Open Access Journal of Toxicology Research Article Volume 1 Issue 2- August 2016 Open Acc J of Toxicol Copyright © All rights are reserved by Chibueze Peter Ihekwereme Development of Respiratory Mucosal Irritation and Toxicity Screening Methods: Comparison of MTT and Colorimetric Resazurin-Based Assay Chibueze Peter Ihekwereme 1,3 , Di Shao 3 , Charles Okechukwu Esimone 2 , RemigiusUchenna Agu 3 * 1 Department of Pharmacology and Toxicology, Nnamdi Azikiwe University, Nigeria 2 Department of Pharmaceutical Microbiology and Biotechnology, Nnamdi Azikiwe University, Nigeria 3 Biopharmaceutics and Drug Delivery Laboratory, Dalhousie University, Canada Submission: June 30, 2016 ; Published: August 05, 2016 *Corresponding author: RemigiusUchenna Agu, Biopharmaceutics and Drug Delivery Laboratory, College of Pharmacy, Dalhousie University, Halifax, NS B3H 3J5, Canada. Introduction There seems to be a conflict between the reported sensitivity of colorimetric resazurin-based assays (CRA) and the wide clinical use of Resazurin Microtiter Assay (REMA). REMA, a liquid culture- based CRA has been endorsed by the World Health Organization for drug susceptibility testing (DST) for Mycobacterium tuberculosis [1]. Though CRA is not popular in respiratory cell culture as fluorimetric resazurin-based assays (FRA) [2,3], its use in DST of M. tuberculosis has been compared with similar assay methods and reported to be accurate [4,5]. Furthermore, the method has been used as a reference method in the evaluation of other assay methods [5]. The inherent advantage of resazurin- based assay (RA) that cells may be re-used (since the dye is not lethal) makes the method more suitable in resource-poor settings where acquisition and sustenance of some cell types poses a challenge. There is need to assess the extent of deficiency of the method as well as determine its relative usefulness especially in resource-poor countries. Though, CRA have previously been judged less sensitive than FRA, supporting data is scarce [2,3,6]. Outcome measure of resazurin-based assay (RA) is taken either by colorimetric or fluorimetric determination [7-11]. Fluorimetric reduction assay (FRA)have earlier been compared with other assay methods [7- Open Acc J of Toxicol 1(2): OAJT.MS.ID.555557 (2016) 001 Abstract Availability of reliable and reproducible assay methods that uses simple tools will facilitate research especially in resource-poor settings. The purpose of this study is to evaluate colorimetric resazurin reduction assay (CRA) as a possible tool in respiratory mucosal cell irritation and toxicity screening. It compared CRA with another well-accepted colorimetric cell viability assay method (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolinium bromide {MTT} assay). Cell viability results of treated cells were obtained using both assay methods and used to determine Z′ factor, signal-to-background (S/B) ratio and IC 50 values. The results show all the Z ‘ values obtained were positive except values from CRA for cells treated with triton-X 100 incubated for 30min and 2h. All the Z ‘ values for MTT shows the method is optimized (0.5< Z′<1.0) while most values for CRA were outside acceptable range (Z′<0, Z′>1). MTT demonstrated large S/B ratio at both 30min and 2h for all the compounds. The range of values obtained for the S/B ratio is 12-57 for MTT and 1-3 for CRA. Furthermore, CRA failed to estimate IC 50 for some of the compounds (BKC, 2h; SDS, 2h; BKC, 30min). The values obtained for Z′ factor, S/B ratio and IC 50 for both assays were at variance. This result demonstrates that CRA performed below acceptable assay standard and therefore may not be an appropriate method for human respiratory cell viability studies. Consequently, we conclude that CRA is not useful in human respiratory mucosal irritation and toxicity studies. Keywords: Calu-3 cells; Colorimetric resazurin assay; Assay performance measures; Respiratory mucosal irritation; Toxicity screening; MTT Abbreviations: CRA: Colorimetric Resazurin Reduction Assay; DST: Drug Susceptibility Testing; REMA: Resazurin Microtiter Assay; FRA: Fluorimetric Resazurin-Based Assays; RA: Resazurin-Based Assay; APM: Assay Performance Measures; HTS: High-Throughput Screening