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Study Design of In Vitro Skin Sensitization - ARE-Nrf2 Luciferase Test Method [KeratinoSens] (OECD 442D)

The KeratinoSens assay is a cell-based reporter gene assay that models the second key event in the Adverse Outcome Pathway (AOP) for dermal sensitization: keratinocyte activation.

It measures the induction of a stably transfected luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene. This makes it a promising alternative to animal testing for skin sensitization assessment.

Here's an overview of the KeratinoSens study design:

Test materials:

Human keratinocyte cell line: The KeratinoSens assay uses a stably transfected human keratinocyte cell line (e.g., HaCaT cells) containing the ARE-luciferase reporter gene construct.
Test chemicals: Chemicals being tested for skin sensitization potential are dissolved in an appropriate solvent.
Positive and negative controls: Known skin sensitizers and non-sensitizers are included as controls to ensure the test system is working properly.

Test Procedure:

Cell seeding: The keratinocyte cells are seeded into 96-well plates.
Treatment: The cells are treated with the test chemicals for 24-48 hours.
Luciferase measurement: After treatment, the cells are lysed, and the luciferase activity is measured using a luminometer.

Data analysis:

The luciferase activity is expressed as relative light units (RLUs).
The fold induction in luciferase activity is calculated by dividing the RLUs of the treated cells by the RLUs of the untreated control cells.
The fold induction is compared to a pre-defined cut-off value to categorize the test chemical as a sensitizer or non-sensitizer.

Interpretation of results:

Chemicals with a fold induction above the cut-off value are considered potential skin sensitizers.
Chemicals with a fold induction below the cut-off value are considered non-sensitizers.

Advantages of KeratinoSens:

Mechanistic: KeratinoSens targets a key event in the AOP for skin sensitization, providing mechanistic insights into the sensitizing potential of chemicals.
Animal-free: KeratinoSens is an in vitro assay, which means it does not require the use of animals.
Fast and cost-effective: KeratinoSens is a relatively quick and inexpensive test compared to animal-based skin sensitization assays.
High throughput: KeratinoSens can be used to test a large number of chemicals in a short period of time.

Limitations of KeratinoSens:

Does not address all key events in skin sensitization: KeratinoSens only assesses the second key event in skin sensitization. It does not address other key events, such as T cell activation and sensitization.
False positives and negatives: KeratinoSens may give false positive or negative results for some chemicals.
Limited to certain types of chemicals: KeratinoSens may not be suitable for testing all types of chemicals, such as metals and metal salts.

Additional References:

https://www.oecd.org/env/test-no-442d-in-vitro-skin-sensitisation-9789264229822-en.htm

KeratinoSens is a valuable tool for screening chemicals for skin sensitization potential. It is a relatively simple and inexpensive test that can be used to quickly identify potential sensitizers. However, it is important to remember that KeratinoSens is not a perfect test and its results should be interpreted in conjunction with other information, such as the chemical structure and known skin sensitization hazards of similar chemicals.