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Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for renewal of the authorisation of Enterococcus lactis NCIMB 11181 (Lactiferm®) as a zootechnical additive for weaned piglets, calves for fattening and calves for rearing. The product under assessment is based on a strain originally identified as Enterococcus faecium. During the current assessment, the active agent has been reclassified as Enterococcus lactis. The additive currently authorised is marketed in two formulations: Lactiferm Basic 50 (a solid formulation to be used in feed), and Lactiferm WS200 (a solid ‘water‐soluble’ formulation to be used in water for drinking). The applicant has provided evidence that the additive currently on the market complies with the existing conditions of authorisation. The Panel concludes that the use of Lactiferm® under the authorised conditions of use remains safe for the target species (calves up to 6 months and weaned piglets up to 35 kg), consumers and the environment. The Lactiferm WS200 formulation of the additive is not irritant to skin or eyes. Owing to the proteinaceous nature of the active agent, both formulations of the additive are considered respiratory sensitisers. It is not possible to conclude on the irritating potential for skin and eyes of the Lactiferm Basic 50 formulation or on the potential of both forms of the additive to cause skin sensitisation. There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

EFSA performs dietary exposure assessments for food‐producing and non‐food‐producing animals to deliver risk assessment for mandates on the presence of contaminants in feed. The CONTAM and FEEDAP Panels identified the need to update the animal dietary exposure assessment model used in those assessments in CONTAM Scientific Opinions since 2011 in cases where insufficient occurrence data are available on species specific compound feeds. The Panels proposed in this statement a series of model diets based on groups of feed materials with the possibility to use different feed materials in their formulation. The Panels considered that the currently proposed model diets cover the need of the CONTAM Panel to assess the dietary exposure of animals to contaminants in feed.

Pancreatic ductal adenocarcinoma has a poor clinical outcome and responses to immunotherapy are suboptimal. Stromal fibroblasts are a dominant but heterogenous population within the tumor microenvironment and therapeutic targeting of stromal subsets may have therapeutic utility. Here, we combine spatial transcriptomics and scRNA-Seq datasets to define the transcriptome of tumor-proximal and tumor-distal cancer-associated fibroblasts (CAFs) and link this to clinical outcome. Tumor-proximal fibroblasts comprise large populations of myofibroblasts, strongly expressed podoplanin, and were enriched for Wnt ligand signaling. In contrast, inflammatory CAFs were dominant within tumor-distal subsets and expressed complement components and the Wnt-inhibitor SFRP2. Poor clinical outcome was correlated with elevated HIF-1α and podoplanin expression whilst expression of inflammatory and complement genes was predictive of extended survival. These findings demonstrate the extreme transcriptional heterogeneity of CAFs and its determination by apposition to tumor. Selective targeting of tumor-proximal subsets, potentially combined with HIF-1α inhibition and immune stimulation, may offer a multi-modal therapeutic approach for this disease. Pancreatic cancer is one of the deadliest and most difficult cancers to treat. It responds poorly to immunotherapy for instance, despite this approach often succeeding in enlisting immune cells to fight tumours in other organs. This may be due, in part, to a type of cell called fibroblasts. Not only do these wrap pancreatic tumours in a dense, protective layer, they also foster complex relationships with the cancerous cells: some fibroblasts may fuel tumour growth, while other may help to contain its spread. These different roles may be linked to spatial location, with fibroblasts adopting different profiles depending on their proximity with cancer calls. For example, certain fibroblasts close to the tumour resemble the myofibroblasts present in healing wounds, while those at the periphery show signs of being involved in inflammation. Being able to specifically eliminate pro-cancer fibroblasts requires a better understanding of the factors that shape the role of these cells, and how to identify them. To examine this problem, Croft et al. relied on tumour samples obtained from pancreatic cancer patients. They mapped out the location of individual fibroblasts in the vicinity of the tumour and analysed their gene activity. These experiments helped to reveal the characteristics of different populations of fibroblasts. For example, they showed that the myofibroblast-like cells closest to the tumour exhibited signs of oxygen deprivation; they also produced podoplanin, a protein known to promote cancer progression. In contrast, cells further from the cancer produced more immune-related proteins. Combining these data with information obtained from patients’ clinical records, Croft et al. found that samples from individuals with worse survival outcomes often featured higher levels of podoplanin and hypoxia. Inflammatory markers, however, were more likely to be present in individuals with good outcomes. Overall, these findings could help to develop ways to selectively target fibroblasts that support the growth of pancreatic cancer. Weakening these cells could in turn make the tumour accessible to immune cells, and more vulnerable to immunotherapies.

This guidance document is intended to assist the applicant in the preparation and the presentation of an application, as foreseen in Article 7.6 of Regulation (EC) No 1831/2003, for the renewal of the authorisation of additives for use in animal nutrition. Draft Endorsed by the FEEDAP Panel 14 November 2019 Submitted for public consultation 29 November 2019 End of public consultation 26 January 2020 Adoption by the FEEDAP Panel 19 November 2020 Implementation date 27 March 2021 This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-1996/full

Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.

This novel study applies targeted functional proteomics to examine tissues and cells obtained from a cohort of individuals with severe obesity who underwent bariatric surgery (BS), using a Reverse-Phase Protein Array (RPPA). In obese individuals, visceral adipose tissue (VAT), but not subcutaneous adipose tissue (SAT), shows activation of DNA damage response (DDR) markers including ATM, ATR, histone H2AX, KAP1, Chk1, and Chk2, alongside senescence markers p16 and p21. Additionally, stress-responsive metabolic markers, such as survivin, mTOR, and PFKFB3, are specifically elevated in VAT, suggesting both cellular stress and metabolic dysregulation. Conversely, peripheral blood mononuclear cells (PBMCs), while exhibiting elevated mTOR and JNK levels, did not present significant changes in DDR or senescence markers. Following BS, unexpected increases in phosphorylated ATM, ATR, and KAP1 levels, but not in Chk1 and Chk2 nor in senescence markers, were observed. This was accompanied by heightened levels of survivin and mTOR, along with improvement in markers of mitochondrial quality and health. This suggests that, following BS, pro-survival pathways involved in cellular adaptation to various stressors and metabolic alterations are activated in circulating PBMCs. Moreover, our findings demonstrate that the DDR has a dual nature. In the case of VAT from individuals with obesity, chronic DDR proves to be harmful, as it is associated with senescence and chronic inflammation. Conversely, after BS, the activation of DDR proteins in PBMCs is associated with a beneficial survival response. This response is characterized by metabolic redesign and improved mitochondrial biogenesis and functionality. This study reveals physiological changes associated with obesity and BS that may aid theragnostic approaches.

Assessing genotoxicity, specifically gene mutations and chromosomal aberrations, is fundamental to chemical risk assessment. Notably, the early identification of an aneugenic mechanism is of crucial importance, allowing, in principle, for a threshold-based risk assessment approach. To investigate this issue while pushing towards innovation in risk assessment by leveraging New Approach Methodologies, in silico approaches stand out as a particularly promising avenue. Building on these premises and given the lack of QSAR models for aneuploidy in the public domain, the present study exploited the genotoxicity-relevant alert lists implemented in the OECD QSAR Toolbox to base the investigation of structure-activity relationships for aneuploidy. To address the lack of relevant structured data resources, a dataset of 65 confirmed aneugenic substances was specifically curated and designed for the study. The results highlighted widely differing performances among the various profilers, confirming a general limited discriminatory power for aneuploidy. On the other hand, a granular analysis of the results from individual structural alerts enabled the successful isolation of some features associated with the aneugenic mode of action. Moreover, a subset of tubulin-binding chemicals was investigated to determine whether targeting a specific protein improves the characterization of toxicological alerts. The findings provide a refined definition of specific toxicity determinants for tubulin binders and serve as a promising tool for early hazard assessment, potentially informing relevant AOPs. While the computational approach appears promising, the overarching challenge that emerges is the limited availability of well-curated experimental data. In fact, reliable data on aneuploidy are scarce and fragmented across the literature. Furthermore, existing compilations of micronucleus study results are often complicated by conflicting interpretations.

The safety of bisphenol A (BPA) due to its adverse effects on the immune system has led to an increasing concern and a significant regulatory shift. The European Food Safety Authority (EFSA) proposed a reduction in the tolerable daily intake (TDI) of BPA in food in their 2023 scientific opinion, highlighting the need for stricter regulations compared to their previous assessment in 2015. This regulatory action has spurred the production of BPA alternatives, raising concerns about their safety due to insufficient toxicological data. Addressing this gap is crucial for ensuring human and environmental health. In this project, multiple genotoxicity endpoints were applied for testing of two regulatory relevant BPA alternatives, bisphenol E (BPE) and bisphenol P (BPP), in different human models: 2D HepG2 liver cells, 3D liver spheroids and primary human peripheral blood lymphocytes. DNA strand breaks and oxidised base lesions were evaluated by the enzyme‐modified version of the comet assay, while clastogenicity and aneugenicity were analysed by the in vitro micronucleus assay (OECD TG 487, 2016), together with cytotoxicity. Development of new advanced models (NAMs), as 3D spheroids, are essential for next‐generation risk assessment (NGRA) in line with the 3R's to replace, reduce or refine animal experiments. In this aspect, validation and standardisation of NAMs are needed to reach regulatory readiness level and development of OECD Test Guidelines. Therefore, a standardisation and pre‐validation of the advanced 3D liver spheroid model was performed by using multiple genotoxicity endpoints and by comparing the obtained results with standard genotoxicity models.

The aim of this paper is to propose and analyze a stabilization-free virtual element method for the non-selfadjoint convection–diffusion eigenvalue problem. The method is based on high order harmonic polynomial projections which are used to approximate the continuous sesquilinear forms. In order to analyze the continuous and discrete eigenvalue problems, we introduce solution operators and we prove convergence in norm. Then, by using the approximation spectral theory for compact non-selfadjoint operators, we show that the scheme provides a correct approximation of the spectrum and prove optimal error estimates for the eigenfunctions and a double order for the eigenvalues. The theoretical analysis considers only the case of quadrilateral meshes. Our study is supported by a series of numerical experiments, that assess the robustness of the method.

New approach methodologies (NAMs) are gaining increasing attention within the scientific community as strategies to reduce the use of animal testing. Their integration into regulatory frameworks for chemical hazard and risk assessment in food and feed would directly support the goals of the European Food Safety Authority (EFSA). Within the framework of the EU‐FORA fellowship programme, this study investigated the use of metabolomic approaches to evaluate cellular responses to genotoxic agents, serving as a proof of concept for the development, standardisation and future regulatory application of metabolomics in genotoxicity testing. Human liver‐derived cells (HepRG) were exposed to two well‐characterised genotoxicants, and both intracellular (endometabolome) and extracellular (exometabolome) metabolites were extracted and analysed using gas chromatography–mass spectrometry (GC–MS). Metabolites associated with energy metabolism, sugar metabolism and antioxidant capacity were identified, and both multivariate and univariate statistical analyses were applied to characterise metabolic profiles. Results obtained under the experimental conditions tested in this pilot study, do not highlight significant differences between exposed and control cultures. Nevertheless, the findings demonstrate the potential of metabolomics to capture biologically relevant pathways, supporting its future application in genotoxicity assessment under alternative experimental conditions and designs.