| Description |
Incorporating biotransformation capabilities into in vitro assays represents one of the most critical challenges in toxicology, facilitating the transition from in vivo models to integrated in vitro strategies. Although emerging technologies show promise, their current limitations in scalability hinder their high-throughput applications. In the short to mid term, externally added biotransformation systems (“BTS”: S9 and microsomal liver fractions) used together with in vitro assays offer viable alternatives. However, despite over 50 years of use, BTS are marred by reproducibility issues, raising concerns about their reliability and raising the question: Are BTS inherently unreliable, or has their reputation been flawed by methodological oversights? This review critically evaluates BTS’ methodological rigor, applying a deep statistical analysis of the scientific literature. We employed Boolean operator searches across scientific literature repositories to curate a database on BTS research in conjunction with relevant in vitro assays, focusing on endocrine disruption, mutagenicity, and genotoxicity end points. Through systematic searches, screening, and eligibility criteria, we identified 229 bibliographic records. Data parametrization and extraction were conducted across 24 domains of BTS relevance and reliability. Methodological reporting rigor was assessed via scoring (reported vs nonreported data items) and revealed a lack of reproducible standards. Numerical measures associated with principal BTS reaction components were subjected to meta-regression analyses. Within the aggregated data set, no statistically significant correlations were found for BTS and related cofactor concentration–response relationships or time-related elements. Finally, descriptive statistics, multiple correspondence analysis, and Apriori algorithm-based relational networks identified qualitative patterns of methodological reporting robustness and deficiencies. In conclusion, these results emphasize shortcomings across the scientific literature in complying with appropriate methodological reporting. We offer evidence-based recommendations, in the form of a conceptual regulatory guidance framework, to enhance research practices, quality, and reproducibility of BTS applications, designed to strengthen the robustness of BTS research and its integration into regulatory-relevant hazard and risk assessment of chemicals.
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