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

Addendum to the
Direct Peptide Reactivity Assay (DPRA)ECVAM Validation Study Report

Addendum to the Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 1 of 4

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

This document provides additional information to the Working Group in charge of thepeer-review of the DPRA validation study to inform its opinion in relation to the resultsof the study with regards to the study secondary goal b) “preliminary consideration of theability of the three tests evaluated in the above mentioned study (including the DPRA) tocontribute to subcategorisation of the skin sensitising chemicals, e.g. into Subcategory 1A
and Subcategory 1B as adopted in the 3rd revised version of the GHS” as the Working
Group felt that this study goal was not sufficiently addressed in the original report.
Within the GHS classification scheme there is the possibility to refine the evaluation ofskin sensitisers on the basis of the potency of the sensitising effect. Skin sensitisers canbe assigned to subcategory 1A “strong sensitisers” or to subcategory 1B “other skinsensitisers” using a weight of evidence approach on the basis of reliable and good quality
evidence from human cases or epidemiological studies and/or observations fromappropriate studies in recognized and officially accepted animal tests.
In the case of the LLNA a threshold defined for the EC3 value is used to subcategoriseskin sensitisers into subcategory 1A (EC3≤2 ) or into subcategory 1B (EC3>2).
The DPRA validation study results have been analysed with respect to the potential of theDPRA to contribute to the GHS subcategorisation by evaluating the extent of agreement
between the GHS subcategorisation based on LLNA results and the DPRA reactivity
class results as represented in the following table.

Addendum to the Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 2 of 4

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

Table 1 Agreement between the predicted reactivity class and the GHS categories
GHS
subcategorisation
based on LLNA
data

MajorA

P&G

Ricerca

IVMU

Benzoquinone+ (1A)HIGH

PPD+ (1A)

HIGH

HIGH
HIGH

HIGHLYSHIGHCL

HIGH
HIGH

Kathon CG+ (1A)HIGH

HIGHLYS

HIGH

HIGH

Formaldehyde+ (1A)

LOW

LOW

MODERATE

LOW

Chloramine T+ (1A)HIGH

HIGHLYSHIGHLYS

HIGH

Chlorpromazine HCl+ (1A)MINIMALMINIMALMINIMALMINIMAL

2-MBT+ (1A)

HIGHHIGHLYS

HIGH

HIGH

Thioglycerol+ (1B)

≥LOWCL

MODERATELYS≥LOWCL≥LOWCL

Imidazolidinyl Urea+ (1B)

MODERATE

MODERATE

MODERATE

MODERATE

Methyl Methacrylate+ (1B)

MODERATE

LOW

Benzyl Salicylate+ (1B)

MINIMALMINIMALMINIMAL

LOW

Benzyl Cinnamate+ (1B)

MINIMALMINIMALMINIMALMINIMAL

R(+)Limonene+ (1B)

LOW

LOW

LOW

LOW

Glycerol- (NC)MINIMALMINIMALMINIMALMINIMAL

DCNB- (NC)MINIMALMINIMALMINIMALMINIMAL

Benzyl Alcohol- (NC)MINIMALMINIMALMINIMAL

LOW

Methyl Salicylate- (NC)MINIMALMINIMALMINIMAL

LOW

Isopropanol- (NC)MINIMALMINIMALMINIMALMINIMAL

Dimethyl Isophthalate- (NC)MINIMALMINIMALMINIMALMINIMAL

4-PABA- (NC)MINIMALMINIMALMINIMALMINIMAL

Xylene- (NC)BMINIMALMINIMALMINIMALMINIMAL

ChemicalA≥MODERATECl MODERATE

final predicted reactivity class based on majority voting

LYS subscript or CL subscript corresponds with co-elution for Lysine and co-elution with CYS/LYS
NC: Not Classified
B
Xylene is a false positive in the LLNA and was assigned to the “no category” by weight of evidence.

Since there are three GHS categories (No Category (NC) and the two subcategories 1Aand 1B) while the DPRA can assign the chemicals to four different reactivity classes, forthe purposes of this analysis, and based on the definition of the GHS category classes, aminimal reactivity in the DPRA was judged to correspond to a NC in the officialclassification, low and moderate reactivity to subcategory 1B and high reactivity
corresponds to subcategory 1A.

Addendum to the Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 3 of 4EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Union Reference Laboratory for Alternative Methods to Animal Testing(ECVAM)

Comparing this subcategorisation to the reference GHS classification of the chemicals
resulted in an overall accuracy (cumulative over the 3 labs) of 79.4%. The accuracy for
P&G and Ricerca were 81.0% and 76.2% for IVMU as presented in table 2.
The chemicals considered by the VMG to fall outside the applicability domain of theDPRA were excluded from the analysis.
Table 2 Overall agreement (cumulative over the three laboratories) of the DPRA
reactivity classes with the 3 GHS categories (NC, 1A and 1B) and extent of agreement
obtained by each laboratory
Referenceclassification

Cumulative
Hi

L-M

P&GMi

Hi

Ricerca
L-M

Mi

Hi

IVMU

L-M

Mi

Hi

L-M

Mi+ (1A) (n=7)

15

3

3

5

1

1

5

1

1

5

1

1+ (1B) (n=6)

0

13

50

4

20

4

20

5

1- (NC) (n=8)

02

2200

80

0

80

2

6Total15

1879.430

5581.0115581.0115876.2

8

Accuracy (%)

On the basis of these results the VMG noted that although the DPRA prediction model
was not designed specifically for the purpose of classifying chemicals into the GHS
categories, there was a significant correlation between the chemical's GHS categories andthe reactivity class assigned by the DPRA: chemicals assigned by the DPRA in the HIGH
reactivity class were always GHS subcategory 1A substances, chemicals assigned by theDPRA to a LOW or MODERATE reactivity class tend to be GHS subcategory 1B
substances and chemicals assigned the MINIMAL reactivity class tend to be notclassified (as was also observed in the analysis of the Sensitiser/Non-sensitiser
predictions).
Despite the fact that it not possible to conclude on the ability of the DPRA to contributeto a skin sensitizer subcategorisation on the basis of this limited sample size, these results
are however encouraging and suggest that the DPRA might have the potential tocontribute to such subcategorisation. This will need to be fully assessed on the basis of alarger set of results, and with the possible contribution of other sources of information(eg. in combination with other test methods).

Addendum to the Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 4 of 4