Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals

A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLCspectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals.

ALEPEE Nathalie;  VIEGAS BARROSO Joao Filipe;  DE SMEDT Ann;  DE WEVER Bart;  HIBATALLAH Jalila;  KLARIC Martina;  MEWES Karsten R.;  MILLET Marion;  PFANNENBECKER Uwe;  TAILHARDAT Magalie;  TEMPLIER Marie;  MCNAMEE Pauline; 

2016-01-15

PERGAMON-ELSEVIER SCIENCE LTD

JRC100141

0887-2333,   

http://www.sciencedirect.com/science/article/pii/S0887233315000223,    https://publications.jrc.ec.europa.eu/repository/handle/JRC100141,   

10.1016/j.tiv.2015.02.005,