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The “totality” of the human exposure is conceived to encompass life-associated endogenous and exogenous aggregate exposures. Process-related contaminants (PRCs) are not only formed in foods by heat processing, but also occur endogenously in the organism as physiological components of energy metabolism, potentially also generated by the human microbiome. To arrive at a comprehensive risk assessment, it is necessary to understand the contribution of in vivo background occurrence as compared to the ingestion from exogenous sources. Hence, this review provides an overview of the knowledge on the contribution of endogenous exposure to the overall exposure to putative genotoxic food contaminants, namely ethanol, acetaldehyde, formaldehyde, acrylamide, acrolein, α,β-unsaturated alkenals, glycation compounds, N-nitroso compounds, ethylene oxide, furans, 2- and 3-MCPD, and glycidyl esters. The evidence discussed herein allows to conclude that endogenous formation of some contaminants appears to contribute substantially to the exposome. This is of critical importance for risk assessment in the cases where endogenous exposure is suspected to outweigh the exogenous one (e.g. formaldehyde and acrolein).

To assess potential differences in the intestinal microbial metabolism of deoxynivalenol (DON) and its acetylated forms 3-Ac-DON and 15-Ac-DON, in vitro anaerobic incubations with intestinal contents from chickens were conducted. Quantitative microbiota characterization was obtained by 16S rRNA sequencing. The data showed substantial differences in the level of different toxin conversions by the microbiota from the different intestinal segments. The transformation rate of DON to its metabolite DOM-1 decreased in the order of cecum > ileum > jejunum, and caecum contents could completely transform DON to DOM-1 within 24 h. However, no transformation appeared in the duodenum. For 3-Ac-DON, the deacetylation rate decreased as follows: duodenum > caecum > ileum > jejunum, and 100% deacetylation was observed in the duodenum within 24 h. The deacetylation of 15-Ac-DON decreased in the order of cecum > duodenum > ileum with no transformation in the jejunum. Some genera may contribute to the transformation of the toxins. Based on the in vitro kinetic parameters and their scaling to the in vivo situation, it was concluded that in the chicken small intestine, the deconjugation of both 3-Ac-DON and 15-Ac-DON will not likely be completed upon full transit. Whether this also holds for humans remains to be established.

Here we show that in tobacco (Nicotiana tabacum cultivar Samsun NN Rx1) the development of Rx1 gene-mediated, symptomless, extreme resistance to Potato virus X (PVX) is preceded by an early, intensive accumulation of the reactive oxygen species (ROS) superoxide (O2·−), evident between 1 and 6 h after inoculation and associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities. This suggests a direct contribution of this ROS to virus restriction during symptomless, extreme resistance. Superoxide inhibition in PVX-inoculated leaves by infiltration of antioxidants (superoxide dismutase [SOD] and catalase [CAT]) partially suppresses extreme resistance in parallel with the appearance of localized leaf necrosis resembling a hypersensitive resistance (HR) response. F1 progeny from crosses of Rx1 and ferritin overproducer (deficient in production of the ROS OH·) tobaccos also display a suppressed extreme resistance to PVX, because significantly increased virus levels are coupled to HR, suggesting a role of the hydroxyl radical (OH·) in this symptomless antiviral defense. In addition, treatment of PVX-susceptible tobacco with a superoxide-generating agent (riboflavin/methionine) results in HR-like symptoms and reduced PVX titers. Finally, by comparing defense responses during PVX-elicited symptomless, extreme resistance and HR-type resistance elicited by Tobacco mosaic virus, we conclude that defense reactions typical of an HR (e.g., induction of cell death/ROS-regulator genes and antioxidants) are early and transient in the course of extreme resistance. Our results demonstrate the contribution of early accumulation of ROS (superoxide, OH·) in limiting PVX replication during symptomless extreme resistance and support earlier findings that virus-elicited HR represents a delayed, slower resistance response than symptomless, extreme resistance.

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.

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.

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.

Per- and polyfluoroalkyl substances (PFAS) are a group of emerging substances that have proved to be persistent and highly bioaccumulative. They are broadly used in various applications and are known for their long-distance migration and toxicity. In this study, 65 recent specimens of a terrestrial apex predator (Common buzzard), freshwater and marine apex predators (Eurasian otter, harbour porpoise, grey seal, harbour seal) and their potential prey (bream, roach, herring, eelpout) from northern Europe (United Kingdom, Germany, the Netherlands and Sweden) were analyzed for the presence of legacy and emerging PFAS, employing a highly sensitive liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method. 56 compounds from 14 classes were measured; 13 perfluoroalkyl carboxylic acids (PFCAs), 7 perfluoroalkyl sulphonic acids (PFSAs), 3 perfluorooctane sulfonamides (FOSAs), 4 perfluoroalkylphosphonic acids (PFAPAs), 3 perfluoroalkylphosphinic acids (PFPi's), 5 telomer alcohols (FTOHs), 2 mono-substituted polyfluorinated phosphate esters (PAPs), 2 di-substituted polyfluorinated phosphate esters (diPAPs), 6 saturated fluorotelomer acids (FTAS), 3 unsaturated fluorotelomer acids (FTUAs), 2 N-Alkyl perfluorooctane sulfonamidoethanols (FOSEs), 3 fluorotelomer sulphonic acids (FTSAs), 2 perfluoroether carboxylic acids (PFECAs) and 1 chlorinated perfluoroether sulphonic acid (Cl-PFESA). All samples were lyophilized before analysis, in order to enhance extraction efficiency, improve the precision and achieve lower detection limits. The analytes were extracted from the dry matrices through generic methods of extraction, using an accelerated solvent extraction (ASE), followed by clean-up through solid phase extraction (SPE). Method detection limits and method quantification limits ranged from 0.02 to 1.25 ng/g wet weight (ww) and from 0.05 to 3.79 ng/g (ww), respectively. Recovery ranged from 40 to 137%. Method precision ranged from 3 to 20 %RSD. The sum of PFAS concentration in apex predators livers ranged from 0.2 to 20.2 μg/g (ww), whereas in the fish species muscle tissues it ranged from 16 to 325 ng/g (ww). All analyzed specimens were primarily contaminated with PFOS, while the three PFPi's included in this study exhibited frequency of appearance (FoA) 100 %. C9 to C13 PFCAs were found at high concentrations in apex predator livers, while the overall PFAS levels in fish fillets also exceeded ecotoxicological thresholds. The findings of our study show a clear association between the PFAS concentrations in apex predators and the geographical origin of the specimens, with samples that were collected in urban and agricultural zones being highly contaminated compared to samples from pristine or semi-pristine areas. The high variety of PFAS and the different PFAS composition in the apex predators and their prey (AP&P) samples is alarming and strengthens the importance of PFAS monitoring across the food chain.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAsʼ pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

The aim of the present thesis was to investigate whether new approach methodologies (NAMs) and especially physiologically based kinetic (PBK) model-based reverse dosimetry using in vitro assays and in silico data, in combination with Monte Carlo simulations or the toxic equivalency factor (TEF) methodology, can be used to derive points of departure (POD) and chemical-specific adjustment factors (CSAFs) for characterizing inter-ethnic, inter-species, and inter-individual differences in toxicokinetics and red blood cell (RBC) acetylcholinesterase (AChE) inhibition by organophosphate (OP) pesticides upon single acute oral exposure.Chapter 1 provided an overview of relevant background information, including an introduction of chlorpyrifos (CPF), diazinon (DZN), and profenofos (PFF) as model compounds, their absorption, distribution, metabolism and excretion (ADME) and toxicity profiles, as well as their reported PODs and health-based guidance values (HBGVs). The aim of the thesis and the NAMs that were applied in the thesis (PBK model-based reverse dosimetry solely, or combined with Monte Carlo simulations or the TEF methodology) were also presented. At the end, the general outline of the thesis was provided.Chapter 2 assessed the inter-ethnic differences in toxicokinetics of CPF and its resulting RBC AChE inhibition between the Chinese and Caucasian populations using PBK model-facilitated reverse dosimetry based on in vitro and in silico data. The results obtained revealed a marked inter-ethnic difference in toxicokinetics of CPF, with a slower CPF bioactivation and faster detoxification of its active metabolite chlorpyrifos oxon (CPO) in the Chinese than in the Caucasian population. This could be explained by the inter-ethnic variation in enzyme profiles and enzyme activities involved in biotransformation of CPF and CPO. The differences resulted in 5- to 6-fold lower predicted POD values of CPF for the Caucasian than the Chinese population, reflecting a lower risk of CPF for the Chinese than the Caucasian at similar levels of exposure. Besides, because of inter-ethnic variation in toxicokinetics of CPF, 2-fold lower cumulative urinary 3,5,6-trichloro-2-pyridinol (TCPy) biomarker levels were observed in Chinese than in Caucasian at similar dose levels, implying the importance of taking possible inter-ethnic differences into account when characterizing CPF exposure for different ethnic groups based on their urinary biomarker levels.Chapter 3 focused on another OP and investigated the inter-species differences between rat and human in PFF-induced RBC AChE inhibition by PBK model-based reverse dosimetry. The obtained results show the predicted POD of human to be 45-fold lower than that of rat, indicating human to be more sensitive than rat to the inhibition from equivalent oral doses of PFF. Such differences appeared mainly due to marked interspecies differences in toxicokinetics of PFF, with rat being more efficient in hepatic and plasma detoxification of PFF than human, while the difference in toxicodynamics appeared limited as reflected by a similar potency of PFF to inhibit rat RBC AChE and human recombinant AChE in vitro. In conclusion, this chapter provided another proof-of-principle that integrating in vitro toxicity data and PBK model-based reverse dosimetry is a promising strategy for non-animal-based safety assessment of OP pesticides.Chapter 4 further studied the applicability of PBK model-based reverse dosimetry to predict the combined inhibitory effect of DZN and its active oxon metabolite diazoxon (DZO) on RBC AChE inhibition by integrating the TEF approach into the model. Use of the TEF coded PBK model enabled calculation of the external exposure dose of DZN that would produce a defined combined free DZN plus DZO concentration expressed in DZO equivalents in human/rat blood, and to translate the observed inhibition effects of DZO in the in vitro concentration-response curve expressed in free in vitro DZO equivalents to the in vivo situation. The obtained results show that DZN contributes substantially to the DZO equivalents, indicating it is of critical importance to take DZN into account in addition to the active metabolite DZO when conducting a risk assessment for DZN. Although no inter-species differences were observed in the in vitro concentration-response curves for rat or human RBC AChE inhibition by DZN or DZO, marked inter-species differences in toxicokinetics were observed, as rat displayed a faster metabolic rate for biotransformation of DZN and DZO than human, resulting in a 6-fold lower POD in human than in rat. In conclusion, this chapter provided a proof-of-principle for derivation of a point of departure (POD) in risk assessment for an OP pesticide for which both the parent OP and its oxon metabolite have the ability to contribute to RBC AChE inhibition.Chapter 5 characterized the inter-individual variation in toxicokinetics of CPF and its resulting RBC AChE inhibition. A CSAF for inter-individual differences in kinetics (HKAF) was quantified on the basis of the PBK model for CPF developed in Caucasian (Chapter 2), in combination with Monte Carlo simulations. To define the metabolic variation in the PBK model, two different approaches were developed and compared, namely a SupersomeTM cytochrome P450 (CYP)-based PBK model approach and a human liver microsome (HLM)-based PBK model approach. The results revealed that bioactivation of CPF exhibits biphasic kinetics due to distinct differences in Km values for CYPs involved in the CPF metabolic pathway, and these kinetic characteristics could be best identified by SupersomeTM CYP. In general, there is a good match between the two approaches, as no substantial differences were found in the predicted HKAF values and the BMDL10 values obtained by the two procedures. The predicted HKAF values for the 99th percentile obtained by the two approaches were slightly higher than the default uncertainty factor (UF) of 3.16, reflecting possible inadequate protection for extremely sensitive adult individuals by the default UF. Altogether, this chapter provided a proof-of-principle for assessing inter-individual variation in toxicokinetics of OP pesticides and its resulting RBC AChE inhibition, and the SupersomeTM CYP-based approach seems to be the preferred option for defining inter-individual differences in CYP-mediated kinetics.Chapter 6 provided an overview of the results and main findings of the present thesis, followed by an in-depth discussion of the results obtained, and the future perspectives. It was concluded that the present thesis generated proofs-of-principle for the use of NAM and especially PBK model-facilitated reverse dosimetry based on in vitro assays and in silico data, in combination with Monte Carlo simulations or the TEF methodology, to derive PODs and CSAF values for defining inter-ethnic, inter-species and inter-individual differences in toxicokinetics and the resulting RBC AChE inhibition following an OP pesticide acute single oral exposure. Altogether, the current work supports the application of such a NAM in regulatory safety decision making on compounds, and contributes to the implementation of the 3Rs (replacement, reduction, and refinement of experimental animal studies), thereby promoting the development of next generation risk assessment.&nbsp

International audience; Lipophilic phycotoxins are secondary metabolites produced by phytoplankton. They can accumulate in edible filtering-shellfish and cause human intoxications, particularly gastrointestinal symptoms. Up to now, the in vitro intestinal effects of these toxins have been mainly investigated on simple monolayers of intestinal cells such as the enterocyte-like Caco-2 cell line. Recently, the combination of Caco-2 cells with mucus secreting HT29-MTX cell line has been also used to mimic the complexity of the human intestinal epithelium. Besides, enteric glial cells (EGC) from the enteric nervous system identified in the gut mucosa have been largely shown to be involved in gut functions. Therefore, using a novel model integrating Caco-2 and HT29-MTX cells co-cultured on inserts with EGC seeded in the basolateral compartment, we examined the toxicological effects of two phycotoxins, pectenotoxin-2 (PTX2) and okadaic acid (OA). Cell viability, morphology, barrier integrity, inflammation, barrier crossing, and the response of some specific glial markers were evaluated using a broad set of methodologies. The toxicity of PTX2 was depicted by a slight decrease of viability and integrity as well as a slight increase of inflammation of the Caco-2/HT29-MTX co-cultures. PTX2 induced some modifications of EGC morphology. OA induced IL-8 release and decreased viability and integrity of Caco-2/HT29-MTX cell monolayers. EGC viability was slightly affected by OA. The presence of EGC reinforced barrier integrity and reduced the inflammatory response of the epithelial barrier following OA exposure. The release of GDNF and BDNF gliomediators by EGC could be implicated in the protection observed.

Scope: Dietary advanced glycation endproducts (AGEs) are associated with negative biological effects, possibly due to accumulation in plasma and tissues and through modulation of inflammation and gut microbiota. Whether these biological consequences are reversible by limiting dietary AGE intake is unknown.Methods and results: Young healthy C57BL/6 mice were fed a standard chow (n = 10) or a baked chow high AGE-diet (n = 10) (similar to 1.8-6.9 fold increased protein-bound N epsilon-(carboxymethyl)lysine (CML), N epsilon-(1-carboxyethyl) lysine (CEL), and N delta-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1)) for 10 weeks or a switch diet with baked chow for 5 weeks followed by 5 weeks of standard chow (n = 10). We assessed accumulation of AGEs in plasma, kidney, and liver and measured inflammatory markers and gut microbial composition. After 10 weeks of baked chow, a substantial panel of AGEs were increased in plasma, liver, and kidney. These increases were normalized after the switch diet. The inflammatory z-score increased after the baked chow diet. Gut microbial composition differed significantly between groups, with enriched Dubosiella spp. dominating these alterations.Conclusion: A high AGE-diet led to an increase of AGEs in plasma, kidney, and liver and to more inflammation and modification of the gut microbiota. These effects were reversed or discontinued by a diet lower in AGEs.

Sewage sludge contains Ag2S-NPs causing NP exposure of soil fauna when sludge is applied as soil amendment. Earthworm bioturbation is an important process affecting many soil functions. Bioturbation may be affected by the presence of Ag2S-NPs, but the earthworm activity itself may also influence the displacement of these NPs that otherwise show little transport in the soil. The aim of this study was to determine effects of Ag2S-NPs on earthworm bioturbation and effect of this bioturbation on the vertical distribution of Ag2S-NPs. Columns (12 cm) of a sandy loamy soil with and without Lumbricus rubellus were prepared with and without 10 mg Ag kg−1, applied as Ag2S-NPs in the top 2 cm of the soil, while artificial rainwater was applied at ∼1.2 mm day−1. The soil columns were sampled at three depths weekly for 28 days and leachate collected from the bottom. Total Ag measurements showed more displacement of Ag to deeper soil layers in the columns with earthworms. The application of rain only did not significantly affect Ag transport in the soil. No Ag was detected in column leachates. X-ray tomography showed that changes in macro porosity and pore size distribution as a result of bioturbation were not different between columns with and without Ag2S-NPs. Earthworm activity was therefore not affected by Ag2S-NPs at the used exposure concentration. Ag concentrations along the columns and the earthworm density allowed the calculation of the bioturbation rate. The effect on the Ag transport in the soil shows that earthworm burrowing activity is a relevant process that must be taken into account when studying the fate of nanoparticles in soils. Earthworm bioturbation plays a more important role than rainfall in the vertical transport of Ag2S-NPs in soil.

Organophosphates have a long history of use as insecticides over the world. The aim of the present study was to investigate the interethnic differences in kinetics, biomarker formation, and in vivo red blood cell acetylcholinesterase inhibition of chlorpyrifos (CPF) in the Chinese and the Caucasian population. To this purpose, physiologically based kinetic models for CPF in both the Chinese and Caucasian population were developed, and used to study time- and dose-dependent interethnic variation in urinary biomarkers and to convert concentration-response curves for red blood cell acetylcholinesterase inhibition to in vivo dose-response curves in these 2 populations by reverse dosimetry. The results obtained revealed a marked interethnic difference in toxicokinetics of CPF, with lower urinary biomarker levels at similar dose levels and slower CPF bioactivation and faster chlorpyrifos-oxon detoxification in the Chinese compared with the Caucasian population, resulting in 5- to 6-fold higher CPF sensitivity of the Caucasian than the Chinese population. These differences might be related to variation in the frequency of single-nucleotide polymorphisms for the major biotransformation enzymes involved. To conclude, the interethnic variation in kinetics of CPF may affect both its biomarker-based exposure assessment and its toxicity and risk assessment and physiologically based kinetic modeling facilitates the characterization and quantification of these interethnic variations.

The island of Bawean, Indonesia, is home to the endemic Bawean warty pig Sus verrucosus blouchi and Bawean deer Axis kuhlii. Despite their threatened status, no long-term monitoring programme is in place for either species. Using random encounter and occupancy modelling based on 4,516 camera-trap days in 2014 and 2015 we aimed to provide population estimates and ecological data, including habitat preferences, for these species. For the Bawean warty pig we estimate an overall population size of 234–467 mature individuals and demonstrate a negative correlation between probability of occupancy and distance from villages. This preference for human-modified habitat has implications for human–wildlife conflict and hunting pressure for this species. The population of the Bawean deer could not be estimated because of the low number of encounters, but we suggest that this indicates the population is considerably smaller than previously reported. As island endemics, the Bawean warty pig and Bawean deer are particularly vulnerable to threats, and appropriate measures for safeguarding the species need to be taken.

The Wood mouse (Apodemus sylvaticus) is a widespread mammalian species that acts as a reservoir host for multiple infections, including zoonotic diseases. Exposure to immunotoxins, like for instance trace metals, may reduce the ability of the host to mount proper responses to pathogens, potentially increasing the transmission and prevalence of infections. Antibody-mediated responses are crucial in preventing and limiting infections, and the quantification of the primary antibody response is considered a sensitive predictor of immunosuppression. The current study aims to investigate effects of cadmium exposure on the antibody-mediated responses of wood mice inhabiting polluted and non-polluted areas in the Netherlands. Wood mice were captured alive at different locations and immunized to sheep red blood cells (SRBC) to induce a primary antibody response. SRBC-specific antibody-producing cells, or plaque forming cells (PFC), were quantified and related to kidney cadmium levels. Differential circulating main leukocyte populations were also characterised. Cadmium concentrations in mice kidneys differed between mice captured at different locations, and increased with individual body mass, likely associated with age-related time of exposure. Effect of cadmium was apparent on the percentages of B cell counts in blood. Because of potential natural immune heterogeneity between wild rodent populations, mice immune responses were analysed and compared grouped by captured locations. Capture location had significant effect on the total counts of white blood cells. Increasing cadmium exposure in wood mice captured from polluted sites was associated with a decrease of splenic PFC counts. This field research shows that wood mice antibody responses can be impaired by cadmium exposure, even at low environmental levels, by affecting B cell functioning mainly. Impaired B cell function can make exposed mice more susceptible to infections, potentially increasing the reservoir function of their populations. It also shows that immunomodulatory effects in the field should be assessed site specifically.

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.

Background Human milk is the gold standard for the nutrition of infants. Human milk oligosaccharides (HMOs) are structurally diverse sugars highly abundant in human milk but not present in infant formula. The interest in HMOs has gradually increased in recent years due to their beneficial functions on infants. Scope and approach Here we summarize the structures and composition of HMOs, and compare the differences of oligosaccharides in human and other mammals. The absorption and metabolism of HMOs are discussed. Then we discuss existing studies on the functional biology of HMOs. Additionally, the structure-function relationships of HMOs are presented. Finally, this review summarizes the oligosaccharides in recent infant formula as well as limitations and future perspectives of HMOs research. Key findings and conclusions The structures and composition of HMOs are affected by many factors. Large amounts of HMOs are metabolized by gut microbiota, and few are absorbed into circulation to perform physiological functions. Evidences from in vitro and in vivo studies, as well as clinical trials suggest that HMOs modulate gut microbiota, reduce pathogen adhesion, modulate epithelial cell and immune responses, reduce the risk of necrotizing enterocolitis and promote brain development and cognition. The specific structure determines different functions. Currently two HMOs, 2′-fucosyllactose and Lacto-N-Neotetraose, have been demonstrated safe and well-tolerated to be added into infant formula. However, the application in HMOs has been limited due to their insufficient availability. Thus, basic and clinical studies of HMOs need to be further elucidated.

Stable isotopes are often used to provide an indication of the trophic level (TL) of species. TLs may be derived by using food-web-specific enrichment factors in combination with a representative baseline species. It is challenging to sample stable isotopes for all species, regions and seasons in Arctic ecosystems, e.g. because of practical constraints. Species-specific TLs derived from a single region may be used as a proxy for TLs for the Arctic as a whole. However, its suitability is hampered by incomplete knowledge on the variation in TLs. We quantified variation in TLs of Arctic species by collating data on stable isotopes across the Arctic, including corresponding fractionation factors and baseline species. These were used to generate TL distributions for species in both pelagic and benthic food webs for four Arctic areas, which were then used to determine intra-sample, intra-study, intra-region and inter-region variation in TLs. Considerable variation in TLs of species between areas was observed. This is likely due to differences in parameter choice in estimating TLs (e.g. choice of baseline species) and seasonal, temporal and spatial influences. TLs between regions were higher than the variance observed within regions, studies or samples. This implies that TLs derived within one region may not be suitable as a proxy for the Arctic as a whole. The TL distributions derived in this study may be useful in bioaccumulation and climate change studies, as these provide insight in the variability of trophic levels of Arctic species.

Previous studies have explored effects of imidacloprid and its metabolites on terrestrial species, such as bees, and indicated the importance of some active metabolites. However, the biotransformation of IMI and the toxicity of its metabolites to aquatic arthropods are largely unknown, especially the mechanisms driving species sensitivity differences and time-cumulative toxicity effects. To assess the potential effects of the metabolization of IMI and the toxicokinetics and toxicity of the metabolite(s) on aquatic arthropods, we first studied the acute toxicity of IMI and relevant metabolites to the mayfly species Cloen dipterum (sensitive to IMI) and the amphipod species Gammarus pulex (less sensitive to IMI). Secondly, toxicokinetic experiments were conducted using both the parent compound and imidacloprid-olefin (IMI-ole), a metabolite assessed as toxic in the acute tests and defined as bioactive. Of the four tested metabolites, only IMI-ole was readily biotransformed from the parent IMI and showed similar toxicity to C. dipterum as IMI. However, C. dipterum was hardly able to eliminate IMI-ole from its body. For G. pulex, IMI-ole was also the only detected metabolite causing toxicity, but the biotransformation of IMI to IMI-ole was slower and lower in G. pulex compared to C. dipterum, and G. pulex eliminated IMI-ole quicker than C. dipterum. Our results on internal kinetics of IMI and IMI-ole, and on biotransformation of IMI indicated that the metabolite IMI-ole was toxic and was rather persistent inside the body tissue of both invertebrate species, especially for C. dipterum. In conclusion, as IMI and IMI-ole have similar toxicity and IMI was replaced rapidly by IMI-ole which in turn was poorly eliminated by C. dipterum, the overall toxicity is a function of dose and time. As a result, no long-term threshold of effects of IMI may exist for C. dipterum as the poor elimination results in an ongoing increase of toxicity over time for mayflies as also found experimentally in previous published papers.

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 flavor ingredients. This publication, the sixth in the series, will summarize the re-evaluation of eight NFCs whose constituent profiles are characterized by significant amounts of eucalyptol and/or other cyclic ethers. This re-evaluation was based on a procedure first published in 2005 and subsequently updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure relies on a complete chemical characterization of the NFC intended for commerce and the organization of its chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of the constituents of the congeneric groups and the NFC under evaluation. Eight NFCs derived from the Eucalyptus, Melaleuca, Origanum, Laurus, Rosmarinus and Salvia genera were 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.

Contamination of food and feed by mycotoxins is considered one of most serious food safety problems in the world, because these fungal metabolites can be teratogenic, mutagenic, carcinogenic and immunosuppressive, and can cause serious damages to animal and human health. Mycotoxins may modulate the gut microbiota with potential consequences for gut and host health. On the other hand, the gut microbiota may metabolize the mycotoxins thereby converting them to a form with different activity. Chemical-microbial interactions can be categorized into two classes: Microbiome Modulation of Toxicity (MMT) and Toxicant Modulation of the Microbiome (TMM). The present review provides a state-of-the-art overview of this bi-directional interaction between the major food-borne mycotoxins such as aflatoxins, ochratoxins, deoxynivalenols and zearalenone present in food, feed and the gut microbiota. In addition, the effect of probiotics on gut microbiota in animals exposed to mycotoxins is summarized. Possible consequences of the role of gut microbiota for the risk assessment of mycotoxins, are also discussed. It is concluded that without taking the role of the gut microbiota into account effects of food-borne mycotoxins on health may be underestimated.

Fructoselysine is formed upon heating during processing of food products, and being a key intermediate in advanced glycation end product formation considered to be potentially hazardous to human health. Human gut microbes can degrade fructoselysine to yield the short chain fatty acid butyrate. However, quantitative information on these biochemical reactions is lacking, and interindividual differences therein are not well established. Anaerobic incubations with pooled and individual human fecal slurries were optimized and applied to derive quantitative kinetic information for these biochemical reactions. Of 16 individuals tested, 11 were fructoselysine metabolizers, with Vmax, Km and kcat-values varying up to 14.6-fold, 9.5-fold, and 4.4-fold, respectively. Following fructoselysine exposure, 10 of these 11 metabolizers produced significantly increased butyrate concentrations, varying up to 8.6-fold. Bacterial taxonomic profiling of the fecal samples revealed differential abundant taxa for these reactions (e.g. families Ruminococcaceae, Christenellaceae), and Ruminococcus_1 showed the strongest correlation with fructoselysine degradation and butyrate production (ρ ≥ 0.8). This study highlights substantial interindividual differences in gut microbial degradation of fructoselysine. The presented method allows for quantification of gut microbial degradation kinetics for foodborne xenobiotics, and interindividual differences therein, which can be used to refine prediction of internal exposure.

International audience; To evaluate the nanoparticle (NP) toxicity, much efforts have been devoted for developing methods to accurately disperse NPs into aqueous suspensions prior to in vitro toxicological studies. As NP toxicity is strongly dependent on their physicochemical properties, NP characterization is a key step for any in vitro toxicological study. This study demonstrates that the static multiple light scattering (SMLS) technique allows for the simultaneous screening of the NP size, agglomeration state, stability and dosimetry in biological media. Batch dispersions of TiO2 P25 NPs in water with various bovine serum albumin (BSA) mass fractions (from 0% to 0.5%) and dilutions of these dispersions into cell culture media were characterized with SMLS. In the batch dispersions, TiO2 NPs are stable and well dispersed for BSA mass fraction lower than 0.2% while agglomeration and rapid settling is observed for higher BSA mass fractions. Paradoxically, when diluted in cell culture media, TiO2 NPs are well dispersed and stable for BSA mass fractions higher than 0.2%. The TiO2 NP dosimetry of these dilutions was evaluated experimentally with SMLS and confronted with numerical approaches. The TiO2 NP bottom concentration evolves far more slowly in the case of the higher BSA mass fraction. Such measurements give valuable insights on the NP fate and transport in biological media to obtain in fine reliable size and dose-cytotoxicity responses.

The present study investigated the developmental toxicity of diethylstilbestrol (DES) in the zebrafish embryotoxicity test (ZET). This was done to investigate whether the ZET would better capture the developmental toxicity of DES than the embryonic stem cells test (EST) that was previously shown to underpredict the DES-induced developmental toxicity as compared to in vivo data, potentially because the EST does not capture late events in the developmental process. The ZET results showed DES-induced growth retardation, cumulative mortality and dysmorphisms (i.e. induction of pericardial edema) in zebrafish embryos while the endogenous ERα agonist 17β-estradiol (E2) showed only growth retardation and cumulative mortality with lower potency compared to DES. Furthermore, the DES-induced pericardial edema formation in zebrafish embryos could be counteracted by co-exposure with ERα antagonist fulvestrant, indicating that the ZET captures the role of ERα in the mode of action underlying the developmental toxicity of DES. Altogether, it is concluded that the ZET differentiates DES from E2 with respect to their developmental toxicity effects, while confirming the role of ERα in mediating the developmental toxicity of DES. Furthermore, comparison to in vivo data revealed that, like the EST, in a quantitative way also the ZET did not capture the relatively high in vivo potency of DES as a developmental toxicant.

Increasing oil and gas activities may substantially increase chemical stress to benthic ecosystems in the Arctic, and it is necessary to evaluate such environmental risks in these systems. Risk assessment procedures for oil‐related compounds (e.g., polycyclic aromatic hydrocarbons [PAHs]) should address differences in exposure between Arctic and temperate benthos. We compare for the first time the bioaccumulation of PAHs by Arctic benthic invertebrate species with that of temperate species, based on their biota–sediment accumulation factors (BSAFs). Measured PAH BSAFs were generally higher in temperate bivalves (Limecola balthica) than in Arctic bivalves (Macoma calcarea), whereas BSAFs in Arctic polychaetes (Nephtys ciliata) were higher than in temperate polychaetes (Alitta virens). Differences in measured BSAFs were explained by species‐specific feeding modes and traits. However, modeled BSAFs revealed that steady state was not likely to be reached in the 28‐d tests for all PAHs and organisms. Due to the low numbers of individuals, most species‐specific parameters were too uncertain to reveal differences between Arctic and temperate species. The results of the present study suggest that data from temperate species could be used as a surrogate for Arctic species in risk assessment. Environ Toxicol Chem 2019;38:883–895. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Highlights • Nixtamalized maize from Mexico showed a high percentage of left censoring for AFB1. • Four samples above the limit of detection, albeit still below the regulatory limit. • Risk assessment reveals public health concern and a priority for risk management. • AFB1 exposure and risks mainly originate from the high consumption of this cereal., Maize is a staple food in Mexico that might contain Aflatoxin B1 (AFB1). Nonetheless, data on the exposure and risk assessment of AFB1 from maize for the Mexican population are limited. The aim of the present study was to analyse the occurrence of AFB1 in Mexican nixtamalized maize samples, and to assess the accompanying exposure and risk. Four out of 88 samples contained AFB1 at levels above the limit of detection (1 ng/g). AFB1 occurrence values obtained in this study and additional occurrence values from literature were combined with available literature data for mean and P95 consumption of maize based products. For a 70 kg body weight person, lower bound and upper bound exposure assessments resulted in estimated daily intakes (EDI) of 0.7–8.5 ng/kg bw/day, based on a mean maize consumption. Based on the P95 maize consumption these EDI values amounted to 3.3–11.7 ng/kg bw/day. The corresponding Margin of Exposure (MOE) values amounted to 257-20 for the mean and 50-15 for the P95 consumers. The estimated increased cancer risks were 9-320 and 43-439 cases/106 individuals/lifetime of 75 years for the mean and P95 consumers, respectively. Altogether, the assessment reveals the need for continued risk management of AFB1 in Mexico.

Due to the unique properties of differently sized and coated silver nanoparticles (AgNPs), they are used in important industrial and biomedical applications. However, their environmental fate in soil ecosystems and potential mechanisms of toxicity remain elusive, especially at the level of transcriptional regulation. We investigated the transcriptome-wide responses of the earthworm Lumbricus rubellus exposed to nine AgNPs differing in surface coating/charge (bovine serum albumin/negative AgNP_BSA, chitosan/positive AgNP_Chit, and polyvinylpyrrolidone/neutral AgNP_PVP) and sizes (20, 35 and 50 nm) at concentrations close to the EC50 value related to reproduction. AgNO3 was used in two concentrations to benchmark the AgNP effects against those of the Ag salt. A correlation was observed between the number of differentially expressed genes (DEGs) and Ag internal body concentration. Only metallothionein was regulated by all treatments. Medium sized AgNPs caused the most pronounced transcriptional responses, while AgNO3 affected the transcriptome less. Medium sized AgNP_BSA exposure caused the most extensive transcriptional responses with 684 DEGs. Gene ontology enrichment analysis of medium sized AgNP_BSA affected DEGs revealed that mitochondrial electron transport, autophagy and phagocytosis, mesoderm and heart development and microtubule organisation were affected. This was also confirmed by gene set enrichment for KEGG pathway analysis, indicating that phagocytosis, autophagy and signalling pathways related to mesoderm formation were significantly up regulated. All AgNP_BSA and AgNP_PVP exposures caused severe down regulation of ribosomal translation, suggesting that the high energy-demanding protein synthesis process is inhibited. Our data confirm the mechanisms previously identified among other animal models and human cell lines. To conclude, coating formulation and particle size severely impact transcriptional responses at a particular nanoparticle size, suggesting diverse mechanistic responses depending on the coating type.

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 flavor ingredients. This publication, fifth in the series, evaluates the safety of NFCs containing linalool and/or other characteristic mono- and sesquiterpenoid tertiary alcohols and esters using the safety evaluation procedure published by the FEMA Expert Panel in 2005 and updated in 2018. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of the chemical constituents of each NFC into well-defined congeneric groups. The safety of each NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of both the constituent congeneric groups and the NFCs. Sixteen NFCs, derived from the Lavandula, Aniba, Elettaria, Daucus, Salvia, Coriandrum, Ribes, Guaiacum/Bulnesia, Citrus, Pogostemon, Melaleuca and Michelia genera, were 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.

ObjectiveCampylobacter jejuni produces a genotoxin, cytolethal distending toxin (CDT), which has DNAse activity and causes DNA double-strand breaks. Although C. jejuni infection has been shown to promote intestinal inflammation, the impact of this bacterium on carcinogenesis has never been examined.DesignGerm-free (GF) ApcMin/+mice, fed with 1% dextran sulfate sodium, were used to test tumorigenesis potential of CDT-producing C. jejuni. Cells and enteroids were exposed to bacterial lysates to determine DNA damage capacity via γH2AX immunofluorescence, comet assay and cell cycle assay. To examine the interplay of CDT-producing C. jejuni, gut microbiome and host in tumorigenesis, colonic RNA-sequencing and faecal 16S rDNA sequencing were performed. Rapamycin was administrated to investigate the prevention of CDT-producing C. jejuni-induced tumorigenesis.ResultsGF ApcMin/+mice colonised with human clinical isolate C. jejuni81–176 developed significantly more and larger tumours when compared with uninfected mice. C. jejuni with a mutated cdtB subunit, mutcdtB, attenuated C. jejuni-induced tumorigenesis in vivo and decreased DNA damage response in cells and enteroids. C. jejuni infection induced expression of hundreds of colonic genes, with 22 genes dependent on the presence of cdtB. The C. jejuni-infected group had a significantly different microbial gene expression profile compared with the mutcdtB group as shown by metatranscriptomic data, and different microbial communities as measured by 16S rDNA sequencing. Finally, rapamycin could diminish the tumorigenic capability of C. jejuni.ConclusionHuman clinical isolate C. jejuni 81–176 promotes colorectal cancer and induces changes in microbial composition and transcriptomic responses, a process dependent on CDT production.

This review provides an overview of results obtained when using proteome analysis for detecting sex-based differences in response to toxicants. It reveals implications to be taken into account when considering the use of proteomics in toxicological studies. It appears that results may differ when studying the same chemical in the same species in different target tissues. Another result of interest is the limited dose-response behavior of differential abundance patterns observed in studies where more than one dose level is tested. It is concluded that use of proteomics to study differences in modes of action of toxic compounds is an active area of research. The examples from use of proteomics to study sex-dependent differences also reveal that further studies are needed to provide reliable insight in modes of action, novel biomarkers or even novel therapies. To eventually reach this aim for this and other toxicological endpoints, it is essential to consider background variability, consequences of timing of toxicant administration, dose-response behavior, relevant species and target organ, species and organ variability and the presence of proteoforms.

Microplastic fibres (MPFs) and nanoplastics (NPs) have the potential to be hazardous to soil organisms. Understanding uptake into organisms is key in assessing these effects, but this is often limited by the analytical challenges to quantify smaller-sized plastics in complex matrices. This study used MPFs and NPs containing inorganic tracers (In, Pd) to quantify uptake in the earthworm Lumbricus terrestris. Following seven days exposure, tracer concentrations were measured in earthworms and faeces. Earthworms exposed to 500 μg MPFs/g soil retained an estimated 32 MPFs in their tissues, while at 5000 μg MPFs/g earthworms retained between 2 and 593 MPFs. High variation in body burdens of MPFs was linked to soil retention in earthworms and reduced faeces production, suggesting egestion was being affected by MPFs. NPs uptake and elimination was also assessed over a more extended time-period of 42 days. After 1 day, NPs were no longer detectable in faeces during the elimination phase. However, some retention of NPs in the earthworms was estimated, not linked to retained soil, indicating not all NPs were eliminated. MPFs and NPs uptake can be quantified in earthworms and both particle types can be retained beyond the depuration period, suggesting the potential for longer-term accumulation. ISSN:0048-9697 ISSN:1879-1026

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.

The metabolic functionality of the gut microbiota contributes to the metabolism and well-being of its host, although detailed insight in the microbiota's metabolism is lacking. Omics technologies could facilitate unraveling metabolism by the gut microbiota. In this study, we performed metabolite profiling of different matrices of the gut, after antibiotic treatment of rats in order to evaluate metabolite changes observed at different dose levels and in different sexes, and to identify the best tissue matrix for further investigations regarding an assessment of metabolic effects of new compounds with antibiotic activity. Three different antibiotics (vancomycin, streptomycin and roxithromycin) were administered orally to rats for 28 days according to the OECD 407 guideline with a subsequent metabolic profiling in feces, cecum content and gut tissue (jejunum, ileum, cecum, colon and rectum). The data were analyzed in the MetaMap®Tox database. Treatment-related effects could be observed in the metabolite profile of feces and cecum content, but not of the different gut tissues. The metabolite profile showed compound specific effects on the microbiome. In line with the activity spectra of the antibiotics tested, vancomycin showed the largest effects, followed by roxithromycin and then by streptomycin for which changes were modest. In general, for all antibiotics the largest changes were observed for the classes of lipids (increase up to 94-fold), bile acids (increase up to 33-fold), amino acids (increase up to 200-fold) and amino acid related (increase up to 348-fold). The most relevant changes in metabolite values were similar in feces and cecum content and among sexes. The results of this targeted analysis indicate that the metabolic profiles of male and female animals in the gut microbiome are comparable. Concluding, taking other samples than feces does not add any extra information. Thus, as a non-invasive sampling method, feces provide a suitable matrix for studies on metabolism by the gut microbiota.

Mass media reports suggest that pangasius (Pangasianodon hypophthalmus) is ‘full of poison’ because it is able to survive in the ‘heavily polluted Mekong River’ and contains pesticides and veterinary treatment chemicals. However, most of these claims are not substantiated with scientific evidence. To assess the safety of pangasius consumption, a full toxicological risk assessment was performed. The results obtained were compared to toxicity claims made in a selection of media reports. Information on contaminant levels encountered in pangasius was collected from the EU Rapid Alert System for Food and Feed (RASFF) database. The toxicological risk assessments do not support any of the toxicological risks suggested in the media. Next, the maximum pangasius consumption that would be considered safe was estimated on the basis of the highest levels of the toxic contaminants reported in Vietnamese pangasius by RASFF and the safety thresholds for the contaminants. The maximum amount of the recalled fillet that could have been consumed without any adverse effects amounted to between 3.4 and 166.7 kg day−1 (lifelong for a 70 kg adult) in the case of pesticide contamination and between 0.613 and 303 kg fillet day−1 in the case of preservatives and antibiotics. It is concluded that consumption of pangasius available on the European market does not pose any concern for the health of the consumer. The analysis presented in this study illustrates that publicly available independent information could help consumers to develop their own well-informed opinion about food safety issues.

This annual review is the third one to highlight recent advances in the study and assessment of biotransformations and bioactivations ( Table 1 ). We followed the same format as the previous years with selection and authoring each section (see Baillie et al. 2016 ; Khojasteh et al. 2017 ). We acknowledge that many universities no longer train students in mechanistic biotransformation studies reflecting a decline in the investment for those efforts by public funded granting institutions. We hope this work serves as a resource to appreciate the knowledge gained each year to understand and hopefully anticipate toxicological outcomes dependent on biotransformations and bioactivations. This effort itself also continues to evolve. I am pleased that Drs. Rietjens and Miller have again contributed to this annual review. We would like to welcome Kaushik Mitra as an author for this year's issue, and we thank Deepak Dalvie for his contributions to last year's edition. We have intentionally maintained a balance of authors such that two come from an academic setting and two come from industry. As always, please drop us a note if you find this review helpful. 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.

The potential developmental toxicity and mode-of-action of fume condensate extracts of bitumen and oxidized asphalt were evaluated in the aryl hydrocarbon receptor (AhR) CALUX assay, the zebrafish embryotoxicity test (ZET), and the mouse embryonic stem cell test (mEST). In the AhR CALUX assay, both fume condensate extracts showed a concentration-dependent AhR induction following 6-h of exposure, but this activity was substantially reduced after 24-h, indicating a transient AhR activation. The main effect observed in the ZET was early embryonic lethality that occurred mostly in the 24 h-post-fertilization (hpf). This typically reflects non-specific toxicity rather than in vitro developmental toxicity of the fume condensate extracts tested since this effect was not seen as a result of the whole cumulative exposure period in the ZET (up to 96 hpf). No malformations were seen in any zebrafish embryo exposed to these fume condensate extracts, although some developed pericardial and/or yolk-sac edemas. Furthermore, both fume condensate extracts tested negative in the mEST. In conclusion, the results show that fume condensate extracts of bitumen and oxidized asphalt do not induce any in vitro developmental toxicity, which is in line with the results observed in the in vivo prenatal developmental toxicity studies performed with the same materials.

Aspergillus fumigatus is the leading cause of invasive aspergillosis, which in immunocompromised patients results in a mortality rate as high as 90%. Earlier studies showed that HbxA is a global regulator in Aspergillus flavus affecting morphological development and secondary metabolism. Here, we determined its role in A. fumigatus, examining whether HbxA influences the regulation of asexual development, natural product biosynthesis, and virulence of this fungus. Our analysis demonstrated that removal of the hbxA gene caused a near-complete loss of conidial production in the mutant strain, as well as a slight reduction in colony growth. Other aspects of asexual development are affected, such as size and germination of conidia. Furthermore, we showed that in A. fumigatus, the loss of hbxA decreased the expression of the brlA central regulatory pathway involved in asexual development, as well as the expression of the “fluffy” genes flbB, flbD, and fluG. HbxA was also found to regulate secondary metabolism, affecting the biosynthesis of multiple natural products, including fumigaclavines, fumiquinazolines, and chaetominine. In addition, using a neutropenic mouse infection model, hbxA was found to negatively impact the virulence of A. fumigatus. IMPORTANCE The number of immunodepressed individuals is increasing, mainly due to the greater life expectancy in immunodepressed patients due to improvements in modern medical treatments. However, this population group is highly susceptible to invasive aspergillosis. This devastating illness, mainly caused by the fungus Aspergillus fumigatus, is associated with mortality rates reaching 90%. Treatment options for this disease are currently limited, and a better understanding of A. fumigatus genetic regulatory mechanisms is paramount for the design of new strategies to prevent or combat this infection. Our work provides new insight into the regulation of the development, metabolism, and virulence of this important opportunistic pathogen. The transcriptional regulatory gene hbxA has a profound effect on A. fumigatus biology, governing multiple aspects of conidial development. This is relevant since conidia are the main source of inoculum in Aspergillus infections. Importantly, hbxA also regulates the biosynthesis of secondary metabolites and the pathogenicity of this fungus.

Background Obesity is a major public health concern worldwide. A sedentary life and a nutritional transition to processed foods and high-calorie diets are contributing factors to obesity. The demand for nutraceutical foods, such as herbal weight-loss products, which offer the potential to counteract obesity, has consequently increased. We hypothesised that Opuntia cladodes consumption could assist weight management in an obesity prevention context. Methods This study was designed to explore the anti-adipogenic effects of lyophilised Opuntia cladode powders (OCP) in an in vitro cellular model for adipocyte differentiation and an in vivo high-fat-diet (HFD)-induced obesity rat model. Two OCP were tested, one from wild species O. streptacantha and the second from the most known species O. ficus-indica. Results Pre-adipocytes 3 T3-F442A were treated by OCP during the differentiation process by insulin. OCP treatment impaired the differentiation in adipocytes, as supported by the decreased triglyceride content and a low glucose uptake, which remained comparable to that observed in undifferentiated controls, suggesting that an anti-adipogenic effect was exerted by OCP. Sprague–Dawley rats were fed with a normal or HFD, supplemented or not with OCP for 8 weeks. OCP treatment slightly reduced body weight gain, liver and abdominal fat weights, improved some obesity-related metabolic parameters and increased triglyceride excretion in the faeces. Taken together, these results showed that OCP might contribute to reduce adipogenesis and fat storage in a HFD context, notably by promoting the faecal excretion of fats. Conclusions Opuntia cladodes may be used as a dietary supplement or potential therapeutic agent in diet-based therapies for weight management to prevent obesity. Graphical abstract

The author would like to thank N. Bakhiya, S. Hessel-Pras, B. Sachse, and B. Dusemund for their support in the chapter about pyrrolizidine alkaloids.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association initiated the safety re-evaluation of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, 4th in a series focusing on the safety evaluation of NFCs, presents an evaluation of NFCs rich in hydroxyallylbenzene and hydroxypropenylbenzene constituents using a procedure initially published in 2005 and updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure requires the characterization of the chemical composition for each NFC and subsequent organization of the constituents into defined congeneric groups. The safety of each NFC is evaluated using the conservative threshold of toxicological concern (TTC) approach together with studies on absorption, metabolism and toxicology of the NFC and its constituent congeneric groups. By the application of this procedure, seven NFCs, derived from clove, cinnamon leaf and West Indian bay leaf were affirmed as “generally recognized as safe (GRAS)” under their conditions of intended use as flavor ingredients. An eighth NFC, an oleoresin of West Indian bay leaf, was affirmed based on its estimated intake, which is below the TTC of 0.15 μg/person per day for compounds with structural alerts for genotoxicity.

The risk assessment of chemical carcinogens is one major task in toxicology. Even though exposure has been mitigated effectively during the last decades, low levels of carcinogenic substances in food and at the workplace are still present and often not completely avoidable. The distinction between genotoxic and non-genotoxic carcinogens has traditionally been regarded as particularly relevant for risk assessment, with the assumption of the existence of no-effect concentrations (threshold levels) in case of the latter group. In contrast, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites are considered to represent risk factors at all concentrations since even one or a few DNA lesions may in principle result in mutations and, thus, increase tumour risk. Within the current document, an updated risk evaluation for genotoxic carcinogens is proposed, based on mechanistic knowledge regarding the substance (group) under investigation, and taking into account recent improvements in analytical techniques used to quantify DNA lesions and mutations as well as “omics” approaches. Furthermore, wherever possible and appropriate, special attention is given to the integration of background levels of the same or comparable DNA lesions. Within part A, fundamental considerations highlight the terms hazard and risk with respect to DNA reactivity of genotoxic agents, as compared to non-genotoxic agents. Also, current methodologies used in genetic toxicology as well as in dosimetry of exposure are described. Special focus is given on the elucidation of modes of action (MOA) and on the relation between DNA damage and cancer risk. Part B addresses specific examples of genotoxic carcinogens, including those humans are exposed to exogenously and endogenously, such as formaldehyde, acetaldehyde and the corresponding alcohols as well as some alkylating agents, ethylene oxide, and acrylamide, but also examples resulting from exogenous sources like aflatoxin B1, allylalkoxybenzenes, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), benzo[a]pyrene and pyrrolizidine alkaloids. Additionally, special attention is given to some carcinogenic metal compounds, which are considered indirect genotoxins, by accelerating mutagenicity via interactions with the cellular response to DNA damage even at low exposure conditions. Part C finally encompasses conclusions and perspectives, suggesting a refined strategy for the assessment of the carcinogenic risk associated with an exposure to genotoxic compounds and addressing research needs.

Chapter 1 introduced the objectives of the thesis and background information regarding the production of nanomaterials (NMs) and their release in the environment. The concepts of environmental and bio-mediated transformations of NMs were defined and their implication on the NM exposure assessment is explained. Silver nanoparticles (Ag-NPs) were selected as model NM because they are widely produced, undergo transformations that represent all the most relevant transformations of NMs in soil and specific analytical methods are available to quantify them at relatively low concentrations. A short review regarding previous toxicological studies of Ag-NPs in soil organisms and the importance of performing toxicokinetic studies was presented. Finally, selected model organisms (earthworms Eisenia fetida and Lumbricus rubellus) were briefly described. Chapter 2 reported a short-term (28 days) toxicokinetic study in E. fetida exposed to Ag-NPs, aged Ag-NPs (Ag2S-NPs), and AgNO3. A one-compartment model was applied to calculate separately the kinetic constants for uptake and elimination of particulate and ionic forms of Ag. The uptake and elimination rate constants for earthworms exposed to pristine Ag-NP or AgNO3 were not significantly different from each other. Uptake rate constants of (hardly dissolvable) Ag2S-NPs which resemble the environmental relevant form of Ag-NPs was significantly lower. spICP-MS analysis demonstrated that ~85% (average of both Ag-NP and AgNO3 treatments) of the Ag within the earthworms was present as ionic Ag, regardless of the actual form of Ag that the earthworms were exposed to. Indeed, the biogenic formation of particulate Ag (~10 % of total Ag accumulated overtime) in earthworms exposed to AgNO3 led to a kinetic pattern of particulate Ag body burden similar to pristine Ag-NPs. NP size analysis and imaging techniques showed evidences that the particles in the tissues were not the same as those to which worms were exposed, highlighting that biotransformation and/or biogenic formation took place also in the case of the Ag-NP exposure. Chapter 3 investigated the influence of dissolution on the uptake of metal NPs in earthworms by the use of bimetallic NPs. E. fetida specimens were exposed to Au core-Ag shell NPs (Au@Ag-NPs) and to a combination of Au-NPs, Ag-NPs, Ag and Au ions containing natural soil for 28 days. Our hypothesis was that Ag shell would dissolve partially or completely and that Au core would not interact with the exposure media and would therefore behave as a tracer of the particulate uptake. Analysis of earthworm tissues showed that concentrations of Ag in the earthworms were not statistically different in organisms exposed to the different forms of Ag. However, the concentration of Au in the earthworms exposed to HAuCl4 (ionic Au) exceeded around twenty times the Au concentrations in the exposures to particulate Au, which did not differ among each other. Mass measurements by spICP-TOFMS provided evidence that the uptake of the metals in their bimetallic particulate form represents approximately 5 % of the total metal amount. Size measurements by spICP-TOFMS showed that the Au core remained similar after the uptake, while the Ag shell increased in thickness suggesting that biotransformation processes took place at the surface of the NPs (e.g. aggregation, adsorption of Ag ions on the surface of existing particles). The study confirmed that dissolution is the main factor driving the uptake of (dissolving) metal NPs in earthworms. Additionally, different uptake patterns resulted from the co-exposure to Au and Ag-NP and Ag+, indicating that the Ag form can lead to different interactions with Au in the soil affecting the uptake in the earthworms. Chapter 4 presented a toxicokinetic study performed to assess the potential impacts of long-term exposure (nine months) on the uptake of pristine Ag-NP, aged Ag-NP (Ag2S-NP) and ionic Ag in earthworms E. fetida. The study was conducted with same species and conditions similar to the short-term experiment which was previously conducted for 4 weeks (chapter 2), in order to allow comparison between the two models. The accumulation of Ag in Ag-NP and AgNO3 exposed earthworms did not statistically differ after nine months exposure. In Ag2S-NPs exposed earthworms, the internalized concentrations were five times lower compared to the other treatments. The Ag concentrations in pore water did not reflect the uptake pattern and metallothionein concentrations were not different from the control group. The overall conclusion of this chapter was that even after a prolonged period of time the uptake kinetic rate constants of Ag-NP and AgNO3 were not statistically different, while the one of Ag2S-NP was statistically significant lower than the other treatments. Additionally, the short-term kinetic rate constants predicted the average bioaccumulation of pristine Ag-NP and AgNO3 in the earthworms exposed for nine months, while the bioaccumulation of Ag2S-NPs in earthworms was under-predicted somewhat. This was likely because the short-term did not take into account the late dissolution of Ag2-NPs. Ag bioaccumulation of Ag2S-NP could not be related to the concentrations of Ag measured at a specific time in pore water. Chapter 5 reports a study which demonstrated that earthworm bioturbation plays an important role in the vertical transport of Ag2S-NPs in soil. In the soil columns, daily rainfall from artificial rain water did not lead to displacement of Ag2S-NPs within 28 days indicating that in the case of hardly soluble metal NPs and unsaturated soil conditions, bio-mediated transport overcomes physical chemical transport. Bioturbation from L. rubellus was quantified by assessing the changes of the macro porosity in the soil columns. Results indicated that earthworms burrowing activity was not affected by the presence of Ag2S-NPs at the experimental concentrations. The study proposed a linear relationship between bioturbation rate and the abundance of earthworms that is applicable to future bioturbation studies. Chapter 6 presented an overall discussion of the results obtained in the thesis, and concluded with the implication of such findings in the risk assessment of NMs.

Human membrane drug transporters are recognized as major actors of pharmacokinetics. Pesticides also interact with human drug transporters, which may have consequences for pesticide toxicokinetics and toxicity. The present review summarizes key findings about this topic. In vitro assays have demonstrated that some pesticides, belonging to various chemical classes, modulate drug transporter activity, regulate transporter expression and/or are substrates, thus bringing the proof of concept for pesticide-transporter relationships. The expected low human concentration of pesticides in response to environmental exposure constitutes a key-parameter to be kept in mind for judging the in vivo relevance of such pesticide-transporter interactions and their consequences for human health. Existing data about interactions of pesticides with drug transporters remain, however, rather sparse; more extensive and systematic characterization of pesticide-transporter relationships, through the use of high throughput in vitro assays and/or in silico methods, is, therefore, required. In addition, consideration of transporter polymorphisms, pesticide mixture effects and physiological and pathological factors governing drug transporter expression may help to better define the in vivo relevance of pesticide-transporter interactions in terms of toxicokinetics and toxicity for humans. © 2019 Society of Chemical Industry.

Diethylstilbestrol (DES) is a synthetic estrogen that has been used between the 1940s and 1970s by pregnant women to prevent miscarriages and premature delivery by stimulating the synthesis of estrogen and progesterone in the placenta. However, use of DES appeared to cause a wide range of adverse effects, such as clear cell vaginal adenocarcinoma in the daughters of women who took the drug, and developmental and reproductive toxicity. These adverse effects have often been attributed to the functional estrogen receptor alpha (ERα), since it has been reported that ERα is needed to induce DES-mediated adverse developmental and reproductive effects in neonates. The question has been raised why DES behaves differently from the endogenous ERα agonist 17β-estradiol (E2), even though the molecular dimensions and binding orientations of DES and E2 to the ERα are almost identical. The research described in this thesis aimed to investigate the possible differences in the estrogenicity and developmental toxicity between DES and E2, using different in vitro and in silico approaches, focussing on the potential role of possible differences in ERα-mediated effects in the underlying mode of action. Accordingly, first the effect of DES and E2 on ERα-mediated reporter gene expression, ERα-mediated T47D breast cancer cell proliferation, and ERα-coregulator interactions and gene expression in T47D cells were evaluated. In addition, the effects of DES and E2 in two alternative developmental toxicity assays (the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) and the zebrafish embyotoxicity test (ZET)) and the potential role of ERα in these effects were evaluated. Finally, possible dose-dependent differences in internal dose levels of DES and E2 were evaluated with help of PBK modelling, in order to elucidate to what extent possible differences in kinetics could play a role in differential in vivo effects of DES and E2. Altogether, the data show that two estrogens E2 and DES differ in their biological effects related to development in a subtle but significant way. At the cellular level, DES and E2 show high similarities in the molecular pathways that relate to ERα-mediated effects with small significant differences that may contribute to the developmental toxicity in part via potential epigenetic effects of DES. The in vitro developmental toxicity assays EST and ZET can discriminate DES from E2 in terms of developmental toxicity, but at the same time do not capture the full mode of action underlying DES-induced developmental toxicity. Finally, it was shown that in addition to the subtle differences in toxicodynamics, substantial differences in internal concentrations (endogenous E2 concentrations compared to predicted DES concentrations in women that took DES as medication), add to the differential in vivo effects of E2 and DES.

Fumonisins (FB1+FB2) and deoxynivalenol (DON) are mycotoxins produced by Fusarium species that might be present in maize and maize products. Knowledge on their occurrence in nixtamalized maize from Mexico together with an accompanying risk assessment are scarce, while nixtamalized maize is an important food in Mexico. This study presents the occurrence of FB1 + FB2 and DON in nixtamalized maize samples collected in Mexico City and analyses their distribution and resulting estimated daily intake for Mexican consumers by a probabilistic approach using a two-dimensional Monte-Carlo simulation. The results obtained reveal that for FB1 + FB2, 47% of the Mexican men and 30% of the Mexican women might exceed the provisional tolerable daily intake (PMTDI) of 2 &micro, g/kg bw/day for fumonisins and for DON, 9% of men and 5% of women would be exceeding the PMTDI of 1 &micro, g/kg bw/day, corresponding to the high consumers. The results raise a flag for risk managers in Mexico, to consider regulations and interventions that lower mycotoxin levels in nixtamalized maize for human consumption.

Currently, wild edible plants are getting into the research focus of both food and pharmaceutical domains as they are considered valuable resources for improving food and nutritional security as well as for bioactive ingredients that can be used for functional foods or managing chronic diseases. Wild edible plants, in addition to their potential nutritional contribution, may provide important health benefits as the health-promoting components of plant based foods, so called nutraceuticals or functional food ingredients, are usually higher in wild plants. However, at the same time, these plants may contain toxic ingredients that may lead to adverse effects. At the current state of the art novel foods have to comply with novel food regulation while safety of the use of botanicals and botanical preparation as food is the responsibility of the provider and not subject to premarket safety evaluation. This implies that a concern requiring adequate consideration of both risks and benefits has to be addressed when introducing wild edible plants as novel components in a regular diet or as herbal medicine or food supplement. The present PhD thesis aims at investigating the possible hazards and potential health benefits of Maerua subcordata (Gilg) DeWolf, a wild plant locally called ‘ashkulebya’ by the Kunama ethnics of Northern Ethiopia, as a novel nutritional resource and/or as a health food/functional food. To meet this aim, the thesis tested the activity of different extracts of M. subcordata in a wide range of in vitro assays for both adverse and beneficial endpoints, along with, phytochemical studies to identify active constituents. Consideration of ethnobotanical data was also part of the study. The phytochemical studies identified glucosinolates and biogenic amines such as quaternary ammonium compounds and guanidine derivatives in the different M. subcordata extracts. The potential health benefits of M. subcordata extracts via their effects on the expression of electrophile-responsive element (EpRE), peroxisome proliferator-activated receptor gamma (PPARg), and nuclear factor kappa B (NF-κB) regulated genes was evaluated. The induction of EpRE-mediated genes have implications of maintaining redox homeostasis. The fruit, leaf, and seed extracts induced the expression of EpRE-mediated genes while induction by the root extract was minimal. Candidate glucosinolates (or their matching isothiocyanates) exhibited strong induction while some biogenic amines exhibited either no significant induction or slight inhibition. Inhibition of NF-κB regulated genes have implications for anti-inflammatory effects while induction of PPARg regulated genes have implications for control of diseases of the metabolic syndrome. The fruit, root, and seed extracts induced PPARγ-mediated gene expression while induction by the leaf extract was minimal. Instead, all extracts inhibited bacterial lipopolysaccharide (LPS) induced nitric oxide production, with the root showing the lowest potency. Selected candidates such as isothiocyanates and some biogenic amines also inhibited the LPS induced nitric oxide production. Equally, a battery of in vitro tests was applied to identify selected endpoints that could be used to assess the potential hazards associated with the use of botanical preparations or parts of M. subcordata. The Ames test and in silico (Derek Nexus) predictions were applied for initial genotoxicity screening whereas the aryl hydrocarbon receptor and the estrogen receptor alpha reporter gene assays as well as the embryonic stem cell test and the zebrafish embryotoxicity test were applied to investigate alerts for developmental toxicity. The overall hazard assessment results indicated that all extracts of M. subcordata do not point to a genotoxicity hazard; fruit, root, and seed extracts do not raise a concern with respect to developmental toxicity; but the data on the leaf extract may point towards a developmental toxicity hazard. In conclusion, the overall results obtained argue in favour of the use of the root, fruit, and seed parts of M. subcordata as (famine) food and/or functional food whereas the leaf part may be used as a herbal medicine. The outcomes of the study may also contribute scientific data to design further in vivo studies on safety and proper utilization of the plant while the methodology applied can be extrapolated to serve as an approach to the risk-benefit assessment of other plant based products.

The aim of the present thesis was to perform the risk and safety assessment of two groups of natural food-borne toxins, namely alkenylbenzenes and pyrrolizidine alkaloids (PAs) for the Chinese population as compared to the overall Caucasian population using methods including the Margin of Exposure (MOE) approach, physiologically based kinetic (PBK) modelling-based reverse dosimetry and the combination of PBK modelling and Monte Carlo simulation. Chapter 1 introduced the model compounds studied in the thesis, namely alkenylbenzenes and 1,2-unsaturated PAs, the aim of the thesis and the strategies applied to perform a mode of action based safety and risk assessment. The regulations and consumer behaviour towards botanical preparations in China and in several EU countries were also presented. In chapter 2, a risk assessment of 10 plant food supplements (PFS), 23 traditional Chinese medicines (TCM) and 38 herbal teas containing alkenylbenzenes obtained at the Chinese market was performed using the Margin of Exposure (MOE) approach. Use of about half of the samples would result in estimated intakes upon daily life-time exposure that give rise to MOE values lower than 10 000, suggesting a potential priority for risk management. For short-term exposure such as two weeks consumption, applying Haber’s rule, only one TCM (TCM 6) still had a MOE value below 10 000. It is concluded that consumption of Chinese botanical preparations raise a concern because of exposure to alkenylbenzenes, especially when exposure is for longer periods of time. Chapter 3 studied the differences in bioactivation and detoxification of the food-borne genotoxic carcinogen estragole between Chinese and Caucasians using a mode of action based PBK modelling approach. The outcomes of the model predictions showed that the detoxification of the proximate carcinogenic metabolite 1’-hydroxyestragole, mainly by conversion to 1’-oxoestragole, was similar in both ethnic groups. For the bioactivation pathway, at realistic daily intakes of estragole, in the Chinese population only 0.02 % of the dose was converted to the ultimate carcinogenic metabolite 1’-sulfooxyestragole, while this value amounted to 0.09 % of the dose in Caucasian subjects. This 4.5-fold difference accompanied by similar rates of detoxification may indicate a lower risk of estragole for the Chinese population at similar levels of exposure. In chapter 4, it was demonstrated that combining in vitro cytotoxicity data obtained with primary rat hepatocytes with PBK modelling-based reverse dosimetry could adequately predict in vivo acute liver toxicity of lasiocarpine and riddelliine for rats. The PBK models for rats for lasiocarpine and riddelliine were developed based on data derived from in silico approaches, incubation experiments using subcellular tissue fractions and literature to define the PBK model parameters. Concentration-response curves obtained from in vitro cytotoxicity assays in primary rat hepatocytes were converted to in vivo dose-response curves for acute liver toxicity by PBK-modelling based reverse dosimetry. From the in vivo dose-response curves thus obtained points of departure (PoDs) were derived that were compared to available literature data on in vivo liver toxicity of lasiocarpine. The predicted PoDs appeared to fall well within the range of PoDs obtained from the available oral single dose in vivo studies. In conclusion, this chapter showed that PBK modelling based-reverse dosimetry can translate in vitro concentration-response curves to in vivo dose-response curves to predict the acute liver toxicity of lasiocarpine and riddelliine in rats. Chapter 5 investigated the inter-species and inter-ethnic human differences in acute liver toxicity of lasiocarpine and riddelliine using the approach developed in chapter 4 for rats. Thus, PBK models for lasiocarpine and riddelliine for the average Chinese and Caucasian were developed, based on the PBK models defined and validated for these PAs in rats (chapter 4). Subsequently, the models were used to convert in vitro toxicity data obtained in pooled Caucasian primary hepatocytes to predict in vivo dose-response curves for acute liver toxicity of lasiocarpine and riddelliine in humans, from which PoDs were derived that were compared to PoDs derived in a similar manner for rats in chapter 4 to obtain insight in inter-species differences. Similarly, the PoDs obtained for the average Chinese and Caucasian were compared to provide insights in inter-ethnic differences. The inter-species differences amounted to 2-fold for lasiocarpine and 8.2-fold for riddelliine with humans being more sensitive than rats. The inter-ethnic human differences varied 2-fold for lasiocarpine and 5-fold for riddelliine with the average Caucasian being more sensitive than the average Chinese. Altogether, this chapter shows a proof-of-principle for a method to predict inter-species and inter-ethnic differences in in vivo liver toxicity for PAs by an alternative testing strategy integrating in vitro cytotoxicity assays with PBK modelling-based reverse dosimetry. Chapter 6 investigated the effect of inter-individual and inter-ethnic (Chinese and Caucasian) kinetic differences in bioactivation of lasiocarpine, and definition of chemical specific adjustment factors (CSAFs) by applying PBK modelling and Monte Carlo simulation. The results revealed an inter-ethnic variation of 2.1-, 3.3- and 4.3-fold when comparing the predicted 7-GS-DHP formation at the GM, 90th and 99th percentile of the Chinese and Caucasian population, respectively, at a dose of level of 8 ng/kg bw lasiocarpine, indicating that the Caucasian population was more sensitive. The CSAFs obtained based on the GM and 90th percentile individuals of the Chinese, Caucasian and the two populations combined were 3.3, 5.2 and 5.7, respectively. The CSAFs obtained based on the GM and the 99th percentile were 8.3, 17.0 and 19.5 for inter-individual variations in the Chinese, the Caucasian population and the two populations combined, respectively. The CSAF values obtained in this chapter indicate that the default safety factor of 3.16 for inter-individual human kinetic differences may not be sufficiently protective for any of the populations. Chapter 7 presents the general discussion based on the results of the present thesis placing the results in a wider perspective while also presenting future perspectives. It is concluded that the present thesis showed an alternative to animal testing approach to define in vivo dose-response curves for risk assessment, based on in silico and in vitro data. It was demonstrated that combining in vitro toxicity data with the kinetic processes integrated into a PBK model adequately predicts in vivo acute liver toxicity of PAs. The obtained results show the possibility to use the method to provide a PoD to perform risk assessment or to define CSAFs for inter-species and intra-species differences. Additionally, this thesis provided insight in the possibilities to build ethnic-specific PBK models that can be used to further refine the risk assessment of compounds that are genotoxic and carcinogenic and also have non-genotoxic endpoints. Moreover, this thesis illustrated that there is a potential priority for risk management for several botanical preparations containing alkenylbenzenes or PAs for the Chinese population when considering daily life-long exposure. Altogether, this thesis provided a proof-of-principle to perform a risk and safety assessment for food-borne toxins based on only in vitro and in silico data.

A total diet study (TDS) was undertaken to estimate the chronic dietary exposure to pesticide residues and health risks for the French infants and young children below 3 years old. As a whole, 516 pesticides and metabolites were analysed in 309 food composite samples including 219 manufactured baby foods and 90 common foods, which cover 97% of infants and young children's diet. These composite samples were prepared using 5,484 food products purchased during all seasons from 2011 to 2012 and processed as consumed. Pesticide residues were detected in 67% of the samples and quantified in 27% of the baby food samples and in 60% of the common foods. Seventy-eight different pesticides were detected and 37 of these quantified at levels ranging from 0.02 to 594 µg/kg. The most frequently detected pesticides (greater than 5% samples) were (1) the fungicides 2-phenylphenol, azoxystrobin, boscalid, captan and its metabolite tetrahydrophthalimide, carbendazim, cyprodinil, difenoconazole, dodine, imazalil, metalaxyl, tebuconazole, thiabendazole, (2) the insecticides acetamiprid, pirimiphos-methyl and thiacloprid, (3) the herbicide metribuzin and (4) the synergist piperonyl butoxide. Dietary intakes were estimated for each of the 705 individuals studied and for 431 pesticides incl. 281 with a toxicological reference value (TRV). In the lower-bound scenario, which tends to underestimate the exposure, the TRV were never exceeded. In the upper-bound scenario that overestimates exposure, the estimated intakes exceeded the TRV for dieldrin and lindane (two persistent organic pollutants) and propylene thiourea, a metabolite of propineb. For these three substances, more sensitive analyses are needed to refine the assessment. For 17 other detected and/or prioritised pesticides, the risk could not be characterised due to the lack of a valid TRV, of certain food analyses or the absence of analytical standards for their metabolites. Keywords: Food safety, Infants and young children, Pesticide residues, Total diet study, Exposure assessment, Risk characterization

International audience; The H2AX histone protein is rapidly phosphorylated at the serine-139 position (gammaH2AX) in response to a broad range of DNA lesions. gammaH2AX induction is one of the earliest events in the DNA damage response (DDR) and plays a central role in sensing and repairing DNA damage. Since its discovery, measuring gammaH2AX formation using numerous methods in in vitro and in vivo experiments has been an attractive endpoint for the detection of genotoxic agents. Our review focuses on validation studies performed using this biomarker to detect the genotoxicity of model chemicals using different methods. To date, nearly two hundred genotoxic and carcinogenic model chemicals have been shown to induce in vitro gammaH2AX in different cell lines by numerous laboratories. Based on 27 published reports comprising 329 tested chemicals, we compared the performance of the gammaH2AX assay with other genotoxic endpoints (Ames assay, micronucleus, HPRT and comet) regularly used for in vitro genotoxicity assessment. Notably, the gammaH2AX assay performs well (91% predictivity) and efficiently differentiates aneugenic and clastogenic compounds when coupled with the pH3 biomarker. Currently, no formal guidelines have been approved for the gammaH2AX assay for regular genotoxicity studies, but we suggest the gammaH2AX biomarker could be used as a new standard genotoxicity assay and discuss its future role in genotoxicity risk assessment.