On the utilisation and characterisation of external biotransformation systems in in vitro toxicology: a critical review of the scientific literature with guidance recommendations

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 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 fifty 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 rigour, 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 endpoints. Through systematic searches, screening, and eligibility criteria, we identified 229 bibliographic records. Data parameterisation and extraction were conducted across 24 domains of BTS relevance and reliability. Methodological reporting rigour was assessed via scoring (reported vs. non-reported data items) and revealed a lack of reproducible standards. Numerical measures associated with principal BTS reaction components were subjected to meta-regression analyses. 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 robustness and deficiencies. In conclusion, these results emphasise 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/677684v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1a6dd9aorg.highwire.dtl.DTLVardef@1ecb22org.highwire.dtl.DTLVardef@1aa3ee3org.highwire.dtl.DTLVardef@6aaf6_HPS_FORMAT_FIGEXP M_FIG C_FIG