Risk assessment of small organoarsenic species in food.

The European Commission asked EFSA for a risk assessment on small organoarsenic species in food. For monomethylarsonic acid MMA(V), decreased body weight resulting from diarrhoea in rats was identified as the critical endpoint and a BMDL10 of 18.2 mg MMA(V)/kg body weight (bw) per day (equivalent to 9.7 mg As/kg bw per day) was calculated as a reference point (RP). For dimethylarsinic acid DMA(V), increased incidence in urinary bladder tumours in rats was identified as the critical endpoint. A BMDL10 of 1.1 mg DMA(V)/kg bw per day (equivalent to 0.6 mg As/kg bw per day) was calculated as an RP. For other small organoarsenic species, the toxicological data are insufficient to identify critical effects and RPs, and they could not be included in the risk assessment. For both MMA(V) and DMA(V), the toxicological database is incomplete and a margin of exposure (MOE) approach was applied for risk characterisation. The highest chronic dietary exposure to DMA(V) was estimated in 'Toddlers', with rice and fish meat as the main contributors across population groups. For MMA(V), the highest chronic dietary exposures were estimated for high consumers of fish meat and processed/preserved fish in 'Infants' and 'Elderly' age class, respectively. For MMA(V), an MOE of ≥ 500 was identified not to raise a health concern. For MMA(V), all MOEs were well above 500 for average and high consumers and thus do not raise a health concern. For DMA(V), an MOE of 10,000 was identified as of low health concern as it is genotoxic and carcinogenic, although the mechanisms of genotoxicity and its role in carcinogenicity of DMA(V) are not fully elucidated. For DMA(V), MOEs were below 10,000 in many cases across dietary surveys and age groups, in particular for some 95th percentile exposures. The Panel considers that this would raise a health concern.

Effects of 2-ethylhexyl diphenyl phosphate (EHDPP) on glycolipid metabolism in male adult zebrafish revealed by targeted lipidomic analyses.

The present study aims to evaluate the effects of 2-ethylhexyldiphenyl phosphate (EHDPP) on glycolipid metabolism in vivo. Adult male zebrafish were exposed to various concentrations (0, 1, 10, 100 and 250 µg/L) of EHDPP for 28 days, and changes in lipid and glucose levels were measured. Results indicated significant liver damages in the 100 and 250 µg/L EHDPP groups, which both exhibited significant decreases in hepatic somatic index (HSI), elevated activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and liver, as well as hepatocyte vacuolation and nuclear pyknosis. Exposure to 100 and 250 µg/L EHDPP led to significant reductions in serum and liver cholesterol (TC), triglycerides (TGs), and lipid droplet deposition, indicating a significant inhibition of EHDPP on hepatic lipid accumulation. Lipidomic analyses manifested that 250 µg/L EHDPP reduced the levels of 103 lipid metabolites which belong to glycerides (TGs, diglycerides, and monoglycerides), fatty acyles (fatty acids), sterol lipids (cholesterol, bile acids), sphingolipids, and glycerophospholipids, and downregulated genes involved in de novo synthesis of fatty acids (fas, acc, srebp1, and dagt2), while upregulated genes involved in fatty acid ß-oxidation (pparα and cpt1). KEGG analyses revealed that EHDPP significantly disrupted glycerolipid metabolism, steroid biosynthesis and fatty acid biosynthesis pathways. Collectively, the results showed that EHDPP induced lipid reduction in zebrafish liver, possibly through inhibiting lipid synthesis and disrupting glycerolipid metabolism. Our findings provide a theoretical basis for evaluating the ecological hazards and health effects of EHDPP on glycolipid metabolism.

Zinc transporters expression profile in professional handball players supplemented with zinc.

INTRODUCTION: Zinc (Zn) deficiency has been described not only on general human health but also within the sports context -as negatively affecting performance-. Thus, Zn status assessment is of great interest for athletes, especially in order to correct deficiency states of this mineral. OBJECTIVE: The overall objective of this work was to assess Zn status in professional handball players during the competitive period (through plasma levels, dietary intake and gene expression of the Zn transporters), as well as to determine the effect of Zn supplementation. METHODS: A total of twenty-two participants were recruited, -twelve belonged to the Control Group (CG) and ten male handball players comprised the experimental group (ATH-G)-, being monitored over a 2-month period with 2 evaluation moments: baseline (i.e., initial conditions) and follow-up (i.e., after 8 weeks of training and competition). Zn intake, plasma Zn levels, and gene expression of Zn transporters were obtained. RESULTS: Plasma Zn levels were higher in ATH-G than in CG at the end of Zn intervention (p ≤ 0.010). Moreover, differences in the gene expression profile of Zn transporters were observed in ATH-G -with the down-regulation of several Zn transporters-, compared to the CG at baseline (p ≤ 0.05). Likewise, differences in the Zn transporters expression were observed in ATH-G at 8 weeks (all, p ≤ 0.001) -with ZnT2, ZnT5, ZIP3, ZIP5, ZIP11, ZIP13 and ZIP14 transporters being up-regulated-. CONCLUSION: Handball players seemed to have different nutritional needs for Zn, with differences in the gene expression of Zn transporters compared to controls. Zn intervention in our athletes may have influenced the expression of Zn transporters, indicating a potential increase in Zn transporters expression to mobilize Zn at the cellular level at 8 weeks of Zn intervention.

Ip3r-Grp75-Vdac and Relevant Ca2+ Signaling Regulate Dietary Palmitic Acid-Induced De Novo Lipogenesis by Mitochondria-Associated ER Membrane (MAM) Recruiting Seipin in Yellow Catfish.

BACKGROUND: The mitochondria-associated endoplasmic reticulum membrane (MAM) is the central hub for endoplasmic reticulum and mitochondria functional communication. It plays a crucial role in hepatic lipid homeostasis. However, even though MAM has been acknowledged to be rich in enzymes that contribute to lipid biosynthesis, no study has yet investigated the exact role of MAM on hepatic neutral lipid synthesis. OBJECTIVES: To address these gaps, this study investigated the systemic control mechanisms of MAM on neutral lipids synthesis by recruiting seipin, focusing on the role of the inositol trisphosphate receptor-1,4,5(Ip3r)-75 kDa glucose-regulated protein (Grp75)-voltage-dependent anion channel (Vdac) complex and their relevant Ca2+ signaling in this process. METHODS: To this end, a model animal for lipid metabolism, yellow catfish (Pelteobagrus fulvidraco), were fed 6 different diets containing a range of palmitic acid (PA) concentrations from 0-150 g/kg in vivo for 10 wk. In vitro, experiments were also conducted to intercept the MAM-mediated Ca2+ signaling in isolated hepatocytes by transfecting them with si-mitochondrial calcium uniporter (mcu). Because mcu was placed in the inner mitochondrial membrane (IMM), si-mcu cannot disrupt MAM's structural integrity. RESULTS: 1. Hepatocellular MAM subproteome analysis indicated excessive dietary PA intake enhanced hepatic MAM structure joined by activating Ip3r-Grp75-Vdac complexes. 2. Dietary PA intake induced hepatic neutral lipid accumulation through MAM recruiting Seipin, which activated lipid droplet biogenesis. Our findings also revealed a previously unidentified mechanism whereby MAM-recruited seipin and controlled hepatic lipid homeostasis, depending on Ip3r-Grp75-Vdac-controlled Ca2+ signaling and not only MAM's structural integrity. CONCLUSIONS: These results offer a novel insight into the MAM-recruited seipin in controlling hepatic lipid synthesis in a MAM structural integrity-dependent and Ca2+ signaling-dependent manner, highlighting the critical contribution of MAM in maintaining hepatic neutral lipid homeostasis.

Risks for animal and human health related to the presence of polychlorinated naphthalenes (PCNs) in feed and food.

EFSA was asked for a scientific opinion on the risks for animal and human health related to the presence of polychlorinated naphthalenes (PCNs) in feed and food. The assessment focused on hexaCNs due to very limited data on other PCN congeners. For hexaCNs in feed, 217 analytical results were used to estimate dietary exposures for food-producing and non-food-producing animals; however, a risk characterisation could not be performed because none of the toxicological studies allowed identification of reference points. The oral repeated dose toxicity studies performed in rats with a hexaCN mixture containing all 10 hexaCNs indicated that the critical target was the haematological system. A BMDL20 of 0.05 mg/kg body weight (bw) per day was identified for a considerable decrease in the platelet count. For hexaCNs in food, 2317 analytical results were used to estimate dietary exposures across dietary surveys and age groups. The highest exposure ranged from 0.91 to 29.8 pg/kg bw per day in general population and from 220 to 559 pg/kg bw per day for breast-fed infants with the highest consumption of breast milk. Applying a margin of exposure (MOE) approach, the estimated MOEs for the high dietary exposures ranged from 1,700,000 to 55,000,000 for the general population and from 90,000 to 230,000 for breast-fed infants with the highest consumption of breast milk. These MOEs are far above the minimum MOE of 2000 that does not raise a health concern. Taking account of the uncertainties affecting the assessment, the Panel concluded with at least 99% certainty that dietary exposure to hexaCNs does not raise a health concern for any of the population groups considered. Due to major limitations in the available data, no assessment was possible for genotoxic effects or for health risks of PCNs other than hexaCNs.

Guidance for the assessment of detoxification processes in feed.

This statement provides scientific guidance on the information needed to support the risk assessment of the detoxification processes applied to products intended for animal feed in line with the acceptability criteria of the Commission Regulation (EU) 2015/786.

Update of the risk assessment of polybrominated diphenyl ethers (PBDEs) in food.

The European Commission asked EFSA to update its 2011 risk assessment on polybrominated diphenyl ethers (PBDEs) in food, focusing on 10 congeners: BDE-28, -47, -49, -99, -100, -138, -153, -154, -183 and ­209. The CONTAM Panel concluded that the neurodevelopmental effects on behaviour and reproductive/developmental effects are the critical effects in rodent studies. For four congeners (BDE-47, -99, -153, -209) the Panel derived Reference Points, i.e. benchmark doses and corresponding lower 95% confidence limits (BMDLs), for endpoint-specific benchmark responses. Since repeated exposure to PBDEs results in accumulation of these chemicals in the body, the Panel estimated the body burden at the BMDL in rodents, and the chronic intake that would lead to the same body burden in humans. For the remaining six congeners no studies were available to identify Reference Points. The Panel concluded that there is scientific basis for inclusion of all 10 congeners in a common assessment group and performed a combined risk assessment. The Panel concluded that the combined margin of exposure (MOET) approach was the most appropriate risk metric and applied a tiered approach to the risk characterisation. Over 84,000 analytical results for the 10 congeners in food were used to estimate the exposure across dietary surveys and age groups of the European population. The most important contributors to the chronic dietary Lower Bound exposure to PBDEs were meat and meat products and fish and seafood. Taking into account the uncertainties affecting the assessment, the Panel concluded that it is likely that current dietary exposure to PBDEs in the European population raises a health concern.

Risks for animal health related to the presence of ergot alkaloids in feed.

The European Commission requested EFSA to provide an update of the 2012 Scientific Opinion of the Panel on Contaminants in the Food Chain (CONTAM) on the risks for animal health related to the presence of ergot alkaloids (EAs) in feed. EAs are produced by several fungi of the Claviceps and Epichloë genera. This Opinion focussed on the 14 EAs produced by C. purpurea (ergocristine, ergotamine, ergocornine, α- and ß-ergocryptine, ergometrine, ergosine and their corresponding 'inine' epimers). Effects observed with EAs from C. africana (mainly dihydroergosine) and Epichloë (ergovaline/-inine) were also evaluated. There is limited information on toxicokinetics in food and non-food producing animals. However, transfer from feed to food of animal origin is negligible. The major effects of EAs are related to vasoconstriction and are exaggerated during extreme temperatures. In addition, EAs cause a decrease in prolactin, resulting in a reduced milk production. Based on the sum of the EAs, the Panel considered the following as Reference Points (RPs) in complete feed for adverse animal health effects: for pigs and piglets 0.6 mg/kg, for chickens for fattening and hens 2.1 and 3.7 mg/kg, respectively, for ducks 0.2 mg/kg, bovines 0.1 mg/kg and sheep 0.3 mg/kg. A total of 19,023 analytical results on EAs (only from C. purpurea) in feed materials and compound feeds were available for the exposure assessment (1580 samples). Dietary exposure was assessed using two feeding scenarios (model diets and compound feeds). Risk characterisation was done for the animals for which an RP could be identified. The CONTAM Panel considers that, based on exposure from model diets, the presence of EAs in feed raises a health concern in piglets, pigs for fattening, sows and bovines, while for chickens for fattening, laying hens, ducks, ovines and caprines, the health concern related to EAs in feed is low.

Update of the risk assessment of inorganic arsenic in food.

The European Commission asked EFSA to update its 2009 risk assessment on arsenic in food carrying out a hazard assessment of inorganic arsenic (iAs) and using the revised exposure assessment issued by EFSA in 2021. Epidemiological studies show that the chronic intake of iAs via diet and/or drinking water is associated with increased risk of several adverse outcomes including cancers of the skin, bladder and lung. The CONTAM Panel used the benchmark dose lower confidence limit based on a benchmark response (BMR) of 5% (relative increase of the background incidence after adjustment for confounders, BMDL05) of 0.06 µg iAs/kg bw per day obtained from a study on skin cancer as a Reference Point (RP). Inorganic As is a genotoxic carcinogen with additional epigenetic effects and the CONTAM Panel applied a margin of exposure (MOE) approach for the risk characterisation. In adults, the MOEs are low (range between 2 and 0.4 for mean consumers and between 0.9 and 0.2 at the 95th percentile exposure, respectively) and as such raise a health concern despite the uncertainties.

Bioaccumulation and thyroid endcrione disruption of 2-ethylhexyl diphenyl phosphate at environmental concentration in zebrafish larvae.

2-ethylhexyl diphenyl phosphate (EHDPP) strongly binds to transthyretin (TTR) and affects the expression of genes involved in the thyroid hormone (TH) pathway in vitro. However, it is still unknown whether EHDPP induces endocrine disruption of THs in vivo. In this study, zebrafish (Danio rerio) embryos (< 2 h post-fertilization (hpf)) were exposed to environmentally relevant concentrations of EHDPP (0, 0.1, 1, 10, and 100 µg·L-1) for 120 h. EHDPP was detected in 120 hpf larvae at concentrations of 0.06, 0.15, 3.71, and 59.77 µg·g-1 dry weight in the 0.1, 1, 10, and 100 µg·L-1 exposure groups, respectively. Zebrafish development and growth were inhibited by EHDPP, as indicated by the increased malformation rate, decreased survival rate, and shortened body length. Exposure to lower concentrations of EHDPP (0.1 and 1 µg·L-1) significantly decreased the whole-body thyroxine (T4) and triiodothyronine (T3) levels and altered the expressions of genes and proteins involved in the hypothalamic-pituitary-thyroid axis. Downregulation of genes related to TH synthesis (nis and tg) and TH metabolism (dio1 and dio2) may be partially responsible for the decreased T4 and T3 levels, respectively. EHDPP exposure also significantly increased the transcription of genes involved in thyroid development (nkx2.1 and pax8), which may stimulate the growth of thyroid primordium to compensate for hypothyroidism. Moreover, EHDPP exposure significantly decreased the gene and protein expression of the transport protein transthyretin (TTR) in a concentration-dependent manner, suggesting a significant inhibitory effect of EHDPP on TTR. Molecular docking results showed that EHDPP and T4 partly share the same mode of action of binding to the TTR protein, which might result in decreased T4 transport due to the binding of EHDPP to the TTR protein. Taken together, our findings indicate that EHDPP can cause TH disruption in zebrafish and help elucidate the mechanisms underlying EHDPP toxicity.

Moderate replacement of fish oil with palmitic acid-stimulated mitochondrial fusion promotes ß-oxidation by Mfn2 interacting with Cpt1α via its GTPase-domain.

The mitochondrial matrix serves as the principal locale for the process of fatty acids (FAs) ß-oxidation. Preserving the integrity and homeostasis of mitochondria, which is accomplished through ongoing fusion and fission events, is of paramount importance for the effective execution of FAs ß-oxidation. There has been no investigation to date into whether and how mitochondrial fusion directly enhances FAs ß-oxidation. The underlying mechanism of a balanced FAs ratio favoring hepatic lipid homeostasis remains largely unclear. To address such gaps, the present study was conducted to investigate the mechanism through which a balanced dietary FAs ratio enhances hepatic FAs ß-oxidation. The investigation specifically focused on the involvement of Mfn2-mediated mitochondrial fusion in the regulation of Cpt1α in this process. In the present study, the yellow catfish (Pelteobagrus fulvidraco), recognized as a model organism for lipid metabolism, were subjected to eight weeks of in vivo feeding with six distinct diets featuring varying FAs ratios. Additionally, in vitro experiments were conducted to inhibit Mfn2-mediated mitochondrial fusion in isolated hepatocytes, achieved through the transfection of hepatocytes with si-mfn2. Further, deletion mutants for both Mfn2 and Cpt1α were constructed to elucidate the critical regions responsible for the interactions between these two proteins within the system. The key findings were: (1) Substituting palmitic acid (PA) for fish oil (FO) proved to be enhanced in reducing hepatic lipid accumulation. This beneficial effect was primarily attributed to the activation of mitochondrial FAs ß-oxidation; (2) The balanced replacement of PA stimulated Mfn2-mediated mitochondrial fusion by diminishing Mfn2 ubiquitination, thereby enhancing its protein retention within the mitochondria; (3) Mfn2-mediated mitochondrial fusion promoted FAs ß-oxidation through direct interaction between Mfn2 and Cpt1α via its GTPase-domains, which is essential for the maintenance of Cpt1 activity. Notably, the present research results unveil a previously undisclosed mechanism wherein Mfn2-mediated mitochondrial fusion promotes FAs ß-oxidation by directly augmenting the capacity for FA transport into mitochondria (MT), in addition to expanding the mitochondrial matrix. This underscores the pivotal role of mitochondrial fusion in preserving hepatic lipid homeostasis. The present results further confirm that these mechanisms are evolutionarily conserved, extending their relevance from fish to mammals.

Risks for animal health related to the presence of ochratoxin A (OTA) in feed.

In 2004, the EFSA Panel on Contaminants in the Food Chain (CONTAM) adopted a Scientific Opinion on the risks to animal health and transfer from feed to food of animal origin related to the presence of ochratoxin A (OTA) in feed. The European Commission requested EFSA to assess newly available scientific information and to update the 2004 Scientific Opinion. OTA is produced by several fungi of the genera Aspergillus and Penicillium. In most animal species it is rapidly and extensively absorbed in the gastro-intestinal tract, binds strongly to plasma albumins and is mainly detoxified to ochratoxin alpha (OTalpha) by ruminal microbiota. In pigs, OTA has been found mainly in liver and kidney. Transfer of OTA from feed to milk in ruminants and donkeys as well as to eggs from poultry is confirmed but low. Overall, OTA impairs function and structure of kidneys and liver, causes immunosuppression and affects the zootechnical performance (e.g. body weight gain, feed/gain ratio, etc.), with monogastric species being more susceptible than ruminants because of limited detoxification to OTalpha. The CONTAM Panel considered as reference point (RP) for adverse animal health effects: for pigs and rabbits 0.01 mg OTA/kg feed, for chickens for fattening and hens 0.03 mg OTA/kg feed. A total of 9,184 analytical results on OTA in feed, expressed in dry matter, were available. Dietary exposure was assessed using different scenarios based on either model diets or compound feed (complete feed or complementary feed plus forage). Risk characterisation was made for the animals for which an RP could be identified. The CONTAM Panel considers that the risk related to OTA in feed for adverse health effects for pigs, chickens for fattening, hens and rabbits is low.

Aryl hydrocarbon receptor utilises cellular zinc signals to maintain the gut epithelial barrier.

Zinc and plant-derived ligands of the aryl hydrocarbon receptor (AHR) are dietary components affecting intestinal epithelial barrier function. Here, we explore whether zinc and the AHR pathway are linked. We show that dietary supplementation with an AHR pre-ligand offers protection against inflammatory bowel disease in a mouse model while protection fails in mice lacking AHR in the intestinal epithelium. AHR agonist treatment is also ineffective in mice fed zinc depleted diet. In human ileum organoids and Caco-2 cells, AHR activation increases total cellular zinc and cytosolic free Zn2+ concentrations through transcription of genes for zinc importers. Tight junction proteins are upregulated through zinc inhibition of nuclear factor kappa-light-chain-enhancer and calpain activity. Our data show that AHR activation by plant-derived dietary ligands improves gut barrier function at least partly via zinc-dependent cellular pathways, suggesting that combined dietary supplementation with AHR ligands and zinc might be effective in preventing inflammatory gut disorders.

Update of the risk assessment of mineral oil hydrocarbons in food.

Mineral oil hydrocarbons (MOH) are composed of saturated hydrocarbons (MOSH) and aromatic hydrocarbons (MOAH). Due to the complexity of the MOH composition, their complete chemical characterisation is not possible. MOSH accumulation is observed in various tissues, with species-specific differences. Formation of liver epithelioid lipogranulomas and inflammation, as well as increased liver and spleen weights, are observed in Fischer 344 (F344) rats, but not in Sprague-Dawley (SD) rats. These effects are related to specific accumulation of wax components in the liver of F344 rats, which is not observed in SD rats or humans. The CONTAM Panel concluded that F344 rats are not an appropriate model for effects of MOSH with wax components. A NOAEL of 236 mg/kg body weight (bw) per day, corresponding to the highest tested dose in F344 rats of a white mineral oil product virtually free of wax components, was selected as relevant reference point (RP). The highest dietary exposure to MOSH was estimated for the young population, with lower bound-upper bound (LB-UB) means and 95th percentiles of 0.085-0.126 and 0.157-0.212 mg/kg bw per day, respectively. Considering a margin of exposure approach, the Panel concluded that the present dietary exposure to MOSH does not raise concern for human health for all age classes. Genotoxicity and carcinogenicity are associated with MOAH with three or more aromatic rings. For this subfraction, a surrogate RP of 0.49 mg/kg bw per day, calculated from data on eight polycyclic aromatic hydrocarbons, was considered. The highest dietary exposure to MOAH was also in the young population, with LB-UB mean and 95th percentile estimations of 0.003-0.031 and 0.011-0.059 mg/kg bw per day, respectively. Based on two scenarios on three or more ring MOAH contents in the diet and lacking toxicological information on effects of 1 and 2 ring MOAH, a possible concern for human health was raised.

Novel mechanism for zinc inducing hepatic lipolysis via the HDAC3-mediated deacetylation of ß-catenin at lysine 311.

Zinc (Zn) is a multipurpose trace element indispensable for vertebrates and possesses essential regulatory roles in lipid metabolism, but the fundamental mechanism remains largely unknown. In the current study, we found that a high-Zn diet significantly increased hepatic Zn content and influenced the expression of Zn transport-relevant genes. Dietary Zn addition facilitated lipolysis, inhibited lipogenesis, and controlled ß-catenin signal; Zn also promoted T-cell factor 7-like 2 (TCF7L2) to interact with ß-catenin and regulating its transcriptional activity, thereby inducing lipolysis and inhibiting lipogenesis; Zn-induced lipid degradation was mediated by histone deacetylase 3 (HDAC3) which was responsible for ß-catenin deacetylation and the regulation of ß-catenin signal under the Zn treatment. Mechanistically, Zn promoted lipid degradation via stimulating HDAC3-mediated deacetylation of ß-catenin at lysine 311 (K311), which enhanced the interaction between ß-catenin and TCF7L2 and then transcriptionally inhibited fatty acid synthase (FAS), 2-acylglycerol O-acyltransferase 2 (MOGAT2), and sterol regulatory element-binding protein 1 (SREBP1) expression, but elevated the mRNA abundance of adipose triglyceride lipase (ATGL), hormone-sensitive lipase a (HSLA) and carnitine palmitoyltransferase 1a1b (CPT1A1B). Overall, our research reveals a novel mechanism into the important roles of HDAC3/ß-catenin pathway in Zn promoting lipolysis and inhibiting lipogenesis, and highlights the essential roles of K311 deacetylation in ß-catenin actions and lipolytic metabolism, and accordingly provides novel insight into the prevention and treatment of steatosis in the vertebrates.

Assessment of the processing conditions which make the Ambrosia seeds non-viable.

The European Commission requested EFSA to provide an assessment of the processing conditions which make Ambrosia seeds non-viable in feed materials and compound feed. This assessment also includes information on a reliable procedure to verify the non-viability of the seeds. Ambrosia seeds are known contaminants in feed with maximum levels set in the Directive 2002/32/EC. The manufacturing processes and processing conditions applied to the feed may affect the viability of the Ambrosia seeds. Therefore, the CONTAM Panel compared these conditions with conditions that have been shown to be sufficient to render Ambrosia seeds non-viable. The Panel concluded with a certainty of 99-100% that solvent extraction and toasting of oilseed meals at temperatures of 120°C with steam injection for 10 min or more will make Ambrosia seeds non-viable. Since milling/grinding feed materials for compound feed of piglets, aquatic species and non-food producing animals would not allow particles of sizes ≥1 mm (the minimum size of viable Ambrosia seeds) passing the grinding process it was considered very likely (with ≥ 90% certainty) that these feeds will not contain viable Ambrosia seeds. In poultry, pig, and possibly cattle feed, particle sizes are ≥ 1 mm and therefore Ambrosia seeds could likely (66-90% certainty) survive the grinding process. Starch and gluten either from corn or wheat wet milling would not contain Ambrosia seeds with 99-100% certainty. Finally, ensiling fresh forages contaminated with A. artemisiifolia seeds for more than 3 months is very likely to render all seeds non-viable. The Panel concluded that a combination of the germination test and a subsequent triphenyl-tetrazolium-chloride (TTC) test will very likely (with ≥ 90% certainty) verify the non-viability of Ambrosia seeds. The Panel recommends that data on the presence of viable Ambrosia seeds before and after the different feed production processes should be generated.

Exploring Dimensionality Reduction Techniques for Deep Learning Driven QSAR Models of Mutagenicity.

Dimensionality reduction techniques are crucial for enabling deep learning driven quantitative structure-activity relationship (QSAR) models to navigate higher dimensional toxicological spaces, however the use of specific techniques is often arbitrary and poorly explored. Six dimensionality techniques (both linear and non-linear) were hence applied to a higher dimensionality mutagenicity dataset and compared in their ability to power a simple deep learning driven QSAR model, following grid searches for optimal hyperparameter values. It was found that comparatively simpler linear techniques, such as principal component analysis (PCA), were sufficient for enabling optimal QSAR model performances, which indicated that the original dataset was at least approximately linearly separable (in accordance with Cover's theorem). However certain non-linear techniques such as kernel PCA and autoencoders performed at closely comparable levels, while (especially in the case of autoencoders) being more widely applicable to potentially non-linearly separable datasets. Analysis of the chemical space, in terms of XLogP and molecular weight, uncovered that the vast majority of testing data occurred within the defined applicability domain, as well as that certain regions were measurably more problematic and antagonised performances. It was however indicated that certain dimensionality reduction techniques were able to facilitate uniquely beneficial navigations of the chemical space.

Zinc pre-exposure improves Zn resistance by demethylation of metallothionein 2 and transcription regulation of zinc-regulatory genes in zebrafish ZF4 cells.

Mild zinc (Zn) pre-exposure can promote Zn resistance of organism, but the underlying molecular mechanisms are largely unknown. Two experiments were performed using zebrafish ZF4 cells, including short-term and long-term Zn pre-exposure experiments. In the short-term test, the cells were pre-exposed to 100 µM Zn for 24 h, transferred into fresh medium with 4.4 µM Zn for 24 h, and then re-exposed to 250 µM Zn. In the long-term test, the cells were pre-exposed to 100 µM Zn intermittently for 10 passages (3 days per passage), transferred into fresh medium with 4.4 µM Zn for 5 passages, and then re-exposed to 250 µM Zn. Both pretreatments resulted in higher resistance to 250 µM Zn. Exposure to 250 µM Zn caused a more than 2-fold increase in Zn content without Zn pretreatment but did not affect Zn content in the Zn pretreated cells. The Zn pretreated cells had low methylation levels of the metal-response element (MRE) at locus -87 in the promoter of mt2 (metallothionein 2). The up-regulated mRNA expression of Zn-regulatory genes (mtf-1, mt2, slc30a1a, slc30a4, slc30a5, slc30a6 and slc30a7) in the long-term Zn pretreated cells and mt2, slc30a4, slc30a6 and slc30a7 in the short-term Zn pretreated cells were observed. Exposure to 250 µM Zn in combination with the Zn pretreatments up-regulated mRNA expression of these genes and reduced methylation levels of the MRE compared with 250 µM Zn alone and the control. Taken together, the data suggested that demethylation of MRE in the promoter of mt2 and transcriptional induction of mt2 and Zn exporter genes offered Zn resistance in fish ZF4 cells. The traditional toxicological evaluation based on continuous exposure may overestimate the risk of fluctuating concentrations of Zn in the environment.

Copper induces liver lipotoxicity disease by up-regulating Nrf2 expression via the activation of MTF-1 and inhibition of SP1/Fyn pathway.

Excessive copper (Cu) intake leads to hepatic lipotoxicity disease, which has adverse effects on health, but the underlying mechanism is unclear. We found that Cu increased lipotoxicity by promoting Nrf2 recruitment to the ARE site in the promoters of five lipogenic genes (g6pd, 6pgd, me, icdh and pparγ). We also found that Cu affected the Nrf2 expression via different pathways: metal regulatory transcription factor 1 (MTF-1) mediated the Cu-induced Nrf2 transcriptional activation; Cu also enhanced the expression of Nrf2 by inhibiting the SP1 expression, which was achieved by inhibiting the negative regulator Fyn of Nrf2. These promoted the enrichment of Nrf2 in the nucleus and ultimately affected lipotoxicity. Thus, for the first time, we elucidated that Cu induced liver lipotoxicity disease by up-regulating Nrf2 expression via the MTF-1 activation and the inhibition of SP1/Fyn pathway. Our study elucidates the Cu-associated obesity and NAFLD for fish and possibly humans.

Risks for human health related to the presence of grayanotoxins in certain honey.

The European Commission asked EFSA for a scientific opinion on the risks for human health of the presence of grayanotoxins (GTXs) in 'certain honey' from Ericaceae plants. The risk assessment included all structurally related grayananes occurring with GTXs in 'certain' honey. Oral exposure is associated with acute intoxication in humans. Acute symptoms affect the muscles, nervous and cardiovascular systems. These may lead to complete atrioventricular block, convulsions, mental confusion, agitation, syncope and respiratory depression. For acute effects, the CONTAM Panel derived a reference point (RP) of 15.3 µg/kg body weight for the sum of GTX I and III based on a BMDL10 for reduced heart rate in rats. A similar relative potency was considered for GTX I. Without chronic toxicity studies, an RP for long-term effects could not be derived. There is evidence for genotoxicity in mice exposed to GTX III or honey containing GTX I and III, showing increased levels of chromosomal damage. The mechanism of genotoxicity is unknown. Without representative occurrence data for the sum of GTX I and III and consumption data from Ericaceae honey, acute dietary exposure was estimated based on selected concentrations for GTX I and III reflecting concentrations measured in 'certain' honeys. Applying a margin of exposure (MOE) approach, the estimated MOEs raised health concerns for acute toxicity. The Panel calculated the highest concentrations for GTX I and III below which no acute effects would be expected following 'certain honey' consumption. The Panel is 75% or more certain that the calculated highest concentration of 0.05 mg for the sum of GTX I and III per kg honey is protective for all age groups regarding acute intoxications. This value does not consider other grayananes in 'certain honey' and does not cover the identified genotoxicity.