Next generation physiologically based kinetic (NG-PBK) models in support of regulatory decision making.

The fields of toxicology and chemical risk assessment seek to reduce, and eventually replace, the use of animals for the prediction of toxicity in humans. In this context, physiologically based kinetic (PBK) modelling based on in vitro and in silico kinetic data has the potential to a play significant role in reducing animal testing, by providing a methodology capable of incorporating in vitro human data to facilitate the development of in vitro to in vivo extrapolation of hazard information. In the present article, we discuss the challenges in: 1) applying PBK modelling to support regulatory decision making under the toxicology and risk-assessment paradigm shift towards animal replacement; 2) constructing PBK models without in vivo animal kinetic data, while relying solely on in vitro or in silico methods for model parameterization; and 3) assessing the validity and credibility of PBK models built largely using non-animal data. The strengths, uncertainties, and limitations of PBK models developed using in vitro or in silico data are discussed in an effort to establish a higher degree of confidence in the application of such models in a regulatory context. The article summarises the outcome of an expert workshop hosted by the European Commission Joint Research Centre (EC-JRC) - European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), on "Physiologically-Based Kinetic modelling in risk assessment - reaching a whole new level in regulatory decision-making" held in Ispra, Italy, in November 2016, along with results from an international survey conducted in 2017 and recently reported activities occurring within the PBK modelling field. The discussions presented herein highlight the potential applications of next generation (NG)-PBK modelling, based on new data streams.

Theoretical and mathematical foundation of the Virtual Cell Based Assay - A review.

There is a need to interpret in vitro concentration-viability data in terms of the actual concentration that the cells are exposed to, rather than the nominal concentration applied to the test system. We have developed a process-based model to simulate the kinetics and dynamics of a chemical compound in cell-based in vitro assays. In the present paper we describe the mathematical equations governing this model as well as the parameters that are needed to run the model. The Virtual Cell Based Assay (VCBA) is an integrated model composed of: [1] a fate and transport model; [2] a cell partitioning model; [3] a cell growth and division model; [4] a toxicity and effects model; [5] the experimental set up. The purpose of the VCBA is to simulate the medium and intracellular concentrations, which can be used on its own to design and interpret in vitro experiments, and in combination with physiologically based kinetic (PBK) models to perform in vitro to in vivo extrapolation. The results can be used in chemical risk assessment to link an external dose to an internal effect or vice versa, using solely in vitro and in silico tools and thereby avoiding animal testing.

Practical use of the Virtual Cell Based Assay: Simulation of repeated exposure experiments in liver cell lines.

The Virtual Cell Based Assay (VCBA) was applied to simulate the long-term (repeat dose) toxic effects of chemicals, including substances in cosmetics and personal care products. The presented model is an extension of the original VCBA for simulation of single exposure and is implemented in a KNIME workflow. This work illustrates the steps taken to simulate the repeated dose effects of two reference compounds, caffeine and amiodarone. Using caffeine, in vitro experimental viability data in single exposure from two human liver cell lines, HepG2 and HepaRG, were measured and used to optimize the VCBA, subsequently repeated exposure simulations were run. Amiodarone was then tested and simulations were performed under repeated exposure conditions in HepaRG. The results show that the VCBA can adequately predict repeated exposure experiments in liver cell lines. The refined VCBA model can be used not only to support the design of long term in vitro experiments but also practical applications in risk assessment. Our model is a step towards the development of in silico predictive approaches to replace, refine, and reduce the in vivo repeated dose systemic toxicity studies in the assessment of human safety.

Development of a standard documentation protocol for communicating exposure models.

An important step in building a computational model is its documentation; a comprehensive and structured documentation can improve the model applicability and transparency in science/research and for regulatory purposes. This is particularly crucial and challenging for environmental and/or human exposure models that aim to establish quantitative relationships between personal exposure levels and their determinants. Exposure models simulate the transport and fate of a contaminant from the source to the receptor and may involve a large set of entities (e.g. all the media the contaminants may pass though). Such complex models are difficult to be described in a comprehensive, unambiguous and accessible way. Bad communication of assumptions, theory, structure and/or parameterization can lead to lack of confidence by the user and it may be source of errors. The goal of this paper is to propose a standard documentation protocol (SDP) for exposure models, i.e. a generic format and a standard structure by which all exposure models could be documented. For this purpose, a CEN (European Committee for Standardisation) workshop was set up with objective to agree on minimum requirements for the amount and type of information to be provided on exposure models documentation along with guidelines for the structure and presentation of the information. The resulting CEN workshop agreement (CWA) was expected to facilitate a more rigorous formulation of exposure models description and the understanding by users. This paper intends to describe the process followed for defining the SDP, the standardisation approach, as well as the main components of the SDP resulting from a wide consultation of interested stakeholders. The main outcome is a CEN CWA which establishes terms and definitions for exposure models and their elements, specifies minimum requirements for the amount and type of information to be documented, and proposes a structure for communicating the documentation to different users.

In vitro-to-in vivo correlation of the skin penetration, liver clearance and hepatotoxicity of caffeine.

This work illustrates the use of Physiologically-Based Toxicokinetic (PBTK) modelling for the healthy Caucasian population in in vitro-to-in vivo correlation of kinetic measures of caffeine skin penetration and liver clearance (based on literature experiments), as well as dose metrics of caffeine-induced measured HepaRG toxicity. We applied a simple correlation factor to quantify the in vitro and in vivo differences in the amount of caffeine permeated through the skin and concentration-time profiles of caffeine in the liver. We developed a multi-scale computational approach by linking the PBTK model with a Virtual Cell-Based Assay to relate an external oral and dermal dose with the measured in vitro HepaRG cell viability. The results revealed higher in vivo skin permeation profiles than those determined in vitro using identical exposure conditions. Liver clearance of caffeine derived from in vitro metabolism rates was found to be much slower than the optimised in vivo clearance with respect to caffeine plasma concentrations. Finally, HepaRG cell viability was shown to remain almost unchanged for external caffeine doses of 5-400 mg for both oral and dermal absorption routes. We modelled single exposure to caffeine only.

Identification of nevadensin as an important herb-based constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect.

Estragole is a natural constituent of several herbs and spices including sweet basil. In rodent bioassays, estragole induces hepatomas, an effect ascribed to estragole bioactivation to 1'-sulfooxyestragole resulting in DNA adduct formation. The present paper identifies nevadensin as a basil constituent able to inhibit DNA adduct formation in rat hepatocytes exposed to the proximate carcinogen 1'-hydroxyestragole and nevadensin. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1'-hydroxyestragole. The Ki for SULT inhibition by nevadensin was 4 nM in male rat and human liver fractions. Furthermore, nevadensin up to 20 microM did not inhibit 1'-hydroxyestragole detoxification by glucuronidation and oxidation. The inhibition of SULT by nevadensin was incorporated into the recently developed physiologically based biokinetic (PBBK) rat and human models for estragole bioactivation and detoxification. The results predict that co-administration of estragole at a level inducing hepatic tumors in vivo (50mg/kg bw) with nevadensin at a molar ratio of 0.06, representing the ratio of their occurrence in basil, results in almost 100% inhibition of the ultimate carcinogen 1'-sulfooxyestragole when assuming 100% uptake of nevadensin. Assuming 1% uptake, inhibition would still amount to more than 83%. Altogether these data point at a nevadensin-mediated inhibition of the formation of the ultimate carcinogenic metabolite of estragole, without reducing the capacity to detoxify 1'-hydroxyestragole via glucuronidation or oxidation. These data also point at a potential reduction of the cancer risk when estragole exposure occurs within a food matrix containing SULT inhibitors compared to what is observed upon exposure to pure estragole.

Maternal cardiovascular function in pregnancies complicated by intrauterine growth restriction.

OBJECTIVE: To investigate maternal cardiovascular function in pregnancies complicated by intrauterine growth restriction (IUGR). METHODS: Maternal echocardiography and ambulatory blood pressure monitoring were performed in pregnancies complicated by IUGR (n = 12) and controls (n = 12), all of whom were normotensive at enrollment. RESULTS: Compared to controls, maternal blood pressure (P = 0.016) and total vascular resistance (P = 0.008) were higher in IUGR pregnancies. Heart rate was lower (P = 0.003), as was systolic function expressed by midwall fractional shortening (P = 0.04). No significant differences between the two groups were observed for left atrial or left ventricular dimensions, nor for left ventricular geometry. Assessment of diastolic function by means of transmitral Doppler flow measurements revealed a significantly longer isovolumetric relaxation time in pregnancies with IUGR (P = 0.006). CONCLUSIONS: In normotensive pregnancies complicated by IUGR, as compared to controls, there is decreased diastolic and systolic maternal cardiac function, and a higher blood pressure.