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.
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/UPLC-spectrophotometry 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.
authors.affiliation: L'Oréal Research & Innovation, Aulnay Sous Bois, France.
authors.forename: N
authors.initials: N
authors.lastname: Alépée
authors.affiliation: Cosmetics Europe, The Personal Care Association, Brussels, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Barroso
authors.affiliation: Janssen Research & Development, Beerse, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: De Smedt
authors.affiliation: Henkel AG & Co. KGaA, Düsseldorf, Germany.
authors.forename: B
authors.initials: B
authors.lastname: De Wever
authors.affiliation: Chanel Parfums Beauté, Neuilly sur Seine, France.
authors.forename: J
authors.initials: J
authors.lastname: Hibatallah
authors.affiliation: Cosmetics Europe, The Personal Care Association, Brussels, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Klaric
authors.affiliation: Henkel AG & Co. KGaA, Düsseldorf, Germany.
authors.forename: K R
authors.initials: KR
authors.lastname: Mewes
authors.affiliation: Laboratoire Pierre Fabre, Castres, France.
authors.forename: M
authors.initials: M
authors.lastname: Millet
authors.affiliation: Beiersdorf AG, Hamburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Pfannenbecker
authors.affiliation: LVMH Recherche, St Jean De Braye Cedex, France.
authors.forename: M
authors.initials: M
authors.lastname: Tailhardat
authors.affiliation: Laboratoire Pierre Fabre, Castres, France.
authors.forename: M
authors.initials: M
authors.lastname: Templier
authors.affiliation: The Procter & Gamble Company, Egham, Surrey, United Kingdom. Electronic address: mcnamee.pm@pg.com.
authors.forename: P
authors.initials: P
authors.lastname: McNamee
mesh_terms: D000826:Animal Testing Alternatives; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000633:toxicity*; D003358:Cosmetics / Q000633:toxicity; D005128:Eye Diseases / Q000139:chemically induced; D005562:Formazans / Q000633:toxicity*; D006801:Humans; D007509:Irritants / Q000633:toxicity; D015203:Reproducibility of Results; D012871:Skin Diseases / Q000139:chemically induced / Q000473:pathology; D023422:Skin Irritancy Tests / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013778:Tetrazolium Salts / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D003358:Cosmetics; D005562:Formazans; D007509:Irritants; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue
keywords: Eye irritation; HPLC/UPLC-spectrophotometry; In vitro RhT test methods; Skin corrosion; Skin irritation