Prediction of dose-hepatotoxic response in humans based on toxicokinetic/toxicodynamic modeling with or without in vivo data: a case study with acetaminophen.

In the present legislations, the use of methods alternative to animal testing is explicitly encouraged, to use animal testing only 'as a last resort' or to ban it. The use of alternative methods to replace kinetics or repeated dose in vivo tests is a challenging issue. We propose here a strategy based on in vitro tests and QSAR (Quantitative Structure Activity Relationship) models to calibrate a dose-response model predicting hepatotoxicity. The dose response consists in calibrating and coupling a PBPK (physiologically-based pharmacokinetic) model with a toxicodynamic model for cell viability. We applied our strategy to acetaminophen and compared three different ways to calibrate the PBPK model: only with in vitro and in silico methods, using rat data or using all available data including data on humans. Some estimates of kinetic parameters differed substantially among the three calibration processes, but, at the end, the three models were quite comparable in terms of liver toxicity predictions and close to the usual range of human overdose. For the model based on alternative methods, the good adequation with the two other models resulted from an overestimated renal elimination rate which compensated for the underestimation of the metabolism rate. Our study points out that toxicokinetics/toxicodynamics approaches, based on alternative methods and modelling only, can predict in vivo liver toxicity with accuracy comparable to in vivo methods.

Toxicokinetics as a key to the integrated toxicity risk assessment based primarily on non-animal approaches.

Toxicokinetics (TK) is the endpoint that informs about the penetration into and fate within the body of a toxic substance, including the possible emergence of metabolites. Traditionally, the data needed to understand those phenomena have been obtained in vivo. Currently, with a drive towards non-animal testing approaches, TK has been identified as a key element to integrate the results from in silico, in vitro and already available in vivo studies. TK is needed to estimate the range of target organ doses that can be expected from realistic human external exposure scenarios. This information is crucial for determining the dose/concentration range that should be used for in vitro testing. Vice versa, TK is necessary to convert the in vitro results, generated at tissue/cell or sub-cellular level, into dose response or potency information relating to the entire target organism, i.e. the human body (in vitro-in vivo extrapolation, IVIVE). Physiologically based toxicokinetic modelling (PBTK) is currently regarded as the most adequate approach to simulate human TK and extrapolate between in vitro and in vivo contexts. The fact that PBTK models are mechanism-based which allows them to be 'generic' to a certain extent (various extrapolations possible) has been critical for their success so far. The need for high-quality in vitro and in silico data on absorption, distribution, metabolism as well as excretion (ADME) as input for PBTK models to predict human dose-response curves is currently a bottleneck for integrative risk assessment.

Application of multielectrode array (MEA) chips for the evaluation of mixtures neurotoxicity.

Cortical neurons grown on multielectrode array (MEA) chips have been shown to be a valuable alternative method to study electrophysiological properties of the central nervous system neurons and to perform functional toxicological screening. Here we studied the effects of binary mixtures on neuronal networks cultured on MEAs. We have considered compounds with similar and different mode-of-action (MoA) to characterize and assess their combined effects. Individual and binary mixture dose-response curves based on spontaneous neuronal activity have been generated and the IC(50) has been considered as the end-point for neurotoxicity assessment. The two classical approaches of mixtures toxicity studies: concentration addition (CA) and independent action (IA) have been applied to compare calculated and experimental results. Nuclear magnetic resonance (NMR) spectroscopy has been employed to confirm no chemical reaction or complexation between mixtures components. The results suggest that both CA and IA are able to predict the toxicity of the mixture and that the combination of in vitro test methods with theoretical dose-response models has a strong potential as an alternative tool for the prediction of mixtures neurotoxicity.

Screening of chemicals for human bioaccumulative potential with a physiologically based toxicokinetic model.

Human bioaccumulative potential is an important element in the risk assessment of chemicals. Due to the high number of synthetic chemicals, there exists the need to develop prioritisation strategies. The purpose of this study was to develop a predictive tool for human bioaccumulation risk assessment that incorporates not only the chemical properties of the compounds, but also the processes that tend to decrease the concentration of the compound such as metabolisation. We used a generic physiologically based toxicokinetic model that based on in vitro human liver metabolism data, minimal renal excretion and a constant exposure was able to assess the bioaccumulative potential of a chemical. The approach has been analysed using literature data on well-known bioaccumulative compounds and liver metabolism data from the ECVAM database and a subset of the ToxCast phase I chemical library-in total 94 compounds covering pharmaceuticals, plant protection products and industrial chemicals. Our results provide further evidence that partitioning properties do not allow for a reliable screening criteria for human chemical hazard. Our model, based on a 100% intestinal absorption assumption, suggests that metabolic clearance, plasma protein-binding properties and renal excretion are the main factors in determining whether bioaccumulation will occur and its amount. It is essential that in vitro metabolic clearance tests with metabolic competent cell lines as well as plasma protein-binding assays be performed for suspected bioaccumulative compounds.

A model-based prioritisation exercise for the European water framework directive.

A model-based prioritisation exercise has been carried out for the Water Framework Directive (WFD) implementation. The approach considers two aspects: the hazard of a certain chemical and its exposure levels, and focuses on aquatic ecosystems, but also takes into account hazards due to secondary poisoning, bioaccumulation through the food chain and potential human health effects. A list provided by EU Member States, Stakeholders and Non-Governmental Organizations comprising 2,034 substances was evaluated according to hazard and exposure criteria. Then 78 substances classified as "of high concern" where analysed and ranked in terms of risk ratio (Predicted Environmental Concentration/Predicted No-Effect Concentration). This exercise has been complemented by a monitoring-based prioritization exercise using data provided by Member States. The proposed approach constitutes the first step in setting the basis for an open modular screening tool that could be used for the next prioritization exercises foreseen by the WFD.

A biology-based dynamic approach for the reconciliation of acute and chronic toxicity tests: application to Daphnia magna.

There is the need to integrate existing toxicity data in a coherent framework for extending their domain of applicability as well as their extrapolation potential. This integration would also reduce time and cost-consuming aspects of these tests and reduce animal usage. In this work, based on data extracted from literature, we have assessed the advantages that a dynamic biology-toxicant fate coupled model for Daphnia magna could provide when assessing toxicity data, in particular, the possibility to obtain from short-term (acute) toxicity test long-term (chronic) toxicity values taking into account the inherent variability of D. magna populations and the multiple sources of data. The results show that this approach overcomes some of the limitations of existing toxicity tests and that the prediction errors are considerably reduced when compared with the factor from 2 to 5 obtained using acute-to-chronic ratios.

Implementation of a 3D coupled hydrodynamic and contaminant fate model for PCDD/Fs in Thau Lagoon (France): the importance of atmospheric sources of contamination.

A 3D hydrodynamic and contaminant fate model was implemented for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in Thau lagoon. The hydrodynamic model was tested against temperature and salinity measurements, while the contaminant fate model was assessed against available data collected at different stations inside the lagoon. The model results allow an assessment of the spatial and temporal variability of the distribution of contaminants in the lagoon, the seasonality of loads and the role of atmospheric deposition for the input of PCDD/Fs. The outcome suggests that air is an important source of PCDD/Fs for this ecosystem, therefore the monitoring of air pollution is very appropriate for assessing the inputs of these contaminants. These results call for the development of integrated environmental protection policies.

Integrated modelling of Polycyclic Aromatic Hydrocarbons in the marine environment: coupling of hydrodynamic, fate and transport, bioaccumulation and planktonic food-web models.

Spatio-temporal variability of pollutants in the environment is a complex phenomenon that requires a combined approach for its analysis. Whereas data on measured levels of contaminants in various environmental compartments is essential, it is not always possible to monitor at the necessary frequency and with the adequate spatial sampling distribution to capture this variability. Therefore a modelling approach able to complement experimental data and close the gaps in the monitoring programs is useful for assessing the contaminant dynamics occurring at different time scales. In this work a 1D water column fate model has been developed and tested for Polycyclic Aromatic Hydrocarbons (PAHs). The model has been coupled with a simple ecological model that includes a bioaccumulation module. Afterwards, the model has been used to study the temporal variability of contaminant concentrations as well as the fluxes between compartments. The results evidence the complex coupling between spatio-temporal scales and its influence on environmental concentration levels.

Modelling the influence of thermal stratification and complete mixing on the distribution and fluxes of polychlorinated biphenyls in the water column of Ispra Bay (Lake Maggiore).

A 1D coupled hydrodynamic and contaminant fate model was applied to simulate the distribution of polychlorinated biphenyls (PCBs) in the Ispra Bay located in the southern part of Lake Maggiore (Italy). The model succeeded in representing the hydrodynamic processes occurring in the lake such as thermal stratification during summer 2005 followed by the complete mixing of the water column in February 2006. The results of the PCB fate model highlighted that these processes play a key role for the settling of particles and consequently for the distribution of PCBs in the water column as well as for the contaminant flux at the sediment-water interface. On the air-water front, the simulations emphasised that the net atmospheric PCB input fluxes are generally more important during the cold season and show peaks during periods of high wet deposition. Finally, the seasonal variability of the distribution of PCB in the water column was assessed.

Joint effects of nutrients and contaminants on the dynamics of a food chain in marine ecosystems.

We analyze the joint effect of contaminants and nutrient loading on population dynamics of marine food chains by means of bifurcation analysis. Contaminant toxicity is assumed to alter mortality of some species with a sigmoidal dose-response relationship. A generic effect of pollutants is to delay transitions to complex dynamical states towards higher nutrient load values, but more counterintuitive consequences arising from indirect effects are described. In particular, the top predator seems to be the species more affected by pollutants, even when contaminant is toxic only to lower trophic levels.

A bioaccumulation model for herbicides in Ulva rigida and Tapes philippinarum in Sacca di Goro lagoon (Northern Adriatic).

A bioaccumulation model to predict concentrations of s-triazine herbicides in the macroalgae Ulva rigida and in clams Tapes philippinarum has been implemented, calibrated and validated. The model uses input data from a 3D biogeochemical model that provides biomasses in the different compartments, i.e. phytoplankton, zooplankton and bacteria; and from a 3D fate model that provides the herbicides concentrations in the water column as well as in the sediments. Simulated data were compared with experimental data collected during a set of sampling campaigns carried out in 2004 and 2005 in the Sacca di Goro lagoon (Northern Adriatic). The model predicts correctly the concentrations of herbicides measured in Ulva rigida and reproduces with good agreement the values of concentration of herbicides found in clams. Furthermore, the simulated spatial and temporal dynamics in the biota compartment, following those of the water and sediments, are also in agreement with the experimental data. This integrated approach combining biogeochemical, fate and bioaccumulation models provide an overall assessment of the importance of the different environmental compartments and it can also support the testing of different management strategies to improve ecosystem state and functioning. Further research is necessary to elucidate the role and importance of the metabolism of these compounds by clams.

Analysis of perfluorooctanoate (PFOA) and other perfluorinated compounds (PFCs) in the River Po watershed in N-Italy.

C7-C11 perfluorinated carboxylates (PFACs) and perfluorooctansulfonate (PFOS) were analysed in selected stretches of the River Po and its major tributaries. Analyses were performed by solid-phase extraction (SPE) with Oasis HLB cartridges and methanol elution followed by LC-MS-MS detection using 13C-labelled internal standards. High concentration levels ( approximately 1.3 microg l(-1)) of perfluorooctanoate (PFOA) were detected in the Tánaro River close to the city Alessandria. After this tributary, levels between 60 and 337 ng l(-1) were measured in the Po River on several occasions. The PFOA concentration close to the river mouth in Ferrara was between 60 and 174 ng l(-1). Using the river discharge flow data in m3 s(-1) at this point (average approximately 920 m3 s(-1) for the year 2006), a mass load of approximately 0.3 kg PFOA per hour or approximately 2.6 tons per year discharged in the Adriatic Sea has been calculated. PFOS concentration levels in the Po River at Ferrara were approximately 10 ng l(-1).

Fate of persistent organic pollutants in the water column: does turbulent mixing matter?

The effect of vertical turbulent mixing on the dynamics of persistent organic pollutants has long been overlooked and its role is still hardly understood. Here we present the first comprehensive analysis of the role of turbulent diffusion on the distribution of those contaminants and its interplay with sinking fluxes. To this end, a 1D dynamic coupled hydrodynamic-contaminant model has been developed and applied to a Mediterranean continental shelf environment. The hydrodynamic sub-model is adapted from COHERENS, the contaminant sub-model is an improvement from the BIODEP model and considers the contaminant in 3 phases: dissolved-colloidal-particulate. The simulation highlights the role of turbulence in determining the POP distribution and variability in the water column. In short, turbulent flux of contaminants strengthens the upward diffusion of sediment entrained contaminants and determines the extent to which inputs from the atmosphere mix into the water column. It acts in parallel with degradation and sinking fluxes, the combined effect yielding a surface enriched - depth depleted - benthic layer enriched region distribution, which presents similarities to reported experimental measures.