LuSens – In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method based on OECD 442D

Short introduction

The ARE reporter gene assay LuSens is a cell based animal free test system that helps to identify the skin sensitisation potential of substances based on OECD guideline 442D. The test system uses a transgenic HaCaT cell line that carries the luciferase reporter gene under control of an ARE-element (Antioxidative Response Element) from rat NADPH quinone oxidoreductase 1 gene. Sensitiser induced luciferase activity can be measured as luminescence and provides quantitative information about the sensitisation potential of a substance in the context of hazard identification (Ramirez et al. 2014).

Study design of OECD

Test setup

• Cytotoxicity range finding test (6 days):
  • Testing of 12 test item concentrations
• Experiment I (6 days):
  • Treatment with a minimum of 6 test item concentrations
  • Measurement of cell viability (MTT) and Luciferase activity
• Experiment II for validation of results of Experiment I(6 days):
  • Treatment with a minimum of 6 test item concentrations
  • Measurement of cell viability (MTT) and Luciferase activity

Results

A test compound is considered to have sensitizing potential when the luciferase induction is above or equal to 1.5 fold compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic tested concentrations whereby at least three tested concentrations must be non-cytotoxic. A test compound is considered to be not having sensitizing potential if the above effects are not observed (OECD 2015).

Bibliography

OECD. 2015. “OECD / OCDE TG 442D - In Vitro Skin Sensitisation : ARE-Nrf2 Luciferase Test Method.” OECD Guidelines for the Testing of Chemicals. Vol. 442 D.

Ramirez, Tzutzuy, Annette Mehling, Susanne N. Kolle, Christoph J. Wruck, Wera Teubner, Tobias Eltze, Alexandra Aumann, Daniel Urbisch, Ben van Ravenzwaay, and Robert Landsiedel. 2014. “LuSens: A Keratinocyte Based ARE Reporter Gene Assay for Use in Integrated Testing Strategies for Skin Sensitization Hazard Identification.” Toxicology in Vitro : An International Journal Published in Association with BIBRA 28 (8). Elsevier Ltd: 1482–97.