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For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

This deliverable describes the overall workflow followed in EuroMix Work Package 2. This workflow, which is proposed to be used as a first tier in-silico risk assessment of chemical mixtures in general, consists of four steps: 1) The first step is the identification of chemicals in food or feed that are of interest for combined exposures. A list of 1598 substances has been identified specifically for the purpose of EuroMix, and is described in D2.1 “Report describing cumulative assessment groups for a broad range of chemicals, based on information extracted from (literature) databases” . This list – the EuroMix Chemical Inventory – is subsequently completed with the results from the in-silico tools and calculations performed in EuroMix Work Package 2, described in detail in Deliverables 2.2, 2.3 and this deliverable (see next steps). 2) Determining the probability that a chemical belongs to a certain Cumulative Assessment Group (CAG). The level at which CAGs need to be determined for mixture risk assessment (level 1, organ toxicity, level 2: organ effect phenotype, level 3: mode of action, i.e. cellular processes, and level 4: mechanism of action, or molecular initiating events) is not established yet, as indications for this should come from the experimental work packages in EuroMix. The present deliverable D2.4 outlines the work-flows using existing QSAR models described in EuroMix D2.2 (Report on the use of in‐silico methods for the prioritisation of substances and mixtures thereof), in combination with the results from detailed molecular docking calculations performed as new research in the EuroMix project and described in D2.3. 3) Assignment of a relative potency factor to each chemical that was considered in the previous step to be part of the CAG of interest. These relative potencies are used to scale the exposures of all mixture components. RPF derivation using Threshold of Toxicological Concern (TTC) concepts, and Molecular Docking results were described in D2.3. Additionally a proposal for the use of read across based on chemical similarity is given in this deliverable. Interpretation of molecular docking based RPFs, as well as the interpretation of in vitro modelling results from WP3 in EuroMix, requires In Vitro to In Vivo Extrapolation. A first (QSAR based) tier to IVIVE is presented in section 6 of this deliverable. 4) Finally the mixture risk is determined by adding up all the scaled exposures of the mixture components that are predicted to be part of the Cumulative Assessment Group. As exposure considerations are not taken into account at all at this stage – for a large part of the EuroMix Inventory no exposure data will be available – the final risk assessment calculations are not part of this Deliverable. This workflow and all the data obtained with the tools and model predictions will be implemented in the EuroMix software tool as part of the EuroMix Work Package 6. The individual tools used in the mixture risk assessment workflow can be used to do prioritizations and rankings, i.e. to find the (predicted) most potent substances for a specific CAG. In section 5 of this Deliverable, these prioritizations have been performed to generate lists of potentially interesting substances - based on their predicted effect / mechanism of action and potency – for further testing in the in-vitro toolbox (EuroMix work package 3).

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Additional file of An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

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Additional file 1:PDF file containing Figures S1, S2, S3, S4 and S5 and Tables S1, S2, S3, S4, S5 and S6, mentioned in the text. (PDF 711 KB)

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