The ADRA Assay: A Solid 3Rs Choice for Skin Sensitization Tests

The Amino Acid Derivative Reactivity Assay (ADRA), a newer in chemico assay is helping reduce even further the use of animals for skin sensitization tests

Skin sensitization-the endpoint regulators use to identify whether chemicals are able to trigger an allergic reaction in individuals-is understandably an important component of any safety assessment. The biological mechanisms underlying skin sensitization have been well defined in an Adverse Outcome Pathway (AOP)1, model, which starts with a trigger event between the chemical and the skin proteins. (Key event 1).

Various in chemico methods address this molecular initiating event: the Direct Peptide Reactivity Assay (DPRA), the Kinetic Direct Peptide Reactivity Assay (kDPRA) and the Amino Acid Derivative Reactivity Assay (ADRA). All are described in OECD test guideline 442C2. The DPRA has been running at Charles River's Den Bosch site since 2015, and the kDPRA was added in 2019. This year we implemented and validated the ADRA. Even though the ADRA assay is not yet included in the OECD test guideline 497 on defined approaches for skin sensitization3, it allows for the evaluation of key event 1 for poorly soluble compounds or compounds otherwise not suited for the DPRA assay in a weight of evidence approach. This means skin sensitization potential of poorly soluble compounds can be determined using a combination of in chemico and in vitro methods instead of using animal tests.

To demonstrate technical proficiency of the ADRA method implemented at our site, ten proficiency substances recommended by OECD test guideline 442C2 were evaluated. The skin sensitizing potential of these compounds have been well-studied making them good candidates for this experiment. The compounds were incubated with model synthetic amino acid derivatives containing either cysteine or lysine for 24 hours at 25°C.

After incubation, the relative amino acid derivative concentration was determined by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 281 nm and 291 nm and the percent amino acid derivative depletion values were calculated. An additional benefit of the ADRA is the use of a fluorescence detector (Ex/Em 284/333 nm) which can be used in case there is co-elution of the test material with the amino acid derivatives. Co-elution is defined as two compounds eluting from the chromatographic column at the same time, making separation and identification of these compounds difficult.

Results of the ADRA test

So, what did we learn from this technical proficiency exercise? When testing reactivity with amino acid derivatives containing either cysteine or lysine, 10 of 10 substances showed the correct ADRA skin sensitization prediction as defined by the OECD 442C guideline. This shows that we successfully implemented the ADRA and demonstrated technical proficiency.

Sixty percent of the animal tests for skin sensitization have already been replaced by a combination of computer models and lab tests. It is expected that the ADRA will further improve the non-animal test strategy by reducing the need for animal tests for poorly soluble compounds.

References:
1. OECD (2012). Series on Testing and Assessment No. 168. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Organisation for Economic Cooperation and Development, Paris
2. OECD (2024). OECD Guideline for the Testing of Chemicals No. 442C: In chemico Skin Sensitisation: Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins. In chemico. Paris, France: Organisation for Economic Cooperation and Development.
3. OECD (2023) OECD Guideline No. 497: Guideline on Defined Approaches for Skin Sensitisation. Paris, France: Organisation for Economic Cooperation and Development.

Milou J.C. Santbergen, PhD, is a Study Director in the In Vitro DMPK group at Charles River Den Bosch. She obtained her PhD in intestinal in vitro models combined with mass spectrometry analysis and is currently working on a wide range of in vitro metabolism studies and skin sensitization assays.

Gido Snaterse, PhD, is a Study Director in the In Vitro DMPK group at Charles River Den Bosch. He has a research background in prostate cancer and steroid hormone metabolism and is now working on skin sensitization assays and analytical method development.

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