The in chemico DPRA test is a new alternative to the white rabbit skin sensitization test. It is based on the premise that the molecular initiating event in skin sensitization is the binding of electrophilic chemicals to nucleophilic sites in epidermal proteins.
In the DPRA test, the test item is incubated with cysteine and/or lysine peptides and adequacy of depletion measured by HPLC.
Reactivity Assays
The direct peptide reactivity assay (DPRA) measures the reaction of chemicals with two synthetic peptides, cysteine and lysine. The DPRA test is an in vitro skin sensitization assay and a key element of the adverse outcome pathway of skin sensitization (AOP).
In the DPRA assay, the peptide-npp resins are pretreated with a deprotection solution of 5,5′-dithiobis-2-nitrobenzoic acid and triisopropyl silane before they are reacted with the chemical under investigation. The resulting mixture is incubated for 24 h at 25 degC, and the peptides are measured by high-pressure liquid chromatography with UV detection. The average peptide depletion data for cysteine and lysine is used to predict the sensitizer status of the test substance.
As a chemistry-based assay, DPRA has significant limitations due to its reliance on reactivity parameters such as the peptide depletion ratio and logkmax values. Using reactivity values to assign a chemical as a sensitiser or non-sensitiser, or to assign potency classification (1A/B/NS), is based on the implicit assumption that a chemical’s reactivity alone can provide information about its overall potency, which may not be valid.
This limitation is especially evident for chemistry-potency applicability domains that involve acyl transfer, Schiff base electrophiles and unreactive chemicals that can sensitise via metabolic or abiotic activation. These chemical classes will still give rise to peptide depletion values, however their logkmax values can no longer be interpreted as representative of a reactivity rate constant and therefore cannot be reliably used to assess a chemical’s potency.
A spectrophotometric method, Spectro-DPRA, has been developed to overcome some of the limitations of DPRA. In a GLP study, Spectro-DPRA was compared with the DPRA, kDPRA and two in vitro cell-based assays to determine its transferability, proficiency, within and between laboratories, and predictive ability, all in compliance with OECD Test Guideline 442C. All three laboratories demonstrated high concordance for evaluating the sensitivity induced by the tested chemicals, with balanced accuracy values in the 80-85% range.
The Spectro-DPRA combines the advantages of the peptide-npp technology with the analytical power of mass spectrometry to detect and characterize peptide adducts. It enables the identification of novel adducts that cannot be discerned with other methods and provides detailed information on the chemical reaction mechanisms involved in the formation of these adducts. This knowledge can help to improve the interpretation of reactivity data and identify potential problems with an assay.
Spectro-DPRA
DPRA is an in vitro test to evaluate skin sensitization by evaluating the chemical’s reaction to two model peptides. Spectro-DPRA is a modified method to increase the throughput and enable it to be used within high throughput screening (HTS) platforms. The spectrophotometric method allows for continuous monitoring of the cysteine/lysine peptide peak, which eliminates the need to sample and analyze the entire peptide mixture. Furthermore, spectro-DPRA eliminates the need to perform protein conjugation and deconjugation steps.
To demonstrate the applicability of Spectro-DPRA, the authors performed a pre-validation study to evaluate transferability and proficiency, as well as within- and between-laboratory reproducibility. Ten chemical substances were tested in a murine local lymph node assay (LLNA), the human cell line activation test (h-CLAT), and the guinea pig maximization test (GPMT). Both Spectro-DPRA and the LLNA showed high concordance between laboratories in assessing the sensitivity of these chemicals, with the exception of unsaturated carbon-carbon double-bond and lipid acids.
This study demonstrates that Spectro-DPRA has the potential to be an effective tool for integrating multiple animal alternatives into a tiered testing framework. This is because DPRA measures Key Event 1, the molecular initiating step of binding to host proteins, while other traditional tests measure different events. Successfully replacing animal testing requires integrated approaches that evaluate a series of different events in non-animal models to identify the risk for allergic contact dermatitis.
Developing a methodology to confidently rescale in vitro results to in vivo data is a critical challenge for researchers looking for alternatives to animal testing. While factors such as sample characteristics are often considered when investigating correlations between in vivo and in vitro data, they are rarely integrated into the rescaling process. This article focuses on a method for accounting for this variability in order to improve confidence in the rescaling process. This is demonstrated using an example of rescaling DPRA reactivity to in vivo local lymph node assay (LLNA) potency classification for the essential oils linalool, citral, p-cymene, and geraniol. Specifically, the authors investigated the impact of adjusting for differences in sample characteristics when rescaling DPRA vs LLNA reactivity and PPRA vs GPMT reactivity to LLNA potency.
Kinetic DPRA
The kinetic direct peptide reactivity assay (kDPRA) is an extension of the DPRA. In this assay, the reaction kinetics of the test substance with a synthetic cysteine-containing heptapeptide are evaluated. The peptide is incubated with the test substance for various reaction times. After the reaction is stopped, the peptide is mixed with monobromobimane, which binds to the remaining unreacted peptide and results in a fluorescent product that can be quantified. The peptide depletion is calculated from the difference between the peptide starting material and the resulting fluorescent product. This assay is capable of predicting GHS category 1A potency without the need for animal testing.
In kDPRA, the test substance is incubated with the synthetic peptide for several different reaction times. The assay is stopped by adding the highly reactive monobromobimane, which cleaves the peptide at the cysteine atom and results in the formation of a fluorescent product that can be quantified.
The assay can be used to determine the reactivity of substances at a variety of concentrations. It is also possible to evaluate unknown chemical mixtures. For example, in one experiment, a series of nine substances was tested at testing concentrations that differed from their LLNA EC3 values. Eight of the nine substances were correctly classified. However, the extremely potent sensitizer oxazolone was incorrectly predicted as non-sensitizing when tested at its low EC3 concentration.
In addition, the kDPRA was evaluated in the presence of surrogate mixtures to see how well it performed when analyzing mixed chemical compounds. The assay was found to perform quite well, even in the presence of surrogate chemicals that have no reactivity with cysteine peptides. This suggests that the reactivity of the peptide is not dependent on its hydrophobicity.
The kDPRA also appears to be suitable for assessing the reactivity of aldehydes and ketones, although it should be noted that some of these substances have very high potency and do not react significantly with the peptide. This could be due to a mechanism other than a SN2 reaction and/or the formation of oxidative species that do not interact with the peptide -SH groups.
DPRA for Skin Sensitization
DPRA is an in chemico method that models the first key event (KE1) in the Adverse Outcome Pathway for skin sensitization of low molecular weight chemicals. The KE1 involves the covalent binding of chemicals to skin proteins. This process is modeled by the reactivity of the test chemical toward two model peptides containing the nucleophilic amino acid residues lysine and cysteine. Chemicals that react with peptides containing these amino acids are more likely to bind to proteins in the skin.
Using the DPRA method, a test chemical is incubated with the two model peptides for 24 hours. The reactivity of the test chemical is determined by determining the percentage depletion of the model peptides (as measured by HPLC-UV). A higher percentage depletion indicates more reactivity. The DPRA is a non-animal alternative to the mouse local lymph node assay (LLNA), and it has been shown to be more effective in predicting skin sensitizers than LLNA.
In a variation on the DPRA method, kinetic DPRA is used to determine the rate of the reactivity of the test compound. This allows for the determination of a rate constant for the depletion of the peptides, which is correlated with sensitizer potency. The kinetic DPRA is a more accurate method for assessing the ability of a test substance to bind to proteins, and it also offers a more comprehensive prediction of a chemical’s reactivity profile.
A new modification on the DPRA is the kinetic direct peptide reactivity assay (kDPRA), which has been developed to provide a rapid and robust method for assessing the reactivity of test compounds toward small heptapeptides containing cysteine or lysine. Unlike the standard DPRA assay, kDPRA does not require long incubation times and is compatible with mixtures such as essential oils. In addition, a spectro-photometric method has been developed to directly quantify the reaction adducts, eliminating the need for the complex LC/MS procedure required by other reactivity assays. direct peptides