Archives of Toxicology

, Volume 93, Issue 8, pp 2295–2305 | Cite as

Quantification of freely dissolved effect concentrations in in vitro cell-based bioassays

  • Luise HennebergerEmail author
  • Marie Mühlenbrink
  • Maria König
  • Rita Schlichting
  • Fabian C. Fischer
  • Beate I. Escher
In vitro systems


Improved understanding of chemical exposure in in vitro bioassays is required for quantitative in vitro–in vivo extrapolation (QIVIVE). In this study, we quantified freely dissolved concentrations in medium sampled from in vitro cell-based bioassays (Cfree,medium) for nine chemicals with different hydrophobicity and speciation at the time point of dosing and after an incubation period of 24 h using solid-phase microextraction. The chemicals were tested in two reporter gene assays, the AREc32 assay indicative of the oxidative stress response and the PPARγ-GeneBLAzer assay that responds to chemicals which bind to the peroxisome proliferator-activated receptor gamma. For seven of the nine chemicals, Cfree,medium did not change significantly over time in both assays and the experimentally determined Cfree,medium generally agreed well with predictions of a mass balance model that describes the partitioning between proteinaceous and lipidous medium constituents, cells and the aqueous phase. Two chemicals showed a decrease of Cfree,medium in the AREc32 assay over time that was probably caused by cellular metabolism. Furthermore, Cfree,medium of the acidic chemical diclofenac deviated from the model predictions by more than a factor of 10 at higher concentrations, which indicates nonlinear binding and saturation of the medium proteins. Bioassay results are typically reported as nominal effect concentrations (ECnom), although it is established that freely dissolved effect concentrations (ECfree) are a better measure for the bioavailable dose and the method developed here provides a simple experimental approach to measure and model ECfree in in vitro bioassay for improved QIVIVE models.


QIVIVE Protein binding Solid-phase microextraction Mass balance models 



We thank Sophia Mälzer for supporting the SPME experiments, Jenny John for cell culturing and the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (Grant No. NC/C017104/1) for funding this work (Phase 1) through the CRACK IT program (Ref CRACKITDC-P1-1).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

204_2019_2498_MOESM1_ESM.pdf (2.5 mb)
Supplementary material 1 (PDF 2557 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Cell ToxicologyHelmholtz Centre for Environmental Research (UFZ)LeipzigGermany
  2. 2.Environmental Toxicology, Center for Applied GeoscienceEberhard Karls University TübingenTübingenGermany

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