•Reliable effect biomarkers are available for most of the relevant MoAs.•Increasing AOP knowledge fosters the use of effect biomarkers in regulatory context.•PBK/D models allow interpretation and ...simulation of biomarkers of effect.•An inter-regulatory setting of effect-based trigger values is demanded.•Effect-biomarkers have in many cases reached a level of maturity ensuring use in mixture assessments.
Effect biomarkers can be used to elucidate relationships between exposure to environmental chemicals and their mixtures with associated health outcomes, but they are often underused, as underlying biological mechanisms are not understood. We aim to provide an overview of available effect biomarkers for monitoring chemical exposures in the general and occupational populations, and highlight their potential in monitoring humans exposed to chemical mixtures. We also discuss the role of the adverse outcome pathway (AOP) framework and physiologically based kinetic and dynamic (PBK/D) modelling to strengthen the understanding of the biological mechanism of effect biomarkers, and in particular for use in regulatory risk assessments. An interdisciplinary network of experts from the European chapter of the International Society for Exposure Science (ISES Europe) and the Organization for Economic Co-operation and Development (OECD) Occupational Biomonitoring activity of Working Parties of Hazard and Exposure Assessment group worked together to map the conventional framework of biomarkers and provided recommendations for their systematic use. We summarized the key aspects of this work here, and discussed these in three parts. Part I, we inventory available effect biomarkers and promising new biomarkers for the general population based on the H2020 Human Biomonitoring for Europe (HBM4EU) initiative. Part II, we provide an overview AOP and PBK/D modelling use that improved the selection and interpretation of effect biomarkers. Part III, we describe the collected expertise from the OECD Occupational Biomonitoring subtask effect biomarkers in prioritizing relevant mode of actions (MoAs) and suitable effect biomarkers. Furthermore, we propose a tiered risk assessment approach for occupational biomonitoring.
Several effect biomarkers, especially for use in occupational settings, are validated. They offer a direct assessment of the overall health risks associated with exposure to chemicals, chemical mixtures and their transformation products. Promising novel effect biomarkers are emerging for biomonitoring of the general population. Efforts are being dedicated to prioritizing molecular and biochemical effect biomarkers that can provide a causal link in exposure-health outcome associations. This mechanistic approach has great potential in improving human health risk assessment. New techniques such as in silico methods (e.g. QSAR, PBK/D modelling) as well as ‘omics data will aid this process.
Our multidisciplinary review represents a starting point for enhancing the identification of effect biomarkers and their mechanistic pathways following the AOP framework. This may help in prioritizing the effect biomarker implementation as well as defining threshold limits for chemical mixtures in a more structured way. Several ex vivo biomarkers have been proposed to evaluate combined effects including genotoxicity and xeno-estrogenicity. There is a regulatory need to derive effect-based trigger values using the increasing mechanistic knowledge coming from the AOP framework to address adverse health effects due to exposure to chemical mixtures. Such a mechanistic strategy would reduce the fragmentation observed in different regulations. It could also stimulate a harmonized use of effect biomarkers in a more comparable way, in particular for risk assessments to chemical mixtures.
•PBK models have helped to facilitate quantitative in vitro to in vivo extrapolation.•PBK modelling has the potential to play a significant role in reducing animal testing.•It is critical to assess ...the validity of PBK models built using non-animal data.•A framework is needed for communicating characteristics and results of PBK modelling.
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.
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.
•The VCBA simulates intracellular concentrations of chemicals under repeated exposure.•This is a step towards animal-free repeated dose testing.•Will aid the refinement of in vitro testing.
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.
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•The VCBA simulates intracellular concentrations of chemicals in an in vitro system.•The VCBA can be used with PBK models to perform in vitro to in vivo extrapolation.•The VCBA can be applied to interpret and refine in vitro experiments.
OBJECTIVE:Non-alcoholic fatty liver disease (NAFLD) may be detected in nearly 40% of subjects with a body mass index > 25, with a dramatic increase over time. Aim of the present observational study ...was to evaluate the prevalence of cardiovascular risk factors in a selected population of patients with primary NAFLD, and to correlate the degree of steatosis and fibrosis with the individual cardiovascular risk.
DESIGN AND METHOD:We investigated 31 patient with primary NAFLD. In all patients an ultrasonographic evaluation of the liver was performed, and the degree of steatosis and fibrosis were established according to validates scores (mild, moderate or severe steatosis by ultrasonography, NAFLD fibrosis score and FIB 4 score according to clinical parameters). Cardiovascular risk was calculated according to 5 different scoresProgetto Cuore of the Italian Health Institute, Framingham score 2004 – ATP III (POINT Total e 10 years risk) Framingham risk score 2008, ACC/AHA ASCVD risk score 2013 and ACC/AHA ASCVD risk score new model 2 2018).
RESULTS:FIB 4 score was closely correlated with ACC/AHA score 2013 and 2018 (p = 0.0038 and 0.0317 respectively) while NAFLD fibrosis score was correlated with ACC/AHA score 2013 and 2018 as well as with Framingham risk score 2008 (p = 0.0109, 0.005 and 0.0223 respectively). Then, the relationship between degrees of steatosis and cardiovascular risk was assessed with the Mann Whitney test. There was a significant difference for three cardiovascular risk scores (ACC/AHA score 2013, 2018 and Framingham risk score 2008) between patients with absent-mild or moderate-severe steatosis (p = 0,0162, 0,0210 and 0,0336 respectively). The χ square test provided similar results, however a significant difference was detected also with Framingham score 2004 10 years risk).
CONCLUSIONS:Our study confirmed the high prevalence cardiovascular risk factors in patients with NAFLD, and demonstrated a correlation between steatosis (evaluated by ultrasonography) and fibrosis (evaluated by clinical indices) severity and cardiovascular risk. Some scores of cardiovascular risk (ACC/AHA ASCVD 2013 e new model 2 2018) are more useful that others in the evaluation of individual cardiovascular risk over time in patients with NAFLD, while the Italian Progetto Cuore score performed poorly.
•We used toxicokinetic modelling in vitro-to-in vivo correlation studies of caffeine.•We performed multiscale modelling.•We compared in vitro and in vivo skin permeation of caffeine.•We compared in ...vitro and in vivo liver clearance of caffeine.•We studied the relationship between external dose and HepaRG cell viability.
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.
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.
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•Improving documentation of exposure models will increase transparency in regulation.•A standard documentation protocol (SDP) for exposure models will improve transparency.•The SDP development was based on a wide consultation of interested stakeholders.•The format and structure of the SDP can facilitate exposure models description.
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