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Following the occurrence of Tetrodotoxins (TTXs) in Europe—a group of neurotoxins identified in Asia, where fatalities occurred after the ingestion of contaminated pufferfish—the EFSA proposed a limit of 44 µg of TTX/kg of shellfish meat in mollusks in 2017, to protect heavy consumers. The limit was based on an acute reference dose (ARfD) derived from the few available data on TTX toxicity. TTX is expected to increase with sea-surface warming; indeed, it has been found in spring/summer in mollusks in Europe, with concentrations often exceeding this limit. Due to the numerous uncertainties of the EFSA’s ARfD, we conducted a systematic review to provide an update on TTX toxicity. Out of 12,741 articles retrieved from PubMed, Science Direct, and Scopus since 2017, only 17 were eligible for data extraction. Our results show that they are not sufficient to modify the EFSA’s conclusions. Furthermore, our analysis of occurrence data in European seafood, to assess the current risk of exposure to TTX, reveals several gaps, such as different LODs/LOQs and seasonal monitoring not allowing comparisons between areas and too few analyzed sites. However, the presence of positive samples exceeding the EFSA limit indicates a potential risk even for general consumers, highlighting the urgency to address these knowledge gaps.

Cyanobacteria commonly form large blooms in waterbodies; they can produce cyanotoxins, with toxic effects on humans and animals, and volatile compounds, causing bad tastes and odors (T&O) at naturally occurring low concentrations. Notwithstanding the large amount of literature on either cyanotoxins or T&O, no review has focused on them at the same time. The present review critically evaluates the recent literature on cyanotoxins and T&O compounds (geosmin, 2-methylisoborneol, β-ionone and β-cyclocitral) to identify research gaps on harmful exposure of humans and animals to both metabolite classes. T&O and cyanotoxins production can be due to the same or common to different cyanobacterial species/strains, with the additional possibility of T&O production by non-cyanobacterial species. The few environmental studies on the co-occurrence of these two groups of metabolites are not sufficient to understand if and how they can co-vary, or influence each other, perhaps stimulating cyanotoxin production. Therefore, T&Os cannot reliably serve as early warning surrogates for cyanotoxins. The scarce data on T&O toxicity seem to indicate a low health risk (but the inhalation of β-cyclocitral deserves more study). However, no data are available on the effects of combined exposure to mixtures of cyanotoxins and T&O compounds and to combinations of T&O compounds; therefore, whether the co-occurrence of cyanotoxins and T&O compounds is a health issue remains an open question.

Introduction : Epidemiological studies in children suggested that in utero exposure to chlorpyrifos (CPF), an organophosphate insecticide, may cause developmental neurotoxicity (DNT). We applied quantitative in vitro – in vivo extrapolation (QIVIVE) based on in vitro concentration and non-choline esterase-dependent effects data combined with Benchmark dose (BMD) modelling to predict oral maternal CPF exposure during pregnancy leading to fetal brain effect concentration. By comparing the results with data from epidemiological studies, we evaluated the contribution of the in vitro endpoints to the mode of action (MoA) for CPF-induced DNT. Methods: A maternal-fetal PBK model built in PK-Sim ® was used to perform QIVIVE predicting CPF concentrations in a pregnant women population at 15 weeks of gestation from cell lysate concentrations obtained in human induced pluripotent stem cell-derived neural stem cells undergoing differentiation towards neurons and glia exposed to CPF for 14 days. The in vitro concentration and effect data were used to perform BMD modelling. Results: The upper BMD was converted into maternal doses which ranged from 3.21 to 271 mg/kg bw/day. Maternal CPF blood levels from epidemiological studies reporting DNT findings in their children were used to estimate oral CPF exposure during pregnancy using the PBK model. It ranged from 0.11 to 140 μg/kg bw/day. Discussion: The effective daily intake doses predicted from the in vitro model were several orders of magnitude higher than exposures estimated from epidemiological studies to induce developmental non-cholinergic neurotoxic responses, which were captured by the analyzed in vitro test battery. These were also higher than the in vivo LOEC for cholinergic effects. Therefore, the quantitative predictive value of the investigated non-choline esterase-dependent effects, although possibly relevant for other chemicals, may not adequately represent potential key events in the MoA for CPF-associated DNT.

Amiodarone is an antiarrhythmic agent inducing adverse effects on the nervous system, among others. We applied physiologically based pharmacokinetic (PBPK) modeling combined with benchmark dose modeling to predict, based on published in vitro data, the in vivo dose of amiodarone which may lead to adverse neurological effects in patients. We performed in vitro–in vivo extrapolation (IVIVE) from concentrations measured in the cell lysate of a rat brain 3D cell model using a validated human PBPK model. Among the observed in vitro effects, inhibition of choline acetyl transferase (ChAT) was selected as a marker for neurotoxicity. By reverse dosimetry, we transformed the in vitro concentration–effect relationship into in vivo effective human doses, using the calculated in vitro area under the curve (AUC) of amiodarone as the pharmacokinetic metric. The upper benchmark dose (BMDU) was calculated and compared with clinical doses eliciting neurological adverse effects in patients. The AUCs in the in vitro brain cell culture after 14-day repeated dosing of nominal concentration equal to 1.25 and 2.5 µM amiodarone were 1.00 and 1.99 µg*h/mL, respectively. The BMDU was 385.4 mg for intravenous converted to 593 mg for oral application using the bioavailability factor of 0.65 as reported in the literature. The predicted dose compares well with neurotoxic doses in patients supporting the hypothesis that impaired ChAT activity may be related to the molecular/cellular mechanisms of amiodarone neurotoxicity. Our study shows that predicting effects from in vitro data together with IVIVE can be used at the initial stage for the evaluation of potential adverse drug reactions and safety assessment in humans.

This review focuses on the risk assessment associated with human exposure to cyanotoxins, secondary metabolites of an ubiquitous group of photosynthetic procariota. Cyanobacteria occurr especially in eutrophic inland and coastal surface waters, where under favorable conditions they attain high densities and may form blooms and scums. Cyanotoxins can be grouped according to their biological effects into hepatotoxins, neurotoxins, cytotoxins, and toxins with irritating potential, also acting on the gastrointestinal system. The chemical and toxicological properties of the main cyanotoxins, relevant for the evaluation of possible risks for human health, are presented. Humans may be exposed to cyanotoxins via several routes, with the oral one being by far the most important, occurring by ingesting contaminated drinking water, food, some dietary supplements, or water during recreational activities. Acute and short-term toxic effects have been associated in humans with exposure to high levels of cyanotoxins in drinking and bathing waters. However, the chronic exposure to low cyanotoxin levels remains a critical issue. This article identifies the actual risky exposure scenarios, provides toxicologically derived reference values, and discusses open issues and research needs.

The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.

EFSA requested the Panel on Plant Protection Products and their Residues (PPR Panel) to produce a Scientific Opinion on the application of physiologically based kinetic (PBK) modelling for the quantitative in vitro to in vivo extrapolation (QIVIVE) of data from the 17‐assay developmental neurotoxicity in vitro battery (DNT IVB) for pesticide active substances. PBK modelling‐supported QIVIVE is essential for the integration of in vitro data in hazard and risk assessment and may be conducted via forward dosimetry (estimating internal exposure from external exposure to a chemical) or reverse dosimetry (deriving an external exposure from an internal exposure). The request was accomplished via targeted expert discussions and EFSA‐internal and ‐external review. A scientifically robust QIVIVE requires accurate characterisation of two interrelated exposure metrics: the in vitro concentration eliciting a biological response (e.g. free or cellular concentration) and the corresponding in vivo internal concentration predicted by PBK modelling that reflects the same biologically relevant exposure. These metrics are influenced by chemical‐specific properties, assay design and physiological variability, requiring a case‐by‐case assessment of uncertainties. Each QIVIVE assessment should include, as a minimum, a low‐tier PBK model using conservative assumptions to avoid underestimation of internal exposure. Where sufficient kinetic data exist, higher tier models may be applied to enhance prediction accuracy. All modelling parameters, assumptions and validation steps must be transparently documented to facilitate regulatory appraisal. QIVIVE outcomes are to be documented in the overall weight of evidence for the DNT assessment. The PPR Panel identified the following three key uncertainty domains: (a) defining the appropriate in vitro exposure metric, (b) predicting internal exposure metrics via PBK modelling and (c) aligning PBK modelling‐derived internal exposure with the in vitro exposure. Addressing these uncertainties will strengthen regulatory confidence in using DNT IVB data for hazard and risk assessment of pesticide active substances.

The margin of exposure (MOE) is a risk assessment tool used to evaluate the safety of substances in food and feed. Adopted by the European Food Safety Authority (EFSA) in 2005, the MOE is calculated as the ratio between a Reference Point (RP) and the estimated exposure. While some regulatory bodies use ‘margin of safety’ (MOS) interchangeably with MOE, others define it differently, leading to inconsistencies in interpretation. To address this, EFSA has standardised its terminology, establishing MOE as a primary metric for safety assessments across human and animal health evaluations. In addition, the meaning and interpretation of terms used to qualify a ‘concern’ is elaborated. The EFSA definitions will come into force from when this statement is published. By refining these definitions and ensuring consistent terminology across sectors, EFSA aims to improve clarity and transparency in its risk assessments, facilitating effective communication.

Increasing toxic cyanobacterial blooms in freshwater demand environmentally friendly solutions to control their growth and toxicity, especially in arid countries, where most drinking water is produced from surface reservoirs. We tested the effects of macrophyte allelochemicals on Microcystis aeruginosa and on the fundamental role of bacteria in nutrient recycling. The effects of Ranunculus aquatilis aqueous extract, the most bioactive of four Moroccan macrophyte extracts, were tested in batch systems on M. aeruginosa growth, toxin production and oxidative stress response and on the ectoenzymatic activity associated with the bacterial community. M. aeruginosa density was reduced by 82.18%, and a significant increase in oxidative stress markers was evidenced in cyanobacterial cells. Microcystin concentration significantly decreased, and they were detected only intracellularly, an important aspect in managing toxic blooms. R. aquatilis extract had no negative effects on associated bacteria. These results confirm a promising use of macrophyte extracts, but they cannot be generalized. The use of the extract on other toxic strains, such as Planktothrix rubescens, Raphidiopsis raciborskii and Chrysosporum ovalisporum, caused a reduction in growth rate but not in cyanotoxin content, increasing toxicity. The need to assess species-specific cyanobacteria responses to verify the efficacy and safety of the extracts for human health and the environment is highlighted.

EFSA asked the Panel on Plant Protection Products and their residues to deliver a Scientific Opinion on testing and interpretation of comparative in vitro metabolism studies for both new active substances and existing ones. The main aim of comparative in vitro metabolism studies of pesticide active substances is to evaluate whether all significant metabolites formed in the human in vitro test system, as a surrogate of the in vivo situation, are also present at comparable level in animal species tested in toxicological studies and, therefore, if their potential toxicity has been appropriately covered by animal studies. The studies may also help to decide which animal model, with regard to a particular compound, is the most relevant for humans. In the experimental strategy, primary hepatocytes in suspension or culture are recommended since hepatocytes are considered the most representative in vitro system for prediction of in vivo metabolites. The experimental design of 3 × 3 × 3 (concentrations, time points, technical replicates, on pooled hepatocytes) will maximise the chance to identify unique (UHM) and disproportionate (DHM) human metabolites. When DHM and UHM are being assessed, test item‐related radioactivity recovery and metabolite profile are the most important parameters. Subsequently, structural characterisation of the assigned metabolites is performed with appropriate analytical techniques. In toxicological assessment of metabolites, the uncertainty factor approach is the first alternative to testing option, followed by new approach methodologies (QSAR, read‐across, in vitro methods), and only if these fail, in vivo animal toxicity studies may be performed. Knowledge of in vitro metabolites in human and animal hepatocytes would enable toxicological evaluation of all metabolites of concern, and, furthermore, add useful pieces of information for detection and evaluation of metabolites in different matrices (crops, livestock, environment), improve biomonitoring efforts via better toxicokinetic understanding, and ultimately, develop regulatory schemes employing physiologically based or physiology‐mimicking in silico and/or in vitro test systems to anticipate the exposure of humans to potentially hazardous substances in plant protection products. This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-6989/full