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The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns. In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium. Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

All chemicals can interfere with cellular membranes and this leads to baseline toxicity, which is the minimal toxicity any chemical elicits. The critical membrane burden is constant for all chemicals; that is, the dosing concentrations to trigger baseline toxicity decrease with increasing hydrophobicity of the chemicals. Quantitative structure-activity relationships, based on hydrophobicity of chemicals, have been established to predict nominal concentrations causing baseline toxicity in human and mammalian cell lines. However, their applicability is limited to hydrophilic neutral compounds. To develop a prediction model that includes more hydrophobic and charged organic chemicals, a mass balance model was applied for mammalian cells (AREc32, AhR-CALUX, PPARγ-BLA, and SH-SY5Y) considering different bioassay conditions. The critical membrane burden for baseline toxicity was converted into nominal concentration causing 10% cytotoxicity by baseline toxicity (IC10,baseline) using a mass balance model whose main chemical input parameter was the liposome-water partition constants (Klip/w) for neutral chemicals or the speciation-corrected Dlip/w(pH 7.4) for ionizable chemicals plus the bioassay-specific protein, lipid, and water contents of cells and media. In these bioassay-specific models, log(1/IC10,baseline) increased with increasing hydrophobicity, and the relationship started to level off at log Dlip/w around 2. The bioassay-specific models were applied to 392 chemicals covering a broad range of hydrophobicity and speciation. Comparing the predicted IC10,baseline and experimental cytotoxicity IC10, known baseline toxicants and many additional chemicals were identified as baseline toxicants, while the others were classified based on specificity of their modes of action in the four cell lines, confirming excess toxicity of some fungicides, antibiotics, and uncouplers. Given the similarity of the bioassay-specific models, we propose a generalized baseline-model for adherent human cell lines: log[1/IC10,baseline (M)] = 1.23 + 4.97 × (1 - e-0.236 log Dlip/w). The derived models for baseline toxicity may serve for specificity analysis in reporter gene and neurotoxicity assays as well as for planning the dosing for cell-based assays.

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.

Suspended particulate matter (SPM) plays an important role in the fate of organic micropollutants in rivers during rain events, when sediments are remobilized and turbid runoff components enter the rivers. Under baseflow conditions, the SPM concentration is low and the contribution of SPM-bound contaminants to the overall risk of organic contaminants in rivers is assumed to be negligible. To challenge this assumption, we explored if SPM may act as a source or sink for all or specific groups of organic chemicals in a small river. The concentrations of over 600 contaminants and the mixture effects stemming from all chemicals in in vitro bioassays were measured for river water, SPM, and the surface sediment after solid-phase extraction or exhaustive solvent extraction. The bioavailable fractions of chemicals and mixture effects were estimated after passive equilibrium sampling of enriched SPM slurries and sediments in the lab. Dissolved compounds dominated the total chemical burden in the water column (water plus SPM) of the river, whereas SPM-bound chemicals contributed up to 46% of the effect burden even if the SPM concentration in rivers was merely 1 mg/L. The equilibrium between water and SPM was still not reached under low-flow conditions with SPM as a source of water contamination. The ratios of SPM-associated to sediment-associated neutral and hydrophobic chemicals as well as the ratios of the mixture effects expressed as bioanalytical equivalent concentrations were close to 1, suggesting that the surface sediment can be used as a proxy for SPM under baseflow conditions when the sampling of a large amount of water to obtain sufficient SPM cannot be realized.

The acetylcholinesterase (AChE) inhibition assay has been frequently applied for environmental monitoring to capture insecticides such as organothiophosphates (OTPs) and carbamates. However, natural organic matter such as dissolved organic carbon (DOC) co-extracted with solid-phase extraction from environmental samples can produce false-negative AChE inhibition in free enzyme-based AChE assays. We evaluated whether disturbance by DOC can be alleviated in a cell-based AChE assay using differentiated human neuroblastoma SH-SY5Y cells. The exposure duration was set at an optimum of 3 h considering the effects of OTPs and carbamates. Because loss to the airspace was expected for the more volatile OTPs (chlorpyrifos, diazinon, and parathion), the chemical loss in this bioassay setup was investigated using solid-phase microextraction followed by chemical analysis. The three OTPs were relatively well retained (loss 34%) during 3 h of exposure in the 384-well plate, but higher losses occurred on prolonged exposure, accompanied by slight cross-contamination of adjacent wells. Inhibition of AChE by paraoxon-ethyl was not altered in the presence of up to 68 mg

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 (Ctotal,medium) and/or freely dissolved medium concentrations (Cfree,medium) were determined for one neutral chemical and 14 IOCs (acids, bases, multifunctional) at concentrations relevant for determination of cytotoxicity and effect. Cfree,medium was measured in two in vitro bioassays at the time of dosing and after 24 h of incubation using solid-phase microextraction. Cfree,medium was maximally 1.7 times lower than the nominal concentrations (Cnom) for the hydrophilic chemicals (caffeine and lamotrigine). For the organic acids (naproxen, ibuprofen, warfarin, and diclofenac), Cfree,medium was by a factor of 4 lower than Cnom at high concentrations, but the ratio was much higher at low concentrations, indicating a nonlinear binding behavior. The experimental Cfree,medium was also compared with Cfree,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 Cfree,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.

High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell line...

The freely dissolved concentration in the assay medium (Cfree) and the total cellular concentration (Ccell) are essential input parameters for quantitative in vitro-to-in vivo extrapolations (QIVIV...

Controlling the exposure of chemicals in in vitro mammalian cell assays is an important prerequisite for the application of in vitro methods in risk and hazard assessment of chemicals. Existing models require numerous physicochemical and system parameters to quantify the effective concentration in the assay. Synthesizing these studies, this article briefly communicates how the protein-rich supplement in the medium can be utilized to adjust constant and quantifiable exposure concentrations without the need for measurements and complex modeling. We present a simplified mass balance equation based on chemical properties and system parameters from openly accessible databases, which can be used to adjust the dose of chemicals in the exposure medium, leading to defined and stable freely dissolved concentrations (Cfree). The proposed framework prevents experimental artifacts associated with the use of cosolvents and medium oversaturation and enables the conversion of in vitro effect data to freely dissolved effect concentrations (ECfree), which can directly be applied in quantitative in vitro to in vivo extrapolation models and compared to other exposure scenarios.

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.

The aim of the current study was to understand and develop models to predict the pH-dependent toxicity of ionizable pharmaceuticals in embryos of the zebrafish Danio rerio. We found a higher uptake and toxicity with increasing neutral fraction of acids (diclofenac, genistein, naproxen, torasemide, and warfarin) and bases (metoprolol and propranolol). Simple mass balance models accounting for the partitioning to lipids and proteins in the zebrafish embryo were found to be suitable to predict the bioconcentration after 96 h of exposure if pH values did not differ much from the internal pH of 7.55. For other pH values, a kinetic ion-trap model for the zebrafish embryo explained the pH dependence of biouptake and toxicity. The total internal lethal concentrations killing 50% of the zebrafish embryos (ILC50) were calculated from the measured BCF and LC50. The resulting ILC50 were independent of external pH. Critical membrane concentrations were deduced by an internal mass balance model, and apart from diclofenac, whose specific toxicity in fish had already been established, all pharmaceuticals were confirmed to act as baseline toxicants in zebrafish.

Discussions are ongoing on which dose metric should be used for quantitative

Concerns are increasing that pharmaceuticals released into the environment pose a risk to nontarget organism such as fish. The fish plasma model is a read-across approach that uses human therapeutic blood plasma concentrations for estimating likely effects in fish. However, the fish plasma model neglects differences in plasma protein binding between fish and humans. Because binding data for fish plasma are scarce, the binding of 12 active pharmaceutical ingredients (APIs; acidic, basic, and neutral) to rainbow trout (Oncorhynchus mykiss) and human plasma was measured using solid-phase microextraction (SPME). The plasma/water distribution ratios (D) of neutral and basic APIs were similar for trout and human plasma, differing by no more than a factor of 2.7 for a given API. For the acidic APIs, the D values of trout plasma were much lower than for human plasma, by up to a factor of 71 for naproxen. The lower affinity of the acidic APIs to trout plasma compared with human plasma suggests that the bioavailability of these APIs is higher in trout. Read-across approaches like the fish plasma model should account for differences in plasma protein binding to avoid over- or underestimation of effects in fish. For the acidic APIs, the effect ratio of the fish plasma model would increase by a factor of 5 to 60 if the unbound plasma concentrations were used to calculate the effect ratio. Environ Toxicol Chem 2020;00:1–10.

Background: High-throughput screening of chemicals with in vitro reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action. However, the validity of some of the reported activities is questionable due to the “cytotoxicity burst,” which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death. Objectives: We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity. Methods: Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes. Quantitative structure–activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation. Measured 10% effect concentrations were related to measured or QSAR-predicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to ∼8,000 chemicals that were tested in six of the high-throughput Tox21 reporter gene assays. Results: Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst. In six Tox21 assays, 37%–87% of the active hits were presumably caused by the cytotoxicity burst (SR

Exposure assessment in

Many environmental pollutants pose a toxicological hazard only after metabolic activation. In vitro bioassays using cell lines or bacteria have often no or reduced metabolic activity, which impedes their use in the risk assessment. To improve the predictive capability of in vitro assays, external metabolization systems like the liver S9 fraction are frequently combined with in vitro toxicity assays. While it is typical for S9 fractions that samples and testing systems are combined in the same exposure system, we propose to separate the metabolism step and toxicity measurement. This allows for a modular combination of metabolic activation by enzymes isolated from rat liver (S9) or a biotechnological alternative (ewoS9R) with in vitro bioassays that lack metabolic capacity. Benzo(a)pyrene and 2-aminoanthracene were used as model compounds to optimize the conditions for the S9 metabolic degradation/activation step. The Ames assay with Salmonella typhimurium strains TA98 and TA100 was applied to validate the set-up of decoupling the S9 activation/metabolism from the bioassay system. S9 protein concentration of 0.25 mgprotein/mL, a supplement of 0.13 mM NADPH and a pre-incubation time of 100 min are recommended for activation of samples prior to dosing them to in vitro bioassays using the regular dosing protocols of the respective bioassay. EwoS9R performed equally well as Moltox S9, which is a step forward in developing true animal-free in vitro bioassays. After pre-incubation with S9 fraction, chemicals induced bacteria revertants in both the TA98 and the TA100 assay as efficiently as the standard Ames assay. The pre-incubation of chemicals with S9 fraction could serve for a wide range of cellular in vitro assays to efficiently combine activation and toxicity measurement, which may greatly facilitate the application of these assays for chemical hazard assessment and monitoring of environmental samples.

Sorption to the polystyrene (PS) of multiwell plates can affect the exposure to organic chemicals over time in in vitro and in vivo bioassays. Experimentally determined diffusion coefficients in PS ( DPS) were in a narrow range of 1.25 to 8.0 · 10-16 m2 s-1 and PS-water partition constants ( KPS/w) ranged from 0.04 to 5.10 log-units for 22 neutral organic chemicals. A kinetic model, which explicitly accounts for diffusion in the plastic, was applied to predict the depletion of neutral organic chemicals from different bioassay media by sorption to various multiwell plate formats. For chemicals with log Kow > 3, the medium concentrations decreased rapidly and considerably in the fish embryo toxicity assay but medium concentrations remained relatively constant in the cell-based bioassays with medium containing 10% fetal bovine serum (FBS), emphasizing the ability of the protein- and lipid-rich medium to compensate for losses by multiwell plate sorption. The PS sorption data may serve not only for exposure assessment in bioassays but also to model the contaminant uptake by and release from plastic packaging material and the chemical transport by PS particles in the environment.

The Microtox assay, a bioluminescence inhibition assay with the marine bacterium Aliivibrio fischeri, is one of the most popular bioassays for assessing the cytotoxicity of organic chemicals, mixtures and environmental samples. Most environmental chemicals act as baseline toxicants in this short-term screening assay, which is typically run with only 30 min of exposure duration. Numerous Quantitative Structure-Activity Relationships (QSARs) exist for the Microtox assay for nonpolar and polar narcosis. However, typical water pollutants, which have highly diverse structures covering a wide range of hydrophobicity and speciation from neutral to anionic and cationic, are often outside the applicability domain of these QSARs. To include all types of environmentally relevant organic pollutants we developed a general baseline toxicity QSAR using liposome-water distribution ratios as descriptors. Previous limitations in availability of experimental liposome-water partition constants were overcome by reliable prediction models based on polyparameter linear free energy relationships for neutral chemicals and the COSMOmic model for charged chemicals. With this QSAR and targeted mixture experiments we could demonstrate that ionisable chemicals fall in the applicability domain. Most investigated water pollutants acted as baseline toxicants in this bioassay, with the few outliers identified as uncouplers or reactive toxicants. The main limitation of the Microtox assay is that chemicals with a high melting point and/or high hydrophobicity were outside of the applicability domain because of their low water solubility. We quantitatively derived a solubility cut-off but also demonstrated with mixture experiments that chemicals inactive on their own can contribute to mixture toxicity, which is highly relevant for complex environmental mixtures, where these chemicals may be present at concentrations below the solubility cut-off.

Traditionally our tools for environmental risk assessment of organic chemicals have been developed for neutral chemicals. However, many commercial chemicals are ionic or ionizable and require different tools and approaches for their assessment. In recent years this task starts to obtain increasing attention but our understanding for their environmental fate is still far behind that for neutral chemicals. This review first gives an overview on the principles that govern ionic partitioning in environmental systems which are more complex than the simple partition processes of neutral chemicals. Second, a summary of our current knowledge on various topics such as bioaccumulation, sorption in soils, and nonspecific-toxicity reveals that ionic species can actually be quite hydrophobic contrary to commonly held beliefs. Eventually, we discuss existing models for the quantitative prediction of organic ions' sorption in soils and biota. We have to assert that the available model tools are quite restricted in their application range compared to neutral chemicals which is due to the higher complexity of the various ionic sorption processes. In order to further advance our understanding more high-quality sorption data are needed with a focus on multivalent and zwitterionic ions in all partition systems as well as cations in biological matrices.

Controlling the exposure of chemicals in

Traditionally our tools for environmental risk assessment of organic chemicals have been developed for neutral chemicals. However, many commercial chemicals are ionic or ionizable and require different tools and approaches for their assessment. In recent years this task starts to obtain increasing attention but our understanding for their environmental fate is still far behind that for neutral chemicals. This review first gives an overview on the principles that govern ionic partitioning in environmental systems which are more complex than the simple partition processes of neutral chemicals. Second, a summary of our current knowledge on various topics such as bioaccumulation, sorption in soils, and nonspecific-toxicity reveals that ionic species can actually be quite hydrophobic contrary to commonly held beliefs. Eventually, we discuss existing models for the quantitative prediction of organic ions’ sorption in soils and biota. We have to assert that the available model tools are quite restricted in their application range compared to neutral chemicals which is due to the higher complexity of the various ionic sorption processes. In order to further advance our understanding more high-quality sorption data are needed with a focus on multivalent and zwitterionic ions in all partition systems as well as cations in biological matrices.

Reliable partitioning data are essential for assessing the bioaccumulation potential and the toxicity of chemicals. In contrast to neutral organic chemicals, the partitioning behavior of ionogenic organic chemicals (IOCs) is still a black box for environmental scientists. Partitioning to serum albumin, the major protein in blood plasma, strongly influences the freely dissolved concentration of many chemicals (including IOCs), which affects their transport and distribution in the body. Because consistent data sets for partitioning of IOCs are rarely available, bovine serum albumin-water partition coefficients (KBSA/w) were measured in this study for 45 anionic and 4 cationic organic chemicals, including various substituted benzoic and naphthoic acids, sulfonates and several pesticides and pharmaceuticals. The results of this study suggest that binding to BSA is substantially influenced by the three-dimensional structure of the chemicals and the position of substitutions on the sorbing molecules. For example, we found a difference of1.5 log units between isomeric chemicals such as 3,4-dichlorobenzoic acid and 2,6-dichlorobenzoic acid, and 1-naphthoic acid and 2-naphthoic acid. Conventional modeling approaches (e.g., based on octanol-water partition coefficients) poorly predict log KBSA/w of organic ions (R(2) ≤ 0.5), partially because they do not capture the observed steric effects. Hence, alternative modeling strategies will be required for accurate prediction of serum albumin-water partition coefficients of organic ions.

The effects measured with in vitro cell-based bioassays are typically reported as nominal effect concentrations (Cnom), but the freely dissolved concentration in the exposure medium (Cw) and the total cellular concentration (Ccell) are considered more quantitative dose metrics that allow extrapolation to the whole-organism level. To predict Cw and Ccell, the partitioning of the test chemicals to medium proteins and lipids and cells has to be known. In this study, we developed a solid-phase microextraction (SPME) method based on C18-coated fibers to quantify the partitioning of diclofenac, 2,4-dichlorophenoxyacetic acid (2,4-D), ibuprofen, naproxen, torasemide, warfarin, and genistein to bovine serum albumin (BSA), phospholipid liposomes, fetal bovine serum (FBS), and cells. For ibuprofen, 2,4-D, naproxen, and warfarin, the partitioning to the SPME fibers was found to be concentration dependent, which had to be considered for the calculation of distribution ratios to biological materials. The sorption isot...

Cellular uptake kinetics are key for understanding time-dependent chemical exposure in in vitro cell assays. Slow cellular uptake kinetics in relation to the total exposure time can considerably reduce the biologically effective dose. In this study, fluorescence microscopy combined with automated image analysis was applied for time-resolved quantification of cellular uptake of 10 neutral, anionic, cationic, and zwitterionic fluorophores in two reporter gene assays. The chemical fluorescence in the medium remained relatively constant during the 24-h assay duration, emphasizing that the proteins and lipids in the fetal bovine serum (FBS) supplemented to the assay medium represent a large reservoir of reversibly bound chemicals with the potential to compensate for chemical depletion by cell uptake, growth, and sorption to well materials. Hence FBS plays a role in stabilizing the cellular dose in a similar way as polymer-based passive dosing, here we term this process as serum-mediated passive dosing (SMPD). Neutral chemicals accumulated in the cells up to 12 times faster than charged chemicals. Increasing medium FBS concentrations accelerated uptake due to FBS-facilitated transport but led to lower cellular concentrations as a result of increased sorption to medium proteins and lipids. In vitro cell exposure results from the interaction of several extra- and intracellular processes, leading to variable and time-dependent exposure between different chemicals and assay setups. The medium FBS plays a crucial role for the thermodynamic equilibria as well as for the cellular uptake kinetics, hence influencing exposure. However, quantification of cellular exposure by an area under the curve (AUC) analysis illustrated that, for the evaluated bioassay setup, current in vitro exposure models that assume instantaneous equilibrium between medium and cells still reflect a realistic exposure because the AUC was typically reduced less than 20% compared to the cellular dose that would result from instantaneous equilibrium.

In this work we combine partition coefficients between water and membrane lipid, storage lipid, the plasma protein albumin as well as structural protein with the tissue dependent fraction of the respective phases in order to obtain a clearer picture on the relevance of various biological tissues for the bioaccumulation of 31 organic anions. Most of the partition coefficients are based on experimental data, supplemented by some predicted ones. The data suggest that the plasma protein, albumin, will be the major sorption matrix in mammals. Only small fractions of the studied chemicals will occur freely dissolved in an organism. For the investigated acids with pKa5, partitioning is dominated by the ionic species rather than the corresponding neutral species. Bioconcentration in fish is not expected to occur for many of these acids unless pH in the aqueous environment is low or specific sorption mechanisms are relevant. In contrast, biomagnification in terrestrial mammals would be expected for most organic anions if they are not sufficiently metabolized. We conclude that sorption is important for the toxicokinetics of ionizable organic chemicals and the dominating sorbing matrices are quite different from those for neutral species.

The in vivo partitioning behavior of ionogenic organic chemicals (IOCs) is of paramount importance for their toxicokinetics and bioaccumulation. Among other proteins, structural proteins including muscle proteins could be an important sorption phase for IOCs, because of their high quantity in the human and other animals' body and their polar nature. Binding data for IOCs to structural proteins are, however, severely limited. Therefore, in this study muscle protein-water partition coefficients (KMP/w) of 51 systematically selected organic anions and cations were determined experimentally. A comparison of the measured KMP/w with bovine serum albumin (BSA)-water partition coefficients showed that anionic chemicals sorb more strongly to BSA than to muscle protein (by up to 3.5 orders of magnitude), while cations sorb similarly to both proteins. Sorption isotherms of selected IOCs to muscle protein are linear (i.e., KMP/w is concentration independent), and KMP/w is only marginally influenced by pH value and salt concentration. Using the obtained data set of KMP/w a polyparameter linear free energy relationship (PP-LFER) model was established. The derived equation fits the data well (R(2) = 0.89, RMSE = 0.29). Finally, it was demonstrated that the in vitro measured KMP/w values of this study have the potential to be used to evaluate tissue-plasma partitioning of IOCs in vivo.