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Session A5: Incorporation of In Vitro-Derived Data on Biokinetics and Toxicodynamics in Risk AssessmentsChairs: Bas Blaauboer (The Netherlands) and Harvey Clewell (USA) A5: The Use of Biokinetic Modeling and In Vitro Data to Reduce the Requirement for in vivo Animal Testing One approach for reducing the requirement for in vivo animal testing is the development of in vitro studies that can provide similar information on toxicity. However, results obtained from in vitro studies are often not directly applicable to the in vivo situation. One of the most obvious differences between the situation in vitro and in vivo is the absence of the processes of absorption, distribution, metabolism, and excretion (i.e., biokinetics) that govern the exposure of the target tissue in the intact organism. Thus, the concentrations to which in vitro systems are exposed may not correspond directly to the actual situation at the target tissue after in vivo exposure. In addition, the occurrence of metabolic activation and/or saturation of specific metabolic pathways or absorption and elimination mechanisms may also become relevant for the toxicity of a compound in vivo. This may lead to misinterpretation of in vitro data if such information is not taken into account. Therefore, the results of in vitro studies of the biological activity of compounds can only properly be interpreted with regard to their implications for the in vivo situation using a biokinetic model. Biokinetic modeling can also contribute to reduction and refinement of animal studies by optimization of study design. Of course, the development of a biokinetic model in itself requires the collection of animal data. However, in the case of physiologically based biokinetic (PBBK) models, the parameters in such a model are, in general, available in the physiological literature or can be determined from limited in vitro experiments. Moreover, in many cases, currently available quantitative structure-activity relationship (QSAR) techniques can be used to estimate chemical properties and biokinetics when the specific data for that chemical is lacking. QSAR techniques may also prove useful in predicting potential target tissues for toxicity so that the appropriate assays of in vitro dynamics (response) can be selected. The use of QSAR and in vitro data to support the development of PBBK models and the use of the resulting models to reduce the requirement for in vivo testing will be demonstrated by several examples. A5: Use of a Perfusion Co-culture System Consisting of Caco-2 and Hep G2 Cell Compartments for the Kinetic Analysis of Benzo[a]Pyrene Toxicity Conventional cytotoxicity tests cannot usually include various metabolic processes in humans. We therefore developed a physiologically based, multi-compartment perfusion coculture system, using a Caco-2 cell monolayer on a semi-permeable membrane and three-dimensional culture of Hep G2 cells. Cocultivation enhanced the cytochrome P450 1A1/2 capacities of both cell lines, particularly under induced conditions. When benzo[a]pyrene (BaP) was loaded to the apical side of the Caco-2 cell layer, the enhanced P450 capacities almost completely degraded the added BaP into various BaP metabolites, including BaP-7,8-diol, a precursor to the ultimate carcinogen of BaP. Although such metabolites are relatively secreted to the apical side, the increased concentration of the ultimate carcinogen led to strong cytotoxicity in Hep G2 cells. Because this system can reproduce such complicated phenomena, it is helpful as a new in vitro experimental system when we understand unknown mechanisms involved in final toxicity in humans and improve numerical simulation models. A5: Prediction of Whole Body Metabolic Clearance of Drugs Through the Combined Use of Slices from Rat Liver, Lung, Kidney, Small Intestine, and Colon The aim of the current study was to investigate whether precision-cut rat tissue slices can be used to predict drug clearance in vivo, using liver, as well as lung, kidney, small intestine, and colon. For this, metabolic clearance of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, and three candidate drugs were calculated after measuring disappearance of these compounds during the incubation of tissue slices. To be sure that the clearance was independent of the substrate concentration, incubations were performed on two concentrations. The total in vitro metabolic clearance was determined by summing the individual in vitro organ clearance values from the slices. Prediction on the basis of the in vitro clearance was found to be reasonably accurate, being 0.14 to 1.78 fold of the corresponding in vivo values. Interestingly, the relative contribution of extra-hepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 50% to 87% of the total metabolic clearance. It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance, and that it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance. A5: Integration of In Vitro-Derived Toxicity Data and Biokinetics in Strategies for Hazard and Risk Assessment of Chemicals Toxicity of a compound for an organism is dependent on the route and the amount (or concentration) of exposure, the way in which the compound is taken up, distributes, and is eliminated from the organism (ADME, kinetics), and the intrinsic properties (reactivity; mode of action, dynamics) of the compound toward the organism. These three elements: exposure, kinetics, and dynamics form the basis of hazard and risk evaluations. Developments in our knowledge of the way in which physico-chemical properties of chemicals (on the one side) and physiological processes in the organism (on the other side) determine a compound's toxicity have greatly increased our understanding of toxicological processes and our ability to interpret experimental results. This has resulted in the development of model systems in which the above-mentioned processes can be described mathematically. Biokinetic modeling is currently of great interest, but the further development of toxicodynamic modeling is equally important. The combination of both allows the estimation of a compound's critical amount/concentration on the critical site of action, which, ideally, would be the basis for hazard and risk assessments. In vitro systems have been extremely useful in studying the molecular basis of a chemical's biological activity, including its mechanism(s) of toxic action. Other achievements include the prediction of biological reactivity on the basis of physico-chemical properties and the construction of structure-activity relationships (QSARs). However, for the incorporation of in vitro–derived data, as well as the results of QSARs, kinetic modeling is indispensable. Thus, biokinetic and toxicodynamic modeling are important (if not crucial) tools in toxicological research, and there are increasing opportunities to incorporate the results of this work in hazard and risk assessments. Their implementation will allow a much more scientifically-based and a better structured risk assessment, which will be to a much lesser extent relying on animal experimentation. A5: Monitoring of Muscle and Connective Tissue Metabolite Concentrations Intramuscularly and Subcutaneously in the Isolated Hemoperfused Porcine Forelimb by Microdialysis The isolated porcine forelimb was proven to be useful as an adequate alternative to investigate the penetration of dermally applied substances. In order to get detailed information on the tissue distribution of penetrating substances, the microdialysis technique was established on the isolated porcine forelimb. Limbs of slaughtered pigs were connected to the perfusion system and perfused under standardized conditions. Samples from catheters, inserted intramuscularly or subcutaneously, were collected continuously and the concentration of metabolites, as well as recovery and flow rate, were determined. Flow rate in the catheters was fairly constant in the connective tissue and in the muscle. Lactate and glycerol concentration in the connective tissue were higher compared to the muscle tissue. In contrast, glucose concentrations in the muscle tissue were higher compared to connective tissue. Pyruvate concentrations were comparable in both tissues. During the assay period, concentrations of all metabolites remained unchanged in both tissues. The results demonstrate that the microdialysis technique is applicable on the isolated porcine forelimb. Because of the constant perfusion and microdialysis conditions, the method is valuable to study the pharmacokinetics of dermally applied substances in different skin layers and intramuscularly.
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