# On the Utilization and Characterization of External Biotransformation Systems in In Vitro Toxicology: A Critical Review of the Scientific Literature with Guidance Recommendations

## TL;DR

This paper reviews the use of external biotransformation systems in in vitro toxicology, highlighting methodological issues and offering guidance to improve reliability and reproducibility.

## Contribution

The paper provides a critical evaluation of biotransformation systems' methodological rigor and proposes a regulatory guidance framework to enhance their use in toxicology.

## Key findings

- Biotransformation systems lack reproducible standards and show no significant correlations in concentration–response relationships.
- Methodological reporting in the literature is inconsistent, affecting the reliability of biotransformation system applications.
- Recommendations are proposed to improve research practices and regulatory integration of biotransformation systems.

## Abstract

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.