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The nominal concentration is generally used to express concentration–effect relationships in in vitro toxicity assays. However, the nominal concentration does not necessarily represent the exposure concentration responsible for the observed effect. Surfactants accumulate at interphases and likely sorb to in vitro system components such as serum protein and well plate plastic. The extent of sorption and the consequences of this sorption on in vitro readouts is largely unknown for these chemicals. The aim of this study was to demonstrate the effect of sorption to in vitro components on the observed cytotoxic potency of benzalkonium chlorides (BAC) varying in alkyl chain length (6–18 carbon atoms, C6–18) in a basal cytotoxicity assay with the rainbow trout gill cell line (RTgill-W1). Cells were exposed for 48 h in 96-well plates to increasing concentration of BACs in exposure medium containing 0, 60 μM bovine serum albumin (BSA) or 10% fetal bovine serum (FBS). Before and after exposure, BAC concentrations in exposure medium were analytically determined. Based on freely dissolved concentrations at the end of the exposure, median effect concentrations (EC50) decreased with increasing alkyl chain length up to 14 carbons. For BAC with alkyl chains of 12 or more carbons, EC50’s based on measured concentrations after exposure in supplement-free medium were up to 25-times lower than EC50’s calculated using nominal concentrations. When BSA or FBS was added to the medium, a decrease in cytotoxic potency of up to 22 times was observed for BAC with alkyl chains of eight or more carbons. The results of this study emphasize the importance of expressing the in vitro readouts as a function of a dose metric that is least influenced by assay setup to compare assay sensitivities and chemical potencies.

Introduction:Reliable in vitroand in silicoassays as alternatives for in vivodevelopmental toxicity studies are urgently needed, for the replacement, reduction and refinement (3Rs) of animal use in toxicological research. Therefore, relevant biomarkers for in vivodevelopmental toxicity in in vitroassays are needed.Areas covered:The present review gives an overview of alternative assays, as described in literature, for in vivodevelopmental toxicity, including the effects (readouts) assessed in these assays. The authors discuss how these data may be used to obtain relevant biomarkers for in vivodevelopmental toxicity, and how in vitroeffect data can be translated to the in vivosituation using physiologically based kinetic (PBK) modeling. Expert opinion:Relevance of readouts in in vitrodevelopmental toxicity assays as predictive biomarkers for in vivodevelopmental toxicity should be evaluated by comparing the obtained in vitroeffect concentrations with in vivointernal concentrations at dose levels causing developmental toxicity. Extrapolation of the in vitroeffect concentrations to in vivodose levels using PBK modeling (i.e., reverse dosimetry) is promising in its use to derive points of departure for risk assessment, enabling the use of in vitrotoxicity data in the safety assessment of compounds.

The application of sandwich-cultured rat hepatocytes for the identification of the hepatic intrinsic clearance of compounds with widely varying extraction ratios was investigated. We previously showed the applicability of this in vitro system, in combination with a model describing molecular diffusion, hepatocyte/medium partition, and nonsaturated metabolism, which resulted in a successful identification of this parameter for tolbutamide. This approach is further validated using the compounds 7-ethoxycoumarin and warfarin, covering a 100-fold range of extraction ratios. Clearance of these two substrates could be reliably determined, but only if the depletion of the parent compound in medium as well as in the hepatocyte sandwich was measured. Sensitivity analyses showed that the time course of depletion of the parent compound in medium, especially for warfarin, is insensitive to the partition and diffusion parameter values, whereas depletion in the hepatocyte sandwich was far more sensitive. When varying the volumes of collagen in the sandwich culture, it appears that the most reliable kinetic parameters could be obtained by fitting the data with the smaller collagen volume and that these parameters obtained from fitting to data of the larger volumes generally cannot be verified satisfactorily with the data of the smaller volumes. The values of hepatic clearance that were obtained after extrapolation of the intrinsic clearance to the hepatic clearance from blood were comparable within a factor of 2 to hepatic clearance data in the literature. This indicates that this sandwich culture and modeling system can be applied for the identification of the hepatic intrinsic clearance rate of the total range from low to high clearance compounds.

An alternative approach is introduced in determining the in vitro intrinsic clearance of slowly metabolized compounds. The longterm sandwich rat hepatocyte culture was exploited, allowing for sufficient substrate depletion to obtain a reliable clearance estimation; in its physiology, it resembles the in vivo liver, thus allowing in vivo extrapolation of the in vitro clearance value. Substrate depletion of tolbutamide and the formation of its metabolites hydroxytolbutamide and carboxytolbutamide were measured in the medium and sandwich layer. Depletion data from the medium were fitted to a mathematical model incorporating system-dependent parameters (diffusion, protein binding, and partitioning) to calculate the hepatocytes' intrinsic clearance. Based on the decrease of the parent compound in the medium, a specific intrinsic clearance value, i.e., clearance per unit of volume of hepatocytes, of 0.085 min(-1) was fitted. This value was in accordance with in vivo and in vitro values from the literature. The model was verified with substrate depletion data from the sandwich layer. Data on metabolite formation showed an incomplete mass balance. A radiochemical experiment revealed the presence of three additional metabolites. These metabolites were analyzed by liquid chromatography-mass spectometry. One was identified as p-tolysulfonylurea. The structure of the other two needs to be elucidated. After the addition of these compounds to the metabolic pattern, the mass balance was completed. The in vitro clearance value was incorporated in a physiologically based pharmacokinetic literature model of tolbutamide that accurately describes the plasma concentration. The approach used in this study successfully predicts the intrinsic clearance of tolbutamide. In addition, the sandwich rat hepatocyte culture also proves to be useful in the identification of metabolic pathways.

Toxicological risk assessment for chemicals is still mainly based on highly standardised protocols for animal experimentation and exposure assessment. However, developments in our knowledge of general physiology, in chemicobiological interactions and in (computer-supported) modelling, have resulted in a tremendous change in our understanding of the molecular mechanisms underlying the toxicity of chemicals. This permits the development of biologically based models, in which the biokinetics as well as the toxicodynamics of compounds can be described. In this paper, the possibilities are discussed of developing systems in which the systemic (acute and chronic) toxicities of chemicals can be quantified without the heavy reliance on animal experiments. By integrating data derived from different sources, predictions of toxicity can be made. Key elements in this integrated approach are the evaluation of chemical functionalities representing structural alerts for toxic actions, the construction of biokinetic models on the basis of non-animal data (for example, tissue–blood partition coefficients, in vitrobiotransformation parameters), tests or batteries of tests for determining basal cytotoxicity, and more-specific tests for evaluating tissue or organ toxicity. It is concluded that this approach is a useful tool for various steps in toxicological hazard and risk assessment, especially for those forms of toxicity for which validated in vitroand other non-animal tests have already been developed.

Data derived from in vitrotoxicity studies are not directly applicable in an assessment of the toxicity of compounds in intact organisms. The major limitation is the lack of knowledge of biokinetic behaviour in vivo.Since the toxicity of a compound will be determined by the critical concentration (or other dose metric) of the critical compound (or a metabolite thereof) at the critical site of toxic action, biokinetic behaviour must be taken into account. Possibilities of biokinetic modelling on the basis of in vitroand other non-animal data are discussed, and the application of the results in hazard and risk-assessment schedules is considered.

The ECVAM Task Force on Integrated Testing Strategies was established in December 1996, with the remit of assessing the current status of integrated toxicity testing, and of making proposals regarding the design and implementation of integrated testing strategies. The first step in an integrated testing strategy is usually to determine the chemical functionality of a substance, on the basis of its structure and physicochemical properties. The biokinetic and dynamic behaviours of the chemical in various in vitrosystems are then assessed. The various elements are then integrated, in either a parallel or a stepwise fashion, to make predictions of the local or systemic toxicity of the chemical of interest. In this report, a generic scheme for local/systemic toxicity, and a specific scheme for target organ toxicity, are proposed. The scope and limitations of the approaches are discussed. The task force hopes that its proposals will stimulate a discussion on the feasibility of this type of approach and it welcomes any feedback. It is planned that the discussion points will be elaborated in a second task force report.

Assay conditions in determining total cytochrome P-450 in four laboratories were compared. Although the determination was derived from the original Omura and Sato method in each laboratory, the four standard protocols differed slightly, resulting in considerable differences in the results. Since the cytochrome P-450 content is usually expressed per mg protein, the protein assay conditions were evaluated as well. Furthermore, we compared the cytochrome P-450 values obtained by the CO- and the dithionite (DT)-difference methods. The effect of a number of variables in the assay was investigated. The influence of the storage temperature of the microsomes was ascertained as well as effects of the gassing time with CO and the time between addition of dithionite, CO-gassing and the recording of the difference spectra. After evaluating these variables a standard operation procedure was established. Using this procedure the interlaboratory coefficient of variation for total cytochrome P-450 was 4.8%, a value which was comparable to the intralaboratory coefficients of variation. The final results also show that the millimolar extinction coefficient for the DT-difference method is higher than for the CO-difference method.

Dioxins are potent inducers of chloracne in humans. This skin aberration can be interpreted as an altered differentiation pattern of acinar sebaceous base cells and a change in the rate of terminal differentiation of the keratinocytes. We measured this rate induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in primary cultures of human keratinocytes. As parameters for differentiation, we quantified the 35S-methionine incorporation into cross-linked envelopes (revealing the total CLE biomass), as well as the number of microscopically visible CLEs. It was shown that TCDD is a very potent inducer of both CLE biomass and number with a half-maximal effect concentration (EC50) of 1.4 nM. CLE biomass was maximally increased 10-fold and the number of cells in culture producing a CLE was increased from 15% in control cultures to maximally 75% of the cells in TCDD-treated cultures. Both effects were Ca2+-dependent and increased with elevated cell density, being optimal in post-confluent cultures. Retinoic acid dose-dependently decreased the effect of 10−8M TCDD, 10−6M having a nearly complete antagonistic action. This interaction of retionic acid with TCDD-induced differentiation was non-competitive. Retinol was equally potent as an antagonist of the TCDD-induced elevation of CLE formation as compared with retinoic acid. Retinyl palmitate and etretinate were not very effective as TCDD antagonists. Supplementation of hydrocortisone suppressed the TCDD-induced keratinocyte differentiation. It was concluded that CLE biomass quantification provides a reliable and sensitive parameter for keratinocyte differentiation. In this in vitro system it is shown that TCDD strongly induces a switch from proliferation to terminal differentiation and that this effect can be antagonized effectively by retinoic acid and retinol.

The cytotoxic events in freshly isolated rat hepatocytes following exposure over 2 h to menadione (2-methyl-1,4-naphthoquinone) and two closely related quinones, 2,3-dimethyl-1,4-naphthoquinone (DMNQ) and 1,4-naphthoquinone (NQ), were examined. These quinones differ in their arylation capacity (NQ > menadione ≫ DMNQ) and in their potential to induce redox cycling (NQ ≈ menadione ≫ DMNQ). The glutathione status (reduced and oxidized glutathione) of the hepatocytes was determined using HPLC after derivatization with monobromobimane. Protein thiols were measured spectrophotometrically and the energy charge of the cells was determined with HPLC using ion pair chromatography. The leakage of lactate dehydrogenase was used as a marker for cell viability. All three quinones caused alterations of the glutathione status of the exposed cells but the effects were markedly different. Exposure to DMNQ resulted in a slow decrease of reduced glutathione and an increase of mixed disulfides. The other two quinones caused an almost complete depletion of reduced glutathione within 5 min. Hepatocytes exposed to NQ accumulated oxidized glutathione whereas menadione-exposed hepatocytes showed increased levels of mixed disulfides. We did not find any effects of DMNQ (200 μM) on protein thiols, energy charge or cell viability. There was a clear difference in the effects of menadione and NQ on protein thiols, energy charge and cell viability: exposure to NQ resulted in a more extensive decrease of protein thiols and energy charge and an earlier onset of lactate dehydrogenase leakage. From our results we conclude that the arylation capacity of a quinone is a determining factor in the cytotoxic potential of such compounds and that the decrease of protein thiols and of the energy charge are critical events preceding loss of cell viability.

The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4±0.6 to 4.8±2.1 (fomesafen) 16.9±2.9 (oxyfluorfen) and 25.9±3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides.

The hamster is known to display very high rates of monooxygenase-mediated biotransformation. In comparison with other species little knowledge has been gathered with respect to the nature of its cytochrome P450 enzymes and their respective inducibility. We studied the consequences of induction of P450 enzymes in rats and Syrian golden hamsters using the regioselective oxidative O-demethylation of the coumarin derivative scoparone. This metabolic conversion indicates differential effects of P450 inducers in the rat, in which various types of inducers cause different shifts in the isoscopoletin/scopoletin metabolite ratio (I/S-ratio). Liver microsomes from hamster not treated with P450 inducers oxidized scoparone much more efficiently than liver microsomes of untreated rats. In rat liver microsomes total demethylation rates of scoparone increased upon in vivo treatment with phenobarbital or ß-naphthoflavone. Phenobarbital reduced the I/S-ratio whereas ß-naphthoflavone caused an increase in this ratio. In hamster liver microsomes both phenobarbital and β-naphthoflavone treatments resulted in a decrease in the I/S ratio. In this species the total scoparone demethylation rate was not much affected by phenobarbital, but β-naphthoflavone caused a huge increase in over-all scoparone biotransformation. In both species, dexamethasone, isoniazid and clofibrate were much less effective. In contrast to the rat, in the hamster the scoparone biotransformation profile cannot be used to differentiate between phenobarbital- or β-naphthoflavone-treated animals.

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands.

The moral value of animals deserves greater recognition in scientific publications

The biokinetic behavior of toluene was modeled in the rat with a physiologically based pharmacokinetic (PB-PK) model. The model was parameterized by using reference physiological parameter values and partition coefficients that were reported earlier from in vitro studies. Biotransformation parameters for toluene, reported from two in vivo and six in vitro studies, were subsequently substituted in the model while keeping all other model parameters constant. Simulations of toluene kinetics, based on these eight biotransformation parameter sets, were compared with empirical data reported on toluene uptake in blood and/or brain tissue after inhalation exposure. It was observed that most empirical data on toluene blood concentrations were adequately predicted by the model for almost each of the eight biotransformation parameter sets. It was also observed that differences between model predictions, based on either in vivo-or in vitro-derived biotransformation parameters, were generally small. It is concluded that the results from most in vitro studies on toluene biotransformation can be applied successfully to predict the kinetics of toluene in vivo. It is also concluded that the brain-blood partition coefficient may be at least as important for the outcome of the model as the biotransformation parameters are. These results support earlier reported findings in the literature on application of in vitro techniques to derive parameters for PB-PK models.