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.
![Study Design of In Vitro Skin Sensitization - ARE-Nrf2 Luciferase Test Method [KeratinoSens] (OECD 442D)](https://www.scs-groups.com/wp-content/uploads/2024/12/In-Vitro-Skin-Sensitization-ARE-Nrf2-Luciferase-Test-Method-KeratinoSens.png "Study Design of In Vitro Skin Sensitization - ARE-Nrf2 Luciferase Test Method [KeratinoSens] (OECD 442D) 1")
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.
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.
Additional Resources:
https://www.oecd.org/env/ehs/testing/replaced-test-guideline-442c-2022.pdf
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