BioPharma Product Testing EN >> Validated Standard Testing Methods >> Direct Peptide Reactivity Assay (DPRA)

Direct Peptide Reactivity Assay (DPRA)

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In Chemico Skin Sensitisation Assay  

The in chemico direct peptide reactivity assay (DPRA) addresses the first molecular key event of the adverse outcome pathway (AOP) of skin sensitisation.1 The UN GHS (United Nations Globally Harmonized System of Classification and Labelling of Chemicals) defines a skin sensitiser as a substance that will cause an allergic response after skin contact.2

The DPRA is validated by the EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) and is performed in accordance with the OECD guidance OECD 442C at Eurofins BioPharma Product Testing Munich GmbH1, 3 with chemicals, cosmetics or personal care products and pharmaceuticals.

The DPRA is one of three test methods (KeratinoSensTM and h-CLAT) for the assessment of skin sensitisation potential.

 

Assessment of Skin Sensitisation Potential with the DPRA

  • The molecular initiating event of skin sensitisation addresses epidermal protein binding and reactivity towards proteins, by mimicking the reaction with artificial peptides.
  • A cysteine and a lysine containing peptide are mixed with the test substance and after 24 hours the sample is analyzed by high-performance liquid chromatography (HPLC) with UV detection at 220 nm.
  • If a depletion or a diminishment of the peptides is detected it can be used for classification of reactivity of chemicals and to discriminate between sensitisers and non-sensitisers.

 

DPRA Test at Eurofins

The molecular initiating event is the covalent binding of electrophilic substances to nucleophilic centers in skin proteins. The ability of a chemical to react with skin proteins is thought to play a key role in the development of skin sensitisation1, 4The test items are incubated with a cysteine and a lysine peptide and their content is determined via HPLC.

 

Procedure

Principle of the DPRA

Protocol

Peptides

Ac-RFAACAA-COOH (Cysteine)

Ac-RFAAKAA-COOH (Lysine)

Analysis

Quantification of cysteine and lysine peptide concentration by high-performance liquid chromatography (HPLC) using UV detection at 220 nm

Concentrations

Test chemical = 100 mM

Cysteine peptide = 10:1 ratio to test chemical

Lysine peptide = 50:1 ratio to test chemical

Exposure time

24 h

Quality controls

Positive control: cinnamic aldehyde (100 mM)

Different reference controls

Co-elution controls of test chemical and positive control

Solvents of test chemical

Acetonitrile, water, acetonitrile / water 1:1, isopropanol, methanol, ethanol, 1,4-butandiol, N,N-dimethyl formamide, tert. butanol

Data delivery

Depletion of cysteine and lysine peptide (%)

Reactivity category

Prediction of sensitising potential

Positive prediction

Depletion of cysteine and lysine peptide is >6.38%

Depletion of cysteine is >13.89% (Cysteine-only)

 

Based on the peptide depletion of cysteine and lysine, chemicals can be classified with the DPRA into four reactivity categories and can be discriminated between sensitisers and non-sensitisers.

Mean of cysteine and lysine depletion (%)

Reactivity class

Prediction

mean % depletion between 0 and 6.38

no or minimal reactivity

negative

mean % depletion between 6.39 and 22.62

low reactivity

positive

mean % depletion between 22.63 and 42.47

moderate reactivity

mean % depletion between 42.48 and 100

high reactivity

Table 1: Classification of reactivity and prediction of sensitising potential dependent on cysteine and lysine peptide depletion (cysteine 1:10 / lysine 1:50 prediction model).2

If lysine has a co-elution or it cannot be evaluated and it is negative, we can use the cysteine for evaluation on its own:

Mean of cysteine depletion (%)

Reactivity class

Prediction

mean % depletion between 0 and 13.89

no or minimal reactivity

negative

mean % depletion between 13.90 and 23.09

low reactivity

positive

mean % depletion between 23.10 and 98.24

moderate reactivity

mean % depletion between 98.25 and 100

high reactivity

Table 2: Classification of reactivity and prediction of sensitising potential dependent on cysteine peptide depletion (cysteine 1:10 prediction model).2

If cysteine has a co-elution or it cannot be evaluated and it is negative, then the result remains inconclusive. A positive result can still be used.

 

Data

Eurofins data for demonstrating technical proficiency of the DPRA

Chemical

Cysteine PPD (OECD)

Lysine PPD (OECD)

Prediction (OECD)

Cysteine PPD (EF)

Lysine PPD (EF)

Prediction (EF)

Non-sensitising Chemicals

1-Butanol

0-7

0-5.5

Negative

0.00

0.72

Negative

6-Methylcoumarin

0-7

0-5.5

Negative

0.00

0.25

Negative

Lactic Acid

0-7

0-5.5

Negative

0.05

0.25

Negative

4-Methoxyacetophenone

0-7

0-5.5

Negative

1.60

1.43

Negative

Sensitising Chemicals

2,4-Dinitrochlorbenzene

90-100

15-45

Positive

100.00

40.18

Positive

Oxazolone

60-80

10-55

Positive

72.24

36.85

Positive

Formaldehyde

30-60

0-24

Positive

49.91

2.42

Positive

Benzylidene acetone

80-100

0-7

Positive

92.75

3.10

Positive

Farnesal

15-55

0-25

Positive

35.78

8.93

Positive

2,3-Butandione

60-100

10-45

Positive

71.87

19.58

Positive

PPD: Percent peptide depletion                           EF = Eurofins Munich GmbH

Table 3: Eurofins data of the DPRA – ten tested proficiency chemicals compared to the data of the OECD guideline.1

In Table 3 the obtained data from the DPRA of four non-sensitising and six sensitising chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline.

 

Eurofins DPRA Data

 

Figure 1: Eurofins DPRA data of the mean peptide depletion of cysteine and lysine compared with literature data 5.

The graph shows the data from the DPRA of 10 tested proficiency chemicals and cinnamic aldehyde in comparison to the data of Gerberick et al., (2007). The generated Eurofins data is quite comparable to the literature data.

 

References

  1. OECD Guidelines for Testing of Chemicals, number 442C “In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)” (adopted: February 04, 2015).
  2. UN (2015), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Sixth revised edition, UN New York and Geneva.
  3. EC EURL-ECVAM (2013). Recommendation on the Direct Peptide Reactivity Assay (DPRA) for skin sensitisation testing.
  4. Troutman JA et al., (2011) The incorporation of lysine into the peroxidase peptide reactivity assay for skin sensitization assessment Toxicol Sci 122(2); 422-436.