There is increasing demand from in vitro method developers, validation bodies, in vitro method end-users as well as receiving authorities and OECD to integrate kinetics information in toxicity testing in order to improve chemical risk assessment. A strategic way to do this is detailed in the "EURL ECVAM strategy for achieving 3Rs impact in the assessment of toxicokinetics and systemic toxicity" (Bessems J et al. 2015). One of the strategic aims of this report urges for the development and applied use of in vitro kinetic methods for absorption, distribution, metabolism and excretion to be used in the future risk assessment approaches using new advanced methodologies (NAMs).
Information on metabolism has been identified as a critical piece of information in integrated test strategies based on non-animal methods. Therefore, the purpose of this report is to describe the preliminary experimental work towards a representative in vitro human hepatic metabolic clearance method that can be ultimately used in future defined approaches for toxicological risk assessment.
The proposed method is a result of the efforts of the EURL ECVAM laboratory team combined with knowledge gathered during (1) a literature search on in vitro human hepatic metabolic clearance methods, (2) an EURL ECVAM call for procedures detailing in vitro human hepatic metabolic clearance methods and (3) an expert meeting to establish the elements critical for in vitro methods that predict human hepatic metabolic clearance. The representative in vitro human hepatic metabolic clearance method was used to assess the in vitro intrinsic clearance of the following three chemicals: Acetaminophen, Diclofenac and Verapamil. Each chemical was added at one concentration to cryopreserved primary human hepatocytes (pooled from 10 donors) and the cells were exposed at different incubation time-points up to 2 h. The concentrations of each chemical over-time were measured by using Ultra-Performance Liquid-Chromatography (UPLC) coupled with (Quadrupole Time-of-Flight) Q-Tof mass spectrometer.
The results obtained show good within-laboratory reproducibility (e.g. between different runs on different days), of the calculated in vitro intrinsic clearance.
The results and experience gained with this experimental in-house effort by Directorate F – Health, Consumers and Reference Materials, F.3 Chemicals Safety and Alternative Methods will support the definition of an EURL ECVAM recommendation on human hepatic metabolic clearance and the subsequent drafting of a proposal for an OECD Guidance Document with the objective to characterise and describe in vitro hepatic metabolic clearance methods in order to facilitate their regulatory uptake and use to support chemical risk assessment.