USE OF HPLC/UPLC INSTEAD OF PHOTOMETRY FOR EVALUATION OF MTT IN IN VITRO IRRITATION ASSAYS BASED ON HUMAN RECONSTRUCTED TISSUE FOR ASSESSMENT OF COLOURED TEST MATERIALS.

This study by researchers at several Procter & Gamble research facilities in the European Union utilized MatTek’s EpiOcular in vitro 3-D human corneal tissue equivalent to demonstrate that the use of HPLC/UPLC technology can extend the applicability of the MTT reduction assay to test materials that are intrinsically coloured and that may interfere with evaluation of the MTT assay using standard photometry. In vitro assays using Human Reconstructed Tissue (HRT; epithelium/epidermis) models for the detection of irritants versus non-irritants represent way forward for alternatives to in vivo irritation tests. In these assays, the irritation potential of a test material is typically determined by measuring cell viability in the treated tissues by means of the colorimetric MTT reduction assay after topical application onto the tissue surface. Cell viability is determined by evaluating enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to blue formazan salt. Reduced MTT is quantified photometrically with the results expressed as % viability in the test material treated tissues relative to the negative control. A decrease in MTT reduction capacity is used as the indicator of potential irritancy according to a prediction model that identifies irritants versus non-irritants based on a cut-off percentage viability in the MTT assay. A known limitation of the MTT test is possible interference with the absorbance measurement of reduced MTT (formazan) for test chemicals that are intrinsically coloured. The aims of the present work were to: 1) develop an analytical approach using High Performance Liquid Chromatography/Ultra Performance Liquid Chromatography (HPLC/UPLC) to detect sepa-rately reduced MTT (formazan) from intrinsically coloured test material; 2) demonstrate, as a proof of principle, that for non-coloured chemicals and for dyes that do not exhibit colour interference the percentage viability results obtained using HPLC/UPLC were comparable with those obtained via standard photometry and 3) apply the HPLC/UPLC method only to evaluation of intrinsically coloured dyes that exhibit colour interference. For the purpose of this work the MatTek EpiOcular™ model was used as an in vitro eye irritation HRT assay (Kaluzhny et al., 2008). Tissues were treated with test material as well as with negative and positive controls. Following this, the tissues were stained with MTT and formazan was then extracted in isopropanol. The extract solutions were used for photometry measurements (standard methodology for analysis of formazan) and then used for analysis by HPLC/UPLC. A selection of chemicals/dyes without intrinsic colour interference as well as 2 dyes identified to interfere with MTT due to their intrinsic colour were tested in this programme. Results from this preliminary programme support the following conclusions: 1) early development of an analytical approach using HPLC/UPLC to detect separately formazan from intrinsically coloured test material has occurred; 2) proof of principle that for non-coloured chemicals and dyes that do not exhibit colour interference the % viability results obtained using HPLC/UPLC are comparable with those obtained using standard photometry and 3) HPLC/UPLC analysis can determine the level of formazan produced in in vitro HRT (epithelium/epidermis) assays for evaluation of intrinsically coloured dyes that exhibit colour interference in situations where analysis by standard photometry is not possible. Therefore, this use of HPLC/UPLC to determine formazan is shown to extend the applicability of such assays to test materials that are intrinsically coloured and that may interfere with evaluation of the MTT assay using standard photometry.

Color interference, Colored test material, Direct dyes, Dyes, EpiOcular, EpiOcular reach protocol, HPLC, High Performance Liquid Chromatography, Killed tissue, MTT assay, Oxidative dyes, UPLC, Ultra Performance Liquid Chromatography

1-Hydroxyethyl-4.5-diminopyrazole, 1-Hydroxyethyl-4.5-diminopyrazole at 2%, Acetone, Aluminum hydroxide, Basic violet 2, Basic violet 2 at 1%, Benzalkonium chloride, Cyclohexanol, Disperse black No. 9, Disperse black No. 9 at 1%, Glycerol, HC blue No 15, Hydroxyethyl-2-nitro-p-toluidine, Hydroxyethyl-2-nitro-p-toluidine at 1.5%, Hydroxyethyl-p-phenylenediamine sulfate, Iminodibenzene, Methy acetate, Methylcydopentane, Quinacrine, Tetraoctylammonium bromide, Tween-20