bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.09.23.677684

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

Lungu-Mitea, S.·Stein Aslund, M.·Reichstein, I.·Pinto-Vidal, F. A.·Schiwy, A.·Hollert, H.·Jacobs, M.·Hilscherova, K.
View original source ↗Export metadata ↓

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 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

Explore related subjects

pharmacology and toxicologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Lungu-Mitea, S., Stein Aslund, M., Reichstein, I., Pinto-Vidal, F. A., Schiwy, A., Hollert, H., Jacobs, M., Hilscherova, K.. 2025-09-25. On the utilisation and characterisation of external biotransformation systems in in vitro toxicology: a critical review of the scientific literature with guidance recommendations. https://doi.org/10.1101/2025.09.23.677684

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Protective effects of heat shock protein 70 induction against global warming by using oriental bezoar and ginseng

The interest in compounds that protect against heat stress-induced damage has been heightened due to world global warming. We found the protective effects of a Japanese natural drug named BG, containing oriental bezoar and ginseng, against heat stress in Drosophila. BG suppressed the heat-induced shortened lifespan and reduced fertility in Drosophila. Interestingly, the protective effects of BG against heat stress were abolished in heat shock protein 70 (HSP70) mutant flies. To see the protective effects in humans, we applied BG on the cytotoxicity in heat-stressed human hepatic cell line, HepG2. BG suppressed heat stress-induced cytotoxicity at 43{degrees}C, and increased HSP70 and heat shock factor 1 (HSF1) mRNA expression in HepG2 cells. These findings indicate that BG protects against heat stress-induced damage via the HSF1/HSP70 pathway and has potential as a therapeutic agent for heat stress-induced disorders, including heatstroke even in human.

pharmacology and toxicology↗
bioRxiv2026

Replacing In Vivo Experiments for PK/PD Target Determination Through In Vitro Time-Kill Experiments and PK/PD Modelling Incorporating Inter-strain Variability: Application to Meropenem Against Pseudomonas aeruginosa

Background. Optimal antibiotic dosing regimens depend on the pharmacokinetic/pharmacodynamic (PK/PD) index that best predicts antibacterial efficacy. PK/PD targets are traditionally determined using murine infection models based on a limited number of bacterial isolates. Objective. This study aimed to investigate whether animal experiments could be replaced by in vitro time-kill experiments performed on a large collection of clinical isolates and analyzed using a modelling approach accounting for inter-strain variability. The proposed framework was evaluated using meropenem against Pseudomonas aeruginosa. Materials and Methods. In vitro time-kill experiments were performed on 66 clinical isolates of P. aeruginosa. A population pharmacodynamic model was developed from experimental data. A murine pharmacokinetic model was reproduced from literature and combined with the pharmacodynamic model to simulate in vivo bacterial burden over time. The relationships between simulated bacterial counts at 24 h and the three main PK/PD indices (fCmax/MIC, fAUC/MIC and %fT>MIC) were characterized using nonlinear mixed-effects Imax models. Results. The PK/PD index showing the strongest correlation with meropenem efficacy at 24 h was %fT>MIC (R2 = 0.989), compared with fAUC/MIC (R2 = 0.373) and fCmax/MIC (R2 = 0.284). These findings are consistent with previous studies using murine thigh infection models. The %fT>MIC target required to achieve a 2-log CFU reduction was estimated at 44%, with substantial inter-strain variability (10th and 90th percentiles: 27% and 71%, respectively). Conclusions. Using meropenem against P. aeruginosa as a proof of concept, we demonstrate that in vitro time-kill experiments combined with pharmacometric modelling can identify the same PK/PD efficacy targets as animal infection models. Moreover, performing experiments on a large panel of clinical isolates enables the quantification of inter-strain variability in PK/PD targets, providing information that may improve their translation to clinical dosing optimization.

pharmacology and toxicology↗
bioRxiv2026

Physicochemical compatibility and stability of urapidil-propofol admixtures during simulated Y-site Administration

Background/Objectives: Urapidil with propofol is clinically efficient against elevated blood pressure during sedation. However, their physicochemical compatibility and emulsion stability upon continuous infusion remain unclear. This study aimed to evaluate different mixing ratios and diluents, thereby proving the safety limits for their co-administration. Methods: Urapidil solutions prepared with either sodium chloride (NS) or glucose injection (GS), and emulsified with propofol at different ratios (v/v), were stored for 12 h. Physical compatibility was assessed by visual inspection, pH, osmolality, mean droplet diameter (MDD), polydispersity index (PDI), zeta potential, and percentage of fat globules larger than 5 m (PFAT5). Chemical stability was quantified using high-performance liquid chromatography. Results: pH and osmolality stabilized. Urapidil hydrochloride and propofol contents remained pure at > 95%, MDD was < 500 nm, and PDI was < 0.2. Urapidil proportion in NS was significantly negatively correlated with the zeta potential. PFAT5 was > 0.05% after 2-8 h. In contrast, in GS at a 1:2 ratio, PFAT5 remained < 0.05%, which increased slightly in the 1:1 group at 8 h. PFAT5 stabilized in the high-propofol group (10:1) under all conditions. Conclusions: The chemical compatibility of the admixture was acceptable after 12 h of storage. However, physical compatibility was influenced by the mixing ratio, diluent type, and storage time. For clinical Y-site co-administration, a 10:1 mixing ratio or dilution in 5% GS is recommended. Enhanced proportions must be mixed with NS, while continuous infusion time must be < 2 h to mitigate fat embolism risk.

pharmacology and toxicology↗