Exposure assessment in in vitro cell-based bioassays is challenging for ionizable organic chemicals (IOCs), because they are present as more than one chemical species in the bioassay medium. Furthermore, compared to neutral organic chemicals, their binding to medium proteins and lipids is driven by more complex molecular interactions. Total medium concentrations (C total,medium ) and/or freely dissolved medium concentrations (C free,medium ) were determined for one neutral chemical and 14 IOCs (acids, bases, multifunctional) at concentrations relevant for determination of cytotoxicity and effect. C free,medium was measured in two in vitro bioassays at the time of dosing and after 24 h of incubation using solid-phase microextraction. C free,medium was maximally 1.7 times lower than the nominal concentrations (C nom ) for the hydrophilic chemicals (caffeine and lamotrigine). For the organic acids (naproxen, ibuprofen, warfarin, and diclofenac), C free,medium was by a factor of 4 lower than C nom at high concentrations, but the ratio was much higher at low concentrations, indicating a nonlinear binding behavior. The experimental C free,medium was also compared with C free,medium predicted with a mass balance model accounting for binding to medium proteins and lipids. The mass balance model performed well for five of the test chemicals (within a factor of 10), but it underestimated C free,medium by up to a factor of 1200 for chemicals that showed nonlinear binding to medium components. These findings emphasize that experimental exposure assessment is required for improved understanding of in vitro toxicity data.
The silicone polymer polydimethysiloxane (PDMS) is a popular passive sampler for in situ and ex situ sampling of hydrophobic organic chemicals. Despite its limited sorptive capacity for polar and ionizable organic chemicals (IOC), IOCs have been found in PDMS when extracting sediment and suspended particulate matter. The pH-dependent partitioning of 190 organics and IOCs covering a range of octanol−water partition constants log K ow from −0.3 to 7.7 was evaluated with a 10-day shaking method using mixtures composed of all chemicals at varying ratios of mass of PDMS to volume of water. This method reproduced the PDMS−water partition constant K PDMS/w of neutral chemicals from the literature and extended the dataset by 93 neutral chemicals. The existing quantitative structure−activity relationship between the log K ow and K PDMS/w could be extended with the measured K PDMS/w linearly to a log K ow of −0.3. Fully charged organics were not taken up into PDMS. Thirty-eight monoprotic organic acids and 42 bases showed negligible uptake of the charged species, and the pH dependence of the apparent D PDMS/w (pH) could be explained by the fraction of neutral species multiplied by the K PDMS/w of the neutral species of these IOCs. Seventeen multiprotic chemicals with up to three acidity constants pK a also showed a pH dependence of D PDMS/w (pH) with the tendency that the neutral and zwitterionic forms showed the highest D PDMS/w (pH). D PDMS/w (pH) of charged species of more hydrophobic multiprotic chemicals such as tetrabromobisphenol A and telmisartan was smaller but not negligible. Since these chemicals show high bioactivity, their contribution to mixture effects has to be considered when testing passive sampling extracts with in vitro bioassays. This work has further implications for understanding the role of microplastic as a vector for organic micropollutants.
Discussions are ongoing on which dose metric should be used for quantitative in vitro-to-in vivo extrapolation (QIVIVE) of in vitro bioassay data. The nominal concentration of the test chemicals is most commonly used and easily accessible, while the concentration freely dissolved in the assay medium is considered to better reflect the bioavailable concentration but is tedious to measure. The aim of this study was to elucidate how much QIVIVE results will differ when using either nominal or freely dissolved concentrations. QIVIVEnom and QIVIVEfree ratios, that is, the ratios of plasma concentrations divided by in vitro effect concentrations, were calculated for 10 pharmaceuticals using previously published nominal and freely dissolved effect concentrations for the activation of the peroxisome proliferator-activated receptor gamma (PPARγ) and the activation of oxidative stress response. The QIVIVEnom ratios were higher than QIVIVEfree ratios by up to a factor of 60. The risk of in vivo effects was classified as being high or low for four chemicals using the QIVIVEnom and for three chemicals using QIVIVEfree ratios. Unambiguous classification was possible for nine chemicals by combining the QIVIVEnom or QIVIVEfree ratios with the respective specificity ratios (SRnom or SRfree) of the in vitro effect data, which helps to identify whether the specific effect was influenced by cytotoxicity. QIVIVEfree models should be preferred as they account for differences in bioavailability between in vitro and in vivo, but QIVIVEnom may still be useful for screening the effects of large numbers of chemicals because it is generally more conservative. The use of SR of the in vitro effect data as a second classification factor is recommended for QIVIVEnom and QIVIVEfree models because a clearer picture can be obtained with respect to the likelihood that a biological effect will occur and that it is not caused by nonspecific cytotoxicity.
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