Subzero project: comparing trace element profiles of enriched mitochondria fractions from frozen and fresh liver tissue.

From organs to subcellular organelles, trace element (TE) homeostasis is fundamental for many physiological processes. While often overlooked in early stages, manifested TE disbalance can have severe health consequences, particularly in the context of aging or pathological conditions. Monitoring TE concentrations at the mitochondrial level could identify organelle-specific imbalances, contributing to targeted diagnostics and a healthier aging process. However, mitochondria isolation from frozen tissue is challenging, as it poses the risk of TE losses from the organelles due to cryodamage, but would significantly ease routine laboratory work. To address this, a novel method to isolate an enriched mitochondria fraction (EMF) from frozen tissue was adapted from already established protocols. Validation of manganese (Mn), iron (Fe), and copper (Cu) quantification via inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) showed sufficiently low quantification limits for EMF TE analysis. Successful mitochondrial enrichment from frozen liver samples was confirmed via immunoblots and transmission electron microscopy (TEM) revealed sufficient structural integrity of the EMFs. No significant differences in EMF TEs between frozen and fresh tissue were evident for Mn and Cu and only slight decreases in EMF Fe. Consequently, EMF TEs were highly comparable for isolates from both tissue states. In application, this method effectively detected dietary differences in EMF Fe of a murine feeding study and identified the disease status in a Wilson disease rat model based on drastically increased EMF Cu. In summary, the present method is suitable for future applications, facilitating sample storage and high-throughput analyses of mitochondrial TEs.

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.

Establishment of a nonradioactive DNA ligation assay and its applications in murine tissues.

As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a method reliably assessing DNA ligation capacity in protein extracts from murine tissues was aimed to establish. To optimize applicability, the use of radioactively labeled substrates was avoided and replaced by fluorescently labeled oligonucleotides. Briefly, tissue extracts were incubated with those complementary oligonucleotides so that in an ensuing gel electrophoresis ligated strands could be separated from unconnected molecules. Originally, the method was intended for use in cerebellum tissue to further elucidate possible mechanisms of neurodegenerative diseases. However, due to its inhomogeneous anatomy, DNA ligation efficiency varied strongly between different cerebellar areas, illuminating the established assay to be suitable only for homogenous organs. Thus, for murine liver tissue sufficient intra- and interday repeatability was shown during validation. In further experiments, the established assay was applied to an animal study comprising young and old (24 and 110 weeks) mice which showed that DNA ligation efficiency was affected by neither sex nor age. Finally, the impact of in vitro addition of the trace elements copper, iron, and zinc on DNA ligation in tissue extracts was investigated. While all three metals inhibited DNA ligation, variations in their potency became evident. In conclusion, the established method can be reliably used for investigation of DNA ligation efficiency in homogenous murine tissues.

Fostering healthy aging through selective nutrition: A long-term comparison of two dietary patterns and their holistic impact on mineral status in middle-aged individuals-A randomized controlled intervention trial in Germany.

Aging is associated with a decline in physiological functions and an increased risk of age-related diseases, emphasizing the importance of identifying dietary strategies for healthy aging. Minerals play a crucial role in maintaining optimal health during aging, making them relevant targets for investigation. Therefore, we aimed to analyze the effect of different dietary pattern on mineral status in the elderly. We included 502 individuals aged 50-80 years in a 36-month randomized controlled trial (RCT) (NutriAct study). This article focuses on the results within the two-year intervention period. NutriAct is not a mineral-modulating-targeted intervention study, rather examining nutrition in the context of healthy aging in general. However, mineral status might be affected in an incidental manner. Participants were assigned to either NutriAct dietary pattern (proportionate intake of total energy consumption (%E) of 35-45 %E carbohydrates, 35-40 %E fats, and 15-25 %E protein) or the German Nutrition Society (DGE) dietary pattern (proportionate intake of total energy consumption (%E) of 55 %E carbohydrates, 30 %E fats, and 15 %E protein), differing in the composition of macronutrients. Data from 368 participants regarding dietary intake (energy, calcium, magnesium, iron, and zinc) and serum mineral concentrations of calcium, magnesium, iron, copper, zinc, selenium, iodine, and manganese, free zinc, and selenoprotein P were analyzed at baseline, as well as after 12 and 24 months to gain comprehensive insight into the characteristics of the mineral status. Additionally, inflammatory status - sensitive to changes in mineral status - was assessed by measurement of C-reactive protein and interleukin-6. At baseline, inadequate dietary mineral intake and low serum concentrations of zinc and selenium were observed in both dietary patterns. Throughout two years, serum zinc concentrations decreased, while an increase of serum selenium, manganese and magnesium concentrations was observable, likely influenced by both dietary interventions. No significant changes were observed for serum calcium, iron, copper, or iodine concentrations. In conclusion, long-term dietary interventions can influence serum mineral concentrations in a middle-aged population. Our findings provide valuable insights into the associations between dietary habits, mineral status, and disease, contributing to dietary strategies for healthy aging.

Single is not combined: The role of Co and Ni bioavailability on toxicity mechanisms in liver and brain cells.

The two trace elements cobalt (Co) and nickel (Ni) are widely distributed in the environment due to the increasing industrial application, for example in lithium-ion batteries. Both metals are known to cause detrimental health impacts to humans when overexposed and both are supposed to be a risk factor for various diseases. The individual toxicity of Co and Ni has been partially investigated, however the underlying mechanisms, as well as the interactions of both remain unknown. In this study, we focused on the treatment of liver carcinoma (HepG2) and astrocytoma (CCF-STTG1) cells as a model for the target sites of these two metals. We investigated their effects in single and combined exposure on cell survival, cell death mechanisms, bioavailability, and the induction of oxidative stress. The combination of CoCl2 and NiCl2 resulted in higher Co levels with subsequent decreased amount of Ni compared to the individual treatment. Only CoCl2 and the combination of both metals led to RONS induction and increased GSSG formation, while apoptosis and necrosis seem to be involved in the cell death mechanisms of both CoCl2 and NiCl2. Collectively, this study demonstrates cell-type specific toxicity, with HepG2 representing the more sensitive cell line. Importantly, combined exposure to CoCl2 and NiCl2 is more toxic than single exposure, which may originate partly from the respective cellular Co and Ni content. Our data imply that the major mechanism of joint toxicity is associated with oxidative stress. More studies are needed to assess toxicity after combined exposure to elements such as Co and Ni to advance an improved hazard prediction for less artificial and more real-life exposure scenarios.

Determination of copper status by five biomarkers in serum of healthy women.

BACKGROUND: The essential trace element copper is relevant for many important physiological processes. Changes in copper homeostasis can result from disease and affect human health. A reliable assessment of copper status by suitable biomarkers may enable fast detection of subtle changes in copper metabolism. To this end, additional biomarkers besides serum copper and ceruloplasmin (CP) concentrations are required. OBJECTIVES: The aim of this study was to investigate the emerging copper biomarkers CP oxidase (CPO) activity, exchangeable copper (CuEXC) and labile copper in serum of healthy women and compare them with the conventional biomarkers total serum copper and CP. METHOD AND MAIN FINDINGS: This observational study determined CPO activity, the non CP-bound copper species CuEXC and labile copper, total serum copper and CP in sera of 110 healthy women. Samples were collected at four time points over a period of 24 weeks. The concentrations of total serum copper and CP were within the reference ranges. The comparison of all five biomarkers provided insight into their relationship, the intra- and inter-individual variability as well as the age dependence. The correlation and Principal Component Analyses (PCA) indicated that CP, CPO activity and total copper correlated well, followed by CuEXC, while the labile copper pool was unrelated to the other parameters. CONCLUSIONS: This study suggests that the non-CP-bound copper species represent copper pools that are differently regulated from total copper or CP-bound copper, making them interesting complementary biomarkers to enable a more complete assessment of body copper status with potential relevance for clinical application.

Postprandial Micronutrient Variability and Bioavailability: An Interventional Meal Study in Young vs. Old Participants.

This study explores age- and time-dependent variations in postprandial micronutrient absorption after a micronutrient-rich intervention meal within the Biomiel (bioavailability of micronutrients in elderly) study. Comprising 43 healthy participants, the study compares young (n = 21; mean age 26.90 years) and old (n = 22; mean age 66.77 years) men and women, analyzing baseline concentrations and six-hour postprandial dynamics of iron (Fe), copper (Cu), zinc (Zn), selenium (Se), iodine (I), free zinc (fZn), vitamin C, retinol, lycopene, ß-carotene, α-tocopherol, and γ-tocopherol, along with 25(OH) vitamin D (quantified only at baseline). Methodologically, quantifications in serum or plasma were performed at baseline and also at 90, 180, 270, and 360 min postprandially. Results reveal higher baseline serum Zn and plasma lycopene concentrations in the young group, whereas Cu, Se, Cu/Zn ratio, 25(OH) vitamin D, α-tocopherol, and γ-tocopherol were higher in old participants. Postprandial variability of Zn, vitamin C, and lycopene showed a strong time-dependency. Age-related differences in postprandial metabolism were observed for Se, Cu, and I. Nevertheless, most of the variance was explained by individuality. Despite some limitations, this study provides insights into postprandial micronutrient metabolism (in serum/plasma), emphasizing the need for further research for a comprehensive understanding of this complex field. Our discoveries offer valuable insights for designing targeted interventions to address and mitigate micronutrient deficiencies in older adults, fostering optimal health and well-being across the lifespan.

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.

Dysfunction in atox-1 and ceruloplasmin alters labile Cu levels and consequently Cu homeostasis in C. elegans.

Copper (Cu) is an essential trace element, however an excess is toxic due to its redox properties. Cu homeostasis therefore needs to be tightly regulated via cellular transporters, storage proteins and exporters. An imbalance in Cu homeostasis has been associated with neurodegenerative disorders such as Wilson's disease, but also Alzheimer's or Parkinson's disease. In our current study, we explored the utility of using Caenorhabditis elegans (C. elegans) as a model of Cu dyshomeostasis. The application of excess Cu dosing and the use of mutants lacking the intracellular Cu chaperone atox-1 and major Cu storage protein ceruloplasmin facilitated the assessment of Cu status, functional markers including total Cu levels, labile Cu levels, Cu distribution and the gene expression of homeostasis-related genes. Our data revealed a decrease in total Cu uptake but an increase in labile Cu levels due to genetic dysfunction, as well as altered gene expression levels of Cu homeostasis-associated genes. In addition, the data uncovered the role ceruloplasmin and atox-1 play in the worm's Cu homeostasis. This study provides insights into suitable functional Cu markers and Cu homeostasis in C. elegans, with a focus on labile Cu levels, a promising marker of Cu dysregulation during disease progression.

Long-term suboptimal dietary trace element supply does not affect trace element homeostasis in murine cerebellum.

The ageing process is associated with alterations of systemic trace element (TE) homeostasis increasing the risk, e.g. neurodegenerative diseases. Here, the impact of long-term modulation of dietary intake of copper, iron, selenium, and zinc was investigated in murine cerebellum. Four- and 40-wk-old mice of both sexes were supplied with different amounts of those TEs for 26 wk. In an adequate supply group, TE concentrations were in accordance with recommendations for laboratory mice while suboptimally supplied animals received only limited amounts of copper, iron, selenium, and zinc. An additional age-adjusted group was fed selenium and zinc in amounts exceeding recommendations. Cerebellar TE concentrations were measured by inductively coupled plasma-tandem mass spectrometry. Furthermore, the expression of genes involved in TE transport, DNA damage response, and DNA repair as well as selected markers of genomic stability [8-oxoguanine, incision efficiency toward 8-oxoguanine, 5-hydroxyuracil, and apurinic/apyrimidinic sites and global DNA (hydroxy)methylation] were analysed. Ageing resulted in a mild increase of iron and copper concentrations in the cerebellum, which was most pronounced in the suboptimally supplied groups. Thus, TE changes in the cerebellum were predominantly driven by age and less by nutritional intervention. Interestingly, deviation from adequate TE supply resulted in higher manganese concentrations of female mice even though the manganese supply itself was not modulated. Parameters of genomic stability were neither affected by age, sex, nor diet. Overall, this study revealed that suboptimal dietary TE supply does not substantially affect TE homeostasis in the murine cerebellum.

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.

Molecular and functional changes in neutrophilic granulocytes induced by nicotine: a systematic review and critical evaluation.

Background: Over 1.1 billion people smoke worldwide. The alkaloid nicotine is a prominent and addictive component of tobacco. In addition to tumors and cardiovascular disorders, tobacco consumption is associated with a variety of chronic-inflammatory diseases. Although neutrophilic granulocytes (neutrophils) play a role in the pathogenesis of many of these diseases, the impact of nicotine on neutrophils has not been systematically reviewed so far. Objectives: The aim of this systematic review was to evaluate the direct influence of nicotine on human neutrophil functions, specifically on cell death/damage, apoptosis, chemotaxis, general motility, adhesion molecule expression, eicosanoid synthesis, cytokine/chemokine expression, formation of neutrophil extracellular traps (NETs), phagocytosis, generation of reactive oxygen species (ROS), net antimicrobial activity, and enzyme release. Material and methods: This review was conducted according to the PRISMA guidelines. A literature search was performed in the databases NCBI Pubmed® and Web of Science™ in February 2023. Inclusion criteria comprised English written research articles, showing in vitro studies on the direct impact of nicotine on specified human neutrophil functions. Results: Of the 532 originally identified articles, data from 34 articles were finally compiled after several evaluation steps. The considered studies highly varied in methodological aspects. While at high concentrations (>3 mmol/l) nicotine started to be cytotoxic to neutrophils, concentrations typically achieved in blood of smokers (in the nmol/l range) applied for long exposure times (24-72h) supported the survival of neutrophils. Smoking-relevant nicotine concentrations also increased the chemotaxis of neutrophils towards several chemoattractants, elevated their production of elastase, lipocalin-2, CXCL8, leukotriene B4 and prostaglandin E2, and reduced their integrin expression. Moreover, while nicotine impaired the neutrophil phagocytotic and anti-microbial activity, a range of studies demonstrated increased NET formation. However, conflicting effects were found on ROS generation, selectin expression and release of ß-glucuronidase and myeloperoxidase. Conclusion: Nicotine seems to support the presence in the tissue and the inflammatory and selected tissue-damaging activity of neutrophils and reduces their antimicrobial functions, suggesting a direct contribution of nicotine to the pathogenesis of chronic-inflammatory diseases via influencing the neutrophil biology.

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.

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.

Less is more: a methodological assessment of extraction techniques for per- and polyfluoroalkyl substances (PFAS) analysis in mammalian tissues.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants. Studying the bioaccumulation in mammalian tissues requires a considerable effort for the PFAS extraction from complex biological matrices. The aim of the current work was to select and optimize the most efficient among common extraction strategies for eleven perfluoroalkyl acids (PFAA). Primary extractions from wild boar tissues (liver, kidney, and lung) were performed with methanol at neutral, acidic, or alkaline conditions, or with methyl-tert-butyl ether (MTBE) after ion-pairing with tetrabutylammonium (TBA) ions. A second purification step was chosen after comparing different solid-phase extraction (SPE) cartridges (Oasis WAX, ENVI-Carb, HybridSPE Phospholipid) and various combinations thereof or dispersive SPE with C18 and ENVI-Carb material. The best extraction efficiencies of the liquid PFAA extraction from tissue homogenates were achieved with methanol alone (recoveries from liver 86.6-114.4%). Further purification of the methanolic extracts using dispersive SPE or Oasis WAX columns decreased recoveries of most PFAA, whereas using pairs of two SPE columns connected in series proved to be more efficient albeit laborious. Highest recoveries for ten out of eleven PFAA were achieved using ENVI-Carb columns (80.3-110.6%). In summary, the simplest extraction methods using methanol and ENVI-Carb columns were also the most efficient. The technique was validated and applied in a proof of principle analysis in human tissue samples.

A Reliable Method Based on Liquid Chromatography-Tandem Mass Spectrometry for the Simultaneous Quantification of Neurotransmitters in Caenorhabditis elegans.

Neurotransmitters like dopamine (DA), serotonin (SRT), γ-aminobutyric acid (GABA) and acetylcholine (ACh) are messenger molecules that play a pivotal role in transmitting excitation between neurons across chemical synapses, thus enabling complex processes in the central nervous system (CNS). Balance in neurotransmitter homeostasis is essential, and altered neurotransmitter levels are associated with various neurological disorders, e.g., loss of dopaminergic neurons (Parkinson's disease) or altered ACh synthesis (Alzheimer's disease). Therefore, it is crucial to possess adequate tools to assess precise neurotransmitter levels, and to apply targeted therapies. An established in vivo model to study neurotoxicity is the model organism Caenorhabditis elegans (C. elegans), as its neurons have been well characterized and functionally are analogous to mammals. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method including a sample preparation assuring neurotransmitter stability, which allows a simultaneous neurotransmitter quantification of DA, SRT, GABA and ACh in C. elegans, but can easily be applied to other matrices. LC-MS/MS combined with isotope-labeled standards is the tool of choice, due to its otherwise unattainable sensitivity and specificity. Using C. elegans together with our analytically validated and verified method provides a powerful tool to evaluate mechanisms of neurotoxicity, and furthermore to identify possible therapeutic approaches.

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.