Cefic-lri Programme | European Chemical Industry Council

ECO36: Paving the way for QIVIVE – from nominal to free to cellular concentrations in in vitro assays

Principal Investigator

Prof. Beate Escher
Helmholtz Centre for Environmental Research UFZ
UFZ Department Cell Toxicology
Permoserstraße 15
04318 Leipzig
Tel: +49 341 235 1244


Prof. Philipp Mayer, Department of Environmental Engineering Technical University of Denmark (DTU), philm@env.dtu.dk
Dr. Nynke Kramer, Institute for Risk Assessment Sciences, Utrecht University, n.i.kramer@uu.nl
Fabian Fisher, PhD candidate, Helmholtz Centre for Environmental Research UFZ, Leipzig, Germany, fabian.fischer@ufz.de
Dr. Luise Hennerberger, PDF, Helmholtz Centre for Environmental Research UFZ, Leipzig, Germany, luise.henneberger@ufz.de


A very important limitation of most in vitro toxicity tests and in vitro toxicity data is the lack of measured exposure levels. Unfortunately, the direct analytical determination of exposure in high-throughput in vitro assays is generally difficult due to the combination of rich media and small well volumes. The rich medium leads to substantial binding of many chemicals, which in turn leads to low freely dissolved fractions of chemicals and may lead to unrealistically high effect concentrations (Groothuis et al., 2015). This asks for analytical techniques that can measure freely dissolved concentrations (Cfree) rather than the traditionally used nominal or total concentrations (Cnominal or Ctotal). Solid Phase Microextraction (SPME) is the most appropriate method for this purpose (Smith and Schäfer, 2016), and has been widely applied in simple systems using vials or 24 well plates (Heringa et al., 2003; Kramer et al., 2012). However, this technique is very difficult to apply correctly within the smaller volume of multiwell plates and for complex, dynamic assays with multiple cell types and co-cultures. While mass balance models have been very beneficial for studying and predicting the fate and exposure of test compounds within in vitro assays (Armitage et al., 2014), it remains imperative to develop and optimize methodology to actually measure this exposure and validate the prediction models and assess the applicability domain of such models. Ultimately, data from in vitro assays can only be used for quantitative in vitro to in vivo extrapolation (QIVIVE) if the freely dissolved and cellular concentrations (Ccell) are known.


The objective of this project is to bundle existing expertise to progress exposure assessment in in vitro bioassays used for high-throughput screening (HTS) in 384- and 1536-well plates and complex in vitro bioassays based on transwell and 3D cultures. Since direct assessment of Cfree by SPME is not feasible in 1536-well plates (typical volume of medium 6 μL) or only possible for chemicals with favourable physicochemical properties in 384-well plates (typical volume of medium 40 μL), we propose a pragmatic approach to characterise the fate of chemicals in the bioassay systems by a combination of measurement of Cfree and binding to system components in larger-volume systems (100 to 1000 μL) and modelling followed by the development of a routine experimental approach that can be applied for HTS on robotic systems. The common denominator of exposure assessment will be SPME methods based on different types of polymers for neutral and ionisable chemicals. These SPME methods will be applied to determine free concentrations (or free fractions) in the assay medium as well as fate processes like evaporation, binding to the plastic of the well plates, degradation, and binding to medium constituents and cells, in which proteins and lipids are the dominant binding phases.

The short-term expected outcomes are experimentally validated models to predict the freely dissolved and cellular effect concentrations ECfree and ECcell for existing in vitro toxicity data, including ToxCast and published data that were based on nominal effect concentrations only, which will then make them amenable for QIVIVE. The long-term benefit of the project will be routine analytical tools that will improve exposure assessment in HTS tests in the future and could revolutionise the application of HTS in risk assessment.

Click here to read the project summary.

Related Publications


Susana Proença, Beate I. Escher, Fabian C. Fischer, Ciar´an Fisher, S´ebastien Gr´egoire, Nicky J. Hewitt, Beate Nicol, Alicia Paini, Nynke I. Kramer. Effective exposure of chemicals in in vitro cell systems: A review of chemical distribution models. ScienceDirect, March 2021.

Fischer FC, Henneberger L, König M, Bittermann K, Linden L, Goss KU, Escher BI. Modeling Exposure in the Tox21 in Vitro Bioassays. Chem Res Toxicol. 2017 May 15;30(5):1197-1208.

Fischer FC, Abele C, Droge STJ, Henneberger L, König M, Schlichting R, Scholz S, Escher BI. Cellular Uptake Kinetics of Neutral and Charged Chemicals in in Vitro Assays Measured by Fluorescence MicroscopyChem Res Toxicol. 2018 Aug 20;31(8):646-657.

Fischer FC, Cirpka OA, Goss KU, Henneberger L, Escher BI. Application of Experimental Polystyrene Partition Constants and Diffusion Coefficients to Predict the Sorption of Neutral Organic Chemicals to Multiwell Plates in in Vivo and in Vitro Bioassays. Environ Sci Technol. 2018 Nov 20;52(22):13511-13522.

Henneberger L, Mühlenbrink M, Fischer FC, Escher BI. C18-Coated Solid-Phase Microextraction Fibers for the Quantification of Partitioning of Organic Acids to Proteins, Lipids, and Cells. Chem Res Toxicol. 2019 Jan 22;32(1):168-178.

Escher BI, Glauch L, König M, Mayer P, Schlichting R. Baseline Toxicity and Volatility Cutoff in Reporter Gene Assays Used for High-Throughput Screening. Chem Res Toxicol. 2019 Aug 19;32(8):1646-1655.

Fischer FC, Henneberger L, Schlichting R, Escher BI. How To Improve the Dosing of Chemicals in High-Throughput in Vitro Mammalian Cell Assays. Chem Res Toxicol. 2019 Aug 19;32(8):1462-1468.

Birch H, Kramer NI, Mayer P. Time-Resolved Freely Dissolved Concentrations of Semivolatile and Hydrophobic Test Chemicals in In Vitro Assays-Measuring High Losses and Crossover by Headspace Solid-Phase Microextraction. Chem Res Toxicol. 2019 Sep 16;32(9):1780-1790.

Henneberger L, Mühlenbrink M, Heinrich DJ, Teixeira A, Nicol B, Escher BI. Experimental Validation of Mass Balance Models for in Vitro Cell-Based Bioassays. Environ Sci Technol. 2020 Jan 21;54(2):1120-1127.


Heidi Birch, Nynke Kramer and Philipp Mayer. High Losses and Crossover of Semi-Volatile and Hydrophobic Test Chemicals in In Vitro Assays Conducted in 96 Well Plates. SETAC North America 40th Annual Meeting, November 2019, Toronto, ON, CA.

Timeline: January 2017 > December 2019

LRI funding: € 500 000

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