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

Direct Peptide Reactivity Assay (DPRA)ECVAM Validation Study Report

January 2012

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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

STUDY OBJECTIVES ...................................................................................................................................... 8
PROJECT PLAN ........................................................................................................................................... 8a.
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.
INTENDED PURPOSE OF THE TEST METHOD ............................................................................................. 24
2.
EVIDENCE DEMONSTRATING THE NEED OF THE TEST METHOD .............................................................. 24
3.
STATUS OF DEVELOPMENT OF THE TEST METHOD .................................................................................. 24
4.
SCIENTIFIC BASIS – BIOLOGICAL AND/OR MECHANISTIC RELEVANCE OF THE DPRA........................... 25
5.
PROTOCOL OF THE TEST METHOD ........................................................................................................... 25
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 1 ............................................................ 31
TRANSFERABILITY (MODULE 3) .................................................................................................................. 33
1.
GENERAL ASPECTS ................................................................................................................................... 33
2.
TRAINING .................................................................................................................................................. 33
3.
TRANSFER OF THE TEST METHOD TO THE NAÏVE LABORATORIES .......................................................... 35
4.
QUALIFICATION RUNS FOR A SUCCESSFUL COMPLETION OF THE TRANSFER PHASE ............................. 37
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 3 ............................................................ 39
WITHIN-LABORATORY REPRODUCIBILITY (MODULE 2) .................................................................... 41
1.

P&G.......................................................................................................................................................... 41
Reproducibility (concordance in predictions).................................................................................... 41
Reproducibility of depletion values for cysteine and lysine................................................................ 42
Reproducibility of the controls values................................................................................................. 44
2.
RICERCA ................................................................................................................................................ 46a.
Reproducibility (concordance in predictions).................................................................................... 46b.
Reproducibility of depletion values for cysteine and lysine................................................................ 47
c.
Reproducibility of controls values....................................................................................................... 48
3.
IVMU ....................................................................................................................................................... 50a.
Reproducibility of the controls values................................................................................................. 50b.
Reproducibility (concordance in predictions).................................................................................... 52c.
Reproducibility of depletion values for cysteine and lysine................................................................ 53
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 2 ............................................................ 54
a.
b.
c.

BETWEEN LABORATORY REPRODUCIBILITY (MODULE 4)................................................................ 55
1.
REPRODUCIBILITY (CONCORDANCE IN PREDICTIONS)........................................................................... 57
2. REPRODUCIBILITY OF DEPLETION VALUES FOR CYSTEINE AND LYSINE (FOR THE 24 CHEMICALS) ......... 60
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 4 ............................................................ 64
PREDICTIVE CAPACITY (MODULE 5) ......................................................................................................... 65
CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 5 ............................................................ 67
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VMG OVERALL CONCLUSIONS AND RECOMMENDATIONS ............................................................... 69
OVERALL CONCLUSIONS ................................................................................................................................... 69
RECOMMENDATIONS ......................................................................................................................................... 71
LIST OF APPENDICES....................................................................................................................................... 72
LIST OF REFERENCES...................................................................................................................................... 73

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Summary
The evaluation of the skin sensitisation potential represents an important component of thesafety assessment of new and existing substances. Traditionally this is achieved with animal
tests such as the Local Lymph Node Assay (LLNA) and the guinea pig tests (Buehler Test andGuinea Pig Maximisation Test).
To date, there is a pressing need for having alternative non-animal methods available to reduceand eliminate the need for animals for this endpoint as advocated by some European
regulations aiming at the protection 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 Direct Peptide Reactivity
Assay (DPRA) was part of a study coordinated by ECVAM in which two other methods (thehuman Cell Line Activation Test (h-CLAT) and the Myeloid U937 Skin Sensitisation Test
(MUSST)) are still in the evaluation process.
The study was designed to allow sound conclusions to be drawn on the transferability andreproducibility (within- and between-laboratories) of the DPRA in view of its future use as partof a non-animal testing strategy for skin sensitisation hazard assessment.
Having reviewed the information generated in the study, the Validation Management Group(VMG) concluded that this appropriately completes the information requirement for modules
1-4 (test definition, within laboratory reproducibility, transferability, between laboratory
reproducibility) and contributes to module 5 (predictive capacity) and module 6 (applicability
domain) of the ECVAM modular approach to validation.
In addition, the VMG concluded that the information generated in the study shows that the
DPRA is a robust and reliable test method. Therefore the VMG supports the use of the DPRA
in a weight-of-evidence approach to support regulatory decision making and the acceptance of
positive DPRA results in the context of specific regulations (e.g. REACH in the EU).
The VMG also considers the DPRA deserves further evaluation as part of an integrated testing
strategy for the full replacement of the in vivo assays for skin sensitisation hazard identificationand for the role it might play in the determination of skin sensitisation potency.

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Background
Skin sensitisation is the toxicological endpoint associated with substances that have theintrinsic ability to cause skin allergy, termed allergic contact dermatitis (ACD) in humans, andrepresents the most common manifestation of immunotoxicity. The identification of the skinsensitisation potential represents an important component of the safety assessment of new andexisting substances including cosmetic ingredients. Current regulatory predictive tests for skinsensitisation rely on the use of animals. These include the traditional guinea pig tests (Buehler
Test and Guinea-pig Maximisation Test (OECD TG 406, TG B06 EU Regulation 440-2008),
and the Local Lymph Node Assay (LLNA, OECD TG 429, TG 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 and Restriction of Chemicals) there is a greater needfor having alternative non-animal methods available 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 although not fully characterised. These include: 1) the ability of thechemical to penetrate the skin and reach the site of haptenation (skin bioavailability), 2) thecovalent binding of the chemical sensitiser to the skin protein (haptenation), 3) the release of
pro-inflammatory signals by epidermal keratinocytes (skin inflammation) 4) the activation andmaturation of Dendritic cells (DC) the skin immunocompetent cells, 5) the migration of DC
from skin to the regional lymph nodes, 6) the expansion of memory T cells (lymphocytes
capable of being stimulated and activated specifically by the haptenised chemical). Progresshas 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 mechanistically-based non-animal tests currently underdevelopment/evaluation is considered to have the potential to function as a stand-alone method
to fully replace the currently used animal tests. Instead it is proposed that a combination ofsuch tests, addressing the key biological mechanisms of skin sensitisation, will be needed toachieve this goal. A comprehensive overview of the currently available methods targeting the
key mechanisms described above was published in 2011 (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
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 (Colipa) associated industries and optimised within Colipa ring trials. These threetest methods are proposed as candidates for regulatory use as part of an integrated alternative
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approach for replacing the current regulatory in vivo tests. How the information generated withthese test methods could potentially be integrated to achieve this goal still has to be
determined.
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.

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Table 1
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

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study ReportInduction: 1 dose(highest concentration tocause mild skin irritation)
Challenge: 1 dose(highest non-irritant dose)

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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 andavailable on request (Appendix 16)

1. Study objectives
In September 2009 a formal validation study of the three above mentioned test methods waslaunched, with the primary overall objective of evaluating their transferability andreliability (reproducibility within and between laboratories) with a view to their future
use in an integrated non-animal approach for replacing the currently used regulatory
animal tests. Evaluation of how the data generated with these tests would be accommodated
within future data integration activities was outside the scope of the current study, though it isrecognised that the availability of high quality non-animal data, such as those generated in the
ECVAM study, will be a prerequisite for such activities.
As secondary goals of the study, the available information and experimental data were used toperform:a) A preliminary evaluation of the ability of the three tests to reliably discriminate skinsensitising (S) from non-sensitising (NS) chemicals as defined by the Globally Harmonised
System (GHS) for the classification and labelling of substances 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 andmixtures.
b) Where possible, a preliminary consideration of the ability of the three tests 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).
The current report, which was prepared by ECVAM with the support of the Validation
Management Group (VMG), represents the outcome of the validation study of the DPRA
where the transferability and reliability were evaluated in three independent laboratories.

2. Project Plan
Prior to the start of the study, a Project Plan was approved by the members of the Validation
Management Team (VMT) and issued. This document was reviewed as required at each VMTmeeting; the final version is annexed to this report. The Project Plan documents the objectives,
coordination and sponsorship of the study; the nature and roles of the study personnel at eachtesting 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 the
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 were required to return a signed declaration

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that they had read and understood the project plan, and that their testing facilities would workin compliance with the provisions set out in this document.
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) taking
into consideration the study’s objectives. In addition, the experimental data produced in the
study contribute to the module 5 on predictive capacity and to the module 6 on applicability
domain, which were in part addressed by the information generated and submitted by the test
method submitter. However, the number of chemicals used in the validation study, which was
set to satisfy the primary goal of the study, is not sufficient on its own to draw robust
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 methods
SOPs. Figure1 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, 2004).

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Figure 1: Schematic Representation of the Study Structure and Organisation
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/quality check/management
Drafting final study reports
Drafting publications

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 laboratory
Timelines/study progression
Study interpretation and conclusions
Study reports and publications

DPRA

MUSST

Chemical Selection Group
(CSG)
•
•

Definition of selection criteria
Chemical selection

h-CLAT

Lead Laboratories

Kao
P&G
•
•
•

ECVAM

•
•
•

L'Oréal•••

SOPs
Training
Testing

SOPs
Training
Testing

Shiseido
•
•
•

SOPs
Training
Testing•

Testing

IVM
•

SOPs
Training
Testing

IVM

Testing

Chemical acquisition,coding and distribution

FICAM
•

Ricerca•

Testing

Testing

Bioassay•

Testing

Bioassay•

Testing

Ricerca•

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

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, since June 2011)
Pierre Aeby (Colipa)
Sebastian Hoffmann (seh consulting + services)
Jon Richmond (dr.jonrichmond: Advice &Consultancy)
Aiba Setsuya (Tohoku University Graduate
School of Medicine)

Lead laboratory Representatives
Procter & Gamble (DPRA)
L’Oréal (MUSST)
Shiseido (h-CLAT)
Kao Corporation (h-CLAT)

Frank G. Gerberick
Jean-Marc Ovigne (up to June 2011),
Nathalie Alépée (since October 2010)
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
Three laboratories participated in the validation of the DPRA and they are listed below.
Procter& Gamble (P&G), in which the test method was developed and which has the mostexperience in performing it, acted as lead laboratory.

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Laboratory 2 was involved in the study as a naïve laboratory which had experience with HPLC
techniques but was never involved with peptide reactivity assays.
Laboratory 3 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 which included demonstrated awareness of the work involved.
The signatory of the tender from Laboratory 3, an international Contract Research
Organisation, indicated that the laboratory is systematically searching the literature for new invitro technologies/methods for inclusion in its portfolio and had already performed some
peptide reactivity work on the basis of information published in the literature. Therefore,
although this laboratory was not involved directly in the development or performance of thespecific DPRA SOP, this laboratory was not considered entirely naïve with respect to peptide
reactivity assays.
Laboratory 1 (Study Director: Leslie Foertsch)
Procter & Gamble Company
Miami Valley Innovation Center
Cincinnati, USA
Laboratory 2 (Study Director: Siegfried Morath)
In-House Validation and Training Laboratory
In-Vitro Methods Unit/ECVAM
Institute for Health and Consumer Protection
Joint Research Centre
European Commission
Ispra, Italy
Laboratory 3 (Study Director: Fabien Marguerite)
Ricerca Biosciences SAS
Saint Germain sur l'Arbresle, France
Note: At the initiation of the study, Ricerca Biosciences was called "MDS Pharma".
d. Quality Systems of the Participating Laboratories
Only Laboratory 3 was fully Good Laboratory Practice (GLP) accredited and subject toinspections by relevant agencies at the time of the blind testing phase. The laboratory declaredthat the blind testing phase (study phases B Stage 1 and Stage 2) had been conducted fully incompliance with the OECD Principles of Good Laboratory Practice and with the Principles ofGood Laboratory Practice as described in the French Official Journal, and that the final reports
had been audited internally. Laboratory 2 was in the formal process of requesting GLP
accreditation during the conduct of the study. However, as Laboratory 2 had not yet been
audited or approved by a national authority, it cannot be deemed to have been GLP accredited
or compliant at the time the study was performed. Laboratory 1 was not GLP accredited.
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For the laboratories participating in the pre-validation study which do not have formally
implemented GLP, the VMG defined and requested the application of a set of quality assurance
requirements considered essential for the acceptance of information and data produced in thevalidation process. These requirements formed part of the Project Plan which was sent to, andaccepted by, all participating laboratories prior to the initiation of the study.
These minimum requirements were:• 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 number of chemicals
that would be required to properly evaluate the performance of the tests in light of the primarystudy goal, i.e. to perform a statistically sound evaluation of the Within Laboratory
Reproducibility (WLR) and of the Between Laboratory Reproducibility (BLR).
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
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calculated to be at least 21 chemicals for the evaluation of the BLR and at least 13 chemicals
for the evaluation of the WLR.
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.
On the basis of the above considerations, the following design was approved by the VMG:••

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

It was deemed that this experimental design would provide the information needed to perform
a sufficiently robust assessment of the WLR and BLR of both the prediction results and the raw
data for the three test methods evaluated.
Study Phases:
The study was structured and conducted in two sequential phases:
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 for an additional review and control point, following
the initiation of the testing phase, in order to verify that no serious issues were arising before
the bulk 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
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to the chair, the CSG was composed of Dr. Luca Tosti (ECVAM); Dr. David Basketter (chairof the study) and Dr. Bill Stokes (NICEATM/ICCVAM). The general strategy for the chemical
selection was presented and approved by the VMG at its 1st meeting. The final list of chemicals
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 use of the experimental data
generated in this study to support future activities on data integrations to achieve ultimately full
replacement, it was deemed important to use a common set of test chemicals for the three 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 of insufficient eligibile chemicals under the primary criterion.
During their respective development and optimisation, the three in vitro methods had been used
to evaluate certain chemicals listed in these databases, as described in the original submissionsof the methods to ECVAM. Acknowledging this, the chemical selection for this study 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 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 for the DPRA.
To ensure parity between the three in vitro methods involved in the validation study, the only
"tested" chemicals that were considered were those already tested by all three methods and thathave 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, achemical previously tested in the DPRA and in the h-CLAT but not in the MUSST.

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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
database yielded 11 eligible chemicals reported as previously tested in all three methods, all ofwhich correctly classified by the three methods. Applying the primary criterion on the 215
untested chemicals found in the original lists resulted in 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, an adequate and practical shortlist.
This list of 43 substances was further reduced to 24 for different pragmatic reasons (seeAppendix 3) and by expert judgment by the CSG. The final selected chemicals covered therange of sensitisation 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
made provision 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 association of structural analogues, unless contrasting skin sensitisation potential
was evident (e.g. 1-Thioglycerol (S) and Glycerol (NS)).
The list of 43 chemicals, and its refinement to the final selection of 24 chemicals (together withdetailed comments about these choices) can be found in Appendix 3.
The final selection included 9 LLNA performance standards (PS) reference chemicals (OECD,
2010a). 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 potency, and the commercial preparation is arecognised source.
Table 2 lists the final set of chemicals selected for the study.

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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+

Chloramine T

127-65-1

Solid+strong

+

0.14

1A

strong+

0.4

1A

Formaldehyde

50-00-0Liquid2Mercaptobenzothiazole+

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

+

+

+

-

-

-

1

Nickel chloride

7718-54-9

Solid

-

no category+

Benzylcinnamate

103-41-3

Solid

+

weak

+

18.4

1B

1B

Imidazolidinylurea

39236-46-9

Solid

+

weak

+

24

1B

R(+)-Limonene

5989-27-5

Liquid

+

weak

+

69

1B

90

Methylmethacrylate

80-62-6

Liquid+weak+

Glycerol

56-81-5

Liquid-no category

-

NC3

1B

2,4Dichloronitrobenzene

611-06-3

Solid-

no category-

NC

Benzyl alcohol

100-51-6

Liquid-no category-

NC

Methylsalicylate

119-36-8

Liquid-no category

-

NC

-

-

-

Isopropanol

67-63-0Liquid-no category

-

NC

-

-

-

Dimethylisophthalate

1459-93-4

Solid

-

no category-NC

4-Aminobenzoic acid150-13-0

Solid-

no category-

Xylene1330-20-7

Liquid

+

weak2

1

False negative in the LLNA

2

False positive in the LLNA

3

NC: Not Classified

NC
95.8

NC

The selection 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
performed in a stratified random sampling procedure, to ensure a consistent distribution ofpotencies in the 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 to the "moderate" category by weight of evidence. Similarly, Xylene, a falsepositive in the LLNA, was assigned the "no category" class (See Appendix 4).

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Figure 2: Stratified random sampling of the 24 chemicals to identify the 9 chemicals thatwere tested once and the 15 chemicals that were tested three times each.

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)NON-SENS.

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

+

+

+

1-Thioglycerol

96-27-5

Liquid+moderate

+

3.6

1B

Dihydroeugenol

2785-87-7

Liquid+moderate

+

6.8

1B

Imidazolidinylurea

39236-46-9

Solid

+

weak

+

24

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-nocategorynocategorynocategory

-

NC

-

NC

-

NC

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Table 4: List of the phase B2 chemicals tested in three independent experiments by eachlaboratory
CAS#

State

LLNA

LLNA
potency
category

GP

EC3

GHS
potency
category

Beryllium sulfate

7787-56-6

Solid

+

extreme

+

0.001

1A

Kathon CG (1.2%
CMI)

26172-554

Liquid+extreme

+

0.009

1A

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

2Mercaptobenzothiazole

149-30-4

Solid+moderate+

Benzylsalicylate

118-58-1

Liquid+moderate++

NON-SENS.

SENSITISERS

Chemical Name

DPRA
R&D
resulthCLAT
R&D
resultMUSST
R&D
result

+

+

+

1A

+

+

+

1.7

1A

+

+

2.9

1B

Nickel chloride

7718-54-9

Solid

-

no
category

Benzylcinnamate

103-41-3

Solid

+

weak

+

18.4

1B

R(+)-Limonene

5989-27-5

Liquid

+

weak

+

69

1B

Methylsalicylate

119-36-8

Liquid-Isopropanol

67-63-0Liquid-Dimethylisophthalate

1459-93-4

Solid

-

4-Aminobenzoic acid150-13-0

Solid-

Xylene1330-20-7

Liquid+nocategorynocategorynocategorynocategory
weak

1B

-

NC

-

-

-

-

NC

-

-

-

-

NC

-

NC
95.8

NC

It is important to note that in view of the primary objective of the blind testing phase, noconsideration was given to the suggested applicability domain of the DPRA (or the other two
in vitro methods evaluated in this validation study) in the chemical selection. The list contains
two metals, Nickel and Beryllium, two well characterised pre-haptens (4-Phenylendiamine andR(+)-Limonene)and a well characterised pro-hapten (, Dihydroeugenol).
Metals and pro-haptens are materials outside the applicability domain identified for the DPRA
by the test developer. However, some pre-haptens are reported to have been correctly
identified (including 4-Phenylendiamine) therefore it cannot be concluded with sufficientconfidence whether or not these substances fall outside the applicability domain of the test
method.
Wherever possible, the selection of chemicals was predicated on a consistency of evidence
from human experience, guinea pig tests and the local lymph node assay (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 inthe LLNA and nickel chloride, a false negative in the same assay. For xylene, the humanevidence of skin sensitisation, while not entirely absent, is of a similar scale to petrolatum anddoes not meet the criteria for classification. For nickel chloride, the human evidence ofsensitisation is abundant.

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Two other points should be noted. First, for a few substances (eg 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). However, this evidence is of such a limited nature (isolatedcase reports), that they are considered to be substances with insufficient evidence to warrant
classification.

5. Chemicals purchase, coding and distribution
Reference document:- Chemical coding, aliquoting and shipping procedures (Appendix 5)
The chemicals used for study phase A Stage II were not supplied by ECVAM, but werepurchased by the trained laboratories on the basis of the instructions reported in the training
and transfer plan issued by P&G and approved by the VMG.
ECVAM was responsible for purchasing, coding and distributing the chemicals for Phase B to
the laboratories participating in the study. The selected chemicals were sourced from Sigma
Aldrich. Aliquots of the chemicals were prepared and properly stored in the chemical
repository of the IVMU 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 the chemicals underwent an assessment of solubility at ECVAM, following the"solvent selection" procedures as described in each of the test methods' SOP. In all cases, atleast one suitable solvent was identified. The solvent(s) identified for each chemical were notcommunicated to the testing facilities, as the SOP required that they performed their own
determination of the suitable solvents to use in their experiments (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, each chemical from the list of 15 was sent to
the laboratories in three separate vials, each assigned a different two-letter code. A number
was also added to the code (1, 2 or 3) to distinguish the three sets of 15 chemicals, and thelaboratories were instructed never to mix chemicals labeled with different numbers in the samerun/experiment. This ensured that the three evaluations of the corresponding chemicals were
performed in different experiments in order to 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,
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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.
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 DPRA.
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 at eachlaboratory not associated with the testing (typically the Safety Officer at each participating
laboratories) with the instruction to return the unopened MSDSs to ECVAM upon completionof the testing phase.
During the testing of the DPRA, no such incident was reported and none of the envelopes had
to be unsealed, and all sealed envelopes were returned to ECVAM upon completion of thevalidation 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:- DPRA reporting template (Appendix 6)- DPRA QC template (Appendix 7)
Prior to the start of the study a standard reporting template was distributed to the participating
laboratories. This template was developed for the study by ECVAM, in collaboration with thelead laboratory, as no standard templates were available at the time of the submission to
ECVAM. The template contained formulae tested by the ECVAM biostatistician in adocumented exercise to quality assure the template before it was distributed. The laboratories
were asked to use the template during the Transfer phase A to communicate their results to thelead laboratory, so that they could familiarise themselves with it and to ensure that the
instructions included in the template were clear and understood.
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
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ECVAM each completed template underwent a formal Quality Check procedure that wasdeveloped in the form of a checklist (See appendix VII). The quality check focused on theacceptance criteria for the run and for each of the chemicals to ensure the results were valid,
and the proper choice of solvent controls used for the calculations of the peptide depletions.
Once completed, the checklist were scanned as a PDF file and added to the Excel sheet as an
object. The template was then added to the official results folder for 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 determined that only data from the valid
experiments would be considered within the statistical analyses, although failed runs andexperiments were also documented in order to report their occurrence.
The statistical analysis on the test method's reproducibility focused on the concordance of thepredictions obtained in terms of the Yes/No predictions and of the specific reactivity classes
assigned. Reproducibility was evaluated with respect to both WLR and BLR.
Additionally, descriptive and inferential statistical analyses were performed on the raw dataobtained, the results of which are provided as additional descriptive information.
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;
3. the proposed used of the in vitro tests (i.e. as a partial replacement method to become
part of a toolbox of tests to be used in combination); and4. the power of the design of the validation study.
In consideration of the above the target performance for this study was set at 80% for the
Between Laboratory Reproducibility and 85% for the Within Laboratory Reproducibility.
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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 developer in which the DPRA showed a between laboratory
reproducibility of 91% (see Appendix 2 for further details).

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Test definition (Module 1)
Reference Documents:- DPRA SOP version 2 (Appendix 8)- DPRA SOP version 3 (Appendix 9)- Gerberick et al (2004) (Appendix 10)- Gerberick et al (2007) (Appendix 11)
1. Intended purpose of the test method
The DPRA is partial replacement in chemico test method designed to be part of a non-animal
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 animal used for skin sensitisation
testing and to the replacement of current regulatory in vivo tests for skin sensitisation hazard
classification and labelling (OECD TG 406, OECD TG 429, OECD TG 442A, OECD TG
442B). The information the DPRA generates can in any case already be used in a weight-ofevidence approach to support regulatory decision making (i.e. to better characterise equivocal
responses in in vivo studies). For example, REACH already permits the use of methods undervalidation for this purpose.
2. Evidence demonstrating the need of the test method
This test method is of importance due to the forthcoming European Union ban on in vivo
testing of cosmetic and toiletry ingredients and products following the publication of theSeventh Amendment to the Cosmetics Directive and for the European Regulation on
Registration, Evaluation, and Authorisation of Chemicals (REACH) that requires evaluation ofa large number of chemicals. The successful validation of the DPRA along with other assays(e.g., cell-based assay) would allow their use in the assessment of a chemical’s skinsensitisation potential and reduce or eliminate the need for animal testing for skin sensitisation.
3. Status of development of the test method
The DPRA was developed by Procter & Gamble.
The initial work to determine if chemical reactivity toward nucleophilic amino acids correlatedwith skin sensitisation potential examined the reactivity of 38 different chemicals with varying
degrees of sensitisation potency: 11 non sensitisers, 7 weak sensitisers, 11 moderate sensitisers,
5 strong sensitisers, and 4 extreme sensitisers.
82 substances (including the 38 chemicals mentioned above) were then evaluated in the courseof development of the Prediction Model (56 chemicals to initially define the Prediction Model,
and further 26 to demonstrate its robustness). For all test materials LLNA test data was alsoavailable.
Additional work was undertaken pre-submission to ECVAM by P&G to provide insights into
the likely within laboratory reproducibility, transferability, and between laboratory
reproducibility. This work included two ring trials (involving the testing of 15 and 28
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chemicals respectively) with the participation of three external laboratories (Kao Corporation,
L’Oréal and Givaudan).
In the test submission to ECVAM (received February 2009) P&G reported data on the test
performance with 135 chemicals (98 sensitisers – 33 extreme/strong, 36 moderate, 29 weak –and 37 non-sensitisers) with reported 86% accuracy (calculated for 133 chemicals since two
chemicals co-eluted with the cysteine peptide and were not included in the accuracy
calculation), and had trained six laboratories in its use.

4. Scientific basis – biological and/or mechanistic relevance of the DPRA
There are a variety of properties that determine whether a chemical can function as a skincontact sensitiser (or allergen) including the ability to penetrate into the skin, react withprotein, and be recognised as antigenic by immune cells. The correlation of protein reactivity
with skin sensitisation potential is well established (Dupuis and Benezra, 1982; Lepoittevin etal, 1998). In fact, Landsteiner and Jacobs (1936) presented the origin of the reactivity
hypothesis in their landmark paper looking at the underlying mechanisms of contact allergy.
Thus, if a chemical is capable of reacting with proteins either directly or after appropriate
biotransformation, then it may have the potential to act as a contact allergen.
The majority of known chemical skin allergens (or their metabolites/oxidation products) haveelectrophilic properties and are able to react with various nucleophiles to form covalent bonds.
In proteins, the side chains of many amino acids contain electron-rich groups, nucleophiles,
capable of reacting with electrophilic allergens. Lysine and cysteine are well characterised as
nucleophiles, but other amino acids containing nucleophilic heteroatoms, such as histidine,
methionine, and tyrosine have been reported to react with electrophiles (Dupuis and Benezra,
1982; Lepoittevin et al, 1998; Ahlfors et al, 2003).
Since protein reactivity is a key step in the induction of ACD it was hypothesised that in vitro
methods based on the detection of protein reactivity could be developed to screen thesensitisation potential of chemicals. Recently, a detailed review on using chemical reactivity
measurement for the identification of skin sensitisers was published as part of an ECVAM
Workshop (Gerberick et al., 2008).
5. Protocol of the test method
The detailed updated DPRA test method protocol used during the testing phase is described inAppendix 8.
The DPRA is a chemistry based assay. Nucleophile-containing synthetic peptides are used toscreen for skin sensitisation potential by measuring peptide depletion following incubation
with test materials (allergens and non-allergens). For the synthetic heptapeptides that containeither cysteine or lysine, the peptide to chemical ratio used is 1:10 and 1:50, respectively.
Following a 24 hour reaction period with the two synthetic peptides, the samples are analysedby HPLC using UV detection to monitor the depletion of peptide following the reaction.
Average peptide depletion data for cysteine and lysine are then used in a classification tree
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model in which chemicals are classified as having minimal, low, moderate or high reactivity(Figure 3). Generally chemicals with moderate to high reactivity are associated with moderate
to strong allergenicity while those categorised as having low to minimal reactivity include
weak and non-sensitisers. However it would be inappropriate to consider that the DPRA, as astand alone method, has the capability to predict a chemical’s sensitisation potency. Reactivity
information is however foreseen to play an important role in a future integrated approach forboth hazard identification and potency prediction.

Cysteine 1:10/Lysine 1:50 Prediction Model
Mean ofCys & Lys
% depletion
Avg Score < 22.62%

Avg Score > 22.62%

Mean ofCys & Lys
% depletion

Mean ofCys & Lys
% depletion
Avg Score > 6.38%
Avg Score < 42.47%

Avg Score < 6.38%

Minimal Reactivity

Low Reactivity

Avg Score > 42.47%

Moderate Reactivity

High Reactivity

Cysteine 1:10-only Prediction Model
Cysteine% Depletion
Cysteine 1:10 > 23.09%

Cysteine 1:10 < 23.09%

Cysteine% Depletion
Cysteine 1:10 < 13.89%

Minimal Reactivity

Cysteine% Depletion
Cysteine 1:10 > 13.89%
Cysteine 1:10 < 98.24%

Low Reactivity

Moderate Reactivity

Cysteine 1:10 > 98.24%

High Reactivity

Figure 3: DPRA Classification tree prediction model
When co-elution of the test material with the lysine peptide prevents a determination of an
accurate depletion value, a prediction model is available to classify the chemical into reactivity
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classes based on the depletion value for the cysteine peptide alone. A prediction model basedon the depletion value of the lysine peptide alone is not available. The test developer reported
in the submission to ECVAM that the reason for this is that the lysine reactivity does not carryenough weight to drive a lysine-only prediction model and that the cysteine depletion value isrequired in order to accurately predict the reactivity classes..

Modifications made to original protocol prior to the initiation of the validation study
The DPRA protocol used in the validation study is almost identical to the protocol originallysubmitted to ECVAM by P&G. Following the assessment made by ECVAM of the original
protocol, minor technical revisions were made in collaboration with P&G prior to the initiationof the validation study. No major modifications were made to the experimental procedure
itself. The minor changes included clarification of sections of the SOP which were felt not tobe sufficiently well described and could thus impact on the interpretation, performance, andoutputs of the protocol. However, it was also deemed important to include additional run
acceptance criteria which were not set out in the original document. These include:
1. The establishment of acceptance criteria for the positive control cinnamic aldehyde to
be used to ensure that the test method is responding with adequate and reproducible
sensitivity to this substance, for which the magnitude of response is well characterised.
For this purpose the range of percent peptide depletion values of the positive control
cinnamic aldhehyde was based on 95% tolerance intervals calculated from the historical
data submitted to ECVAM for both the cysteine and the lysine peptides.
2. The application of acceptance criteria for the Reference Control C replicate injections(peptide alone dissolved in the appropriate solvent) in the analysis sequence according
to which the mean of the peptide concentrations in the three replicates should
correspond to the theoretical concentration of (0.50mM) plus or minus 10%. This
acceptance criterion has to be used as a run acceptance criterion to verify accuracy ofthe peptide concentrations since the related peptide peak area is then used in theformula to calculate the Percent Peptide Depletion.
As described above, all modifications to the SOP, including the addition of acceptance criteria
not originally foreseen, were made in close collaboration with the lead laboratory to make surethat such modifications/additions did not invalidate or compromise the historical data
generated with the DPRA prior to the submission to ECVAM.
Additional minor technical revisions were made before the start of the blind testing phase inorder to incorporate the experience and knowledge gained during the transfer of the method.
The revised protocol (Version 2, see Appendix 8) was then used by all testing facilities for thegeneration of the results with the coded chemicals.
A summary of the protocol revisions is outlined in Table 5:

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Table 5: Description of the main modifications made to each version of the SOP.
Elementsofthe Version 1
submitted SOP which Released prior tounderwent revisions
initiation of the study

Solubilisation
procedure

theVersion 2
Released prior to theinitiation of the blind
testingphase
toinclude the knowledge
acquired from thetransfer phase

Version 3
Finalversionreleased at the endof the study for
future use.

Included criteria for
estimating peak purity
based on area ratio of
220/258nm for thoselaboratory
equippedwith a PhotodiodeArray detector.

Additionofpractical examples
ofco-elution
chromatograms in
the annexes of the
SOP

Thesectiononthesolubilisation procedure wasmodifiedto
improveconsistency in the choice ofsolvents by including atiered approach for thesolvent choice instead of asimple list of compatible
solvents.

Detection of co-elution

Co-elution controls havebeen included in the first
analysis sequence to assist intheidentificationof
instances of co-elution of thetest chemicals with the
peptides in order to include
more objective rules onwhen to determine coelution had taken place.
Positive
control For both the cysteine and theacceptance criteria
lysine peptides, acceptancecriteria based on 95%
tolerance intervals calculated
from the historical datasubmitted to ECVAM havebeen established for the
positive control cinnamic
aldehyde. Such criteria havebeen included in the SOP
and are used as run
acceptance criteria.

The upper bound of the Revision of theacceptance range for bounds for thethe cysteine percentage acceptance criteriaof depletion induced bycinnamic aldehyde wasincreased from 96.6 to
100 as one laboratory
consistently generated
values outside theoriginal range.

Inadditionmaximum
standard deviations admitted
for
positive
control
replicates
have
been
includedReference
controls Included maximum CV
acceptance criteria
admittedforreference
controls replicated to check
Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

Revision of thebounds for theacceptance criteria
Page 28 of 74

stability of the peptides overtime

Results

Included acceptance criteria
for reference controls C in
the analysis sequence set as
theirtheoreticalvalues(0.50mM) plus or minus
10%.
The data analysis andcalculations section wasrevised to include additional
acceptance criteria for the
run and test chemicals’
results.

Includedadditional
instructions on how todetermine reactivity forchemicals co-eluting
with the peptide

Included additional
clarifications in thesectionabout
negative depletion
valuesIncluded instructions on how
to deal with negative
depletion values.
Analysis sequence
Reagents

Inclusionofanexample of analysis
sequence.
Inclusionofaprovision to test eachbatch of acetonitrile to
be
used
in
the
experiments(during
the transfer of themethod some batches
revealed to impact
peptidestability,
particularly cysteine),

Removed Synbiosci
as suggested peptide
supplier.
Included expiration
date for ammonium
acetate buffer.

During the conduct of the study the two trained laboratories experienced problems in meetingthe criteria described above under 1. and 2.
Accepting that these criteria were not described in the original SOP and were defined on thebasis of data generated by a single laboratory, the VMG decided in one circumstance to relax
the acceptance range for the positive control results and to accept results from runs which had
been considered invalid because either the acceptance criteria for the positive control or thereference control C were not met. The rationale for this VMG decision is documented below inthe relevant sections (see Ricerca results in module 3, and IVMU results in module 4).
Known limitations and drawbacks of the test method
A limitation of the test method is that the test substance needs to be dissolved in a suitable
solvent. The DPRA SOP includes a tiered approach for the selection of the appropriate solvent
where acetonitrile and water are respectively the first and second choice solvents since they areused as mobile phases for the HPLC analysis. In case the test chemical is not soluble in
Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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acetonitrile or water, other solvents are suggested: these include isopropanol, DMSO, andcombinations of the solvents described above. The solvent choice provided should allow thetesting of the vast majority of chemicals. In fact P&G reported in their submission that so far in
their experience they faced solubility problems with only one chemical (squaric acid).
For the purpose of the current study none of the chemicals selected had to be discarded becauseof solvent incompatibility as a result of the solubility assessment performed by ECVAM (seeSection 5a above). This was also consistent with the fact that no problems were subsequently
encountered by the laboratories during the solubility assessment for the blind testing phase
Another limitation of the DPRA is that some chemicals, or the reaction products formed
following incubation with the peptide, may interfere with the peptide determination because
they elute at the same time of the peptide, a phenomenon referred to as co-elution. Since in the
DPRA the HPLC separation occurs by gradient elution (i.e. the mobile phase composition ischanged during the separation process), retention time of a chemical may be influenced by thesolvent mixing system which vary in different instruments. For this reason, and as was
confirmed during the current study (see section on Module 4, below), co-elution with the
peptide can be systematically observed in one laboratory but not necessarily observed in theothers if different instruments are used.
In the DPRA, in cases of co-elution of the chemical with the cysteine peptide alone or withboth the cysteine and the lysine peptides an accurate calculation of the peptide depletion cannot
be performed, however the depletion can always be estimated as described in the relevant
section of the SOP (see page 20 of Annex 8) and the conclusion for this chemical adapted
accordingly. In the case of co-elution with the lysine peptide, the value for the lysine peptide
depletion is ignored and a different prediction model (using only the cysteine peptide
depletion) is used. The DPRA can thus accommodate most cases of co-elution.
In their submission to ECVAM, P&G reported that in the set of 135 chemicals they have tested
in-house 2 chemicals co-eluted with the cysteine peptide and 5 chemicals co-eluted with thelysine peptide.
Applicability domain of the test method
The DPRA test method has been shown by the test submitter in their submission to ECVAM to
be applicable to a broad range of chemicals covering relevant chemical classes, reaction
mechanisms, skin sensitisation potency (as determined with in vivo studies) and physicochemical properties.
However the DPRA test method does not contain a metabolic/bioactivation system, therefore
pre-haptens (i.e. chemicals requiring biochemical activation) and pro-haptens (i.e. chemicals
requiring enzymatic activation) are not systematically detected. In addition, metals which
sensitise via non-covalent bonding mechanisms or interact with amino acids other than cysteine
and lysine (e.g. nickel) fall outside the applicability domain.
In their submission to ECVAM, P&G reported that of the 133 chemicals tested, 13 sensitisers
have been classified as non-sensitisers and 6 non-sensitisers have been classified as sensitisers(Table 6). Of the 13 sensitisers classified as non-sensitisers, seven of them are weak sensitisers(e.g., α-hexylcinnamaldehyde, benzyl benzoate). A few of the sensitisers classified as nonsensitisers are also considered by P&G to be pro-haptens (e.g., 3-dimethylaminopropylamine,
dimethyl benz[a]anthracene). For oxalic acid which is categorised as weak sensitiser in the
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LLNA, P&G suggests a revision of the animal in vivo data since the chemical does not containapparent structural alerts for sensitisation nor do human data exist to classify it as a sensitiser.
Among the 6 non-sensitisers classified as sensitisers Procter & Gamble identified compounds
believed to have reactive properties (e.g., 2-hydroxypropyl methacrylate, 1-bromobutane)
despite the fact that they are not known to induce sensitisation.
Table 6: DPRA misclassifications as reported by P&G in their submission to ECVAM.
Sensitisers classified as non-sensitisers
•
•
•
•
•
•
•

α -Hexylcinnamaldehyde
α -Amyl cinnamaldehyde
Benzyl benzoate
2,2,6,6,-Tetramethyl-3,5-heptanedione
Oxalic acid
Nonanoyl chloride
Dimethyl benz[a]anthracene••

Squaric acid diethyl ester
3-Dimethylaminopropylamine••

Tartaric acid
Ethylenediamine••

Chlorooctadecane
Cis-6-nonenal

P&G comments
Compound is a weak sensitiser.
Compound is a weak sensitiser.
Compound is a weak sensitiser.
Compound is a weak sensitiser.
Compound is a weak sensitiser.
Compound is a moderate sensitiser
Compound is a strong sensitiser and isbelieved to be a prohapten
Compound is a strong sensitiser
Compound is a moderate sensitiser andis believed to be a prohapten
Compound is a moderate sensitiser
Compound is a moderate sensitiser andis believed to be a prohapten
Compound is a weak sensitiser
Compound is a weak sensitiser

Non-Sensitisers classified as sensitisers•

2-Acetylcyclohexanone•

2-Hydroxypropyl methacrylate•

1-bromobutane•

1-Iodohexane•

Saccharin

•

Furil

Compound is categorised as low
reactivity
Compound is categorised as low
reactivity
Compound is categorised as low
reactivity
Compound is categorised as low
reactivity
Compound is categorised as low
reactivity
Compound is categorised as highreactivity

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Conclusion of the Validation Management Team on Module 1
The DPRA protocol proved to be generally robust for the purposes of this study, only minor
clarifications were made to the SOP during the course of the study in relation to specificelements of the procedure and the data interpretation to minimise the sources of variability.
The additions in the sections describing the analysis sequence, data recording and analysis andcalculation of results were introduced largely to resolve ambiguities and minor omissions in theoriginal SOP in order to improve clarity and consistency of data generation and interpretation.
To improve consistency of choice of solvent, a strategy was provided for solvent selection
resulting in concordance across the three laboratories with respect to 22 of 24 chemicals
subsequently tested (see section on Module 4, below).
Co-elution of peptide and test chemical or reaction products was known from the outset tooccur in some instances, and more detailed guidance was included to ensure recognition of thisproblem when it occurred in practice.
The upper bound of the acceptance range for the cysteine peptide depletion induced bycinnamic aldehyde, which was originally set on the basis of the lead laboratory’s historicalfindings, was revised prior to the testing phase taking into account the experience of the otherlaboratories. The aim was to avoid wrongly discarding runs which were generating goodquality results.
The acceptance ranges for the values of reference control C in the different solvents were
pragmatically established by taking over the acceptance range for reference controls A that was
included in the SOP submitted to ECVAM by P&G. During the blind testing phase one of thelaboratories generated a number of runs in which the reference control C values wereconsistently marginally below the preset lower limit.
Acknowledging the limitations of the way those acceptance criteria were set, allowed for
acceptance of these results and their inclusion in the statistical analysis with no negative
discernable impact on the reliability and repeatability of the test (see section on Module 2,
below). The VMG believes that the acceptance range should be revised for future use of the
DPRA (see Recommendations section).
The issue related to the significance of the source/batch of acetonitrile used for the test was
unexpected (see section on Module 3, below), and was resolved with the inclusion of apreliminary acetonitrile suitability experiment in the SOP.
Following these procedural clarifications to the SOP, the VMG believes the supporting
documents (including the original submission to ECVAM and associated scientificpublications) and the current study findings adequately demonstrate the intended purpose, theneed for, the status of development, and the scientific and mechanistic basis and relevance of
the DPRA test method.
In conclusion, the VMG believes that Module 1, Test Method Definition, is satisfied.
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Transferability (Module 3)
Reference documents:- DPRA Training and Transfer Plan (Appendix 12)- Training reports for Ricerca and IVMU (Appendices 13-14)- List of additional available documents filed for the study and available on request(Appendix 16)
1. General aspects
The DPRA procedure can be performed in analytical chemistry laboratories equipped withstandard high performance liquid chromatography (HPLC) instrumentation and with laboratory
personnel trained and experienced in HPLC. All apparatus/instruments and reagents needed forthe performance of the method are readily available commercially.
P&G being the lead laboratory and having developed the method was responsible for both thetraining of the personnel at the other testing facilities participating in the study and foroverseeing and providing advice during the test method transfer in order to make sure that theprocedure for performing the DPRA as described in the SOP was clearly understood andproperly implemented.
P&G provided training to the study personnel of Ricerca and IVMU at P&G (Phase A Stage I).
The trained personnel were then responsible for the transfer of the test method to their own
laboratories under the supervision of the lead laboratory P&G (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 laboratory on the basis of its 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. The chemicals used for these qualification runs, aswell as the criteria for a successful achievement of the transfer of the method, were selected by
the lead laboratory, and approved by the VMG prior to the initiation 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 12). All chemicals were tested uncoded, and the results sent directly to the leadlaboratories for evaluation.
2. Training
Ricerca and IVMU personnel received theoretical and procedural training at P&G. The training
sessions for Ricerca and IVMU staff took place on March 2-4 and on March 15-17 2010,
respectively. The training included a theoretical component with discussion on the different
aspects of the SOP and a practical part were the trainees were given the opportunity to setup an
actual DPRA test run for cysteine and lysine depletion, to apply the prediction model and toanalyse the results.

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The theoretical part focused on specific aspects of the procedure including the revisions madeto the SOP during the study preparatory phase and described in DPRA SOP version 1. Items
discussed include:• The use of a photodiode array detector and/or the co-elution controls to help determine
peptide peak identity and the presence of co-elution.
• Importance of peptide purity not exceeding 90-95% (to avoid precipitation).
• Solubility assessments and the order of solvents used in the “Solubility Assessment”
section of the SOP.
• Description of the Reference Controls A, B, and C and how they are prepared and used.
• Revisions made to the Data Analysis & Calculations section. This includes thecalculation of CV, use of the Reference Controls and additional calculations of peptide
concentration.
• Use of the prediction models and when to use the cysteine 1:10-only Prediction Model.
• Useof acceptance criteria from the amended SOP. Specific acceptance criteria werediscussed as well as when an entire assay needs to be repeated and when a single test
chemical evaluation needs to be repeated.
For the practical part, the following test chemicals were chosen by P&G for testing by thetrainees
ChemicalCAS

In vivo Potencyp-phenylenediamine
3,3,5trimethylhexanoyl
chloride, 98%
Glyoxal, 99%
Citral, 95%
Imidazolidinyl urea
95%
Glycerol, 99%

106-50-3
36727-29-4

StrongModerate

Sigma
Aldrichcatalog number
P6001
422959107-22-2
5392-40-5
39236-46-9

ModerateWeak
Weak

128465
C83007
I5133

56-81-5

Non-sensitiser

G9012

In the interest of time, the training chemicals described above and peptides were pre-weighedby the trainer the afternoon before the training session and stored at 4oC. The buffers for
peptide dissolution were also prepared in advance by the lead laboratory. All other aspects ofassay setup were performed on site by the trainee.
Due to software problems, the HPLC’s were unable to be run during the training session held
for Ricerca. Ricerca was therefore asked to purchase the training chemicals and set the assay
up in their own laboratory. As described below (see “Qualification of Ricerca”), this issue had
no influence on the transfer of the method to Ricerca.
The data obtained during the training session for IVMU correlated well with P&G’s historical
data (see Appendix 13). All six chemicals were classified correctly as “sensitiser” or “nonsensitiser.” Five out of the six chemicals were predicted into the same reactivity category ascompared to historical data, the only exception being Citral which was predicted as moderate

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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in the training run and as high in P&G’s historical database. The acceptance criteria describedin the SOP were met and the triplicate values for each chemical were highly reproducible.
Trainings sessions for Ricerca and IVMU have been judged to be successful from by thetrainer and the trainees as reported in the Statement on training outcome in the corresponding
Training Reports (Appendices 13 and 14).
3. Transfer of the test method to the naïve laboratories
As part of the Training and Transfer plan, P&G requested that trainees then used the training
chemicals to establish the assay in their laboratories. They were asked to perform three valid
runs and report their data back to the trainer prior to beginning the official Transfer Phase.
Ricerca reported its data back to P&G on April 15, 2010. The main concern observed in the
results was that, despite results which were otherwise very consistent with the historical datafrom P&G, the cysteine depletion values for the positive control cinnamic aldehyde obtained at
Ricerca were consistently marginally above the upper limit (set at 96.6% in the DPRA SOP
version 1), which led the VMG to remove the upper bound for the acceptance criteria andaccept cysteine depletions up to 100% for the testing phase.
A stability issue was encountered with the cysteine peptide (observed in test results withcysteine control solutions) during the implementation of the DPRA to the naïve laboratories.
This stability issue appeared as a progressive decrease of the integration peak area over time of
the cysteine peptide in absence of any test item induced depletion reaction. A problem with thequality/purity of the cysteine peptide used was initially suspected, but difference sources ofcysteine peptide were evaluated and showed the same phenomenon.
Ricerca then performed further experiments to investigate the cause of the cysteine peptide
decrease. The peptide cysteine (Reference control A and STD1 from the SOP, see Appendix 8)
was maintained at room temperature using the Fisher Acetonitrile (originally purchased by
Ricerca) and a fresh batch of Acetonitrile from Fluka, and analysed by HPLC for 48 hours (seeFigure 4, taken from the Transfer Report from Ricerca (See Appendix 16)). The results
suggest that the source/batch of the acetonitrile used during the peptide depletion reaction was
the cause of this problem. As the precise cause of this phenomenon could not be determined,
the SOP was modified to require preliminary testing to be performed prior to conducting the
DPRA procedure in order to test the suitability of new batches of acetonitrile.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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Figure 4: Decrease of the integration peak area (220 nm) over time of the cysteine peptide
in absence of test item-induced depletion reaction
Establishment of the DPRA at the IVMU laboratory started in June 2010 and continued until
December 2010. The IVMU laboratory experienced problems in fulfilling the reference
controls acceptance criteria. The mean peptide concentration for the reference controls for boththe cysteine and the lysine peptides, were for most of the initial experiments outside therequested range (0.50 +/- 0.05 mM). Furthermore, the variability (expressed as %CV) of thereference control values was relatively high compared to what was normally achieved by thelead laboratory. Experiments performed with the use of a different batch and a different
supplier of acetonitrile (see above) presented the same problems. To identify the reasons forthese atypical findings, the IVMU undertook systematic troubleshooting under the supervisionof the lead laboratory.
In order to exclude the possibility that the peptides used were the source of the problems P&G
supplied the IVMU from their own batch of cysteine peptide (purchased from RS Synthesis)
and asked the IVMU to verify injection repeatability by injecting the cysteine peptide at twohour intervals. Despite the change of peptide supplier the calculated %CV was still too high(6.47%). To verify whether or not such high variability was associated with the peptide
stability, the same experiment was performed with salicylic acid, a more stable compound.
This resulted in an improvement in the repeatability (2.65%CV) which was consideredhowever to be still high for HPLC standards, indicating that the problem might be due to the
HPLC instrumentation setup. At that point P&G provided the IVMU laboratory with a smaller
injection loop and a smaller syringe for their HPLC instrument. Changing the injection volume
from a partial loop injection (5μL) to a full injection loop (10μL) achieved a repeatability of
0.91%CV, which was deemed to be appropriate. Following this change, in December 2010 the
IVMU performed the three valid runs requested by the lead laboratory to progress to thequalification runs.

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4. Qualification runs for a successful completion of the transfer phase
To prove successful method transfer before being allowed to proceed to the blind testing phase(phase B), Ricerca and IVMU had to perform qualification analysis with 15 test items ofknown identity (see table 7) in two independent runs with each peptide, and meet the transfer
acceptance criteria as defined in the Transfer Plan issued by the lead laboratory and approvedby the VMG. Experimental data were submitted to the lead laboratory for evaluation using the“data reporting template” provided during the training session.
Test chemicals, peptides and all other reagents were purchased by the individual laboratories.
Table 7. Test chemicals used for the qualification runs of the DPRA transfer
ChemicalsCAS

In vivo potency

p-Benzoquinone, 98%
2,4-Dinitrochlorobenzene, 99%
Oxazolone, >90%
Formaldehyde, 37%
2-Phenylpropionaldehyde, 98%
Diethyl maleate, 97%
Benzylideneacetone, 99%
Farnesal, >85%

106-51-4
97-00-7
15646-46-5
50-00-0
93-53-8
141-05-9
122-57-6
19317-11-4

StrongStrongStrongStrongModerate
Moderate
ModerateWeak

2,3-Butanedione, 97%
4-Allylanisol, 98%
Hydroxycitronellal, 95%

431-03-8
140-67-0
107-75-5

Weak
Weak
Weak

Butanol, 99.4%
6-Methylcoumarin, 99%
Lactic acid, 85%
4-Methoxyacetophenone, 99%

71-36-3
92-48-8
50-21-5100-06-1

Non-sensitiser
Non-sensitiser
Non-sensitiser
Non-sensitiser

Sigma
Aldrichcatalog number
B10358237329
E0753
F15587241369
D97703147885
W401900SAMPLE
13530
A29208
W258318SAMPLE
360465
M36203252476117374The criteria for successful transfer were:••

Each assay run must meet all acceptance criteria as described in the SOP in order tobe considered a valid run.
In each run, at least 14/15 chemicals should be correctly categorised as “sensitiser”
or “non-sensitiser” with at least 13/15 chemicals being assigned a reactivity
category that is the same, or no more than one above or below, as P&G’s historical
data. Each independent run must meet these criteria, and misclassifications may
occur in any potency category. The historical data from P&G was not revealed to
the naïve laboratories beforehand.

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Qualification of Ricerca
Transfer experiments at Ricerca were performed in April-May 2010 and the final quality
audited report was released on 29th June 2010. Informed by the feedback received by the leadlaboratory and on the basis of the results provided in the study report, the VMG officiallyapproved the successful transfer of the DPRA at Ricerca, satisfied that all the criteria for a
successful method transfer had been met (See Table 7). On July 15th 2010 the VMG formallycommunicated to Ricerca that its transfer report was approved.
RUN 1 (29-04-2010)
ChemicalCinnamic aldehyde
p-Benzoquinone
2,4-Dinitrochlorobenzene
Oxazolone
Formaldehyde
2-Phenylpropionaldehyde
Diethyl maleate
Benzylideneacetone
Farnesal
2,3-Butanedione
4-Allylanisol
Hydroxycitronellal
Butanol,
6-Methylcoumarin
Lactic acid
4-Methoxyacetophenone

Mean
Cysteine%
depletion
99.9
99.4
99.4
75.0
50.9
100.0
100.0
94.1
23.9
80.4
17.1
31.7
0.4
0.6
0.1
0.5

Mean
Lysine%Depletion
64.9
91.4
30.5
53.7
4.3
18.4
85.0
1.9
16.8
24.1
0.2
12.6
0.0
0.4
0.1
0.1

Mean
%
Cys+Lysdepletion
82.4
95.4
65.0
64.3
27.6
59.2
92.5
48.0
20.4
52.2
8.6
22.1
0.22
0.47
0.12
0.31

RUN 2 (05-05-2010)
Assigned
Reactivity
class
High
High
High
High
Moderate
High
High
High
Low
High
Low
Low
MinimalMinimalMinimalMinimalMean
Cysteine%
depletion
100.0
99.6
99.5
75.2
47.8
100.0
100.0
94.2
20.9
78.5
18.4
22.4
0.1
0.0
0.0
2.0

Mean
Lysine%Depletion
66.0
91.3
24.7
47.9
5.3
18.9
82.2
1.8
18.7
22.7
0.2
10.5
0.0
0.1
0.2
0.0

Mean
Cys+Lysdepletion

Assigned
Reactivity
class

83.0
95.4
62.1
61.5
26.5
59.5
91.1
48.0
19.8
50.6
9.3
16.4
0.06
0.03
0.08
1.01

High
High
High
High
Moderate
High
High
High
Low
High
Low
Low
MinimalMinimalMinimalMinimalP&G
historical
reactivity
class
High
High
High
High
Moderate
High
High
High
Low
High
Low
Low
MinimalMinimalMinimalMinimalTable 7: DPRA-Ricerca qualification run results

Qualification of IVMU
Transfer experiments at IVMU were performed in January-February 2011. In both independentruns with the cysteine peptide, the criterion for reference controls C (the reference control used
to check peptide stability over the analysis time) was not met as the mean peptide
concentration fell marginally outside the requested range (0.50 +/- 0.05 mM) with a lowest
mean value of 0.424 mM reported when acetonitrile was used to solubilise the peptide.
The lead laboratory did not feel that fulfilling this empirical criterion, as originally specified,
was essential for completion of the transfer phase to be deemed successful. Informed by the
feedback received by the lead laboratory and on the basis of the results provided in the IVMU
study report (Table 8), the VMG officially approved on 17 March 2011 the successful transferof the DPRA at the IVMU laboratory.
In view of the difficulties they faced during the implementation of the method, the IVMU
laboratory requested a review of values for the following acceptance criteria:a) Lower bound for mean of lysine peptide depletion values of the three replicates for cinnamic
aldehyde (positive control)

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b) Range of acceptability of mean cysteine peptide concentration of reference control C(dissolved in acetonitrile and water)
However, the VMG did not consider the evidence at the time with respect to one naïve test
facility to be sufficient to adopt the suggested changes and required that the original acceptancecriteria should be maintained for the blind testing phase.
It is important to note that P&G communicated to the laboratories the chemicals’ reactivity
category only after the full set of experiments had been performed and the experimental data
sent to the lead laboratory for assessment and evaluation. This additional information was thenused by the laboratory for the finalisation of the transfer reports.

ChemicalCinnamic aldehyde
p-Benzoquinone
2,4-Dinitrochlorobenzene
Oxazolone
Formaldehyde
2-Phenylpropionaldehyde
Diethyl maleate
Benzylideneacetone
Farnesal
2,3-Butanedione
4-Allylanisol
Hydroxycitronellal
Butanol,
6-Methylcoumarin
Lactic acid
4-Methoxyacetophenone

RUN 1

RUN 1

RUN 2

RUN 2

(19-01-2011)(12-01-2011)(09-02-2011)(02-02-2011)

Mean
Cysteine%
depletion
79.9
100.0
100.0
77.5
40.4
62.9
100.0
93.2
21.8
77.1
22.7
28.3
1.3
3.0
1.0
1.9

Mean
Lysine%Depletion
40.5
84.4
19.3
Int
6.9
20.0
Int
Int
8.4
21.7
0.4
20.8
0.0
0.8
0.0
0.5

Mean
Cysteine%
depletion
75.1
100
100
76.6
38.8
54.4
99.8
93.7
30.6
79.1
31.4
43.9
2.3
3.1
6.1
3.9

Mean
Lysine%Depletion
41.1
87.7
26.4
Int
18.8
29.7
60.0
Int
12.9
17.7
2.4
22.6
0.0
1.9
0.0
0.2

Mean
%
Cys+Lysdepletion
60.2
92.2
59.65
23.66
41.43
15.07
49.37
11.57
24.53
0.67
1.89
0.51
1.19

Assigned
Reactivity
class
High
High
High
Moderate*
Moderate
Moderate
High*
Moderate*
Low
High
Low
Moderate
MinimalMinimalMinimalMinimalMean
Cys+Lysdepletion

Assigned
Reactivity
class

P&G
historical

58.1
93.84
63.22
28.82
42.07
79.93
21.73
48.37
16.93
33.26
1.15
2.49
3.04
2.05

High
High
High
Moderate*
Moderate
Moderate
High
Moderate*
Low
High
Low
Moderate
MinimalMinimalMinimalMinimal

High
High
High
High
Moderate
High
High
High
Low
High
Low
Low
MinimalMinimalMinimalMinimal*Determined with the cysteine prediction model because of interference of the chemical with the lysine
peptideTable 8: DPRA-IVMU qualification run results

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Conclusion of the Validation Management Team on Module 3
The VMG concluded that the DPRA test method was successfully transferred from the leadlaboratory to the other two laboratories. All the problems experienced by the naïve laboratories
during the transfer phase proved to be due either to reagents or instrument configuration whichwere resolved and addressed in the revised SOP where appropriate. Concerning the acceptancecriteria that were set prior to the initiation of the study both laboratories experienced problems
in fully meeting some of them despite generating results otherwise consistent with P&G
historical data. In the case of the upper bound for the positive control cysteine depletion the
VMG agreed to remove it since it was already very close to 100% and since the upper bound isless relevant for the sensitivity of the test. In the case of the lower bounds for the lysine
depletion for the positive control as well as for the lower bound for the reference controls C,
the VMG concluded that there was insufficient evidence at the time to justify any modification.
Despite being HPLC based, the DPRA test method does not require strict adherence to themore stringent and demanding criteria required for the implementation of bioanalyticalmethods for quantitative determination of test materials and metabolites, therefore it is easier toimplement. However, as demonstrated by some of the issues encountered during this transfer
phase and the solutions that were implemented, performance of the DPRA assay requires adetailed understanding of the HPLC techniques and strict adherence to the specified equipment
and procedural details.
In conclusion the VMG considers that the DPRA can be readily transferred among properly
equipped and staffed laboratories. The techniques involved are commonly used in analyticallaboratories. Experienced personnel can readily be trained in the test method, and the necessary
equipment and supplies can be readily obtained. The DPRA SOP is clearly written and theanalysis can be performed without difficulties.

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Within-laboratory reproducibility (Module 2)
Reference document: Statistical report (Appendix 15)
List of additional documents filed for the study and available on
request (Appendix 16)
The within laboratory reproducibility was assessed with data generated with a subset of 15
chemicals tested in three independent experiments in each laboratory (Study Phase B2). As
described in the SOP, each experiment is composed of one run to evaluate the cysteine
depletion and another run to evaluate the lysine depletion. The SOP states that up to 25
chemicals can be accommodated within a single run, therefore the 15 chemicals were always
tested within the same run. As already described in the coding/decoding section, a number wasadded to the codes to distinguish the three sets of 15 chemicals, and the laboratories were
instructed never to include chemicals with different numbers in their codes in the samerun/experiment.
The data are presented laboratory by laboratory and the three required independent assessmentsof each chemical are referred to as experiment 1, experiment 2, and experiment 3.
The main determinant of the test method's reliability assessment was the concordance ofclassification, sensitiser (S) versus non-sensitiser (NS), which were determined from thepeptide depletion values. Furthermore the concordance of classification with regard to the 4
reactivity classes was considered. Additionally, descriptive and inferential statistical analyses(ANOVA) were performed on the raw peptide depletion data. However, because of the limited
number of replicates (n=3), the results of the inferential tests applied cannot be consistently
interpreted and are only considered as additional descriptive information. With a small numberof replicates, violations of ANOVA assumptions (normal distribution of the errors andhomogeneity of variances) are difficult to assess and confirm.
1. P&Ga. Reproducibility (concordance in predictions)
In relation to the primary aim, the reproducibility in terms of classification S versus NS, for 11of the 15 chemicals the same prediction was obtained in the 3 independent experiments
resulting in a WLR of 73.3% (Table 9, left side). For the assignment to a reactivity class, 10 of
15 chemicals were assigned the same reactivity class in all 3 experiments. Note that in all casesof disagreement, the difference in the reactivity class assignment was only of one class, e.g.
Formaldehyde (Table 9, right side), was classified as either LOW (twice) or MODERATE(once).

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TABLE 9. Phase B2 P&G: concordance in predictions between the three independent
experiments for the subset of 15 chemicals
Seq.
numberChemical

10

Kathon CG

11

Beryllium Sulphate

12

Formaldehyde

24.5

19.9

13

Chloramine T

100.0

100.0

14

Chlorpromazine HCl

3.3

3.3

15

2-MBT

100.0

100.0

16

Benzyl Salicylate

3.9

2.2

17

Benzyl Cinnamate

5.1

1.6

18

R(+)Limonene

7.9

19

Methyl Salicylate

2022

Isopropanol
Dimethyl
Isophthalate
4-PABA

23
24

21

P&G (mean pept depl,
A%)

Agreement

Exp 1

Exp 2

Exp 3

2 classes

Exp1

Exp2

Exp 3

4 classes

100.0

100.0

100.0

Yes

HIGHLys

HIGHLys

HIGHLys

Yes

10.5

12.3

15.9

Yes

LOW

LOW

LOW

Yes

22.2

Yes

MODERATE

LOW

LOW

No

100.0

Yes

HIGHLys

HIGHLys

HIGHLys

Yes

2.8

Yes

MINIMALMINIMALMINIMAL

Yes

100.0

Yes

HIGHLys

HIGHLys

HIGHLys

Yes

7.9

No

MINIMALMINIMAL

LOW

No

3.1

Yes

MINIMALMINIMALMINIMAL

Yes

1.9

6.6

No

LOW

MINIMAL

LOW

No

5.1

3.1

7.1

No

MINIMALMINIMAL

LOW

No

5.2

3.6

5.8

Yes

MINIMALMINIMALMINIMAL

Yes

6.3

1.1

5.1

Yes

MINIMALLysMINIMALLysMINIMALLys

Yes

5.6

2.4

5.6

Yes

MINIMALMINIMALMINIMAL

Yes

Nickel Chloride

0.6

1.0

0.8

Yes

MINIMALMINIMALMINIMALYes

Xylene

5.2

2.3

6.9

No

MINIMALMINIMAL

LOW

No

P&G (4 reactivity classes)

Agreement

A

Values represent the mean cysteine and lysine peptide depletion. In case of co-elution with the lysine peptide (indicated by the
LYS subscript in the columns with the reactivity classes) the value represents the the cysteine peptide depletion. Mean depletion
values with an orange background correspond to a sensitiser prediction, those with a green background correspond to a nonsensitiser prediction.

b. Reproducibility of depletion values for cysteine and lysine
The cysteine and lysine peptide depletions for each chemical were compared between the 3
independent experiments by one-way ANOVA. The results for the cysteine depletion arepresented in Table 10 and the results for lysine depletion are presented in TABLE 11. ANOVA
revealed no differences in mean cysteine or lysine depletion between the three independent
experiments, except for Formaldehyde. For this chemical, although the differences in the mean
cysteine depletions reached statistical significance, this had no impact on the finalclassification as the chemical was predicted as sensitiser in all three independent experiments.
The variability within an experiment was very small, therefore minor differences between theexperiments resulted in a statistically significant difference in mean cysteine depletion for thischemical.

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Table 10. Phase B2 P&G: within laboratory variability of the cysteine depletion values
Seq.
numberChemicalCysteine depletion (%)

Cysteine depl (%) p-value

1

Experiment 1

Experiment 2

Experiment 3

Betweenexperiment100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

NA25.5 ± 5.1

0.362

40.6 ± 4.0<0.001*

10

Kathon CG

100.0 ± 0.011Beryllium Sulphate

20.8 ± 10.4
C

24.6 ± 5.5
36.6 ± 1.2

31.0 ± 7.6
A40.6 ± 1.2

B12

Formaldehyde

44.7 ± 1.0

13Chloramine T

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0NA14Chlorpromazine HCl

6.6 ± 8.2

6.4 ± 11.0

5.5 ± 5.2

6.2 ± 0.5

0.988

15

2-MBT

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

NA16

Benzyl Salicylate

7.8 ± 7.2

2.8 ± 4.2

14.4 ± 6.1

8.3 ± 5.8

0.134

17

Benzyl Cinnamate

10.0 ± 9.8

2.0 ± 3.4

5.8 ± 5.9

5.9 ± 4.0

0.414

18

R(+)Limonene

15.5 ± 4.1

2.5 ± 2.4

12.2 ± 6.2

10.1 ± 6.8

0.029

19

Methyl Salicylate

9.1 ± 8.0

3.4 ± 5.1

11.3 ± 4.9

7.9 ± 4.1

0.330

20

Isopropanol

9.9 ± 9.2

5.0 ± 5.8

10.8 ± 4.0

8.5 ± 3.1

0.551

21

Dimethyl Isophthalate

6.3 ± 8.3

1.1 ± 2.0

5.1 ± 4.6

4.2 ± 2.7

0.534

22

4-PABA

10.7 ± 8.4

3.9 ± 4.2

10.6 ± 8.1

8.4 ± 3.9

0.457

23

Nickel Chloride

1.2 ± 2.0

0.0 ± 0.0

0.0 ± 0.0

0.4 ± 0.7

0.422

24

Xylene9.6 ± 8.4

2.5 ± 4.1

12.1 ± 7.6

8.1 ± 5.0

0.289

Values presented as mean of the three replicates within the same run ± SD, n=3
1

One-way ANOVA critical α-level = 0.027 (corrected for the number of hypothesis tested), *mean values with a different subscript
are significantly different from each other (Tukey post-hoc all comparisons)
NA: ANOVA not performed since all individual values were equal

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Table 11. Phase B2 P&G: within laboratory variability of the lysine depletion values
Seq.
ChemicalnumberLysine depletion (%)

Lysine depl (%)

Experiment 1

Experiment 2

Experiment 3

Betweenexperimentp-value

10

Kathon CG

4.8 ± 5.0

11.1 ± 5.4

3.5 ± 3.6

6.5 ± 4.1

0.789

11

Beryllium Sulphate

0.1 ± 0.1

0.0 ± 0.0

0.8 ± 1.3

0.3 ± 0.4

0.288

12

Formaldehyde

4.3 ± 4.7

3.2 ± 1.2

3.8 ± 1.5

3.8 ± 0.5

0.841

13

Chloramine T

14

Chlorpromazine HCl

0.0 ± 0.0

0.1 ± 0.2

0.0 ± 0.0

0.0 ± 0.1

NA

15

2-MBT

3.5 ± 2.8

8.7 ± 5.2

9.0 ± 6.7

7.1 ± 3.1

0.211

16

Benzyl Salicylate

0.0 ± 0.0

1.6 ± 1.3

1.5 ± 0.7

1.0 ± 0.9

0.093

17

Benzyl Cinnamate

0.2 ± 0.4

1.1 ± 1.2

0.3 ± 0.3

0.6 ± 0.5

0.923

18

R(+)Limonene

0.3 ± 0.5

1.3 ± 0.9

1.0 ± 0.4

0.9 ± 0.5

0.262

19

Methyl Salicylate

1.0 ± 0.8

2.9 ± 1.5

3.0 ± 0.2

2.3 ± 1.1

0.052

20

Isopropanol

0.5 ± 0.8

2.3 ± 3.2

0.7 ± 0.6

1.2 ± 1.0

0.905

21

Dimethyl Isophthalate

22

4-PABA

0.4 ± 0.7

0.9 ± 0.6

0.6 ± 0.2

0.6 ± 0.3

0.718

23

Nickel Chloride

0.1 ± 0.2

2.0 ± 2.3

1.5 ± 1.8

1.2 ± 1.0

0.338

24

Xylene0.8 ± 1.4

2.1 ± 1.9

1.6 ± 1.5

1.5 ± 0.6

0.548

1

Values presented as mean of the three replicates within the same run ± SD, n=3
1

One-way ANOVA, critical α-level = 0.027 (corrected for the number of hypothesis tested)

Chemicals with a grey background correspond with lysine co-elution. When it was possible to estimate a depletion value, thisvalue is reported in the table.

c. Reproducibility of the controls values
Before the results were accepted for further analysis, it was determined whether the acceptancecriteria for reference controls A and C and for the positive control cinnamic aldehyde were
met. According to the SOP, the mean cysteine and lysine concentration for reference control A,
reference control C in water and reference control C in acetonitrile should be between 0.45 mM
and 0.55 mM. The cysteine depletion for the positive control should be between 60.8% and100% and the lysine depletion between 40.2% and 69.4%.
The individual values within each of the runs are shown in Figure 5. The mean peptide
concentration of the reference controls and the mean peptide depletion of the positive control
were always within the predefined ranges (Table 12 and Table 13).

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Figure 5. Peptide concentration for the different reference controls and peptide depletion for the
positive control (individual data) for each of the individual runs performed at P&G. Run 1
represents the phase B1 experiment and runs 2 , 3 and 4 represent the phase B2 experiments. The greylines correspond with the lower and upper threshold for the mean peptide concentration (reference
controls) or for the mean peptide depletion (positive control).
Table 12. Cysteine concentration of the reference controls and cysteine depletion of the positive
control for the independent runs at P&G.
Lab

Experiment

Cysteine concentration (mM)

Cysteine depletion

Ctrl A

Ctrl C water

Ctrl C acetonitrile

Postive control

P&G

Phase B1-1

0.47 ± 0.01

0.45 ± 0.02

0.45 ± 0.03

72.99 ± 0.6

P&G

Phase B2-2

0.51 ± 0.01

0.46 ± 0.02

0.45 ± 0.04

80.33 ± 1.0

P&G

Phase B2-3

0.50 ± 0.01

0.55 ± 0.03

0.54 ± 0.01

72.67 ± 1.6

P&G

Phase B2-4

0.50 ± 0.01

0.46 ± 0.01

0.46 ± 0.01

76.82 ± 0.4

Values are presented as mean of the three replicates within the same run ± SD, n=3

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Table 13. Lysine concentration of the reference controls and lysine depletion of the positive
control for the independent runs at P&G.
Lab

Experiment

Lysine concentration (mM)

Lysine depletion

Ctrl A

Ctrl C water

Ctrl C acetonitrile

Postive control

P&G

Phase B1-1

0.46 ± 0.00

0.46 ± 0.02

0.47 ± 0.02

53.7 ± 3.1

P&G

Phase B2-2

0.50 ± 0.00

0.49 ± 0.00

0.50 ± 0.00

63.1 ± 1.8

P&G

Phase B2-3

0.51 ± 0.00

0.48 ± 0.01

0.49 ± 0.01

67.6 ± 3.3

P&G

Phase B2-4

0.50 ± 0.00

0.48 ± 0.01

0.49 ± 0.02

64.4 ± 1.1

Values are presented as mean of the three replicates within the same run ± SD, n=3

2. RICERCA
a. Reproducibility (concordance in predictions)
In relation to the primary aim, the reproducibility in terms of the classification S versus NS, forall of the 15 chemicals the same prediction was obtained in the 3 independent experiments
resulting in a WLR of 100% .(Table 14, left side). For the assignment to a reactivity class, the
15 chemicals were assigned the same reactivity class in all 3 experiments (Table 14, right side).
Table 14. Phase B2 Ricerca: concordance in predictions between the three independent
experiments for the subset of 15 chemicals
Seq.
numberChemicalRicerca (mean pept depl,
A%)

Agreement

Exp 2

Exp 3

Exp 4

2 classes

Exp 2

Exp 3

Exp 4

4 classes

53.3

52.6

54.1

Yes

HIGH

HIGH

HIGH

Yes

MINIMALMINIMAL

Ricerca (4 reactivity classes)

10

Kathon CG

11

Beryllium Sulphate

1.1

0.4

1.4

Yes

12

Formaldehyde

26.0

26.9

25.3

Yes

13

100.0

100.0

100.0

Yes

HIGHLys

2.0

3.7

2.7

Yes

MINIMAL

15

Chloramine T
Chlorpromazine
HCl
2-MBT

51.2

51.3

50.8

Yes

HIGH

16

Benzyl Salicylate

0.2

0.0

0.3

Yes

MINIMAL17

Benzyl Cinnamate

1.0

1.4

0.3

Yes

MINIMAL

18

R(+)Limonene

7.1

10.2

9.2

Yes

LOW

19

Methyl Salicylate

0.8

0.9

0.7

Yes

20

0.4

0.8

3.1

2.5

0.8

22

Isopropanol
Dimethyl
Isophthalate
4-PABA

2.4

23

Nickel Chloride

7.0

24

Xylene

0.4

0.3

14

21

Agreement

MINIMAL

Yes

MODERATEYes

HIGHLys

HIGHLys

Yes

MINIMALMINIMAL

Yes

HIGH

HIGH

Yes

MINIMALMINIMALYes

MINIMALMINIMAL

Yes

LOW

LOW

Yes

MINIMALMINIMALMINIMALYes

Yes

MINIMALMINIMALMINIMAL

Yes

2.5

Yes

MINIMALMINIMALMINIMAL

Yes

1.1

0.4

Yes

MINIMALMINIMALMINIMAL

Yes

10.2

11.5

Yes

LOW

LOW

LOW

Yes

0

Yes

MINIMALMINIMALMINIMAL

Yes

MODERATE MODERATEA

Values represent the mean cysteine and lysine peptide depletion. In case of co-elution with the lysine peptide (indicated by the
LYS subscript in the columns with the reactivity classes) the value represents the the cysteine peptide depletion. Mean depletion
values with an orange background correspond to a sensitiser prediction, those with a green background correspond to a nonsensitiser prediction.

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b. Reproducibility of depletion values for cysteine and lysine
The cysteine and lysine peptide depletions for each chemical were compared between the 3
independent experiments by one-way ANOVA. The results for the cysteine depletion arepresented in Table 15 and the results for lysine depletion are presented in Table 16. ANOVA
revealed no statistically significant differences in mean cysteine and lysine depletion between
the three independent experiments, except for Isopropanol (cysteine) and Kathon CG, 2-MBT
and Dimethyl Isophthalate (lysine). For these chemicals, differences in the mean depletion
were small in absolute terms but reached statistical significance because the variability of thetriplicates within the runs was very small. Understandably this had no impact on the finalclassifications of the chemicals or on the assigned reactivity class.
Table 15. Phase B2 Ricerca: within laboratory variability of the cysteine depletion
Seq.
Chemicalnumber10

Kathon CG

Cysteine depletion (%)

Cysteine depl (%) p-value

Experiment 1

Experiment 2

Experiment 3

Betweenexperiment100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0NA11Beryllium Sulphate

2.0 ± 2.2

0.7 ± 1.3

2.6 ± 1.9

1.8 ± 1.0

0.418

12

Formaldehyde

49.6 ± 1.6

51.4 ± 2.0

48.6 ± 2.8

49.9 ± 1.4

0.347

13

Chloramine T

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0

100.0 ± 0.0NA14Chlorpromazine HCl

15

2-MBT

3.9 ± 2.4

7.4 ± 1.9

5.4 ± 4.4

5.5 ± 1.7

0.435

100.0 ± 0.0

98.7 ± 2.2

100.0 ± 0.0

99.6 ± 0.7

0.422

16

Benzyl Salicylate

0.2 ± 0.3

0.0 ± 0.1

0.7 ± 1.2

0.3 ± 0.3

0.553

17

Benzyl Cinnamate

1.4 ± 1.3

2.5 ± 1.6

0.7 ± 1.2

1.5 ± 0.9

0.314

18

R(+)Limonene

13.6 ± 2.0

18.8 ± 2.5

18.4 ± 1.1

16.9 ± 2.9

0.030

19

Methyl Salicylate

0.3 ± 0.6

0.2 ± 0.4

0.4 ± 0.6

0.3 ± 0.1

0.953

20

Isopropanol

0.3 ± 0.5

2.5 ± 3.2

<0.001*21

Dimethyl Isophthalate

0.7 ± 0.8

0.4 ± 0.2

0.697

22

4-PABA

4.6 ± 2.1

1.4 ± 1.2

0.9 ± 1.4

2.3 ± 2.0

0.590

23

Nickel Chloride

13.9 ± 4.7

20.4 ± 5.7

22.9 ± 6.2

19.1 ± 4.6

0.204

24

Xylene0.5 ± 0.9

0.3 ± 0.6

0.0 ± 0.0

0.3 ± 0.3

0.603

A

1.0 ± 1.0A

6.1 ± 1.2

0.4 ± 0.7

0.2 ± 0.4

B

1

Values presented as mean of the three replicates within the same run ± SD, n=3
1

One-way ANOVA critical α-level = 0.027 (corrected for the number of hypothesis tested), *mean values with a different subscript
are significantly different from each other (Tukey post-hoc all comparisons)
NA: ANOVA testing not performed since all individual values were equal

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Table 16. Phase B2 Ricerca: within laboratory variability of the lysine depletion
Seq.
ChemicalnumberLysine depletion (%)
Experiment 2

10

B

Experiment 3

Kathon CG

6.6± 0.711Beryllium Sulphate

0.1± 0.1

0.1± 0.1

12Formaldehyde

2.3± 0.3

2.5± 0.4

13

Chloramine T

14

Chlorpromazine HCl

0.0± 0.0

5.2± 0.8

Lysine depl (%)
Experiment 4AB

0.0± 0.0A

B

Betweenexperiment
6.7 ± 1.5

0.005*

0.2± 0.1

0.1 ± 0.1

0.340

2.0± 0.3

2.3 ± 0.2

0.213

0.0 ± 0.0NA

0.0± 0.0
B

2-MBT

2.3± 0.5

2.6 ± 1.2

<0.001*

16

Benzyl Salicylate

0.1± 0.1

0.0± 0.0

0.0± 0.0

0.0 ± 0.0

0.079

17

Benzyl Cinnamate

0.6± 0.1

0.2± 0.3

0.0± 0.0

0.3 ± 0.3

0.038

18

R(+)Limonene

0.5± 0.3

1.5± 1.0

0.0± 0.1

0.7 ± 0.7

0.073

19

Methyl Salicylate

1.3± 0.3

1.6± 0.3

1.0± 0.0

1.3 ± 0.3

0.059

20

Isopropanol

0.5± 0.1

0.4 ± 0.3

0.096

3.4 ± 1.9

<0.001*0.6± 0.5B

1.2± 0.5

1.6± 0.2A1

15

4.2± 0.7

3.9± 0.2

8.2± 0.4

p-value0.0± 0.0A

4.7± 0.5

B21

Dimethyl Isophthalate

22

4-PABA0.1± 0.1

0.7± 0.4

0.0± 0.0

0.3 ± 0.4

0.030

23

Nickel Chloride

0.0± 0.0

0.0± 0.1

0.0± 0.0

0.0 ± 0.0

0.422

24

Xylene0.2± 0.1

0.2± 0.2

0.0± 0.0

0.1 ± 0.1

0.171

Values presented as mean of the three replicates within the same run ± SD, n=3
1

One-way ANOVA, *mean values with a different subscript are significantly different from each other (Tukey post-hoc allcomparisons)
NA: ANOVA testing not performed since all individual values were equal
Chemicals with a grey background correspond with lysine co-elution. When it was possible to estimate a depletion value, thisvalue is reported in the table.

c. Reproducibility of controls values
The individual values within each of the runs are shown in Figure 6. The mean peptide
concentration of the reference controls and the mean peptide depletion of the positive control
were always within the predefined ranges (Table 17 and Table 18).

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Figure 6. Peptide concentration for the different reference controls and peptide depletion for the
positive control (individual data) for each of the individual runs performed at Ricerca. Run 1
represents the phase B1 experiment and runs 2, 3 and 4 represent the phase B2 experiments. The greylines correspond with the lower and upper threshold for the mean peptide concentration (reference
controls) or for the mean peptide depletion (positive control).
Table 17. Cysteine concentration of the reference controls and cysteine depletion of the positive
control for the independent runs at Ricerca
Lab

Experiment

Cysteine concentration (mM)

Cysteine depletion

Ctrl A

Ctrl C water

Ctrl C acetonitrile

Postive control

RicercaPhase B1-1

0.51 ± 0.00

0.47 ± 0.01

0.48 ± 0.00

100.00 ± 0.0

RicercaPhase B2-2

0.50 ± 0.00

0.46 ± 0.01

0.47 ± 0.01

97.80 ± 0.4

RicercaPhase B2-3

0.50 ± 0.00

0.47 ± 0.01

0.48 ± 0.00

100.00 ± 0.0

RicercaPhase B2-4

0.50 ± 0.00

0.45 ± 0.01

0.46 ± 0.01

100.00 ± 0.0

Values are presented as mean of the three replicates within the same run ± SD, n=3

Table 18. Lysine concentration of the reference controls and lysine depletion of the positive
control for the independent runs at Ricerca
Lab

Experiment

Lysine concentration (mM)

Lysine depletion

Ctrl A

Ctrl C water

Ctrl C acetonitrile

Postive control

RicercaPhase B1-1

0.50 ± 0.00

0.49 ± 0.00

0.50 ± 0.00

68.5 ± 1.2

RicercaPhase B2-2

0.50 ± 0.00

0.49 ± 0.00

0.50 ± 0.00

66.9 ± 2.8

RicercaPhase B2-3

0.51 ± 0.00

0.50 ± 0.00

0.51 ± 0.00

47.9 ± 1.8

RicercaPhase B2-4

0.50 ± 0.00

0.49 ± 0.00

0.50 ± 0.00

65.8 ± 2.1

Values are presented as mean of the three replicates within the same run ± SD, n=3

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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3. IVMU
Having been alerted to issues with meeting the run acceptance criteria, the VMG examined thecontrol values first to determine whether to proceed with analysis of the results or to request
retesting. Only once that decision was taken did the VMG proceed to review the experimental
data.
a. Reproducibility of the controls values
The individual values within each of the runs are shown in Figure 7. The mean peptide
concentration of the reference controls and the mean peptide depletion of the positive control
are presented in Table 19 and Table 20. The mean cysteine concentration for reference control
C was for some runs outside the requested range (0.50 +/- 0.05 mM) and for the other runs themean values were very close to the lower boundary. The mean lysine depletion for the positive
control was also outside the requested range in one of the three runs (< 40.2%). The mean
lysine depletion for the other runs was also close to the lower boundary.

Figure 7. Peptide concentration for the different reference controls and peptide depletion for the
positive control (individual data) for each of the individual runs performed at IVMU. Run 1
represents the phase B1 experiment and runs 2, 3 and 4 represent the phase B2 experiments. The greylines correspond with the lower and upper threshold for the mean peptide concentration (reference
controls) or for the mean peptide depletion (positive control).

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Table 19. Cysteine concentration of the reference controls and cysteine depletion of the positive
control for the independent runs at IVMU
Lab

Experiment

Cysteine concentration (mM)

Cysteine depletion

Ctrl A

Ctrl C water

Ctrl C acetonitrile

Postive control

IVMU

Phase B1-1

0.50 ± 0.00

0.45 ± 0.02

0.46 ± 0.01

73.50 ± 2.9

IVMU

Phase B2-2

0.50 ± 0.00

0.44 ± 0.01

0.44 ± 0.01

95.00 ± 0.6

IVMU

Phase B2-3

0.49 ± 0.00

0.45 ± 0.02

0.44 ± 0.04

77.50 ± 0.8

IVMU

Phase B2-4

0.50 ± 0.00

0.47 ± 0.02

0.47 ± 0.01

83.50 ± 1.1

Values are presented as mean of the three replicates within the same run ± SD, n=3
Values in red: acceptance criteria not met

Table 20. Lysine concentration of the reference controls and lysine depletion of the positive
control for the independent runs at IVMU
Lab

Experiment

Lysine concentration (mM)

Lysine depletion

Ctrl A

Ctrl C water

Ctrl C acetonitrile

Postive control

IVMU

Phase B1-1

0.51 ± 0.00

0.50 ± 0.00

0.51 ± 0.00

43.4 ± 3.0

IVMU

Phase B2-2

0.50 ± 0.00

0.49 ± 0.00

0.51 ± 0.00

42.0 ± 2.2

IVMU

Phase B2-3

0.50 ± 0.01

0.49 ± 0.00

0.50 ± 0.00

39.8 ± 2.1

IVMU

Phase B2-4

0.52 ± 0.00

0.50 ± 0.00

0.52 ± 0.00

41.9 ± 3.0

Values are presented as mean of the three replicates within the same run ± SD, n=3
Values in red: acceptance criteria not met

The findings were reviewed and discussed by the Validation Management Group, taking into
account the fact that:•At the IVMU laboratory, the values for the positive control and the reference control C
both in the valid and the invalid runs were systematically and consistently very close to
the lower limit of the acceptance range.

•

The reason reported by IVMU for the difficulties in meeting the acceptance criterion
for the positive control was a small but systematic co-elution observed with cinnamic
aldehyde and the lysine peptide, suggesting that the range for this criterion might not beappropriate for all laboratories as they were based on the experience and results of thelead laboratory alone.

•

The second acceptance criterion which the IVMU found difficult to meet, reference
control C, was set by the VMG at the initiation of the study and was based on thecriteria set by P&G for the reference controls A in the system suitability section.
However, unlike reference control A where the three replicates are analysed one after
the other soon after the samples are prepared, the triplicates of reference control C are
measured at different time points in the analysis sequence, with the first one towards
the beginning and the last one at the very end. Therefore, these controls are more prone
to variations caused by the incubation period, as was indeed observed in all laboratories(see figures 5, 6 and 7 for the variability of the reference controls C for cysteine
measurements compared to reference controls A), and suggesting that the range
assigned for reference control C might have been too narrow in practice.

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•

The issue had already been highlighted during the transfer phase. At that time, theVMG acknowledged the potential issue but concluded that there was insufficientevidence to justify modifications to the acceptance criteria (see Module 3, above).

•

No results from the other laboratories were rejected because of these criteria.

•

It was felt that minimal value would be gained by generating numerous additional(possibly) invalid runs at IVMU when it was already clear that the acceptance criteria
would need to be revised at the end of the study.

The VMG therefore decided to instruct the IVMU lab not to undertake supplementary testingin order to avoid generating additional invalid runs and instead to use the results generated so
far for the statistical evaluations.
b. Reproducibility (concordance in predictions)
In relation to the primary aim, the reproducibility in terms of the classification S versus NS, for
13 of the 15 chemicals the same prediction was obtained in the 3 independent experiments
resulting in a WLR of 86.7% .(Table 21, left side). For the assignment to a reactivity class, 11of the 15 chemicals were assigned the same reactivity class in all 3 experiments (Table 21,
right side). Note that in case of disagreement, all reported differences were never more than asingle reactivity class.
Table 21. Phase B2 IVMU: concordance in predictions between the three independent
experiments for the subset of 15 chemicals
Seq.
numberChemical

2 classes

Exp 1

Exp 2

10

Kathon CG

46.8

46.5

93.5

Yes

HIGH

HIGH

Exp 3
MODERATE

11

Beryllium Sulphate

1.7

1.5

1.2

Yes

MINIMALMINIMALMINIMALYes

12

Formaldehyde

19.5

15.4

19.0

Yes

LOW

LOW

LOW

Yes

13

Chloramine T

79.5

79.0

77.1

Yes

HIGH

HIGH

HIGH

Yes

14

Chlorpromazine HCl

4.3

2.0

5.7

Yes

MINIMALMINIMALMINIMAL

Yes

15

2-MBT

50.0

50.0

50.0

Yes

HIGH

HIGH

HIGH

Yes

16

Benzyl Salicylate

8.5

5.7

6.9

No

LOW

MINIMAL

LOW

No

17

Benzyl Cinnamate

9.4

2.5

4.9

No

LOW

MINIMALMINIMAL

No

18

R(+)Limonene

26.0

16.8

17.4

Yes

MODERATE

LOW

LOW

No

19

Methyl Salicylate

12.8

12.2

11.6

Yes

LOW

LOW

LOW

Yes

20

Isopropanol

0.9

0.4

4.8

Yes

IVMU (mean peptAdepl, %)Exp Exp Exp
12
3

Agreement

IVMU (4 reactivity classes)

Agreement
4 classes
Lys

No

MINIMALMINIMALMINIMALMINIMALMINIMALMINIMALMINIMALMINIMAL

Yes

Yes

21

Dimethyl Isophthalate

2.4

0.0

2.1

22

4-PABA

2.3

0.4

0.5

Yes

MINIMAL

23

Nickel Chloride

4.0

3.2

4.3

Yes

MINIMALMINIMALMINIMALYes

24

Xylene

1.3

0.0

0.6

Yes

MINIMALMINIMALMINIMAL

Yes

Yes

Lys

Lys

Lys

Yes

A

Values represent the mean cysteine and lysine peptide depletion. In case of co-elution with the lysine peptide (indicated by the
LYS subscript in the columns with the reactivity classes) the value represents the the cysteine peptide depletion. Mean depletion
values with an orange background correspond to a sensitiser prediction, those with a green background correspond to a nonsensitiser prediction.

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c. Reproducibility of depletion values for cysteine and lysine
The cysteine and lysine peptide depletions for each chemical were compared between the 3
independent experiments by one-way ANOVA. The results for the cysteine depletion arepresented in Table 22 and the results for lysine depletion are presented in Table 23. ANOVA
revealed differences between the three independent experiments, for four chemicals in themean cysteine peptide depletion values and for four chemical in the mean lysine peptide
depletion values. For only one of these chemicals, (Benzyl Cinnamate which showedsignificant differences in the mean cysteine depletion values), this resulted in different S/NSclassification between the 3 independent experiments.
Table 22. Phase B2 IVMU: within laboratory variability of the cysteine depletion values
Seq.
ChemicalnumberCysteine depletion (%)

Cysteine depl (%) p-value

Experiment 1

Experiment 2

Experiment 3

Betweenexperiment93.0± 0.593.5± 1.3

93.4 ± 0.3

0.702

2.7 ± 0.3

0.962

34.8 ± 3.8<0.001*

10

Kathon CG

93.5± 0.211Beryllium Sulphate

2.6± 2.2

3.0± 3.9
B

Formaldehyde

37.3± 1.2

13

Chloramine T

100.0± 0.0

100.0± 0.0

100.0± 0.0

100.0 ± 0.0NA14Chlorpromazine HCl

8.5± 3.2

3.9± 3.8

11.4± 2.6

7.9 ± 3.8

0.077

15

2-MBT

100.0± 0.0

100.0± 0.0

100.0± 0.0

100.0 ± 0.0

NABenzyl Salicylate

4.5± 3.8

17

Benzyl Cinnamate

13.2± 4.1

B
B

18

R(+)Limonene

51.8± 3.4

19

Methyl Salicylate

1.0± 1.3

36.7± 0.5

B

12

16

30.5± 0.8

2.4± 2.6A

0.0± 0.0

2.4± 2.40.0± 0.0A

33.5± 3.0A

2.3 ± 2.20.185

3.9± 3.6A

5.7 ± 6.8

0.005*

33.0± 2.3

A

39.5 ± 10.7

<0.001*0.5 ± 0.50.390

0.0± 0.0A

0.0± 0.0

0.4± 0.8A

9.3± 2.4

B

20

Isopropanol

1.5± 1.8

3.6 ± 5.0

0.001*

21

Dimethyl Isophthalate

2.4± 2.1

0.0± 0.0

2.1± 2.2

1.5 ± 1.3

0.281

22

4-PABA

4.6± 3.5

0.7± 1.3

1.0± 1.0

2.1 ± 2.2

0.135

23

Nickel Chloride

8.1± 4.9

6.3± 5.9

8.5± 5.8

7.6 ± 1.1

0.883

24

Xylene2.7± 2.3

0.0± 0.0

1.1± 1.5

1.3 ± 1.3

0.198

1

Values presented as mean of the three replicates within the same run ± SD, n=3
1

One-way ANOVA critical α-level = 0.027 (corrected for the number of hypothesis tested), *mean values with a different subscript
are significantly different from each other (Tukey post-hoc all comparisons)
NA: ANOVA testing not performed since all individual values were equal

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Table 23. Phase B2 IVMU: within laboratory variability of the lysine depletion
Seq.
ChemicalnumberLysine depletion (%)

Lysine depl (%)

1

Betweenexperiment

Experiment 1

Experiment 2

Kathon CG

0.0± 0.0

0.0± 0.011Beryllium Sulphate

0.9± 0.8

0.3 ± 0.5

0.081

12

Formaldehyde

1.8± 0.2C

0.3± 0.2A

1.3± 0.1B

1.1 ± 0.7

<0.001*

13

Chloramine T

59.0± 0.4B58.1± 0.9B54.2± 1.1

A

57.1 ± 2.5<0.001*

14

Chlorpromazine HCl

0.0± 0.0

0.0 ± 0.0

NA

15

2-MBT

0.0± 0.0

0.0± 0.0

0.0± 0.0

0.0 ± 0.0

NA

16

Benzyl Salicylate

12.5± 1.8

11.4± 2.7

11.3± 0.9

11.7 ± 0.7

0.728

17

Benzyl Cinnamate

5.6± 1.3

10

Experiment 3

p-value

0.0 ± 0.0

0.0± 0.0

0.0± 0.0

0.0± 0.0

0.0± 0.0

5.0± 1.8

6.0± 0.8

0.0± 0.0A

24.4± 0.6BNA

5.5 ± 0.50.682

1.8± 0.2B

0.6 ± 1.0

<0.001*22.9± 0.4

A

24.0 ± 1.0

0.009*

0.4 ± 0.4

0.038

18

R(+)Limonene

0.1± 0.2

A

19

Methyl Salicylate

24.6± 0.5

B20

Isopropanol

0.2± 0.3

21

Dimethyl Isophthalate

22

4-PABA

0.0± 0.0

0.0± 0.0

0.0± 0.0

0.0 ± 0.0NA23

Nickel Chloride

0.0± 0.0

0.0± 0.0

0.0± 0.0

0.0 ± 0.0NA24

Xylene0.0± 0.0

0.0± 0.0

0.1± 0.1

0.0 ± 0.1

0.079

0.9± 0.3

0.2± 0.1

NA

Values presented as mean of the three replicates within the same run ± SD, n=3
1

One-way ANOVA critical α-level = 0.027 (corrected for the number of hypothesis tested), *mean values with a different subscript
are significantly different from each other (Tukey post-hoc all comparisons)
NA: ANOVA testing not performed since all individual values were equal
Chemicals with a grey background correspond with lysine co-elution. When it was possible to estimate a depletion value, thisvalue is reported in the table.

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) between the three independent experiments. The WLR for P&G, Ricerca, andIVMU for the S/NS predictions were 73.3%, 100% and 86.7%, respectively. When 4 reactivity
classes were considered, the WLR was 66.7% for P&G, 100% for Ricerca, and 73.3% for
IVMU. Notably, in case of inconsistency all reported differences were never more than a single
reactivity class.
Overall, the VMG agreed, on the basis of the test results and their statistical evaluation, that the
average WLR for sensitiser (S) versus non-sensitiser (NS) from the three laboratories (87%)
successfully met the target performance proposed at the onset of the study (85%). The goodaverage WLR of 80% for the four reactivity classes further supported the conclusion of the
VMG that the DPRA demonstrated good within-laboratory reproducibility.
As an additional note, both of the trained laboratories achieved higher reproducibility than thelead laboratory, fully consistent with the VMG conclusions for Module 3.

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Between laboratory reproducibility (Module 4)
Reference document: Statistical report (Appendix 15)
List of additional documents filed for the study and available on
request (Appendix 16)
Note on the solubilisation procedures:
It was decided by the VMG prior to the initiation of the study that the participating laboratories
would not be instructed on which solvent to use to solubilise the coded chemicals. The VMG
considered the solvent selection procedures to be an integral part of the test method SOP, to beevaluated for reproducibility together with the rest of the procedure.
The SOP provided a tiered solvent selection strategy. The results of the study showed that thesame solvent was chosen by all laboratories for 22 of the 24 chemicals (see Table 24 below).
Unlike the other two laboratories, Ricerca reported that chemicals Kathon CG and
Chlorpromazine HCl were not soluble in acetonitrile during their solubilisation assessment.
However the different solvents used by Ricerca for these two chemicals had no effect on theclassification sensitisers/non sensitisers or on the assignment of the reactivity class comparedto the other laboratories (see below).
Note on the coelution:
As discussed in the test definition module, in the DPRA some chemicals, or the reaction
products formed following incubation with the peptide, may interfere with the peptide
determination because they elute at the same time of the peptide. In some cases this seems to
be instrument dependent: if different instruments are used (as was the case for this study where
the laboratories were equipped with HPLC instruments from different producers) thisphenomenon may be systematically observed in one laboratory but not necessarily in theothers.
Table 25 reports the co-elution observed by the different laboratories for either the cysteine
peptide or the lysine peptide during the blind testing phase and shows that the patterns of coelution were not consistent between the laboratories, but were reproducible at each site (withthe single exception of Kathon CG at IVMU, where co-elution was reported in only one of theexperiments).

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Table 24. Solvent selection for each of the chemicals by the three laboratories. The
inconsistent choices are highlighted in grey.
Seq.
numberChemical

P&G

IVMU

Ricerca1

Benzoquinone

Acetonitrile

Acetonitrile

Acetonitrile

2

PPD

Acetonitrile

Acetonitrile

Acetonitrile3

Dihydroeugenol

Acetonitrile

Acetonitrile

Acetonitrile4

Thioglycerol

Acetonitrile

Acetonitrile

Acetonitrile

5

Imidazolidinyl Urea

Water

Water

Water

6

Methyl Methacrylate

Acetonitrile

Acetonitrile

Acetonitrile

7

GlycerolAcetonitrile

Acetonitrile

Acetonitrile

8

DCNB

Acetonitrile

Acetonitrile

Acetonitrile

9

Benzyl Alcohol

Acetonitrile

Acetonitrile

Acetonitrile

10

Kathon CG

Acetonitrile

Acetonitrile

Water

11

Beryllium Sulphate

Water

Water

Water

12

Formaldehyde

Acetonitrile

Acetonitrile

Acetonitrile

13

Chloramine T

Water

Water

Water

14

Chlorpromazine HCl

Acetonitrile

Acetonitrile

Water

15

2-MBT

Acetonitrile

Acetonitrile

Acetonitrile

16

Benzyl Salicylate

Acetonitrile

Acetonitrile

Acetonitrile

17

Benzyl Cinnamate

Acetonitrile

Acetonitrile

Acetonitrile

18

R(+)Limonene

Acetonitrile

Acetonitrile

Acetonitrile

19

Methyl Salicylate

Acetonitrile

Acetonitrile

Acetonitrile

20

Isopropanol

Acetonitrile

Acetonitrile

Acetonitrile

21

Dimethyl Isophthalate

Acetonitrile

Acetonitrile

Acetonitrile

22

4-PABA

Acetonitrile

Acetonitrile

Acetonitrile

23

Nickel Chloride

Water

Water

Water

24

XyleneAcetonitrile

Acetonitrile

Acetonitrile

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Table 25. Co-elution reported for each of the chemicals in the three laboratories.
Seq.
number1
2

Chemical

P&G

IVMU

Benzoquinone

-

-

LYS

PPD

-

-

CYS/LYS

3

Dihydroeugenol

4

Thioglycerol

5

Ricerca

-

-

LYS

LYS

CYS/LYS

CYS/LYS

Imidazolidinyl Urea

-

-

-

6

Methyl Methacrylate

CYS/LYS-

CYS/LYS

7

Glycerol

-

-

-

8

DCNB

-

-

-

9

Benzyl Alcohol

-

-

10

Kathon CG

-

LYS

LYS

LYS

-

-

LYS

-

-

-

11

Beryllium Sulphate

-

-

-

-

-

-

-

-

12

Formaldehyde

-

-

-

-

-

-

-

-

-

13

Chloramine T

LYS

LYS

LYS

-

-

-

LYS

LYS

LYS

14

Chlorpromazine HCl

15

2-MBT

-

-

-

-

-

-

-

-

-

LYS

LYS

LYS

-

-

-

-

-

-

16

Benzyl Salicylate

-

-

-

-

-

-

-

-

-

17

Benzyl Cinnamate

-

-

-

-

-

-

-

-

-

18

R(+)Limonene

-

-

-

-

-

-

-

-

-

19

Methyl Salicylate

-

-

-

-

-

-

-

-

-

20

Isopropanol
Dimethyl
Isophthalate
4-PABA

-

-

-

-

-

-

-

-

-

LYS

LYS

LYS

LYS

LYS

LYS

-

-

-

-

-

-

-

-

-

-

-

-

23

Nickel Chloride

-

-

-

-

-

-

-

-

-

24

Xylene

-

-

-

-

-

-

-

-

-

21
22

Of the nine chemicals shown above to have different reported patterns of co-elution, only one(Dihydroeugenol) was not consistently classified (sensitisers/non sensitisers) by the threelaboratories, demonstrating that the DPRA test method and prediction model are robust enoughto accommodate different instances of co-elution caused by the specific equipment used andthe fact that in the case of the cysteine co-elution the depletion values have to be estimatedrather then measured.
1. Reproducibility (concordance in predictions)
The between laboratory reproducibility was assessed on the basis of the 24 chemicals tested (9chemicals tested once and 15 chemicals tested 3 times in each laboratory). The main focus of
the evaluation of the between-laboratory reproducibility was on the concordance of thepredictions sensitisers (S) versus non-sensitisers (NS) and for the assignment to one of the four
reactivity classes. As discussed in the WLR section, descriptive and inferential statisticalanalyses were also performed on the raw peptide depletion data (ANOVA) but were not relied
upon by the VMG to determine to what extent the primary objective of the study had beensatisfied.
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 classification obtained using the
Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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median depletion values. For example, for R(+)Limonene (P&G) the depletion values were
1.9, 6.6, and 7.9. The median equals 6.6 which corresponds to a sensitiser classification and theassignment to a low reactivity class.
Eighteen of the 24 chemicals were consistently classified (S/NS) by the 3 laboratories resulting
in a BLR of 75% (Table 26). The BLR for the pair-wise comparisons was 87% for
P&G/Ricerca (13/15 chemicals), and 67% for P&G/IVMU and Ricerca/IVMU (10/15
chemicals).
An overview of the peptide depletions for the individual experiments and the assigned
reactivity classes (4 classes) are also given in Table 26.
For 15 out of the 24 chemicals the laboratories assigned the same reactivity class resulting in a
BLR of 62.5%. As for the within-laboratory reproducibility in case of disagreement, thedifference in the reactivity class assignment was only of one class.
Note that for two
chemicals (thioglycerol and methyl methacrylate) not all the laboratories could assign adefinitive reactivity class because of co-elution with the cysteine peptide. Despite this, thelaboratories consistently and correctly classified these chemicals as sensitisers.
As an important observation, the previously tested substances gave in this study consistent
results with the historical data from P&G (see right column of table 26).

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Table 26. Phase B1: concordance in S versus NS predictions between the laboratories
Seq.
ChemicalnumberA

Agreement

IVMU

2 classes

Peptide depletion (%)
P&G

Ricerca

4 reactivity classes
P&G

Ricerca

1 experiment

Agreement

P&G

IVMU

4 classes

historical

1 experiment

1

Benzoquinone

92.3

99.4LYS

92.5

Yes

HIGH

HIGHLYS

HIGH

Yes

95.0

2

PPD

53.6

52.3C/L

65.0

Yes

HIGH

HIGHC/L

HIGH

Yes

58.2

3

Dihydroeugenol

4.4

4.0LYS

9.2

No

MINIMALMINIMAL

LOW

No

4

23.7LYS

17.0C/L

16.7C/L

Yes

MODERATELYS≥LOWC/L≥LOWC/L

No

42.1

38.5

31.9

Yes

MODERATE

MODERATE

MODERATE

Yes

12.3

29.8C/L

23.7

Yes

LOW

≥MODERATEC/L MODERATE

No

7

Thioglycerol
Imidazolidinyl
Urea
MethylMethacrylate
Glycerol

1.7

0.6

0.9

Yes

MINIMALMINIMALMINIMAL

Yes

8

DCNB

3.9

3.3

1.7

Yes

MINIMALMINIMALMINIMALYes

9

Benzyl Alcohol

1.4

1.1

14.0

No

MINIMALMINIMAL

LOW

No

10

Kathon CG
Beryllium
Sulphate
Formaldehyde

100.0LYS

53.3

46.8

Yes

HIGHLYS

HIGH

HIGH

Yes

12.3

1.1

1.5

No

LOW

MINIMALMINIMAL

No

22.2

26.0

19.0

Yes

LOW

MODERATE

LOW

No

Chloramine T
100.0LYS 100.0LYS
Chlorpromazine
3.3
2.7
HCl
2-MBT
100.0LYS
51.3
Benzyl3.9
0.2
Salicylate
Benzyl3.1
1.0
Cinnamate
R(+)Limonene
6.6
9.2
Methyl5.1
0.8
Salicylate
Isopropanol
5.2
0.8
Dimethyl
5.1LYS
2.5LYS
Isophthalate
4-PABA
5.6
1.1

79.0

Yes

HIGHLYSHIGHLYS

HIGH

Yes

4.3

Yes

MINIMALMINIMALMINIMAL

Yes

50.0

Yes

HIGHLYS

HIGH

HIGH

Yes

6.9

No

MINIMALMINIMAL

LOW

No

4.9

Yes

MINIMALMINIMALMINIMAL

Yes

17.4

Yes

LOW

LOW

LOW

Yes

12.2

No

MINIMALMINIMAL

LOW

No

0.9

0.9

Yes

MINIMALMINIMALMINIMAL

Yes

0.0

2.1

Yes

MINIMALMINIMALMINIMAL

Yes

0.5

Yes

MINIMALMINIMALMINIMAL

Yes

23

Nickel Chloride

0.9

10.2

4.0

No

MINIMALLOW

MINIMALNo

24

Xylene

5.2

0.4

0.6

Yes

MINIMALMINIMALMINIMAL

Yes

5
6Median of 3 experiments

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

26.8

1.0

Median of 3 experiments
51.5

35.8

48.7

A
Values represent the peptide depletion (Chemical 1 to 9) or median peptide depletion of 3 experiments (chemical 10 to 24) In case of coelution with the lysine peptide (indicated by the LYS subscript in the columns with the reactivity classes) the value represents the the cysteine
peptide depletion. C/L indicates co-elution with both peptides. Depletion values with an orange background correspond to a sensitiser
prediction, those with a green background correspond to a non-sensitiser prediction.

Table 27 provides a complete overview of the results for the 24 chemicals generated by thethree laboratories. Note that for the 15 chemicals tested three times the assigned reactivity
classes were always the same or one-off with the only exception of limonene which was
assigned to three different classes (minimal, low and medium reactivity). Limonene is a known
pre-hapten easily activated by air oxidation and this might have been the cause of thesedifferences.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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TABLE 27. Phase B1: concordance in reactivity class (4 classes) within and between laboratories
Seq.
numberMean peptide depletion (%)
Chemical

P&G
Exp 1/2

1

Exp 3

A

Majority

Ricerca
Exp 4

Exp 1/2

Exp 3

IVMU
Exp 4

Exp
1/2

Exp 3

agreement
Exp 4

(%)

Benzoquinone

92.3

99.4LYS

92.5

Yes

2

PPD

53.6

52.3C/L

65.0

Yes

3

Dihydroeugenol

4.4

4.0LYS

9.2

No

4

Thioglycerol

23.7LYS

17.0C/L

16.7CLNo

5

Imidazolidinyl Urea

42.1

38.5

31.9

Yes

6

Methyl Methacrylate

12.3

29.8C/L

23.7

No

7

Glycerol

1.7

0.6

0.9

Yes

8

DCNB

3.9

3.3

1.7

Yes

9

Benzyl Alcohol

10

Kathon CG

1.4

1.1

100.0LYS 100.0LYS 100.0LYS

53.3

14.0
52.6

54.1

46.8

No
46.5

93.5LYS

Yes

11

Beryllium Sulphate

10.5

12.3

15.9

1.1

0.4

1.4

1.7

1.5

1.2

No

12

Formaldehyde

24.5

19.9

22.2

26.0

26.9

25.3

19.5

15.4

19.0

No

13

Chloramine T

14

Chlorpromazine HCl

15

2-MBT

16

Benzyl Salicylate

3.9

2.2

7.9

17

Benzyl Cinnamate

5.1

1.6

3.1

18

R(+)Limonene

7.9

1.9

6.6

19

Methyl Salicylate

5.1

3.1

20

5.2

22

Isopropanol
Dimethyl
Isophthalate
4-PABA

23

Nickel Chloride

24

Xylene21100.0LYS 100.0LYS 100.0LYS 100.0LYS 100.0LYS 100.0LYS

79.5

79.0

77.1

Yes

2.0

3.7

2.7

4.3

2.0

5.7

Yes

51.2

51.3

50.8

50.0

50.0

50.0

Yes

0.2

0.0

0.3

8.5

5.7

6.9

No

1.0

1.4

0.3

9.4

2.5

4.9

Yes

7.1

10.2

9.2

26.0

16.8

17.4

Yes

7.1

0.8

0.9

0.7

12.8

12.2

11.6

No

3.6

5.8

0.4

0.8

3.1

0.9

0.4

4.8

Yes

6.3LYS

1.1LYS

5.1LYS

2.5

0.8

2.5

2.4LYS 0.0LYS

2.1LYS

Yes

5.6

2.4

5.6

2.4

1.1

0.4

2.3

0.4

0.5

Yes

0.6

1.0

0.8

7.0

10.2

11.5

4.0

3.2

4.3

No

5.2

2.3

6.9

0.4

0.3

0

1.3

0.0

0.6

Yes

3.3

3.3

2.8

100.0LYS 100.0LYS 100.0LYS

A

Values represent the mean peptide depletion. In case of co-elution with the lysine peptide (indicated by the LYS subscript in thecolumns with the reactivity classes) the value represents the cysteine peptide depletion. C/L indicates co-elution with both
peptides, in these cases the depletion values are estimated. The background of the mean depletion values corresponds with thefollowing reactivity classes: dark orange = high, orange = moderate, light orange = low and green = minimal reactivity.
Exp. 1 corresponds to the 9 chemicals tested once in study phase B1, Exp 2, 3 and 4 correspond to the three independent
experiments performed by the laboratories with the set of 15 chemicals.

2. Reproducibility of depletion values for cysteine and lysine (for the 24 chemicals)
For information purposes, statistics for the mean cysteine and lysine depletion are shown in
Table 28 and Table 29. Differences of means were tested with ANOVA. For 9 chemicals nosignificant differences were observed between the mean cysteine depletions obtained by thedifferent laboratories. Chloramine T and 2-MBT resulted in 100% depletion in all the repeatedexperiments with one exception; therefore ANOVA testing was not appropriate in this case.
The lysine depletion values were generally very low for the different chemicals (Table 29) andonly 4 chemicals showed no significant differences in mean lysine depletion between the labs.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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The variability within the experiments was also low in the majority of the cases which resulted
in significant differences between and within the laboratories for 17 of the 24 chemicals.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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Table 28. Within and between laboratory variability of the cysteine depletionSeq.
number Chemical

P&G
Exp 1

1

1
2
3

Benzoquinone
PPD
Dihydroeugenol

4

Thioglycerol

5
6
7
8
9

Imidazolidinyl Urea
Methyl Methacrylate
Glycerol
DCNB
Benzyl Alcohol

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

Kathon CG
Beryllium Sulphate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2-MBT
Benzyl Salicylate
Benzyl Cinnamate
R(+)Limonene
Methyl Salicylate
Isopropanol
Dimethyl Isophthalate
4-PABA
Nickel Chloride
XyleneExp 2

Mean SD Mean
94.8 6.7
92.3 10.1
6.9 6.1
23.7
58.5
21
2.5
6.9

5.3
5.1
9.6
4.4
4

2.8

3.8

Ricerca
Exp 3

Exp 4

Exp 1

Exp 2

IVMU

Exp 3

Exp 4

Exp 1

SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean
99.4 0.1
99.9
85.1 10
100
4 1.2
11.2
12.3 3.8
55.9 1.1
45.1 4.4
1.2 0.5
6.5 1.5
2 0.9

Exp 2

Between lab
Exp 3

Exp 4

SD Mean SD Mean SD Mean SD Mean
0.2
98
0
92.5
5.2
7.4

47.8
42.1
1.8
3.5

(n=1)
2.8
4.5
1.6
3.9

2.3

2.5

SD ANOVA
2.8
NS
7.5
NS
3.6
NS

18
54.1
36.1
1.8
5.6

8.1
5.6
13.1
0.7
1.9

NS
Sign
Sign
NS
NS

2.4

0.4

NS

100
0
20.8 10.4
44.7
1
100
0
6.6 8.2
100
0
7.8 7.2
10 9.815.5 4.1
9.1
8
9.9 9.2
6.3 8.3
10.7 8.4
1.2
2

100 0
24.6 5.5
36.6 1.2
100 0
6.4 11100 0
2.8 4.2
2 3.4
2.5 2.4
3.4 5.1
5 5.8
1.1 2
3.9 4.2
0 0

100 0
31 7.640.6 1.2
100 0
5.5 5.2
100 0
14.4 6.1
5.8 5.9
12.2 6.2
11.3 4.9
10.8 4
5.1 4.6
10.6 8.1
0 0

100 0
2 2.2
49.6 1.6
100 0
3.9 2.4100 0
0.2 0.3
1.4 1.3
13.6 20.3 0.6
0.3 0.5
0.7 0.8
4.6 2.1
13.9 4.7

100 0
0.7 1.3
51.4 2100 0
7.4 1.9
98.7 2.2
0 0.1
2.5 1.6
18.8 2.5
0.2 0.4
1 1
0.4 0.7
1.4 1.2
20.4 5.7

100 0
2.6 1.9
48.6 2.8
100 0
5.4 4.4
100 0
0.7 1.2
0.7 1.2
18.4 1.1
0.4 0.6
6.1 1.2
0.2 0.4
0.9 1.4
22.9 6.2

93.5 0.2
2.6 2.2
37.3 1.2
100 0
8.5 3.2
100 0
4.5 3.8
13.2 4.1
51.8 3.4
1 1.3
1.5 1.8
2.4 2.1
4.6 3.5
8.1 4.9

93 0.5
3 3.9
30.5 0.8
100 0
3.9 3.8
100 0
0 0
0 0
33.5 30 0
0 0
0 0
0.7 1.3
6.3 5.9

93.5 1.3
2.4 2.6
36.7 0.5
100 011.4 2.6
100 0
2.4 2.4
3.9 3.6
33 2.30.4 0.8
9.3 2.4
2.1 2.2
1 1
8.5 5.8

97.8
10
41.8
100
6.6
99.9
3.6
4.4
22.1
2.9
4.9
2
4.3
9

3.8
13.4
7.6
0
1.2
0.3
4.2
2.5
15.4
4.4
3.2
1.9
3.6
9.4

Sign
Sign
Sign
NA
NS
NA
Sign
Sign
Sign
Sign
Sign
NS
Sign
Sign

9.6

2.5 4.1

12.1 7.6

0.5 0.9

0.3 0.60

2.7 2.30

1.1 1.5

3.2

4.2

Sign

8.4

0

0

1

ANOVA F-test with critical α-level = 0.024 (corrected for number of hypothesis tested); NS: not significant (p>0.024), Sign: significantly different (p<0.024)

NA: ANOVA testing not performed since all individual values were equal; Chemicals with a grey background correspond with cysteine co-elution. When it was possible to estimate a depletion value,
this value is reported in the table.
Exp. 1 corresponds to the 9 chemicals tested once in study phase B1, Exp 2, 3 and 4 correspond to the three independent experiments performed by the laboratories with the set of 15 chemicals.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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Table 29. Within and between laboratory variability of the lysine depletionSeq.
number Chemical

P&G
Exp 1

1

Mean
89.7
14.9
1.8

Exp 2

Ricerca
Exp 3

Exp 4

Exp 1

SD Mean SD Mean SD Mean SD Mean
0.5
13.2
3.4
19.6
1.9
3.3

Exp 3

Exp 4

Exp 1

SD Mean SD Mean SD Mean SD Mean
2.5
85.0
0.9
29.9
0.3
7.1

1
2
3

Benzoquinone
PPD
Dihydroeugenol

4

Thioglycerol

28.4 2.3

21.8 1.4

5
6
7
8
9

Imidazolidinyl Urea
Methyl Methacrylate
Glycerol
DCNB
Benzyl Alcohol

25.7
3.7
0.8
0.8
0.0

21.1
14.5
0.0
0.0
0.1

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

Kathon CG
Beryllium Sulphate
Formaldehyde
Chloramine T
Chlorpromazine HCl
2-MBT
Benzyl Salicylate
Benzyl Cinnamate
R(+)Limonene
Methyl Salicylate
Isopropanol
Dimethyl Isophthalate
4-PABA
Nickel Chloride
Xylene

0.5
2.5
0.7
1.4
0.0

Exp 2

IVMU

0.4
0.4
0.1
0.0
0.2

4.8
0.1
4.3

5.0 11.1 5.4
0.1 0.0 0.0
4.7 3.2 1.2

3.5
0.8
3.8

3.6
1.3
1.5

6.6
0.1
2.3

0.7
0.1
0.3

5.2
0.1
2.5

0.8
0.1
0.4

8.2
0.2
2.0

0.4
0.1
0.3

0.0
3.5
0.0
0.2
0.3
1.0
0.5

0.0
2.8
0.0
0.4
0.5
0.8
0.8

0.1
8.7
1.6
1.1
1.3
2.9
2.3

0.2
5.2
1.3
1.2
0.9
1.5
3.2

0.0
9.0
1.5
0.3
1.0
3.0
0.7

0.0
6.7
0.7
0.3
0.4
0.2
0.6

0.4
0.1
0.8

0.7
0.2
1.4

0.9
2.0
2.1

0.6
2.3
1.9

0.6
1.5
1.6

0.2
1.8
1.5

0.0
2.3
0.1
0.6
0.5
1.3
0.5
4.2
0.1
0.0
0.2

0.0
0.5
0.1
0.1
0.3
0.3
0.1
0.7
0.1
0.0
0.1

0.0
3.9
0.0
0.2
1.5
1.6
0.6
1.2
0.7
0.0
0.2

0.0
0.2
0.0
0.3
1.0
0.3
0.5
0.5
0.4
0.1
0.2

0.0
1.6
0.0
0.0
0.0
1.0
0.0
4.7
0.0
0.0
0.0

0.0
0.2
0.0
0.0
0.1
0.0
0.0
0.5
0.0
0.0
0.0

Exp 2

Between lab
Exp 3

Exp 4

SD Mean SD Mean SD Mean SD Mean
4.8
62.6
3.4
21.5
1.6
4.1

SD ANOVA
42.9
Sign
7.7
Sign
2.7
Sign

33.4

1.1

27.9

5.8

Sign

15.9
5.3
0.1
0.0
25.6

0.9
0.7
0.2
0.0
0.6

20.9
7.8
0.3
0.3
8.6

4.9
5.9
0.4
0.5
14.8

Sign
Sign
NS
NS
Sign

4.4
0.2
2.4
57.1
0.0
3.2
4.3
2.1
0.7
9.2
0.7
3.4
0.3
0.4
0.6

3.8
0.1
1.3

Sign
NS
Sign
NA
NA
Sign
Sign
Sign
Sign
Sign
NS
NA
Sign
Sign
Sign

0.0
0.9
1.8
59.0
0.0
0.0
12.5
5.6
0.1
24.6
0.2

0.0 0.0 0.0
0.8 0.0 0.0 0.0 0.0
0.2 0.3 0.2 1.3 0.1
0.4 58.1 0.9 54.2 1.1
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0
1.8 11.4 2.7 11.3 0.9
1.3 5.0 1.8 6.0 0.8
0.2 0.0 0.0 1.8 0.2
0.5 24.4 0.6 22.9 0.4
0.3 0.9 0.3 0.2 0.1

0.0
0.0
0.0

0.0
0.0
0.0

0.0
0.0
0.0

0.0
0.0
0.0

0.0
0.0
0.1

0.0
0.0
0.1

0.0
3.6
6.5
3.0
0.1
12.8
0.4
0.3
0.7
0.8

1

ANOVA F-test with critical α-level = 0.024 (corrected for number of hypothesis tested); NS: not significant (p>0.024), Sign: significantly different (p<0.024)

NA: ANOVA testing not performed since all individual values were equal; Chemicals with a grey background correspond with lysine co-elution. When it was possible to estimate a depletion value,
this value is reported in the table.
Exp. 1 corresponds to the 9 chemicals tested once in study phase B1, Exp 2, 3 and 4 correspond to the three independent experiments performed by the laboratories with the set of 15 chemicals.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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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 thethree laboratories. The BLR for the S/NS prediction was 75%. When 4 reactivity classes
where considered the BLR was 62.5%
The VMG agreed that the BLR results (75%) did not meet the target performance proposed at
the onset of the study (80%). However, the VMG regarded the BLR result to be neverthelessacceptable bearing in mind the proposed use of the DPRA, as part of an integrated testing
strategy for full replacement. In arriving at this conclusion, the VMG also took note of thefact that 80% between-laboratory reproducibility is considered an acceptable target
performance for stand alone full replacement methods (OECD, 2010b).
Furthermore, the VMG notes that exclusion of the three substances outside the applicability
domain (dihydroeugenol, beryllium sulfate and nickel chloride, see below) would haveincreased the BLR to 87.5%.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

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Predictive Capacity (Module 5)
Reference document: Statistical report (Appendix 15)
List of additional documents filed for the study and available on
request (Appendix 16)
An overview of the predicted classification and the reference classification is presented inTable 30.
When the test chemicals were selected for the current study, three of the 24 chemicals were
known to fall outside the suggested applicability domain of the DPRA: dihydroeugenol which
is well characterised pro-hapten, and beryllium sulfate and nickel chloride which are metalsalts.
Therefore, the analysis of the predictive capacity was performed using the results from theremaining 21 chemicals.
The predictive capacity was evaluated for each laboratory (Table 31). For chemicals that were
tested three times in each laboratory, the median was selected as the final conclusion for eachlaboratory, i.e. in the case of R(+)Limonene (P&G) that was two times predicted S and once
NS by P&G, the final prediction for this laboratory was S.
This resulted in an accuracy for S/NS classification of 85.7% for both P&G and Ricerca and
81% for IVMU. The sensitivity and specificity for P&G and Ricerca were 76.9% and 100%,
respectively. The sensitivity and specificity for IVMU were 84.6% and 75% respectively.
The overall accuracy (cumulative over three labs) was 84.1% with sensitivity of 79.5%, andspecificity of 91.7%. Note that these numbers are for a total of 63 results, representing theresults from the 21 chemicals tested in each of the three laboratories.
Alternatively, the predictive parameters were calculated by assigning the median classificationof the three laboratories to each chemical. These results are shown in table 32. In this case theoverall accuracy was 85.7% with a sensitivity of 76.9% and a specificity of 100%.
While the accuracies of both approaches are very similar, sensitivity and specificity arebalanced differently. While the median approach has the advantages that it maintains the
sample size of 21 and that it allows calculating confidence intervals, it reduces the available
information to some extent. The cumulative approach increases the sample size by considering
the individual, but dependent laboratory classifications per chemical. Therefore, confidence
intervals are not reported.
The VMG preferred to focus on the cumulative approach because the resulting estimates weredeemed to better reflect the predictive capacity of the DPRA. Especially, the specificity of
100% of the median approach was considered misleading as one laboratory produced also
false positive results (see Table 31).
As already mentioned, chemicals previously tested in the DPRA were consistently andaccurately identified (96.3%; 26/27 cumulative). Accuracy for chemicals not previously tested
Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

Page 65 of 74

was 77.8% (28/36 cumulative). These latter chemicals should contribute to a future and morecomprehensive assessment of predictive accuracy, since no meaningful conclusions on thisparameter can be drawn from this limited sample size.
Table 30. Agreement between the predicted class and the reference class for each of thechemicals
Seq.
ChemicalnumberTest referenceA
prev result

P&G

Ricerca

IVMU

Exp 1 Exp 2 Exp 3 Exp 4 Exp 1 Exp 2 Exp 3 Exp 4

1
2

Benzoquinone
PPD

Y
Y

+ (1A)+ (1A)SSSS

Exp 1 Exp 2 Exp 3 Exp 4

S

LYS

S

CL

3

Dihydroeugenol*N+ (1B)NSNS

4

ThioglycerolN+ (1B)SS

5

Imidazolidinyl Urea

Y

+ (1B)S

S

S

6

Methyl MethacrylateN+ (1B)

S

S

CL

S

7

GlycerolY- (NC)

NS

NS

NS

8

DCNB

N

- (NC)

NS

NS

NS

9

Benzyl AlcoholN- (NC)

NS

LYS

CL

S

LYS

S

CL

NS

LYS
B

S
B

Major

CL

B

Major

Major

10

Kathon CG

Y+ (1A)

S

SLys

SLys

SLys

S

S

S

S

S

S

S

S

11

Beryllium Sulphate*N+ (1A)

S

S

S

S

NS

NS

NS

NS

NS

NS

NS

NS

12

FormaldehydeY+ (1A)

S

S

S

S

S

S

S

S

S

S

S

S

S

S

13

Chloramine T

N+ (1A)

S

SLys

SLys

SLys

S

S

Lys

S

S

S

S

14

Chlorpromazine HClN+ (1A)

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

15

2-MBT

Y

+ (1A)

S

SLys

SLys

SLys

S

S

S

S

S

S

S

S

16

Benzyl SalicylateN+ (1B)

NS

NS

NS

S

NS

NS

NS

NS

S

S

NS

S

17

Benzyl CinnamateN+ (1B)

NS

NS

NS

NS

NS

NS

NS

NS

NS

S

NS

NS

18

R(+)LimoneneN+ (1B)

S

S

NS

S

S

S

S

S

S

S

S

S

19

Methyl SalicylateY- (NC)

NS

NS

NS

S

NS

NS

NS

NS

S

S

S

S

20

IsopropanolY- (NC)

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

21

Dimethyl
IsophthalateN- (NC)NS NSLys NSLys NSLys

NS

NS

NS

NS

NS NSLys NSLys NSLys

22

4-PABAN- (NC)

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

23

Nickel Chloride*N+ (NA)

NS

NS

NS

NS

S

S

S

S

NS

NS

NS

NS

24

XyleneN- (NA)

NS

NS

NS

S

NS

NS

NS

NS

NS

NS

NS

NS

A

Indication if the chemical was tested previously (Y: yes) or not (N: no)B

final predicted class based on majority voting

Lys

Lys

*outside the applicability domain
LYS subscript or CL subscript corresponds with co-elution for Lysine and co-elution with CYS/LYS

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Table 31. Overall predictive capacity of the DPRA (cumulative over the 3 labs) andpredictive capacity for each laboratory for the chemicals falling into the applicability
domainReference result+ (n=13)

Cumulative

P&G

Ricerca

IVMU

+

-

+

-

+

-

+

-

31

8

10

3

10

3

11

2

- (n=8)

2

22

0

8

0

8

2

6

Total

33

30

10

11

10

11

13

8

Sensitivity (95%
79.5
A
CI)
Specificity (95%91.7
CI)
Accuracy
84.1
A
Wilson CI’s based on the score test

76.9 (49.7-91.8)

76.9 (49.791.8)

84.6 (57.895.7)

100 (67.6-100)

100 (67.6-100)

75 (40.9-92.9)

85.7

85.7

81.0

Table 32. Overall predictive capacity of the DPRA (considering the median of the resultsin each laboratory) for the chemicals falling into the applicability domain
Reference result

Median+-+ (n=13)10

3- (n=8)

0

8

Total

10

11

Sensitivity (95% CI)

76.9 (49.7-91.8)

Specificity (95% CI)

100 (67.6-100)

Accuracy

85.7

In addition, cross referencing the reactivity class results from Table 26 in Module 4 to the
GHS sub-categories, it is noted that when a chemical was consistently assigned by the DPRA
in the HIGH reactivity class, it was always a GHS category 1A substance.

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Conclusion of the Validation Management Team on Module 5
In our study, the sensitivity of the DPRA was 79.5% and the specificity was 91.7%, resulting
in an accuracy of 84.1%. The VMG concludes that this outcome is consistent with thesubmitted and published information on the predictive capacity of the DPRA (see Table 33).
However, it is important to note that this assessment of predictive capacity forms only asecondary goal of the present validation study, not least since the limited sample size, which
was defined for the assessment of the within and between laboratory reproducibility, does not
permit a robust conclusion to be drawn. In addition, it has been anticipated that the in vitro
assays in the current validation study are most likely to form a part of an integrated testing
strategy (ITS) for hazard identification. It is envisaged that predictive capacity will need to beassessed on the basis of the information generated by a future ITS. The information above
must be understood in this context.

Table 33. Performance of the DPRA as evaluated from the results submitted to ECVAM by P&G

Predicted
Classification(based on classification treemodel)

ChemicalClassification(based on
LLNA)

NonSensitiser
30

Sensitiser

total

6

36

Sensitiser

13

84

97

Total

43

90

133

Non-Sensitiser

table statistics for the shadowed 2 x 2
tablesensitivity:
specificity:
positive predictivity:
negative predictivity:
accuracy:

87%
83%
93%
70%
86%

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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 DPRA with a number of coded chemicals thatwere judged by the VMG to be suitable to permit robust conclusions to be drawn.
The VMG considers that the information generated in the study completes the information
requirement for modules 1-4 (test definition, within laboratory reproducibility, transferability,
between laboratory reproducibility) of the ECVAM modular approach to validation (Hartunget al., 2007). In addition the information generated contributes to module 5 (predictive
capacity) and module 6 (applicability domain) for which a substantial body of information isalready available as evidenced in the material submitted to ECVAM and published in thescientific literature.

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

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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
DPRA test method.
An improved, well-detailed and robust SOP is available.

2

Withinlaboratory
reproducibility

The overall within laboratory reproducibility was satisfactory andmet the target performance. The results of the within laboratory
reproducibility for the four reactivity classes further support thisconclusion.
The test method was shown to be transferable between
laboratories.

3

Transferability

4

Betweenlaboratory
reproducibility

5

Predictive
capacity

Training, and demonstration of competence, in the conduct of theassay is however considered important especially for laboratories
which are not familiar with peptide reactivity assays.
The between laboratory reproducibility, despite not fully meetingthe target performance critieria, is considered to be acceptable forthe proposed use of the DPRA (i.e. as part of an integreated testing
strategy).
Complete evaluation of the predictive capacity was not one of thegoals of this study. The VMG notes that the predictive capacity of
the DPRA for the set of chemicals evaluated in the study is
consistent with submitted and published information.

Overall, the VMG concludes that the information generated in this validation study shows thatthe DPRA is a robust and reliable test method. Consequently:• Information generated by the DPRA 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
DPRA result should be considered sufficient to classify a test material as a skinsensitiser.

Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

Page 70 of 74• The DPRA is suitable for further evaluation as a component of an ITS for full
replacement of the in vivo assays for skin sensitisation hazard identification.
• Reliable categorisation into one of the four reactivity classes may play a role in thedetermination of skin sensitisation potency, including GHS sub-categorisation.

Recommendations
The predictive accuracy of the DPRA should be evaluated in terms of its inclusion into 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 obtainedwith the chemicals tested previously it is suggested that existing/historical results are taken
into account for future formal evaluations on the predictive capacity.
GHS sub-categorisation of sensitisers should form part of a wider assessment, it is envisaged
that DPRA reactivity categories will provide useful information for this purpose.
The experience gained within this validation trial suggests that the run acceptance criteria
might be enhanced by their adaptation, by the lead laboratory, to take account of theinformation from other laboratories generated in this study and in other DPRA work.
In view of the known limitations of the DPRA, including those related to the absence of ametabolic competent activity and the issue of co-elution in the HPLC analysis which canaffect the accurate determination of a chemical’s reactivity, the VMG encourages continuing
investment in the next generation peptide reactivity assays which offer an opportunity toovercome these limitations.

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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
Appendix 16

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
DPRA results reporting template
DPRA results Quality Check template
DPRA SOP version 2
DPRA SOP version 3
Gerberick et al, 2004
Gerberick et al, 2007
DPRA Training and Transfer Plan version 2
IVMU Training report
Ricerca Training report
Statistical report
List of additional documents filed for the study and available on request

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List of References
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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,
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Ahlfors, S.R., Sterner, O. & Hansson, C. (2003). Reactivity of contact allergenic haptens toamino acid residues in a model carrier peptide, and characterization of formed peptide-hapten
adducts. Skin Pharmacology and Applied Skin Physiology 16, 59-68.
Dupuis, G. & Benezra, C. (1982). Allergic contact dermatitis to simple chemicals: a molecularapproach. New York & Basel: Marcel Dekker Inc.
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Lepoittevin, A. Natsch, J-M. Ovigne, C. Rovida, H. Sakaguchi and T. Schultz. 2008. Chemical
reactivity measurement and the predictive identification of skin sensitizers: The report andrecommendations of ECVAM Workshop 64. ATLA 36:215-242.
Hartung, T., Bremer, S., Casati, S., Coecke, S., Corvi, R., Fortaner, S., Gribaldo, L., Halder,
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(2004). A modular approach to the ECVAM principles on test validity. ATLA 32, pp. 467–
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Landsteiner, K. & Jacobs, J. (1936). Studies on the sensitization of animals with simple
chemical compounds. Journal of Experimental Medicine 64, 625-639.
Lepoittevin J.-P., Basketter D.A., Goossens A., Karlberg A.-T. (1998) Allergic contact
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OECD (2005). Guidance document on the validation and international acceptance of new or
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OECD (2010a). TG 429: Skin Sensitisation: Local Lymph Node Assay (LLNA) OECD
Guidelines for the Testing of Chemicals, Section 4: Health Effects. (ISBN: 978-92-6407-1100).
OECD (2010b). TG 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test
Method OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects. (ISBN :9789264090958).
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UN (2009). Globally Harmonised System (GHS) of classification and labelling of chemicals.
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