Quantifying processes determining the free concentration of phenanthrene in basal cytotoxicity assays
Open accessAbstract
Difficulties may arise when extrapolating in vitro derived toxicity data to in vivo toxicity data because of the high variability and occasional low sensitivity of in vitro results. Differences in the free concentration of a test compound between in vitro and in vivo systems and between different in vitro systems may in part explain this variability and sensitivity difference. The aim of this study was to determine what assay components influence the free concentration of phenanthrene in a Balb/c 3T3 and RTgill-W1 MTT assay. Partition coefficients of phenanthrene to serum, well plate plastic, cells, and headspace were measured and subsequently used to model the free concentration of the compound in vitro. The estimated free concentration was compared to the free concentration measured in the assays using solid phase microextraction (SPME). Results indicate that the free concentration of phenanthrene, a relatively volatile and hydrophobic compound, is significantly reduced in a typical in vitro setup as it binds to matrices such as serum protein and well plate plastic. A reduction in free concentration due to increasing serum protein levels is accompanied by an increase in the median effect concentration (EC(50)) and can be modeled, with the exception of evaporation, using the partition coefficients of the compound to assay components.
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In vitro assays often test nominal concentrations that may not reflect freely available, intracellular or in vivo-relevant concentrations.
“In vitro assays often test nominal concentrations that may not reflect freely available, intracellular or in vivo-relevant concentrations (Dimitrijevic et al., 2022; Kramer et al., 2012).”
From Cytotoxicity across OECD methods is not a single endpoint: context-dependent roles, variability and implications for decision-making (2026) · cites this paper as Kramer et al., 2012
The indexed Abstract (no page given) reports that phenanthrene’s free concentration is reduced in a typical in vitro setup by binding to serum protein and well-plate plastic, and that free concentrations can differ between in vitro and in vivo systems. This supports the concern about nominal versus freely available and in vivo-relevant concentrations, but the passage does not establish a mismatch with intracellular concentrations. (LLM, conf=0.82)
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Coecke, S., Balls, M., Bowe, G., Davis, J., Gstraunthaler, G., Hartung, T., Hay, R., Merten, O.-W., Price, A., Schechtman, L., et al. (2005) · Alternatives to Laboratory Animals · doi:10.1177/026119290503300313 · Cited by 371 · see citing works
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Guidance Document on Good In Vitro Method Practices (GIVIMP)
OECD (2018) · OECD Series on Testing and Assessment · doi:10.1787/9789264304796-en · Cited by 161 · see citing works
Brinkmann, M., Preuss, T. G., & Hollert, H. (2016) · Advances in Biochemical Engineering/Biotechnology · doi:10.1007/10_2015_5015 · Cited by 15 · see citing works
Thoma, T., Löhr, L., Demuth, P., Hambruch, N., Oesch, F., & Landsiedel, R. (2026) · NAM Journal · doi:10.1016/j.namjnl.2026.100126
- Authors
- Kramer, N. I., Krismartina, M., Rico-Rico, Á., Blaauboer, B. J., & Hermens, J. L. M.
- Affiliations
- Utrecht University (NL)
- Corresponding author
- Kramer, Nynke I.
- Venue
- Chemical Research in Toxicology
- Publisher
- American Chemical Society (ACS)
- Published
- 2012-02-08
- Volume
- 25
- Issue
- 2
- Pages
- 436-445
- ISSN
- 0893-228X (ISSN-L)
- 1520-5010
- OpenAlex
- W2162097198
- Cited by
- 156
- References
- 1
- Citations by year
- 2026: 13 · 2025: 10 · 2024: 6 · 2023: 7 · 2022: 8 · 2021: 8 · 2020: 14 · 2019: 13 · 2018: 26 · 2017: 9 · 2016: 9 · 2015: 12 · 2014: 9 · 2013: 11 · 2012: 1
- FWCI
- 6.88
- Type
- journal article
- Language
- en
- Open access
- green
- hdl.handle.net
- Licence
- cc-by-nc-sa
- Locations
- Chemical Research in Toxicology (publishedVersion)
- PubMed (publishedVersion)
- DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion)
- LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, cc-by-nc-sa)
- Utrecht University Repository (Utrecht University) (submittedVersion)
- Topics
- Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Environmental Toxicology and Ecotoxicology
- Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
- Keywords
- phenanthrene, partition coefficient, plasma protein binding, headspace, cytotoxicity assay, in vitro-in vivo extrapolation, solid-phase microextraction, EC50, hydrophobic compounds, evaporation
- MeSH terms
- Adsorption, Animals, Cell Line, Cell Survival, Culture Media, Fishes, Models, Theoretical, Phenanthrenes, Plastics, Toxicity Tests, NIH 3T3 Cells, Mice
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