Relating In Vitro to In Vivo: The Importance of Bioavailability

Joop LM Hermens1, Nynke I Kramer1, Todd Gouin2

1. Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands2. SEAC, Unilever, Colworth Science Park, Sharnbrook, Bedfordshire, United Kingdom

E-mail contact: todd.gouin@unilever.com

Background and SummaryIn vitro assays are often applied for unravelling toxicological mechanisms of action. To extendthe use of these assays to hazard characterization and perform quantitative in vitro to in vivoextrapolations (QIVIVE), the dose in the in vitro test should be linked to a dose in a wholeanimal study [1, 2]. Nominal concentrations from in vitro tests may be appropriate for “simplecompounds”, but for more hydrophobic or volatile chemicals. Information about the dose atthe target is needed because this concentrations may decrease significantly during the courseof thee assay (Fig. 1) [3-6]. The freely dissolved concentration is one example of a dose metricthat better link in vitro (cell medium) to in vivo (blood) [3, 4].

References:1. Louisse, J.; de Jong, E.; van de Sandt, J. J. M.; Blaauboer, B. J.; Woutersen, R. A.; Piersma, A. H.; Rietjens, I.; Verwei, M., The use of In vitro toxicity data and physiologically based kinetic modeling to predict dose-response curves for in vivo developmental toxicity of glycol ethers in rat and man. Toxicol. Sci. 2010, 118, (2), 470-484.2. Wetmore, B. A.; Wambaugh, J. F.; Ferguson, S. S.; Sochaski, M. A.; Rotroff, D. M.; Freeman, K.; Clewell, H. J.; Dix, D. J.; Andersen, M. E.; Houck, K. A.; Allen, B.; Judson, R. S.; Singh, R.; Kavlock, R. J.; Richard, A. M.; Thomas, R. S., Integration of Dosimetry, Exposure, and High-Throughput Screening Data in Chemical Toxicity Assessment. Toxicol. Sci. 2012, 125, (1), 157-174.3. Gulden, M.; Seibert, H., Influence of protein binding and lipophilicity on the distribution of chemical compounds in in vitro systems. Toxicol. in Vitro 1997, 11, (5), 479-83.4. Heringa, M. B.; Schreurs, R. H. M. M.; Busser, F.; van der Saag, P.; van der Burg, B.; Hermens, J. L. M., Toward more useful in vitro toxicity data with measured free concentrations. Environ. Sci. Technol. 2004, 38, 6263-6270.5. Tanneberger, K.; Rico-Rico, A.; Kramer, N. I.; Busser, F. J. M.; Hermens, J. L. M.; Schirmer, K., Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays. Environ. Sci. Technol. 2010, 44, (12), 4775-4781.6. Groothuis, F. A.; Heringa, M. B.; Nicol, B.; Hermens, J. L. M.; Blaauboer, B. J.; Kramer, N. I., Dose metric considerations in in vitro assays to improve quantitative in vitro-in vivo dose extrapolations. Toxicology 2015, 332, 30-40.7. Kramer, N. I.; Krismartina, M.; Rico-Rico, A.; Blaauboer, B. J.; Hermens, J. L. M., Quantifying processes determining the free concentration of phenanthrene in basal cytotoxicity assays. Chem. Res. Toxicol. 2012, 25, (2), 436-445.8. Mayer, P.; Holmstrup, M., Passive dosing of soil invertebrates with polycyclic aromatic hydrocarbons: Limited chemical activity explains toxicity cutoff. Environ. Sci. Technol. 2008, 42, (19), 7516-7521.9. Kramer, N. I.; Busser, F. J. M.; Oosterwijk, M. T. T.; Schirmer, K.; Escher, B. I.; Hermens, J. L. M., Development of a partition-controlled dosing system for cell assays. Chem. Res. Toxicol. 2010, 23, (11), 1806-1814.10. Armitage, J. M.; Wania, F.; Arnot, J. A., Application of mass balance models and the chemical activity concept to facilitate the use of in vitro toxicity data for risk assessment. Environ. Sci. Technol. 2014, 48, (16), 9770-9779.11. Chen, Y.; Geurts, M.; Sjollema, S. B.; Kramer, N. I.; Hermens, J. L. M.; Droge, S. T. J., Acute toxicity of the cationic surfactant C12-benzalkonium in different bioassays: How test design affects bioavailability and effect concentrations. Environ. Toxicol. Chem. 2014, 33, (3), 606-615.12. Droge, S. T. J.; Hermens, J. L. M.; Rabone, J.; Gutsell, S.; Hodges, G., Phospholipophilicity of CxHyN+ amines: chromatographic descriptors and molecular simulations for understanding partitioning into membranes. Environmental Science-Processes & Impacts 2016, 18, (8), 1011-1023.13. Peltenburg, H.; Bosman, I. J.; Hermens, J. L. M., Sensitive determination of plasma protein binding of cationic drugs using mixed-mode solid-phase microextraction. J. Pharm. Biomed. Anal. 2015, 115, 534-542.14. Henneberger, L.; Goss, K. U.; Endo, S., Partitioning of Organic Ions to Muscle Protein: Experimental Data, Modeling, and Implications for in Vivo Distribution of Organic Ions. Environ. Sci. Technol. 2016, 50, (13), 7029-7036.15. Stadnicka-Michalak, J.; Tanneberger, K.; Schirmer, K.; Ashauer, R., Measured and Modeled Toxicokinetics in Cultured Fish Cells and Application to In Vitro - In Vivo Toxicity Extrapolation. Plos One 2014, 9, (3).16. Jager, T.; Albert, C.; Preuss, T. G.; Ashauer, R., General Unified Threshold Model of Survival - a toxicokinetic-toxicodynamic framework for ecotoxicology. Environ. Sci. Technol. 2011, 45, (7), 2529-2540.

AcknowledgementThe work presented here was performed in several projects that were funded by EU FP6/7 (Acutetox, Predict-IV) and by CEFIC (CellSens).

There are several options to improve characterization of the dose in in vitro assays, including:• Measurement of the total and freely dissolved concentration in medium [4, 7]• Controlling the free concentration in an in vitro assay [8, 9]• Exposure modelling [10]

Here we present a few examples form earlier work at Utrecht University to show the relevance of this topic and examples of some of thesolutions. Moreover, recommendations for future research are identified.

Example 1: Total versus free concentration in an in vitro test with increasing serum levels in the medium - Heringa et al. [4]

• C free is a more suitable dose parameter• C free is independent of assay setup and more closely related to the

“intrinsic dose”

Example 2: Controlling the dose in an in vitro assay via passive dosing – Kramer et al. [9]

• concentration in medium is constant during the entire exposure period

• with solvent spiking, the concentration decreased by <95% of the initial concentration (see also Tanneberger et al. [5])

The concept of free concentration in in vitro-in vivo extrapolations to meet 3R principles in toxicity testing:

Challenges

Experimental• expand applicability domain: ionic and very hydrophobic

compounds [11-14]• bring in time as a variable [15]• include dynamics (TK-TD modelling) [16].

Modeling• refine modeling (concentration, non-equilibrium)• more complex compoundsAlso see CEFIC LRI project ECO36

• the scheme includes many detailed aspects• need for quantifying individual processes• link with AOP (adverse outcome pathways)

Fig. 1

test chemicals: benzo(a)pyrene, 1,2-dichlorobenzene and 1,2,3-trichlorobenzene in vitro assays: EROD (CYP1A induction) and basal cytotoxicity (CFDA-AM) assays RTL-W1 and RTgill-W1 cells

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Utrecht University