The direct peptide reactivity assay (DPRA) is used to contribute to the assessment of the skin sensitisation potential of chemicals.
The method models the covalent binding of a chemical to skin proteins (haptenation) which represents the first key event (KE1) of the skin sensitisation Adverse Outcome Pathway (AOP), also called Molecular Initiating Event (MIE), by quantifying the reactivity of chemicals towards synthetic peptides.
The DPRA was developed by Procter & Gamble in the US and has been evaluated in a EURL ECVAM coordinated validation study and subsequently underwent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC).
The method is adopted by the OECD as TG 442C and by the EU as TM B59.
The results of the validation study, the peer review of ESAC and our recommendations can be found on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.Read more about the Direct Peptide Reactivity Assay (DPRA) on TSAR
[collapsed]Skin sensitisation is the regulatory endpoint aiming at the identification of chemicals able to elicit an allergic response in susceptible individuals.
Following repeated exposure to a sensitising agent, the adverse health effect of allergic contact dermatitis (ACD) may be provoked. Thus the development of ACD is characterised by two distinct phases:
a) the induction of specialised immunological memory following the initial exposure to an allergen, called sensitisation and
b) elicitation of the clinical allergic response following subsequent exposure to the allergen.[/collapse]
The Direct Peptide Reactivity Assay (DPRA)
[collapsed]The DPRA mimics the process of haptenation, i.e. the covalent binding of low-molecular weight substances ("haptens") to proteins. This process is considered a key mechanism through which chemicals (or their metabolites) become antigenic.
The test is termed an in chemico method - that is to say it does not involve the use of cells but it is based on a chemical reaction.
Specifically the DPRA addresses the reactivity of the test chemical with either cysteine or lysine (amino acids that are part of synthetic peptides) following 24 hours of incubation. The measurement used in the test is the rate of depletion of these peptides and is assessed via a widely available HPLC-UV method.
The resulting data is then used to classify chemicals as having minimal, low, medium or high reactivity. This in turn gives an indication of likely skin sensitisation potential of a chemical.
The method has some limitations. It cannot be used to assess chemicals that are pro-haptens - i.e. chemicals that normally require metabolic conversion before interacting with skin proteins. This is because the method has no metabolic component.[/collapse]
Animal testing replacement
[collapsed]The test method addresses only one mechanisms of the cascade of events that leads to the development of skin sensitisation.
On its own the method is unlikely to replace any one particular animal test method. Rather, it is expected to be used in conjunction with other methods to be able to replace the current animal tests.
Such complimentary information may come from other testing methods that address other key events involved in skin sensitisation. Examples of such non-animal, alternative methods might include the human Cell Line Activation Test (h-CLAT) or the KeratinoSens assay.
Currently skin sensitisation tests in animals are described here.[/collapse]
[collapsed]The DPRA method was submitted to EURL ECVAM in the first quarter of 2009 by Procter & Gamble. The method had previously been subjected to a variety of evaluation studies. On the basis of the information submitted the method was judged to be sufficiently mature enough to enter the EURL ECVAM validation process.
The validation of the method was conducted in the period November 2009 to November 2011.
The validation study was designed to evaluate the transferability and reliability of the method when challenged with 24 coded (blinded) chemicals. Secondary objectives included a preliminary evaluation of the ability of the method to reliably discriminate skin sensitising and non-sensitising chemicals.
The experimental design foresaw the testing of 24 coded test substances in three independent laboratories for the assessment of between-laboratory reproducibility. A subset of the test items were selected for additional testing to assess within-laboratory reproducibility.
Transferability was also assessed in the process of establishing the methods in each of the laboratories that took part.
In addition, it was foreseen that the generated data could be used to give only a preliminary assessment of predictive capacity since the DPRA cannot be used as one-to-one replacement of the animal tests.[/collapse]
Validation study outcomes
[collapsed]It was found that the DPRA testing method is transferable to suitably equipped laboratories that have adequate experience in setting up and running HPLC-UV methods.
The results of this study indicate that the within- and between-laboratory reproducibility of the method is 87% and 75% respectively.
Although full evaluation of the predictive capacity of the method was beyond the scope of the study design, the analysis from this study suggests the accuracy of DPRA towards distinguishing sensitisers from non-sensitisers was 82% (based on a sensitivity of 76%, specificity of 92%). This rate is in agreement with a number of previous published studies.[/collapse]
EURL ECVAM recommendation
[collapsed]On the basis of the validation study performed, and reports from the scientific literature, EURL ECVAM made the following main recommendations:
- That data generated with the DPRA method should prove valuable as part of integrated approaches to testing and assessment (IATA) together with complimentary information (e.g. other in vitro data, QSAR and/or read-across predictions).
- In addition to supporting identification of sensitisers/non-sensitisers, the DPRA may also be able to contribute to the assessment of sensitising potency, e.g. by supporting, within an integrated approach, the subcategorisation of sensitisers. More work however is required to determine to which extent DPRA results relate to potency categories.
- We fully support the development of an OECD test guideline for this method.
- DPRA data should be considered before any animal experiments are performed and that such data should be used in the context of weight-of-evidence judgements.[/collapse]