Direct Peptide Reactivity Assay is used in cosmetic applications for the characterization of the skin sensitizing potential of a substance, framed by OECD Guideline no 442.
The molecular initiating event (MIE) in skin sensitization is a binding between epidermal proteins and the sensitizing chemical substance. MIE is part of the adverse outcome pathway (AOP) of skin sensitization.
It is thanks to the properties of Cysteine peptide and Lysine peptide that the chemical binding will be able to take place, so these synthetic heptapeptides will mimic the reaction of a skin exposed to a substance.
Binding between nucleophilic proteins and electrophile substance will be measured by High Performance Liquid Chromatography (HPLC). Therefore, the decrease in Cysteine peptide and Lysine peptide levels will be a sign of sensitizing event. Depending on the rate of depletion, the sensitizing character of a molecule will be determined (see table at the bottom of the page).
In chemico DPRA test also has wider applications such as hazard classification in cosmetics, but also for pharmaceuticals and biocides. It is a good alternative to animal experimentation.
DPRA as an alternative to LLNA tests on animals
The DPRA test is a major advance in characterization of sensitizing elements. It is a non-animal approach. Skin sensitization studies are the cause of death of hundreds of thousands of mice worldwide each year. The DPRA test is therefore a preferred alternative to animal studies such as mouse local lymph node assay (LLNA). On the other hand it is a more effective method, as 22% of skin sensitizers are not detected with the LLNA test, and half of the sensitizers are classified in the wrong category.
As a basis for ‘integrated approaches to testing and assessment’ (IATA), the OECD has therefore developed the DPRA test via the adverse outcome pathways (AOP). OECD has thus enabled the DPRA test to become a new element of choice in the animal study of sensitisers, as a harmonised and validated test.
BACKGROUND: Cosmetics are normally composed of various ingredients. Some cosmetic ingredients can act as chemical haptens reacting toward proteins or peptides of human skin and they can provoke an immunologic reaction, called as skin sensitization. This haptenation process is very important step of inducing skin sensitization and evaluating the sensitizing potentials of cosmetic ingredients is very important for consumer safety. Therefore, animal alternative methods focusing on monitoring haptenation potential are undergoing vigorous research. To examine the further usefulness of spectrophotometric methods to monitor reactivity of chemicals toward peptides for cosmetic ingredients.
OBJECTIVE: Evaluating an alternative method for detecting the skin reactivity of chemicals.
METHOD/RESULTS: Forty chemicals (25 sensitizers and 15 non-sensitizers) were reacted with 2 synthetic peptides, e.g., the cysteine peptides (Ac-RFAACAA-COOH) with free thiol group and the lysine peptides (Ac-RFAAKAA-COOH) with free amine group. Unreacted peptides can be detected after incubating with 5,5′-dithiobis-2-nitrobenzoic acid or fluorescamine™ as detection reagents for free thiol and amine group, respectively. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine peptides or more than 30% depletion of lysine peptides.
CONCLUSION: The sensitivity, specificity, and accuracy were 80.0%, 86.7% and 82.5%, respectively. These results demonstrate that spectrophotometric methods can be an easy, fast, and high-throughput screening tools predicting the skin sensitization potential of chemical including cosmetic ingredient.
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