Direct Chemical Interference with the MTT viability assay: Identification and mitigation strategies in New Approach Methodologies (NAMs)

The reduction of the vital dye 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) is commonly used to assess the cellular viability and proliferation and thus to evaluate the cytotoxicity of discrete chemicals, complex mixtures, new drug candidates or finished cosmetic/personal care products. The MTT assay plays a central role in New Approach Methodologies (NAMs) due to its relatively simple design, short execution time and high throughput versatility. However, certain chemicals can directly interfere with the colorimetric MTT endpoint in the absence of a metabolically active biological system, potentially affecting data interpretation. In this manuscript, we identified and analyzed peer-reviewed manuscripts reporting chemical classes or product categories with direct MTT interference effects and compiled them into an accessible reference list. We further discuss the mitigation strategies and alternative assays evaluated and/or incorporated into the validated NAM-based guidelines to improve the reliability of the data analysis and interpretation. As a case study, we discuss about an established mitigation strategy incorporated in validated NAMs that address ocular and dermal safety endpoints and based on human reconstructed tissue models. This manuscript provides a practical collection of information to support study design, identification of potential MTT-interfering compounds, and selection of appropriate mitigation strategies when MTT is used as a viability endpoint in NAM-based safety assessments.