EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Union Reference Laboratory for Alternative Methods to Animal Testing(ECVAM)

Human Cell Line Activation Test
(h-CLAT)
Validation Study Reporthuman Cell Line Activation Test (h-CLAT) Validation Study Report

Page 1 of 74

SUMMARY.............................................................................................................................................................. 4
BACKGROUND...................................................................................................................................................... 5
MANAGEMENT OF THE STUDY....................................................................................................................... 81.
2.

STUDY OBJECTIVES .................................................................................................................................... 8
PROJECT PLAN ........................................................................................................................................... 9a.
Structure of the study ............................................................................................................................ 9b.
Validation Management Group .......................................................................................................... 10c.
Laboratories......................................................................................................................................... 11d.
Quality Systems of the Participating Laboratories ............................................................................. 123.
STUDY EXPERIMENTAL DESIGN AND SAMPLE SIZE ................................................................................. 13
4.
SELECTION OF TEST CHEMICALS ............................................................................................................ 14
5.
CHEMICALS PURCHASE, CODING AND DISTRIBUTION ............................................................................. 20a.
Solvent Compatibility assessment........................................................................................................ 20b.
Coding/Decoding ................................................................................................................................. 20
6.
DATA MANAGEMENT ................................................................................................................................ 21
7.
STATISTICAL ANALYSIS OF EXPERIMENTAL DATA ................................................................................ 22a.
Analysis performed on the experimental data .................................................................................... 22b.
Criteria for Assessing the Study Outcome .......................................................................................... 22
TEST DEFINITION (MODULE 1) .................................................................................................................... 24
1.
2.
3.
4.
5.

INTENDED PURPOSE OF THE TEST METHOD ............................................................................................. 24
EVIDENCE DEMONSTRATING THE NEED OF THE TEST METHOD .............................................................. 24
STATUS OF DEVELOPMENT OF THE TEST METHOD .................................................................................. 24
SCIENTIFIC BASIS – BIOLOGICAL AND/OR MECHANISTIC RELEVANCE OF THE H-CLAT....................... 25
PROTOCOL OF THE TEST METHOD ........................................................................................................... 25a)Selection of the solvent........................................................................................................................ 25b)
Determination of the CV75 ................................................................................................................. 26c)
Evaluation runs ................................................................................................................................... 26d)
Modifications made to original protocol prior to the initiation of the validation study .................... 28e)
Acceptance criteria .............................................................................................................................. 32
f)
Requirement for a third evaluation run.............................................................................................. 32
6.
KNOWN TECHNICAL LIMITATIONS AND DRAWBACKS OF THE TEST METHOD ........................................ 32
7.
LIMITATIONS IN APPLICABILITY .............................................................................................................. 33
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 1................................... 34
TRANSFERABILITY (MODULE 3) .................................................................................................................. 35
1.
2.
3.

GENERAL ASPECTS ................................................................................................................................... 35
TRAINING .................................................................................................................................................. 35
TRANSFER OF THE TEST METHOD TO THE NAÏVE LABORATORIES .......................................................... 36

CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 3................................... 41
WITHIN-LABORATORY REPRODUCIBILITY (MODULE 2) .................................................................... 42
1.

KAO ........................................................................................................................................................... 42
Reproducibility (concordance of predictions).................................................................................... 42
Reproducibility of CV75 values........................................................................................................... 43
Reproducibility of EC values............................................................................................................... 44
2.
SHISEIDO ................................................................................................................................................... 45a.
Reproducibility (concordance in predictions).................................................................................... 45b.
Reproducibility of CV75 values........................................................................................................... 45c.
Reproducibility of EC values............................................................................................................... 46
3.
BIOASSAY .................................................................................................................................................. 47a.
Reproducibility (concordance in predictions)..................................................................................... 47b.
Reproducibility of CV75 values........................................................................................................... 48
c.
Reproducibility of EC values............................................................................................................... 49
a.
b.
c.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 2 of 74

4.

EURL ECVAM........................................................................................................................................ 50
Reproducibility (concordance in predictions)..................................................................................... 50
Reproducibility of CV75 values........................................................................................................... 50
Reproducibility of EC values............................................................................................................... 51
SUMMARY ........................................................................................................................................................... 52
a.
b.
c.

CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 2................................... 53
BETWEEN LABORATORY REPRODUCIBILITY (MODULE 4)................................................................ 54
1.
2.
A)
B)
C)

REPRODUCIBILITY (CONCORDANCE IN PREDICTIONS)........................................................................... 54
OTHER CONSIDERATIONS ......................................................................................................................... 56
SOLVENT SELECTION................................................................................................................................ 56
DETERMINATION OF THE CV75............................................................................................................... 57
REPRODUCIBILITY OF THE EC150 AND EC200 VALUES ......................................................................... 58

CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 4................................... 60
PREDICTIVE CAPACITY (MODULE 5) ......................................................................................................... 61
POTENCY SUBCATEGORISATION ........................................................................................................................ 63
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 5................................... 64
ADDITIONAL OBSERVATIONS ...................................................................................................................... 65
LOG KOW AND APPLICABILITY DOMAIN OF THE H-CLAT............................................................................... 65
RUN ACCEPTANCE CRITERIA ............................................................................................................................. 65
CONTROL RESULTS ............................................................................................................................................ 66
VMG OVERALL CONCLUSIONS AND RECOMMENDATIONS ............................................................... 67
OVERALL CONCLUSIONS ................................................................................................................................... 67
RECOMMENDATIONS ......................................................................................................................................... 69
LIST OF APPENDICES....................................................................................................................................... 70
LIST OF REFERENCES...................................................................................................................................... 71

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 3 of 74

Summary
The evaluation of skin sensitisation potential represents an important component of the safetyassessment of new and existing substances. Traditionally this has required animal tests such as
the murine Local Lymph Node Assay (LLNA) and/or guinea pig tests (Buehler Test andGuinea Pig Maximisation Test).
There is a pressing need for alternative non-animal methods to reduce and ultimately replace
animal tests for this endpoint as required by some European regulations aiming at theprotection of human health and the environment (i.e. Cosmetics Regulation and REACH).
Several mechanistically-based non-animal test methods for the assessment of skin sensitisation
are currently under development/evaluation. The validation of the human Cell Line Activation
Test (h-CLAT) was part of a larger study coordinated by the European Union Reference
Laboratory for Alternatives to Animal Testing (EURL-ECVAM), including two other methods:
the Direct Peptide Reactivity Assay (DPRA) and the Myeloid U937 Skin Sensitisation Test
(MUSST).
The study reported here was designed to allow sound conclusions to be drawn on thetransferability and reproducibility (within- and between-laboratories) of the h-CLAT in view of
its potential for future use as part of non-animal integrated testing strategies (ITS) for skinsensitisation hazard assessment.
Having reviewed the information generated in the course of this study, the Validation
Management Group (VMG) concluded that the findings satisfy the information requirement for
the evaluation of validation modules 1-4 (test definition, within laboratory reproducibility,transferability, between laboratory reproducibility) and, in addition, contributes to module 5(predictive capacity) and module 6 (applicability domain) of the ECVAM modular approach tovalidation.
The VMG concluded that the information generated in the study shows that the h-CLAT is arobust and reliable test method. Therefore, the VMG supports the consideration and use of theh-CLAT in non-animal Integrated Testing Strategies (ITS)/approaches to support regulatory
decision making.
The VMG also considers the h-CLAT merits further evaluation as a component in ITS for
replacing the in vivo assays for skin sensitisation hazard identification and for the role it mightplay in the determination of skin sensitisation potency.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 4 of 74

Background
Skin sensitisation is the toxicological endpoint associated with substances that have theintrinsic ability to cause skin allergy, leading to the disease allergic contact dermatitis (ACD)
in humans, and which represents the most common manifestation of immunotoxicity tochemicals. The identification of skin sensitisation potential represents an important componentof the safety assessment of new and existing substances including cosmetic ingredients.
Current regulatory predictive tests for skin sensitisation rely on the use of animals. These
include guinea pig tests (Buehler Test and Guinea-pig Maximisation Test (OECD TG 406; TM
B06, EU Regulation 440-2008), and the murine Local Lymph Node Assay (LLNA, OECD TG
429; TM B42, EU Regulation 440-2008) including non-radio-isotopic variants (OECD TG
422A and OECD TG 422B). Summary details of the animal tests currently in use are provided
in Table 1. The LLNA is considered a reduction/refinement method with respect to the
traditional guinea-pig tests and of greater value in generating skin sensitisation potency
information which is required for full risk assessment to establish safe levels of human
exposure. With the entry into force of the 7th Amendment to the Cosmetics Directive and the
new European chemicals regulation REACH (Registration, Evaluation, Authorisation andRestriction of Chemicals) there is an urgent need for validated alternative non-animal methods
for this endpoint.
Skin sensitisation is a delayed-type hypersensitivity reaction induced by low molecular weightreactive chemicals. It develops in two distinct phases; the induction phase which sensitises theimmune system for an allergic response and the elicitation phase which occurs following a
subsequent contact with the allergen and which leads to the clinical signs and symptoms ofallergic contact dermatitis (ACD) in humans or contact hypersensitivity (CHS) in the rodent
models. The key underlying biological mechanisms of the induction of skin sensitisation are
relatively well understood as recently documented by the OECD in its report on: “The Adverse
Outcome Pathway (AOP) for Skin Sensitisation Initiated by Covalent Binding to Proteins”(OECD 2012a; 2012b). These include: 1) the ability of the chemical to penetrate the skin andreach the site of haptenation (skin bioavailability), 2) the covalent binding of the chemicalsensitiser to the skin protein (haptenation), 3) the release of pro-inflammatory signals by
epidermal keratinocytes (skin inflammation) 4) the activation and maturation of Dendritic cells(DC) the skin immunocompetent cells, 5) the migration of DC from skin to the regional lymph
nodes, 6) the presentation by DCs of the haptenated protein to T cells and the clonal expansionof memory T cells (lymphocytes capable of being stimulated and activated specifically by thehaptenated protein). Progress has been made in recent years in the development of
mechanistically-based alternative methods for hazard identification some of which might also
be able to contribute to potency characterisation. However none of the mechanistically-based
non-animal tests currently under development/evaluation is considered to have the potential tofunction as a stand-alone method to fully replace the currently used animal tests. Instead it is
proposed that a combination of such mechanistic tests, addressing the key biological events ofskin sensitisation, will be needed to achieve this goal. A comprehensive overview of thecurrently available methods targeting the key mechanisms described above was published in2010 (Adler et al., 2010).
In the first quarter of 2009 three partial replacement methods for skin sensitisation testing wereformally submitted to ECVAM for evaluation. These methods, namely the Direct Peptide
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 5 of 74

Reactivity Assay (DPRA), the human Cell Line Activation Test (h-CLAT) and the Myeloid
U937 Skin Sensitisation Test (MUSST) were developed by the European Cosmetics
Association (Cosmetics Europe, former Colipa) associated industries and optimised within
Cosmetics Europe ring trials. These three test methods were proposed as potential candidates
for regulatory use as part of integrated alternative testing approaches for replacing the current
regulatory in vivo tests. How the information generated with these test methods could
potentially be integrated to achieve this goal still has to be fully determined, and did not form
part of the validation study reported here.
Following the evaluation of the information provided on these test methods and after review ofthe submitted protocols (i.e. SOPs) ECVAM concluded that they were sufficiently developedand standardised to be included in the ECVAM validation process.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 6 of 74

Table 1: Overview of regulatory accepted animal tests

OECD Test Guidelines for
Skin Sensitisation
406: Guinea Pig
Maximisation Test
(GPMT)

406: Buehler Test

Skin
sensitisation
phasescoveredInduction +elicitation

Induction +elicitation

No ofanimals
in control
group
5

No of animals
in treatment
group

Test
duration(days)

Endpoint

Classification
criteria

10

23-25

Skin reactions(erythema/
oedema)Positive reaction
in at least 30%of the animals in
the treatment
group

10

20

30-32

Skin reactions(erythema/
oedema)Positive reaction
in at least 15%of the animals in
the treatment
group

At least 3 doses (highest
dose should not givesystemic toxicity and/or
excessive local irritation)

4

4

6

Stimulation
Index (SI) >3 atany dose.

Topical application

At least 3 doses (highest
dose should not givesystemic toxicity and/or
excessive local irritation)

4

4

8

Topical application

At least 3 doses (highest
dose should not givesystemic toxicity and/or
excessive local irritation)

4

4

6

Cellularproliferation inauricular lymph
nodes measured
by radioactive
labelling
Cellularproliferation inauricular lymph
nodes quantified
by determinationof ATP content
Cellularproliferation inauricular lymph
nodes quantified
by determinationof BrdU
incorporation

Animalspecies

Adjuvant

Exposure

Dose levels

Guineapig

Y(Freund’s Complete
Adjuvant-FCA)

Induction: intradermal
injections (day 0) andtopical application (day5-7 and day 6-8)

Induction: 1 dose(highest concentration tocause mild-to moderateskin irritation)

Challenge: topicalapplication (day 20-22)
by occluded patch

Challenge: 1 dose(highest non-irritant dose)

Guineapig

NRe-challenge: possible
Induction:
topical application (day0, day 6-8 and day 1315)
Challenge: topicalapplication (day 27-29)

429: Local Lymph Node
Assay

Induction

Mouse

N

442A: Local Lymph Node
Assay: DA

Induction

Mouse

N
Pre-treatment with
1% Sodium Lauryl
Sulphate (SLS)

442B: Local Lymph Node
Assay: BrdU Elisa

Induction

Mouse

Re-challenge: possible
Topical application

human Cell Line Activation Test (h-CLAT) Validation Study ReportInduction: 1 dose(highest concentration tocause mild skin irritation)
Challenge: 1 dose(highest non-irritant dose)

Page 7 of 74

Stimulation
Index (SI) ≥1.8
at any dose.

Stimulation
Index (SI) ≥1.6
at any dose.

Management of the Study
Reference documents:
Project Plan (Appendix 1)
List of additional available documents filed for the study and available on
request (Appendix 15)

1. Study objectives
1.1 Primary objectives
In September 2009 a formal validation study of the h-CLAT was launched, with the primary
overall objective of evaluating its transferability and reliability (reproducibility withinand between-laboratories) with a view to its future use in integrated non-animal
approaches for replacing the currently used regulatory animal tests.
1.2 Secondary objectives
As secondary goals of the study, the information and experimental data generated during theevaluation of transferability and reliability were used to perform:a) A preliminary evaluation of the ability of the h-CLAT to reliably discriminate skinsensitising (S) from non-sensitising (NS) chemicals as defined by the Globally Harmonised
System (GHS) of Classification and Labelling of Chemicals for skin sensitisation (category 1;
no category) and as implemented in the European Commission Regulation (EC) No 1272/2008(EC, 2008) on classification, labelling and packaging (CLP) of substances and mixtures.
b) Where possible, a preliminary consideration of the ability of the h-CLAT to contribute to
sub-categorisation of skin sensitising chemicals, e.g. into Sub-category 1A and Sub-category
1B as adopted in the 3rd revised version of the GHS (UN, 2009).
1.3 Other considerations
The current report, which was prepared by EURL ECVAM with the support of the Validation
Management Group (VMG), presents the outcome of the validation study of the h-CLAT,
during which the transferability and reliability were independently evaluated in four separate
laboratories.
Evaluation of how the data generated with the h-CLAT might be accommodated within future
testing strategies and data integration activities was outside the scope of the current study,
though it is recognised that the availability of high quality non-animal data, such as thosegenerated in the ECVAM study, is a prerequisite for such activities.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 8 of 74

2. Project Plan
Prior to the start of the study, a Project Plan was approved and issued by the Validation
Management Team (VMT). This document was reviewed as required at each VMT meeting;the final version is annexed to this report (see Appendix 1). The Project Plan documents theobjectives, coordination and sponsorship of the study; the nature and roles of the study
personnel at each testing site; the minimum quality assurance systems required in the case of
non-GLP laboratories; the nature and deliverables of the different study phases; as well as
instructions regarding the reception, handling and storage of the test chemicals.
Prior to the start of the training phase, the Project Plan was sent to all testing sites for theirinformation, approval and implementation. They each returned a signed declaration attestingthat they had read and understood the project plan, and that their testing facilities would workin compliance with the provisions set out in the Project Plan.
a. Structure of the study
This validation study was organised to generate information relevant to modules 1-4 (1: test
definition, 2: within laboratory reproducibility, 3: transferability, 4: between laboratory
reproducibility) of the ECVAM modular approach to validation (Hartung et al., 2004) in linewith the study’s stated objectives. In addition, the experimental data produced in the study also
contribute to module 5 on predictive capacity and to module 6 on applicability domain, whichwere in part addressed by the information generated and submitted to ECVAM by the test
method submitter. However, the number of chemicals used in this validation study, which was
set to satisfy the primary goal of the study, is not sufficient on its own to draw robust or
definitive conclusions on these last two modules.
The study was entirely coordinated by ECVAM with participation from NICEATM-ICCVAM
and JaCVAM via the VMT with regard to study design, chemical selection, and test method
SOPs. Figure 1 illustrates how the validation study was organised with respect to the
management, the test methods included, the participating laboratories, the selection, coding andsupply of the test chemicals and the data collection and statistical analyses. Full details of the
management, sponsorship, coordination, timings, responsibility and overall set-up of the study
are provided in the Project Plan (Appendix 1). The organisation and conduct of the study was
performed in compliance with the principles laid down in the OECD guidance document on
test method validation (OECD, 2005).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 9 of 74

Coordinator ECVAM is responsible for

Liaisons•Establishment of/support to VMG•Establishment of/oversight of CSG•Study communication and coordination•Subcontracts•Organising meetings•Organising QC audits (if needed)•Data collection•Data management•Data quality check•Drafting final study reports

Biostatistics•Support chemical selection•Provide codes for chemicals•Approve spreadsheets•Analyse data
•Provide statistical report(s)•ICCVAM/NICEATM•JaCVAM
Validation Management Group (VMG) approves•Study goal and project plan•Study protocols/SOPs and amendments•Outcome of QC audits•Chemical selection•Reports from the laboratories•Timelines/study progression•Study interpretation and conclusions•Final study reports and publications

Lead Laboratories

Kao

•SOPs

•Training•Testing
Shiseido•SOPs•Training•Testing

ECVAM

Chemical Selection Group (CSG)•Definition of selection criteria•Chemical selection

Contract
Laboratory
Bioassay
Testing

EURL ECVAM
Testing

Chemical acquisition, coding
and distribution

Figure 1: Schematic representation of the study structure and organisation.

b. Validation Management GroupAn expert independent Validation Management Group (VMG) was established by ECVAM. Itsrole was to ensure that the study objectives and goals were clearly defined, to guide andfacilitate the validation process, to take study management decisions as the study progressed, toevaluate the results and to draw conclusions regarding the outcome of the study with respect tothe study goals. David Basketter was appointed as chair of the VMG because of his
acknowledged expertise in the field.
In addition to the VMG, representatives from the lead laboratories were involved in a subset ofthe discussions, together with liaisons from other validation bodies, ICCVAM/NICEATM(USA), and JaCVAM (Japan). This extended group is referred to as the "Validation
Management Team" (VMT).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 10 of 74

The strategic decisions, including the selection and approval of the test chemicals to be used in
the study, were taken by the VMG only. The liaisons were involved in all discussions. The leadlaboratories representatives were not involved in discussions related to the selection of thechemicals.
Validation Management Team Composition:
Validation Management GroupChair
Co-chairStudy Coordinator
Chair of the Chemical Selection Group
Statistician

Industry representative
External expert
External expert

David Basketter (DABMEB Consultancy Ltd)Silvia Casati (ECVAM)
Alexandre Angers (ECVAM)
Thomas Cole (ECVAM)
André Kleensang (ECVAM, up to September2010), Anna Compagnoni (ECVAM, up to
January 2011), Els Adriaens (Adriaens
Consulting, from June 2011)
Pierre Aeby (Cosmetics Europe (ex Colipa))
Sebastian Hoffmann (seh consulting + services)
Jon Richmond (dr.jonrichmond: Advice &Consultancy)

Lead laboratories Representatives
Shiseido (h-CLAT)
Kao Corporation (h-CLAT)

Takao Ashikaga
Hitoshi Sakaguchi

Liaisons
JaCVAMNICEATMICCVAM

Hajime Kojima; alternate Yasuo Ohno
William S. Stokes; alternate Eleni Salicru (up to
May 2011); alternate Judy Strickland (since June
2011)
Joanna M. Matheson; alternate Abigail Jacobs

c. Laboratories
Four laboratories listed below participated in the validation of the h-CLAT. Kao Corporationand Shiseido, in whose laboratories the test method was jointly developed and which have themost experience in performing it, acted as lead laboratories.
Laboratory 3 was involved in the study as a naïve laboratory which had experience with flow
cytometry.
Laboratory 4 was selected through an open call for tender published by the Institute for Healthand Consumer Protection of the JRC. The criteria for selection were primarily based on thetechnical merit of the tender including the information provided on the laboratories awarenessof the nature of the work involved.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 11 of 74

Laboratory 1 (Study Director: Hitoshi Sakaguchi)
Global R&D, Safety and Microbial
Kao Corporation
2606 Akabane
Ichikai-Machi, Haga-Gun
Tochigi 321-3497 Japan
Laboratory 2 (Study Director: Takao Ashikaga)
Shiseido Quality Assessment Center
2-12-1, Fukuura, Kanazawa-ku, Yokohama
236-8643, Japan
Laboratory 3 (Study Director: Ingrid Langezaal)
The European Union Reference Laboratory for Alternatives to Animal Testing (EURLECVAM) GLP Test Facility (as from May 2012, former IVMU and VAM).
Institute for Health and Consumer Protection
Joint Research Centre
European Commission
Ispra, Italy
Laboratory 4 (Study Director: Axel Hohenstein)
Bioassay GmbHIm Neuenheimer Feld 515
69120 Heidelberg
Germanyd. Quality Systems of the Participating Laboratories
Laboratories 1 and 2 were not OECD Good Laboratory Practice (GLP) compliant. Laboratory
3 was in the formal process of requesting the OECD GLP compliance status during the conductof the study and used this study as a pilot for the OECD GLP compliance monitoring.
However, as Laboratory 3 had not yet been monitored by the OECD GLP monitoring
authorities, it cannot be deemed to have been OECD GLP compliant at the time the study was
performed. Only Laboratory 4 was fully OECD GLP compliant and subject to inspections byrelevant agencies however the study was not conducted under full GLP compliance at thislaboratory.
For the laboratories participating in the validation study which were not formally GLP
compliant, the VMG defined and requested the application of a minimum set of quality
assurance requirements considered essential for the acceptance of information and data
produced in the validation process. These requirements formed part of the Project Plan which
was sent to, and accepted by, all participating laboratories prior to the initiation of the study.
These minimum requirements were:

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 12 of 74

• Qualified personnel, and appropriate facilities, equipment and materials shall be provided.
• Records of the qualifications, training and experience, and a job description for eachprofessional and technical individual, shall be maintained.
• For each study, an individual with appropriate qualifications, training and experience shall be
appointed to be responsible for its overall conduct and for any report issued.
• Instruments used for the generation of experimental data shall be inspected regularly, cleaned,maintained and calibrated according to established SOPs, if available, or to manufacturers'
instructions. Records of these processes shall be kept, and made available for inspection onrequest.
• Reagents shall be labelled, as appropriate, to indicate their source, identity, concentration andstability. The labelling shall include the preparation and expiry dates, and specific storage
conditions.
• All data generated during a study shall be recorded directly, promptly and legibly by theindividual(s) responsible. These entries shall be attributable and dated.
• All changes to data shall be identified with the date and the identity of the individual
responsible, and a reason for the change shall be documented and explained at the time.

3. Study Experimental Design and sample size
Reference document: Experimental design (Appendix 2)
The number and nature of the chemicals involved in the training and transfer phases were at
the discretion of the lead laboratories.
For the blind testing phase, the ECVAM biostatisticians calculated the minimum number ofchemicals that would be required to properly evaluate the performance of the tests in light ofthe primary study goal, i.e. to perform a statistically sound evaluation of the Within Laboratory
Reproducibility (WLR) and of the Between Laboratory Reproducibility (BLR).
To ensure coverage of the range of sensitisation potencies, and since it was expected that weak
and moderate sensitisers would be more informative for the evaluation of the reproducibility ofthe test methods under consideration; the ratio of sensitisers to non-sensitisers was set to 2:1.
Preliminary parameters for the statistical analysis and evaluation of the WLR and the BLR
were defined at the beginning of the process taking into consideration the expected proportionof concordant classifications derived from the data reported in the test submissions to ECVAM(see Appendix 2 for full details). From these parameters, the number of chemicals required was
calculated to be at least 21 chemicals for the evaluation of the BLR and at least 13 chemicals
for the evaluation of the WLR.
Informed by the calculations performed by the biostatistician, the VMG felt it prudent, as thechemical set was used to evaluate three different in vitro test methods, to increase the number
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 13 of 74

of chemicals with respect to the calculated minimum number. This would allow for potential
reduction of the sample size, e.g. the exclusion of chemicals that might fall outside theapplicability of some of the test methods (e.g. metals in case of the DPRA).
On the basis of the above considerations, the following design was approved by the VMG:••

BLR - For evaluation of the BLR, 24 chemicals tested once in every laboratory (16sensitisers and 8 non-sensitisers).
WLR - For evaluation of the WLR, a subset of 15 chemicals (10 sensitisers and 5 nonsensitisers) from those used for the evaluation of the BLR, tested two additional times
in each laboratory, the same subset being tested at every site.

The VMG considered this experimental design appropriate and adequate for providing theinformation needed to perform a sufficiently robust assessment of the WLR and BLR based on
the concordance of classification, sensitizer (S) versus non-sensitisers (NS).
Study Phases:
The study was structured and conducted in two sequential phases, with the performance of thetest system being reviewed by the VMG between each phase (and sub-phase):
Phase A: training of the participating laboratories (phase A1), test method transfer to thetrained laboratories and verification of the Test Method Protocols (phase A2).
Phase B: assessment of the protocol performance by testing chemicals, under blind conditions,in all the laboratories.
Since 15 of the 24 chemicals selected would be tested three times in each laboratory (toevaluate the WLR, see above), and 9 would be tested only once, it was decided to separate thePhase B into two phases: first, Phase B Stage I with the 9 chemicals being tested once at eachsite, and Phase B Stage II for the remaining 15 chemicals tested three times at each site. The
laboratories were required to prepare and submit an interim report at the end of the Phase B
Stage I, with the results being evaluated by the VMG before allowing the laboratories toproceed to the next phase. This provided an additional review and control point, following theinitiation of the testing phase, in order to verify that no serious issues were arising before thebulk of the testing was performed.

4. Selection of Test Chemicals
Reference documents:
Chemical Selection report (Appendix 3)
Selection of the Phase B Stage I chemicals (Appendix 4)
The test chemicals for validation study were selected by an independent Chemicals Selection
Group (CSG) appointed by ECVAM and chaired by Dr. Thomas Cole (ECVAM). In addition
to the chair, the CSG was composed of Dr. Luca Tosti (ECVAM); Dr. David Basketter (chairhuman Cell Line Activation Test (h-CLAT) Validation Study Report

Page 14 of 74

of the VMG) and Dr. Bill Stokes (NICEATM/ICCVAM). The general strategy for thechemical selection was presented and approved by the VMG at its 1st meeting. The final list ofchemicals was presented and endorsed by the VMG at its 4th meeting, before the initiation of
the blind testing phase.
To facilitate the chemical selection process and in view of the intended use of the experimental
data generated in this study to support future activities on data integrations to achieveultimately full replacement, the VMG decided to use a common set of test chemicals for thethree test methods under evaluation.
Two recognised databases provided a convenient master source of authoritative data for
selection of eligible substances:
1) The ICCVAM database of 103 chemicals, subsequently supplemented with unpublishedadditions, provided by NICEATM.
2) The LLNA database of 341 chemicals, extracted by the CSG from published compilations(Gerberick et al., 2005; Kern et al., 2010).
A primary eligibility criterion for the chemical selection was the availability of robust in vivo
data to allow a proper comparative evaluation of the in vitro results. In particular, availabilityof both LLNA and GPMT in vivo data, with concordance of their corresponding skinsensitisation classification as an assurance of quality, formed the basis for short-listing
candidate chemicals. Availability of accepted human data was adopted as a secondary criterion,
in cases where there would otherwise be an insufficient number of sub-sets of eligible
chemicals as determined by the primary criterion.
During their respective development and optimisation, the three in vitro methods had been used
to evaluate certain of the chemicals listed in these databases, as described in the originalsubmissions of the methods to ECVAM. Acknowledging this, the chemical selection for thisstudy was designed to include:- A small quota of "tested" substances (i.e. substances reported as being previously tested by
the method in the original submission to ECVAM)- A majority of "untested" substances (i.e. substances not being reported as previously tested
in the original submission to ECVAM)
The preferred ratio of tested to untested chemicals was set in advance at around 1:2 by the
VMG. The final selection was composed of 9 tested chemicals and 15 untested chemicals forthe h-CLAT.
To ensure parity between the three in vitro methods (DPRA, h-CLAT and MUSST) evaluatedin the validation study, the only "tested" chemicals that were considered were those alreadytested by all three methods and that have been correctly predicted by each method with respect
to the in vivo classifications. The only exception to this criterion was the inclusion in the final
list of 2-Mercaptobenzothiazole, a chemical previously tested in the DPRA and in the h-CLAT
but not in the MUSST.
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 15 of 74

Inclusion in the final list of a proportion of chemicals already successfully tested provided an
opportunity to confirm the reproducibility of the test method with these chemicals when tested
under blind conditions and by other laboratories.
Applying the primary criterion (available and concordant LLNA and GPMT data), the source
databases yielded 11 eligible chemicals reported as previously tested in all three methods, all ofwhich were correctly classified by the three methods. Applying the primary criterion to the 215
untested chemicals found in the original lists identified another 24 eligible substances.
Therefore, in collaboration with NICEATM, 8 additional untested chemicals were identified
from an unpublished updated version of the ICCVAM database, increasing the total number ofcandidates to 43, to provide an adequate and practical shortlist.
This list of 43 substances was reduced to 24 for different pragmatic reasons (see Appendix 3)and by expert judgment by the CSG. The final selected chemicals covered the range ofsensitisation potency (i.e.: extreme, strong, moderate, weak). Inclusion of a small subset of
substances known to be misclassified or classified inconsistently by the in vivo tests madeprovision for evaluating whether these limitations were shared by the in vitro tests.
Furthermore, the chemical selection aimed for a balance of physical states(solid versus liquid)
and avoided structural analogues, unless contrasting skin sensitisation potential was evident(e.g. 1-Thioglycerol (S) and Glycerol (NS)).
The shortlist of 43 chemicals and its refinement to the final selection of 24 chemicals (together
with detailed comments about the choices made) can be found in Appendix 3.
The final selection included 9 LLNA performance standards (PS) reference chemicals (OECD,2010). In particular, nickel chloride and xylene (both with ambiguous or inconsistent in vivo
classification from LLNA and GPMT, but known human response) were considered eligible
under the secondary selection criterion. Nickel chloride (human positive, GPMT positive,
LLNA negative) is accepted as a PS true positive reference chemical (i.e., LLNA false
negative). Xylene (human negative, LLNA positive) is accepted as a PS true negative reference
chemical (i.e., LLNA false positive). In addition, Kathon CG, a commercial aqueous mixture
including 1.2% CMI (5-chloro-2-methyl-4-isothiazolin-3-one) was selected, making exception
to a general preference for pure substances with discrete chemical composition. CMI is a
LLNA PS reference chemical of extreme sensitisation potency, and the Kathon CG commercial
preparation is a recognised source.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 16 of 74

NON-SENSITISERSSENSITISERS

Table 2: List of the 24 chemicals selected for the coded testing phase.
Chemical Name

CAS#

State

LLNA

LLNA
potency
category

GP

EC3

GHS
potency
category

DPRA
R&D
resulthCLAT
R&D
resultMUSST
R&D
resultBeryllium sulfate

7787-56-6

Solid

+

extreme

+

0.001

1A

Kathon CG (1.2%
CMI)

26172-55-4

Liquid+extreme

+

0.009

1A

+

+

+

Benzoquinone

106-51-4

Solid+extreme+0.0099

1A

+

+

+

4-Phenylenediamine

106-50-3

Solid+strong

+

0.11

1A

+

+

+

Chlorpromazine HCl

69-09-0

Solid+strongChloramine T

127-65-1

Solid+strong

+

0.14

1A

+

0.4

1A

Formaldehyde

50-00-0Liquid+2Mercaptobenzothiazole

strong

+

0.61

1A

+

+

+

149-30-4

Solid+moderate

+

1.7

1A

+

+

+

+

+

-

-

-

Benzylsalicylate

118-58-1

Liquid+moderate

+

2.9

1B

1-Thioglycerol

96-27-5

Liquid+moderate

+

3.6

1B

Dihydroeugenol

2785-87-7

Liquid+moderate

+

6.8

1B

+

Nickel chloride

7718-54-9

Solid

-

no
category1

Benzylcinnamate

103-41-3

Solid

+

weak

+

18.4

1B

Imidazolidinylurea

39236-46-9

Solid

+

weak

+

24

1B

R(+)-Limonene

5989-27-5

Liquid

+

weak

+

69

1B

Methylmethacrylate

80-62-6

Liquid

+

weak

+

90

1B

Glycerol

56-81-5

Liquid

-2,4Dichloronitrobenzene

611-06-3

Solid-

Benzyl alcohol

100-51-6

Liquid-Methylsalicylate

119-36-8

Liquid-Isopropanol

67-63-0Liquid-Dimethylisophthalate

1459-93-4

Solid

-

4-Aminobenzoic acid150-13-0

Solid-

Xylene1330-20-7

Liquid+1

False negative in the LLNA

2

False positive in the LLNA

3

NC: Not Classified

nocategorynocategorynocategorynocategorynocategorynocategorynocategory

1B

-

NC3

-

NC

-

NC

-

NC

-

-

-

-

NC

-

-

-

-

NC

-

NC

weak2

95.8

NC

The subset of 9 of the 24 chemicals which were to be tested only once at each site (as 15
chemicals were shown to be sufficient for the evaluation of the WLR, see above) was selectedby a stratified random sampling procedure, to ensure a consistent distribution of potencies inthe subsets of 15 and 9 chemicals (see Figure 2 and Appendix 4 for details). For the purpose of
the stratification, the chemical Nickel chloride, false-negative in the LLNA, was assigned tothe "moderate" category by weight of evidence. Similarly, Xylene, a false-positive in the
LLNA, was assigned the "no category" class (see Appendix 4).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 17 of 74

3 Extreme

9 chemicals tested once ineach laboratory(Phase B Stage I)

4 Strong

5 Moderate

4 Weak15 chemicals tested threetimes in each laboratory(Phase B Stage II)
8 Not Classified

Final list of Chemicals (24)Figure 2: Stratified random sampling of the 24 chemicals to identify the 9 chemicals that were tested
once and the 15 chemicals that were tested three times each.

SENSITISERS

Table 3: List of the phase B1 chemicals tested only once in each laboratory.
Chemical Name

CAS#

State

LLNA

LLNA
potency
category

GP

EC3

GHS
potency
category

DPRA
R&D
resulthCLAT
R&D
resultMUSST
R&D
resultBenzoquinone

106-51-4

Solid+extreme+0.0099

1A

+

+

+

4-Phenylenediamine

106-50-3

Solid+strong

+

0.11

1A

+

+

+

96-27-5

Liquid+moderate

+

3.6

1B

Liquid+moderate

+

6.8

1B

Solid

+

weak

+

24

1B

+

+

+

Liquid

+

weak

+

90

1B
-

-

-

1-Thioglycerol
Dihydroeugenol
Imidazolidinylurea

NON-SENS.

Methylmethacrylate2785-877
3923646-9
80-62-6Glycerol

56-81-5

Liquid

-2,4Dichloronitrobenzene

611-06-3

Solid-

Benzyl alcohol

100-51-6

Liquid-nocategorynocategorynocategory

-

NC

-

NC

-

NC

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 18 of 74

Table 4: List of the phase B2 chemicals tested in three independent experiments by each laboratory.
Chemical NameSENSITISERS

State

LLNA

LLNA
potency
category

GP

EC3

GHS
potency
category

Solid+extreme

+

0.001

1A

Liquid+extreme

+

0.009

1A

DPRA
R&D
resulthCLAT
R&D
resultMUSST
R&D
result++++Kathon CG (1.2%
CMI)

7787-566
2617255-4

Chlorpromazine HCl

69-09-0

Solid+strong

+

0.14

1A

Chloramine T

127-65-1

Solid+strong+

0.4

1A

Formaldehyde

50-00-0Liquid+strong

+

0.61

1A

+

+

2Mercaptobenzothiazole

149-30-4

Solid+moderate

+

1.7

1A

+

+

Benzylsalicylate

118-58-1

Liquid+moderate

+

2.9

1B

Nickel chloride

7718-549

Solid

-

no
category+

Benzylcinnamate

103-41-3

Solid

+

weak

+

18.4

1B

R(+)-Limonene

5989-275

Liquid

+

weak

+

69

1B

Beryllium sulfate

NON-SENS.

CAS#

Methylsalicylate

119-36-8

Liquid-Isopropanol

67-63-0Liquid-Dimethylisophthalate

1459-934

Solid-

4-Aminobenzoic acid150-13-0

Solid-

Xylene1330-207

Liquid+nocategorynocategorynocategorynocategory

1B

-

NC

-

-

-

-

NC

-

-

-

-

NC

-

NC

weak

95.8

NC

It is important to note that in view of the primary objective (i.e. assessment of the reliability ofthe test method) of the blind testing phase, no consideration was given to the suggested
applicability domain of the h-CLAT (or the other two in vitro methods evaluated in thevalidation study) in the chemical selection. For example the list contains two wellcharacterised pre-haptens (4-Phenylendiamine and R(+)-Limonene) and a well characterised
pro-hapten (Dihydroeugenol).
Wherever possible, the selection of chemicals was predicated on a consistency of evidence
from human experience, guinea pig tests and the LLNA. Thus for the majority of chemicals
chosen as skin sensitisers, clear human evidence of allergic contact dermatitis was supported
by positive guinea pig maximisation test and/or Buehler test results coupled with a positive
LLNA. Two clear exceptions to this were xylene, a false positive in the LLNA and nickel
chloride, a false negative in the same assay. For xylene, the human evidence of skinsensitisation, while not entirely absent, is of a similar scale to petrolatum and does not meet thecriteria for classification as a skin sensitiser. For nickel chloride, the human evidence of skinsensitisation is abundant.
Two other points should be noted. First, for a few substances (e.g. benzoquinone anddichloronitrobenzene) human data is very limited, but does not contradict the results fromanimal tests. Second, for some of the chemicals which have a “Not Classified” categorisation,
there is evidence that they are human sensitisers (benzyl alcohol, isopropanol, xylene, methyl
salicylate, 4-aminobenzoic acid) and this may be kept in mind when interpreting the test resultswith respect to the test's predictive capacity. However, important also to remember that thisevidence is of limited nature (e.g. isolated case reports), that they are considered to be
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 19 of 74

substances with insufficient human evidence to warrant classification according to REACH
and United Nations GHS guidelines.

5. Chemicals purchase, coding and distribution
Reference document:
Chemical coding, sampling and shipping procedures (Appendix 5)
The chemicals used for the transfer phase (study phase A Stage II) were purchased by thetrained laboratories on the basis of the instructions reported in the training and transfer planissued by Kao and Shiseido and approved by the VMG.
ECVAM was responsible for purchasing, coding and distributing the chemicals for the blind
testing phase (Phase B) to the laboratories participating in the study. The selected chemicals
were sourced from Sigma Aldrich. Aliquots of the chemicals were prepared, coded, labelled
and properly stored in the chemical repository of EURL ECVAM before distribution to the test
laboratories.
a. Solvent Compatibility assessment
To avoid possible problems with the solubility of the test chemicals during the blind testing
phase, all chemicals underwent an assessment of solubility at ECVAM, following the "solvent
selection" procedures as described in the test methods' SOP. In all cases, at least one suitable
solvent was identified. The solvent(s) identified for each chemical was not communicated tothe testing facilities, since the SOP required the testing laboratory to be responsible for theidentification of the suitable solvent(s) (see below).
b. Coding/Decoding
A randomly generated code was assigned to each aliquot, unique for each method, laboratory
and experiment. For the assessment of the WLR, three separate vials of each chemical fromthe list of 15 were sent to the laboratories, each assigned a different two-letter code. A number
added to the code (1, 2 or 3) distinguished the three sets of 15 chemicals to be used for theindividual experiments, and the laboratories were instructed never to mix chemicals labeledwith different experiment numbers in the same run. This ensured that the three evaluations of
the corresponding chemicals were performed in independent runs in order to best provide data
suitable for a proper evaluation of the WLR.
The codes for all chemical aliquots were recorded in a database (Excel spreadsheet format)
prepared and maintained by the Chemical Selection Group. The identity of the chemicals towhich the codes were assigned was not disclosed to the laboratories, and was kept confidentialfrom the VMG and the biostatisticians until the end of the study. Copies of the tables, showing
the codes assigned to the same chemicals but which do not identify the chemicals by name,were prepared to be given to the biostatistician for analysis of the reproducibility, and to the
ECVAM study coordinator(s) to assist the VMG consideration of the experimental data. A
detailed description of the chemical coding and distribution procedures applied is provided inAppendix 5.
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 20 of 74

The Chemical Selection Group provided the VMG with the final decoding list for thechemicals only once all the experimental data had been generated by the laboratories, quality
checked by ECVAM and analysed by the biostatistician for the assessment of the WLR and
BLR. The decoding list was then used by the VMG and the biostatistician to analyse and assess
the information generated in this study on the predictive capacity of the h-CLAT.
c. Emergency procedure implemented at the laboratories during the blind testing
phase.
An emergency procedure was established to allow the laboratories to obtain the necessary
chemical safety information in the event of an accident. Individual sealed envelopes, eachcontaining a Material Safety Data Sheet (MSDS) relating to one specific chemical and labelledwith the corresponding code, were sent with the test chemicals to a named recipient notassociated with the testing (typically the Safety Officer) at each laboratory, with instructions toreturn the unopened MSDSs to ECVAM upon completion of the testing phase.
During the testing of the h-CLAT, no such incident was reported and none of the envelopes
had to be unsealed, and all envelopes were returned to ECVAM, sealed, upon completion of
the validation study.
The laboratories were instructed to treat all coded test chemicals as potential sensitisers and to
dispose of laboratory waste as toxic waste.

6. Data management
Reference documents:
h-CLAT reporting templates (Appendix 6 and 7)
h-CLAT QC template (Appendix 8)
Prior to the start of the study standard reporting templates were prepared and distributed to theparticipating laboratories. These templates were developed by the lead laboratories, andcomprised three different Excel documents:• A template to report the cytotoxicity assays, and calculate the CV75 values (Appendix 6)• A template to report the evaluation runs and calculate the RFI values for all tested
concentrations (Appendix 7)
In addition the laboratories were provided a template they could use to compile the results ofthe three evaluation runs required to apply the prediction model (summary template).
Following a request from the laboratories, another template was prepared by the leadlaboratories to assist in the calculations of the EC150 and EC200 values
The templates each contained formulae verified by the ECVAM biostatistician in adocumented exercise before they were distributed. The laboratories were asked to use thetemplates during the Transfer phase to compile and communicate their results to the leadlaboratories. In this way they could familiarise themselves with them and this ensured that thehuman Cell Line Activation Test (h-CLAT) Validation Study Report

Page 21 of 74

instructions included in the templates were clear and understood before the start of the blind
testing.
All the results from the Phase B were submitted by the laboratories directly and exclusively to
ECVAM by e-mail. Since the templates were not sent protected/locked, upon receipt at
ECVAM each completed template was formally quality controlled according to a checklist (seeAppendix 8). The quality check focused on the acceptance criteria for the run and for each of
the chemicals’ data to ensure the results were valid, and confirm the correct selection of theCV75 concentration. Once completed, the checklists were scanned as a PDF file. The
templates and checklists were then added to the official results folder of the study.
For the statistical analyses, a summary template was designed by the statistician, and the results
were transferred to this template by ECVAM. Preparation of this summary template containedinternal checks that ensured that no transcription errors were made in the transfer of the results.
As an additional check, the final conclusions/outcomes for each chemical were then comparedto the conclusions/outcomes in the reports sent by the laboratories.

7. Statistical Analysis of Experimental Data
Reference document: Experimental Design (Appendix 2)
a. Analysis performed on the experimental data
A detailed statistical analysis plan was produced and agreed by the VMG before the start of thetesting phase (see Appendix 2 for full details). It was stipulated that only data from the valid
experiments would be considered for statistical analyses. Failed runs and experiments werealso reported in order to assess their occurrence and frequency.
The statistical analysis on the test method's reproducibility focused on the concordance ofclassification, sensitizer (S) versus non-sensitisers (NS). Reproducibility was evaluated withrespect to both WLR and BLR.
Additionally the raw data were analysed using descriptive statistical analysis however the
information generated by this analysis was not used by the VMG, on its own, to draw
conclusions on the WLR and BLR of the h-CLAT.
b. Criteria for Assessing the Study Outcome
The VMG considered it appropriate to define in advance indicative assessment criteria to be
used to enable a transparent judgment and decision on the performance of the test methods inconsideration of the study primary goal.
When defining such indicative criteria the following factors were considered important:
1. the background and specific objectives of the validation study;
2. the standards of performance that can realistically be expected from an in vitro test andstandards of performance which have been considered acceptable in previous validation
studies;
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 22 of 74

3. the proposed used of the in vitro tests (i.e. as a partial replacement method to become
part of a testing strategy or a toolbox of tests to be used in combination); and4. the statistical power of the design of the validation study.
In consideration of the above, and in particular the anticipated use of such test methods within
integrated testing strategies or a toolbox of tests to be used in combination, the target
performance for this study was set at 80% for the Between Laboratory Reproducibility and
85% for the Within Laboratory Reproducibility.
The target performance informed the sample size calculation and is consistent with thereproducibility values, in terms of reproducibility in the concordance of predictions, elaborated
from the data submitted by the test developers, in which the h-CLAT showed a between
laboratory reproducibility of 87% (see Appendix 2 for further details).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 23 of 74

Test definition (Module 1)
Reference Documents:
h-CLAT SOPs (Appendices 9 and 10)
1. Intended purpose of the test method
The h-CLAT is a potential partial replacement in vitro test method designed to be part of a
non-animal test battery or integrated testing strategy for assessing the skin sensitisation
potential of chemicals. As such it could contribute to the reduction of the number of animals
used for skin sensitisation testing and to the replacement of current regulatory in vivo tests forskin sensitisation hazard classification and labelling (OECD TG 406, OECD TG 429, OECD
TG 442A, OECD TG 442B). The information the h-CLAT generates can already be used in aweight of evidence approach to support regulatory decision making (e.g. to better characterise
equivocal responses in in vivo studies). For example, REACH permits the use of methods
under validation for this purpose.
2. Evidence demonstrating the need of the test method
This test method is of importance in regard of the European Union ban on in vivo testing ofcosmetic and toiletry ingredients and products implemented by the Seventh Amendment to the
Cosmetics Directive and for the European Regulation on Registration, Evaluation, andAuthorisation of Chemicals (REACH) that requires evaluation of a large number of chemicals.
The successful validation of the h-CLAT and other in vitro assays (e.g. assessing peptide
reactivity) would support their use in the assessment of a chemical’s skin sensitisation potential
and reduce or eliminate the need for animal testing for skin sensitisation.
3. Status of development of the test method
The h-CLAT was developed in a collaborative effort by Kao & Shiseido.
Extensive work had been undertaken since 2003 to optimise the h-CLAT protocol. This
included four inter-laboratory studies, during which critical aspects of the h-CLAT protocol
were investigated and optimal procedural conditions (including criteria for the selection of asuitable lot of cells and Fetal Bovine Serum (FBS) were established (Sakaguchi et al., 2005;
Ashikaga et al., 2008; Kosaka et al., 2008; Sono et al., 2008; Mizuno et al., 2008). In thecontext of this optimisation work the h-CLAT has previously been successfully transferred to 5
Japanese laboratories. Subsequently, the technology was transferred to an additional 3laboratories for the purpose of a ring trial organised by Cosmetics Europe (formerly Colipa)(Sakaguchi et al., 2010). This study aimed at providing insights in the likely within-laboratory
reproducibility, transferability, and between laboratory reproducibility of the test method byevaluating a set of 15 substances with varying degrees of sensitisation potency, including nonsensitisers.
In their test submission to ECVAM (received in April 2009) Kao and Shiseido reported test
data for 100 chemicals (72 sensitisers: 8 extreme, 16 strong, 25 moderate, 23 weak and 28
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 24 of 74

non-sensitisers according to LLNA results) with an accuracy of 84% (sensitivity 88% andspecificity 75%). When compared to human data the h-CLAT was reported to have an
accuracy of 80% (sensitivity 83% and specificity 69%).

4. Scientific basis – biological and/or mechanistic relevance of the h-CLAT
Immature epidermal dendritic cells, the Langerhans cells (LCs), and dermal dendritic cells(DCs) are important mediators in the skin sensitisation process since they are capable ofpresenting the hapten-protein conjugate to responsive T lymphocytes in the lymph nodes
draining the site of exposure (Kimber and Cumberbatch, 1992). The maturation process of
LCs and DCs from antigen processing cells to antigen presenting cells is considered a Key
Event in the acquisition of skin sensitisation as also documented in the Skin Sensitisation
Adverse Outcome Pathway recently developed by the OECD (OECD, 2012a, 2012b).
This maturation process involves the modulation of the expression of cell surface phenotypicmarkers, 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 DC-like
immortalised cell-lines to screen the skin sensitization potential of chemicals.
As described in detail below, the h-CLAT measures by flow-cytometry the modulation of
CD86 and CD54 protein markers on the surface of THP-1 cells following controlled exposure
to the chemical. THP-1 is a human monocytic leukemia immortalised cell line used as asurrogate of DCs.
5. Protocol of the test method
The detailed updated h-CLAT test method protocol used during the Phase BII blind testing
phase is described in Appendix 9.
The h-CLAT is an in vitro cell based assay in which the expression of CD86 and CD54 surface
markers in THP-1 cells are quantified by flow cytometric analysis following 24 hour exposure
to 8 concentrations of the test substance. A propidium iodide (PI) viability assay is performed
prior to the main experiment for the calculation of the CV75 value (estimated concentration oftest substance yielding 75% cell viability) which is then used for the selection of the final
concentrations.
a) Selection of the solvent
The h-CLAT SOP prescribes a procedure to select the appropriate solvent.
In this study, since the three vials containing each of the chemicals that were tested three times
in each laboratory (10-24) were coded independently, the laboratories had to repeat the solvent
selection procedures three times for these chemicals. The choice of solvent was, for thehuman Cell Line Activation Test (h-CLAT) Validation Study Report

Page 25 of 74

majority of cases, consistent within and between the laboratories (see Table 15), suggestingthat the procedure in the SOP is adequately described.
When they occurred, differences in solvent selection did not correlate with differences in
predictions obtained.
b) Determination of the CV75
To conclude on the sensitising potential of a test substance based on the induction of cellsurface markers, the h-CLAT requires the evaluation of a specific range of chemicals
concentrations, which is based on the CV75 value, i.e. the concentration of chemical thatallows 75% of THP-1 cell survival (25% cell toxicity).
The h-CLAT SOP contains a section that explains how this value should be derived, using flow
cytometry and Propidium Iodine (PI) staining.
c) Evaluation runs
One complete experiment requires the generation of three independent evaluation runs. Eachevaluation run of the h-CLAT measures the induction of two independent cell-surface markers:
CD54 and CD86.
Eight concentrations of chemicals are tested, based on the calculated CV75, for a final rangethat corresponds to 1.2xCV75 (highest dose) to 0.335xCV75 (smallest dose). The content ofeach well of treated cells is split in three aliquots for staining with three different antibodies:•

FITC-labelled anti-CD86, to measure the expression (Mean fluorescence Intensity,
MFI) of the CD86 cell marker•

FITC-labelled anti-CD54, to measure the expression (MFI) of the CD54 cell marker•

FITC-labelled mouse IgGI ("isotype"), used to correct the fluorescence CD86 andCD54

For each of the chemical concentration, two values are then calculated: the CD86 and theCD54 RFI (relative fluorescence intensities compared to an untreated control well), using thefollowing formula:
RFI= MFI of chemical-treated cells – MFI of chemical treated isotype control cells X100
MFI of vehicle control cells – MFI of vehicle isotype control cells
The whole evaluation run (each with the 8 test chemical concentrations and the staining for thetwo markers) is repeated THREE TIMES, in independent runs, and a final prediction is derived
based on the obtained RFI values using the following rule:human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 26 of 74

If the RFI of CD86 is ≥ 150 at any dose in at least 2 of 3 independent evaluation runs
AND/OR
If the RFI of CD54 is ≥ 200 at any dose in at least 2 of 3 independent evaluation runs,
THEN
The chemical is considered to be as a sensitiser.
Otherwise, the chemical is considered to be a non-sensitiser.
The following two tables show examples how the prediction model is applied:

For chemical D3SS, the three runs were negative for CD86, and only the second run showed
induction of CD54 above the threshold (200) indicated by the red numbers. As the conditions
to be considered a sensitiser are not met, the h-CLAT classifies this chemical as non-sensitiser.
In contrast, for chemical D3QX, the chemical is labelled as a sensitiser since two of the threeruns showed induction of CD54 above the threshold.
For chemicals considered to be sensitisers, this threshold-based prediction model allows thecalculation of two Effective Concentrations (EC) values, EC150 for CD86 and EC200 for
CD54, i.e. the concentration at which the chemical induces a RFI of 150 or 200. Note thatsince the prediction model requires only at least one of the two markers to be positive, it is notalways possible to derive both EC150 and EC200 values for positive chemicals.
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 27 of 74

The SOP submitted to ECVAM did not contain descriptions or formulae to calculate therelevant EC values. A proposal was subsequently made by the lead laboratories and, with theconsent of the VMG, included in the SOP for the blind testing phase covering most of thepossible concentration-response scenarios. Although the h-CLAT SOP was not developed or
adapted with the aim of a precise derivation of EC values, the EC values obtained during thestudies are reported in the within- and between-laboratory reproducibility sections, below.
d) Modifications made to original protocol prior to the initiation of thevalidation study
Following the ECVAM assessment of the original h-CLAT protocol, revisions to the SOP weremade in collaboration with Kao and Shiseido prior to the initiation of the validation study toclarify sections of the SOP which were felt not clear enough and could thus impact on theinterpretation and performance of the protocol. This work resulted in the release of SOP
Version 2 which Kao and Shiseido then used to train the naïve laboratories (Study Phase AI).
SOP Version 3 aimed to clarify procedural aspects following the training stage and subsequentdiscussions with the laboratories and was released for use during the transfer of the method(Study Phase AII). SOP Version 4, then 5, were prepared and issued based on the experience ofthe transfer phase at IVMU and Bioassay. Version 5 was released for use during the blind
testing phases. However, following the experience of Phase B1, one clarification, resulting in
Version 6, was added to Version 5 to address special cases that occurred during the testing
phase but were not correctly covered by Version 5 of the SOP.
The SOP includes a requirement for the adoption of the evaluation runs that produced anegative response: the viability at the highest concentration should be less than 90%. This ismeant to prevent the possibility that, in the event of an incorrect CV75 determination,
inappropriate concentrations are used in the evaluation runs. When cell viability criteria are
not met, the SOP requires the run to be considered invalid and the CV75 re-evaluated.
However, some chemicals are not cytotoxic even at the maximum soluble or allowed
concentrations (1000 µg/ml in DMSO, and 5000 μg/ml in saline). The instructions to reevaluate the CV75 for these chemicals in the event they produced negative results wasquestioned (the issue was reported by one of the study directors). Communications with thelead laboratories clarified that, historically, the results in these cases were accepted, and thiswas clarified in the SOP (version 6) and applied for this study.
All modifications to the SOP were made in close collaboration with the lead laboratories tomake sure that such modifications did not affect the outputs of the h-CLAT procedure itself, inways which could invalidate or compromise the historical data generated with the h-CLAT
prior to the submission to ECVAM.
Version 7 was prepared and released by the VMG at the end of the validation study to include
the provision to allow performing more than one run with the same chemical on the same day
and the conditions for this. This version of the SOP is the one suggested for future use of the hCLAT.
A summary of the protocol revisions is outlined in Table 5.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 28 of 74

Table 5: Description of the main modifications made to each version of the SOP.

I. Establishing themethodVersion 1

Version 2

Version 3

Version 4

Version 5

Version 6

Version 7

Added list ofreagents

Clarifiedconcentrations ofPenicillin andStreptomycin

Added BSA,
volumetric flasks,
and glass vials
suppliers

Added that morethan oneconcentration ofchemicals can beused in thereactivity checks,
with instructions

Added that if in
the first
reactivity check
SLS is positive,
the reactivity
check can be
repeated before
dismissing thebatch of cells.

Changed thenegative chemicalin the reactivity
check from SLS
to Lactic acid
although testing
SLS was still
requested

Removedrequirement totest SLS in thereactivity check.

Added cells
thawing, freezing
and maintenanceinstructions

Clarifiedcentrifugation
speedsAdded cell
doubling timeevaluation
procedures
2. Dose finding
assay (PI assay)

Clarified
reactivity checkprocedure
Added sample
plate setup for
CV75
determination
assay
Added cell
viability
calculation
formulaAdded minimuml
chemicalconcentrations fordose-finding assay

Clarifiedchemicalssolubilisation
procedure

Added maximum
number of thechemicals that canbe tested at thesame in the dosefinding assay

Clarified that thecells and reagentsshould be kept in
the cold and in thedark during thestaining
procedures

Added example
flow cytometry
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 29 of 74

plotsSet the number ofCV75determination
assays to be
performed
3. Experiment(measuring
CD86/CD54
expression)

Clarification of
the CD86/CD54
staining
procedure,
including sample
plate setup andmaster mixes
preparations
Clarification of
the flow
cytometryacquisition
procedure,
including
Excitation andEmission
wavelengths
Description of thecytometer plot
and gate settings,
includingexamples.
Instructions on

Clarified starting
dose inCD86/CD54
staining when
CV75 could not be
determined

Clarifiedchemicalssolubilisation
procedure
Clarified that thecells and reagentsshould be kept in
the cold and in thedark during thestaining
procedures
Clarified that thetwo independent
PI assays shouldbe performed on
different days
Clarified that thenegative controls
should include
both a medium
control sample
and a vehicle

human Cell Line Activation Test (h-CLAT) Validation Study Report

Clarification that
the CD86/CD54
inductionmeasurement
should be doneusing the samebatch of THP-1
cells as used for
the CV75
determination
Added that morethan one dose of
positive control
can be used forthe experiment.
Added setting
instructions for
the FACSAria(BectonDickinson) flow
cytometer
Added
instructions ofPage 30 of 74

Clarification on
the possibility toperform morethan one run per
chemical on thesame day and theconditions forthis.

what to do in caseof unqualified
tests

control

4. Data analysis
and interpretation

Clarification thatthe cell viability
at the highest does
should be morethan 90% only inthe case ofnegative results.
Inclusion of a
maximumallowed numberof runs allowed totest a given
chemical.

propermaintenance ofthe flow
cytometers
Added
requirement for
qualified testing:In the DMSO
vehicle control,
RFI values
compared to themedium control
of both CD86 andCD54 should notexceed thepositive criteria("CD86 > 150 and
CD54 > 200")
Added section on"abnormal values"

5. Data reporting

Instructions on
how to calculate
EC150 and
EC200
Instructions fordata reporting.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 31 of 74

Added
requirement for
qualifiedtesting: For
both medium
and DMSO
controls, theMFI ratio of
both CD86 andCD54 toisotype control
should be >105%.

Clarified thatwhen 5000µg/mL in saline,
1000 μg/mL in
DMSO, orhighest soluble
dose is used as
the maximal test
concentration
instead of CV75based dose, thedata for test
chemical are
acceptedregardless of thecell viabilitye) Acceptance criteria
The h-CLAT SOP, as submitted to ECVAM by the lead laboratories, contained a set of
acceptance criteria for the evaluation runs to determine whether the results obtained are valid.
Following the VMG evaluation of the SOP prior to the start of the study, it was not felt
necessary to propose additional criteria and no modification was made to this section of the
SOP.
The testing laboratories raised no issues with respect to meeting these criteria during the study
as demonstrated by the relatively low rate of invalid runs which was consistent between thelaboratories (see Tables 19 and 20).
f) Requirement for a third evaluation run
The h-CLAT SOP requires three independent evaluations runs to be performed to apply theprediction model. Since the classification is driven by the majority of the conclusions of thethree runs (i.e. at least two out three) the VMG discussed prior to the start of the study whether
the third run was required when the first two runs give consistent conclusions (i.e. twonegatives or two positives runs) since in that case the result of the third run can have no effecton the final classification. Although it was appreciated that eliminating the requirement of thethird run in such cases would decrease the amount of work involved, the VMG did notconsider necessary to modify this element of the h-CLAT SOP, thus maintaining therequirement for a third run. This allowed the generation a full dataset for the chemicals
evaluated in the study.
Additional analysis requested and performed in parallel by Japanese biostatisticians, (seeAppendix 13) suggests that the third run is important in case of positive predictions to refine
the derivation of the EC values. This may be important for the purpose of using the h-CLAT
for informing potency predictions.
However, the VMG agreed that, for future use of the h-CLAT in cases where it is used forhazard identification purposes, the SOP could be adapted to eliminate the need for the third
evaluation run in case the first two runs are consistent without compromising test sensitivity
and specificity.
6. Known technical limitations and drawbacks of the test method
The following limitations were reported by the lead laboratories in their submission to
ECVAM:-The test method requires expensive equipment (flow cytometer).

-

When cell viability is less than 50%, flow cytometric analysis cannot be conducted
correctly because of artefacts arising from diffuse labelling cytoplasmic structures due
to cell membrane destruction.

-

To avoid osmotic stress, the maximum concentration of test chemical cannot exceed
5000 μg/mL.

-

Since the h-CLAT uses a fluorescently labelled antibody, self-fluorescence of test
chemicals might interfere with the flow cytometry acquisition (e.g. Abietic acid).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 32 of 74

-In the h-CLAT test method the test substance needs to be dissolved in a solvent
compatible with cell culture conditions (i.e. saline or DMSO). In the submission to
ECVAM chemicals reported not to be correctly classified because of solubility
problems include: Hexil cinnamic aldehyde, Abietic acid and Phthalic anhydride.

In relation to this last point it is important to note that for the purpose of the current study noneof the chemicals selected had to be discarded because of solvent incompatibility consistent
with the preliminary solubility assessment performed by ECVAM (see Section 5a above). This
was also consistent with the fact that no major problem was subsequently encountered by thelaboratories during the solubility assessment for the blind testing phase.
7. Limitations in applicability
Some chemicals, designated “pro-haptens”, require a metabolic transformation step to act as
haptens capable of activating the immune system. If the in vitro test system (i.e. the THP-1
cells in the h-CLAT assay) does not provide adequate metabolic activity, the detection of suchpro-haptens may be impaired. The THP-1 cells used as the test system in the h-CLAT assay
have indeed been reported by the protocol submitter and others (for example Hennen et al.,2011; Chipinda et al., 2011) to possess only a limited or no metabolic activity. Moreover,some chemicals (designated “pre-haptens”) need a non enzymatic oxidation step to act as
haptens. Depending on the chemistry involved and the oxidation conditions pertaining during
the performance of the assay, such molecules might not be transformed into active haptens andwill thus not be detected as potential sensitizers by the h-CLAT assay. For example, Benzoyl
peroxide, Isoeugenol and Abietic acid are reported to be false negatives in the h-CLAT.
Assays using a threshold based prediction model (h-CLAT uses EC150 for CD86 or EC200
for CD54) have an inherent limitation in the detection of some weak sensitisers. The cell
activation induced by very weak sensitizers may remain just below the thresholds set for apositive result and the molecules will not be correctly classified (e.g. 1-Bromohexane,
Cyclamen aldehyde, Butyl glycidyl ether).
Finally, a general limitation of assays using suspension cell culture techniques concerns thesolubility of the test substance in an aqueous environment compatible with cell survival.
Therefore, when no toxic effect is observed up to the highest test concentration allowed in theassay, a careful examination of the solubility data should be performed to ensure that the test
substance has indeed been solubilised. Negative test results obtained with poorly or insoluble
substances should not be considered sufficient for classification decisions.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 33 of 74

Conclusion of the Validation Management Team on Module 1
The h-CLAT test method addresses a biological mechanism, the modulation of the expressionof cell surface phenotypic markers associated with the maturation of DCs, which is considered
to be a key step in the skin sensitisation AOP.
Information provided by the h-CLAT on a chemical’s potential to activate dendritic-like cells
in vitro can complement information generated by other non animal approaches (in silico, in
chemico, in vitro) designed to address chemical/biological mechanisms preceding andfollowing the activation of DC in the sequence of events leading to the induction of skinsensitisation. This supports the use of the h-CLAT as mechanistically relevant element of atesting strategy for skin sensitsation.
The need for the method in the context of current EU regulatory requirements is evident. The
proposed use of the methods as mechanistically-based partial replacement method to be usedin conjunction with other data for hazard identification purposes is plausible.
The h-CLAT protocol (as revised prior to the initiation of the blind testing phase) proved to begenerally robust for the purposes of this study, only minor clarifications were made to the SOP
during the course of the study in relation to specific elements of the procedure and the datainterpretation to minimise the sources of variability.
The amendments introduced were largely to resolve ambiguities and minor omissions in theoriginal SOP in order to improve clarity and consistency of data generation and interpretation.
The VMG believes that the procedural clarifications to the h-CLAT original SOP, thesupporting documents (including the original submission to ECVAM and associated scientificpublications) and the current study findings adequately demonstrate the status of development
and optimisation, the mechanistic basis, the intended use and, the regulatory relevance of theh-CLAT.
In conclusion, the VMG believes that Module 1, Test Method Definition, is now satisfied.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 34 of 74

Transferability (Module 3)
Reference documents:
h-CLAT Training and Transfer Plan (11)List of additional available documents filed for the study and available on
request (Appendix 15)
1. General aspects
The h-CLAT can be performed in laboratories equipped with and experienced in cell culture
and flow cytometry techniques. All apparatus/instruments and reagents needed for the
performance of the method are readily available commercially.
Kao and Shiseido, being the lead laboratories and having jointly developed the method, were
responsible for both the training of the personnel at the other testing facilities participating inthe study and for overseeing and providing advice during the test method transfer in order tomake sure that the procedure for performing the h-CLAT as described in the SOP was clearly
understood and properly implemented.
Specialists from both Kao and Shiseido provided and evaluated training to the study personnelof Bioassay and EURL ECVAM at the Food and Drug Safety Center of the Hatano ResearchInstitute, Kanagawa, Japan (Phase A Stage I). The newly trained personnel were thenresponsible for the transfer of the test method to their own laboratories under the supervisionof the lead laboratories (Phase A Stage II).
The schedule for the training of these laboratories as well as the details of the transfer
experiments were drafted by the lead laboratories on the basis of their experience with the test
method. To demonstrate successful method transfer, the laboratories had to perform the test
method procedure by testing in-house a number of chemicals and meet the transfer acceptancecriteria as defined in the Transfer Plan (Appendix 11). The chemicals used for thesequalification runs, as well as the criteria for a successful achievement of the transfer of themethod, were selected by the lead laboratories, and approved by the VMG prior to theinitiation of the training.
The chemicals used for this phase A were not supplied by ECVAM, but were purchased by thetrained laboratories on the basis of the instructions set out in the training and transfer plan
(Appendix 11). All chemicals were tested uncoded, and the results sent directly to the leadlaboratories for evaluation.
2. Training
Bioassay and EURL ECVAM personnel received theoretical and procedural training at the
Hatano Research Institute. The training sessions took place between March 1st and March 5th,
2010. The training included a theoretical component with discussions on different theoreticalaspects (SOP; critical aspects of the procedure; data analysis and reporting), and practicalsessions where the trainees observed and performed the different elements of the h-CLAT

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 35 of 74

SOP (PI assays, cell passaging, plating and treatment, flow cytometry staining andacquisition).
For the practical training sessions, two test chemicals were chosen by the lead laboratories fortesting by the trainees: DNCB and SLS
Dose finding assay (cytotoxicity test):
To determine the appropriate test concentrations of the two sample chemicals (DNCB as
positive chemical and SLS as negative chemical), cytotoxicity testing was conducted by eachlab following a demonstration. The trainees from each laboratory conducted this experimentand the mean values were calculated to derive the test application concentrations. The CV75
of DNCB was 6.9 (6.6 and 7.3) mg/mL. The CV75 of SLS was 39.9 (38.1 and 41.8) mg/mL.
The value for EURL ECVAM and Bioassay were very similar, and consistent with leadlaboratories' historical data.
CD86 and CD54 measurements:
The trainees from each laboratory conducted CD86/CD54 measurement of DNCB and of
SLS-treated THP-1 cells. In terms of DNCB, the requirement for qualified testing was
satisfied in all experiments. The results of both naïve laboratories were, correctly, positive. For
SLS, the expression levels of both CD86 and CD54 were below the cut-off points.
Trainings sessions for Bioassay and EURL ECVAM were judged to be successful by thetrainers in a Statement on training outcome in the Training Report.
3. Transfer of the test method to the naïve laboratories
As part of the Training and Transfer plan, Kao and Shiseido requested that trainees thenacquire the following list of chemicals to evaluate the establishment of the assay in theirlaboratories:
Table 6: List of transfer chemicals for the h-CLAT.
Chemical nameabbreviation

Cas#

Commercial source

Cat#

LLNA
EC3

Potency category

2,4-Dinitrochlorobenzene

DNCB

97-00-7

Aldrich2373290.04

Extreme

Nickel sulfate

NiSO4

10101-97-0

Sigma-Aldrich

N4882

4.8

Moderate

Phenylacetoaldehyde

PAA

122-78-1

Aldrich1073953Moderate

Sodium lauryl sulfate

SLS

151-21-3

Sigma-Aldrich

L6026

14

Weak (false positive)

Lactic acidLA50-21-5

Sigma

L6661

NC

Non-sensitizer

The transfer of the h-CLAT was separated into phases covering the different steps of the SOP.
After each phase, the laboratories were asked to report the results to the lead laboratories foranalyses and permission to proceed.
The schedule was planned as follows:
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 36 of 74

Week
1-2
3-4
5
6-7
8-9

Experiments
Doubling time, cell viability, reactivity
Cytotoxicity test for the 5 chemicals
CD86 and CD54 expression (MFI raw data)and cell viability of control (untreated) cells
Valid data (3 runs) for DNCB and SLS
Valid data (3 runs) for the other threechemicals

The success of the transfer was judged based on the results obtained for the fivechemicals, with the following criteria:
All three sensitizers (DNCB, Nickel Sulfate and phenylacetaldehyde) must produce a
positive response in both CD86 and CD54 (i.e. for both CD86 and CD54, at least 2 out of
3 runs should be positive).
Both non-sensitizers (SLS, Lactic acid) must produce a negative response in both CD86
and CD54 (i.e. for both CD86 and CD54, at least 2 out of 3 runs should be negative).
Note that, in the case of the positive chemicals, the transfer criteria is different from, and more
stringent than, the h-CLAT prediction model, where only one of the two markers has to bepositive for the chemical to be declared positive (sensitiser).
The sections below summarize the results of the transfer phases at Bioassay and EURLECVAM. Details on the results can be found on the corresponding transfer reports, available
upon request (see Appendix 15).
1. Transfer to Bioassay.
Having allowed the laboratory the necessary time to acquire all the reagents andbuy/expand/bank the THP-1 cells, the transfer experiments (described in the table above)
started on the 10th of May, 2010. No major issues arose during the first steps of the transfer
phase. DNCB, SLS, NiSO4 and PAA were correctly evaluated in consecutive runs.
However, problems arose in the testing of Lactic Acid, as the second and third runs showed
unexpected CD54 activation. Eight additional runs were required to solve the issue and obtain
the data required in the transfer plan. Comparison of different lactic acid batches did notsuggest that this was an issue with a particular batch. A site visit of lead laboratories
representatives was organised to investigate the problem at Bioassay. The visit did not
identify major problems in the performance of the assay.
The main procedural issue arising from the site visit related to the setup of the flow cytometryat Bioassay, which used a micro plate autosampler. Since with this system the time lap
between samples acquisition is longer than without the autosampler, the problem encountered
might have been due to prolonged sample storage at room temperature leading to variations in
the RPMI control results that might have caused the unexpected results with Lactic Acid.
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 37 of 74

To remedy this problem, Bioassay subsequently tested a single substance per plate, with its
own control wells. While one plate was being analysed, the other one(s) were kept on ice inthe dark. This setup reduced the throughput of the testing at Bioassay, but allowed thecompletion of the transfer phase, and was maintained during the whole testing phase.
Additional runs performed following the visit confirmed the consistent negative results with
Lactic Acid, leading to approval of the transfer at Bioassay by the lead laboratories. Based on
this recommendation of the lead laboratories, the VMG approved the transfer of the h-CLAT
at Bioassay on the 16th of December 2010.
2. Transfer to EURL ECVAM
Having allowed time to acquire all the reagents and buy/expand/bank the THP-1 cells, thetransfer experiments (described in the table above) started on 26th of April 2010. No major
issues arose during the first steps of the transfer phase and the testing of the sensitisingchemicals. However, difficulties in obtaining negative results for the non-sensitising
substances (in particular SLS) led to additional experiments and required a site visit from leadlaboratories representatives in October 2010. The visit failed to identify the cause of the
problem.
It was suggested that the problem could be due to the higher sensitivity of the FACS Aria flow
cytometer used by EURL ECVAM. An important difference between the FACS Aria andother flow cytometers used in the validation study is that the FACS Aria uses a fully digitalprocessing of the signals, while the flow cytometers used by the other participating
laboratories are analog. Despite this difficulty in obtaining consistent negative results with
SLS, the lead laboratories felt that, as the issue was limited to this chemical, the h-CLAT had
been successfully transferred at EURL ECVAM. Based on this recommendation, the VMG
approved the transfer on the 24th of January, 2011.
The issue of the digital versus analog cytometer was subsequently investigated by additional
studies from the lead laboratories (see additional considerations).
3. Second Transfer to EURL ECVAM
At the time they were given permission to proceed on the basis of the results above, EURLECVAM was in the process of acquiring a new flow cytometer, a FACS CANTO II.
EURL ECVAM requested permission to delay the start of the testing phase in order to use thismachine instead of the FACS Aria, as they felt it would greatly increase the throughput of thetesting and still allow EURL ECVAM to complete the testing in the requested timeframe.
This request was discussed by the VMG during the 5th VMG meeting, and agreed to, with thefollowing conditions:••

All the testing for the blind testing phases (BI and BII) should be performed using thesame flow cytometer
To use the new cytometer for phase B, a full set of successful transfer experiments
should first be performed with the new FACS CANTO II machine.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 38 of 74

•

Initiation, and completion, of Phase B testing should not be unreasonably delayed.

EURL ECVAM successfully transferred the h-CLAT to the new flow cytometer between the
14th of February and the 11th of March 2011, and was given the official permission to start thetesting on April 11th, 2011. No problems surfaced during this second set of transfer
experiments.

4. Additional considerationsA) Although the exact value of the CV75 concentrations determined by the laboratories were
not part of the criteria established for successful transfer, it was observed that the values werevery reproducible, as shown in Table 7.
Table 7: Summary of the CV75 values obtained for the 5 transfer chemicals during the Transfer phaseof the study.

Chemical

Bioassay

DNCB
SLS
NiSO4
Lactic Acid
PAA

5.8
46.7 / 60.6
160.2
2707
33.1

EURL ECVAM(FACS Aria)
9.7 / 13.5
60.2 / 58.1
198.0 / 216.5
2822.8 / 2990.2
52.6

EURL ECVAM(FACS CANTO II)
8.7
54.7
156.7
3010.646.6B) In its transfer report, EURL ECVAM recommended the VMG to consult a flow cytometry
expert to consider in more detail the possible differences in sensitivity between analogue anddigital instruments, as this was felt to be a factor potentially causing the frequent positive
results obtained by EURL ECVAM with the non-sensitising chemicals (all the otherlaboratories in the study, as well as the laboratories that developed the h-CLAT, use analog
flow cytometers).
Following discussions during the 6th VMT meeting, Kao and Shiseido agreed to investigate
this issue and to discuss with the Japanese branch of Becton Dickinson. In addition,
experiments were conducted in the lead laboratories to compare the results from an analog
flow cytometer (BD FACS Calibur) and a digital flow cytometer (BD FACS Canto II), testing
DNCB and SLS.
The results of these discussions and study was presented during the 9th VMT meeting, where
the lead laboratory representatives concluded that no significant differences were observed
when comparing the results generated by both cytometers (seeFigure 3).

Interestingly the results show that for SLS higher concentrations result in a positive signal,emphasizing the importance of correctly determining the CV75 for this chemical.
human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 39 of 74

Figure 3: Results of the study comparing the FACS CantoII (digital) and FACS Calibur (analog)
cytometers.

DNCB and SLS are also used in the h-CLAT for the qualification of freshly thawed cultures of
THP-1 cells ("reactivity checks") prior to using them for experiments. In view of the dose
response curves observed for these chemicals, i.e. the fact that SLS can become positive and
DNCB negative at high doses, the VMG, at the suggestion of the lead laboratories, authorizeda modification of the reactivity checks procedure to allow each laboratory to determine theiroptimal chemical concentration to be used, based on an evaluation of the CV75. In addition,
the lead laboratories proposed the use of Lactic Acid instead of SLS in the reactivity checks,
as Lactic Acid showed, historically, a lower rate of false positive results with the h-CLAT.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 40 of 74

Conclusion of the Validation Management Team on Module 3
The VMG agreed that both laboratories succeeded in transferring the h-CLAT protocol to theirfacilities and, in the case of EURL ECVAM, using two different flow cytometers.
The settings of the flow cytometer posed no particular challenges, and were set after the initial
reactivity checks experiments. The different batches of THP-1 cells purchased by thelaboratories caused no concerns for the success of the transfer, consistent with publishedinformation (Kosaka et al., 2008).
Both laboratories generated the required experiments in 3 successive runs for the three
sensitisers. The difficulties at both sites related to the negative chemicals, SLS and Lactic
Acid, which were solved following site visits by representatives of the lead laboratories. Thisemphasizes the importance of transfer experiments in new laboratories that would include
these chemicals to ensure correct implementation of the protocol.
In general the VMG recommends the use of a careful step-wise approach similar to the oneused in the study for future implementations of the test method in new laboratories.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 41 of 74

Within-laboratory reproducibility (Module 2)
Reference documents:
Statistical report (Appendix 12)
List of additional documents filed for the study and available on request(Appendix 15)
The within laboratory reproducibility was assessed using the data generated with a subset of
15 chemicals tested in three independent experiments in each laboratory (Study Phase B2). As
described in the h-CLAT SOP, each of the independent experiments is composed of threevalid evaluation runs to derive a final conclusion.
To minimise bias in the generation of the results for the assessment of the WLR thelaboratories were provided with 3 different vials for each chemical (with different codes), one
for each experiment.
For this reason the laboratories had to perform a separate solvent determination (see section on
BLR) and CV75 determination prior to the performance of the evaluation runs; that is, it wasdone on three separate occasions for each chemical.
The WLR of the h-CLAT was assessed on the concordance of classification, sensitiser (S)versus non-sensitiser (NS) between the three independent experiments. Additionally,
descriptive analyses were performed to evaluate the reproducibility of the three respective
CV75 values and on the calculated EC values, where appropriate. The descriptive analysis ispresented for information only and was not used by the VMG, on its own, to draw conclusions
on the WLR of the h-CLAT
The data are presented laboratory by laboratory and the three independent assessments of eachchemical required for the evaluation of the WLR are referred to as experiment 1, experiment2, and experiment 3.
1. Kaoa. Reproducibility (concordance of predictions)
In relation to the primary objective, the reproducibility in terms of classification S versus NS,
the same prediction in the 3 independent experiments was obtained for 13 of the 15 chemicals
resulting in a WLR of 86.7%. The two chemicals for which the classification was notconcordant across experiments were Benzyl salicylate (chem.16) and Methyl salicylate (chem.
19).
Chemical

Prediction (run1, run2, run3)

10

Exp 1
PPP

Kathon CG (CMI/MI)

Exp 2
PPP

human Cell Line Activation Test (h-CLAT) Validation Study Report

Exp 3
PPP

Agreement
Betweenexperiment
Yes

Page 42 of 74

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

Beryllium sulfate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2-Mercaptobenzothiazole
Benzyl salicylate
Benzyl cinnamate
R(+)- Limonene
Methyl salicylate
Isopropanol
Dimethyl isophthalate
4-Aminobenzoic acid
Nickel chloride
Xylene

NNN
PPP
PPP
PPP
PPP
NNN
NNN
PPP
PPN
NNN
NPN
NNN
PPP
NNN

NNN
PPP
PPP
PPP
PPP
PPN
NNN
PPP
NNN
NNN
NNN
PNN
PPP
NNN

NNN
PPP
PPP
PPP
PPP
NPN
NNN
PPP
NPP
NNN
NPN
NNN
PPP
NNN

Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes

Table 8: concordance in the classifications sensitisers (S) verus non-sensitiser (NS) obtained in three
independent experiments at Kao with the 15 chemicals (chem. 10-24) used for the evalution of the
WLR.

b. Reproducibility of CV75 values
Figure 4 shows the distribution of the CV75 determinations performed as part of the three
independent experiments for each of the 15 chemicals. CV75 values for chemicals 11(Beryllium sulphate), 17 (Benzyl cinnamate), 20 (Isopropanol), 21 (Dimethyl isophthalate), 22(4-Aminobenzoic acid) and 24 (Xylene) could not be derived because of their low cytotoxicity
and for this reason the highest concentration used for these chemicals was the maximum one
allowed by the SOP (i.e. 5000 µg/mL in medium/saline or 1000 µg/mL in DMSO), or the
maximum soluble concentration (chemical 21, Dimethyl isophthalate, whose maximum
concentration tested was 500 µg/mL in DMSO). For more information refer to Appendix 12(Statistical report).
Notwithstanding that different cell batches and passage numbers have been used, thecalculated CV75 are consistent across experiments.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 43 of 74

Figure 4: CV75 determinations expressed in µg/mL performed within three individual experiments forthe 15 chemicals (chem. 10-24). Each dot corresponds to the mean of 2 or more independent CV75
determinations.

c. Reproducibility of EC values
The concentration estimated to produce an RFI of 150 or 200 for CD86 (EC150) and CD54
(EC200), respectively are presented in Figure 5. For more information, refer to Appendix 12(Statistical report).
For CD86, 10/15 (66.7%) chemicals were consistently negative (RFI<150 for allconcentrations) or positive (RFI ≥150 for at least one concentration) in the three independent
experiments. For CD54, 12/15 (80%) chemicals were consistently negative (RFI<200 for allconcentrations) or positive (RFI ≥200 for at least one concentration) in the three independent
experiments.
Where it was possible to calculate them, the EC150 and EC200 values were very reproducible.
Raw RFI data and the corresponding viability for all concentrations for each chemical arepresented in Annex II of the statistical report (see Appendix 12).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 44 of 74

Figure 5: EC150 (CD86) and EC200 (CD54) concentrations for the three independent experiments
performed at Kao

2. Shiseido
a. Reproducibility (concordance in predictions)
In relation to the primary aim, the reproducibility in terms of classification S versus NS, thesame prediction in the 3 independent experiments was obtained for 12 of the 15 chemicals
resulting in a WLR of 80%. The three chemicals for which the classification was notconcordant across experiments were Kathon CG (chem. 10), Beryllium Sulphate (chem. 11)
and Formaldehyde (chem. 12).
Chemical
10
11
12
13

Prediction (run1, run2, run3)
Kathon CG (CMI/MI)
Beryllium sulfate
Formaldehyde
Chloramine T

Exp 1
PPNA
NPP
NNP
PPP

Exp 2
PPP
PPN
PPP
PPP

human Cell Line Activation Test (h-CLAT) Validation Study Report

Exp 3
PPP
NPN
PPP
PPN

Agreement
BetweenExperiments
No
No
No
Yes

Page 45 of 74

14
15
16
17
18
19
20
21
22
23
24

Chlorpromazine HCl
2-Mercaptobenzothiazole
Benzyl salicylate
Benzyl cinnamate
R(+)- Limonene
Methyl salicylate
Isopropanol
Dimethyl isophthalate
4-Aminobenzoic acid
Nickel chloride
Xylene

PPP
PPP
NNN
NPP
PPP
NNP
NNN
NNN
NNN
PPP
NNP

PPP
PPP
PNN
PPN
PPP
PNN
PNN
NNN
NNN
PPP
NNN

PPP
PPP
NNP
PPP
PPP
NNN
NNN
NNN
NNN
PPP
NNN

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

Table 9: concordance in the classifications sensitisers (S) verus non-sensitiser (NS) obtained in three
independent experiments at Shiseido with the 15 chemicals (chem. 10-24) used for the evalution of the
WLR. A This chemical resulted positive in one experiment for the CD54 an in another for the CD86,
therefore the final prediction corresponds with a non-sensitizer.

b. Reproducibility of CV75 values
Figure 6 shows the distribution of the CV75 determinations performed as part of the three
independent experiments for each of the 15 chemicals. CV75 values for chemical 11(Beryllium sulphate), could be determined only in experiments 2 and 3 since in experiment 1
the same chemical did not show sufficient cytotoxicity. Also for chemicals 20 (Isopropanol),
22 (4- Aminobenzoic acid) and 24 (Xylene) the CV75 values could not be derived because of
their low cytotoxicity and for this reason the highest concentration used for these chemicals
was the maximum one allowed by the SOP (i.e. 5000 µg/mL in medium/ saline or 1000
µg/mL in DMSO). For more information, refer to Appendix 12 (Statistical report).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 46 of 74

Figure 6: CV75 determinations expressed in µg/mL performed within three individual experiments forthe 15 chemicals (chem. 10-24). Each dot corresponds to the mean of 2 or more independent CV75
determinations.

c. Reproducibility of EC values
The concentration estimated to produce an RFI of 150 or 200 for CD86 (EC150) and CD54
(EC200), respectively are presented in Figure 7. For more information, refer to Appendix 12(Statistical report).
For CD86, 9/15 (60%) chemicals were consistently negative (RFI<150 for all concentrations)or positive (RFI ≥150 for at least one concentration) in the three independent experiments. For
CD54, 11/15 (73%) chemicals were consistently negative (RFI<200 for all concentrations) or
positive (RFI ≥200 for at least one concentration) in the three independent experiments.
Where it was possible to calculate them, the EC150 and EC200 values were very reproducible.
Raw RFI data and the corresponding viability for all concentrations for each chemical arepresented in Annex II of the statistical report (see Appendix 12).

Figure 7: EC150 (CD86) and EC200 (CD54) concentration for the three independent experiments
performed at Shiseido.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 47 of 74

3. Bioassay
a. Reproducibility (concordance in predictions)
In relation to the primary aim, the reproducibility in terms of classification S versus NS, thesame prediction in the 3 independent experiments was obtained for 11 of the 15 chemicals
resulting in a WLR of 73.3%. The four chemicals for which the classification was notconcordant across experiments were Formaldehyde (chem. 12), Benzyl salicylate (chem. 16),
Methyl salicylate (chem. 19) and Xylene (chem. 24).
Chemical

Prediction (run1, run2, run3)

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

Exp 1
PPP
NNP
NPN
PPP
PPP
PPP
NPN
NNN
PPP
PPP
NNP
PNN
NNN
PPP
PPN

Kathon CG (CMI/MI)
Beryllium sulfate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2-Mercaptobenzothiazole
Benzyl salicylate
Benzyl cinnamate
R(+)- Limonene
Methyl salicylate
Isopropanol
Dimethyl isophthalate
4-Aminobenzoic acid
Nickel chloride
Xylene

Exp 2
PPP
NNN
PPP
PPP
PPP
PPP
NNN
NNN
PPP
NNN
NNN
NNN
NNN
PPP
NNP

Exp 3
PPN
NNN
PPP
PPP
NPP
PPP
PPN
NNN
PPP
NPN
NNN
NPN
NNN
PPP
PNN

Agreement
BetweenExperiments
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
No

Table 10: concordance in the classifications sensitisers (S) verus non-sensitiser (NS) obtained in three
independent experiments at Bioassay with the 15 chemicals (chem. 10-24) used for the evalution of the
WLR.

b. Reproducibility of CV75 values
Figure 8 shows the distribution of the CV75 determinations performed as part of the three
independent experiments for each of the 15 chemicals. CV75 values for chemicals 11(Beryllium sulphate), 17 (Benzyl cinnamate), 20 (Isopropanol), 22 (4-Aminobenzoic acid) and24 (Xylene) could not be derived because of their low cytotoxicity and for this reason thehighest concentration used for these chemicals was the maximum one allowed by the SOP (i.e.
5000 µg/mL in medium/saline or 1000 µg/mL in DMSO), or the maximum soluble
concentration (chemical 11, Beryllium sulphate, whose maximum concentration tested was
500 µg/mL in DMSO). CV75 values chemical 21 (Dimethyl isophthalate) could behuman Cell Line Activation Test (h-CLAT) Validation Study Report

Page 48 of 74

determined only in experiments 2 and 3 since in experiment 1 the chemical did not show
sufficient cytotoxicity. For more information, refer to Appendix 12 (Statistical report).

Figure 8: CV75 determinations expressed in µg/mL performed within three individual experiments forthe 15 chemicals (chem 10-24). Each dot corresponds to the mean of 2 or more independent CV75
determinations.

c. Reproducibility of EC values
The concentration estimated to produce an RFI of 150 or 200 for CD86 (EC150) and CD54
(EC200), respectively are presented in Figure 9. For more information, refer to Appendix 12(Statistical report).
For CD86, 11/15 (73.3%) chemicals were always negative (RFI<150 for all concentrations) or
positive (RFI ≥150 for at least one concentration) in the three independent experiments. For
CD54, 9/15 (60%) chemicals were always negative (RFI<200 for all concentrations) or
positive (RFI ≥200 for at least one concentration) in the three independent experiments.
Raw RFI data and the corresponding viability for all concentrations for each chemical arepresented in Annex II of the statistical report (see Appendix 12).
Where it was possible to calculate them, the EC150 and EC200 values were very reproducible.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 49 of 74

Figure 9: EC150 (CD86) and EC200 (CD54) concentration for the three independent experiments
performed at Bioassay.

4. EURL ECVAM

a. Reproducibility (concordance in predictions)
In relation to the primary aim, the reproducibility in terms of classification S versus NS, thesame prediction in the 3 independent experiments was obtained for 12 of the 15 chemicals
resulting in a WLR of 80%. The three chemicals for which the classification was notconcordant across experiments were Chlorpromazine hydrochloride (chem. 14), Benzyl
cinnamate (chem. 17), and Dimethyl isophthalate (chem. 24).
Chemical
10
11
12
13
14

Prediction (run1, run2, run3)
Kathon CG (CMI/MI)
Beryllium sulfate
Formaldehyde
Chloramine T
Chlorpromazine HCl

Exp 1
PPP
PNP
PPP
PPP
PPP

Exp 2
PPN
PPP
PPP
PPP
PPP

human Cell Line Activation Test (h-CLAT) Validation Study Report

Exp 3
PPP
PPN
PPP
PPP
NPN

Agreement
BetweenExperiments
Yes
Yes
Yes
Yes
No

Page 50 of 74

15
16
17
18
19
20
21
22
23
CHEM 24

2-Mercaptobenzothiazole
Benzyl salicylate
Benzyl cinnamate
R(+)- Limonene
Methyl salicylate
Isopropanol
Dimethyl isophthalate
4-Aminobenzoic acid
Nickel chloride
Xylene

PPP
PPP
PPP
PPP
PPP
NNN
NNP
NNN
PPP
PPP

PPP
PPP
NNP
PPP
PPP
NNN
PPN
NNN
PPP
PPP

PPP
PPP
NNN
PPP
PNP
NNN
NPPA
NNP
PPP
PPP

Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes

Table 11: concordance in the classifications sensitisers (S) verus non-sensitiser (NS) obtained in three
independent experiments at EURL ECVAM with the 15 chemicals (chem. 10-24) used for theevalution of the WLR. AThis chemical resulted positive in one experiment for the CD54 an in anotherfor the CD86, therefore the final prediction corresponds with a non-sensitizer.

b. Reproducibility of CV75 values
Figure 10 shows the distribution of the CV75 determinations performed within each of thethree independent experiments for each of the 15 chemicals. CV75 values for chemicals 18(R(+)- Limonene), 20 (Isopropanol), 22 (4-Aminobenzoic acid) and 24 (Xylene) could not bederived because of their low cytotoxicity and for this reason the highest concentration used forthese chemicals was the maximum one allowed by the SOP (i.e. 5000 µg/mL in medium/saline or 1000 µg/mL in DMSO), or the maximum soluble concentration (chemical 18, R(+)Limonene, whose maximum concentration tested was 120 µg/mL in DMSO). CV75 values forchemical 11 (Beryllium sulphate), chemical 17 (Benzyl cinnamate) and chemical 21(Dimethyl isophthalate) could be determined only in experiments 2, 1-2, and 1-2 respectively
since in the other experiments the chemicals did not show sufficient cytotoxicity. For more
information, refer to Appendix 12 (Statistical report).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 51 of 74

Figure 10: CV75 determinations expressed in µg/mL performed within three individual experiments
for the 15 chemicals (chem. 10-24). Each dot corresponds to the mean of 2 or more independent CV75
determinations.

c. Reproducibility of EC values
The concentration estimated to produce an RFI of 150 or 200 for CD86 (EC150) and CD54
(EC200), respectively are presented in Figure 11. For more information, refer to Appendix 12(Statistical report)
For CD86, 12/15 (80%) chemicals were always negative (RFI <150 for all concentrations) or
positive (RFI ≥150 for at least one concentration) in the three independent experiments. For
CD54, 14/15 (93.3%) chemicals were always negative (RFI <200 for all concentrations) or
positive (RFI ≥200 for at least one concentration) in the three independent experiments.
Raw RFI data and the corresponding viability for all concentrations for each chemical arepresented in Annex II of the statistical report (see Appendix 12).
Where it was possible to calculate them, the values of the EC150 and EC200 values were very
reproducible.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 52 of 74

Figure 11: EC150 (CD86) and EC200 (CD54) concentration for the three independent
experiments performed at EURL ECVAM.

Summary
The following table shows the chemicals that were not consistently classified within thelaboratories. Of the eight chemicals which were not consistently classified in one laboratory
three of them (Formaldehyde, Benzyl salicylate and Methyl salicylate), gave discordant results
also in a second laboratory.
Table 12: chemicals with inconsistent classifications within laboratories.
Kao

Shiseido
Kathon CG
Beryllium sulphate
Formaldehyde

WLR
Bioassay

EURL ECVAM

Formaldehyde
Chlorpromazine HCl

Benzyl salicylate

Benzyl salicylate
Benzyl cinnamate

Methyl salicylate

Methyl salicylate
XyleneDimethyl isophtalate

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 53 of 74

Conclusion of the Validation Management Team on Module 2
The main focus of the within laboratory reproducibility (WLR) for the subset of 15 chemicals
in each laboratory was on the concordance of the predictions sensitiser (S) versus nonsensitiser (NS) as determined by the results of three independent experiments. The WLR for
Kao, Shiseido, EURL ECVAM and Bioassay for the S/NS predictions were 86.7%, 80%, 80%
and 73.3% respectively.
Only one of the participating laboratories met the target performance proposed at the outset ofthe study (85%).
However, the VMG agreed, that the obtained WLR was sufficient considering the potential
use of the method in a toolbox or integrated testing strategies, taking into consideration thefact that:• The BLR met the target performance set by the VMG prior to the initiation of the blind
testing phase (see section below)• There is a consistency in the chemicals that showed discrepancies in the WLR and theBLR (see table 14).
• It was noted that some of the chemicals responsible for the discrepancies are suggested
to fall outside the applicability domain of the h-CLAT (in relation to the Log Kow, seesection on predictive capacity).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 54 of 74

Between laboratory reproducibility (Module 4)
Reference documents:
Statistical report (Appendix 12)
List of additional documents filed for the study and available on request(Appendix 15)
1. Reproducibility (concordance in predictions)
The between laboratory reproducibility (BLR) was assessed on the basis of the results
generated with 24 chemicals (9 chemicals tested once and 15 chemicals tested independently 3
times in each laboratory). The main focus of the evaluation of the between-laboratory
reproducibility was on the concordance of the predictions sensitisers (S) versus non-sensitisers(NS). As discussed in the WLR section, descriptive statistical analyses were also performed on
the EC values, but were not considered by the VMG, on their own, to determine to what extent
the primary objective of the study had been satisfied.
For the evaluation of the BLR, the final prediction for the chemicals that were tested 3 times(chemicals 10 to 24) in each laboratory was based on the median classification obtained in thatlaboratory. For example, for Chlorpromazine hydrochloride at EURL ECVAM the predictions
obtained were "sensitiser", "sensitiser" and "non-sensitiser", so the median classification was"sensitiser".
Nineteen of the 24 chemicals were consistently classified (S or NS) by the 4 laboratories
resulting in a BLR of 79.2% (Table 13). The five chemicals which were not consistently
classified by the laboratories were: Beryllium sulphate (chem. 11), Benzyl salicylate (chem.
16), Benzyl cinnamate (chem. 17), Methyl salicylate (chem. 19) and Xylene (chem. 24).
Since BLR is usually reported less stringently as the consistency of prediction between 3laboratories, which is one fewer than the number of testing laboratories in this study due to theparticipation of 2 lead laboratories, the VMG suggested, in the experimental design document
drafted prior to the initiation of the testing phase, that two BLR values would be derived: onecomparing the consistency of the two naïve laboratories with the first lead laboratory (Kao)and the second comparing the consistency of the two naïve laboratories with the other leadlaboratory (Shiseido). The values obtained in these comparisons (83.3% and 79.2%) arecomparable to the reproducibility value calculated when all four laboratories are considered
together.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 55 of 74

Table 13: Concordance in S versus NS predictions between the laboratories.
Kao

Shiseido

Bioassay

EURL
ECVAM

Agree

Kao versus
BA &
EURL
ECVAM

1
2
3
4
5
6
7
8
9
10

Benzoquinone
PPD
Dihydroeugenol
Thioglycerol
Imidazolidinyl urea
Methyl methacrylate
Glycerol
DCNB
Benzyl alcohol
Kathon CG (CMI/MI)

S
S
S
S
S
NS
NS
S
S
S

S
S
S
S
S
NS
NS
S
S
S

S
S
S
S
S
NS
NS
S
S
S

S
S
S
S
S
NS
NS
S
S
S

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

Shi
versus
BA &
EURL
ECVA
M
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

11
12
13
14
15

Beryllium sulfate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2-Mercaptobenzothiazole

NS
S
S
S
S

S
S
S
S
S

NS
S
S
S
S

S
S
S
S
S

No
Yes
Yes
Yes
Yes

No
Yes
Yes
Yes
Yes

No
Yes
Yes
Yes
Yes

16
17
18

Benzyl salicylate
Benzyl cinnamate
R(+)- Limonene

NS
NS
S

NS
S
S

NS
NS
S

S
NS
S

No
No
Yes

No
Yes
Yes

No
No
Yes

19
20
21
22
23

Methyl salicylate
Isopropanol
Dimethyl isophthalate
4-Aminobenzoic acid
Nickel chloride

S
NS
NS
NS
S

NS
NS
NS
NS
S

NS
NS
NS
NS
S

S
NS
NS
NS
S

No
Yes
Yes
Yes
Yes

No
Yes
Yes
Yes
Yes

No
Yes
Yes
Yes
Yes

24

Xylene

NS

NS

NS

S

No

No

No

79.2%

83.3%

79.2%

Interestingly all of the five chemicals which were not consistently classified between thelaboratories gave also discordant results, within at least one of the laboratories, in the three
independent experiments performed for the evaluation of the WLR (see Table 12). As
observed earlier, this tends to suggest that the observed performance may be enhanced bycareful determination and consideration of the applicability domain of the h-CLAT.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 56 of 74

Table 14: chemicals with inconsistent classifications within and between laboratories.
Kao

Shiseido
Kathon CG
Beryllium sulphate
Formaldehyde

WLR
Bioassay

BLR
EURL ECVAM
Berillium sulphate

Formaldehyde
Chlorpromazine HCl

Benzyl salicylate

Benzyl salicylate

Benzyl salicylate
Benzyl cinnamate
Methyl salicylate
XyleneBenzyl cinnamate
Methyl salicylate

Methyl salicylate
XyleneDimethyl isophtalate

2. Other considerationsa) Solvent selection
It was decided by the VMG prior to the initiation of the study, and after the preliminary
ECVAM confirmation of solubility had been performed, that the participating laboratories
would not be instructed by the VMG on which solvent to use to solubilise the codedchemicals. The VMG considered the solvent selection procedures to be an integral part of thetest method SOP, to be evaluated for reproducibility together with the rest of the procedure.
The SOP provided a tiered solvent selection strategy. Since the chemicals that were tested
three times in each laboratory (9-24) were coded independently, the laboratories had to repeat
the solvent selection procedures for each test (that is, three separate, independent
determinations for each chemical).
The choice of solvent was, for the majority, consistent within and between the laboratories,
suggesting that the procedure in the SOP is adequately described.
Discrepancies in the prediction obtained did not correlate with different solvents chosen.
Table 15: Solvent selection for each of the chemicals by the four laboratories. Cells with grey
background correspond with different vehicles used for dissolving the same chemical within and/or
between laboratories.
Chemical
1
2
3
4
5
6
7
8

Benzoquinone
PPD
Dihydroeugenol
Thioglycerol
Imidazolidinyl urea
Methyl methacrylate
Glycerol
DCNB

Kao

Shiseido

Bioassay

VAM

DMSO

DMSO

SalineDMSO

SalineSaline

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

SalineSalineSalineSalineSalineSalineSalineSaline

DMSO

DMSO

DMSO

DMSO

SalineSalineSalineSaline

DMSO

DMSO

DMSO

DMSO

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 57 of 74

9Benzyl alcohol

10

SalineSalineKathon CG (CMI/MI) DMSO (1) Saline (2,3) Saline
DMSO (2,3) Saline (1) Saline
DMSO (2,3) Saline (1) DMSO (3) Saline (1,2)
Beryllium sulfate
DMSO(3)
Saline(1,2)
SalineSalineSalineFormaldehyde
SalineSalineSalineSalineChloramine T
SalineSalineChlorpromazine HCl DMSO (3) Saline (1,2) Saline
2DMSO
DMSO
DMSO
DMSO
Mercaptobenzothiazole
DMSO
DMSO
DMSO
DMSO
Benzyl salicylate
DMSO
DMSO
DMSO
DMSO
Benzyl cinnamate
DMSO
DMSO
DMSO
DMSO
R(+)- Limonene
DMSO
DMSO
DMSO
DMSO
Methyl salicylate
SalineSalineSalineSalineIsopropanol
DMSO (1,3) Saline (2) DMSODMSO
Dimethyl isophthalate DMSO
DMSO
DMSO
DMSO
4-Aminobenzoic acid DMSOSalineDMSO(1)Saline(2,3)
SalineSalineNickel chloride
DMSO
DMSO
DMSO
DMSO
Xylene

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

DMSO

DMSO

DMSO

DMSO

b) Determination of the CV75

To correctly conclude on the skin sensitisation potential of a test substance based on the
induction of cell surface markers, the h-CLAT requires the exposure of the cells to a specificrange of chemicals concentrations, the selection of which is based on the CV75 values for thischemical, i.e. the concentration of chemical that allows 75% of THP-1 cell survival (25% cell
toxicity).
The SOP of the h-CLAT contains a section that explains how this value should be derived,
using flow cytometry and Propidium Iodine (PI). At least two experiments are needed todetermine a CV75 value to be used in the evaluation runs.
Although this value is not strictly part of the h-CLAT prediction model, it can have an effecton the final conclusion, as the induction of the cell markers by a chemical is highly dependent
on its concentration. Efforts were made by the VMG prior to the initiation of the study toclarify and optimize this section in the SOP, and the CV75 determined by each of thelaboratories for the blind tested chemicals were compiled. As for the solvent selection, since
the replicates used to evaluate the WLR were coded independently, the laboratories had to
apply the CV75 determination procedure for each of three independent experiments. Figure 12
show the CV75 obtained by each laboratory during the study. For more details, refer toAppendix 12 (Statistical report).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 58 of 74

Figure 12: CV75 determinations expressed in µg/mL performed within the individual experiments byeach laboratory for the 24 chemicals. Each dot corresponds to the mean of 2 or more independent
CV75 determinations.

The determination of the CV75 was not considered by the VMG for the formal evaluation of
the BLR. However, considering that the laboratories were using a different lot of the THP-1
cells, the calculated CV75 for each chemical proved to be highly consistent within and acrosslaboratories suggesting that the procedure described in the SOP for defining the dose range tobe used in the evaluation runs is robust and can be consistently reproduced when performed indifferent laboratories.
c) Reproducibility of the EC150 and EC200 values
A summary of the EC150 concentrations (CD86) and EC200 concentrations (CD54) is given
in Figure 13. In case a chemical resulted in an RFI ≥ 150 for at least one concentration, thecorresponding EC150 concentrations obtained in the four laboratories were generally of thesame magnitude. For some chemicals the threshold value of RFI ≥ 150 was not obtained in alllaboratories; this was the case for chemical 3, 10, 13, 15, and 18. The EC200 concentrations
were also of the same magnitude in the four laboratories except for some chemicals were thethreshold value of RFI ≥ 200 was not reached in all laboratories; this was the case forchemical 8, 11, 17, 19, and 24.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 59 of 74

Figure 13: EC150 (CD86) and EC200 (CD54) concentration for the independent experiments of thedifferent laboratories. Phase B1 (chemical 1 to 9), every chemical was tested in one experiment. PhaseB2 (chemical 10 to 24), every chemical was tested in three independent experiments.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 60 of 74

Conclusion of the Validation Management Team on Module 4

The main focus of the between laboratory reproducibility (BLR) for the 24 chemicals was on
the concordance of the predictions sensitisers (S) versus non-sensitisers (NS) between the four
laboratories.
Since two of the laboratories were lead laboratories, the VMG decided prior to the initiation of
the testing phase to evaluate the BLR by comparing the two naïve laboratories with each of thetwo lead laboratories separately (see Appendix 2, Experimental Design). The BLR for the
S/NS prediction of the two naïve laboratories and Kao was 83.3%, while the BLR for the twonaïve laboratories and Shiseido was 79.2%, for an average of 81%.
The BLR comparing all four laboratories was 79.2%.
The VMG agreed that the BLR results were adequate for the proposed use of the test as acomponent of integrated testing strategies for skin sensitisation testing.
The values were also consistent with the target performance proposed at the onset of the study(80%).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 61 of 74

Predictive Capacity (Module 5)
Reference documents:
Statistical report (Appendix 12)
Analysis of h-CLAT historical data to categorise chemicals in three classes(Appendix 13)
Analysis of h-CLAT validation study data to categorise chemicals in threeclasses (Appendix 14)
List of additional documents filed for the study and available on request(Appendix 15)
The assessment of the predictive capacity forms only a secondary goal of the present
validation study, not least since the sample size was determined for the purpose of satisfying
the primary study goal and does not permit robust conclusions to be drawn on the predictive
capacity of the h-CLAT.
The analysis of the predictive capacity was performed using the results from all the 24
chemicals. An overview of the predicted classification and the reference classification ispresented in Table 16.
The predictive capacity was evaluated for each laboratory (Table 17). As for the evaluation of
the BLR, for chemicals that were tested three times in each laboratory, the medianclassification for each laboratory was chosen for this analysis.
This resulted in an accuracy for S/NS classifications of 76% for all laboratories (Table 18),
with individual results ranging from 70.8% (Kao) to 83.3% (Shiseido). The two naïve
laboratories each had an accuracy of 75%.
The nine chemicals previously tested in the h-CLAT were all consistently and accurately
identified with the only exception of Methyl Salicylate (misclassified at Kao and EURLECVAM) suggesting that the behaviour of the h-CLAT in this study was consistent withhistorical and published information. Accuracy for the 15 chemicals not previously tested was
65% (39 out of 60, 15 chemicals in 4 laboratories). The results for these latter chemicals
should contribute to a future and more comprehensive assessment of predictive accuracy,
since no meaningful or robust conclusions on this parameter can be drawn from the limited
sample size of this study alone.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 62 of 74

Table 16: Agreement between the predicted class and the reference class.
ChemicalR&D
Kao Shiseido

1
2
3
4
5
6
7
8
9

Benzoquinone
PPD
Dihydroeugenol
Thioglycerol
Imidazolidinyl urea
Methyl methacrylate
Glycerol
DCNB
Benzyl alcohol

10
11
12
13
14

Kathon CG (CMI/MI)
Beryllium sulfate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2Mercaptobenzothiazole
Benzyl salicylate
Benzyl cinnamate
R(+)- Limonene
Methyl salicylate
Isopropanol
Dimethyl isophthalate
4-Aminobenzoic acid
Nickel chloride
Xylene

15
16
17
18
19
20
21
22
23
24

S
S

S
NS

S
S

S

NS
NS

Kao

S
S
S
S
S
NS
NS
S
S

Shiseido

Bioassay

EURL
ECVAM

Prediction based on 1 experiment
S
S
S
S
S
S
S
S
S
S
NS
NS
NS
NS
S
S
S
S

S
S
S
S
S
NS
NS
S
S

Reference+ (1A)+ (1A)+ (1B)+ (1B)+ (1B)+ (1B)- (NC)- (NC)- (NC)

Prediction based on median of 3 experimentsS
S (2/3)SS
NSS (2/3)NSSSS (2/3)S (2/3)
S
S
S
S
S
S
S
S
S (2/3)+ (1A)+ (1A)+ (1A)+ (1A)+ (1A)S

S

S

S+ (1A)NS (2/3)
NSS
S (2/3)
NS
NS
NS
S
NS

NS
S
S
NS
NS
NS
NS
S
NS

NS (2/3)
NSS
NS (2/3)
NSNSNSS
NS (2/3)

S
NS (2/3)
SS
NSNS (2/3)
NSS
S+ (1B)+ (1B)+ (1B)- (NC)- (NC)- (NC)- (NC)+ (NA)- (NA)

Chemical 10 to 24: (2/3) means that two times the same prediction was obtained, in case nothing is mentioned the same
prediction was obtained for the three independent experiments.

Table 17: Predictive capacity of the h-CLAT for each laboratory.
Reference result

Kao+ (n=16)- (n=8)
Total

+
12
3
15

Sensitivity
Specificity

75.0
62.5

Shiseido
4
5
9

+
14
2
16

Bioassay
2
6
8

87.5
75.0

human Cell Line Activation Test (h-CLAT) Validation Study Report

+
12
2
14
75.0
75.0

4
6
10

EURL
ECVAM
+
14
2
4
4
18
6
87.5
50.0
Page 63 of 74

Accuracy
70.8
83.3
75.0
75.0
Table 18: Overall predictive capacity of the h-CLAT (cumulative over the four laboratories).
Reference result+ (n=64)- (n=32)
Total

Cumulative
+
52
12
11
21
63
33

Sensitivity
Specificity
Accuracy

81.3
65.6
76.0

Potency subcategorisation
Within the GHS classification scheme, there is the possibility to refine the evaluation of skinsensitisers on the basis of their potency. Skin sensitisers can be assigned to subcategory 1A“strong sensitisers” or to subcategory 1B “other skin sensitisers” using a weight of evidence
approach on the basis of reliable and good quality data from human cases or epidemiologicalstudies and/or observations from appropriate studies in recognised and accepted animal tests.
In the case of the LLNA a threshold defined for the EC3 value is used to subcategorise skinsensitisers into the two subcategories.
In order to address this secondary study goal the VMG made a request to Dr Omori and Dr
Yoshimura, statisticians based in Japan, to analyse the lead laboratories' h-CLAT historical
data and propose a prediction model for potency subcategorization. The proposal that wasmade uses the MIT, i.e. the lowest value between the calculated EC150 and EC200 of eachchemical to assign it to a GHS subcategory (see Appendix 13). Specifically if the MIT isequal or less than 13μg/mL then the chemical would be classified as category 1A; otherwise it
would be classified as 1B. Applying the MIT cut-off of 13μg/mL to the historical data theaccuracy in correctly assigning a chemical to one of the three categories is reported to be 72%.
This proposal is similar to the one that has been previously published (Nukada et al., 2012),
however the MIT cut-off value used is different.
When applied to the results of the validation, a MIT cut-off value of 13μg/mL gave aconcordant rate of 56.5% in assigning a chemical to one of the three categories (NScorresponding to the GHS NC category = Not Classified and subcategories 1A and 1B). A
MIT of 16 μg/mL applied to the study results produced a slightly more accurate classification(58%) (see Appendix 14).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 64 of 74

Conclusion of the Validation Management Team on Module 5
Secondary goal a) A preliminary evaluation of the ability of the h-CLAT to reliably
discriminate skin sensitising (S) from non-sensitising (NS) chemicals
In our study, the sensitivity of the h-CLAT was 81.3% and the specificity was 65.6%,
resulting in an accuracy of 76%. The VMG concludes that, in the current study, the predictive
accuracy is lower than previously published information on the predictive capacity of the hCLAT (Accuracy of 84%, with sensitivity of 88% and specificity of 75% (Ashikaga et al.,2010). However, the VMG notes that, in the current study, all the GHS 1A chemicals (withthe exception of Beryllium sulphate) were accurately and consistently identified as sensitisers.
It is important to note that the chemicals chosen for the study reported here included a subsetof chemicals previously reported as tested in the h-CLAT, and that those chemicals gave
consistent classifications compared to the published information.
The assessment of predictive capacity forms only a secondary goal of the present validation
study, not least since the sample size was defined for the assessment of the within and between
laboratory reproducibility and does not permit a robust conclusion to be drawn for predictive
capacity.
In addition, it has been anticipated that the in vitro assays designed to address a specific eventof the skin sensitisation pathway are expected to be used as part of a toolbox or an integrated
testing strategy (ITS). It is envisaged that predictive capacity will need to be assessed on thebasis of the information generated by a future ITS. The information presented in this reportmust be understood and considered in this context.
Secondary goal b) Preliminary consideration of the ability of the h-CLAT to contributeto sub-categorisation of skin sensitising chemicals, e.g. into Sub-category 1A and Subcategory 1B as adopted in the 3rd revised version of the GHS (UN, 2009).
Based on the accuracy of GHS subcategorisation of the proposal made by Dr Omori and Dr
Yoshimura, the VMG agreed that the initial results were encouraging, and that further
evaluations will be necessary to determine how information generated with the h-CLAT cansuccessfully contribute to potency subcategorisation, as described above, in the context of an
integrated testing strategy (ITS).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 65 of 74

Additional observations
Log Kow and applicability domain of the h-CLAT
Consideration is being given to better define the applicability domain of the h-CLAT. A
proposal was recently made by the lead laboratories to consider relatively low water solublecompounds, i.e. chemicals with a Log Kow higher than 3.5, to be prone to false negative
results in the h-CLAT and thus to fall outside the applicability of the method (Takenouchi etal., accepted for publication). In our study, the chemicals with a log Kow higher than 3.5 aretabulated below.
14
16
17
18

Chemical
Chlorpromazine hydrochloride
Benzyl salicylate
Benzyl cinnamate
R(+)-Limonene

1

Predicted data generated using the US Environmental
http://www.epa.gov/opptintr/exposure/pubs/episuite.htm

Log Kow1
5.4
4.31
4.06
4.57
Protection

Agency’s

EPISuite™,

Of the four chemicals with a Log Kow higher than 3.5, Chlorpromazine hydrochloride andR(+)-Limonene (chem.14 and 18) have been consistently and accurately classified assensitisers by all the four laboratories whereas Benzyl salicylate and Benzyl cinnamate (chem.
16 and 17) were in the majority of cases false negatives with discordant prediction in one of
the four laboratories (EURL ECVAM and Shiseido respectively).
Run acceptance criteria
The h-CLAT SOP describes a set of acceptance criteria for the evaluation runs to determine
whether the results are valid. For a given run to be considered valid the following conditions
should be met otherwise the run should be discarded and the chemical re-tested.
Viability:-

Medium controls: viability should be > 90%.
DMSO controls: viability should be > 90%.
DNCB controls: viability should be > 50%

CD54 and CD86 RFI values:
DMSO should be negative for both markers
DNCB should be positive for both markers
The following table (Table 19) shows the occurrence of invalid runs per laboratory for theblind testing phases (phase BI and BII).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 66 of 74

Table 19: Overview of valid and invalid runs by laboratory.

Laboratory

Kao
Shiseido
Bioassay
EURL ECVAM

Number of runs
Total
69
102
174
69

Proportion
invalid

Valid
65
99
162
64

Invalid
4
3
12
5

5.8%
2.9%
6.9%
7.2%

The following table (Table 20) shows a breakdown of the reasons for the invalidation of theruns by laboratory and by acceptance criteria.

Table 20: Invalid runs - reason for rejection.
Laboratory
MediumKao
Shiseido
Bioassay
EURL ECVAM
Total runs

Viability
DMSO

DNCB
1

DMSO
CD86 CD54
1

2

2

1

4
3
8

DNCB
CD86
CD54
3
1
3
8
2
1
6
12

The proportion of invalid runs was generally and consistently low between laboratories(<7.5%) and for the majority of the runs that were rejected the acceptance criteria were not
met because the positive control (DNCB) gave RFI values below the threshold.

Control results
A complete analysis of the control results obtained for all the runs generated during the study
can be found in Annex I of the statistical report (Appendix 12).

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 67 of 74

VMG overall conclusions and recommendations
Overall Conclusions
The primary aim of this validation study was to assess the transferability, within laboratory
and between laboratory reproducibility of the h-CLAT with a number of relevant codedchemicals that were judged by the VMG to be suitable and sufficiently challenging to permit
robust conclusions to be drawn.
The VMG considers that the information generated in the study completes and satisfies theinformation requirements for modules 1-4 (test definition, within laboratory reproducibility,transferability, between laboratory reproducibility) of the ECVAM modular approach tovalidation (Hartung et al., 2004). In addition the information generated contributes to, butdoes not on its own satisfy, module 5 (predictive capacity) and module 6 (applicability
domain) for which a substantial body of information is already available as evidenced in thematerial submitted to ECVAM and published in the scientific literature.

The main conclusions of the VMG in relation to each module are set out in the table below:

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 68 of 74

Module1

Test definition

Summary & Conclusions
Both the existing body of evidence (original submission to
ECVAM including scientific publications) and the current study
findings adequately demonstrate the intended purpose, the needfor, the status of development, and the mechanistic basis of the hCLAT test method.
An improved, well-detailed and robust SOP is available.

2

Withinlaboratory
reproducibility

The overall within laboratory reproducibility was considered to be
acceptable for the proposed use of the h-CLAT (i.e. as part of an
integrated testing strategy).
The test method was shown to be transferable between
laboratories.

3

Transferability

Training and demonstration of competence in the conduct of theassay is however considered important. In particular, the chemicals
used during this study's transfer phase should be considered.

4

Betweenlaboratory
reproducibility

The between laboratory reproducibility was acceptable.

5

Predictive
capacityComplete evaluation of the predictive capacity was not one of thegoals of this study.

Overall, the VMG concludes that the information generated in this validation study shows thatthe h-CLAT is a robust and reliable test method that can contribute to the determination of thesensitisation potential of substances. Consequently:• Information generated by the h-CLAT can already be used in a weight-of-evidence
approach to support regulatory decision making, e.g. to characterise equivocal
responses in in vivo studies (e.g. conflicting results from multiple studies).
• For the purposes of some regulations (for example REACH in the EU) a positive hCLAT result should be considered sufficient to classify a test material as a skinsensitiser.
• The h-CLAT is suitable for further evaluation as a component of a toolbox or an ITS
for full replacement of the in vivo assays for skin sensitisation hazard identification.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 69 of 74• The consistent determination of EC values observed for the two markers suggest thatthey may play a role in the determination of skin sensitisation potency, including GHS
sub-categorisation.

Recommendations• The predictive accuracy of the h-CLAT should be evaluated in terms of its contribution
to an integrated testing strategy for full replacement of current in vivo hazard
identification assays.
• Considering the outcome of the study and in particular the consistency of the results
obtained with the chemicals tested previously, and the fact that the submitted SOP wasamended during the study solely to provide clarifications on the procedure that was
described, it is suggested that existing/historical results are taken into account for
future formal evaluations on the predictive capacity of this test methods.
• GHS sub-categorisation of sensitisers should form part of a wider assessment, as on thebasis of these results it is envisaged that h-CLAT EC150 and EC200 values mightprovide useful information contributing to this purpose.
• For hazard classification purposes, the SOP can be adapted to reduce resource costs byeliminating the need for a third evaluation run in case the first two runs are consistent.
• Further consideration of the applicability domain of the h-CLAT and its place in
integrated testing strategies are merited.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 70 of 74

List of Appendices

Appendix 1
Appendix 2
Appendix 3
Appendix 4
Appendix 5
Appendix 6
Appendix 7
Appendix 8
Appendix 9
Appendix 10
Appendix 11
Appendix 12
Appendix 13
Appendix 14
Appendix 15

Project Plan version 5
Experimental Design version 1
Chemical Selection report
Report on the selection of the 9 chemicals for Phase BI
Chemical coding, sampling and distribution procedures
Template for CV75 determination
Template for evaluation runs
h-CLAT results Quality Check templateh-CLAT SOP version 6
h-CLAT SOP version 7
h-CLAT Training and Transfer Plan version 2
Statistical report
Analysis of h-CLAT historical data to categorise chemicals in three classes
Analysis of h-CLAT validation study data to categorise chemicals in three classes
List of additional documents filed for the study and available on request

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 71 of 74

List of References
Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau
A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E,
Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S,
Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa
J, Leite SB, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, PothA, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, vanDelft J, van Loveren H, Vinken M, Worth A, Zaldivar JM. (2011). Alternative (non-animal) methods
for cosmetics testing: current status and future prospects-2010. Archives of Toxicology 85(5):367-485.
Ashikaga, T., Sakaguchi, H., Okamoto, K., Mizuno, M., Sato, J., Yamada, T., Yoshida, M., Ota, N.,
Hasegawa, S., Kodama, T., Okamoto, Y., Kuwahara, H., Kosaka, N., Sono, S., Ohno, Y. (2008).
Assessment of the human Cell Line Activation Test (h-CLAT) for skin sensitization; results of the firstJapanese inter-laboratory study. AATEX, 13: 27–35.
Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama
N, Itagaki H. (2010). A comparative evaluation of in vitro skin sensitisation tests: the human cell-line
activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim, 38: 275-84.
Chipinda I, Ruwona TB, Templeton SP, Siegel PD. (2011). Use of the human monocytic leukemia
THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten
sensitizers. Toxicology. 27:135-43.
EC (2008). Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16
December 2008 on classification, labelling and packaging of substances and mixtures, amending andrepealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006.
Official Journal No L 353, p. 0001-1355.
Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, Patlewicz GY, Basketter DA.
(2005). Compilation of historical local lymph node data for evaluation of skin sensitization alternative
methods. Dermatitis. 16: 157-202.
Hartung, T., Bremer, S., Casati, S., Coecke, S., Corvi, R., Fortaner, S., Gribaldo, L., Halder, M.,
Hoffmann, S., Janusch Roi, A., Prieto, P., Sabbioni, E., Scott, L., Worth, A. and Zuang, V. (2004). A
modular approach to the ECVAM principles on test validity. ATLA 32, pp. 467–472.
Hennen, J., Aeby, P., Goebel, C., Schettgen, T., Oberli, A., Kalmes, M., Blömeke, B., (2011. Cross
talk between keratinocytes and dendritic cells: impact on the prediction ofsensitization. Toxicol Sci.
123: 501-10.
Kern PS, Gerberick GF, Ryan CA, Kimber I, Aptula A, Basketter DA. (2010). Local lymph node data
for the evaluation of skin sensitization alternatives: a second compilation. Dermatitis. 21: 8-32.
Kimber I, Cumberbatch M. (1992). Dendritic cells and cutaneous immune responses to chemicalallergens. Toxicol Appl Pharmacol. 117: 137-46. Review.
Kosaka, N., Okamoto, Y., Mizuno, M., Yamada, T., Yoshida, M., Kodama, T., Sakiko, S., Ashikaga,T., Sato, J., Ota, N., Hasegawa, S., Okamoto, Y., Kuwahara, H., Sakaguchi, H., Ohno, Y. (2008). A
study of the criteria for selection of THP-1 cells in the human Cell Line Activation Test (h-CLAT).
Results of 2nd Japanese inter-laboratory study. AATEX, 13: 55-62.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 72 of 74

Mizuno, M., Yoshida, M., Kodama, T., Kosaka, N., Okamoto, K., Sono, S., Yamada, T., Hasegawa, S.,
Ashikaga, T., Kuwahara, H., Sakaguchi, H., Sato, J., Ota, N., Okamoto,Y., Ohno, Y. (2008). Effects ofpre-culture conditions on the human Cell Line Activation Test (h-CLAT) results: Results of the 4thJapanese inter-laboratory study. AATEX 13: 70-82.
Nukada, Y., Ashikaga, T., Miyazawa, M., Hirota, M., Sakaguchi, H., Sasa, H., Nishiyama, N. (2012).
Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT)
and an attempt at classifying skin sensitization potency. Toxicology In Vitro. 26: 1150-60.
OECD (2005). Guidance document on the validation and international acceptance of new or updated
test methods for hazard assessment. OECD series on testing and assessment No. 34.
ENV/JM/MONO(2005)14.Accessible at: http://www.oecd.org/
OECD (2010). OECD Guideline for the Testing of Chemicals No. 429: Skin Sensitisation: Local
Lymph Node Assay. Paris, France: Organisation for Economic Cooperation and Development.
Accessible at: http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicalssection-4-health-effects_20745788
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. Accessible at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/P
ART1&docLanguage=EnOECD (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. Series on Testing and Assessment No. 168. Accessible at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/P
ART2&docLanguage=En
Sakaguchi H, Ashikaga T, Miyazawa M, Yoshida Y, Ito Y, Yoneyama K, Hirota M, Itagaki H, Toyoda
H, Suzuki H. (2006). Development of an in vitro skin sensitization test using human cell lines; human
Cell Line Activation Test (h-CLAT). II. An inter laboratory study of the h-CLAT. Toxicol In Vitro.
20:774-84.
Sakaguchi H, Ryan C, Ovigne JM, Schroeder KR, Ashikaga T. (2010). Predicting skin sensitization
potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in theEuropean Cosmetics Association (COLIPA) ring trials. Toxicol In Vitro. 24:1810-20.
dos Santos GG, Reinders J, Ouwehand K, Rustemeyer T, Scheper RJ, Gibbs S. (2009). Progress on thedevelopment of human in vitro dendritic cell based assays for assessment of the sensitizing potential of
a compound. Toxicol Appl Pharmacol. 236: 372-82.
Takenouchi, O., Miyazawa, M., Saito, K., Ashikaga, T., Sakaguchi, H. Predictive performance of thehuman Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-water partition
coefficients. Journal of Toxicological Sciences. Accepted for publication.
UN (2009). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Part 3:
Health Hazards. New York, NY, USA, and Geneva, Switzerland. United Nations Economic
Commission for Europe. Accessible at:
http://www.unece.org/trans/danger/publi/ghs/ghs_rev03/03files_e.html
Yamada, T., Kosaka, N., Okamoto, Y., Mizuno, M., Sato, J., Yoshida, M., Ota, N., Kodama, T.,Okamoto, Y., Kuwahara, H., Sakaguchi, H., Hasegawa, S., Ashikaga, T., Ohno, Y. (2008). A study onhuman Cell Line Activation Test (h-CLAT) Validation Study Report

Page 73 of 74

serum difference on test results in the human Cell Line Activation Test (h-CLAT): Results of the 3rdJapanese inter-laboratory study. AATEX 13: 63:69.

human Cell Line Activation Test (h-CLAT) Validation Study Report

Page 74 of 74