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A novel, integrated, in vitro gastrointestinal (GI) system is presented to study oral bioavailability parameters of small molecules. Three compartments were combined into one hyphenated, flow-through set-up. In the first compartment, a compound was exposed dynamically to enzymatic digestion in three consecutive microreactors, mimicking the processes of the mouth, stomach, and intestine. The resulting solution (chyme) continued to the second compartment, a flow-through barrier model of the intestinal epithelium allowing absorption of the compound and metabolites thereof. The composition of the effluents from the barrier model were analysed either offline by electrospray-ionisation-mass spectrometry (ESI–MS), or online in the final compartment using chip-based ESI–MS. Two model drugs, omeprazole and verapamil, were used to test the integrated model. Omeprazole was shown to be broken down upon treatment with gastric acid, but reached the cell barrier unharmed when introduced to the system in a manner emulating an enteric-coated formulation. In contrast, verapamil was unaffected by digestion. Finally, a reduced uptake of verapamil was observed when verapamil was introduced to the system dissolved in apple juice, a simple food matrix. It is envisaged that this integrated, compartmentalised GI system has potential for enabling future research in the fields of pharmacology, toxicology, and nutrition. [ABSTRACT FROM AUTHOR]

Gut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models. [ABSTRACT FROM AUTHOR]

In oral bioavailability studies, evaluation of the absorption and transport of drugs and food components across the intestinal barrier is crucial. Advances in the field of organ-on-a-chip technology have resulted in a dynamic gut-on-a-chip model that better mimics the in vivo microenvironment of the intestine. Despite a few recent integration attempts, ensuring a biologically relevant microenvironment while coupling with a fully online detection system still represents a major challenge. Herein, we designed an online technique to measure drug permeability and analyse unknown product formation across an intestinal epithelial layer of Caco-2 and HT29-MTX cells cultured on a flow-through Transwell system, while ensuring the quality and relevance of the biological model. Chip-based ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was coupled to the dynamic Transwell system via a series of switching valves, thus allowing alternating measurements of the apical and basolateral sides of the in vitro model. Two trap columns were integrated for online sample pre-treatment and compatibility enhancement. Temporal analysis of the intestinal permeability was successfully demonstrated using verapamil as a model drug and ergotamine epimers as a model for natural toxins present in foods. Evidence was obtained that our newly developed dynamic system provided reliable results versus classical static in vitro models, and moreover, for the first time, epimer-specific transport is shown for ergotamine. Finally, initial experiments with the drug granisetron suggest that metabolic activity can be studied as well, thus highlighting the versatility of the bio-integrated online analysis system developed. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel intestinal in vitro co-culture model was developed to study the active transport in the inflamed intestine. • The active transport of primary bile acids is reduced in the inflamed co-cultures compared to healthy co-cultures. • Deoxynivalenol increases pro-inflammatory cytokine secretion and reduces bile acid transport in an inflamed intestine. • Local activation of immune cells reinforces the direct pro-inflammatory effects of DON on epithelial cells in intestine. The fungal secondary metabolite deoxynivalenol (DON) that can contaminate cereal-based food products not only induces inflammation but also reduces bile acid absorption by a healthy human intestine. Bile acid malabsorption is commonly observed in individuals with an inflamed intestine. Here we studied the effects of DON on inflammation and primary bile acid transport using an in vitro model for an inflamed intestine. An inflamed intestinal in vitro model was established by co-culturing a Caco-2 cell-layer and LPS-pre-stimulated THP-1 macrophages in Transwells. We observed a decreased transport of 5 primary bile acids across inflamed co-cultures compared to healthy co-cultures but not of chenodeoxycholic acid. DON exposure further reduced the transport of the affected primary bile acids across the inflamed co-cultures. DON exposure also enhanced the secretion of pro-inflammatory cytokines in the inflamed co-cultures, while it did not increase the pro-inflammatory cytokines secretion from LPS-pre-stimulated THP-1 monocultures. Exposure of Caco-2 cell-layers to pro-inflammatory cytokines or THP-1 conditioned media partly mimicked the DON-induced effects of the co-culture model. Local activation of intestinal immune cells reinforces the direct pro-inflammatory effects of DON on intestinal epithelial cells. This affects the bile acid intestinal kinetics in an inflamed intestine. [ABSTRACT FROM AUTHOR]

Background: Upon ingestion, nanoparticles can interact with the intestinal epithelial barrier potentially resulting in systemic uptake of nanoparticles. Nanoparticle properties have been described to influence the protein corona formation and subsequent cellular adhesion, uptake and transport. Here, we aimed to study the effects of nanoparticle size and surface chemistry on the protein corona formation and subsequent cellular adhesion, uptake and transport. Caco-2 intestinal cells, were exposed to negatively charged polystyrene nanoparticles (PSNPs) (50 and 200 nm), functionalized with sulfone or carboxyl groups, at nine nominal concentrations (15–250 μg/ml) for 10 up to 120 min. The protein coronas were analysed by LC–MS/MS. Results: Subtle differences in the protein composition of the two PSNPs with different surface chemistry were noted. High-content imaging analysis demonstrated that sulfone PSNPs were associated with the cells to a significantly higher extent than the other PSNPs. The apparent cellular adhesion and uptake of 200 nm PSNPs was not significantly increased compared to 50 nm PSNPs with the same surface charge and chemistry. Surface chemistry outweighs the impact of size on the observed PSNP cellular associations. Also transport of the sulfone PSNPs through the monolayer of cells was significantly higher than that of carboxyl PSNPs. Conclusions: The results suggest that the composition of the protein corona and the PSNP surface chemistry influences cellular adhesion, uptake and monolayer transport, which might be predictive of the intestinal transport potency of NPs. Keywords: Nanoparticles, High throughput screening, Cellular adhesion and uptake, Label-free LC–MS/MS, Quantitative proteomics [ABSTRACT FROM AUTHOR]

Hydroxyanthraquinones that can be present in traditional Chinese medicine (TCM) and herbal extracts have claimed beneficial intestinal effects. We examined the ability of a panel hydroxyanthraquinones, and methanolic extracts from selected TCM and herbal granules to activate Nrf2-EpRE mediated gene expression using a reporter-gene assay. The results indicate that purpurin, aloe-emodin, 2-hydroxy-3-methylanthraquinone and rhein induced Nrf2 mediated gene expressions with a high induction factor (IFs>10), with BMCL 10 values (the lower confidence limit of the concentration giving 10% added response above background) of 16 μM, 1.1 μM, 23 μM and 2.3 μM, respectively, while aurantio-obtusin, obtusifolin, rubiadin 1-methyl ether and emodin were less potent (IFs<5), with BMCL 10 values for added response above background level of 4.6 μM, 15 μM, 9.8 μM and 3.8 μM, respectively. All TCM extracts and the herbal extracts of Aloe Vera , Polygonum multiflorum , Rubia (cordifolia) and Rheum officinale activated the Nrf2-EpRE pathway. Of the TCM extracts, Chuan-Xin-Lian-Kang-Yan-Pian was the most potent Nrf2-inducer. LC-MS/MS analysis indicated the presence of selected hydroxyanthraquinones in the extracts and herbs, in part explaining their Nrf2-EpRE mediated activity. In conclusion, different hydroxyanthraquinones have different potencies of Nrf2 activation. The Nrf2 activation by extracts from TCM and herbs can be partially explained by the presence of selected hydroxyanthraquinones. • The 16 hydroxyanthraquinones have a different potency to induce Nrf2-EpRE mediated gene expression in the Nrf2 CALUX assay. • Purpurin, aloe-emodin, 2-hydroxy-3-methylanthraquinone and rhein induced a high Nrf2 activation. • Methanolic extracts of herbal granules and TCMs induced EpRE mediated gene expression in the Nrf2 CALUX assay. • Nrf2-activated hydroxyanthraquinones in methanolic extracts of herbal granules and TCMs were quantified by LC-MS/MS. • Hydroxyanthraquinones partly account for Nrf2 activation of methanolic extracts of herbal granules and TCMs. [ABSTRACT FROM AUTHOR]

Nanotechnology has the potential to innovate the agricultural, feed and food sectors (hereinafter referred to as agri/feed/food). Applications that are marketed already include nano-encapsulated agrochemicals or nutrients, antimicrobial nanoparticles and active and intelligent food packaging. Many nano-enabled products are currently under research and development, and may enter the market in the near future. As for any other regulated product, applicants applying for market approval have to demonstrate the safe use of such new products without posing undue safety risks to the consumer and the environment. Several countries all over the world have been active in examining the appropriateness of their regulatory frameworks for dealing with nanotechnologies. As a consequence of this, different approaches have been taken in regulating nano-based products in agri/feed/food. The EU, along with Switzerland, were identified to be the only world region where nano-specific provisions have been incorporated in existing legislation, while in other regions nanomaterials are regulated more implicitly by mainly building on guidance for industry. This paper presents an overview and discusses the state of the art of different regulatory measures for nanomaterials in agri/feed/food, including legislation and guidance for safety assessment in EU and non-EU countries. [ABSTRACT FROM AUTHOR]

To obtain insight in translocation of nanoparticles across the placental barrier, translocation was studied for one positively and two negatively charged polystyrene nanoparticles (PS-NPs) of similar size in an in vitro model. The model consisted of BeWo b30 cells, derived from a human choriocarcinoma grown on a transwell insert forming a cell layer that separates an apical from a basolateral compartment. PS-NPs were characterized with respect to size, surface charge, morphology and protein corona. Translocation of PS-NPs was not related to PS-NP charge. Two PS-NPs were translocated across the BeWo transwell model to a lower extent than amoxicillin, a model compound known to be translocated over the placental barrier to only a limited extent, whereas one PS-NP showed a slightly higher translocation. Studies on the effect of transporter inhibitors on the translocation of the PS-NPs indicated that their translocation was not mediated by known transporters and mainly dependent on passive diffusion. It is concluded that the BeWo b30 model can be used as an efficient method to get an initial qualitative impression about the capacity of NPs to translocate across the placental barrier and set priorities in further in vivo studies on translocation of NPs to the fetus. [ABSTRACT FROM AUTHOR]

The increasing use of nanoparticles in products likely results in increased exposure of both workers and consumers. Because of their small size, there are concerns that nanoparticles unintentionally cross the barriers of the human body. Several in vivo rodent studies show that, dependent on the exposure route, time, and concentration, and their characteristics, nanoparticles can cross the lung, gut, skin, and placental barrier. This review aims to evaluate the performance of in vitro models that mimic the barriers of the human body, with a focus on the lung, gut, skin, and placental barrier. For these barriers, in vitro models of varying complexity are available, ranging from single-cell-type monolayer to multi-cell (3D) models. Only a few studies are available that allow comparison of the in vitro translocation to in vivo data. This situation could change since the availability of analytical detection techniques is no longer a limiting factor for this comparison. We conclude that to further develop in vitro models to be used in risk assessment, the current strategy to improve the models to more closely mimic the human situation by using co-cultures of different cell types and microfluidic approaches to better control the tissue microenvironments are essential. At the current state of the art, the in vitro models do not yet allow prediction of absolute transfer rates but they do support the definition of relative transfer rates and can thus help to reduce animal testing by setting priorities for subsequent in vivo testing. [ABSTRACT FROM AUTHOR]

The application of nanomaterials is leading to innovative developments in industry, agriculture, consumer products, and food and related sectors. However, due to the special properties of these materials there are concerns about their safety, especially because of our limited knowledge of human health effects and the fact that constantly new nanomaterials and applications thereof are being produced. The development of analytical techniques is a key element to understand the benefits as well as the risks of the application of such materials. In this study, a method is developed and validated for sizing and quantifying nano-silver in chicken meat using single particle inductive coupled plasma mass spectrometry (ICP-MS). Samples are processed using an enzymatic digestion followed by dilution of the digest and instrumental analysis of the diluted digest using single particle ICP-MS. Validation of the method in the concentration of 5-25 mg/kg 60-nm silver nanoparticles showed good performance with respect to trueness (98-99 % for size, 91-101 % for concentration), repeatability (<2 % for size, <11 % for concentration), and reproducibility (<6 % for size, <16 % for concentration). The response of the method is linear, and a detection limit as low as 0.1 mg/kg can be obtained. Additional experiments showed that the method is robust and that digests are stable for 3 weeks at 4 °C. Once diluted for single particle ICP-MS analysis, the stability is limited. Finally, it was shown that nano-silver in chicken meat is not stable. Silver nanoparticles dissolved and were transformed into silver sulfide. While this has implications for the form in which nano-silver will be present in real-life meat samples, the developed method will be able to determine the presence and quantity of nanoparticle silver in such samples. [ABSTRACT FROM AUTHOR]

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. • Flagellin increased IL-8 apical secretion in all tested in vitro intestinal models. • IL-8 secretion was lower basolaterally than apically except in HT29-p cells. • Poly (I:C) increased IL-8 secretion in HT29-p cells only. • Di-culture model of Caco-2/HT29-MTX has no additional value to study immunomodulation. [ABSTRACT FROM AUTHOR]

The global burden of Inflammatory bowel disease (IBD) has been rising over the last decades. IBD is an intestinal disorder with a complex and largely unknown etiology. The disease is characterized by a chronically inflamed gastrointestinal tract, with intermittent phases of exacerbation and remission. This compromised intestinal barrier can contribute to, enhance, or even enable the toxicity of drugs, food-borne chemicals and particulate matter. This review discusses whether the rising prevalence of IBD in our society warrants the consideration of IBD patients as a specific population group in toxicological safety assessment. Various in vivo, ex vivo and in vitro models are discussed that can simulate hallmarks of IBD and may be used to study the effects of prevalent intestinal inflammation on the hazards of these various toxicants. In conclusion, risk assessments based on healthy individuals may not sufficiently cover IBD patient safety and it is suggested to consider this susceptible subgroup of the population in future toxicological assessments., (© 2024. The Author(s).)

Since their introduction into agriculture, the toxicity of organophosphate (OP) pesticides has been widely studied in animal models. However, next generation risk assessment (NGRA) intends to maximize the use of novel approach methodologies based on in vitro and in silico methods. Therefore, this study describes the development and evaluation of a generic physiologically based kinetic (PBK) model for acute exposure to OP pesticides in rats and humans using quantitative structure property relationships and data from published in vitro studies. The models were evaluated using in vivo studies from the literature for chlorpyrifos, diazinon, fenitrothion, methyl-parathion, ethyl-parathion, dimethoate, chlorfenvinphos, and profenofos. Evaluation was performed by comparing simulated and in vivo observed time profiles for blood, plasma, or urinary concentrations and other toxicokinetic parameters. Of simulated concentration-time profiles, 87 and 91% were within a 5-fold difference from observed toxicokinetic data from rat and human studies, respectively. Only for dimethyl-organophosphates further refinement of the model is required. It is concluded that the developed generic PBK model provides a new tool to assess species differences in rat and human kinetics of OP pesticides. This approach provides a means to perform NGRA for these compounds and could also be adopted for other classes of compounds.

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

Background: Micro- and nanoplastics (MNPs) represent one of the most widespread environmental pollutants of the twenty-first century to which all humans are orally exposed. Upon ingestion, MNPs pass harsh biochemical conditions within the gastrointestinal tract, causing a unique protein corona on the MNP surface. Little is known about the digestion-associated protein corona and its impact on the cellular uptake of MNPs. Here, we systematically studied the influence of gastrointestinal digestion on the cellular uptake of neutral and charged polystyrene MNPs using THP-1-derived macrophages., Results: The protein corona composition was quantified using LC‒MS-MS-based proteomics, and the cellular uptake of MNPs was determined using flow cytometry and confocal microscopy. Gastrointestinal digestion resulted in a distinct protein corona on MNPs that was retained in serum-containing cell culture medium. Digestion increased the uptake of uncharged MNPs below 500 nm by 4.0-6.1-fold but did not affect the uptake of larger sized or charged MNPs. Forty proteins showed a good correlation between protein abundance and MNP uptake, including coagulation factors, apolipoproteins and vitronectin., Conclusion: This study provides quantitative data on the presence of gastrointestinal proteins on MNPs and relates this to cellular uptake, underpinning the need to include the protein corona in hazard assessment of MNPs., (© 2024. The Author(s).)

Current points of departure used to derive health-based guidance values for deoxynivalenol (DON) are based on studies in laboratory animals. Here, an animal-free testing approach was adopted in which a reverse dosimetry physiologically based kinetic (PBK) modeling is used to predict in vivo dose response curves for DON's effects on intestinal pro-inflammatory cytokine secretion and intestinal bile acid reabsorption in humans from concentration-effect relationships for DON in vitro. The calculated doses for inducing a 5% added effect above the background level (ED 5 ) of DON for increasing IL-1β secretion in intestinal tissue and for increasing the amounts in the colon lumen of glycochenodeoxycholic acid (GCDCA) were 246 and 36 μg/kg bw/day, respectively. These in vitro-in silico-derived ED 5 values were compared to human dietary DON exposure levels, indicating that the risk of DON's effects on these end points occurring in various human populations cannot be excluded. This in vitro-in silico approach provides a novel testing strategy for hazard and risk assessment without using laboratory animals.

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

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

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

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

The trichothecene toxin deoxynivalenol (DON) is a ribotoxic mycotoxin that contaminates cereal-based food. DON binds to ribosomes, thereby inhibiting protein translation and activating stress mitogen-activated protein kinases (MAPK). The activation of MAPK induces pro-inflammatory cytokine production. Emerging evidence showed that DON decreased bile acid reabsorption and apical sodium-dependent bile acid transporter (ASBT) expression in Caco-2 cell layers. We hypothesized that the effect of DON on decreased ASBT mRNA expression is regulated via pro-inflammatory cytokines. We observed that MAPK inhibitors prevented DON to induce IL-8 secretion and prevented the DON-induced downregulation of ASBT mRNA expression. However, DON-induced taurocholic acid (TCA) transport reduction was not prevent by the MAPK inhibitors. We next observed a similarity between the activity of the non-inflammatory ribotoxin cycloheximide and DON to decrease TCA transport, which is consistent with their common ability to inhibit protein synthesis. Together, our results suggest that DON-induced TCA malabsorption is regulated by MAPK activation-induced pro-inflammatory cytokine production and protein synthesis inhibition, both of which are initiated by DON binding to the ribosomes which therefore is the molecular initiating event for the adverse outcome of bile acid malabsorption. This study provides insights into the mechanism of ribotoxins-induced bile acid malabsorption in human intestine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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

There is a need for standardized in vitro models emulating the functionalities of the human intestinal tract to study human intestinal health without the use of laboratory animals. The Caco-2 cell line is a well-accepted and highly characterized intestinal barrier model, which has been intensively used to study intestinal (drug) transport, host-microbe interactions and chemical or drug toxicity. This cell line has been cultured in different in vitro models, ranging from simple static to complex dynamic microfluidic models. We aimed to investigate the effect of these different in vitro experimental variables on gene expression. To this end, we systematically collected and extracted data from studies in which transcriptome analyses were performed on Caco-2 cells grown on permeable membranes. A collection of 13 studies comprising 100 samples revealed a weak association of experimental variables with overall as well as individual gene expression. This can be explained by the large heterogeneity in cell culture practice, or the lack of adequate reporting thereof, as suggested by our systematic analysis of experimental parameters not included in the main analysis. Given the rapidly increasing use of in vitro cell culture models, including more advanced (micro) fluidic models, our analysis reinforces the need for improved, standardized reporting protocols. Additionally, our systematic analysis serves as a template for future comparative studies on in vitro transcriptome and other experimental data., (© 2023. The Author(s).)

Plastic particles in the nanometer range-called nanoplastics-are environmental contaminants with growing public health concern. As plastic particles are present in water, soil, air and food, human exposure via intestine and lung is unavoidable, but possible health effects are still to be elucidated. To better understand the Mode of Action of plastic particles, it is key to use experimental models that best reflect human physiology. Novel assessment methods like advanced cell models and several alternative approaches are currently used and developed in the scientific community. So far, the use of cancer cell line-based models is the standard approach regarding in vitro nanotoxicology. However, among the many advantages of the use of cancer cell lines, there are also disadvantages that might favor other approaches. In this review, we compare cell line-based models with stem cell-based in vitro models of the human intestine and lung. In the context of nanoplastics research, we highlight the advantages that come with the use of stem cells. Further, the specific challenges of testing nanoplastics in vitro are discussed. Although the use of stem cell-based models can be demanding, we conclude that, depending on the research question, stem cells in combination with advanced exposure strategies might be a more suitable approach than cancer cell lines when it comes to toxicological investigation of nanoplastics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Busch, Brouwer, Aalderink, Bredeck, Kämpfer, Schins and Bouwmeester.)

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., (© 2022. The Author(s).)

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA ("biomaterial risk management") an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe 3 O 4 -PEG-PLGA nanomaterials while Ag and TiO 2 "benchmark" nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe 3 O 4 -PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO 2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Martin, de Haan, Miro Estruch, Eder, Marzi, Schnekenburger, Blosi, Costa, Antonello, Bergamaschi, Riganti, Beal, Carrière, Taché, Hutchison, Malone, Young, Campagnolo, La Civita, Pietroiusti, Devineau, Baeza, Boland, Zong, Ichihara, Fadeel and Bouwmeester.)

New approach methodologies predicting human cardiotoxicity are of interest to support or even replace in vivo-based drug safety testing. The present study presents an in vitro-in silico approach to predict the effect of inter-individual and inter-ethnic kinetic variations in the cardiotoxicity of R- and S-methadone in the Caucasian and the Chinese population. In vitro cardiotoxicity data, and metabolic data obtained from two approaches, using either individual human liver microsomes or recombinant cytochrome P450 enzymes (rCYPs), were integrated with physiologically based kinetic (PBK) models and Monte Carlo simulations to predict inter-individual and inter-ethnic variations in methadone-induced cardiotoxicity. Chemical specific adjustment factors were defined and used to derive dose-response curves for the sensitive individuals. Our simulations indicated that Chinese are more sensitive towards methadone-induced cardiotoxicity with Margin of Safety values being generally two-fold lower than those for Caucasians for both methadone enantiomers. Individual PBK models using microsomes and PBK models using rCYPs combined with Monte Carlo simulations predicted similar inter-individual and inter-ethnic variations in methadone-induced cardiotoxicity. The present study illustrates how inter-individual and inter-ethnic variations in cardiotoxicity can be predicted by combining in vitro toxicity and metabolic data, PBK modelling and Monte Carlo simulations. The novel methodology can be used to enhance cardiac safety evaluations and risk assessment of chemicals., (© 2022. The Author(s).)

Microplastics have been documented in drinking water, but their effects on human health from ingestion, or the concentrations at which those effects begin to manifest, are not established. Here, we report on the outcome of a virtual expert workshop conducted between October 2020 and October 2021 in which a comprehensive review of mammalian hazard studies was conducted. A key objective of this assessment was to evaluate the feasibility and confidence in deriving a human health-based threshold value to inform development of the State of California's monitoring and management strategy for microplastics in drinking water. A tiered approach was adopted to evaluate the quality and reliability of studies identified from a review of the peer-reviewed scientific literature. A total of 41 in vitro and 31 in vivo studies using mammals were identified and subjected to a Tier 1 screening and prioritization exercise, which was based on an evaluation of how each of the studies addressed various quality criteria. Prioritized studies were identified largely based on their application and reporting of dose-response relationships. Given that methods for extrapolating between in vitro and in vivo systems are currently lacking, only oral exposure in vivo studies were identified as fit-for-purpose within the context of this workshop. Twelve mammalian toxicity studies were prioritized and subjected to a Tier 2 qualitative evaluation by external experts. Of the 12 studies, 7 report adverse effects on male and female reproductive systems, while 5 reported effects on various other physiological endpoints. It is notable that the majority of studies (83%) subjected to Tier 2 evaluation report results from exposure to a single polymer type (polystyrene spheres), representing a size range of 0.040 to 20 µm. No single study met all desired quality criteria, but collectively toxicological effects with respect to biomarkers of inflammation and oxidative stress represented a consistent trend. While it was possible to derive a conservative screening level to inform monitoring activities, it was not possible to extrapolate a human-health-based threshold value for microplastics, which is largely due to concerns regarding the relative quality and reliability of current data, but also due to the inability to extrapolate data from studies using monodisperse plastic particles, such as polystyrene spheres to an environmentally relevant exposure of microplastics. Nevertheless, a conservative screening level value was used to estimate a volume of drinking water (1000 L) that could be used to support monitoring activities and improve our overall understanding of exposure in California's drinking water. In order to increase confidence in our ability to derive a human-health-based threshold value in the future, several research recommendations are provided, with an emphasis towards strengthening how toxicity studies should be conducted in the future and an improved understanding of human exposure to microplastics, insights critically important to better inform future risk assessments., Supplementary Information: The online version contains supplementary material available at 10.1186/s43591-022-00030-6., Competing Interests: Competing interestsThe following authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: Christine Lemieux, Elaine Khan, Scott Coffin, Steve Weisberg, Katja Teerds, Pauliina Damdimopoulou. The following authors declare the following financial interests/personal relationships which may be considered as potential competing interests: • Ludovic Hermabessiere reports financial support was provided by Environment and Climate Change Canada; • Hans Bouwmeester is actively involved in research on the safety of nano and microplastics currently in two projects: Momentum (funded by the Dutch research organization ZonMW and Health Holland) and Plasticheal (EU H2020). • Martin Wagner reports a relationship with The European Parliament, SAPEA and European Commission Group of Chief Scientific Advisors, The World Bank, the Food Packaging Forum, and Hold Norge Rent that includes travel reimbursement. Martin Wagner reports a relationship with the Norwegian Committee for Food and the Environment that included travel reimbursement and consulting. • Susanne Brander reports financial support was provided by National Science Foundation. Susanne Brander reports a relationship with Ocean Science Trust that includes: consulting or advisory, and received compensation as a panelist for a call for proposals from the European Union in fall 2020, on the topic of microplastic / nanoplastics and human health. • Todd Gouin reports a relationship with World Health Organization that includes: consulting or advisory. Todd Gouin reports a relationship with European Centre for Ecotoxicology and Toxicology of Chemicals that includes: consulting or advisory. Todd Gouin reports a relationship with European Food Safety Authority that includes: travel reimbursement. Todd Gouin reports a relationship with International Life Sciences Institute Europe ILSI that includes: non-financial support. Todd Gouin reports a relationship with Environment and Climate Change Canada that includes: consulting or advisory. Todd Gouin reports a relationship with American Chemistry Council Chemical Products and Technology Division that includes: consulting or advisory. Todd Gouin reports a relationship with Horizon 2020 that includes: funding grants. • AAK reports a relationship with World Health Organization, European Food Safety Authority, United Nations and SAPEA & European Commission Group of Chief Scientific Advisors that includes consulting or advisory, and financial support in the form of funding grants or travel reimbursement. AAK also acknowledges research funding from the European Chemical Industry Council, the Dutch Research Council, the Norwegian Research Council and Rijkswaterstaat Water Traffic and Living Environment. All research is carried out under the Dutch code of conduct for scientific integrity., (© The Author(s) 2022.)

The development of non-animal-based new approach methodologies (NAMs) for chemical risk assessment and safety evaluation is urgently needed. The aim of the present study was to investigate the applicability of an in vitro-in silico approach to predict human cardiotoxicity of the herbal alkaloid ibogaine and its metabolite noribogaine, which are promising anti-addiction drugs. Physiologically based kinetic (PBK) models were developed using in silico-derived parameters and biokinetic data obtained from in vitro liver microsomal incubations and Caco-2 transport studies. Human induced pluripotent stem cell-derived cardiomyocytes combined with a multi-electrode array (MEA) assay were used to determine in vitro concentration-dependent cardiotoxicity reflected by prolongation of field potential duration, which was subsequently translated to in vivo dose-dependent prolongation of the QTc (heart rate corrected duration from ventricular depolarization to repolarization) using PBK modeling-based reverse dosimetry. Results showed that the predictions matched well with in vivo kinetic data and QTc data for ibogaine and noribogaine available in the literature, indicating a good performance of the NAM. Benchmark dose analysis of the predicted dose response curves adequately predicted the onset of in vivo cardiotoxicity detected by QTc prolongation upon oral exposure to ibogaine and noribogaine. The present study provides an additional proof-of-principle of using PBK modeling-based reverse dosimetry as a NAM to predict human cardiotoxicity.

Development of novel testing strategies to detect adverse human health effects is of interest to replace in vivo-based drug and chemical safety testing. The aim of the present study was to investigate whether physiologically based kinetic (PBK) modeling-facilitated conversion of in vitro toxicity data is an adequate approach to predict in vivo cardiotoxicity in humans. To enable evaluation of predictions made, methadone was selected as the model compound, being a compound for which data on both kinetics and cardiotoxicity in humans are available. A PBK model for methadone in humans was developed and evaluated against available kinetic data presenting an adequate match. Use of the developed PBK model to convert concentration-response curves for the effect of methadone on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in the so-called multi electrode array (MEA) assay resulted in predictions for in vivo dose-response curves for methadone-induced cardiotoxicity that matched the available in vivo data. The results also revealed differences in protein plasma binding of methadone to be a potential factor underlying variation between individuals with respect to sensitivity towards the cardiotoxic effects of methadone. The present study provides a proof-of-principle of using PBK modeling-based reverse dosimetry of in vitro data for the prediction of cardiotoxicity in humans, providing a novel testing strategy in cardiac safety studies.

Background: Silver nanoparticles (AgNPs) are used extensively in various consumer products because of their antimicrobial potential. This requires insight in their potential hazards and risks including adverse effects during pregnancy on the developing fetus. Using a combination of the BeWo b30 placental transport model and the mouse embryonic stem cell test (EST), we investigated the capability of pristine AgNPs with different surface chemistries and aged AgNPs (silver sulfide (Ag 2 S) NPs) to cross the placental barrier and induce developmental toxicity. The uptake/association and transport of AgNPs through the BeWo b30 was characterized using ICP-MS and single particle (sp)ICP-MS at different time points. The developmental toxicity of the AgNPs was investigated by characterizing their potential to inhibit the differentiation of mouse embryonic stem cells (mESCs) into beating cardiomyocytes., Results: The AgNPs are able to cross the BeWo b30 cell layer to a level that was limited and dependent on their surface chemistry. In the EST, no in vitro developmental toxicity was observed as the effects on differentiation of the mESCs were only detected at cytotoxic concentrations. The aged AgNPs were significantly less cytotoxic, less bioavailable and did not induce developmental toxicity., Conclusions: Pristine AgNPs are capable to cross the placental barrier to an extent that is influenced by their surface chemistry and that this transport is likely low but not negligible. Next to that, the tested AgNPs have low intrinsic potencies for developmental toxicity. The combination of the BeWo b30 model with the EST is of added value in developmental toxicity screening and prioritization of AgNPs.

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.

Societies worldwide are investing considerable resources into the safe development and use of nanomaterials. Although each of these protective efforts is crucial for governing the risks of nanomaterials, they are insufficient in isolation. What is missing is a more integrative governance approach that goes beyond legislation. Development of this approach must be evidence based and involve key stakeholders to ensure acceptance by end users. The challenge is to develop a framework that coordinates the variety of actors involved in nanotechnology and civil society to facilitate consideration of the complex issues that occur in this rapidly evolving research and development area. Here, we propose three sets of essential elements required to generate an effective risk governance framework for nanomaterials. (1) Advanced tools to facilitate risk-based decision making, including an assessment of the needs of users regarding risk assessment, mitigation, and transfer. (2) An integrated model of predicted human behavior and decision making concerning nanomaterial risks. (3) Legal and other (nano-specific and general) regulatory requirements to ensure compliance and to stimulate proactive approaches to safety. The implementation of such an approach should facilitate and motivate good practice for the various stakeholders to allow the safe and sustainable future development of nanotechnology., (© 2017 Society for Risk Analysis.)

Inclusion of alternative methods that replace, reduce, or refine (3R) animal testing within regulatory safety evaluations of chemicals generally faces many hurdles. The goal of the current work is to i) collect responses from key stakeholders involved in food safety evaluations on what they consider the most relevant factors that influence the acceptance and use of 3R methods and to ii) use these responses to formulate activities needed to increase the acceptance and use of 3R methods, particularly for kinetics. The stakeholders were contacted by e-mail for their opinions, asking the respondents to write down three barriers and/or drivers and scoring these by distributing 5 points over the three factors. The main barriers that obtained the highest aggregated scores were i) uncertain predictability 3R methods/lack of validation, ii) insufficient guidance regulators/industry and iii) insufficient harmonization of legislation. The major driver identified was the possibility of 3R methods to provide more mechanistic information. Based on the results, recommendations are given to enhance the acceptance and application of 3R toxicokinetic methods in food safety evaluations. These include steering of regulatory data requirements as well as creating (funding) opportunities for development and validation of alternative methods for kinetics and development of guidances., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Ingestion of engineered nanomaterials is inevitable due to their addition to food and prevalence in food packaging and domestic products such as toothpaste and sun cream. In the absence of robust dosimetry and particokinetic data, it is currently challenging to accurately assess the potential toxicity of food-borne nanomaterials. Herein, we review current understanding of gastrointestinal uptake mechanisms, consider some data on the potential for toxicity of the most commonly encountered classes of food-borne nanomaterials (including TiO 2 , SiO 2 , ZnO, and Ag nanoparticles), and discuss the potential impact of the luminal environment on nanoparticle properties and toxicity. Much of our current understanding of gastrointestinal nanotoxicology is derived from increasingly sophisticated epithelial models that augment in vivo studies. In addition to considering the direct effects of food-borne nanomaterials on gastrointestinal tissues, including the potential role of chronic nanoparticle exposure in development of inflammatory diseases, we also discuss the potential for food-borne nanomaterials to disturb the normal balance of microbiota within the gastrointestinal tract. The latter possibility warrants close attention given the increasing awareness of the critical role of microbiota in human health and the known impact of some food-borne nanomaterials on bacterial viability. WIREs Nanomed Nanobiotechnol 2018, 10:e1481. doi: 10.1002/wnan.1481 This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials., (© 2017 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.)

Governments invest in "key enabling technologies," such as nanotechnology, to solve societal challenges and boost the economy. At the same time, governmental agencies demand risk reduction to prohibit any often unknown adverse effects, and industrial parties demand smart approaches to reduce uncertainties. Responsible research and innovation (RRI) is therefore a central theme in policy making. Risk analysis and technology assessment, together referred to as "RATA," can provide a basis to assess human, environmental, and societal risks of new technological developments during the various stages of technological development. This assessment can help both governmental authorities and innovative industry to move forward in a sustainable manner. Here we describe the developed procedures and products and our experiences to bring RATA in practice within a large Dutch nanotechnology consortium. This is an example of how to put responsible innovation in practice as an integrated part of a research program, how to increase awareness of RATA, and how to help technology developers perform and use RATA. Integr Environ Assess Manag 2018;14:9-16. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC)., (© 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

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

While control banding has been identified as a suitable framework for the evaluation and the determination of potential human health risks associated with exposure to nanomaterials (NMs), the approach currently lacks any implementation that enjoys widespread support. Large inconsistencies in characterisation data, toxicological measurements and exposure scenarios make it difficult to map and compare the risk associated with NMs based on physicochemical data, concentration and exposure route. Here we demonstrate the use of Bayesian networks as a reliable tool for NM risk estimation. This tool is tractable, accessible and scalable. Most importantly, it captures a broad span of data types, from complete, high quality data sets through to data sets with missing data and/or values with a relatively high spread of probability distribution. The tool is able to learn iteratively in order to further refine forecasts as the quality of data available improves. We demonstrate how this risk measurement approach works on NMs with varying degrees of risk potential, namely, carbon nanotubes, silver and titanium dioxide. The results afford even non-experts an accurate picture of the occupational risk probabilities associated with these NMs and, in doing so, demonstrated how NM risk can be evaluated into a tractable, quantitative risk comparator.

The likelihood of oral exposure to nanoparticles (NPs) is increasing, and it is necessary to evaluate the oral bioavailability of NPs. In vitro approaches could help reducing animal studies, but validation against in vivo studies is essential. Previously, we assessed the translocation of 50 nm polystyrene NPs of different charges (neutral, positive and negative) using a Caco-2/HT29-MTX in vitro intestinal translocation model. The NPs translocated in a surface charge-dependent manner. The present study aimed to validate this in vitro intestinal model by an in vivo study. For this, rats were orally exposed to a single dose of these polystyrene NPs and the uptake in organs was determined. A negatively charged NP was taken up more than other NPs, with the highest amounts in kidney (37.4 µg/g tissue), heart (52.8 µg/g tissue), stomach wall (98.3 µg/g tissue) and small intestinal wall (94.4 µg/g tissue). This partly confirms our in vitro findings, where the same NPs translocated to the highest extent. The estimated bioavailability of different types of NPs ranged from 0.2 to 1.7 % in vivo, which was much lower than in vitro (1.6-12.3 %). Therefore, the integrated in vitro model cannot be used for a direct prediction of the bioavailability of orally administered NPs. However, the model can be used for prioritizing NPs before further in vivo testing for risk assessment.

Due to new, previously unknown, properties attributed to engineered nanoparticles many new products are introduced in the agro-food area. Nanotechnologies cover many aspects, such as disease treatment, food security, new materials for pathogen detection, packaging materials and delivery systems. As with most new and evolving technologies, potential benefits are emphasized, while little is known on safety of the application of nanotechnologies in the agro-food sector. This review gives an overview of scientific issues that need to be addressed with priority in order to improve the risk assessment for nanoparticles in food. The following research topics are considered to contribute pivotally to risk assessment of nanotechnologies and nanoparticles in food products. Set a definition for NPs to facilitate regulatory discussions, prioritization of research and exchange of study results. Develop analytical tools for the characterization of nanoparticles in complex biological matrices like food. Establish relevant dose metrics for nanoparticles used for both interpretation of scientific studies as well as regulatory frameworks. Search for deviant behavior (kinetics) and novel effects (toxicity) of nanoparticles and assess the validity of currently used test systems following oral exposure. Estimate the consumer exposure to nanoparticles.

There is evidence for neurodevelopment disturbances in schizophrenia. In rats, a neonatal basolateral amygdala lesion induces behavioural features in adults reminiscent of the symptomatology of schizophrenia. Dopamine plays a key role in the pathogenesis of schizophrenia, and cannabis use has been implicated in the risk for developing schizophrenia. The effects of an excitotoxic, bilateral basolateral amygdala lesion on postnatal days 7 or 21 were compared when the rats were adult. The behavioural response to a novelty challenge and the level of dopamine receptors and cannabinoid receptors in the brain using in-vitro autoradiography was determined. In brain tissue punches concentrations of monoamines and metabolites were determined by high-performance liquid chromatography. The neonatal lesion, but not the later lesion induced behavioural hyperactivity and biochemical effects. The neonatal lesion reduced the density of dopamine D2-like, but not D3-, and less markely D1-like receptors and increased dopamine turnover. These effects were observed in the mesolimbic, but not in the striatal regions. In contrast, density of cannabinoid receptors was increased in the striatal, but not the mesolimbic regions of these animals. Noradrenergic neurotransmission was reduced in both regions. The present findings contribute to the idea that the neonatal basolateral amygdala lesion induces features in adults reminiscent of the neurodevelopmental disturbances in schizophrenia, with a focus on the amygdala-prefrontal cortex-nucleus accumbens circuit.