Non-animal approaches for toxicokinetics in risk evaluations of food chemicals

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs versus that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics, thereby increasing regulatory acceptance of non-animal kinetic data

Expert opinions on the acceptance of alternative methods in food safety evaluations : Formulating recommendations to increase acceptance of non-animal methods for kinetics

Inclusion of alternative methods that replace, reduce, or refine (3R) animal testing within regulatory safety evaluations of chemicals generally faces many hurdles. The goal of the current work is to i) collect responses from key stakeholders involved in food safety evaluations on what they consider the most relevant factors that influence the acceptance and use of 3R methods and to ii) use these responses to formulate activities needed to increase the acceptance and use of 3R methods, particularly for kinetics. The stakeholders were contacted by e-mail for their opinions, asking the respondents to write down three barriers and/or drivers and scoring these by distributing 5 points over the three factors. The main barriers that obtained the highest aggregated scores were i) uncertain predictability 3R methods/lack of validation, ii) insufficient guidance regulators/industry and iii) insufficient harmonization of legislation. The major driver identified was the possibility of 3R methods to provide more mechanistic information. Based on the results, recommendations are given to enhance the acceptance and application of 3R toxicokinetic methods in food safety evaluations. These include steering of regulatory data requirements as well as creating (funding) opportunities for development and validation of alternative methods for kinetics and development of guidances

Correction: Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

In the original publication of the article, the dilution of DAPT in sentence “To promote intestinal differentiation,…” was published with an error. The “5 mM DAPT” should be “5 μM DAPT”. The original article has been updated with the correct dilution.</p

Expert opinions on the acceptance of alternative methods in food safety evaluations: Formulating recommendations to increase acceptance of non-animal methods for kinetics

Inclusion of alternative methods that replace, reduce, or refine (3R) animal testing within regulatory safety evaluations of chemicals generally faces many hurdles. The goal of the current work is to i) collect responses from key stakeholders involved in food safety evaluations on what they consider the most relevant factors that influence the acceptance and use of 3R methods and to ii) use these responses to formulate activities needed to increase the acceptance and use of 3R methods, particularly for kinetics. The stakeholders were contacted by e-mail for their opinions, asking the respondents to write down three barriers and/or drivers and scoring these by distributing 5 points over the three factors. The main barriers that obtained the highest aggregated scores were i) uncertain predictability 3R methods/lack of validation, ii) insufficient guidance regulators/industry and iii) insufficient harmonization of legislation. The major driver identified was the possibility of 3R methods to provide more mechanistic information. Based on the results, recommendations are given to enhance the acceptance and application of 3R toxicokinetic methods in food safety evaluations. These include steering of regulatory data requirements as well as creating (funding) opportunities for development and validation of alternative methods for kinetics and development of guidances

Bioassay-directed analysis-based identification of relevant pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC–MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC–Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes

Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.</p

Quantitative in vitro-to-in vivo extrapolation (QIVIVE) of estrogenic and anti-androgenic potencies of BPA and BADGE analogues

The goal of the present study was to obtain an in vivo relevant prioritization method for the endocrine potencies of different polycarbonate monomers, by combining in vitro bioassay data with physiologically based kinetic (PBK) modelling. PBK models were developed for a selection of monomers, including bisphenol A (BPA), two bisphenol F (BPF) isomers and four different bisphenol A diglycidyl ethers (BADGEs), using in vitro input data. With these models, the plasma concentrations of the compounds were simulated, providing means to estimate the dose levels at which the in vitro endocrine effect concentrations are reached. The results revealed that, whereas the in vitro relative potencies of different BADGEs (predominantly anti-androgenic effects) can be up to fourfold higher than BPA, the estimated in vivo potencies based on the oral equivalent doses are one to two orders of magnitude lower than BPA because of fast detoxification of the BADGEs. In contrast, the relative potencies of 2,2-BPF and 4,4-BPF increase when accounting for the in vivo availability. 4,4-BPF is estimated to be fivefold more potent than BPA in humans in vivo in inducing estrogenic effects and both 2,2-BPF and 4,4-BPF are estimated to be, respectively, 7 and 11-fold more potent in inducing anti-androgenic effects. These relative potencies were considered to be first-tier estimates, particularly given that the potential influence of intestinal metabolism on the in vivo availability was not accounted for. Overall, it can be concluded that both 2,2-BPF and 4,4-BPF are priority compounds

Perfluoroalkyl substances (PFASs) decrease the expression of recombination-activating genes (RAG1 and RAG2) in human B lymphoma Namalwa cells

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse effects, including hepatotoxicity, developmental toxicity and immunotoxicity. So far, little information is available about the mechanisms underlying the toxicity of PFASs, including those related to their immunotoxicity. Reported immunotoxic effects of PFASs include decreased antibody responses in experimental animals and humans, indicating that PFASs may, among others, affect B cell function. In the present study, we first assessed the effects of PFOA on the transcriptome of the human Namalwa B cell line using RNA seq analysis. Gene expression changes, analyzed using Ingenuity Pathway Analysis, pointed to various cellular processes affected by PFOA, including ‘B cell development’ and ‘Primary immunodeficiency signaling’. Interestingly, PFOA decreased the expression of RAG1 and RAG2, genes involved in immunoglobulin and T cell receptor V(D)J recombination. As a next step, time- and concentration-dependent changes in the expression of RAG1 and RAG2 upon exposure to PFOA, PFNA, PFHxS and PFOS were studied through RT-qPCR analysis. Analysis with the concentration–response modeling software PROAST resulted in the following potency ranking: PFNA > PFOA > PFOS > PFHxS. Altogether, the present in vitro study provides insights into the effects of selected PFASs on B cells, identifying RAG1 and RAG2 expression as possible relevant targets that may play a role in the immunotoxicity of PFASs

Determination of genotoxic potencies of pyrrolizidine alkaloids in HepaRG cells using the γH2AX assay

Pyrrolizidine alkaloids (PAs) are secondary metabolites from plants that have been found in substantial amounts in herbal supplements, infusions and teas. Several PAs cause cancer in animal bioassays, mediated via a genotoxic mode of action, but for the majority of the PAs, carcinogenicity data are lacking. It is assumed in the risk assessment that all PAs have the same potency as riddelliine, which is considered to be one of the most potent carcinogenic PAs in rats. This may overestimate the risks, since many PAs are expected to have lower potencies. In this study we determined the concentration-dependent genotoxicity of 37 PAs representing different chemical classes using the γH2AX in cell western assay in HepaRG human liver cells. Based on these in vitro data, PAs were grouped into different potency classes. The group with the highest potency consists particularly of open diester PAs and cyclic diester PAs (including riddelliine). The group of the least potent or non-active PAs includes the monoester PAs, non-esterified necine bases, PA N-oxides, and the unsaturated PA trachelanthamine. This study reveals differences in in vitro genotoxic potencies of PAs, supporting that the assumption that all PAs have a similar potency as riddelliine is rather conservative.</p