Abstract During the assessment of cannabidiol (CBD) as a novel food, in 2022 the NDA Panel identified significant data gaps. Concerns focused on potential adverse effects on the liver, gastrointestinal tract, endocrine, nervous and reproductive systems. Literature searches covering animal and human studies from the previous Statement until June 2024 confirmed the persistence of these gaps, as many of the new studies suffer from methodological limitations, including non‐standardised protocols, short durations and concomitant treatment with medicine. Pharmacokinetic studies confirmed that CBD's bioavailability is variable, influenced by delivery matrix and food intake. Its ability to cross the placenta and accumulate systemically raises further safety concerns. Animal studies revealed consistent liver toxicity, with liver weight and histopathological changes emerging as sensitive endpoints. Human trials indicated hepatotoxic potential, particularly when CBD is used in combination with other medications. Gastrointestinal effects were reported at higher doses, while neurological and psychiatric safety data remain insufficient. Animal studies on reproductive toxicity reinforced the concern regarding this endpoint. Neurodevelopmental effects following prenatal exposure were observed, suggesting long‐lasting, sex‐specific outcomes. Endocrine disruptions were noted, including altered thyroid hormone levels and adrenal histopathology. No studies addressed immunotoxicity, though CBD's interaction with immune pathways warrants caution. The Panel performed benchmark dose modelling based on GLP‐compliant subchronic studies to identify a toxicological reference point. By applying an uncertainty factor of 400, a provisional safe dose of 0.0275 mg/kg bw per day (approximately 2 mg/day for a 70 kg adult) was derived. This provisional safe dose applies solely to food supplement formulations with CBD purity ≥ 98%, without nanoparticles, for which the production process is considered safe and genotoxicity is ruled out. The Panel concludes that, based on all available data, the safety of CBD for individuals under 25 years of age, pregnant or lactating women, and those on concurrent medications, cannot be established.

Read-across is frequently used in chemical risk assessment to predict the toxicological properties of data-poor compounds instead of performing new animal tests. The selection of relevant source com­pounds (SCs) for read-across to data-poor target compounds (TCs) is one of the main challenges, particularly for endpoints such as chronic and developmental toxicity, where the mechanisms leading to the observed apical toxicological findings are often unknown. In this study, the predic­tivities of using chemical, biological and/or metabolite similarity to inform read-across strategies were compared. Existing data from two reference compound groups, i.e., the pesticide classes of 27 triazoles and 8 triazinyl-sulfonylureas, were used to assess the performance of the three approaches and their modular combinations to identify relevant SCs from a large database of 468 pesticide active compounds. Metabolite similarity yielded high positive predictive values (PPV) of 85-100% while sensitivity was relatively low (12-65%). Basing metabolite similarity assessment on observed or predicted metabolites yielded comparable results. Chemical and biological simi­larity alone were less effective in separating SCs from irrelevant compounds. Modular approaches combining, e.g., chemical and metabolic similarity, enhanced SC selection (PPV up to 100%). This research underscores the potential of metabolite data to strengthen read-across justifications, thereby contributing to regulatory compliance and safety evaluations. We propose the integration of metabolite similarity assessment into an existing EU-ToxRisk workflow to enhance the reliability of read-across for chemical risk assessments.

Current chemical risk assessment uses a default uncertainty factor (UF) of 3.16 for toxicodynamic (TD) variability in humans. The objective was to create a systematic evidence map (SEM) of the human variability in TD by identifying and organizing the available empirical data to assess if a further refinement of the default UF of 3.16 for TD can be achieved. PubMed and Web of Science™ were searched from 2004 to 2023. Studies were screened according to the eligibility criteria. Inclusion criteria included studies, where TD could be separated from toxicokinetics (TK) to exclude an impact of TK on TD variability. The literature search retrieved 2408 studies. Manual screening identified 23 in vitro studies assessing human TD variability quantitively, of which only seven in vitro studies provided quantitative estimates of a TD variability factor. No in vivo study met the inclusion criteria. Several studies found TD UF of 3.16 not covering human variability; others did. However, the data were heterogeneous, and variability in Points of Departure (PODs) and methods used to estimate TD variability complicated comparisons across studies. A standardized approach for TDVFs determination is identified. This SEM underscores the scarcity of data assessing human variability in TD, while omitting the influence of TK.

Recent advancements in food research alongside the growing interest in new sources with enhanced nutritional value have led to an increasing development of food products derived from microalgae. Some of these products fall under the category of novel foods (NFs) in the European Union (EU) and their safety must be evaluated by the European Food Safety Authority (EFSA) before being authorised on the EU market. By August 2024, EFSA had evaluated eleven NFs derived from microalgae, including oils rich in docosahexaenoic acid (DHA) from Schizochytrium spp., whole biomass of the microalga Euglena gracilis and its derivative beta-glucan polymer (paramylon), ethanolic extract from Phaeodactylum tricornutum and oleoresin rich in astaxanthin from Haematococcus pluvialis. One of the key scientific requirements for the safety assessment of these products is the characterisation of the microalga strain, including its unambiguous taxonomic identification at species level and pathogenicity. The "Qualified Presumption of Safety" (QPS) status of the microalgae also plays a significant role in determining the safety assessment approach to be applied. Other relevant requirements comprise a thorough chemical characterisation (e.g., biotoxins, undesirable substances, heavy metals) together with microbiological and nutritional characterisation of the product, description of the manufacturing process and a toxicological and allergenicity assessment. By illustrating examples of NF that consist of, are isolated from or are produced by microalgae we highlight the main requirements needed for their safety assessment alongside the challenges encountered. Taking into account the continuous evolution of the microalga sector leading to innovative products, we also extend these requirements to the safety assessment of microalgal proteins, considering potential future mandates to assess algae-derived proteins as NFs by EFSA.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on synthetic cannabidiol as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF which is subject of the application is trans-cannabidiol (CBD), produced by chemical synthesis and proposed to be used in food supplements at a level of 30 mg/day. The target population is the general population, excluding pregnant and lactating women. During the risk assessment, the Panel identified a number of data gaps, which needed to be addressed by the applicant. Therefore, an EFSA request for additional information was sent to the applicant. The requested data concerned the identity, the production process, the compositional data, the specifications, the genotoxicity, the reproductive and developmental toxicity and the human data of the NF. Despite being contacted several times, the applicant did not reply to EFSA's requests for additional data. Based on the available data, the Panel concludes that the safety of the NF, i.e. synthetic cannabidiol, cannot be established.

The Mela Rosa Marchigiana (MRM) pulp callus, kept alive since 2017 by monthly sub-cultures, was investigated and morphologically described. In the present paper, the MRM callus ethanolic Extract (MRME) was prepared from one of the 2022 callus sub-cultures and analysed by GC-MS and GC-FID. The investigation was carried out to test whether a long-term culture could affect either the amount or the bioactivity (or both) of the triterpenes produced by the callus. To this aim, the 2022 extract (MRME 2022) was compared to the extract prepared a few years ago (i.e., MRME 2019). Obtained results showed that the composition of both extracts was almost the same, thus revealing that the callus culture efficiency of triterpenes production was nearly unchanged over time. When analysing the biological effect of the extract, all experimental tests, including cell-free, in vitro, ex vivo, and in vivo assays, confirmed that MRME 2022 significantly protected cells, tissues, or whole organisms from oxidative stress-induced damage. Present results could lead to future studies to test MRME as a possible ingredient of a nutraceutical formulation for healthy aging or for the prevention of oxidative stress-associated pathologies.

The anti-inflammatory activity of the "Mela Rosa Marchigiana" (MRM) pulp callus ethanol Extract (MRME) was tested in different cellular models including (i) LPS-treated RAW 264.7; (ii) HUVEC exposed to short-term high Glucose (HG, 45 mM) or normal glucose (NG, 5 mM) concentrations; and (iii) HG (30 mN) and LPS-treated U937. To evaluate the anti-inflammatory effect of the extract, Nitric Oxide (NO) production was measured in RAW 264.6 cells, while IL-8, IL-1 ss ss and MCP-1 expressions, along with modulation of some inflammation- or senescence-associated miRNAs (miR-21, miR-126, miR-17 and miR-217) were assessed in HUVECs and/or U937. MRME treatment reduced pro-inflammatory markers amount, suggesting a decreased generalised inflammatory response. Present findings indicate that MRME can contrast senescence- or HG-induced inflammation. Moreover, in HG- and LPS-induced inflammation, MRME reduced U937 monocyte activation by inhibiting mitochondrial respiration and inflammatory markers. Finally, the inhibitory potential of MRME on the digestive enzymes alpha-glucosidase, alpha-amylase and lipase activity was investigated. Our results support the idea that MRME has a positive effect on inhibition of endothelial/macrophage dysfunction under HG/senescence inflammatory conditions. Furthermore, the obtained results showed a modest inhibitory power of the extract (IC50 values: 2.98 +/- 0.24, 1.77 +/- 0.15, and 2.06 +/- 0.31 mg/ml, respectively), which, however, could be of some help in decreasing the absorption of glucose and triglycerides.

Following the adoption of Regulation (EU) No 1169/2011 on food information to consumers, the European Commission requested EFSA to update its 'Guidance on safety evaluation of sources of nutrients and bioavailability of the nutrient from the sources' regarding the scientific principles and data requirements for the scientific assessment of all new forms of micronutrients and to derive a conversion factor for new micronutrient sources or forms of micronutrients to be authorised for addition to foods, including food supplements. This guidance outlines the scientific principles that the NDA Panel will consider for the assessment of the safety and the quantification of the relative bioavailability of new sources of micronutrients, which applicants are requested to consider when preparing their applications. It also outlines the data requirements for dossiers. Applicants should integrate the data presented in different sections to provide their overall considerations on how the information provided supports the safety of the new micronutrient source and the quantification of its relative bioavailability compared to a reference source under the proposed conditions of use. As preparatory work for the development of this guidance, EFSA launched an Expert Survey and held an online workshop on 9th March 2023 inviting scientific input from stakeholders and scientific experts, the report of which is now available online in the EFSA's webpage.

Scorpions are the oldest known arachnids and include some of the earliest invertebrates to have become fully terrestrial, with fossil records dating back to the Silurian period approximately 444–419 million years ago. Scorpion anatomy features two main segments: a frontal prosoma, to which eight legs are attached, and an opisthosoma, divided in mesosoma as the main body and metasoma as the tail. Taxonomically, scorpions belong to the class Arachnida which includes spiders (order: Araneae), ticks and mites (order: Acarina) among other orders, and form the order Scorpiones. Over 2200 species have been described and these are distributed all over the earth with the exception of Antarctica. Relatively few scorpion genera are considered of public health importance with regards to clinical consequences of their stings and these are mostly found in Africa, Middle East and Central-Southern America. Such genera of clinical relevance include Androctonus, Buthus, Centruroides, Hemiscorpius, Hottentotta, Leiurus, Odontobuthus, Parabuthus and Tityus and around 30 species. Scorpion venoms encode toxins, primarily acting on sodium and potassium ion channels which are responsible for clinical effects resulting in the depolarization of excitable nerve and muscle cells. In addition, scorpion venoms also contain non-toxic metalloproteinases, calcium channel and chloride channel toxins, bradykinin-potentiating peptides, serine protease inhibitors, phospholipase A2 enzymes as well as defensins and antimicrobial peptides. Scorpion envenomation and associated symptoms are usually classified as Class I-mild, Class II-moderate and Class III-severe. The clinical management of scorpion stings is described here within a protocol.

The webinar series and workshop titled “Trust Your Gut: Establishing Confidence in Gastrointestinal Models – An Overview of the State of the Science and Contexts of Use” was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and pro­cesses occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of “New Approach Methodologies” (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today’s regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free “Next Generation Risk Assessment” (NGRA).

This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8441/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8440/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8437/full.

Background: New Approach Methodologies (NAMs) comprise in silico and in vitro methods applied as alternative to animal testing. Even though NAMs are already fully implemented as research tools, their use in regulatory risk assessments (RA) is limited currently. To promote the regulatory uptake/acceptance of NAMs, a paradigm shift in risk assessment approaches, and a proper dialogue between risk assessors and risk managers is needed. Scope and approach: Several reviews addressed the use of NAMs for chemical RA in generic terms, but without providing specific considerations on their use for food/feed safety assessments. Therefore, in this review, we give insights on the potential use of NAMs for regulatory purposes in the EU. We summarise relevant projects and activities on NAMs coordinated by the European Food Safety Authority (EFSA), which is the agency of the Eu-ropean Union that contributes to the safety of the European food and feed chain. The review informs on future developments on the use of NAMs in human health chemical RA, and touches on their use for the assessment of protein toxicity and allergenicity, as well as environmental risks. Main findings and conclusions: Reducing animal testing and filling some RA gaps via NAMs is almost a reality. Moreover, there is a growing body of evidence confirming that the inclusion of mechanistic information im-proves risk assessments. EFSA's projects address the main challenge of using intermediate effects observed in non-animal models for safety assessments, especially those linked to adverse effects that are insufficiently covered or uncovered by animal apical endpoints.