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Bisphenol A (BPA), a synthetic chemical widely used in the production of polycarbonate plastic and epoxy resins, has been associated with a variety of adverse effects in humans including metabolic, immunological, reproductive, and neurodevelopmental effects, raising concern about its health impact. In the EU, it has been classified as toxic to reproduction and as an endocrine disruptor and was thus included in the candidate list of substances of very high concern (SVHC). On this basis, its use has been banned or restricted in some products. As a consequence, industries turned to bisphenol alternatives, such as bisphenol S (BPS) and bisphenol F (BPF), which are now found in various consumer products, as well as in human matrices at a global scale. However, due to their toxicity, these two bisphenols are in the process of being regulated. Other BPA alternatives, whose potential toxicity remains largely unknown due to a knowledge gap, have also started to be used in manufacturing processes. The gradual restriction of the use of BPA underscores the importance of understanding the potential risks associated with its alternatives to avoid regrettable substitutions. This review aims to summarize the current knowledge on the potential hazards related to BPA alternatives prioritized by European Regulatory Agencies based on their regulatory relevance and selected to be studied under the European Partnership for the Assessment of Risks from Chemicals (PARC): BPE, BPAP, BPP, BPZ, BPS-MAE, and TCBPA. The focus is on data related to toxicokinetic, endocrine disruption, immunotoxicity, developmental neurotoxicity, and genotoxicity/carcinogenicity, which were considered the most relevant endpoints to assess the hazard related to those substances. The goal here is to identify the data gaps in BPA alternatives toxicology and hence formulate the future directions that will be taken in the frame of the PARC project, which seeks also to enhance chemical risk assessment methodologies using new approach methodologies (NAMs).

Introduction: Communities affected by large scale and long lasting industrial contamination are often keen to understand whether their health has been impaired by such contamination. This requires answers that integrate environmental public health and environmental justice perspectives. At these sites, exposure scenarios from environmental contamination over time by multiple chemicals, often involving different environmental matrices, are complex and challenging to reconstruct., Methods: An approach for describing the health of such communities in association with environmental contamination is presented, with the methods applied across the three domains of environmental contamination, population exposure and toxicology, environmental and social epidemiology, and environmental public health communication. The approach is described with examples from its application to the case study of Porto Torres, a town with a substantial industrially conditioned evolution., Results: Activities in the field of environmental contamination, population exposure and toxicology focus on the collection and systematization of available contamination data, the identification of priority pollutants based on their toxicological profiles, the qualitative assessment of the likelihood of exposure for the population to priority pollutants and their known health effects. Environmental and social epidemiology methods are applied to describe the health profiles and socioeconomic conditions of the local population, taking into account multiple health outcomes from local information systems and considering specific diseases based on exposure and toxicological assessments. The environmental public health communication methods are directed to produce a communication plan and for its implementation through interaction with local institutional and social actors. The interpretation of health profiles benefits from a transdisciplinary analysis of the results., Discussion: The proposed approach combines the needs of environmental public health and environmental justice allowing the integration of multidisciplinary knowledge to define recommendations for reducing and/or preventing hazardous environmental exposures and adverse health effects, stimulating the interactions between stakeholders, and making the study results more accessible to citizens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pasetto, Zona, Marsili, Buratti, Iavarone, Soggiu and Testai.)

The focus on implementation of systematic review (SR) principles in chemical risk assessments (CRAs) is growing as it has the potential to advance the rigour and transparency of the CRAs. However, the SR and CRA communities use their own specific terminologies. Understanding the meaning of core SR and CRA terms and where they overlap is critical for application of SR methods and principles in CRAs. Moreover, it will increase the possibility for cross-sectorial collaboration, avoid misunderstandings, and improve communication among risk assessors, researchers, and policy makers. We present a process for the cross-mapping of core CRA terms and core SR terms. Core terms for study appraisal, evidence synthesis and integration used in the SR and CRA communities will be included. The outcome will be an overview of how core SR terms map onto core CRA terms and vice versa, and a description of the relationship and conceptual overlap between the terms. The cross-mapping is divided in three phases, where in the first phase the core SR and CRA terms will be identified. In the second phase, existing SR and CRA definitions will be mapped. In the third phase, descriptions of the relationship and conceptual overlap between the terms will be derived. The third phase will include weekly one-hour online meetings for SR and CRA experts., Competing Interests: Declaration of interests Completed declaration of interest forms for each author are available as supplementary materials. The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this protocol.

A large majority of chemicals is converted into metabolites through xenobiotic-metabolising enzymes. Metabolites may present a spectrum of characteristics varying from similar to vastly different compared with the parent compound in terms of both toxicokinetics and toxicodynamics. In the pesticide arena, the role of metabolism and metabolites is increasingly recognised as a significant factor particularly for the design and interpretation of mammalian toxicological studies and in the toxicity assessment of pesticide/metabolite-associated issues for hazard characterization and risk assessment purposes, including the role of metabolites as parts in various residues in ecotoxicological adversities. This is of particular relevance to pesticide metabolites that are unique to humans in comparison with metabolites found in in vitro or in vivo animal studies, but also to disproportionate metabolites (quantitative differences) between humans and mammalian species. Presence of unique or disproportionate metabolites may underlie potential toxicological concerns. This review aims to present the current state-of-the-art of comparative metabolism and metabolites in pesticide research for hazard and risk assessment, including One Health perspectives, and future research needs based on the experiences gained at the European Food Safety Authority., Competing Interests: JoL, JeL, MP and JP were employed by the European Food Safety Authority (EFSA). SC and CB were employed by the European Commission Joint Research Centre (JRC). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Pelkonen, Abass, Parra Morte, Panzarea, Testai, Rudaz, Louisse, Gundert-Remy, Wolterink, Jean-Lou CM, Coecke and Bernasconi.)

This protocol describes the design and development of a tool for evaluation of the internal validity of in vitro studies, which is needed to include the data as evidence in systematic reviews and chemical risk assessments. The tool will be designed specifically to be applied to cell culture studies, including, but not restricted to, studies meeting the new approach methodology (NAM) definition. The tool is called INVITES-IN (IN VITro Experimental Studies INternal validity). In this protocol, three of the four studies that will be performed to create the release version of INVITES-IN are described. In the first study, evaluation of existing assessment tools will be combined with focus group discussions to identify how characteristics of the design or conduct of an in vitro study can affect its internal validity. Bias domains and items considered to be of relevance for in vitro studies will be identified. In the second study, group agreement on internal validity domains and items of importance for in vitro studies will be identified via a modified Delphi methodology. In the third study, the draft version of the tool will be created, based on the data on relevance and importance of bias domains and items collected in Studies 1 and 2. A separate protocol will be prepared for the fourth study, which includes the user testing and validation of the tool, and collection of users' experience.

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., Competing Interests: The authors declare no conflicts of interest.

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., Competing Interests: Author Abdulkarim Najjar were employed by the company Beiersdorf AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Algharably, Di Consiglio, Testai, Pistollato, Bal-Price, Najjar, Kreutz and Gundert-Remy.)

Microcystins constitute a group of over 200 variants and are increasingly considered as emerging toxins in food and feed safety, particularly with regards to sea-food and fish consumption. Toxicity of MCs is congener-specific, being characterised by different acute potencies, likely related to the differential activity of metabolic enzymes and transporters proteins involved in their cellular uptake. However, the active transport of MCs across intestinal membranes has not been fully elucidated. Our results, obtained using a fit for purpose 3D human reconstructed intestinal epithelium, provide new information on the complex mechanisms involved in the absorption of 5 MC variants': it is indeed characterised by the equilibrium between uptake and extrusion, since the selected congeners are substrates of both influx and efflux proteins. In the range of tested nominal concentrations (10-40 µM) fully representative of relevant exposure scenarios, none of the active tested transporters were saturated. The comparison of permeability (Papp) values of MCs variants highlighted a dose independent relationship for MC-LR, -YR and -RR (Papp x 10 -7 ranged from 2.95 to 3.54 cm/s), whereas -LW and-LF showed a dose dependent increase in permeability reaching Papp values which were similar to the other congeners at 40 µM. MC-RR, -LR, -YR show absorption values around 5% of the administered dose. Due to their lipophilicity, MC-LW and -LF were also detected within the cellular compartment. The intestinal uptake was only partially attributable to OATPs, suggesting the involvement of additional transporters. Regarding the efflux proteins, MCs are not P-gp substrates whereas MRP2 and to a lesser extent Breast cancer resistance protein are active in their extrusion. Despite the presence of GST proteins, as an indication of metabolic competence, in the intestinal tissue, MC-conjugates were never detected in our experimental settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Turco, Santori, Buratti, Dorne and Testai.)

Physiologically based kinetic (PBK) modeling has been increasingly used since the beginning of the 21st century to support dose selection to be used in preclinical and clinical safety studies in the pharmaceutical sector. For chemical safety assessment, the use of PBK has also found interest, however, to a smaller extent, although an internationally agreed document was published already in 2010 (IPCS/WHO), but at that time, PBK modeling was based mostly on in vivo data as the example in the IPCS/WHO document indicates. Recently, the OECD has published a guidance document which set standards on how to characterize, validate, and report PBK models for regulatory purposes. In the past few years, we gained experience on using in vitro data for performing quantitative in vitro - in vivo extrapolation (QIVIVE), in which biokinetic data play a crucial role to obtain a realistic estimation of human exposure. In addition, pharmaco-/toxicodynamic aspects have been introduced into the approach. Here, three examples with different drugs/chemicals are described, in which different approaches have been applied. The lessons we learned from the exercise are as follows: 1) in vitro conditions should be considered and compared to the in vivo situation, particularly for protein binding; 2) in vitro inhibition of metabolizing enzymes by the formed metabolites should be taken into consideration; and 3) it is important to extrapolate from the in vitro measured intracellular concentration and not from the nominal concentration to the tissue/organ concentration to come up with an appropriate QIVIVE for the relevant adverse effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Algharably, Di Consiglio, Testai, Pistollato, Mielke and Gundert-Remy.)

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., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

This guidance document provides harmonised and flexible methodologies to apply scientific criteria and prioritisation methods for grouping chemicals into assessment groups for human risk assessment of combined exposure to multiple chemicals. In the context of EFSA's risk assessments, the problem formulation step defines the chemicals to be assessed in the terms of reference usually through regulatory criteria often set by risk managers based on legislative requirements. Scientific criteria such as hazard-driven criteria can be used to group these chemicals into assessment groups. In this guidance document, a framework is proposed to apply hazard-driven criteria for grouping of chemicals into assessment groups using mechanistic information on toxicity as the gold standard where available (i.e. common mode of action or adverse outcome pathway) through a structured weight of evidence approach. However, when such mechanistic data are not available, grouping may be performed using a common adverse outcome. Toxicokinetic data can also be useful for grouping, particularly when metabolism information is available for a class of compounds and common toxicologically relevant metabolites are shared. In addition, prioritisation methods provide means to identify low-priority chemicals and reduce the number of chemicals in an assessment group. Prioritisation methods include combined risk-based approaches, risk-based approaches for single chemicals and exposure-driven approaches. Case studies have been provided to illustrate the practical application of hazard-driven criteria and the use of prioritisation methods for grouping of chemicals in assessment groups. Recommendations for future work are discussed., (© 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The open source database "OpenCYP database" has been developed based on the results of extensive literature searches from the peer-reviewed literature. OpenCYP provides data on human variability on baseline of activities and polymophism frequencies for selected cytochrome P-450 isoforms (CYP1A2, CYP2A6, CYP2D6, CYP3A4/3A5 and CYP3A7) in healthy adult populations from world populations. CYP enzymatic activities were generally expressed as the metabolic ratio (MR) between an unchanged probe drug and its metabolite(s) in urine or plasma measured in healthy adults. Data on other age groups were very limited and fragmented, constituting an important data gap. Quantitative comparisons were often hampered by the different experimental conditions used. However, variability was quite limited for CYP1A2, using caffeine as a probe substrate, with a symmetrical distribution of metabolic activity values. For CYP3A4, human variability was dependent on the probe substrate itself with low variability when data considering the dextromethorphan/demethilathed metabolite MR were used and large variability when the urinary 6β-hydroxycortisol/cortisol ratio was used. The largest variability in CYP activity was shown for CYP2D6 activity, after oral dosing of dextromethorphan, for which genetic polymorphisms are well characterised and constitute a significant source of variability. It is foreseen that the OpenCYP database can contribute to promising tools to support the further development of QIVIVE and PBK models for human risk assessment of chemicals particularly when combined with information on isoform-specific content in cells using proteomic approaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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.

Background: The etiopathogenesis of amyotrophic lateral sclerosis (ALS) is still largely unknown., Methods: We performed a case-control study (33 cases and 35 controls) in Umbria, Italy. We investigated associations between common lifestyle, clinical factors, as well as environmental exposures potentially implicated with ALS onset. Face-to-face interviews were carried out. All cases were recruited and diagnosed according to El Escorial criteria. Case-control comparisons were made for educational and residential status, occupational exposures, and clinical and lifestyle factors prior to cases' dates of diagnosis., Results: Our results showed an increased risk of ALS for subjects chronically exposed to raw water use (odds ratio (OR) = 6.55, 95% confidence interval (CI): 2.24-19.12). Garden activities showed a tight association with ALS as well, very likely as a consequence of chronic raw water exposure. Indeed, we could exclude an impact for pesticides, as no significant differences were observed in pesticide exposure in the two groups interviewed. However, cases were more often exposed to fertilizers. After adjustment for age, sex, and heavy physical activities, exposure to raw water was still associated with increased ALS risk (OR = 4.74, 95% CI: 1.33-16.85)., Discussion: These findings suggest an association between ALS and exposure to raw water, which should be further investigated for the presence of chemicals interfering with nervous system functionality.

The input into the QIVIVE and Physiologically-Based kinetic and dynamic models of drug metabolising enzymes performance and their inter-individual differences significantly improve the modelling performance, supporting the development and integration of alternative approaches to animal testing. Bayesian meta-analyses allow generating and integrating statistical distributions with human in vitro metabolism data for quantitative in vitro-in vivo extrapolation. Such data are lacking on glutathione-S-transferases (GSTs). This paper reports for the first time results on the human variability of GST activities in healthy individuals, their tissue localisation and the frequencies of their major polymorphic variants by means of extensive literature search, data collection, data base creation and meta-analysis. A limited number of papers focussed on in vivo GST inter-individual differences in humans. Ex-vivo total GST activity without discriminating amongst isozymes is generally reported, resulting in a high inter-individual variability. The highest levels of cytosolic GSTs in humans are measured in the kidney, liver, adrenal glands and blood. The frequencies of GST polymorphisms for cytosolic isozymes in populations of different geographical ancestry were also presented. Bayesian meta-analyses to derive GST-related uncertainty factors provided uncertain estimates, due to the limited database. Considering the relevance of GST activities and their pivotal role in cellular adaptive response mechanisms to chemical stressors, further studies are needed to identify GST probe substrates for specific isozymes and quantify inter-individual differences., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

For some complex toxicological endpoints, chemical safety assessment has conventionally relied on animal testing. Apart from the ethical issues, also scientific considerations have been raised concerning the traditional approach, highlighting the importance for considering real life exposure scenario. Implementation of flexible testing strategies, integrating multiple sources of information, including in vitro reliable test methods and in vitro biokinetics, would enhance the relevance of the obtained results. Such an approach could be pivotal in the evaluation of developmental neurotoxicity (DNT), especially when applied to human cell-based models, mimicking key neurodevelopmental processes, relevant to human brain development. Here, we integrated the kinetic behaviour with the toxicodynamic alterations of chlorpyrifos (CPF), such as in vitro endpoints specific for DNT evaluation, after repeated exposure during differentiation of human neural stem cells into a mixed culture of neurons and astrocytes. The upregulation of some cytochrome P450 and glutathione S-transferase genes during neuronal differentiation and the formation of the two major CPF metabolites (due to bioactivation and detoxification) supported the metabolic competence of the used in vitro model. The alterations in the number of synapses, neurite outgrowth, brain derived neurotrophic factor, the proportion of neurons and astrocytes, as well as spontaneous electrical activity correlated well with the CPF ability to enter the cells and be bioactivated to CPF-oxon. Overall, our results confirm that combining in vitro biokinetics and assays to evaluate effects on neurodevelopmental endpoints in human cells should be regarded as a key strategy for a quantitative characterization of DNT effects., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently available evidence supports that the predominant route of human-to-human transmission of the SARS-CoV-2 is through respiratory droplets. Indirect hands contact with surfaces contaminated by infectious droplets subsequently touching the mouth, nose or eyes seems to be another route of an indirect contact transmission. Persistence of the virus on different surfaces and other materials has been reported in recent studies: SARS-CoV-2 was more stable on plastic and stainless steel than on copper and cardboard. Viable virus was detected up to 72 h after application to different surfaces, although infectivity decay was also observed. This evidence suggests the likelihood that waste generated from patients affected by COVID-19 or subjects in quarantine treated in private houses or in areas different from hospitals and medical centres could be contaminated by SARS-CoV-2. Consequently, waste streams may represent a route for viral spreading being a potential risk also for the operators directly involved in the different phases of waste management. To address this concern, a specific multidisciplinary working group was settled by the Italian National Institute of Health (ISS) during the COVID-19 emergency, in order to establish guidelines related to solid waste collection, delivering, withdrawal, transport, treatment and disposal. Temporary stop of waste sorting, instructions for the population on how to package waste, instructions for Companies and operators for the adoption of adequate personal protection equipment (PPE), the use and sanitation of proper vehicles were among the main recommendations provided to the community by publications of freely downloadable reports and infographics in layman language. Incineration, sterilization and properly managed landfills were identified as the facilities to be preferentially adopted for the treatment of this kind of waste, considering the main inactivation strategies of SARS-CoV-2 (e.g. treatment length > 9 days and temperature > 70 °C for more than 5 min)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Data on the bioactivation of Phosmet (Pho), a phthalimide-derived organophosphate pesticide (OPT), to the neurotoxic metabolite Phosmet-oxon (PhOx) in human are not available. The characterization of the reaction in single human recombinant CYPs evidenced that the ranking of the intrinsic clearances was: 2C18>2C19>2B6>2C9>1A1>1A2>2D6>3A4>2A6. Considering the average human hepatic content, CYP2C19 contributed for the great majority (60%) at relevant exposure concentrations, while CYP2C9 (33%) and CYP3A4 (31%) were relevant at high substrate concentration. The dose-dependent role of the active isoforms was confirmed in human liver microsomes by using selective CYP inhibitors. This prominent role of CYP2C in oxon formation was not shared by other OPTs. The pre-systemic Pho bioactivation measured in human intestinal microsomes was relevant accounting for ¼ of that measured in the liver showing two reaction phases catalysed by CYP2C and CYP3A4. Phosmet efficiently inhibited CPF bioactivation and detoxication, with Ki values (≈30 μM) relevant to pesticide concentrations achievable in the human liver, while the opposite is unlikely (Ki ≈ 160 μM) at the actual exposure levels, depending on the peculiar isoform-specific Pho bioactivation. Kinetic information in humans can support the development of quantitative in vitro/in vivo extrapolation and in silico models for risk assessment refinement for single and multiple pesticides., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The European Commission has established a priority list of 15 additives contained in cigarettes and roll-your-own tobacco subject to enhanced reporting obligations. The European Union (EU) Tobacco Products Directive (TPD) prescribes that Member States shall require manufacturers and importers of tobacco products to carry out comprehensive studies on these additives to assess their contribution to any of the properties listed in Article 6 of the TPD: toxicity or addictiveness, characterizing flavor, inhalation facilitation, nicotine uptake, and carcinogenic, mutagenic, or toxic for reproduction. The Scientific Committee on Health, Environmental, and Emerging Risks (SCHEER) has provided guidance on the type and criteria for comprehensive studies, and on the most suitable methodologies to test these 15 tobacco additives as well as additives on future updated lists. The SCHEER proposes a stepwise strategy as the most pragmatic and efficient way to assess the effects of tobacco additives. In addition to proposing specific steps and tests to be considered by industry, some general criteria were also identified such as no comparative testing (testing cigarettes with and without the additive) and no animal studies. As tobacco additives have no benefits for health, but rather may promote use of and addiction to an extremely toxic product, a risk-benefit analysis is not the appropriate paradigm for assessing the additive. When comprehensive studies confirm that additives have any of the properties listed in Article 6 of the TPD, regulatory actions should be considered. If uncertainties cannot be solved by comprehensive studies, the SCHEER recommends that the assessors consider the worst-case evaluation., Implications: In this article, the SCHEER proposes a stepwise strategy to assess (1) the toxic and addictive effects, (2) the characterizing flavor, and (3) facilitating inhalation properties of tobacco additives. The proposed steps and tests provide guidance to (1) Member State on which comprehensive studies should be requested and (2) tobacco industry on which strategy of testing should be applied to address the request and to prepare reports to be sent to the relevant authorities for the evaluation of tobacco additives "safety" to comply with the Tobacco Products Directive 2014/40/EU., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cyanotoxins, among which >200 variants of Microcystins (MC), constitute an emerging issue in food safety. Microcystins (MC) toxicity is congener-specific; however, the in vitro inhibition of PP1/PP2A (the key molecular event of MC toxicity) by single MC variants is comparable and MC toxicokinetics seems to be the critical point. Here, the variability in GSH conjugation catalysed by human recombinant enzymes and human hepatic cytosol has been compared between hydrophilic (MC-LR and MC-RR) and hydrophobic (MC-LW, MC-YR and MC-LF) variants, according to measured logPow. In vitro detoxication reaction (spontaneous plus enzymatic) is favored by the variant hydrophilicity, with MC-LF very poorly detoxified. With MC-YR and -LW the spontaneous reaction always gave the major contribution, whereas with MC-LR and -RR the enzymatic reaction became by far predominant when GSH was depleted. Consequently, the well-known GST polymorphisms seems not to be the major driver for potential human variability in susceptibility towards the MC-toxicity, except for MC-RR and -LR when GSH is depleted. Looking at these results and literature data, MC-RR (the least cytotoxic and acutely toxic in rodents) is the more hydrophilic, has the lowest OATP-mediated hepatic uptake and the highest detoxication efficiency. The opposite is true for the most lipophilic MC-LF: once entered in the cells with the highest uptake, it is very poorly detoxified, and resulted as the most toxic in various cell types. MC-dependent TK should be considered in order to estimate the variability in toxicity and to support the use of quantitative in vitro-in vivo extrapolation models of single toxins and their mixtures co-occurring in the environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human exposure to per- and polyfluorinated alkyl substances (PFASs) is a major public health concern because in the last decades several cases of overexposure of people to PFASs, in particular through contaminated water, occurred worldwide. In 2013-2017 a PFAS drinking water contamination was discovered and investigated in northern Italy (Veneto region) and high PFAS serum levels were detected in exposed people. 629 subjects were enrolled: 257 residing in municipalities in the areas under impact, 250 residing in municipalities in areas at presumed background exposure and 122 farmers living in contaminated rural areas producing and consuming own livestock and vegetables and frequently using well water. The highest PFAS serum concentrations (median PFOA concentrations 40 ng/g) were found in the subgroup of farmers. The main factors influencing PFAS serum levels of farmers were residence area and the related extent of drinking water contamination, gender, years of residence in the municipalities, well water consumption and consumption of own produced food. PFOA serum concentrations in farmers residing in the areas of the Veneto region impacted by PFAS contamination are among the highest found worldwide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Climate changes affect social and environmental health determinants such as clean air, ecosystems health, safe drinking water and safe sufficient food. Globally, people at greatest risk of adverse health effects associated with climate change include children, the elderly and other vulnerable groups. Temperature-related death and illness, extreme events, polluted or stressed ecosystems represent relevant issues raising concern for both health and economic consequences. The aim of the Symposium "Health and Climate Change" (Istituto Superiore di Sanità, Rome 3-5 December 2018) was to promote an inter-sectoral and multidisciplinary approach to estimate and prevent climate change-related events as well as to call the authorities to put in place measures to reduce adverse health effects. At the end of the Symposium the Rome International Charter on Health and Climate Change was presented. It includes a series of actions and recommendations, discussed and shared by all the participants, intended to inform policy makers and all the stakeholders involved in the management of climate changes.

Triflumuron (TFM) is a benzoylurea insecticide commonly used in Tunisian agriculture and around the world to control crop pests and flies as a promising alternative to conventional insecticides for its arthropod specificity and low toxicity. From the evidence available in animal models, it can be expected that the metabolism of TFM is catalyzed by cytochrome P450 (CYP) and esterases. However, no data are available on human metabolism of TFM with regards to phase I metabolism and CYP isoform specificity. Hence, this manuscript describes experimental investigations to underpin in vitro phase I TFM metabolism in human samples for the first time. TFM biotransformation by recombinant human CYPs was characterized, then human liver microsomes (HLM) and chemical specific inhibitors have been used to identify the relative contribution of CYPs and esterases. Our results showed that all CYP isoforms were able to metabolize TFM with different affinity and efficiency. The relative contribution based both on the kinetic parameters and the CYP hepatic content was 3A4 > >2C9 > 2C8 > 2A6 > 1A2 > 2B6 > 2D6 > 2C19 > 2C18 > 1A1 at low TFM concentration, whilst at high TFM concentration it was 1A2 > >2C9 = 3A4 = 2A6 > 2C19 > 2B6 = 2C8 > 2D6 > 1A1 > 2C18. Experiments with HLMs confirmed the involvement of the most relevant CYPs in the presence of specific chemical inhibitors with a catalytic efficiency (Cliapp) lower by an order of magnitude compared with recombinant enzymes. Esterases were also relevant to the overall TFM kinetics and metabolism, with catalytic efficiency higher than that of CYPs. It is foreseen that such isoform-specific information in humans will further support in silico models for the refinement of the human risk assessment of single pesticides or mixtures., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This Guidance document describes harmonised risk assessment methodologies for combined exposure to multiple chemicals for all relevant areas within EFSA's remit, i.e. human health, animal health and ecological areas. First, a short review of the key terms, scientific basis for combined exposure risk assessment and approaches to assessing (eco)toxicology is given, including existing frameworks for these risk assessments. This background was evaluated, resulting in a harmonised framework for risk assessment of combined exposure to multiple chemicals. The framework is based on the risk assessment steps (problem formulation, exposure assessment, hazard identification and characterisation, and risk characterisation including uncertainty analysis), with tiered and stepwise approaches for both whole mixture approaches and component-based approaches. Specific considerations are given to component-based approaches including the grouping of chemicals into common assessment groups, the use of dose addition as a default assumption, approaches to integrate evidence of interactions and the refinement of assessment groups. Case studies are annexed in this guidance document to explore the feasibility and spectrum of applications of the proposed methods and approaches for human and animal health and ecological risk assessment. The Scientific Committee considers that this Guidance is fit for purpose for risk assessments of combined exposure to multiple chemicals and should be applied in all relevant areas of EFSA's work. Future work and research are recommended., (© 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

Hepatic metabolic clearance is one of the most important factors driving the overall kinetics of chemicals including substances used in various product categories such as pesticides, biocides, pharmaceuticals, and cosmetics. A large number of in vitro systems from purified isozymes and subcellular organelles to hepatocytes in simple cultures and in complex scaffold setups are available for measuring hepatic metabolic clearance for different applications. However, there is currently no approach for systematically characterising and comparing these in vitro methods in terms of their design, applicability and performance. To address this, existing knowledge in the field of in vitro human hepatic metabolic clearance methods was gathered and analysed in order to establish a framework to systematically characterise methods based on a set of relevant components. An analogous framework would be also applicable for non-human in vitro systems. The components are associated with the biological test systems used (e.g. subcellular or cells), the in vitro method (e.g. number of cells, test item solubility), related analytical techniques, data interpretation methods (based on substrate depletion/metabolite formation), and performance assessments (precision and accuracy of clearance measurements). To facilitate the regulatory acceptance of this class of methods, it is intended that the framework provide the basis of harmonisation work within the OECD., (Copyright © 2018. Published by Elsevier Ltd.)

Competing Interests: Competing interests: None to be declared.

In 2013 a contamination of drinking water by perfluoroalkylated substances (PFASs) was discovered in areas of the Veneto Region (northern Italy). In this study the exposure to PFASs of people living in the aforesaid areas was characterized: contaminant serum concentrations were measured and compared with those of a control population group living in neighboring areas at background exposure (based on available drinking water data). The enrolled population was also genotyped for the OATP1A2*3 allelic variant, possibly affecting PFAS excretion and hence the internal dose. The difference in PFAS concentrations between exposed and not exposed subjects was significantly larger for nine of the 12 substances analyzed, and confirmed that water contamination had resulted in an appreciable high exposure of the residing population over time. Within the group of exposed subjects, subgroups at different exposure levels were identified. The contamination of drinking water of the residence area was found to be the main factor influencing PFAS serum levels; in addition to water contamination, other relevant influencing factors were sex, the years of residence and raising own livestock. No relationship with the genetic trait for the studied renal transporter was evidenced. These results provide a baseline characterization of PFAS exposure of the monitored population groups for further studies, planned to be carried out in the near future., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cyanobacteria thrive in many aquatic environments, where they can produce cyanotoxins with different toxicological profile. Anthropic pressure and climate changes are causing the expansion in terms of time and space of their blooms, increasing the concerns for human health in several exposure scenarios. Here the update of the Italian guidelines for the management of cyanobacterial blooms in bathing water is presented. A risk-based approach has been developed according to the current scientific knowledge on cyanobacteria distribution in the Italian Lakes and on chemical, toxicological and epidemiological aspects of different cyanotoxins, summarized in the first part of the paper. Oral, dermal and inhalation exposure to cyanotoxins, during recreational activities, are individually examined, to develop a framework of thresholds and actions aimed at preventing harmful effects for bathers. Guidelines, also by comparing international guidance values and/or guidelines, provide criteria to plan environmental monitoring activities, health surveillance and public communication systems. Finally the still important scientific gaps and research needs are highlighted., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cyanobacteria were present on the earth 3.5 billion years ago; since then they have colonized almost all terrestrial and aquatic ecosystems. They produce a high number of bioactive molecules, among which some are cyanotoxins. Cyanobacterial growth at high densities, forming blooms, is increasing in extension and frequency, following anthropogenic activities and climate changes, giving rise to some concern for human health and animal life exposed to cyanotoxins. Numerous cases of lethal poisonings have been associated with cyanotoxins ingestion in wild animal and livestock. In humans few episodes of lethal or severe human poisonings have been recorded after acute or short-term exposure, but the repeated/chronic exposure to low cyanotoxin levels remains a critical issue. The properties of the most frequently detected cyanotoxins (namely, microcystins, nodularins, cylindrospermopsin and neurotoxins) are here critically reviewed, describing for each toxin the available information on producing organisms, biosynthesis/genetic and occurrence, with a focus on the toxicological profile (including kinetics, acute systemic toxicity, mechanism and mode of action, local effects, repeated toxicity, genotoxicity, carcinogenicity, reproductive toxicity; human health effects and epidemiological studies; animal poisoning) with the derivation of health-based values and considerations on the risks for human health.

Vico Lake, a volcanic meso-eutrophic lake in Central Italy, whose water is used for drinking and recreational activities, experienced the presence of the microcystins (MC) producing cyanobacterium Planktothrix rubescens. In order to assess the human health risks and to provide the local health authorities with a scientific basis for planning tailored monitoring activities, we studied P. rubescens ecology and toxicity for two years. P. rubescens generally dominated the phytoplankton community, alternating with Limnothrix redekei, potentially toxic. P. rubescens was distributed throughout the water column during winter; in summer it produced intense blooms where drinking water is collected (-20 m); here MC were detected all year round (0.5-5 μg/L), with implications for drinking water quality. In surface waters, MC posed no risk for recreational activities in summer, while in winter surface blooms and foams (containing up to 56 μg MC/L) can represent a risk for people and children practicing water sports and for animals consuming raw water. Total phosphorus, phosphate and inorganic nitrogen were not relevant to predict densities nor toxicity; however, a strong correlation between P. rubescens density and aminopeptidase ectoenzymatic activity, an enzyme involved in protein degradation, suggested a role of organic nitrogen for this species. The fraction of potentially toxic population, determined both as mcyB(+)/16SrDNA (10-100%) and as the MC/mcyB(+) cells (0.03-0.79 pg MC/cell), was much more variable than usually observed for P. rubescens. Differently from other Italian and European lakes, the correlation between cell density or the mcyB(+) cells and MC explained only ∼50 and 30% of MC variability, respectively: for Vico Lake, monitoring only cell or the mcyB(+) cell density is not sufficient to predict MC concentrations, and consequently to protect population health. Finally, during a winter bloom one site has been sampled weekly, showing that monthly sampling during such a phase could greatly underestimate the 'hazard'. Our results highlight the need to adopt a stepwise monitoring activity, considering the lake and the cyanobacteria specific features. This activity should be complemented with communication to the public and involvement of stakeholders., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cyanobacteria are ubiquitous photosynthetic micro-organisms forming blooms and scums in surface water; among them some species can produce cyanotoxins giving rise to some concern for human health and animal life. To date, more than 65 cyanobacterial neurotoxins have been described, of which the most studied are the groups of anatoxins and saxitoxins (STXs), comprising many different variants. In freshwaters, the hepatotoxic microcystins represent the most frequently detected cyanotoxin: on this basis, it could appear that neurotoxins are less relevant, but the low frequency of detection may partially reflect an a priori choice of target analytes, the low method sensitivity and the lack of certified standards. Cyanobacterial neurotoxins target cholinergic synapses or voltage-gated ion channels, blocking skeletal and respiratory muscles, thus leading to death by respiratory failure. This review reports and analyzes the available literature data on environmental occurrence of cyanobacterial neurotoxic alkaloids, namely anatoxins and STXs, their biosynthesis, toxicology and epidemiology, derivation of guidance values and action limits. These data are used as the basis to assess the risk posed to human health, identify critical exposure scenarios and highlight the major data gaps and research needs.

This special issue of Toxicology in Vitro is dedicated to disseminating the results of the EU-funded collaborative project "Profiling the toxicity of new drugs: a non animal-based approach integrating toxicodynamics and biokinetics" (Predict-IV; Grant 202222). The project's overall aim was to develop strategies to improve the assessment of drug safety in the early stage of development and late discovery phase, by an intelligent combination of non animal-based test systems, cell biology, mechanistic toxicology and in silico modeling, in a rapid and cost effective manner. This overview introduces the scope and overall achievements of Predict-IV., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The limited value of in vitro toxicity data for the in vivo extrapolation has been often attributed to the lack of kinetic data. Here the in vitro kinetics of amiodarone (AMI) and its mono-N-desethyl (MDEA) metabolite was determined and modelled in primary human hepatocytes (PHH) and HepaRG cells, after single and repeated administration of clinically relevant concentrations. AMI bioavailability was influenced by adsorption to the plastic and the presence of protein in the medium (e.g. 10% serum protein reduced the uptake by half in HepaRG cells). The cell uptake was quick (within 3h), AMI metabolism was efficient and a dynamic equilibrium was reached in about a week after multiple dosing. In HepaRG cells the metabolic clearance was higher than in PHH and increased over time, as well as CYP3A4. The interindividual variability in MDEA production in PHHs was not proportional to the differences in CYP3A4 activities, suggesting the involvement of other CYPs and/or AMI-related CYP inhibition. After repeated treatment AMI showed a slight potential for bioaccumulation, whereas much higher intracellular MDEA levels accumulated over time, especially in the HepaRG cells, associated with occurrence of phospholipidosis. The knowledge of in vitro biokinetics is important to transform an actual in vitro concentration-effect into an in vivo dose-effect relationship by using appropriate modelling, thus improving the in vitro-to-in vivo extrapolation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The aim of the EU FP7 Predict-IV project was to improve the predictivity of in vitro assays for unwanted effects of drugs after repeated dosing. The project assessed the added benefit of integrating long-lived in vitro organotypic cell systems with 'omics' technologies and in silico modelling, including systems biology and pharmacokinetic assessments. RPTEC/TERT1 kidney cells, primary rat and human hepatocytes, HepaRG liver cells and 2D and 3D primary brain cultures were dosed daily or every other day for 14 days to a selection of drugs varying in their mechanism of pharmacological action. Since concentration-effect relationships not only depend on the activity of the drug or the sensitivity of the target, but also on the distribution of compounds in the in vitro system, the concentration of a selection of drugs in cells, microtitre plate plastic and medium was measured over time. Results, reviewed in this paper, indicate that lipophilic drugs bind significantly to plastic labware. A few drugs, including less lipophilic drugs, bind to cell-attachment matrices. Chemicals that reach high concentrations in cells, including cyclosporin A and amiodarone, significantly accumulate over time after repeated dosing, partly explaining their increased toxicity after repeated dosing, compared to a single dose., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24 h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14 days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Common in vitro toxicity testing often neglects the fate and intracellular concentration of tested compounds, potentially limiting the predictability of in vitro results for in vivo extrapolation. We used in vitro long-term cultures of primary rat (PRH) and human hepatocytes (PHH) and HepaRG cells to characterise and model the biokinetic profile of ibuprofen (IBU) after single and daily repeated exposure (14 days) to two concentrations. A cross-model comparison was carried out at 100μM, roughly corresponding to the human therapeutic plasma concentration. Our results showed that IBU uptake was rapid and a dynamic equilibrium was reached within 1 or 2 days. All three cell systems efficiently metabolised IBU. In terms of species-differences, our data mirrored known in vivo results. Although no bioaccumulation was observed, IBU intracellular concentration was higher in PRH due to a 10-fold lower metabolic clearance compared to the human-derived cells. In HepaRG cells, IBU metabolism increased over time, but was not related to the treatment. In PHH, a low CYP2C9 activity, the major IBU-metabolising CYP, led to an increased cytotoxicity. A high inter-individual variability was seen in PHH, whereas HepaRG cells and PRH were more reproducible models. Although the concentrations of IBU in PRH over time differed from the concentrations found in human cells under similar exposure conditions., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The accepted pathway for MC biotransformation is GSH conjugation, occurring either spontaneously or catalyzed by GST. In the present work, the already available information on human MC metabolism have been expanded and the capacity of human GST to conjugate MC-LR has been confirmed in human liver cytosol. At physiological GSH content the spontaneous reaction predominated on the enzymatic one; the prevalence of the enzymatic reaction occurred following GSH depletion, and the shift was detectable at higher GSH levels, the lower was MC concentration. However, at low MC-LR concentrations (≤10μM), representative of repeated oral exposure, the relevance of the enzymatic reaction became predominant at GSH concentration between 1 and 2mM. MC-LR conjugate was detectable at ≥0.5mM GSH, whereas, with 10μM MC-RR detectable levels of conjugate were observed at 0.05mM GSH, a 10-fold lower concentration. Overall, our data indicate that MC-RR is more efficiently conjugated than MC-LR, especially at low concentrations. Cytosol samples from rat and mouse were used to characterize GSH conjugation of MC-LR and MC-RR, and to check for possible species differences. At physiological GSH content, in both rodent species the enzymatic reaction accounted for half of the total conjugate formation, reducing the impact of spontaneous reaction with respect to human. Rat and mouse GST showed similar MC-LR and-RR GSH conjugation, but a two-fold higher catalytic efficiency than human sample. This is mainly due to higher affinity for the substrate, with Kmapp values being an order of magnitude lower in the animal models than in human liver cytosol. More pronounced differences in the metabolism of the two variants were evidenced in rodents than in humans., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

According to EU Regulation No. 1223/2009/CE cosmetic products for daily use can contain 'technically unavoidable traces' of metals. This definition is too vague. Authorities should set well-defined limits, considering the risks associated with metal contamination of personal care products (PCPs). This paper characterizes the risk arising from a number of metals (antimony, arsenic, cadmium, cobalt, chromium, mercury, nickel, lead) that may occur in 'unavoidable traces" in raw materials and, consequently, in PCPs. A 'worst case scenario' was adopted, based on the following assumptions: (i) the individual ingredients contained the maximum amount in traces allowed for each metal; (ii) the hypothetical PCP was produced exclusively with that single ingredient; (iii) when absorption through the skin was not known, data related to oral absorption were used. Risk characterization was performed calculating the Systemic Exposure Dosage (SED) and the Margin of Safety (MoS=NOAEL or BMDL10/SED). Exposure to the allegedly 'technically unavoidable' maximum amounts of metals in cosmetic ingredients resulted in MoSs exceeding 100 (safety threshold) with one exception. This suggests that the availability of experimental dermal absorption rates could enable significant improvement in MoS, thus increasing safety levels. Although results are reassuring, the authors recommend minimization of contamination, according to the state of the art of manufacturing methods., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cyanotoxins (CTX) are widely produced by several cyanobacteria (CB), increasingly spreading in most water bodies and terrestrial habitats, and represent a risk for human health. CB are prokaryotes, and although mostly autotrophic, several examples of heterotrophy in symbiotic relationship with different organisms have been described. In addition to the known routes of exposure, it has been hypothesized that CB might 'colonize' human intestine with relevant implications for human health. Colonization is a complex process and requires specific features of the possible invaders. Still, a short-term persistence as living and toxin-producing organisms within the intestinal lumen of the host could represent an 'internal' source of exposure to CTX. In this work we ran microcosm experiments (4-18days), looking at Microcystis aeruginosa PCC7806 resistance and cyanotoxin-producing capabilities in darkness, 37°C, pH 2, and subsequent recovery in a rich medium, in darkness, 37°C, in the presence of enteric bacteria, mimicking few important features of the gastrointestinal environment. We measured cyanobacterial populations and growth, microcystin (MC) production and the presence of mcyB gene. M. aeruginosa could grow in the dark at 37°C up to 17days, and survive at pH 2 at a rate between 30% and 70%, depending on the age and toxicity of the starting culture. Cell lysis resulted in a substantial amounts of MC released, not degraded at gastric pH. Following the acidic passage, still in the dark at 37°C, M. aeruginosa restarted to grow within 24h for the next 3-4days, independently on the presence of intestinal bacteria, maintaining the MC cell quota and mcyB gene. Our results show new features of CB: a significant resistance of M. aeruginosa in conditions far from its optimal one, that is an environment mimicking some of the important characteristics of human gastrointestinal tract, suggesting the possibility of an internal source of exposure to CTX, with implications for the risk assessment., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Information on toxicokinetics is critical for animal-free human risk assessment. Human external exposure must be translated into human tissue doses and compared with in vitro actual cell exposure associated to effects (in vitro-in vivo comparison). Data on absorption, distribution, metabolism and excretion in humans (ADME) could be generated using in vitro and QSAR tools. Physiologically-based toxicokinetic (PBTK) computer modelling could serve to integrate disparate in vitro and in silico findings. However, there are only few freely-available PBTK platforms currently available. And although some ADME parameters can be reasonably estimated in vitro or in silico, important gaps exist. Examples include unknown or limited applicability domains and lack of (high-throughput) tools to measure penetration of barriers, partitioning between blood and tissues and metabolic clearance. This paper is based on a joint EPAA--EURL ECVAM expert meeting. It provides a state-of-the-art overview of the availability of PBTK platforms as well as the in vitro and in silico methods to parameterise basic (Tier 1) PBTK models. Five high-priority issues are presented that provide the prerequisites for wider use of non-animal based PBTK modelling for animal-free chemical risk assessment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Some recent EU Regulations have focused on the potential risks posed by the presence of endocrine disrupters (ED) into the environment. However there are conflicting opinions on how to assess the risk from exposure to these molecules that can reversibly modulate hormonal activity, endocrine active substances (EAS) rather than causing irreversible damage (ED). The present paper attempts to discuss that perturbation of normal endocrine homeostasis in itself may not be an adverse effect, since the endocrine system is naturally dynamic and responsive to various stimuli as part of its normal function and it is modulated according to the characteristic trend of the dose-response curve. EDs should be evaluated using a weight-of-evidence (WoE) approach. If a chemical meets the criteria to be defined as an ED in experimental animals, the relevance of observed effects to the human then needs to be addressed. Hazard-based risk management is therefore not justified since does not meet the criteria for a sound scientifically based assessment., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Palytoxin (PLTX) is a highly toxic hydrophilic polyether detected in several edible marine organisms from intra-tropical areas, where seafood poisoning were reported. Symptoms usually start with gastro-intestinal malaise, often accompanied by myalgia, muscular cramps, dyspnea and, sometimes, arrhythmias. Monitoring programs in the Mediterranean Sea have detected PLTX-like molecules in edible mollusks and echinoderms. Despite the potential exposure of the human population and its high toxic potential, the toxicological profile of the molecule is still an issue. Thus, the effects of repeated oral administration of PLTX in mice were investigated. Seven days of PLTX administration caused lethality and toxic effects at doses ≥ 30 μg/kg/day. A NOAEL was estimated equal to 3 μg/kg/day, indicating a quite steep dose-response curve. This value, due to the limited number of animal tested, is provisional, although represents a sound basis for further testing. Macroscopic alterations at gastrointestinal level (gastric ulcers and intestinal fluid accumulation) were observed in mice dead during the treatment period. Histological analysis highlighted severe inflammation, locally associated with necrosis, at pulmonary level, as well as hyper-eosinophilia and fiber separation in myocardium. A cardiac damage was supported by the in vitro effect of the toxin on cardiomyocytes, indicating a severe and irreversible impairment of their electrical properties: electrophysiological recordings detected a progressive cell depolarization, arrest of action potentials and beating., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Toxicokinetics (TK) is the endpoint that informs about the penetration into and fate within the body of a toxic substance, including the possible emergence of metabolites. Traditionally, the data needed to understand those phenomena have been obtained in vivo. Currently, with a drive towards non-animal testing approaches, TK has been identified as a key element to integrate the results from in silico, in vitro and already available in vivo studies. TK is needed to estimate the range of target organ doses that can be expected from realistic human external exposure scenarios. This information is crucial for determining the dose/concentration range that should be used for in vitro testing. Vice versa, TK is necessary to convert the in vitro results, generated at tissue/cell or sub-cellular level, into dose response or potency information relating to the entire target organism, i.e. the human body (in vitro-in vivo extrapolation, IVIVE). Physiologically based toxicokinetic modelling (PBTK) is currently regarded as the most adequate approach to simulate human TK and extrapolate between in vitro and in vivo contexts. The fact that PBTK models are mechanism-based which allows them to be 'generic' to a certain extent (various extrapolations possible) has been critical for their success so far. The need for high-quality in vitro and in silico data on absorption, distribution, metabolism as well as excretion (ADME) as input for PBTK models to predict human dose-response curves is currently a bottleneck for integrative risk assessment., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)