 2014 validation study.
--

Ring trial 2013:

21 test substances were evaluated within L'Oréal.



Two independent experiments by L’Oréal (reviewed by EURL ECVAM), followed by anadditional independent experiment conducted by L’Oréal.



Based on the L’Oréal data, 20/21 substances were concordant (WLR = 95.2 %).
Chlorobenzene was the outlier. A plausible explanation was provided: poor solubility
and reactivity with cell membranes.



The within laboratory reproducibility was very good as far as the overall binary
prediction (Y/N) is concerned. However, discordant results between the independent
experiments may occur and may be laboratory dependent.

Validation study 2014:

15 test substances were evaluated within four laboratories.



Three independent experiments per laboratory, three differently coded samples
provided.

Based on the WLRs for each of the participating laboratories (100 %, 93.3 %, 73.3 %and 100 %), an average WLR of 91.7 % was obtained.



Outliers were explained but the ESAC WG was not satisfied by some of the
explanations (See Section 11).

7.2 Conclusion on within-laboratory reproducibility as assessed by the study
Overall, the WLR is very good, however a lower reproducibility was observed with one of the naïve
laboratories. This may highlight the importance of proper training. Since proper training
opportunities were provided by the test developer, a lower reproducibility may also indicate
additional challenges for a naïve laboratory trying to apply the method.
In spite of this one laboratory performing weaker than the others, the ESAC WG considers the WLR
comparable, arguably better, than recently validated methods listed as OECD test guidelines.

8. Transferability (Module 3)
8.1 Quality of design and analysis of the transfer phase
The test substances for the transfer phase were carefully selected following predefined criteria.
A detailed training protocol was provided. One (hands-on) training week (included discussion of the
protocol) was organised at L’Oréal. In house training at the CRO was necessary. Each naïve laboratory
was expected to perform at least 2 runs per week for 3-6 weeks. The criteria describing a successfultransfer were predefined, and include, among others, compliance with the U-SENS™ acceptancecriteria, accuracy in selecting/discarding controls and stable CD86 expression, reproducibility.

ESAC WORKING GROUP REPORTPage | 34

8.2 Conclusion on transferability to a naïve laboratory / naïve laboratories as assessed by
the study
Based on the provided transfer report, the transfer of the test method to the participating
laboratories can be considered to have been successful.

9. Between-laboratory reproducibility (BLR) (Module 4)
9.1 Assessment of reproducibility in different laboratories
The BLR was assessed based on data from the 2013 ring trial and the 2014 validation study.
--

Ring trial 2013:

14 test substances were evaluated, of which the majority were sensitisers withcharacteristics potentially interfering with test performance.



Two independent experiments (reviewed by EURL ECVAM) by the four laboratories,
followed by an additional independent experiment conducted by L’Oréal.



Based on the data from the four laboratories, an average BLR of 79 % was obtained(11/14 substances concordant in all four laboratories).

Validation study 2014:

24 test substances were evaluated, the majority being non-sensitisers.



Three independent experiments per laboratory, three differently coded samples
provided.

Based on the WLRs for each of the participating laboratories, an average BLR of
87.5 % (21/24 substances concordant in all four laboratories) was obtained.

Taking the data from the 2013 and 2014 studies together, the U-SENS™ exhibited a BLR of 84 % interms of Y/N score.
Explanations were provided for most of the substances not resulting in reproducible classifications.
The ESAC WG does not fully agree with these explanations (See Section 11).

9.2 Conclusion on between-laboratory reproducibility as assessed by the study
Overall, the BLR is very good as far as the overall binary classification (Y/N) is concerned. However,discordant results between the independent experiments may occur and may be laboratory
dependent.
The studies also hint issues with substances of low solubility and volatile substances (see WLR
studies), but also with substances with the potential to cause colour interference.

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 U-SENS™ was assessed on the basis of the 2013 ring trial, the 2014validation study and the assessment of 175 test substances by L’Oréal only.
ESAC WORKING GROUP REPORTPage | 35

The substances were selected on the basis of availability of robust animal and/or human clinical data
(Basketter et al., 2014).
Various ingredient classes (29 % fragrances, 15 % preservatives, 8 % dyes) and protein binding(chemical reactivity) classes (65 % belonging to at least one of the five classes) were represented.
1. 2013 ring trial:-

Not provided

2. 2014 validation study:-

Reference data were LLNA data-

Predictive capacity on 24 substances:

3. 2013 ring trial + 2014 validation study-

Reference data were LLNA data-

Predictive capacity on 38 substances:oSensitivity: 97 % (95 %, 100 %, 100 %, 95 %)oSpecificity: 89 % (100 %, 74 %, 89 %, 95 %)oAccuracy: 93 % (97 %, 87 %, 95 %, 95 %)4. L’Oréal 175 substances, including 35 of the substances used in the previous studies.
-

166 substances with LLNA data, 101 substances with human data and 92 substances with
LLNA and human data.

-

Overall performance:oSensitivity: 95 %oSpecificity: 65 %oAccuracy: 85 %-The sensitivity against potent and human categories 1-4 was 100 %.

-Against human data (N=101) with human class 5 chemicals considered as sensitisers:oSensitivity: 89 % (95 %)oSpecificity: 79 % (59 %)oAccuracy: 88 % (83 %)The values in brackets show the values with human class 5 chemicals are considered as nonsensitisers as proposed by Basketter et al. (2014).
-

Against substances with both human and LLNA data (N=92):oSensitivity: 90 % (92 %)oSpecificity: 60 % (55 %)oAccuracy: 85 % (84 %)Values obtained against LLNA are shown in brackets.

ESAC WORKING GROUP REPORTPage | 36

10.2 Overall relevance (biological relevance and accuracy) of the test method in view ofthe purpose
The data from the 2013 ring trial and the 2014 validation study suggest that the predictive capacity isgood as far as the overall binary classification (Y/N) is concerned. The assay seems to have a goodsensitivity perhaps at the expense of specificity. This conclusion is underpinned by the low specificityof the test method when applied to the larger dataset of 175 substances. Nevertheless, overall, thetest method's accuracy is comparable with accepted in vitro methods.
Where directly compared, performance against human data and LLNA data was very similar.
Explanations for the false positive and false negative results were provided. False positives seem tobe associated with membrane disrupting substances. The studies also lend credence to the potential
for issues with substances of low solubility and volatile substances (see Section 11). These
characteristics seem to affect primarily the specificity of the U-SENS™.

11. Applicability domain (Module 6)
11.1 Appropriateness of study design to conclude on applicability domain, limitations andexclusions
The primary goal of the study was to assess transferability and reproducibility (WLR and BLR) of thetest method. Neither the 2013 ring trial nor the 2014 validation study were designed to provide
extensive information on the applicability domain of the method.
However, these studies in combination with the large study on 175 substances covered a broad
range of potency and chemical reactivity classes. Analysis of these data revealed that outliers did notcorrespond with a particular potency or chemical reactivity class. Prehaptens were correctly
identified.
The test method submitters provided a list of physico-chemical characteristics that are believed to beoutside the applicability domain of the test method: substances of low solubility, rapid oxidation,
volatile substances, substances disrupting cell membranes and substances with colour interference.
However, the data show that 8/8 lipophilic substances were correctly predicted (although all positivein vivo). Thus, the applicability domain of the test method seems to include this type of substanceswith reduced solubility in cell culture medium. In addition, analysis of all the data revealed that alldyes were correctly classified.
The ESAC WG disagrees with the explanation by the VMT that benzyl alcohol may be wrongly
classified because it is a volatile fragrance. This was apparently a problem in one laboratory only, thesame laboratory that showed a lower reproducibility. Furthermore, in order to define a characteristic(e.g. volatility) as a problem, it is necessary to look at the entire group of chemicals having the samecharacteristic. The VMT also fails to explain satisfactorily why this laboratory failed to accurately
score lactic acid, streptomycin and polyethylene glycol, even after application of the set of 'six rules'.
Overall the presented evidence seems to indicate that volatile substances or poorly soluble
substances may require additional attention in the technology transfer and training phase, as
improper handling of such substances during exposure could affect the reproducibility of the results
rather than their accuracy.

ESAC WORKING GROUP REPORTPage | 37

11.2 Quality of the description of applicability domain, limitations, exclusions
In depth analysis of the data revealed potential issues with substances of low solubility or stability inan aqueous environment, substances interfering with flow cytometry analysis, volatile substancesand substances disrupting cell membranes. These limitations are likely to be very similar to othersubmerged cell culture assays (e.g. h-CLAT, KeratinoSens™).
Specific comments:
1. Solubility:
The ESAC WG noted the limited choice of solvents. Not all of the test substances may have been
sufficiently soluble in either RPMI or DMSO, and speculates to what extent this affects (e.g. in
unexperienced laboratories) the reproducibility of the test method.
2. Volatility:
The use of volatility to exclude substances from the applicability domain requires analysis of theentire data to set appropriate criteria. It is likely to apply only for a small subset of chemicals withhigh volatility coupled with low reactivity (e.g. methyl methacrylate).
Against that background it is unlikely that benzyl alcohol is an outlier for reasons of volatility.
3. Chemical classes used to define the 'six rules'.
It is of concern that the 'six rules' were trained to fix INC predictions towards positive results. It is notclear to what extent the training set of chemicals is representative in chemical space.
4. The ESAC WG noticed that Streptomycin is an unusual substance that is normally negative in
predictive tests despite of being a well-recognised human sensitiser.

12. Performance standards (Module 7)12.1 Adequacy of the proposed Essential Test Method Components
Not applicable.

12.2 Adequacy of proposed Reference Chemicals
Not applicable.

12.3 Adequacy of proposed performance target values
Not applicable.

13. Readiness for standardised use
13.1 Assessment of the readiness for regulatory purposes
The provided data (good WLR, BLR and predictive capacity) suggest that the U-SENS™ test method
may be a useful component of a strategy for the regulatory testing and assessment of the skinsensitising potential of substances belonging to the applicability domain defined in the validation
study.
ESAC WORKING GROUP REPORTPage | 38

The major concern of the ESAC WG is the complexity of the prediction model and the uncertainty of
the applicability domain of the 'six rules' for deciding on INC results. In addition, the 'six rules' appear
to be highly sensitive but without specificity. The ESAC WG considers these to be an obstacle forregulatory readiness.

13.2 Assessment of the readiness for other uses
The provided data (good WLR, BLR and predictive capacity) suggest that the U-SENS™ test method
can be used for screening and early decision-making during product development.

13.3 Critical aspects impacting on standardised use
Proper training is required.

13.4 Gap analysis
None identified.

14. Other considerations
None.

15. Conclusions on the study
15.1 ESAC WG summary of the results and conclusions of the study
The VMT concluded that the WLR, BLR and predictive capacity are very good as far as the overall
binary classification (Y/N) is concerned. The transfer of the test method to the participating
laboratories was successful.
Where directly compared, performance against human data and LLNA data was comparable.

15.2 Extent to which study conclusions are justified by the study results alone
The scientific and regulatory rational of the method was clearly formulated. The study design was
reasonable.
The ESAC WG agrees with the VMT that the WLR and BLR are very good, however a lower
reproducibility was observed with one of the naïve laboratories. This may highlight the importance ofproper training. Since proper training opportunities were provided by the test developer, a lower
reproducibility may also indicate additional challenges for a naïve laboratory trying to apply themethod.
Based on the analyses data published in the statistical report and its amendments submitted to EURLECVAM (Attachments 18b, 18m and 18n), the predictive capacity is good as far as the overall binaryclassification (Y/N) is concerned. The assay seems to have a good sensitivity perhaps at the expenseof specificity. Overall, the test method's accuracy is comparable with accepted in vitro methods.
The prediction model was found to be complex and the documentation was insufficiently clear toenable proper understanding of the conclusions drawn by the test submitter from INC results. The
ESAC WORKING GROUP REPORTPage | 39

ESAC WG has the concern that the 'six rules' used to resolve INC outcomes were fitting the outliers
without having a reasonable biological and/or chemical rational. There was no evidence of thepredictive value beyond the INC data set used to train the rules. The data obtained in the validation
study suggest that the 'six rules' have limited accuracy (sensitivity: 90-100 %; specificity: 33 %) andhave no added value to a simple decision where all INC results are considered positive. Indeed,
considering all INC results as sensitiser by default instead of applying the 'six rules' would slightly
decrease the number of false negatives with only an equally small increase of false positives.
Where directly compared, performance against human data and LLNA data appears comparable.

15.3 Extent to which conclusions are plausible in the context of existing information
The ESAC WG was not fully convinced about the explanations for the false positive and false negative
results. For example, some of these explanations were not consistent as they appeared to belaboratory specific.
All data taken together, a broad range of potency and chemical reactivity classes was covered.
Analysis of these data revealed that outliers did not correspond with a particular potency or chemical
reactivity class. Pre-haptens were correctly identified. Overall the presented evidence seems toindicate that volatile substances or poorly soluble substances may require additional attention in thetechnology transfer and training phase, as improper handling of such substances during exposure
could affect the reproducibility of the results rather than the accuracy.
The prediction model was made complex by the introduction of the 'six rules' to be applied on INC
results. The ESAC WG has concerns about the rules, as the data do not provide evidence supporting
any predictive value. The high sensitivity (90-100 %) and very low specificity (33 %) of the 'six rules'
per se indicates that the rules have no added value to a simple decision where all INC results are
considered positive.
The provided data suggest that the U-SENS™ test method may be useful as a component of an IATA
for skin sensitisation or as a tool for screening and early decision-making during productdevelopment, for the assessment of chemicals belonging to the applicability domain defined in thevalidation study. The major concern of the ESAC WG is the complexity of the prediction model andthe uncertainty of the value of the 'six rules' for deciding on INC results. The ESAC WG considers thisan obstacle for regulatory readiness.

16. Recommendations
16.1 General recommendations
1. Use 'U-SENS' consistently in order to make it transparent which test has been validated.

16.2 Specific recommendations (e.g. concerning improvement of SOPs)
1. Upon request from the ESAC WG, the test developer provided a detailed flow chart showing how
the decision about a substance has to be made. It is suggested to make such a flow chart an
integrated part of the protocol (SOP).
2. On the basis of further analysis showing that the 'six rules' appear to change INC results to'sensitisers' in the majority of cases, the test developers are recommended to consider removing
these 'six rules' from the prediction model and to consider any inconclusive result as positive by
default. This would significantly simplify the prediction model and would allow standardising the
ESAC WORKING GROUP REPORTPage | 40

maximum number of runs per experiment to three. Considering the validation dataset, modifying theprediction model and the maximum number of runs as suggested above would have little impact on
the predictive capacity of the U-SENS™ test method and could lead to a decrease in testing
requirements of about 8 %.

17. References
Aptula AO, Roberts DW (2006). Mechanistic applicability domains for non-animal-based prediction of
toxicological end points: general principles and application to reactive toxicity. Chem. Res. Toxicol.
19: 1097-1105.
Basketter DA, Alépée N, Ashikaga T, Barroso J, Gilmour N, Goebel C, Hibatallah J, Hoffmann S, Kern P,
Martinozzi-Teissier S, Maxwell G, Reisinger K, Sakaguchi H, Schepky A, Tailhardat M, TemplierM.(2014). Categorization of chemicals according to their relative human skin sensitization potency.
Dermatitis 25: 11-21.
EC (2008). Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of
substances and mixtures. Official Journal of the European Union, L353.
OECD (2010). Skin Sensitization: Local Lymph Node Assay. OECD Guideline for the Testing ofChemicals No. 429, OECD, Paris.
OECD (2012). The adverse outcome pathway for skin sensitisation initiated by covalent binding toproteins. Part 1: Scientific evidence. Series on Testing and Assessment No. 168, OECD, Paris.
OECD (2016). In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT). OECD Guideline for
the Testing of Chemicals No. 442E, OECD, Paris.
UN (2015). United Nations Globally Harmonized System of Classification and Labelling of Chemicals(GHS), Sixth revised edition, UN New York and Geneva.

ESAC WORKING GROUP REPORTPage | 41

ESAC WORKING GROUP REPORTPage | 42

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ISBN 978-92-79-63200-6

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