EURL ECVAM Recommendation on the
Direct Peptide Reactivity Assay (DPRA)
for Skin Sensitisation Testing

October 2013

Report EUR 25946 EN

European Commission
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Institute for Health and Consumer Protection (IHCP)
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EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)EURL ECVAM RECOMMENDATION
on the Direct Peptide Reactivity Assay (DPRA) for
Skin Sensitisation Testing

November 2013

ACKNOWLEDGEMENTS
This Recommendation was prepared by the EU Reference Laboratory for Alternatives to Animal
Testing (EURL ECVAM), part of the Institute for Health and Consumer Protection (IHCP),
Directorate-General Joint Research Centre (DG JRC) of the European Commission.
The Recommendation was drafted on the basis of the ESAC Opinion and ESAC Working Group
Report summarising the detailed scientific peer review of the EURL ECVAM-coordinated study
on the Direct Peptide Reactivity Assay (DPRA). The Recommendation further benefitted fromcomments and suggestions received from members of PARERE (EURL ECVAM's advisory bodyfor Preliminary Assessment of Regulatory Relevance that brings together representatives ofMember State regulatory bodies as well as EU agencies including ECHA, EFSA and EMA), andESTAF (EURL ECVAM's Stakeholder Forum). Input was also provided by partner organisations of
EURL ECVAM in the framework of the International Collaboration on Alternative Test Methods(ICATM), and by the general public.
Coordinator/Project leader of the validation study was Silvia Casati. Coordinator of the ESAC
Peer Review and EURL ECVAM Recommendation was Claudius Griesinger.

Comments or enquiries related to this EURL ECVAM Recommendation should be sent to:
EU Reference Laboratory for Alternatives to Animal Testing
European Commission DG Joint Research Centre,
Institute for Health and Consumer Protection,
Via E. Fermi 2749,
I-21027 Ispra (VA), Italy.
e-mail: JRC-ECVAM-CONTACT@ec.europa.eu

3TABLE OF CONTENTSEXECUTIVE SUMMARY ....................................................................................................... 5
1. Introduction ................................................................................................................... 7
2. Test Method definition .................................................................................................. 8
3. Overall performance of the Direct Peptide Reactivity Assay ....................................... 9
4. Limitations.................................................................................................................... 10
5. Suggested regulatory use ............................................................................................ 11
6. Follow-up activities recommended by EURL ECVAM ................................................. 13
References ........................................................................................................................ 14
Annex 1ESAC Opinion .................................................................................... 17
Annex 2EURL ECVAM request for ESAC advice ............................................ 25

BACKGROUND TO EURL ECVAM RECOMMENDATIONS
The aim of a EURL ECVAM Recommendation is to provide EURL ECVAM views on the validity ofthe test method in question, to advise on possible regulatory applicability, limitations andproper scientific use of the test method, and to suggest possible follow-up activities in view ofaddressing knowledge gaps.
During the development of its Recommendation, EURL ECVAM consults with its advisory bodyfor Preliminary Assessment of Regulatory Relevance (PARERE) and its EURL ECVAM Stakeholder
Forum (ESTAF). Moreover, EURL ECVAM consults with other Commission services and its
international validation partner organisations of the International Cooperation on Alternative
Test Methods (ICATM). Before finalising its recommendations, EURL ECVAM also invites
comments from the general public and, if applicable, from the test method submitter.

4

EXECUTIVE SUMMARYEURL ECVAM fully endorses the ESAC opinion (Annex I) on the ECVAM-coordinated validation
study of the Direct Peptide Reactivity Assay (DPRA) that assessed mainly protocol transferability
and within- and between-laboratory reproducibility. The study was conducted in view of the
DPRA's possible use as a component of an integrated approach for testing the potential ofchemicals to cause skin sensitisation resulting in Allergic Contact Dermatitis (ACD). On the basisof the ESAC Opinion on the DPRA study, EURL ECVAM makes the following Recommendations:(1) Haptenation, i.e. the covalent binding of low-molecular weight substances ("haptens") toproteins present in skin is considered a prominent mechanism through which chemicals or
their metabolites become antigenic. Haptenation has been described as a "molecular
initiating event" in the OECD Adverse Outcome Pathway (AOP) for skin sensitisation whichsummarises the key events known to be involved in chemically-induced ACD (OECD, 2012).
Therefore, information from peptide reactivity assays such as the DPRA is relevant for theassessment of the skin sensitisation potential of chemicals.
(2) The EURL ECVAM study showed that the DPRA is transferable to suitably equippedlaboratories that are proficient in high performance liquid chromatography (HPLC) analysis
and the results obtained demonstrated within- and between-laboratory reproducibility of
87% and 75%, respectively.
(3) Full evaluation of the predictive capacity and applicability domain of the DPRA were outside
the scope of the EURL ECVAM study. However, based on the study results and excluding
metal compounds for which the test is not applicable, the accuracy of the DPRA for
distinguishing sensitisers from non-sensitisers was 82% (sensitivity of 76%, specificity of92%) which is in agreement with published information from previous studies (Gerberick etal., 2007; Bauch et al., 2012; Natsch et al., 2013).
(4) In addition to supporting identification of sensitisers/non-sensitisers, the DPRA may also beable to contribute to the assessment of sensitising potency, e.g. by supporting, within an
integrated approach, the subcategorisation of sensitisers according to the United Nations
Globally Harmonised System of Classification and Labelling of Chemicals (UN GHS; UN,2011). More work however is required to determine to which extent DPRA results relate topotency categories.
(5) As the DPRA is an in chemico test method lacking metabolic capacity, substances that
require metabolic (pro-haptens) or abiotic activation (pre-haptens) may not be detected by
the DPRA. In addition, the DPRA specifically detects peptide reactivity associated with lysine
and cysteine amino acids. These factors should be taken into account when considering
negative results.
(6) In view of the mechanistic complexity of skin sensitisation, DPRA data should always be
considered in combination with other information in the context of integrated approaches
such as Weight of Evidence (WoE) or Integrated Testing Strategies (ITS). Complementary
information may be derived from test methods addressing other key events involved in skinsensitisation (OECD 2012) as well as non-testing methods including read-across information.
Thus, EURL ECVAM recommends the development of integrated approaches for identifying
and characterising skin sensitisation hazard (EURL ECVAM 2013) and potency. These
approaches should be tailored to specific needs (e.g. depending on various sectorial andregulatory requirements).
5(7) To support development of integrated approaches employing peptide reactivity assays(such as DPRA) and other sources of information, the applicability of the DPRA should befurther characterised, e.g. through a retrospective analysis of existing data and, in case ofidentified data gaps, by generating additional information through targeted prospective
testing. In particular, as pre-haptens are not consistently misclassified by the DPRA, theassay's applicability to these substances should be further investigated. Additionally,
attention should be given to substances with electrophilic residues that react preferentiallywith amino acids other than cysteine or lysine.
(8) Respecting the provision of Directive 2010/63/EU (EU, 2010) on the protection of animals
used for scientific purposes, before embarking on animal experiments to identify substanceswith skin sensitisation potential, data from the DPRA test method should be considered in
combination with complementary information in order to reduce and possibly avoid animal
testing. In agreement with the provision of Annex XI point 1.2 of the REACH Regulation (EU,2006) data from non-standard testing methods, such as the DPRA, may be used to adapt thestandard information requirements in the context of Weight of Evidence judgments.

6

1. Introduction
1) The assessment of skin sensitisation potential is an important component in the safety
evaluation of substances and represents a standard requirement of legislation on
chemicals in the EU. These include: the Classification Labelling and Packaging of substancesand mixtures (CLP) Regulation (EU, 2008a), the REACH Regulation (EU, 2006), the Plant
Protection Products (PPP) Regulation (EU, 2009a), the Biocides Regulation (EU, 2012) andthe Cosmetics Directive (EU, 2009b). As outlined in the EURL ECVAM Strategy for
Replacement of Animal Testing for Skin Sensitisation Hazard Identification andClassification (EURL ECVAM, 2013), determining the skin sensitisation hazard properties ofsubstances is a key requirement satisfying already the majority of regulatory needs, e.g.
under the CLP and REACH Regulations in the EU. Other regulatory contexts can require anunderstanding of the relative potency of skin sensitisers with regard to both induction as
well as elicitation of contact dermatitis in order to support a full risk assessment andappropriate risk management measures (e.g. setting of appropriate thresholds).
2) Currently there are only in vivo regulatory accepted test methods to generate datasatisfying regulatory requirements on skin sensitisation. For instance, in the frameworks of
the Organisation for Economic Cooperation and Development (OECD) and the EU Test
Methods Regulation (EU, 2008b), there are four accepted guidelines, describing: theBuehler Test and Guinea-pig Maximisation Test (GPMT), TG406 (OECD, 1992; EU test
method B.6), the Local Lymph Node Assay (LLNA), TG429 (OECD, 2010a; EU test method
B.42) and its non-radio-isotopic variants, the Local Lymph Node Assay: DA and the Local
Lymph Node Assay: BrdU Elisa , TG 422A and TG 422B respectively (OECD, 2010b; OECD
2010c).
3) The key mechanistic events underpinning the skin sensitisation process that leads toAllergic Contact Dermatitis (ACD) in humans are well understood and have been recently
summarised in the OECD report on “The Adverse Outcome Pathway (AOP) for Skin
Sensitisation Initiated by Covalent Binding to Proteins“(OECD 2012). These include 1) thecovalent binding of the chemical to the skin protein (haptenation), 2) events inkeratinocytes including the production of danger signals and release of pro-inflammatory
mediators 3) the maturation and mobilisation of dendritic cells (DC), the immunocompetent cells in the skin, and 5) the antigen presentation to naïve T-cells and theproliferation of memory T-cells. Considerable progress has been made in recent years
towards the development of alternative non-animal methods that address these key
events. It is plausible that the initial event of haptenation is the major determinant of theskin sensitisation process and thus the protein-binding properties of a chemical should be
intrinsically linked to its sensitisation potential and potency (Roberts & Aptula, 2008).
4) There is general agreement within the scientific community that, in the near future, it isunlikely that one single alternative method will be able to provide sufficient information to
replace the use of animals for this endpoint (Adler et al., 2011). Instead it is held that
information from different alternative testing and non-testing methods used in
combination will need to be integrated to address this health endpoint (Jowsey et al.,2006; Adler et al., 2011). These methods should address different key events leading toskin sensitisation thus covering the mechanistic complexity of this endpoint. Nevertheless,it should not be ruled out a priori that skin sensitisation testing may, in the future, be
addressed by one single test method.
75) EURL ECVAM coordinated a validation study of DPRA following a modular approach(Hartung et al., 2004) which had the following objectives:To fully assess the reliability of the DPRA protocol, i.e. its transferability and withinand between-laboratory reproducibility.
To conduct a preliminary evaluation of the ability of the DPRA to discriminate skinsensitising from non-sensitising chemicals as defined by the Globally Harmonised
System (GHS) for the classification and labelling of substances for skin sensitisation
and as implemented in the European Union CLP Regulation concerning bothsubstances and mixtures. Characterisation of preliminary predictive capacity was
performed in view of determining the potential contribution of the method tocontribute to decisions on hazard within integrated approaches.
To consider the ability of the DPRA to contribute to sub-categorisation of skinsensitising chemicals, e.g. into Sub-category 1A and Sub-category 1B as adopted inthe 3rd revised version of the GHS.

6) After completion of the study and finalisation of the Validation Study Report (EC-ECVAM2012), EURL ECVAM requested the ECVAM Scientific Advisory Committee (ESAC) to provide
an ESAC Opinion on the study. An ESAC Working group (WG) was subsequently establishedwhich drafted an ESAC WG report, which then formed the basis of the ESAC Opinion (seeannex) adopted by the ESAC on 17. 12. 2012.

2. Test Method definition
7) The correlation of protein reactivity with skin sensitisation potential is well recognised(Landsteiner and Jacobs, 1936; Dupuis & Benezra, 1982; Lepottevin et al., 1998). Chemical
covalent binding to nucleophilic centres in skin proteins is regarded to be the molecular
initiating event in the skin sensitisation AOP (OECD, 2012) without which skin sensitisation
would not occur. Thus, chemicals capable of reacting with proteins either directly or after
biotic or abiotic transformation may have the potential to act as a contact allergen. Itshould be noted that, in its current design, the DPRA does not provide a measure ofreaction rate constant.
8) The DPRA is an in chemico method which addresses peptide reactivity by measuring
depletion of synthetic heptapeptides containing either cysteine or lysine following 24
hours incubation with the test substance. Depletion of the peptide in the reaction mixture
is measured by HPLC using UV detection. Average peptide depletion data for cysteine andlysine are then interpreted by using a classification model developed on the basis of adataset of chemicals with known reactivity properties, in which chemicals are classified ashaving minimal, low, moderate or high reactivity. Substances with low to high reactivity areassociated with substances that have skin sensitisation potential while those categorised ashaving minimal reactivity are considered to lack skin sensitisation potential. Due to theabsence of a metabolic competent system in the assay, the DPRA is not suitable for theevaluation of pro-haptens, which require metabolic activation to act as sensitisers. The
ability of the DPRA to detect pre-haptens, which require abiotic activation (e.g. through8

oxidation by air), is not clear, although some pre-haptens are reported to be correctly
identified by the DPRA.
9) As a result of the ECVAM-coordinated study (EC EURL ECVAM, 2012), the standardised
protocol was found to be transferable and reproducible within and between laboratories
experienced in HPLC analysis. Some minor aspects of the protocol have been refined as aresult of the experienced gained in the validation study.
10) EURL ECVAM will publish in its DataBase service for ALternative Methods (DB-ALM, see
http://ecvam-dbalm.jrc.ec.europa.eu), a comprehensive protocol including a detailed
description of the test method and all necessary technical details needed by an end-userlaboratory to implement it in a self-sufficient manner.

3. Overall performance of the Direct Peptide Reactivity Assay
Reference data
A key criterion employed for selecting the validation test chemicals was availability of highquality in vivo testing data from the murine LLNA and the GPMT with concordant classificationfrom these two assays. The set of chemicals used in the study consisted of one third of nonsensitisers and two thirds of sensitisers with a balanced representation of potency classes(weak, moderate strong and extreme). Reference chemicals from the LLNA performancestandards (OECD 2010a) were included in the chemical set. Additional details can be found in
the Validation Study Report (EC EURL ECVAM, 2012).
When interpreting the data of alternative methods, such as the DPRA, that have been largely
developed and validated using animal reference data such as LLNA or GPMT the limitations of
the reference data should be kept in mind. For instance, the predictive relevance of reference
animal tests may not fully reflect the situation in the species of interest, i.e. humans. Notably,an evaluation of the LLNA in comparison to human data has shown an accuracy of about 72%(Anderson et al., 2011), i.e. there is a risk of false negative and false positive results. Moreover
there is indication that the LLNA is deficient in detecting low to moderate sensitisers as well as
metals and organometal compounds (EC, 2000).
Transferability and Reproducibility11) On the basis of the results obtained during the study, it is evident that the DPRA can bereadily transferred to new laboratories that are properly equipped and experienced with
HPLC instruments and techniques.
12) The assessment of the reproducibility was performed on the basis of concordance inclassification (sensitiser/non-sensitiser). The experimental data generated in the study
indicate that the within-laboratory reproducibility (ranging from 73% in the lead laboratory
to 100% in one of the two naïve laboratory) and the between-laboratory reproducibility(75%) are acceptable for the proposed future use of the DPRA (i.e. in combination withother complementary methods).

9Preliminary evaluation of predictive capacity based on the ring trial data
13) Full evaluation of the predictive capacity of the DPRA was not within the scope of the EURLECVAM study. However, the accuracy of the DPRA for dichotomous classification(sensitiser/non-sensitiser) on the basis of all 24 chemicals tested (including two metals,
one pro-hapten, dihydroeugenol, and two pre-haptens, 4-phenylendiamine and R(+)Limonene), was 79% (sensitivity=71%, specificity=92%). When excluding the two metalcompounds (which are considered outside the applicability domain and can be readily
excluded from testing during practical application of the assay), the accuracy was 82%(76% sensitivity and 92% specificity). Thus, the predictive capacity determined in the study
is consistent with published information from a larger set of data (Gerberick et al., 2007).
Importantly, substances reported as false negatives in the EURL ECVAM study weregenerally substances with a low sensitisation potency in vivo.
14) In relation to the ability of the DPRA to categorise substances in reactivity classes, datafrom the validation study does not support the use of the DPRA as a standalone method
for potency categorisation. This is consistent with published information. However the
study results indicate that the assignment of a chemical to a DPRA reactivity category may
have the potential to contribute to the determination of its potency.

4. Limitations
4.1 Technical limitations
15) Solubility of test substances: Peptide depletion values for substances with limited
solubility in the solvents prescribed by the DPRA SOP cannot be derived with sufficientaccuracy. Despite the fact that all the chemicals selected for the EURL ECVAM study were
found to be compatible with the test system, limitations with the testing of insoluble
chemicals have been reported in the submission to ECVAM.
16) Co-elution: In those instances, mainly attributable to specific instrument settings, where
the test substance or the reaction products elute at the same time as the peptide (coelution), an accurate measurement of peptide depletion cannot be made. The DPRA SOP
provides instructions on how to approach different instances of co-elution, allowing incertain cases an estimation of the peptide depletion and reactivity class assignment.
However there might be circumstances were this type of approximation is not appropriate.
17) Cysteine dimerisation: Accurate determination of peptide depletion can also be hamperedby substances promoting the oxidation of the thiol group in the cysteine peptide that leads
to the formation of the cystine dimer. In such cases depletion of the peptide would beoverestimated.
4.2 Limitations with regard to applicability
The following limitations of DPRA should be taken into consideration:1018) Restriction to lysine and cysteine: The DPRA is designed to measure reactivity of theelectrophile towards two amino acids: the thiol group of cysteine and the primary aminogroups of lysine. As other amino acids are not present in the assay, chemicals withpreferential reactivity towards amino acids other than cysteine or lysine (e.g. nucleophilic
sites in histidine), may lead to false negative results when tested in the DPRA. However,when considering this limitation, it should be also kept in mind that the relative
percentages of substances reacting preferably with amino acids other than cysteine andlysine is at present unclear and that the cysteine and lysine peptides represent softer toharder model nucleophiles (OECD, 2012; Schwöbel et al., 2011) which would coverdifferent reaction mechanisms19) Metal compounds: DPRA is not designed to accommodate the spectrum of reaction
mechanisms considered to be associated with sensitising metals. For example Nickel, themost important metal allergen, is postulated to form coordination bonds with nucleophilic
residues in histidine. However, metal compounds can be readily excluded from testing
based on chemical structure and, therefore, this limitation can be easily addressed bysimply avoiding the DPRA for the testing of metal compounds.
20) Pro-haptens and pre-haptens: The DPRA is not designed to detect the sensitisingproperties of pro-haptens which require bioactivation, or pre-haptens which require
abiotic transformation. Nevertheless, pre-haptens are in some cases reported to becorrectly detected as sensitisers by the DPRA, as was the case in the EURL ECVAM study(n=2). However, the reasons why specific pre-haptens are detected while others are notremain unclear. To address the issue of pre- and pro-haptens, in silico expert systems suchas TIMES-SS (Patlewicz et al., 2007; Roberts et al., 2007) and the OECD QSAR toolbox(www.qsartoolbox.org) could prove useful. Notably, a variation of the DPRA including an
additional protocol step which mimics oxidative activation (co-incubation with horseradish
peroxidase and hydrogen peroxide) to detect pre- and pro- haptens is under development.
(Gerberick et al., 2009).
21) Oxidation: Some substances that have oxidative properties (e.g. oxidative colourants)
without necessarily causing haptenation may lead to possible false positive results whentested in the DPRA.

5. Suggested regulatory use22) Due to the complexity of the mechanisms underlying skin sensitisation, it is likely that
information from different methods (in silico, in chemico, in vitro) is needed to reduce orreplace the need for animal testing, both for hazard identification and potency
characterisation purposes. The DPRA is a reliable test method that provides information on
peptide reactivity, which is considered to be the molecular initiating event of skinsensitisation (OECD 2012). Therefore, peptide depletion values generated with the DPRA
could be used to support read-across from chemical analogues or combined withinformation from other non-animal methods in the context of a Weight of Evidence (WoE)
approach or Integrated Testing Strategy (ITS). The extent of information needed tocomplement a DPRA result will depend on the intended application (e.g. hazard
11

identification, classification or potency assessment) and context (availability and quality ofother information).
23) For the purpose of hazard identification (i.e. identifying substances with sensitisingpotential), it is plausible that an unequivocal depletion value in the DPRA combined withthe presence of a structural alert or positive QSAR prediction for skin sensitisation may
prove sufficient for decision making, thus justifying the waiving of an animal test. To
conclude on the absence of sensitising potential, additional information would be needed
to increase confidence, such as in vitro data on downstream events. In any case a negative
DPRA result should be interpreted with care, taking into consideration the possibility offalse negatives due to (1) possible reactivity with amino acid residues other than cysteine
and lysine, (2) the lack of metabolic capacity of the assay leading to possible
misclassification of pro-haptens as well as (3) the uncertain capacity of the DPRA tocorrectly pick up pre-haptens. For hazard assessment purposes, possible uses of DPRA datain the context of a WoE or ITS have been reported in several scientific publications (Ball etal., 2011; Bauch et al., 2012).
24) Use of results generated with the DPRA for potency prediction has also been proposed(Jaworska et al., 2011; Nukada et al., 2012). Results from the EURL ECVAM study showedthat the limited number of substances with high DPRA reactivity fell into Category 1A of
the UN GHS (UN, 2011), suggesting the potential application of DPRA results for potency
sub-categorisation. However, further efforts are required to explore how DPRA data may
support potency assessment, possibly in combination with data from other methods.
25) When employed within an integrated approach, the DPRA may be useful to satisfy
information requirements for Cosmetics (Regulation EC/1223/2009), Chemicals (Regulation
EC/1907/2006), Biocides (EC/528/2012) and Plant Protection Products (Regulation
EC/1107/2009).

12

6. Follow-up activities recommended by EURL ECVAM(1)

In view of further testing with the DPRA, EURL ECVAM recommends that the revised
protocolavailableat
EURL
ECVAM's
DB-ALM
service(http://ecvamdbalm.jrc.ec.europa.eu) be used.

(2)

The predictive capacity of the DPRA for assessing the sensitisation potential of substances
should be further evaluated in the context of its use as part of integrated approaches totesting and assessment.

(3)

DPRA data should be analysed to understand the potential of the method to contribute to
the potency assessment of substances, including sub-categorisation according to GHS (i.e.
categories 1A and 1B). Use of existing human data and data from the LLNA are likely to be
useful for this purpose. A study correlating DPRA data with potency categories derived
from LLNA has recently been published (Natsch et al., 2013).

(4)

To support ITS development and to increase confidence in the DPRA method, additional
prospective testing with the DPRA should be tailored towards better understanding of its
applicability domain to better define how the method performs with (a) weak sensitisers,(b) pre-haptens and (c) chemicals that have selective reactivity towards amino acids otherthan cysteine and lysine. The reason for false positive predictions also deserves further
investigation.

(5)

EURL ECVAM supports the development of an OECD Test Guideline for the DPRA. A
project proposal has already been submitted to the OECD and included in the OECD 2012
work program. As this test is best employed in combination with complementary
methods, it should be considered in the current initiative being undertaken at OECD todevelop a guidance document on Integrated Approaches for Testing and Assessment(IATA) for skin sensitisation.

(6)

EURL ECVAM recommends continued investment in the development of the next
generation of peptide reactivity assays which can potentially address some of thelimitations of the DPRA method.

13

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EU (2009a). Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21
October 2009 concerning the placing of plant protection products on the market and repealing
Council Directives 79/117/EEC and 91/414/EEC Official Journal No L 309, p. 01-50.
EU (2009b). Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30
November 2009 on cosmetic products. Official Journal No L 342, p. 01-59.
EU (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September
2010 on the protection of animals used for scientific purposes. Official Journal L 276, 20/10/2010
P. 0033 – 0079.
EU (2012). Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May
2012 concerning the making available on the market and use of biocidal products. Official Journal
L 167, 27.6.2012, p. 1–123
Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP., (2007). Quantificationof chemical peptide reactivity for screening contact allergens: a classification tree model
approach. Toxicological Sciences 97, 417-427.
Gerberick GF, Troutman JA, Foertsch LM, Vassallo JD, Quijano M, Dobson RL, Goebel C,
Lepoittevin JP (2009) Investigation of peptide reactivity of pro-hapten skin sensitizers using aperoxidase-peroxide oxidation system. Toxicol Sci. Nov;112(1):164-74.
Hartung T, Bremer S, Casati S, Coecke S, corvi R, Fortaner S, Gribaldo L, Halder M, Hoffman S, Roi
AJ, Prieto P, Sabbioni E, Scott L, Worth A, Zuang V. (2004). A modular approach to the ECVAM
principles on test validity. Alternatives to Laboratory Animals 32, 467-472.
Jaworska J, Harol A, Kern PS, Gerberick GF, (2011). Integrating non-animal test information into an
adaptive testing strategy - skin sensitization proof of concept case. ALTEX 28, 211-225.
Jowsey IR, Basketter DA, Westmoreland C, Kimber I (2006). A future approach to measuring
relative skin sensitising potency: a proposal. Journal of Applied Toxicology 26: 341-350.
Landsteiner, K. & Jacobs, J., (1936). Studies on the sensitization of animals with simple chemicalcompounds. Journal of Experimental Medicine 64, 625-639.
Lepoittevin J-P, Basketter DA, Goossens A, Karlberg A-T, (1998). Allergic contact dermatitis: themolecular basis. Springer, Berlin.
Natsch, A, Ryan C.A., Foertsch L., Emter R, Jaworska J, Gerberick F, Kern P (2013) A dataset on 145
chemicals tested in alternative assays for skin sensitisation undergoing prevalidation. Journal ofApplied Toxicology. Epub, ahead of print.
Nukada Y, Miyazawa M, Kazutoshi S, Sakaguchi H, Nishiyama N., 2012. Data integration of nonanimal tests for the development of a test battery to predict the skin sensitizing potential andpotency of chemicals. Toxicology In Vitro 27, 609-618.
15

OECD (1992). OECD Guideline for the Testing of Chemicals No. 406: Skin Sensitisation. Paris,
France: Organisation for Economic Cooperation and Development. Online at:
http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section4-health-effects_20745788
OECD (2010a). OECD Guideline for the Testing of Chemicals No. 429: Skin Sensitisation: Local
Lymph Node Assay. Paris, France: Organisation for Economic Cooperation and Development.
Online at: http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-ofchemicals-section-4 health-effects_20745788
OECD (2010b). OECD Guideline for the Testing of Chemicals No. 442a Skin Sensitisation: Local
Lymph Node Assay DA. Paris, France: Organisation for Economic Cooperation and Development.
Online at: http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-ofchemicals-section-4-health-effects_20745788
OECD (2010c). OECD Guideline for the Testing of Chemicals No. 442b: Skin Sensitisation: Local
Lymph Node Assay BrdU-ELISA. Paris, France: Organisation for Economic Cooperation andDevelopment. Online at: http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-thetesting-of-chemicals-section-4-health-effects_20745788
OECD (2012). The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding
to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 168. Online at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)
10/PART1&docLanguage=En
Patlewicz G, Dimitrov SD, Low LK, Kern PS, Dimitrova GD, Comber MIH, Aptula AO, Phillips RD,
Niemela J, Madsen C, Wedebye EB, Roberts DW, Bailey PT, Mekenyan OG (2007) TIMES-SS – A
promising tool for the assessment of skin sensitization hazard. A characterisation with respect
to the OECD Validation Principles for (Q)SARs. Reg Toxicol Pharmacol 48: 225–239
Roberts DW, Patlewicz G, Dimitrov SD, Low LK, Aptula AO, Kern PS, Dimitrova GD, Comber MI,
Phillips RD, Niemelä J, Madsen C, Wedebye EB, Bailey PT, Mekenyan OG. (2007) TIMES-SS--amechanistic evaluation of an external validation study using reaction chemistry principles. Chem
Res Toxicol. 2007 Sep;20(9):1321-30. Epub 2007 Aug 23.
Roberts DW, Aptula AO. Determinants of skin sensitisation potential. J Appl Toxicol. 2008
Apr;28(3):377-87.
Schwöbel JA, Koleva YK, Enoch SJ, Bajot F, Hewitt M, Madden JC, Roberts DW, Schultz TW,
Cronin MT (2011) Measurement and estimation of electrophilic reactivity for predictive
toxicology. Chem Rev. Apr 13;111(4):2562-96.
UN (2011). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Part 3:
Health and Environmental Hazards. New York, NY, USA, and Geneva, Switzerland. United Nations
Economic Commission for Europe. Online at:
http://www.unece.org/trans/danger/publi/ghs/ghs_rev04/04files_e.html

16

ANNEX 1

ESAC OPINION

Annex 1ESAC Opinion

Opinion of the EURL ECVAM Scientific Advisory Committee (EASC) on the
ECVAM-led study of the Direct Peptide Reactivity Assay (DPRA)
Ispra, 17 December 2012

Summary of the ESAC Opinion
The ESAC was asked to provide an opinion on an EURL-ECVAM led prevalidation study assessing
the transferability and reproducibility (within- and between-laboratories) of the DPRA test
method (primary objective of the study) in view of its possible future use as part of a nonanimal testing strategy for skin sensitization. The study had also been designed to provide
preliminary information on a) the predictive capacity of the test method and b) its potential use
for contributing to sub categorisation of sensitizing chemicals.
The ESAC considered the scientific work presented of good quality. Overall, the conclusions
made by the working group (WG) correspond well with the conclusions formulated in the reportby the Validation Management Group (VMG).








The within laboratory reproducibility (WLR) of the test method with respect toconcordance of classification (S/NS) was considered acceptable.
The data were considered strong enough to support transferability of the test to
properly equipped, trained and staffed laboratories with the appropriate analyticalcapabilities.
The between laboratory reproducibility (BLR) of the test method with respect toconcordance of classification was considered sufficient when compounds outside
the applicability domain were excluded.
The preliminary predictive capacity as evaluated in this study is consistent with the
published data (references 6, 7).
The potential for use of DPRA reactivity information in potency sub-categorisation
requires further examination with a larger dataset.

The ESAC had some questions about the statistical calculations underlying the determination ofan adequate sample size to analyse reproducibility as a primary study goal. The ESAC wasconcerned that possible limitations of the assay were not described in sufficient detail in thevalidation study report.
The predictive capacity, applicability domain and limitations of the test are not defined yet, but
the available data suggest that the test is a useful tool for early decision making during productdevelopment (screening) and a component in a weight-of-evidence (WoE) approach or
integrated testing strategy (ITS).
The ESAC recommends that the possible limitations of the DPRA should be further investigatedspecifically in relation to pre-/pro-haptens, either by additional prospective testing or throughanalysis of existing information as there may be a risk of false negative results associated withthese chemicals.

17

ANNEX 1

1.

ESAC OPINION

Mandate of the ESAC

The opinion of ESAC should support ECVAM with respect to the development ofrecommendations regarding the reliability (transferability, within and between laboratory
reproducibility) of the DPRA and the potential regulatory use of the test method.
(1) Study design – transferability, reliability and relevance

The ESAC was requested to review whether the prevalidation study was conducted
appropriately in view of the objective of the study:

Reproducibility of the DPRA method within one laboratory (WLR);

Transferability to other laboratories;

Reproducibility in other laboratories (BLR);

Predictive capacity of the test method.

With respect to the design and conduct of the study, the following issues were to beaddressed:
 Clarity of the test definition (module 1)
 Clarity of the definition of the study objective
 Appropriateness of the study design in view of study objective
 Appropriateness of the study execution:
 Appropriateness of the statistical analysis used for analysing WLR, transferability,
BLR and (preliminary) predictive capacity.

(2) Conclusions of the study
The ESAC was requested to assess the justification and plausibility ofReproducibility (WLR and BLR) and transferability;
Preliminary predictive capacity;
Possible gaps between study design and study conclusions which remain to beaddressed in view of the suggested conclusions/use;
Applicability and possible limitations of the test method, in particular in view of its
potential use within an ITS for sensitisation.

(3) Possible contribution of test method to integrated approach
The ESAC is requested (a) to evaluate, on the basis of the data submitted in the validation study,
the possible use of the test method (also within a strategy) to identify skin sensitisers, (b) tomake additional recommendations (as required) on the proper scientific use of the test method
within such a strategy taking specific aspects of this method into account (e.g. applicability,
limitations etc.) and (c) to identify possible further information required (i.e. are there gaps) tobe able to conclude on the plausibility of the suggested use (including within an ITS).

18

ANNEX 1

2.

ESAC OPINION

Detailed opinion of the ESAC

The ESAC was asked to provide an opinion on an EURL-ECVAM led study assessing thetransferability and reproducibility (within- and between-laboratories) of the DPRA (primaryobjective of the study) in view of its possible future use as part of a non-animal testing strategy
for skin sensitization. The study had also been designed to provide preliminary information on a)the predictive capacity of the test method and b) its potential use for contributing to subcategorisation of sensitizing chemicals.
(1) Study design – transferability, reliability and relevance.
The WLR was assessed at the level of concordance in prediction (S/NS). WLR for the threelaboratories was in the range from 73% to 100% . The lowest value derived from the leadlaboratory. The ESAC concluded that, in the context of the study, and in view of the fact thatboth naive laboratories exceeded the target of 85% as chosen by the VMG, the WLR was
sufficient.










The definition of the reproducibility target (85%) was based upon i) the backgroundand specific objectives of the validation study; ii) the standards of performance thatcan realistically be expected from an in vitro test and standards of performance
which have been considered acceptable in previous validation studies; iii) 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); and iv) the power of thedesign of the validation study.
Transferability activities were divided into Training, Transferability and Qualification
Runs The WLR was formulated for each partner to include 1) concordance inprediction, 2) depletion values for cysteine and lysine, as well as 3) control values.
The data were considered strong enough to support transferability of the test to
properly equipped, trained and staffed laboratories with the appropriate analyticalcapabilities.
During the transfer and blind testing phase one laboratory had difficulties inmeeting the acceptance criteria defined in the SOP, due to the Reference Control C
being marginally outside the acceptance criteria. The cause of the problem could
not be identified. The ESAC recommends that these acceptance criteria should be
re-examined.
The BLR was assessed in terms of 1) concordance in prediction and 2) depletion
values for cysteine and lysine. Eighteen of the 24 chemicals were consistently
classified (S/NS) by the three laboratories resulting in a BLR reproducibility of 75%,
which is below the target (80%). The reproducibility assessment included 3
chemicals (beryllium sulphate, nickel chloride and dihydroeugenol) that wereconsidered by the VMG as outside the applicability domain of the test. The
exclusion of these three substances considered to fall outside the applicability
domain would lead to a BLR of 87.5%. For 15 out of the 24 chemicals thelaboratories assigned the same reactivity class resulting in a BLR of 62.5%. Data
variability was observed for results from chemicals with low or no reactivity.
The secondary goals included a preliminary evaluation of the ability of the test to
discriminate skin sensitizers from non-sensitizers, and a preliminary considerationof the ability to contribute to sub-categorization of skin sensitising chemicals (GHS
sub-category 1A and 1B). The validation study report (VSR) did not present asummary of the predictive capacity based on all 24 chemicals tested, since the VMG
19

ANNEX 1



ESAC OPINION
judged three of them (beryllium sulphate, nickel chloride, dihydroeugenol) to falloutside the applicability domain. The two pre-haptens (4-phenylendeiamine andR(+)-Limonene) were included in the analysis as the VMG felt that there wasinsufficient evidence to exclude them from the evaluation of the predictive
performance. The predictive capacity for all 24 substances was 77.8% (sensitivity:
70.8%; specificity: 91,7%) while 82.4% (sensitivity: 73.5%; specificity: 91,7%) for the
19 substances (since the WG felt that other two substances would fall outside theapplicability domain, PPD and limonene).
The project was described and designed in clearly recognizable and well describedphases including Test Definition (Module 1), Transferability (Module 3), Within
Laboratory Reproducibility (WLR) (Module 2), Between Laboratory Reproducibility(BLR) (Module 4) and Predictive Capacity (Module 5).
Overall, the chosen statistical approach was considered appropriate. In thecalculations of suitable sample size it was not clear for the ESAC why a power of
75% was chosen for the BLR, especially as a more conventional 80% power wasused for the WLR. However, since more chemicals than the minimum number were
tested in both cases the actual power of the study was considered sufficient.

(2) Conclusions of the studyOverall, the study design and the quality of the selected chemicals (N=24) wereconsidered appropriate for the purpose of addressing the first objective of thestudy: Assessing the WLR and BLR of the DPRA.
 Overall, the conclusions made by the WG correspond well with the conclusions
drawn by the VMG as described in the VSR, indicating that these conclusions aresupported by the results shown in the report.
 The WLR of the test method with respect to concordance of classification (S/NS)
met the target of 85% and was considered sufficient for the purpose of thisstudy.
 The data were considered strong enough to support transferability of the test to
properly equipped, trained and staffed laboratories with the appropriate
analytical capabilities.
In spite of a BLR (75%) below the target of 80%, the BLR of the test method with respect
to concordance of classification was considered sufficient after the removal of thecompounds outside the applicability domain. The potential for use of DPRA reactivity
information in potency sub-categorisation requires further examination with a larger
dataset.
 The number of chemicals (N=24) did not provide support for a firm conclusion
about the predictive capacity of the test method. The preliminary data were,
however, considered promising.
 The number of chemicals did not allow drawing a conclusion about theapplicability domain of the test. Empirically the applicability domain seems to
exclude pre-/pro-haptens and metal salts.
Chemicals that preferably react with amino acids other then cysteine and lysine
may fall outside the applicability domain. In addition, some pre-/pro-haptens werereported as correctly identified. Finally, the data seem to indicate that the test
method has problems identifying weak sensitizers. The uncertainty about theapplicability domain may result in an unacceptable level of false negative results.

20

ANNEX 1

ESAC OPINION(3) Possible use of the test method within an integrated approach
As outlined in the VSR and the ECVAM request for ESAC advice, the DPRA cannot be used as astand-alone test method in a regulatory context but should be considered for use in an
Integrated Testing Strategy (ITS). On the basis of the present report, especially negative
outcomes have to be considered with care.


As pre-haptens are not consistently correctly predicted by the DPRA, there remains
uncertainty about whether to consider pre-haptens as part of the applicability
domain of the method or not.
Unless there are sufficiently accurate assays available identifying chemicals as pre/pro-haptens in view of excluding them from routine testing using the DPRA, suchcompounds will be tested in the DPRA and may cause false negative results.
The selection of cysteine and lysine-containing peptides selects for the majority, but
not all, reactive chemicals.

Regarding reactivity class, the data obtained did not support the possibility to use DPRA as astand-alone test method for potency classification. This is in agreement with the statement of
the VMG that the assay should be further evaluated for its capacity to "contribute" to a potencyclassification (VSR page 8).
Information generated by the DPRA can be used to support regulatory decision making whenused in the context of a weight-of-evidence approach or ITS. It is important to use the test in acontext that allows confident conclusions about the protein-reactivity of the chemical,
especially when the chemical in question is negative in the DPRA. As such the method may be
helpful to address testing requirements of e.g. the REACH legislation and the 7 th Amendment of
the Cosmetic Directive.
Its inclusion into future ITSs can be considered for the purpose of an eventual full replacementof current in vivo hazard identification assays.
Recommendations:
The DPRA addresses a key mechanism (haptenation) in the development of skinsensitization/allergic contact dermatitis. Overall the provided data support transferability andreproducibility of the test to qualified laboratories. The predictive capacity of the test is notdefined yet, but the preliminary data profiles the test as a useful tool for early decision making
during product development (screening) and a component in a weight-of-evidence approach or
ITS for safety/hazard assessment.
The ESAC recommends that the limitations of the DPRA (risk of false negative results) arefurther investigated specifically in relation to pre-/pro-haptens either by additional prospective
testing or through analysis of existing information.

21

ANNEX 1
3.

ESAC OPINION

Informative background to the Mandate and Opinion

Skin sensitizsation is the toxicological endpoint associated with substances that have theintrinsic ability to cause Allergic Contact Dermatitis, ACD in humans. ACD represents the mostcommon manifestation of immunotoxicity in humans, i.e. adverse effects of xenobiotics
involving the immune system. The identification of the skin sensitization potential represents
an important component of the safety assessment of any new substance and especially for
those intended for topical application (e.g. cosmetics). Current regulatory predictive tests forskin sensitization rely on the use of animals, these include:a) the traditional guinea pig tests: Buehler Test and Guinea-pig Maximisation Test (OECDTG 406, Ref.1),b) the Local Lymph Node Assay (LLNA, OECD TG 429, Ref.2) and its recently OECD adopted
non-radioactive variants (OECD TG 422A, Ref.3 and OECD TG 422B, Ref.4).
Despite the progress that has been made in the development of alternative methods for skinsensitisation hazard identification, there are currently no validated methods available. In
addition none of the tests currently under development/evaluation is able to fully characterise
the relative potency of sensitising substances and therefore, none of these assays is considereda stand-alone method, capable of fully replacing current animal procedures, in particular as
regards to cosmetics.
The current view therefore is to combine different test methods in order to address different
key mechanisms of skin sensitisation: skin bioavailability, haptenation (the protein binding ofchemicals which triggers immunological responses), epidermal inflammation, dendritic cell
activation and migration, T cell proliferation. Test methods are currently under development
which have been specifically designed to address these key mechanistic steps involved in skinsensitisation. Before these test methods can be routinely used, e.g. in ITSs, their capacity to
produce reproducible results needs to be demonstrated as a first step.
The Direct Peptide Reactivity Assay DPRA is addressing one of the key upstream events in the
cascade of mechanisms leading to the induction of skin sensitisation. It measures the ability ofchemicals to react with proteins (haptenation). There is good evidence that haptenation is adeterminant step in the induction of skin sensitisation. Chemical allergens are usually low
molecular weight chemicals which are not immunogenic per se. However, chemical allergens (ortheir metabolites, oxidation products) have electrophilic properties that allow them to bindcovalently with the nucleophilic side chains of amino acids of skin proteins to form an
immunogenic conjugate. Already in 1936 this correlation between the reactivity of chemicals
with proteins and their skin sensitisation potential was described (Landsteiner and Jacobs,
Ref.5) and has in the meantime been extensively described in the literature. This knowledge isbeing exploited for the development of several in chemico reactivity assays with relevance forthe testing of sensitisation potential, amongst these the DPRA assay.
The DPRA is designed to screen the sensitisation potential of chemicals by measuring peptide
depletion with UV-HPLC, following incubation of the test chemicals with synthetic
heptapeptides containing either cysteine (peptide/chemical ratio in the reaction mixture 1:10)
or lysine residues (peptide/chemical ratio in the reaction mixture 1:50) (Gerberick 2004, Ref.6).
The average of peptide depletion values for cysteine and lysine are used to classify chemicals
into four reactivity categories: minimal, low, moderate and high reactivity (Gerberick 2007,
Ref.7). Based on the known correlation between haptenation/chemical reactivity andsensitisation potential, it is assumed that these reactivity classes as predicted by the DPRA may
contribute to the characterisation of sensitiser potency.
The possible predictive capacity of the DPRA is supported by the data of the original DPRA
submission. On the basis of 133 chemicals, the DPRA classified chemicals as sensitisers or nonsensitisers (in relation to LLNA data) with an accuracy of 86% (87% sensitivity, 83% specificity).
22

ANNEX 1

4.

ESAC OPINION

References1.

OECD, Organisation for Economic Cooperation and Development (1992) Skin Sensitisation
Guidelines for Testing of Chemicals No. 406, Paris

2.

OECD, Organisation for Economic Cooperation and Development (2002) The Local Lymph
Node Assay. Guidelines for Testing of Chemicals No. 429, Paris

3.

OECD, Organisation for Economic Cooperation and Development (2010a) Skin Sensitization:
Local Lymph Node Assay: DA, Guidelines for Testing of Chemicals No. 442A, Paris

4.

OECD, Organisation for Economic Cooperation and Development(2010b) Skin
Sensitization: Local Lymph Node Assay: BrdU-ELISA, Guidelines for Testing of Chemicals No.
442B, Paris

5.

Landsteiner K & Jacobs J (1936) Studies on the sensitisation of animals with simple
chemical compounds. Journal of Experimental Medicine 64, 625-639

6.

Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP, (2004)
Development of a peptide reactivity assay for screening contact allergens. Toxicol Sci. 81;
332-43.

7.

Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP, (2007).
Quantification of chemical peptide reactivity for screening contact allergens: a classification
tree model approach. Toxicol Sci. 97, 417-27.

23

ANNEX 1

ESAC OPINIONEURL ECVAM Scientific Advisory Committee
















Dr. Nathalie ALÉPÉE
Dr. David BASKETTER
Dr. Neil CARMICHAEL
Prof. Jacques R. CHRETIEN
Prof. Lucio G. COSTA
Dr. Rodger CURREN
Prof. A. Wallace HAYES
Prof. Coenraad HENDRIKSEN
Dr. Dagmar JÍROVÁ
Prof. Walter PFALLER
Dr. Erwin ROGGEN
Prof. Vera ROGIERS
Dr. Andrea SEILER
Prof. Kristin SCHIRMER
Prof. Ruud A. WOUTERSEN

ESAC Working Group Skin Sensitisation









Dr. Erwin ROGGEN (ESAC member, Chair of ESAC WG and rapporteur)
Prof. Walter PFALLER (ESAC member, ESAC Vice Chair)Prof. A. Wallace HAYES (ESAC member)Dr. Maja ALECSIC (external expert)
Dr. Emanuela CORSINI (external expert)
Dr. David LOVELL (external expert)
Dr. Michael WOOLHISER (external expert)
Prof. Yong HEO (external expert, ICATM nomination)

Coordination
 Dr. Claudius GRIESINGER (EURL ECVAM Coordinator for ESAC peer reviews and EURL ECVAM
recommendations) Dr. Alexandre ANGERS (specific support)

24

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

Annex 2EURL ECVAM request for ESAC advice

EURL ECVAM request for ESAC advice on an ECVAM-coordinated study
concerning the transferability and reliability of the Direct Peptide
Reactivity Assay (DPRA) for skin sensitisation testing
1.
TYPE OF REQUEST
Request Type

Identify request ("YES")

1) ESAC Peer Review
of a Prevalidation Study or Validation StudyYES

If R1)applies please specify further:►Prevalidation StudyYES
At present (January 2012) ECVAM is conducting a
study of three test methods for skin sensitisation
testing: 1) the Direct Peptide Reactivity Assay(DPRA), 2) the human Cell Line Activation Test (hCLAT) and 3) the Myeloid U937 Skin Sensitisation
Test (MUSST).
The study assesses transferability andreproducibility of these test methods in view of
their possible future use (e.g. as partial replacement
methods) within an integrated approach for skinsensitisation hazard identification aiming at the full
replacement of the currently used regulatory in vivo
assays for this purpose. In addition the data
generated in this study will inform possible futureevaluations on the predictive capacity of theseassays.
While assessment of the h-CLAT and MUSST test
methods are foreseen to be completed in 2012, theevaluation of the DPRA test method was finalised in2011 and the adopted Validation Study Report isforeseen to be available by January 2012. ESAC
review will commence in February 2012 employing
the same ESAC WG which is currently (January2012) peer reviewing the KeratioSens submission.

Prospective Validation Study

No

Retrospective Validation StudyNo

Validation Study based on PerformanceStandards

No2) Scientific Advice on a test method submitted to ECVAM for
validation(e.g. the test method's biological relevance etc.)

No3) Other Scientific Advice

No25

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE(e.g. on test methods, their use; on technical issues such as cell
culturing, stem cells etc.)

2.
TITLE OF STUDY OR PROJECT FOR WHICH SCIENTIFIC ADVICE OF THE ESAC IS
REQUESTED
Prevalidation of the Direct Peptide Reactivity Assay (DPRA) for skin sensitisation testing3.
BRIEF DESCRIPTION OF THE STUDY OR PROJECT
1) Background to skin sensitization and current predictive tests
Skin sensitisation is the toxicological endpoint associated with substances that have the intrinsic
ability to cause Allergic Contact Dermatitis, ACD in humans. ACD represents the most common
manifestation of immunotoxicity in humans, i.e. adverse effects of xenobiotics involving theimmune system. The identification of the skin sensitization potential represents an importantcomponent of the safety assessment of any new substance and especially for those intended for
topical application (e.g. cosmetics). Current regulatory predictive tests for skin sensitization relyon the use of animals, these include:a) the traditional guinea pig tests: Buehler Test and Guinea-pig Maximisation Test (OECDTG 406, Ref.1),b) the Local Lymph Node Assay (LLNA, OECD TG 429, Ref.2) and its recently OECD adopted
non-radioactive variants (OECD TG 422A, Ref.3 and OECD TG 422B, Ref.4).
Despite the progress that has been made in the development of alternative methods for skinsensitisation hazard identification, there are currently no validated methods available. In
addition none of the tests currently under development/evaluation is able to fully characterise
the relative potency of sensitising substances and therefore, none of these assays is considereda stand-alone method, capable of fully replacing current animal procedures, in particular as
regards to cosmetics.
The current view therefore is to combine different test methods in order to address different
key mechanisms of skin sensitisation: skin bioavailability, haptenation (the protein binding ofchemicals which triggers immunological responses), epidermal inflammation, dendritic cell
activation and migration, T cell proliferation. Test methods are currently under development
which have been specifically designed to address these key mechanistic steps involved in skinsensitisation. Before these test methods can be routinely used, e.g. in integrated testing
strategies, their capacity to produce reproducible results needs to be demonstrated as a firststep.

2) Background to the DPRA, h-CLAT, MUSST
DPRA:
The Direct Peptide Reactivity Assay DPRA is addressing one of the key upstream events in the
cascade of mechanisms leading to the induction of skin sensitisation. It measures the ability ofchemicals to react with proteins (haptenation). There is good evidence that haptenation is adeterminant step in the induction of skin sensitisation. Chemical allergens are usually low
molecular weight chemicals which are not immunogenic per se. However, chemical allergens (ortheir metabolites, oxidation products) have electrophilic properties that allow them to bindcovalently with the nucleophilic side chains of amino acids of skin proteins to form an
immunogenic conjugate. Already in 1936 this correlation between the reactivity of chemicals
with proteins and their skin sensitisation potential was described (Landsteiner and Jacobs,

26

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

Ref.5) and has in the meantime been extensively described in the literature. This knowledge isbeing exploited for the development of several in chemico reactivity assays with relevance forthe testing of sensitisation potential, amongst these the DPRA assay.
The DPRA is designed to screen the sensitisation potential of chemicals by measuring peptide
depletion with UV-HPLC, following incubation of the test chemicals with synthetic
heptapeptides containing either cysteine (peptide/chemical ratio in the reaction mixture 1:10)
or lysine residues (peptide/chemical ratio in the reaction mixture 1:50) (Gerberick 2004, Ref.6).
The average of peptide depletion values for cysteine and lysine are used to classify chemicals
into four reactivity categories: minimal, low, moderate and high reactivity (Gerberick 2007,
Ref.7). Based on the known correlation between haptenation/chemical reactivity andsensitisation potential, it is assumed that these reactivity classes as predicted by the DPRA may
contribute to the characterisation of sensitiser potency.
The possible predictive capacity of the DPRA is supported by the data of the original DPRA
submission. On the basis of 133 chemicals, the DPRA classified chemicals as sensitisers or nonsensitisers (in relation to LLNA data) with an accuracy of 86% (87% sensitivity, 83% specificity).
h-CLAT & MUSST
The h-CLAT and MUSST are based on the use of Dendritic Cell (DC)-like cell lines. Using flow
cytometry, these test methods monitor the induction of cell surface markers associated with DC
activation, following exposure to the chemical. In the MUSST, changes in CD86 expression in the
U937 cell line are detected; in the h-CLAT modulation of both CD86 and CD54 expression arerecorded in THP-1 cells.

3) Study goals and design
In the first quarter of 2009 the DPRA was formally submitted to ECVAM together with other two
test methods namely the human Cell Line Activation Test (h-CLAT) and the Myeloid U937 Skin
Sensitisation Test (MUSST), developed by companies associated with the European Cosmetics
Association (Colipa) and optimized within Colipa ring trials. Following detailed scientificassessment of the information submitted, ECVAM judged the three methods to be mature
enough to enter the ECVAM validation process.
In September 2009 a formal study on the three above mentioned test methods was launched,with the main overall objective to evaluate their transferability and reliability (reproducibility
within and between laboratories) when challenged with 24 coded chemicals.
As a secondary goal of the study, the experimental data will be used to perform:a) A preliminary evaluation of the ability of each of the three tests to reliably discriminate skinsensitising (S) from non-sensitising (NS) chemicals as defined by the Globally Harmonised
System (GHS, Ref. 6) 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 (Ref.8) on classification, labelling and packaging (CLP) of substances and mixtures.
b) Where possible, a preliminary consideration of the ability of each of the three tests tocontribute to sub-categorisation of skin sensitising chemicals, e.g. into Sub-category 1A (strong
sensitisers) and Sub-category 1B (other sensitisers) as adopted in the 3rd revised version of the
GHS.
The study experimental design foresees the testing of the 24 coded test items in each of thethree participating laboratories for the assessment of the between-laboratory reproducibility. A
subset of 15 of these chemicals are being tested two additional times in each laboratory for the27

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

evaluation of within-laboratory reproducibility.
With respect to ECVAM's modular approach of validation (Hartung et al., 2004, Ref.10) the
study will provide information on module 1) test definition, module 2) within laboratory
reproducibility, module 3) transferability and module 4) between laboratory reproducibility. In
addition, the data generated will provide preliminary information on module 5) predictive
capacity; however, the number of chemicals tested is based on statistical considerations related
to the evaluation of the reproducibility, and a larger sample size would be required for module
5 to be considered fulfilled.
References
8. OECD, Organisation for Economic Cooperation and Development (1992) Skin
Sensitisation Guidelines for Testing of Chemicals No. 406, Paris
9. OECD, Organisation for Economic Cooperation and Development (2002) The Local
Lymph Node Assay. Guidelines for Testing of Chemicals No. 429, Paris
10. OECD, Organisation for Economic Cooperation and Development (2010a) Skin
Sensitization: Local Lymph Node Assay: DA, Guidelines for Testing of Chemicals No.
442A, Paris
11. OECD, Organisation for Economic Cooperation and Development (2010b) Skin
Sensitization: Local Lymph Node Assay: BrdU-ELISA, Guidelines for Testing of Chemicals
No. 442B, Paris
12. Landsteiner K & Jacobs J (1936) Studies on the sensitisation of animals with simple
chemical compounds. Journal of Experimental Medicine 64, 625-639
13. Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP, (2004)
Development of a peptide reactivity assay for screening contact allergens. Toxicol Sci.
81; 332-43.
14. Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP, (2007).
Quantification of chemical peptide reactivity for screening contact allergens: aclassification tree model approach. Toxicol Sci. 97, 417-27.
15. Anon. (2003). Globally Harmonised System of Classification and Labelling of Chemicals(GHS). Part 3: Health and Environmental Hazards, pp. 151-158. New York, NY, USA, andGeneva, Switzerland: United Nations Organisation.
16. EU (2008b) Regulation (EC) No 1272/2008 (16 December 2008) of the European
Parliament and of the Council on classification, labelling and packaging of substancesand mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, andamending Regulation (EC) No 1907/2006. Official Journal of the European Union L 353,
(31/12/2008) p. 1-1355.
17. Hartung, T., Bremer, S., Casati, S., Coecke, S., Corvi, R., Fortaner, S., Gribaldo, L., Halder,
M., Hoffmann, S., Roi A.J., Prieto, P., Sabbioni, E., Scott, L., Worth, A. and Zuang. V.
(2004) A Modular Approach to the ECVAM Principles on Test Validity. ATLA 32, 467-72.
4.
4.1

OBJECTIVES, QUESTIONS, TIMELINESOBJECTIVE

Objective
Why does ECVAM
require advice on
the current issue?

The opinion of ESAC should support ECVAM with respect to the development ofrecommendations regarding the reliability (transferability, within and between
laboratory reproducibility) of the DPRA and the potential regulatory use of the test
method.

28

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICEIn addition the ESAC should advice with regard to the possible necessary further
work required (in relation to predictive capacity, applicability, limitations of thetest method) to assess the potential contribution of the DPRA to a future (not yet
designed) testing strategy or test battery that would aim to achieve full
replacement of the currently used animal tests for skin sensitisation hazard
assessment.
Moreover, based on the evaluation of the data submitted, the ESAC should
provide advice on the potential usefulness of the DPRA test method within atesting strategy for skin sensitisation testing and the proper scientific use of thetest method within such a testing strategy (e.g. with respect to its specificapplicability and limitations). It is explicitly noted that the ESAC is not requested tosuggest the precise placing of the submitted method in a hypothetical ITS, butrather to provide advice on the characteristics of the method relevant for its
subsequent integration into an ITS at a later point in time (i.e. when otherbuildings blocks of such an ITS are known).
4.2
QUESTION(S) TO BE ADDRESSEDQuestions
1) DESIGN & CONDUCT OF STUDY: The ESAC is requested to review whether the
What are theprevalidation study was conducted appropriately in view of the objective of thequestions andstudy. The study objective was to assess
issues that should (1) the reproducibility of the DPRA method within one laboratory
be addressed in(2) its transferability to other laboratories
view of achieving(3) its reproducibility in other laboratories (BLR).
the objective of(4) Furthermore, the study aimed at assessing, in a preliminary manner, thethe advice?
predictive capacity of the test method
When reviewing the design and conduct of the study, the following issues should
be addressed in particular:
Clarity of the test definition (module 1)
Clarity of the definition of the study objective
Appropriateness of the study design in view of study objective, inter alia:
Is the number of tested chemicals (24) sufficient for the purposes of the study?
Are the reference data used for assessing in particular the predictive capacity
appropriate and of good quality?
Was the identification of chemicals conducted in an appropriate manner (i.e.
presence or absence of selection criteria, justification etc.)?
Is the adverse effect range of the selected chemicals appropriate for the purposeof the study
In case of gaps (chemical class etc.) – are these justified?
Is the number of laboratories sufficient?
Appropriateness of the study execution (e.g. were there pre-defined acceptancecriteria, were these respected? How were exceptions / deviations handled? Wereprovisions specified for retesting? Was the number of repetitions sufficient? etc.)
Appropriateness of the statistical analysis used for analysing WLR, transferability,
BLR and (preliminary) predictive capacity.
2) CONCLUSIONS OF STUDY: The ESAC is requested to assess whether theconclusions, as presented in the Validation Study Report, are substantiated by the
information generated in the study and are plausible with respect to existing
information and current views (e.g. literature).
In particular:

29

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

Are the conclusions on reproducibility (WLR and BLR) as well as transferability
justified and plausible?
Are the conclusions on preliminary predictive capacity justified and plausible withrespect to existing information
Are there possible gaps between study design and study conclusions which
remain to be addressed in view of the suggested conclusions / use (see also point3)?
Do the data generated with this defined set of chemicals and available existing
evidence provide sufficient information on applicability and possible limitationsof the test method, in particular in view of its potential use within an ITS for
sensitisation?
3) SUGGESTED USE OF THE TEST METHOD: The ESAC is requested (a) to evaluate,on the basis of the data submitted in the validation study, the possible use of thetest method (also within a strategy) to identify skin sensitisers, (b) to makeadditional recommendations (as required) on the proper scientific use of the test
method within such a strategy taking specific aspects of this method into account(e.g. applicability, limitations etc.) and (c) to identify possible further information
required (i.e. are there gaps) to be able to conclude on the plausibility of thesuggested use (including within an ITS).

30

ANNEX 2

4.3
TIMELINES
Timelines
concerning thisrequestWhen does
ECVAM require
the advice?

ECVAM REQUEST FOR ESAC ADVICE

Timeline
Finalised ESAC Opinion required by:
Request to be presented to ESAC bywritten procedure (e.g. due tourgency) prior to the next ESAC
Request to be presented to ESAC at
ESAC plenary meeting

Indication
ESAC 37, 6/7 October 2012 or before
through written procedure
YES, before ESAC 36 (20/21 March
2012)
NO

5.
ECVAM PROPOSALS ON HOW TO ADDRESS THE REQUEST WITHIN ESAC
5.1
ECVAM PROPOSAL REGARDING REQUEST-RELATED STRUCTURES REQUIREDSpecific
Structure(s) required
Required according to ECVAM? (YES/NO)
structures
required within
1) ESAC Rapporteur
NO
ESAC to address2) ESAC Working GroupYES
the request
3) Invited Experts
YES
Does the advice
Ad 3): If yes – list names andrequire an ESAC
affiliations of suggested
working group, an
experts to be invited andESAC rapporteur
specify whether these areetc.?
member of the EEP
If other than above (1-3):

5.2
DELIVERABLES AS PROPOSED BY ECVAM
Deliverables
Title of deliverable other than
What deliverables ESAC opinion(other than the
D1 ESAC Rapporteur Report
ESAC opinion) are and draft opinion
required forD2 ESAC Peer Review Reportaddressing theand draft opinion
request?
If other than above (D1-D2):

31

Required? (YES/NO)
NO
YES
NO

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

6.
LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESACCount Description of document
Available (YES/NO)
1
OECD, Organisation for Economic
YES
Cooperation and Development(1992) Skin Sensitisation Guidelines
for Testing of Chemicals No. 406,
Paris
2OECD, Organisation for Economic
YES
Cooperation and Development(2002) The Local Lymph Node Assay.
Guidelines for Testing of Chemicals
No. 429, Paris
3
OECD, Organisation for Economic
YES
Cooperation and Development(2010a) Skin Sensitization: Local
Lymph Node Assay: DA, Guidelines
for Testing of Chemicals No. 442A,
Paris
4
OECD, Organisation for Economic
YES
Cooperation and Development(2010b) Skin Sensitization: Local
Lymph Node Assay: BrdU-ELISA,
Guidelines for Testing of Chemicals
No. 442B, Paris
5
Gerberick GF, Vassallo JD, Bailey RE,
YES
Chaney JG, Morrall SW, LepoittevinJP, (2004) Development of a peptide
reactivity assay for screening contact
allergens. Toxicol Sci. 81; 332-43.
6
Gerberick GF, Vassallo JD, Foertsch
YESLM, Price BB, Chaney JG, LepoittevinJP, (2007). Quantification of chemical
peptide reactivity for screening
contact allergens: a classification
tree model approach. Toxicol Sci. 97,
417-27.

32

File name
ER2011-03_TG 406

ER2011-03_TG 429

ER2011-03_TG 442A

ER2011-03_TG 442B

ER2011-03_scientificpaper on DPRA

ER201-03_DPRAClassification Tree
Model

ANNEX 2

7.

ECVAM REQUEST FOR ESAC ADVICETERMS OF REFERENCE OF THE ESAC WORKING GROUP

7.1
ESTABLISHMENT OF THE ESAC WORKING GROUP
During its 34th meeting on March 22-23 the ESAC plenary unanimously decided to establish an
ESAC Working Group Sensitisation charged with the detailed scientific review of four test
methods for skin sensitisation.
7.2
TITLE OF THE ESAC WORKING GROUP
Full title:
ESAC Working Group on Skin Sensitisation Test Methods
Abbreviated title:
ESAC WG Sensitisation
7.3
MANDATE OF THE ESAC WG
The EWG is requested to conduct a scientific review of the relevant studies concerning four skinsensitisation test methods (DPRA, MUSST, h-CLAT, Keratinosens). The review needs to address
the questions put forward to ESAC by ECVAM.
The review should focus on the appropriateness of design and conduct of the study in view ofthe study objective and should provide an appraisal to which extent the conclusions of the
Validation Management Team (VMT) / test method submitter are substantiated by the
information generated during the study and how the information generated relates to thescientific background available.
7.4
DELIVERABLE OF THE ESAC WG
The ESAC WG is requested to deliver to the chair of the ESAC and the ESAC Secretariat a
detailed ESAC Working Group Report outlining its analyses and conclusions. A reporting
template has been appended (Appendix 1) intended to facilitate the drafting of the report.
The conclusions drawn in the report should be based preferably on consensus. If no consensus
can be achieved, the report should clearly outline the differences in the appraisals and provide
appropriate scientific justifications.
The WG is further asked to prepare a draft ESAC opinion as basis for the discussions by theentire ESAC, which shall adopt its opinion to the extent possible by consensus and on the basisof the ESAC WG report as well as all documents that were made available to the WG as well as
to all ESAC members.
7.5
PROPOSED TIMELINES OF THE ESAC WG
The following timelines have been proposed by ECVAM. These should be agreed upon during
the face-to-face meeting (Item 1 in the table):
Item Proposed date/time
ActionDeliverable
1
1-3 February
Face to face meeting
1)Input on draft
mandate2) Initial drafting ofreport2
20. / 21. March
Progress report of WG Chair at
Presentation / oral
ESAC36summary3
14 MayFinal report to be delivered to
Final draft report
ESAC Coordinator/Secretariat.
4
8 JuneFeedback from ESAC to WG on
Feedback on final draft
draft report (written procedure)
report529 JuneFinal draft report available
Final report33

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

7.6
QUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WG
The ESAC WG is requested to address the three questions posed to the ESAC which have beenbroken down further in more specific questions by Secretariat (see section 4.2) and werediscussed with the ESAC WG and approved by the ESAC.
When preparing the final ESAC WG report to address these questions, the ESAC WG isrequested to use a pre-defined reporting template. This template (see appendix 1) follows
ECVAM's modular approach and addresses to which extent the standard information
requirements have been addressed by the study. In addition, the template allows for addressing
the specific questions outlined in section 4.2. The Secretariat will provide guidance if necessary.
APPENDIX 1REPORTING STRUCTURE FOR THE ESAC WG REPORTThe following suggested structure follows the ECVAM information requirements ("modules") for
scientific review following validation and allows at the same time for the description of theanalysis and conclusions concerning more specific questions. A template has been created on
the basis of the structure below and this template will be made available to the ESAC.
The template can be used for various types of validation studies (e.g. prospective full studies,retrospective studies, performance-based studies and prevalidation studies). Depending on the
study type and the objective of the study, not all sections may be applicable. However, forreasons of consistency and to clearly identify which information requirements have not been
sufficiently addressed by a specific study, this template is uniformly used for the evaluation ofvalidation studies. The current template is version 5.

END OF EURL ECVAM RECOMMENDATION

34

European Commission
EUR 26383– Joint Research Centre – Institute for Health and Consumer Protection
Title: EUR 26383 - EURL ECVAM Recommendation on the Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation
Testing
Luxembourg: Publications Office of the European Union2013 – 36 pp. – 21.0 x 29.7 cm
EUR – Scientific and Technical Research series –ISSN 1831-9424 (online)
ISBN 978-92-79-34886-0 (pdf)
doi:10.2788/48229

Abstract
Identification of the skin sensitisation hazard of chemicals has traditionally relied on the use of animals. Progress in thedevelopment of alternative methods has been prompted by the increasing knowledge of the key biological mechanisms
underlying this human health effect, as documented by the OECD's recent report summarising the key biological events
leading to skin sensitisation ("Adverse Outcome Pathway" (AOP) for skin sensitisation). The molecular initiating event
defined within this AOP is the covalent binding of chemicals with skin proteins. Thus peptide reactivity assays may
provide valuable information in the context of integrated approaches such as Weight of Evidence (WoE) or Integrated
Testing Strategies (ITS) for skin sensitisation hazard and safety assessment. Based on these considerations, EURL ECVAM
coordinated a validation study on the Direct Peptide Reactivity Assay (DPRA) addressing mainly the test method’stransferability and within- and between-laboratory reproducibility. Following independent scientific peer review by the
EURL ECVAM’s Scientific Advisory Committee (ESAC) and having considered the input from regulators, stakeholders,international partners and the general public, EURL ECVAM concluded that the DPRA may prove a valuable componentof a WoE or ITS for skin sensitisation hazard assessment. In addition to this, the DPRA may also be able to contribute to
the assessment of sensitising potency, e.g. by supporting sub-categorisation of sensitisers according to UN GHS.
However it is recognised that further efforts are required to explore how DPRA data may contribute to potency
assessment.

35

LB-NA-26383-EN-Nz

As the Commission’s in-house science service, the Joint Research Centre’s mission is to provide
EU policies with independent, evidence-based scientific and technical support throughout thewhole policy cycle.
Working in close cooperation with policy Directorates-General, the JRC addresses key societal
challenges while stimulating innovation through developing new standards, methods and tools,
and sharing and transferring its know-how to the Member States and international community.
Key policy areas include: environment and climate change; energy and transport; agriculture
and food security; health and consumer protection; information society and digital agenda;
safety and security including nuclear; all supported through a cross-cutting and multidisciplinary approach.

36