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The future of risk assessment cannot neglect to consider the vast literature produced through the application of new approach methodologies (NAMs). This, however, constitutes a challenge for the risk assessor, as the availability of data in this context is huge and heterogeneous both for the methods applied and the standardisation and quality of the results. The integration of results generated from NAMs is hence only feasible under some degree of automation of the risk assessment workflow, specifically for searching, extracting and integrating such results in “AOP-like” knowledge networks (AOP – Adverse Outcome Pathway). Artificial intelligence (AI) with its state-of-the-art methods and tools is one of the most promising sources to support automation of manual tasks among modern technologies. The present paper illustrates the results of the exploration of possible applications of AI to achieve this goal. After the introduction of an evaluation framework to quantitatively assess these tools and methods, the results of the implementation of six selected case studies with the support of a selection of such tools in a dedicated workflow are presented. A qualitative survey of the state-of-the-art tools and methods, which also incorporates the experience gathered during the case study implementation, is then presented. Finally, recommendations are formulated which address the main aspects identified through the case study implementation that should, in the opinion of the authors, be pursued by EFSA in the context of its SPIDO NAMs and AI roadmaps. In summary, potentials for AI tool support could be identified throughout the workflow. Although many of the tasks can be supported by (semi-)automation, experience showed that subject matter experts need to be involved in all workflow steps.

Aloe-emodin, a natural hydroxyanthraquinone, exerts both adverse and protective effects. This study aimed at investigating these potential effects of aloe-emodin in humans upon the use of food supplements and herbal medicines using a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach. For this, PBK models in rats and humans were established for aloe-emodin including its active metabolite rhein and used to convert in vitro data on hepatotoxicity, nephrotoxicity, reactive oxidative species (ROS) generation, and Nrf2 induction to corresponding in vivo dose-response curves, from which points of departure (PODs) were derived by BMD analysis. The derived PODs were subsequently compared to the estimated daily intakes (EDIs) resulting from the use of food supplements or herbal medicines. It is concluded that the dose levels of aloe-emodin from food supplements or herbal medicines are unlikely to induce toxicity, ROS generation, or Nrf2 activation in liver and kidney.

Current climate trends are likely to expand the geographic distribution of the toxigenic microalgae and concomitant phycotoxins, making intoxications by such toxins a global phenomenon. Among various phycotoxins, saxitoxin (STX) acts as a neurotoxin that might cause severe neurological symptoms in mammals following consumptions of contaminated seafood. To derive a point of departure (POD) for human health risk assessment upon acute neurotoxicity induced by oral STX exposure, a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed. The PBK models for rats, mice, and humans were built using parameters from the literature, in vitro experiments, and in silico predictions. Available in vitro toxicity data for STX were converted to in vivo dose-response curves via the PBK models established for these three species, and POD values were derived from the predicted curves and compared to reported in vivo toxicity data. Interspecies differences in acute STX toxicity between rodents and humans were found, and they appeared to be mainly due to differences in toxicokinetics. The described approach resulted in adequate predictions for acute oral STX exposure, indicating that new approach methodologies, when appropriately integrated, can be used in a 3R-based chemical risk assessment paradigm.

Purpurin is a major anthraquinone present in the roots of Rubia cordifolia (madder). Purpurin is known to activate Nrf2 (Nuclear transcription factor erythroid 2-related factor 2) EpRE (electrophile responsive element) mediated gene expression as a potential beneficial effect. This study aimed to elucidate the balance between the electrophilicity or pro-oxidant activity of purpurin underlying the Nrf2 induction. For this, Nrf2 activation with modified intracellular glutathione (GSH) levels was measured in an Nrf2 CALUX reporter gene assay. In addition, both cell-free and intracellular ROS formation of purpurin with modified (intracellular) GSH levels at different pH were quantified using the DCF-DA assay. GSH adduct formation was evaluated by UPLC and LC-TOF-MS analysis. GSH and GSSG levels following purpurin incubations were quantified by LC-MS/MS. We show that Nrf2 induction by purpurin was significantly increased in cells with buthionine sulfoximine depleted GSH levels, while Nrf2 induction was decreased upon incubation of the cells with N-acetylcysteine being a precursor of GSH. In cell-free incubations, ROS formation increased with increasing pH pointing at a role for the deprotonated form of purpurin. Upon incubations of purpurin with GSH at physiological pH, GSH adduct formation appeared negligible (<1.5% of the added purpurin). The addition of GSH resulted in conversion of GSH to GSSG and significantly reduced the ROS formation. Together these results demonstrate that Nrf2 induction by purpurin originates from intracellular ROS formation and not from its electrophilicity, which becomes especially relevant when intracellular GSH levels can no longer scavenge the ROS. The present study demonstrated that the efficiency of intracellular Nrf2 activation by purpurin and related anthraquinones will depend on (i) their pKa and level of deprotonation at the intracellular pH, (ii) the oxidation potential of their deprotonated form and (iii) the intracellular GSH level

Bile acid homeostasis is vital for numerous metabolic and immune functions in humans. The enterohepatic circulation of bile acids is extremely efficient, with ~95% of the intestinal bile acids being reabsorbed. Disturbing intestinal bile acid uptake is expected to substantially affect intestinal and systemic bile acid levels. Here, we aimed to predict the effects of apical sodium-dependent bile acid transporter (ASBT)-inhibition on systemic plasma levels. For this, we combined the in vitro Caco-2 cell transport assays with physiologically based (PBK) modeling. For this proof-of-principle study we used the selective ASBT-inhibitor odevixibat (ODE) as a model compound. Caco-2 cells grown on culture inserts were used to obtain transport kinetic parameters of glycocholic acid (GCA). The apparent Michaelis Menten constant (Km,app), apparent maximal intestinal transport rate (Vmax,app) and ODE’s inhibitory constant (Ki) were determined for GCA. These kinetic parameters were incorporated in a PBK model and used to predict the ASBT inhibition effects on plasma bile acid levels. GCA is transported over Caco-2 cells in an active and sodium-dependent manner, indicating the presence of functional ASBT. ODE inhibited GCA transport dose-dependently. The PBK model predicted that oral doses of ODE reduced conjugated bile acid levels in plasma. Our simulations match in vivo data and provide a first proof-of-principle for the incorporation of active intestinal bile acid uptake in a bile acid PBK model. This approach could in future be of use to predict the effects of other ASBT-inhibitors on plasma and intestinal bile acid levels.

Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes. ThermoBE4 exhibits a three times larger window of activity compared with the corresponding SpyCas9 base editor (BE4), which may be an advantage for gene mutagenesis applications. Hence, ThermoCas9 provides an alternative platform that expands the targeting scope of both genome and base editing in human cells.

Plastic particles in the nanometer range–called nanoplastics–are environmental contaminants with growing public health concern. As plastic particles are present in water, soil, air and food, human exposure via intestine and lung is unavoidable, but possible health effects are still to be elucidated. To better understand the Mode of Action of plastic particles, it is key to use experimental models that best reflect human physiology. Novel assessment methods like advanced cell models and several alternative approaches are currently used and developed in the scientific community. So far, the use of cancer cell line-based models is the standard approach regarding in vitro nanotoxicology. However, among the many advantages of the use of cancer cell lines, there are also disadvantages that might favor other approaches. In this review, we compare cell line-based models with stem cell-based in vitro models of the human intestine and lung. In the context of nanoplastics research, we highlight the advantages that come with the use of stem cells. Further, the specific challenges of testing nanoplastics in vitro are discussed. Although the use of stem cell-based models can be demanding, we conclude that, depending on the research question, stem cells in combination with advanced exposure strategies might be a more suitable approach than cancer cell lines when it comes to toxicological investigation of nanoplastics.

Worldwide use of organophosphate pesticides as agricultural chemicals aims to maintain a stable food supply, while their toxicity remains a major public health concern. A common mechanism of acute neurotoxicity following organophosphate pesticide exposure is the inhibition of acetylcholinesterase (AChE). To support Next Generation Risk Assessment for public health upon acute neurotoxicity induced by organophosphate pesticides, physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed in this study, with fenitrothion (FNT) as an exemplary organophosphate pesticide. Rat and human PBK models were parametrized with data derived from in silico predictions and in vitro incubations. Then, PBK model-based QIVIVE was performed to convert species-specific concentration-dependent AChE inhibition obtained from in vitro blood assays to corresponding in vivo dose-response curves, from which points of departure (PODs) were derived. The obtained values for rats and humans were comparable with reported no-observed-adverse-effect levels (NOAELs). Humans were found to be more susceptible than rats toward erythrocyte AChE inhibition induced by acute FNT exposure due to interspecies differences in toxicokinetics and toxicodynamics. The described approach adequately predicts toxicokinetics and acute toxicity of FNT, providing a proof-of-principle for applying this approach in a 3R-based chemical risk assessment paradigm.