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Little is known about the bioaccumulation potential of manufactured nanomaterials (MNs). For traditional chemicals, the Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 305, bioaccumulation in fish is often used. However, for MNs, there are no approved processes to trigger or waive this test, or consider alternatives to vertebrate animals. The aim of the present study was to conduct a meta-analysis of existing data sets on particle properties and bioaccumulation in earthworms to understand what particle metrics could be used as a trigger for bioaccumulation testing. An apparent steady state tissue concentration of metal from MNs exposure in the earthworm (Eisenia fetida) was evident following exposures to Ag nanoparticles (NPs), CuO NPs and CdTe quantum dots (QDs). This allowed the derivation of nano bioaccumulation factors (nBAFs), calculated using soil and earthworm tissue metal concentrations. A prediction equation using all the particle metrics correlated with BAFs was possible. Similarly, nano biomagnification factors (nBMFs) were calculated in the rainbow trout (Oncorhynchus mykiss) tissue, relative to the concentration of total metals in the fish diet. Pearson's correlations were found to be significant, with p < 0.05 for nBMFs for the liver, mid intestine, hind intestine and kidney relative to the earthworm tissue nBAFs. Together these data indicate that bioaccumulation measurements in earthworms for metallic MNs could be predictive of those values in fish, and that there is scope to predict the bioaccumulation potential of MNs with confidence from a few simple particle metrics.

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.

The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.

Biotransformation is one of the main mechanisms used by the body to eliminate drugs. As drug molecules become more complicated, the involvement of drug metabolizing enzymes increases beyond those that are typically studied, such as the cytochrome P450 enzymes. In this review, we try to capture the many outstanding articles that were published in the past year in the field of biotransformation (see Table 1). We have divided the articles into two categories of (1) metabolites and drug metabolizing enzymes, and (2) bioactivation and safety. This annual review is the fifth of its kind since 2016 (Baillie et al. 2016; Khojasteh et al. 2017, 2018, 2019). This effort in itself also continues to evolve. We have followed the same format we used in previous years in terms of the selection of articles and the authoring of each section. I am pleased of the continued support of Rietjens, Miller, Zhang, Driscoll and Mitra to this review. We would like to welcome Klarissa D. Jackson as a new author for this year's issue. We strive to maintain a balance of authors from academic and industry settings. We would be pleased to hear your opinions of our commentary, and we extend an invitation to anyone who would like to contribute to a future edition of this review. Cyrus Khojasteh, on behalf of the authors.

The structure of the gut barrier and luminal chemistry in non-mammalian vertebrates and invertebrates has been given little attention with respect to the dietary uptake of engineered nanomaterials (ENMs). This review compares the diversity of gut anatomy in selected species used for regulatory toxicity testing, especially in relation to gut lumen chemistry and the behaviour of ENMs, and the gut as a barrier to ENMs. High ionic strength, the presence of divalent ions and organic matter promote particle aggregation in the lumen. The redox chemistry of the gut offers reducing conditions for ENM transformation, and corona formation will depend on the gut contents. Areas of low pH in the gut lumen in several species will promote the dissolution of metallic ENMs. There is a protective unstirred layer over the surface of the epithelium that may concentrate ENMs. Some organisms, especially vertebrates, can slough mucus to remove this adsorbed nanomaterial and lower bioavailability. Invertebrates also have protective layers of cuticle or peritrophic membranes that will modulate ENM uptake. Paracellular uptake of ENMs is unlikely. Transcellular uptake via vesicular-dependent pathways remains the most likely route across the gut epithelium. Most species have receptor-mediated endocytosis pathways and/or macropinocytosis in the gut epithelium. Crucially, many invertebrates have another potential pathway via 'intracellular digestion' uptake routes leading into the gut epithelium, and with gut associated immune cells being a potential route for ENM translocation across the epithelium. The basal lamina provides another barrier prior to the internal compartments of many animals. The features of the gut lumen and epithelium can limit the uptake of ENMs across the gut barrier in vivo, although some ENMs are detected in the tissues. Invertebrates also have the ability for biogenic mineral formation at the nano scale inside tissues. In conclusion, despite the diverse structural anatomies of the gut barrier of animals, some common features in the gut lumen chemistry tend to promote particle aggregation and settling onto the gut surface. The functional anatomy ensures the gut remains a formidable barrier to ENMs, and with some potential novel uptake processes in invertebrates that are not present in vertebrate animals.

International audience; Pesticides are now recognised to interact with drug transporters, but only few data are available on this issue for carbamate pesticides, a widely-used class of agrochemicals, to which humans are highly exposed. The present study was therefore designed to determine whether four representative carbamate pesticides, i.e., the insecticides aminocarb and carbofuran, the herbicide chlorpropham and the fungicide propamocarb, may impair activities of main drug transporters implicated in pharmacokinetics.The interactions of carbamates with solute carrier and ATP-binding cassette transporters were investigated using cultured transporter-overexpressing cells, reference substrates and spectrofluorimetry-, liquid chomatography/tandem mass spectrometry- or radioactivity-based methods.Aminocarb and carbofuran exerted no or minimal effects on transporter activities, whereas chlorpropham inhibited BCRP and OAT3 activities and propamocarb decreased those of OCT1 and OCT2, but cis-stimulated that of MATE2-K. Such alterations of transporters however required chlorpropham/propamocarb concentrations in the 5-50 µM range, likely not relevant to environmental exposure. Trans-stimulation assays and propamocarb accumulation experiments additionally suggested that propamocarb is not a substrate for OCT1, OCT2 and MATE2-K.These data indicate that some carbamate pesticides can interact in vitro with some drug transporters, but only when used at concentrations higher than those expected to occur in environmentally-exposed humans.

Bile acids (BA) fulfill a wide range of physiological functions, but are also involved in pathologies, such as cholestasis. Cholestasis is characterized by an intrahepatic accumulation of BAs and subsequent spillage to the systemic circulation. The aim of the present study was to develop physiologically based kinetic (PBK) models that would provide a tool to predict dose-dependent BA accumulation in humans upon treatment with a Bile Salt Export Pump (BSEP) inhibitor. We developed a PBK model describing the BA homeostasis using glycochenodeoxycholic acid as an exemplary BA. Population wide distributions of BSEP abundances were incorporated in the PBK model using Markov Chain Monte Carlo simulations, and alternatively the total amount of BAs was scaled empirically to describe interindividual differences in plasma BA levels. Next, the effects of the BSEP inhibitor bosentan on the BA levels were simulated. The PBK model developed adequately predicted the in vivo BA dynamics. Both the Markov Chain Monte Carlo simulations based on a distribution of BSEP abundances and empirical scaling of the total BA pool readily described the variations within and between data in human volunteers. Bosentan treatment disproportionally increased the maximum BA concentration in individuals with a large total BA pool or low BSEP abundance. Especially individuals having a large total BA pool size and a low BSEP abundance were predicted to be at risk for rapid saturation of BSEP and subsequent intrahepatic BA accumulation. This model provides a first estimate of personalized safe therapeutic external dose levels of compounds with BSEP-inhibitory properties.

Trace metals, such as mercury (Hg) and lead (Pb), have been reported to be immunotoxic to animals, however, relatively little is known about their effects and modes of action for avian species, especially waterfowl, which are frequently linked to the transmission of avian influenza infections. Given the fact that wild waterfowl are natural reservoirs for avian influenza viruses, impaired immune competence and pathogen resistance due to trace metal exposure in wild waterfowl may raise the risks of infections and might result in avian influenza outbreaks. Therefore, it is crucial to investigate the modulatory effects of trace metals, namely Hg(II) and Pb(II) in this thesis, on the immune responses of waterfowl. The objective of this thesis was to investigate whether realistic environmental levels of Hg(II) and Pb(II) exposure could modulate the immune responses in waterfowl upon a viral-like immune challenge and to explore the underlying modes of action. With the stepwise approach, we were able to compare the effects of Hg(II) and Pb(II) in the field study with the controlled in vivo and in vitro studies. The major effects of Hg(II) and Pb(II) were promoting inflammation and impairing B-lymphocyte functions upon a viral-like immune challenge. This disturbance of the avian immune system potentially hampers the immune response upon viral infections, which might cause a higher risk of infection for individuals and even prevalence of diseases in the population as a whole. Altogether, this thesis indicates that even at low environmentally relevant levels, Hg(II) and Pb(II) exposure affected the immune responses in waterfowl upon a viral-like immune challenge, mainly by promoting inflammation and impairing B-cell functions. The current study emphasises the potential need to include immunomodulatory effects on birds in the environmental risk assessment of chemicals. This is relevant not only for the protection of wildlife populations, but also towards sustaining environmental and human health.

Toxicology is moving away from animal testing towards in vitro tools to assess chemical safety. This new testing framework requires a quantitative method, i.e. kinetic modelling, which extrapolates effective concentrations in vitro to a bioequivalent human dose in vivo and which can be applied on “high throughput screening” of a wide variety of chemicals. Generic physiologically based kinetic (PBK) models help account for the role of toxicokinetics in setting human toxic exposure levels. Furthermore these models may be parameterized only on in silico QSARs and in vitro metabolism assays, thereby circumventing the use of in vivo toxicokinetics for this purpose. Though several such models exist their applicability domains have yet to be comprehensively assessed. This study extends previous evaluations of the PBK model IndusChemFate and compares it with its more complex biological complement (“TNO Model”). Both models were evaluated with a broad span of chemicals, varying regarding physicochemical properties. The results reveal that the “simpler” performed best, illustrating that IndusChemFate can be a useful first-tier for simulating toxicokinetics based on QSARs and in vitro parameters. Finally, proper quantitative in vitro to in vivo extrapolation conditions were illustrated starting with acetaminophen induced in vitro cytotoxicity in human HepaRG cells.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients in food. In this publication, tenth in the series, NFCs containing a high percentage of at least one naturally occurring allylalkoxybenzene constituent with a suspected concern for genotoxicity and/or carcinogenicity are evaluated. In a related paper, ninth in the series, NFCs containing anethole and/or eugenol and relatively low percentages of these allylalkoxybenzenes are evaluated. The Panel applies the threshold of toxicological concern (TTC) concept and evaluates relevant toxicology data on the NFCs and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s), the estimated intake of the constituent is compared to the TTC for compounds with structural alerts for genotoxicity and when exceeded, a margin of exposure (MOE) is calculated. BMDL10 values are derived from benchmark dose analyses using Bayesian model averaging for safrole, estragole and methyl eugenol using EPA's BMDS software version 3.2. BMDL10 values for myristicin, elemicin and parsley apiole were estimated by read-across using relative potency factors. Margins of safety for each constituent congeneric group and MOEs for each allylalkoxybenzene constituent for each NFC were determined that indicate no safety concern. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food. Ten NFCs, derived from basil, estragon (tarragon), mace, nutmeg, parsley and Canadian snakeroot were determined or affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

The FEMA Expert Panel program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavoring ingredients in food has resulted in the publication of an updated constituent-based procedure as well as publications on the safety evaluation of many botanical-derived NFCs. This publication, ninth in the series and related to the ninth publication, describes the affirmation of the generally recognized as safe (GRAS) status for NFCs with propenylhydroxybenzene and allylalkoxybenzene constituents under their conditions of intended use as flavoring ingredients added to food. The Panel's procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology for the NFCs themselves and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s) with suspected genotoxic potential, the estimated intake of the individual constituent is compared to the TTC for compounds with structural alerts for genotoxicity and if exceeded, a margin of exposure is calculated using BMDL10 values derived from benchmark dose analyses using Bayesian model averaging, as presented in the tenth article of the series. Safety evaluations for NFCs derived from allspice, anise seed, star anise, sweet fennel seed and pimento leaves were conducted and their GRAS status was affirmed for use as flavoring ingredients. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food.

International audience; Mycotoxins and their metabolites are a family of compounds that contains a great diversity of both structure and biological properties. Information on their toxicity is spread within several databases and in scientific literature. Due to the number of molecules and their structure diversity, the cost and time required for hazard evaluation of each compound is unrealistic. In that purpose, new approach methodologies (NAMs) can be applied to evaluate such large set of molecules. Among them, quantitative structure-activity relationship (QSAR) in silico models could be useful to predict the mutagenic and carcinogenic properties of mycotoxins. First, a complete list of 904 mycotoxins and metabolites was built. Then, some known mycotoxins were used to determine the best QSAR tools for mutagenicity and carcinogenicity predictions. The best tool was further applied to the whole list of 904 mycotoxins. At the end, 95 mycotoxins were identified as both mutagen and carcinogen and should be prioritized for further evaluation.

Bile acid metabolism and transport are critical to maintain bile acid homeostasis and host health. In this study, it was investigated if effects on intestinal bile acid deconjugation and transport can be quantified in vitro model systems using mixtures of bile acids instead of studying individual bile acids. To this end deconjugation of mixtures of selected bile acids in anaerobic rat or human fecal incubations and the effect of the antibiotic tobramycin on these reactions was studied. In addition, the effect of tobramycin on the transport of the bile acids in isolation or in a mixture across Caco-2 cell layers was characterized. The results demonstrate that both the reduction of bile acid deconjugation and transport by tobramycin can be adequately detected in vitro systems using a mixture of bile acids, thus eliminating the need to characterize the effects for each bile acid in separate experiments. Subtle differences between the experiments with single or combined bile acids point at mutual competitive interactions and indicate that the use of bile acid mixtures is preferred over use of single bile acid given that also in vivo bile acids occurs in mixtures.

In this study, the ability of a new in vitro/in silico quantitative in vitro–in vivo extrapolation (QIVIVE) methodology was assessed to predict the in vivo neurotoxicity of tetrodotoxin (TTX) in rodents. In vitro concentration–response data of TTX obtained in a multielectrode array assay with primary rat neonatal cortical cells and in an effect study with mouse neuro-2a cells were quantitatively extrapolated into in vivo dose–response data, using newly developed physiologically based kinetic (PBK) models for TTX in rats and mice. Incorporating a kidney compartment accounting for active renal excretion in the PBK models proved to be essential for its performance. To evaluate the predictions, QIVIVE-derived dose–response data were compared with in vivo data on neurotoxicity in rats and mice upon oral and parenteral dosing. The results revealed that for both rats and mice the predicted dose–response data matched the data from available in vivo studies well. It is concluded that PBK modeling-based reserve dosimetry of in vitro TTX effect data can adequately predict the in vivo neurotoxicity of TTX in rodents, providing a novel proof-of-principle for this methodology.

In next generation risk assessment (NGRA), non-animal approaches are used to quantify the chemical concentrations required to trigger bioactivity responses, in order to assure safe levels of human exposure. A limitation of many in vitro bioactivity assays, which are used in an NGRA context as new approach methodologies (NAMs), is that toxicokinetics, including biotransformation, are not adequately captured. The present study aimed to include, as a proof of principle, the bioactivity of the metabolite hydroxyflutamide (HF) in an NGRA approach to evaluate the safety of the anti-androgen flutamide (FLU), using the AR-CALUX assay to derive the NAM point of departure (PoD). The NGRA approach applied also included PBK modelling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE). The PBK model describing FLU and HF kinetics in humans was developed using GastroPlus™ and validated against human pharmacokinetic data. PBK model-facilitated QIVIVE was performed to translate the in vitro AR-CALUX derived concentration-response data to a corresponding in vivo dose-response curve for the anti-androgenicity of FLU, excluding and including the activity of HF (-HF and +HF, respectively). The in vivo benchmark dose 5% lower confidence limits (BMDL05) derived from the predicted in vivo dose-response curves for FLU, revealed a 440-fold lower BMDL05 when taking the bioactivity of HF into account. Subsequent comparison of the predicted BMDL05 values to the human therapeutic doses and historical animal derived PoDs, revealed that PBK modelling-facilitated QIVIVE that includes the bioactivity of the active metabolite is protective and provides a more appropriate PoD to assure human safety via NGRA, whereas excluding this would potentially result in an underestimation of the risk of FLU exposure in humans.

For manufactured nanomaterials (MNs), given the breadth of forms produced, it is not ethical or practical to test all materials using vertebrates. This study aimed to show how alternative methods could predict the in vivo bioaccumulation potential of MNs in fish. This included exploring the physico-chemical properties of MNs as predictors of bioaccumulation, using the ex vivo gut sac technique to measure total metal uptake, and an in chemico digestibility assay to simulate the bioaccessible metal in the gut lumen of fish. An apparent plateau in net metal accumulation by rainbow trout was evident from data on dietary exposures to CuO nanoparticles (NPs), Ag NPs or Ag2S NPs in vivo. From the metal concentrations in the tissues compared to the diet, it was possible to derive nano biomagnification factors (nBMFs). The nBMF for the liver showed the best correlations with the physico-chemical parameters, with a significant correlation to the particle dissolution rate (Spearman's correlation, p < 0.01). Moreover, there was a significant relationship between the total metal released in the stomach compartment of the digestibility assay and the total metal concentration in the liver of trout in vivo (Pearson's correlation coefficient, p = 0.02), suggesting the in chemico digestibility assay can predict bioaccumulation potential. The ex vivo gut sac technique also gave good correlations to in vivo results, with r2 values between 0.8-0.9. In conclusion, the meta-analyses supports the development of an integrated and tiered approach to bioaccumulation testing that considers the 3Rs (replacement, reduction, refinement) and minimises the use of the fish bioaccumulation test (OECD TG 305), for nanomaterials. This journal is

Organ-on-chip (OoC) technology is full of engineering and biological challenges, but it has the potential to revolutionize the Next-Generation Risk Assessment of novel ingredients for consumer products and chemicals. A successful incorporation of OoC technology into the Next-Generation Risk Assessment toolbox depends on the robustness of the microfluidic devices and the organ tissue models used. Recent advances in standardized device manufacturing, organ tissue cultivation and growth protocols offer the ability to bridge the gaps towards the implementation of organ-on-chip technology. Next-Generation Risk Assessment is an exposure-led and hypothesis-driven tiered approach to risk assessment using detailed human exposure information and the application of appropriate new (non-animal) toxicological testing approaches. Organ-on-chip presents a promising in vitro approach by combining human cell culturing with dynamic microfluidics to improve physiological emulation. Here, we critically review commercial organ-on-chip devices, as well as recent tissue culture model studies of the skin, intestinal barrier and liver as the main metabolic organ to be used on-chip for Next-Generation Risk Assessment. Finally, microfluidically linked tissue combinations such as skin–liver and intestine–liver in organ-on-chip devices are reviewed as they form a relevant aspect for advancing toxicokinetic and toxicodynamic studies. We point to recent achievements and challenges to overcome, to advance non-animal, human-relevant safety studies.

Pyrrolizidine alkaloids (PAs) are a large group of plant constituents of which especially the 1,2- unsaturated PAs raise a concern because of their liver toxicity and potential genotoxic carcinogenicity. This toxicity of PAs depends on their kinetics. Differences in absorption, distribution, metabolism, and excretion (ADME) characteristics of PAs may substantially alter the relative toxicity of PAs. As a result, kinetics will also affect relative potency (REP) values. The present review summarizes the current state-of-the art on PA kinetics and resulting consequences for toxicity and illustrates how physiologically-based kinetic (PBK) modelling can be applied to take kinetics into account when defining the relative differences in toxicity between PAs in the in vivo situation. We conclude that toxicokinetics play an important role in the overall toxicity of pyrrolizidine alkaloids. and that kinetics should therefore be considered when defining REP values for combined risk assessment. New approach methodologies (NAMs) can be of use to quantify these kinetic differences between PAs and their N-oxides, thus contributing to the 3Rs (Replacement, Reduction and Refinement) in animal studies.

Raw data and the outputs of openGUTS for paper Size- and sex-related sensitivity differences of aquatic crustaceans to imidacloprid.

The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 μM BaA and the developmental retardations induced by 15 μM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.

Field collected aquatic invertebrates are often used as test organisms in the refinement of the standard Tier 1 risk assessment of various pollutants. This approach can provide insights into the effects of pollutants on the natural environment. However, researchers often pragmatically select test organisms of a specific sex and/or size, which may not represent the sensitivity of the whole population. To investigate such intraspecies sensitivity differences, we performed standard acute toxicity and toxicokinetic tests with different size classes and sex of Gammarus pulex and Asellus aquaticus. Furthermore, toxicokinetics and toxicodynamics models were used to understand the mechanism of the intraspecies sensitivity differences. We used neonates, juveniles and male and female adults in separate dedicated experiments, in which we exposed the animals to imidacloprid and its bioactive metabolite, imidacloprid-olefin. For both species, we found that neonates were the most sensitive group. For G. pulex, the sensitivity decreased linearly with size, which can be explained by the size-related uptake rate constant in the toxicokinetic process and size-related threshold value in the toxicodynamic process. For A. aquaticus, female adults were least sensitive to imidacloprid, which could be explained by a low internal biotransformation of imidacloprid to imidacloprid-olefin. Besides, imidacloprid-olefin was more toxic than imidacloprid to A. aquaticus, with differences being 8.4 times for females and 2.7 times for males. In conclusion, we established size-related sensitivity differences for G. pulex and sex-related sensitivity for A. aquaticus, and intraspecies differences can be explained by both toxicokinetic and toxicodynamic processes. Our findings suggest that to protect populations in the field, we should consider the size and sex of focal organisms and that a pragmatic selection of test organisms of equal size and/or sex can underestimate the sensitivities of populations in the field.

Potential consequences of combined exposure to the selected food-borne alkenylbenzenes safrole and estragole or their proximate carcinogenic 1′-hydroxy metabolites were evaluated in vitro and in silico. HepG2 cells were exposed to 1′-hydroxyestragole and 1′-hydroxysafrole individually or in equipotent combination subsequently detecting cytotoxicity and DNA adduct formation. Results indicate that concentration addition adequately describes the cytotoxic effects and no statistically significant differences were shown in the level of formation of the major DNA adducts. Furthermore, physiologically based kinetic modeling revealed that at normal dietary intake the concentration of the parent compounds and their 1′-hydroxymetabolites remain substantially below the Km values for the respective bioactivation and detoxification reactions providing further support for the fact that the simultaneous presence of the two carcinogens or of their proximate carcinogenic 1′-hydroxy metabolites may not affect their DNA adduct formation. Overall, these results point at the absence of interactions upon combined exposure to selected food-borne alkenylbenzenes at realistic dietary levels of intake.

Pesticide use is essential for control of pests in horticultural crops and adequate production of food supplies for the ever increasing world population as well as for control of vector-borne diseases. Currently, OPs are among the most widely used pesticides in agriculture, and their residues have been found in various foods. Regarding human risk assessment, the points of departure (PODs) to set health based guidance values have been derived from data from animal studies, mostly on OP-induced acetylcholinesterase (AChE) inhibition. The use of such animal data is undesirable due to economical, ethical and scientific limitations. The main aim of this thesis was to develop non-animal approaches for the hazard assessment of OPs to be applied in human risk assessment and application of these novel approach methods (NAMs) to predict the potential of the OPs to inhibit AChE in vivo upon acute exposure, also taking combined exposure into account. Furthermore, this thesis aimed to obtain data on the occurrence of OPs on commonly consumed vegetables in Kenya, as such data, and related estimations of human exposure, are lacking. In the first step, commonly consumed vegetables in Kenya were screened for OPs (and carbamates) and the accompanying exposure and related health risks assessed. A total of 90 samples were analyzed by liquid chromatography/high-resolution tandem mass spectrometry. Residues of acephate, chlorpyrifos (CPF), methamidophos, omethoate and profenofos (PFF) were found in 22% of the samples, at levels ranging from 10 to 1343 μg/kg. The EU MRLs for these OPs was exceeded in 21%, 10%, 8% and 22% of the samples of French beans, kales, spinach, and tomatoes, respectively. CPF levels on spinach were calculated to result in an acute Health Quotient (HQ) of 3.3 and 2.2 for children and adults, respectively, upon high consumption, implying that potential health risks with respect to acute dietary exposure cannot be excluded. Calculations for chronic dietary exposure showed that all chronic HQs were below 1. The Health Index (HI) for the pesticides, i.e. the sum of the chronic HQs, was 0.54 and 0.34 for children and adults, respectively, implying no direct health concern upon chronic average consumption of the vegetables assessed..In the next step, a combined in vitro-in silico approach to predict AChE inhibition by the OP profenofos (PFF) in rats and humans by applying physiologically based kinetic (PBK) modelling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) was developed. A PBK model was developed for both species. PBK model parameter values for PFF conversion to 4-bromo-2-chlorophenol (BCP; detoxification pathway) were derived from in vitro incubations with liver microsomes, liver cytosol, and plasma from rats (catalytic efficiencies of 1.1, 2.8, and 0.19 ml/min/mg protein, respectively) and humans (catalytic efficiencies of 0.17, 0.79, and 0.063 ml/min/mg protein, respectively), whereas other chemical-related PBK model parameter values were derived using in silico calculations. The rat PBK model was evaluated against literature data on urinary excretion of conjugated BCP. Concentration-dependent inhibition of rat and human AChE by PFF was determined in vitro and these data were translated with the PBK models to predicted dose-dependent AChE inhibition in rats and humans in vivo. Comparing predicted dose-dependent AChE inhibition in rats to literature data on PFF-induced AChE inhibition revealed an accurate prediction of in vivo effect levels. Comparison of rat predictions (BMDL10 of predicted dose-response data of 0.45 mg/kg bw) and human predictions (BMDL10 of predicted dose-response data of 0.01 mg/kg bw) suggests that humans are more sensitive than rats, being mainly due to differences in kinetics. Results obtained demonstrate that in vivo AChE inhibition upon acute exposure to PFF was closely predicted in rats, indicating the potential of this NAM in chemical hazard assessment.In order to consider OP-mixture effects, this study assessed combination effects of PFF and chlorpyrifos’ (CPF) toxic metabolite chlorpyrifos oxon (CPO) on AChE inhibition in vitro, and the effects of CPF and CPO on PFF detoxification by human liver microsomes, cytosol, and plasma. PFF was 199-fold less potent than CPO in AChE inhibition, and combined effects of CPO and PFF followed principles of dose addition. CPF and CPO affected PFF detoxification to BCP in a non-competitive manner at high concentrations that are not expected to be reached in vivo. The PBK model of PFF was extended to include the description of CPF and CPO kinetics. PFF concentrations in the model were expressed as CPO-equivalents allowing prediction of total internal CPO-equivalents upon combined exposure to PFF and CPF. Different exposure scenarios were applied for which PBK model-predicted internal (unbound) CPO-equivalents were compared to CPO concentrations affecting AChE activity in vitro, to assess whether in vivo effects on AChE activity are expected. These results reveals that AChE inhibition upon combined exposure to these OPs follows principles of dose addition, and that no effects of CPF on PFF detoxification are expected.Finally, this study determined human interindividual differences in PFF detoxification based on the combined in vitro-in silico approach. To that end, PFF conversion to BCP was studied in in vitro incubations with liver S9 fractions of 25 different Caucasian donors and plasma samples of 25 different Caucasian donors. The obtained information on the variation in the in vitro kinetic constants Vmax and Km was applied in the PBK model using a Monte Carlo simulation modelling approach. Analysis of the predicted internal maximum concentrations of the simulated population indicated a 1.4- and 1.5-fold difference between the geometric mean (GM) and the 90th and 99th percentile, respectively, of the simulated population, being smaller than the default uncertainty factor accounting for human variability in toxicokinetics of 3.16. Concentration-response curves for inhibition of human AChE determined in vitro were translated with the PBK model to predicted dose-dependent AChE inhibition in humans in vivo. In line with the differences in Cmax values, the 99th percentile of the simulated population was predicted to be 1.5-fold more sensitive to PFF-induced AChE inhibition than the GM of the simulated population based on the BMDL10 values derived from predicted dose-response data for the virtual population. This predicted BMDL10 of 0.0057 mg/kg bw (99th percentile of population) is comparable to an ARfD reported by EFSA (0.005 mg/kg bw) based on a German evaluation, and the ARfD reported by EPA (0.002 mg/kg bw), but much lower than the ARfD adopted by JMPR (1 mg/kg bw). In conclusion, integrating PBK modelling with Monte Carlo simulations using human in vitro data provides a powerful strategy to quantify human interindividual variation in kinetics, which can be used to refine the hazard characterization of OPs.Altogether, the present thesis presents a NAM-based hazard assessment of OP-induced AChE inhibition upon single or combined exposure to CPF and PFF. Since a generic bottom-up PBK modelling approach was applied, the approach can be easily extended to include more OPs allowing a mechanism-based hazard and risk assessment of combined OP exposure.&nbsp

Due to the widespread application of food-relevant inorganic nanomaterials, the gastrointestinal tract is potentially exposed to these materials. Gut-on-chip in vitro model systems are proposed for the investigation of compound toxicity as they better recapitulate the in vivo human intestinal environment than static models, due to the added shear stresses associated with the flow of medium in line with what cells experience in vivo. We aimed to compare the cellular responses of intestinal epithelial Caco-2 cells at the gene expression level upon TiO2 (E171) and ZnO (NM110) nanomaterial exposure when cultured under dynamic and static conditions. For this, we applied whole genome transcriptome analyses. Differentially expressed genes and related biological processes revealed culture condition specific responses upon exposure to TiO2 and ZnO nanomaterials. The materials had more effects on cells cultured in the gut-on-chip when compared to the static model, indicating that shear stress might be a major factor in cell susceptibility. This is the first report on application of a gut-on-chip system to evaluate cellular responses upon TiO2 and ZnO nanomaterials compared to a static system and extends current knowledge on nanomaterial-cell interactions and toxicity assessment. Dynamically cultured cells appear to be more sensitive and the gut-on-chip might thus be an attractive model to be used more extensively in the toxicological hazard characterization.

Glycation products comprise a heterogenous group of compounds which can be formed upon thermal processing of food products and include advanced glycation end products (AGEs), such as carboxymethyllysine, and their precursors such as the Amadori product fructoselysine and α-dicarbonyl compounds like methylglyoxal, glyoxal and 3-deoxyglucosone. Besides being present in exogenous dietary sources, glycation products can also be produced endogenously inside the human body. AGEs have been associated with the development of multiple adverse health effects, such as diabetes and cardiovascular diseases, but whether exposure to exogenous dietary AGEs and their precursors contributes to these effects remains debated. In this thesis, an overview was presented of the toxicokinetics and toxicodynamics of exogenous and endogenous AGEs and their precursors which are of relevance to consider when evaluating whether exposure to exogenous dietary glycation products can contribute to the adverse health effects associated with AGEs. It was concluded that better characterization of the tested AGEs and precursors, and to distinguish between their low molecular mass (LMM) and high molecular mass (HMM) forms, as well as quantification of the exposure from endogenous formation relative to that resulting from dietary intake are all of importance to provide a definite conclusion on whether dietary exposure to glycation products can contribute to the adverse health effects associated to AGEs. In addition, it was concluded that part of the AGEs and their precursors remain unabsorbed upon dietary exposure and can reach the colon. Consequently at least part of the AGEs and their precursors can interact with the gut microbiota in a bi-directional manner. This can affect the toxicokinetics of the AGEs and their precursors but on the other hand can also result in exerted toxicodynamic effects by altering the gut microbiota composition and possibly function. The aim of this project was to characterize these interactions of the gut microbiota with selected dietary glycation products and vice versa, and to characterize inter- and intraindividual differences in gut microbial reactions using the Amadori product fructoselysine and the AGE carboxymethyllysine as model compounds. This was achieved by the application of an in vitro model where collected individual human fecal samples were incubated with the substrate of interest. Michaelis-Menten kinetic parameters could be obtained, in addition to information on metabolite formation (short chain fatty acid; SCFA). In addition to interindividual differences, intra-individual differences were quantified as well of microbial degradation of the AGE carboxymethyllysine and its precursor fructoselysine. The gut microbiota is a dynamic ecosystem and thus intraindividual temporal differences are of possible relevance for its functioning. The results revealed pronounced inter- and intraindividual variation in both carboxymethyllysine and fructoselysine degradation while the two degradation rates were not correlated (R2=0.08), showing the need to evaluate toxicokinetics for individual AGEs and precursors instead of evaluating them on a group level. In addition, multiple bacterial genera were correlated with the individuals’ carboxymethyllysine and fructoselysine degradation activities, corroborating that showing that the potential to convert fructoselysine and carboxymethyllysine may originate from multiple bacterial genera. The effect of different dietary exposures on microbial degradation was evaluated by comparing functional differences in microbial fructoselysine degradation between breast-fed (BF) and formula-fed (FF) infants, in view of their different exclusive diets and consequent different dietary fructoselysine exposures. This was done by evaluating a publicly available metagenome dataset analysis with metagenome assembled genomes (MAGs) from infant fecal samples which showed that genes involved in microbial fructoselysine degradation were present in multiple taxa in both BF and FF infant fecal samples but were higher prevalent in fecal samples from FF infants compared to the BF infants. Further collection of individual fecal samples from exclusively BF and FF infants showed that both groups were able to degrade fructoselysine anaerobically but fecal samples from the FF infants resulted in a significantly higher degradation activity compared to the BF infants. This indicated that the infant gut microbiota adapts towards dietary fructoselysine exposure. This dynamic adaptive aspect of the gut microbiota was also observed in mice which were exposed to a heated diet high in AGEs which resulted in an altered gut microbiota composition compared to mice exposed to the control diet. Exposure to the heated diet high in AGEs followed by the control diet (i.e. the switch group) showed that the altered gut microbiota composition was reversible and adapted to the dietary exposure. This reversibility was also observed for the accumulation of the tested AGEs in plasma, kidney and liver (as analyzed in their protein-bound and free form), a result that also pointed to at least partial bioavailability of the dietary AGEs and/or their precursors that appeared to be enriched in the heated chow diet.Overall, it can be concluded that the bi-directional relation of the gut microbiota with exogenous AGEs and their precursors is of relevance when evaluating their toxicokinetic and toxicodynamic characteristics. To accurately evaluate the hazards and risks of dietary exposure to AGEs and precursors, better characterization and quantification of the applied test substances and biological outcomes, and also of the exposure from endogenous formation relative to that resulting from dietary intake, are essential.&nbsp

The use of herbal supplements for sexual improvement and recreational purposes is a rather common practice in Ghana. Although the high accessibility, acceptability and assumed benefits of herbal products has promoted growth in the herbal industry, the presence of adulterants and other contaminants in these products is a major health concern.In this thesis, selected herbal supplements were initially screened using the PDE-Glo bioassay to determine their inhibition potentials against PDE-5 enzyme activity, the mode of action underlying their effect on erectile function. Results based on the bioassay revealed that about 90% of the samples were able to inhibit PDE-5 enzyme activity at various degrees, with estimated intakes of 25 samples (62.5%) pointing at daily doses higher than 25mg sildenafil equivalents and 9 (22.5%) of these even at doses higher than the maximum recommended daily intake of 100mg sildenafil equivalents. Further investigations were carried out to confirm if the observed effects were due to inherent plant constituents or merely the result of added synthetic PDE-5 enzyme inhibitors.Further chemical analysis revealed significant amounts of sildenafil and other approved PDE-5i in 13 out of the 40 supplements. Based on data from the chemical analysis, the estimated daily intakes (EDIs) of six out of the 13 even resulted in EDIs above 100 mg sildenafil equivalents. Only one sample contained a natural ingredient (icariin) potentially able to inhibit PDE-5 enzyme activity, but its concentration (0.013 mg/g) was too low to explain the observed response in the bioassay. The estimated concentrations of PDE5 inhibitors in 35 supplements as derived from the bioassay results, were in line with those of the LC-MS/MS analysis, while discrepancies were observed for 5 supplements. A tiered approach for one of the latter supplements using the PDE-Glo bioassay to select the positive fraction and further examination with LC-MS/MS and 1H-NMR revealed the presence of hydroxythiohomosildenafil, a sildenafil analogue not yet included in the LC-MS reference library. These results highlight the importance of the bioassay to detect unknown PDE-5 inhibitors.To further investigate the safety of the herbal supplements, the presence of contaminants such as polycyclic aromatic hydrocarbons (PAHs), dioxins (polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs)) and dl-PCBs possibly introduced into the products as a result of contaminated raw materials or during processing were also investigated. Selected supplements were initially screened using the DR CALUX® bioassay in order to identify the presence of stable (acid-silica clean-up and 48 hrs exposure) and less stable (without acid-silica clean-up and 4 hrs exposure) aryl hydrocarbon receptor (AhR)-agonists. The results revealed that the selected supplements contained only low levels of stable AhR-agonists like dioxins and dl-PCBs, while some contained less stable AhR-agonists like PAHs.Ten supplements selected for confirmation with GC-HRMS analysis contained PCDD/Fs and dl-PCBs at levels varying from 0.01 to 0.19 pg toxic equivalents (TEQ)/g, while the level of the sum of 4 polycyclic aromatic hydrocarbons (Σ4PAHs), representing less stable AhR agonists, ranged from not detected (ND) to 25.5 ng/g. These concentrations were in line with the responses observed in the DR CALUX® bioassay. The concentration of PCDD/Fs and dl-PCBs corresponded to EDIs ranging from 0.001 to 0.12 pg TEQ/kg bw/week for a 70 kg bw consumer, which is below the established tolerable weekly intake (TWI) of 2 pg TEQ/kg bw/week, thus indicating low concern for consumers’ health. Similarly, the EDIs based on the detected Σ4PAHs in supplements ranged from 7.2 to 111 ng/day, or 0.1 to 1.6 ng/kg bw/day, which compared to carcinogenic effects in animals corresponds to margin of exposure (MOE) values above 10,000, indicating a low health concern.Finally, the presence of pyrrolizidine alkaloids (PAs) in the 40 supplements as a result of contaminated raw materials and/or the unintentional co-harvesting of raw materials with PA containing weeds, was examined using liquid chromatography mass spectrometry (LC/MS). Up to 17 different PAs were identified in 19 out of the 40 samples. In decreasing order, the most detected PAs were rinderine (in 15 samples), its N-oxide (10), intermedine (11), its N-oxide (11), lycopsamine (8) and its N-oxide (5). The total sum of PAs in samples ranged from 0.005 to 3.2 µg/g, resulting in EDIs from 0.01 to 12 µg/day, corresponding to exposures from 0.0002 to 0.2 µg/kg bw/day for a 70 kilogram bodyweight person. When compared to a BMDL10 of 237 µg/kg bw/day for liver tumours in rats treated with riddelliine, the margin of exposure (MOE) values ranged from 1200 to 1,400,000 with 8 samples showing MOE values below 10,000, thus indicating a health concern upon long-term regular use of these 8 supplements.The presence of contaminants and especially added adulterants in the selected supplements compromises their purity and safety, the latter depending on the concentrations present. These findings are indicative of what is pertaining to the numerous herbal products available on the Ghanaian market. The practical approaches employed in this thesis, using a combination of a bioassay and chemical analysis to determine the presence and concentrations of added pharmaceuticals and undesirable contaminants is expected to be applicable in most laboratories. These approaches can be applied in investigating other herbal products available on the Ghanaian market.The adulteration of herbal products with active pharmaceutical ingredients and the presence of contaminants continues to be a problem for regulatory agencies globally including Ghana. In the present thesis, the presence of intentionally added adulterants in the selected herbal products shed a light on on-going practices in Ghana and provides some answers to the numerous reported casualties and deaths of consumers of sexual enhancing herbal products sold on the Ghanaian markets.&nbsp

Intestinal epithelial cells and the intestinal microbiota are in a mutualistic relationship that is dependent on communication. This communication is multifaceted, but one aspect is communication through compounds produced by the microbiota such as the short-chain fatty acids (SCFAs) butyrate, propionate and acetate. Studying the effects of SCFAs and especially butyrate in intestinal epithelial cell lines like Caco-2 cells has been proven problematic. In contrast to the in vivo intestinal epithelium, Caco-2 cells do not use butyrate as an energy source, leading to a build-up of butyrate. Therefore, we used human induced pluripotent stem cell derived intestinal epithelial cells, grown as a cell layer, to study the effects of butyrate, propionate and acetate on whole genome gene expression in the cells. For this, cells were exposed to concentrations of 1 and 10 mM of the individual short-chain fatty acids for 24 hours. Unique gene expression profiles were observed for each of the SCFAs in a concentration-dependent manner. Evaluation on both an individual gene level and pathway level showed that butyrate induced the biggest effects followed by propionate and then acetate. Several known effects of SCFAs on intestinal cells were confirmed, such as effects on metabolism and immune responses. The changes in metabolic pathways in the intestinal epithelial cell layers in this study demonstrate that there is a switch in energy source from glucose to SCFAs, thus induced pluripotent stem cell derived intestinal epithelial cell are responding in a similar manner to SCFAs as in vivo intestinal tissues. Overall design: Induced pluripotent stem cell derived intestinal epithelial cells were exposed to butyric acid (NaB; 1 or 10mM), acetic acid (NaA 1 or 10 mM), propionic acid, (NaP 1 or 10 mM) or control treatment (water) for 24 hours, and subjected to gene expression profiling by RNA-sequencing.

Due to the widespread application of food-relevant inorganic nanomaterials, the gastrointestinal tract is potentially exposed to these materials. Gut-on-chip in vitro systems are proposed for the investigation of compound toxicity as they better recapitulate the in vivo human intestinal environment than static models, due to the added shear stresses associated with the flow of the medium. We aimed to compare cellular responses of intestinal epithelial Caco-2 cells at the gene expression level upon TiO2 (E171) and ZnO (NM110) nanomaterial exposure when cultured under dynamic and conventionally applied static conditions. Whole-genome transcriptome analyses upon exposure of the cells to TiO2 and ZnO nanomaterials revealed differentially expressed genes and related biological processes that were culture condition specific. The total number of differentially expressed genes (p < 0.01) and affected pathways (p < 0.05 and FDR < 0.25) after nanomaterial exposure was higher under dynamic culture conditions than under static conditions for both nanomaterials. The observed increase in nanomaterial-induced responses in the gut-on-chip model indicates that shear stress might be a major factor in cell susceptibility. This is the first report on the application of a gut-on-chip system in which gene expression responses upon TiO2 and ZnO nanomaterial exposure are evaluated and compared to a static system. It extends current knowledge on nanomaterial toxicity assessment and the influence of a dynamic environment on cellular responses. Application of the gut-on-chip system resulted in higher sensitivity of the cells and might thus be an attractive system for use in the toxicological hazard characterization of nanomaterials.

Movalli, Paola et al., The chemical industry is the leading sector in the EU in terms of added value. However, contaminants pose a major threat and significant costs to the environment and human health. While EU legislation and international conventions aim to reduce this threat, regulators struggle to assess and manage chemical risks, given the vast number of substances involved and the lack of data on exposure and hazards. The European Green Deal sets a ‘zero pollution ambition for a toxic free environment’ by 2050 and the EU Chemicals Strategy calls for increased monitoring of chemicals in the environment. Monitoring of contaminants in biota can, inter alia: provide regulators with early warning of bioaccumulation problems with chemicals of emerging concern; trigger risk assessment of persistent, bioaccumulative and toxic substances; enable risk assessment of chemical mixtures in biota; enable risk assessment of mixtures; and enable assessment of the effectiveness of risk management measures and of chemicals regulations overall. A number of these purposes are to be addressed under the recently launched European Partnership for Risk Assessment of Chemicals (PARC). Apex predators are of particular value to biomonitoring. Securing sufficient data at European scale implies large-scale, long-term monitoring and a steady supply of large numbers of fresh apex predator tissue samples from across Europe. Natural science collections are very well-placed to supply these. Pan-European monitoring requires effective coordination among field organisations, collections and analytical laboratories for the flow of required specimens, processing and storage of specimens and tissue samples, contaminant analyses delivering pan-European data sets, and provision of specimen and population contextual data. Collections are well-placed to coordinate this. The COST Action European Raptor Biomonitoring Facility provides a well-developed model showing how this can work, integrating a European Raptor Biomonitoring Scheme, Specimen Bank and Sampling Programme. Simultaneously, the EU-funded LIFE APEX has demonstrated a range of regulatory applications using cutting-edge analytical techniques. PARC plans to make best use of such sampling and biomonitoring programmes. Collections are poised to play a critical role in supporting PARC objectives and thereby contribute to delivery of the EU’s zero-pollution ambition., This paper was carried out in collaboration between ‘European Raptor Biomonitoring Facility’ (COST Action 16224), and the project ‘LIFE APEX—Systematic use of contaminant data from apex predators and their prey in chemicals management’ (LIFE17 ENV/SK/000355). European Raptor Biomonitoring Facility is supported by COST (European Cooperation in Science and Technology) and funded by the Horizon 2020 Framework Programme of the European Union. LIFE APEX is funded by the LIFE Programme, the EU’s funding instrument for the environment and climate action.

Au regard du nombre d’animaux de compagnie en France, de leur médicalisation croissante et des interactions rapprochées avec leurs propriétaires, la question de l’exposition humaine accidentelle aux médicaments vétérinaires mérite d’être soulevée. En s’intéressant aux effets indésirables (EI) survenant chez l’humain consécutivement à l’usage de médicaments vétérinaires, la pharmacovigilance vétérinaire joue un rôle clé dans l’évaluation et l’identification des risques encourus par les utilisateurs. Une étude rétrospective des déclarations d’EI déclarés chez l’être humain en 2021 et collectées dans la base de pharmacovigilance de l’Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail-Agence nationale du médicament vétérinaire (Anses-ANMV) a été menée afin de mettre en évidence les pratiques et les médicaments les plus à risque, et de proposer des mesures de prévention.

The intestine fulfills roles in the uptake of nutrients and water regulation and acts as a gatekeeper for the intestinal microbiome. For the latter, the intestinal gut barrier system is able to respond to a broad range of bacterial antigens, generally through Toll-like receptor (TLR) signaling pathways. To test the capacity of various in vitro intestinal models, we studied IL-8 secretion, as a marker of pro-inflammatory response through the TLR pathway, in a Caco-2 monoculture, Caco-2/HT29-MTX di-culture, Caco-2/HT29-MTX/HMVEC-d tri-culture and in a HT29-p monoculture in response to exposure to various TLR agonists. Twenty-one-day-old differentiated cells in Transwells were exposed to Pam3CSK4 (TLR1/2), lipopolysaccharide (TLR4), single-stranded RNA (TLR7/8), Poly(i:C) (TLR3) and flagellin (TLR5) for 24 h. In all systems IL-8 secretion was increased in response to flagellin exposure, with HT29-p cells also responding to Poly(I:C) exposure. All other agonists did not induce an IL-8 response in the tested in vitro models, indicating that the specific TLRs are either not present or not functional in these models. This highlights the need for careful selection of in vitro models when studying intestinal immune responses and the need for improved in vitro models that better recapitulate intestinal immune responses.

Legislation and regulations around the globe currently use a mixture of hazard- and risk-based approaches for ensuring the safety of food. This chapter focusses on the pros and cons of hazard- versus risk-based approaches to food safety regulation providing examples to illustrate the concepts and arguments provided. Hazard-based approaches only perform the first of the four steps that are part of risk assessment including hazard identification, hazard characterization, exposure assessment, and risk assessment. Hazard-based approaches are, for example, common practice in the field of allergenicity or genotoxicity, and also show a tendency to be increasingly applied as a result of the need for the replacement, reduction, and refinement (3Rs) of animal testing, resulting in use of a wide range of in vitro assays elucidating hazards but not risks. Recent developments, however, indicate a need for risk-based approaches even in the field of genotoxicity testing and allergenicity, while physiologically-based kinetic modeling is able to translate in vitro data to the in vivo situation enabling a risk rather than a hazard-based approach. Risk management and risk communication based on hazard assessment lead to unnecessary risk mitigation actions and consumer fear. Risk perception and the impact of globalization are key considerations for the future. It is concluded that future developments should be directed at moving away from hazard- toward risk-based evaluations and educating the risk assessors, risk managers, and the public on the fact that a hazard does not translate into a risk at certain levels of exposure, even for genotoxic carcinogens or allergens.

Un risque environnemental nouvellement identifié avec la flunixine méglumine va conduire à des modifications de RCP des médicaments concernés. Il s'agit d'un risque sur la faune sauvage (oiseaux charognards en particulier).

Bile acid homeostasis plays an important role in many biological activities through the bile–liver–gut axis. In this study, two in vitro models were applied to further elucidate the mode of action underlying reported in vivo bile acid changes induced by antibiotics (colistin sulfate, tobramycin, meropenem trihydrate, and doripenem hydrate). 16S rRNA analysis of rat fecal samples anaerobically incubated with these antibiotics showed that especially tobramycin induced changes in the gut microbiota. Furthermore, tobramycin was shown to inhibit the microbial deconjugation of taurocholic acid (TCA) and the transport of TCA over an in vitro Caco-2 cell layer used as a model to mimic intestinal bile acid reuptake. The effects induced by the antibiotics in the in vitro model systems provide novel and complementary insight explaining the effects of the antibiotics on microbiota and fecal bile acid levels upon 28-day in vivo treatment of rats. In particular, our results provide insight in the mode(s) of action underlying the increased levels of TCA in the feces upon tobramycin exposure. Altogether, the results of the present study provide a proof-of-principle on how in vitro models can be used to elucidate in vivo effects on bile acid homeostasis, and to obtain insight in the mode(s) of action underlying the effect of an antibiotic, in this case tobramycin, on bile acid homeostasis via effects on intestinal bile acid metabolism and reuptake.

Les situations d’exposition professionnelle aux nanoparticules sont multiples. Les nombreuses applications des silices amorphes synthétiques font que les travailleurs sont particulièrement exposés par voie respiratoire lors des différentes phases de production, qui comprennent notamment le transfert, l’échantillonnage, la pesée, la mise en suspension mais aussi le conditionnement, l’emballage, le stockage et le transport des produits. Dans ce contexte, nous cherchons à mieux caractériser leurs effets sur la santé, notamment leur éventuel rôle d’adjuvant dans le déclenchement de réactions allergiques.

Traditional toxicity risk assessment approaches have until recently focussed mainly on histochemical readouts for cell death. Modern toxicology methods attempt to deduce a mechanistic understanding of pathways involved in the development of toxicity, by using transcriptomics and other big data-driven methods such as high-content screening. Here, we used a recently described optimised method to differentiate human induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs), to assess their potential to classify hepatotoxic and non-hepatotoxic chemicals and their use in mechanistic toxicity studies. The iPSC-HLCs could accurately classify chemicals causing acute hepatocellular injury, and the transcriptomics data on treated HLCs obtained by TempO-Seq technology linked the cytotoxicity to cellular stress pathways, including oxidative stress and unfolded protein response (UPR). Induction of these stress pathways in response to amiodarone, diclofenac, and ibuprofen, was demonstrated to be concentration and time dependent. The transcriptomics data on diclofenac-treated HLCs were found to be more sensitive in detecting differentially expressed genes in response to treatment, as compared to existing datasets of other diclofenac-treated in vitro hepatocyte models. Hence iPSC-HLCs generated by transcription factor overexpression and in metabolically optimised medium appear suitable for chemical toxicity detection as well as mechanistic toxicity studies. ispartof: TOXICS vol:10 issue:1 ispartof: location:Switzerland status: published

Respiratory irritation is an important human health endpoint in chemical risk assessment. There are two established modes of action of respiratory irritation, 1) sensory irritation mediated by the interaction with sensory neurons, potentially stimulating trigeminal nerve, and 2) direct tissue irritation. The aim of our research was to, develop a QSAR method to predict human respiratory irritants, and to potentially reduce the reliance on animal testing for the identification of respiratory irritants. Compounds are classified as irritating based on combined evidence from different types of toxicological data, including inhalation studies with acute and repeated exposure. The curated project database comprised 1997 organic substances, 1553 being classified as irritating and 444 as non-irritating. A comparison of machine learning approaches, including Logistic Regression (LR), Random Forests (RFs), and Gradient Boosted Decision Trees (GBTs), showed, the best classification was obtained by GBTs. The LR model resulted in an area under the curve (AUC) of 0.65, while the optimal performance for both RFs and GBTs gives an AUC of 0.71. In addition to the classification and the information on the applicability domain, the web-based tool provides a list of structurally similar analogues together with their experimental data to facilitate expert review for read-across purposes.

Mineral oil aromatic hydrocarbons (MOAH) have been presented as a modern emerging food safety concern by the European Food Safety Authority (EFSA). MOAH is defined as a chromatographic fraction in the analysis of mineral oil hydrocarbons (MOH) in food and does not represent well defined substances or mixtures. The sources of foodborne MOAH include environmental contamination and intentional uses of mineral oils in consumer products or production processes. Some non-substituted PAHs are known to be genotoxic carcinogens and have been extensively studied, however the majority of the compounds represented by MOAH are highly alkylated aromatic hydrocarbons for which the toxicological data are scarce. Potential concerns for genotoxicity, carcinogenicity and developmental toxicity upon MOAH exposure have been raised. However, the lack of toxicological data on related alkyl substituted polycyclic aromatic hydrocarbons (PAHs) seriously hampers a proper hazard assessment and therefore challenges the risk assessment of MOAH.The present thesis aims at (1) providing data on oxidative metabolism and resulting toxicities of a series of the selected non-substituted and alkyl-substituted aromatic hydrocarbons using in vitro assays and (2) developing a physiologically-based kinetic (PBK) modeling- facilitated quantitative in vitro-in vivo-extrapolation (QIVIVE) approach for prediction of in vivo toxicity of selected PAH. The latter is needed to define data for risk assessment as a proof of principle for using new approach methodologies (NAMs). It was hypothesized (1) that alkylated PAHs will be metabolized preferably on the alkyl side chain rather than on the condensed aromatic rings which can be expected to facilitate their excretion rather than their bioactivation to potentially DNA-reactive metabolites, and (2) that the presence of an additional bay region like aromatic motif introduced by methyl substitution may affect the genotoxic potential of the PAHs.

Our modern food chain may contain natural toxins from plants, either due to their natural presence in the plant-derived food or in the raw material used for food production or because these constituents are added during the regular production process, or present as contaminants. The present chapter presents an overview of situations where natural toxins from plants may raise concern, illustrated by examples of relevant structural features, toxic modes of action, their safety or risk assessment, and potential regulations related to their occurrence in the modern food chain. It is concluded that the safety concerns in part arise from the fact that botanicals and botanical preparations are considered food and thus a priori not subject to premarket evaluation and/or quality control for their safety in use. The chapter ends with a discussion presenting existing data gaps and research directions.

Inherited retinal diseases (IRDs) are a group of neurodegenerative disorders that lead to photoreceptor cell death and eventually blindness. IRDs are characterised by a high genetic heterogeneity, making it imperative to design mutation-independent therapies. Mutations in a number of IRD disease genes have been associated with a rise of cyclic 3’,5’-guanosine monophosphate (cGMP) levels in photoreceptors. Accordingly, the cGMP-dependent protein kinase (PKG) has emerged as a new potential target for the mutation-independent treatment of IRDs. However, the substrates of PKG and the downstream degenerative pathways triggered by its activity have yet to be determined. Here, we performed kinome activity profiling of different murine organotypic retinal explant cultures (diseased rd1 and wild-type controls) using multiplex peptide microarrays to identify proteins whose phosphorylation was significantly altered by PKG activity. In addition, we tested the downstream effect of a known PKG inhibitor CN03 in these organotypic retina cultures. Among the PKG substrates were potassium channels belonging to the Kv1 family (KCNA3, KCNA6), cyclic AMP-responsive element-binding protein 1 (CREB1), DNA topoisomerase 2-α (TOP2A), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (F263), and the glutamate ionotropic receptor kainate 2 (GRIK2). The retinal expression of these PKG targets was further confirmed by immunofluorescence and could be assigned to various neuronal cell types, including photoreceptors, horizontal cells, and ganglion cells. Taken together, this study confirmed the key role of PKG in photoreceptor cell death and identified new downstream targets of cGMP/PKG signalling that will improve the understanding of the degenerative mechanisms underlying IRDs.

This internal request followed on from the report on the “Inventory of alternatives to antibiotics aimed at reducing their use in animal husbandry” (Request No 2013-SA-0122) and from one of the conclusions of the report on the “Assessment of marketing authorisation applications for herbal veterinary medicinal products” (Request No 2014-SA-0081) concerning the primary challenge for MA applications for these substances: the lack of an appropriate maximum residue limit (MRL) status for the large majority of plants of interest in veterinary medicine. The issue of the MRL status of plants and herbal preparations, including essential oils (EOs), is fundamental for the preventive and curative phytotherapy and aromatherapy treatment of food-producing animals, both when assessing MA application dossiers and when prescribing a product for off-label use, for example an extemporaneous herbal preparation (principle of the “therapeutic cascade”, Art. L5143-4 of the French Public Health Code). In addition, the use of phyto/aromatherapy is becoming more and more widespread on farms, in response to the development of organic agriculture and the need to reduce the use of antibiotics (One Health, Ecoantibio plan), and also due to the development of xenobiotic resistance in all pathogens.The development of phyto/aromatherapy in food-producing animals requires a prior MRL assessment of plants and herbal preparations, including EOs, in order to guarantee a safe consumer exposure level. This assessment is the responsibility of the European Medicines Agency (EMA). In a context where efforts are being made to control resistance to antibiotics and other classes of xenobiotics and to find therapeutic alternatives, and in response to the development of organic agriculture, possibilities for assessing hazards and risks to consumers need to be studied, to meet the expectations of farmers, veterinarians and consumers. It is important to note that in phyto/aromatherapy, the definition and quality of products are essential. Strict botanical identification of the plant used is a prerequisite, as is the knowledge of its origin. Differences in varieties, cultivars or chemotypes, geographic locations and harvest periods are likely to induce widely varying compositions. Plant parts have to be defined. In addition, it is necessary to clearly define preparations. For extracts, the method used to treat the plant raw material, the extraction solvent and process (extraction temperature, duration, etc.), and the drug/extract ratio need to be defined. These factors influence the qualitative and quantitative chemical composition of preparations as well as their therapeutic potential and even their toxicity. Any purification processes implemented have to be defined. For aromatherapy, the recognised methods for extracting EOs for therapeutic purposes are defined in pharmacopoeias. These products generally have complex compositions and can sometimes contain more than 100 compounds. EOs can be rectified (crystallisation, distillation, fractioning, etc.), which adds another determining factor. As a result, the MRL approach (according to Regulation (EC) No 470/2009) appears to be unsuitable. Therefore, an internal request on plants and herbal preparations, including EOs, of interest in veterinary medicine seemed necessary to assess the risks to consumer health. The objective was to propose a consumer risk assessment approach taking into account the specific characteristics of these herbal products. The work carried out involved a preliminary review of the state of knowledge on plants and EOs of interest for phyto/aromatherapy in food-producing animals, in order to establish human health risk profiles using:- available data from the development of monographs for herbal medicinal products for human use;- available data from assessments of plants in other regulations, in particular those on animal feed and plant protection products;- the identification of plants and herbal preparations, including EOs, similar to those considered by EMA as not posing any risks to consumer health (listed in Table 1 of Regulation (EU) No 37/2010);- the identification of plants and herbal preparations, including EOs, whose toxicity is known in humans and that are also likely to pose a risk to consumers if used in veterinary phyto/aromatherapy.In conclusion, this work aimed to provide insights for the adoption of a tailored approach for granting an MRL status for plants and herbal preparations, including EOs.This work did not examine the efficacy or the benefit/risk ratio of plants used in veterinary medicine. It was a first step before a comprehensive assessment of the consumer health risks associated with the plants and herbal preparations, including EOs, used in phyto/aromatherapy for food-producing animals.[Related Request Nos 2014-SA-0081 and 2013-SA-0122], Cette autosaisine est une continuité du rapport « État des lieux des alternatives aux antibiotiques en vue de diminuer leur usage en élevage » (Anses 2018) et de l’une des conclusions du rapport « Evaluation des demandes d’autorisation de mise sur le marché (AMM) des médicaments à base de plantes » (Anses 2016) concernant la principale difficulté relative à leur demande d’autorisation d’AMM : l’absence de statut LMR (limites maximales de résidus) adéquat pour une grande majorité de plantes d’intérêt en médecine vétérinaire. Les LMR correspondent à des seuils réglementaires de résidus des substances contenues dans les médicaments vétérinaires que l’on retrouve dans les denrées alimentaires issues d’animaux traités.Elles sont définies pour une substance, une espèce et un tissu ou une denrée donnée. Elles visent à garantir un niveau d’exposition sans risque pour les consommateurs, ainsi qu’une absence d’impact technologique.Concernant la classification des substances pharmacologiquement actives administrées chez des animaux producteurs de denrées au regard des LMR, le règlement (UE) n°37/2010 comporte 2 tableaux :- le tableau 1 correspondant aux substances autorisées (avec la possibilité de restrictions d’usage et/ou d’espèces) ;- le tableau 2 correspondant aux substances interdites (lorsqu’aucune LMR ne peut être fixée).Certaines substances sont considérées, après évaluation par l’Agence européenne du médicament (EMA), comme ne relevant pas du champ d’application des LMR telles que définies dans le règlement (CE) n°470/2009 et sont alors inscrites sur une liste nommée « Out of scope » du comité du médicament vétérinaire de l’EMA ou Committee for Medicinal Products for Veterinary Use (CVMP) (EMA 2020c). Il s’agit notamment de substances naturellement présentes dans l’organisme ou de substances entrant dans l’alimentation humaine et qui ne présentent pas de danger pour la santé du consommateurSi le futur médicament vétérinaire à base de plantes est destiné à des animaux producteurs de denrées, chaque plante, préparation de plante ou huile essentielle (H.E.) qui le compose doit faire l’objet d’une inscription dans le tableau 1 du règlement (UE) n°37/2010 ou être inscrite dans la liste « out of scope ». Ainsi, la question du statut LMR des H.E. et des plantes est fondamentale pour le traitement préventif et curatif en ayant recours à la phytothérapie et l’aromathérapie, des animaux producteurs de denrées, tant dans le cadre de l’évaluation du dossier d’AMM que pour recourir à une prescription hors AMM par exemple en cas de prescription d’une préparation extemporanée à base de plantes (« principe de la cascade thérapeutique » art. L5143-4 du Code de la santé publique (CSP)).De plus, l’utilisation de la phyto / aromathérapie est en plein essor dans les élevages, en réponse au développement de l’agriculture biologique, à la nécessité de réduire l’utilisation d’antibiotiques (One Health4, Ecoantibio) et en raison du développement de résistances aux xénobiotiques de l’ensemble des pathogènes.Cependant, la grande majorité des plantes, préparations de plantes et H.E. fréquemment utilisées en médecine vétérinaire n’ont pas fait l’objet d’une évaluation au sens du règlement (CE) n°470/2009 et ne peuvent donc pas, à l’heure actuelle, entrer dans la composition de médicaments vétérinaires destinés à des animaux producteurs de denrées, ni être prescrites dans le cadre de la « cascade thérapeutique » (art. L.5143-4 du CSP) par un vétérinaire. Cependant, des produits à base de plantes médicinales sont souvent administrés aux animaux, en tant qu’aliments complémentaires ou encore sous forme alimentaire. On doit également rappeler que de nombreux emplois de la phytothérapie vétérinaire sont fondés sur une longue tradition d’usage anciennement très ancrée dans le monde rural.A ce jour, seules 2 spécialités vétérinaires à base de plantes à destination des animaux producteurs de denrées bénéficient d’une AMM en France : COTHIVET® et APILIFE VAR®. Chacune des substances entrant dans la composition de ces médicaments possède un statut LMR.Le développement de la phyto / aromathérapie chez les animaux producteurs de denrées requiert au préalable l’évaluation LMR des plantes et préparations de plantes dont les H.E. afin de garantir un niveau d’exposition sans risque pour les consommateurs. Cette évaluation est de la compétence de l’EMA. Dans le contexte de lutte contre la résistance aux antibiotiques et à d’autres familles de xénobiotiques, de recherche d’alternatives thérapeutiques, et en réponse au développement de l’agriculture biologique, les possibilités d’évaluation du danger et des risques pour le consommateur doivent donc être étudiées, pour répondre aux attentes des éleveurs, de nombreux vétérinaires et des consommateurs.Il est important de noter qu’en phyto / aromathérapie, la définition et la qualité des produits sont primordiales. L’identification botanique rigoureuse de la plante utilisée est un prérequis, ainsi que son origine. Des différences de variétés, de cultivars ou de chimiotype (CT), de localisation géographique, de période de récolte des plantes sont susceptibles d’induire des variations conséquentes de composition. Les parties de plantes doivent être définies. De même, une définition précise des préparations est nécessaire. Pour les extraits, le traitement de la matière première végétale, le solvant d’extraction et le procédé d’obtention (température, durée d’extraction…) ainsi que le ratio drogue / extrait (DER) doivent être définis. Ces facteurs conditionnent la composition chimique qualitative et quantitative des préparations, leurpotentiel thérapeutique voire leur toxicité. D’éventuels procédés de purification doivent être définis s’ils sont mis en œuvre. Pour l’aromathérapie (utilisation d’H.E.) les procédés d’obtention reconnus dans le cadre thérapeutique sont définis à la pharmacopée (EDQM 2019). Ces produits sont généralement de composition complexe et pourront parfois contenir plus d'une centaine de composés. Les H.E. peuvent être rectifiées (cristallisation, distillation fractionnée…), ce qui apporte un facteur supplémentaire de variation.De ce fait, l’approche LMR (selon le règlement (CE) n°470/2009) se révèle peu adaptée.Ainsi, une autosaisine relative aux plantes et préparations de plantes dont les H.E. d’intérêt en médecine vétérinaire semble nécessaire pour évaluer les risques pour la santé du consommateur. L’objectif est de proposer une approche permettant d’évaluer le risque pour le consommateur en tenant compte des particularités des produits à base de plantes.[Saisine liée « 2014-SA-0081 - évaluation des demandes d’AMM des médicaments à base de plantes » et « 2013-SA-0122 - état des lieux des alternatives aux antibiotiques en vue de diminuer leur usage en élevage »]

Green tea, which is mainly manufactured from buds and leaves of Camellia sinensis, is one of the most widely consumed beverages around the world. Catechins are the most abundant bioactive constituents in green teas, amounting up to 30% of the total dry weight. (-)-Epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC) are the four major catechins among others. These catechins are believed to be responsible for various beneficial health effects that have been ascribed to green tea consumption. Though the modes of action underlying these health-promoting effects can be complicated and have not been fully understood, the Kelch-like ECH-associated protein 1/Nuclear factor E2-related factor 2 (Keap1/Nrf2) regulatory network has been reported to play a role. In the present thesis, in vitro anaerobic fecal incubation was used to characterize the microbial conversion of green tea catechins EC and EGCG. Results obtained show phenylpropane-2-ols, phenyl-valerolactones, gallic acid, pyrogallol, phenyl-valeric acids as the major metabolites, with substantial interindividual differences. Moreover, intraindividual difference in the formation of the major EC colonic metabolite 5-(3’,4’-dihydroxyphenyl)-γ-valerolactone (3,4-diHPV) was also observed, though the interindividual difference was larger than intraindividual difference. Furthermore, quantitative microbiota characterization in the fecal samples, achieved by 16S rRNA analysis, revealed substantial differences in microbiota compositions among different individuals. Correlations between specific microbial abundance and formation of certain metabolites were established. The Nrf2 signaling activation by EGCG, EC and their major microbial metabolites were tested using reporter gene assays, RT-qPCR, and proteomics technic. Results show EGCG and the microbial metabolites 3,4-diHPV and pyrogallol can induce concentration-dependent induction of Nrf2 pathway. Moreover, metabolites 3,4-diHPV and pyrogallol exhibited better Nrf2-activation potency than their respective parent compounds, EGCG and EC. Finally, the thesis also developed a human PBK model for EGCG, with sub-models for its major microbial metabolites gallic acid and pyrogallol. The model enabled prediction of in vivo kinetics of EGCG, gallic acid and pyrogallol which allowed the translation of the obtained in vitro concentration-response curves from the U2OS-Nrf2 CALUX reporter gene assay to in vivo dose-response curves for Nrf2 activation, and comparison of these data to estimated daily intake levels. Results obtained reveal, by comparison to literature data on EGCG kinetics, that the developed PBK model could adequately predict in vivo time-dependent blood concentrations of EGCG after either a single or repeated oral administration(s) of EGCG under both fasting and non-fasting conditions. The predicted in vivo dose response curve revealed that at daily intake levels of green tea or EGCG supplements, the resulting blood Cmax of EGCG was in the sub-micromolar range, concentrations at which Nrf2 activation was shown to be limited. Moreover, blood Cmax values of gallic acid and pyrogallol upon intake of EGCG were predicted to be less than 1.5% of the Cmax of EGCG, indicating that despite their higher potential for Nrf2 activation, their contribution to the overall systemic Nrf2 pathway induction upon EGCG exposure is expected to be limited. In contrast, concentrations of these metabolites in the intestinal tract may reach levels that are, expressed in EGCG equivalents for Nrf2 induction, higher than that of EGCG, and high enough to activate Nrf2 gene transcription. Taken together, combining in vitro data with a human PBK model allowed the prediction of a dose-response curve for EGCG induced Nrf2-mediated gene expression in humans, and provided insight into the contribution of gut microbial metabolites to this effect. It also provided a proof-of-principle for a NAM to study the in vivo effects of bioactive phytochemicals without the need for human intervention studies.

Reliable hepatic in vitro systems are crucial for the safety assessment of xenobiotics. Certain xenobiotics decrease the hepatic bile efflux, which can ultimately result in cholestasis. Preclinical animal models and the currently available in vitro systems poorly predict a xenobiotic's cholestatic potential. Here, we compared the phenotype and capacity of three liver derived in vitro systems to emulate human functionality to synthesize and secrete bile acids (BAs). To this end, basal BA production of sandwich cultured human hepatocytes (SCHHs), HepaRG cells (HepaRGs) and hepatocyte-like intrahepatic cholangiocyte organoids (ICO-heps) were analysed, and the effect of the known BSEP (Bile Salt Export Pump)-inhibitors bosentan and lopinavir on BA disposition in SCHHs and HepaRGs was quantified. RT-qPCR of selected target genes involved in maturation status, synthesis, transport and conjugation of BAs was performed to mechanistically underpin the observed differences in BA homeostasis. ICO-heps produced a (very) low amount of BAs. SCHHs are a powerful tool in cholestasis-testing due to their high basal BA production and high transporter expression compared to the other models tested. HepaRGs were responsive to both selected BSEP-inhibitors and produced a BA profile that is most similar to the human in vivo situation, making them a suitable and practical candidate for cholestasis-testing.

Nanoscale objects are not new; they have been known to exist for decades. Yet, it was the ability of scientists to see and engineer nanostructures via self- or directed assembly in the 1980s that catalyzed their rapid development. Nanotechnology has now evolved into a convergent discipline involving a variety of sciences (physical, chemical, biological, engineering, and electronic) designed to understand and manipulate structures and devices at nanoscale. The use of nano-based consumer products is growing rapidly and many such products are available in the market. To date, more than 1800 consumer products that are self-identified by the manufacturers as containing nanotechnology are included in the public database (Project on Emerging Nanotechnologies, 2018). Nano-based goods are projected by various sources to be an estimated $2.6 trillion global industry by 2014 (ScienceDaily, 2007) and a nano-dominated future is not too distant.