Title: 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
Authors: ALEPEE NathalieVIEGAS BARROSO JOAO FILIPEDE SMEDT AnnDE WEVER BartHIBATALLAH JalilaKLARIC MartinaMEWES Karsten R.MILLET MarionPFANNENBECKER UweTAILHARDAT MagalieTEMPLIER MarieMCNAMEE Pauline
Citation: TOXICOLOGY IN VITRO vol. 29 no. 4 p. 741-761
Publisher: PERGAMON-ELSEVIER SCIENCE LTD
Publication Year: 2015
JRC N°: JRC100141
ISSN: 0887-2333
URI: http://www.sciencedirect.com/science/article/pii/S0887233315000223
http://publications.jrc.ec.europa.eu/repository/handle/JRC100141
DOI: 10.1016/j.tiv.2015.02.005
Type: Articles in periodicals and books
Abstract: 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.
JRC Directorate:Institute for Health and Consumer Protection Historical Collection

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