Perfluoroalkyl substances (PFASs) are substrates of the renal human organic anion transporter 4 (OAT4).

Poly- and perfluoroalkyl substances (PFASs) are omnipresent in the environment and have been shown to accumulate in humans. Most PFASs are not biotransformed in animals and humans, so that elimination is largely dependent on non-metabolic clearance via bile and urine. Accumulation of certain PFASs in humans may relate to their reabsorption from the pre-urine by transporter proteins in the proximal tubules of the kidney, such as URAT1 and OAT4. The present study assessed the in vitro transport of 7 PFASs (PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS and PFOS) applying URAT1- or OAT4-transfected human embryonic kidney (HEK) cells. Virtually no transport of PFASs could be measured in URAT1-transfected HEK cells. All PFASs, except PFBS, showed clear uptake in OAT4-transfected HEK cells. In addition, these in vitro results were further supported by in silico docking and molecular dynamic simulation studies assessing transporter-ligand interactions. Information on OAT4-mediated transport may provide insight into the accumulation potential of PFASs in humans, but other kinetic aspects may play a role and should also be taken into account. Quantitative information on all relevant kinetic processes should be integrated in physiologically based kinetic (PBK) models, to predict congener-specific accumulation of PFASs in humans in a more accurate manner.

OECD harmonised template 201: Structuring and reporting mechanistic information to foster the integration of new approach methodologies for hazard and risk assessment of chemicals.

In the European Union, the Chemicals Strategy for Sustainability (CSS) highlights the need to enhance the identification and assessment of substances of concern while reducing animal testing, thus fostering the development and use of New Approach Methodologies (NAMs) such as in silico, in vitro and in chemico. In the United States, the Tox21 strategy aims at shifting toxicological assessments away from traditional animal studies towards target-specific, mechanism-based and biological observations mainly obtained by using NAMs. Many other jurisdictions around the world are also increasing the use of NAMs. Hence, the provision of dedicated non-animal toxicological data and reporting formats as a basis for chemical risk assessment is necessary. Harmonising data reporting is crucial when aiming at re-using and sharing data for chemical risk assessment across jurisdictions. The OECD has developed a series of OECD Harmonised Templates (OHT), which are standard data formats designed for reporting information used for the risk assessment of chemicals relevant to their intrinsic properties, including effects on human health (e.g., toxicokinetics, skin sensitisation, repeated dose toxicity) and the environment (e.g., toxicity to test species and wildlife, biodegradation in soil, metabolism of residues in crops). The objective of this paper is to demonstrate the applicability of the OHT standard format for reporting information under various chemical risk assessment regimes, and to provide users with practical guidance on the use of OHT 201, in particular to report test results on intermediate effects and mechanistic information.

The VEGA Tool to Check the Applicability Domain Gives Greater Confidence in the Prediction of In Silico Models.

A sound assessment of in silico models and their applicability domain can support the use of new approach methodologies (NAMs) in chemical risk assessment and requires increasing the users' confidence in this approach. Several approaches have been proposed to evaluate the applicability domain of such models, but their prediction power still needs a thorough assessment. In this context, the VEGA tool capable of assessing the applicability domain of in silico models is examined for a range of toxicological endpoints. The VEGA tool evaluates chemical structures and other features related to the predicted endpoints and is efficient in measuring applicability domain, enabling the user to identify less accurate predictions. This is demonstrated with many models addressing different endpoints, towards toxicity of relevance to human health, ecotoxicological endpoints, environmental fate, physicochemical and toxicokinetic properties, for both regression models and classifiers.

Virtual Extensive Read-Across: A New Open-Access Software for Chemical Read-Across and Its Application to the Carcinogenicity Assessment of Botanicals.

Read-across applies the principle of similarity to identify the most similar substances to represent a given target substance in data-poor situations. However, differences between the target and the source substances exist. The present study aims to screen and assess the effect of the key components in a molecule which may escape the evaluation for read-across based only on the most similar substance(s) using a new open-access software: Virtual Extensive Read-Across (VERA). VERA provides a means to assess similarity between chemicals using structural alerts specific to the property, pre-defined molecular groups and structural similarity. The software finds the most similar compounds with a certain feature, e.g., structural alerts and molecular groups, and provides clusters of similar substances while comparing these similar substances within different clusters. Carcinogenicity is a complex endpoint with several mechanisms, requiring resource intensive experimental bioassays and a large number of animals; as such, the use of read-across as part of new approach methodologies would support carcinogenicity assessment. To test the VERA software, carcinogenicity was selected as the endpoint of interest for a range of botanicals. VERA correctly labelled 70% of the botanicals, indicating the most similar substances and the main features associated with carcinogenicity.

Integrated In Silico Models for the Prediction of No-Observed-(Adverse)-Effect Levels and Lowest-Observed-(Adverse)-Effect Levels in Rats for Sub-chronic Repeated-Dose Toxicity.

Repeated-dose toxicity (RDT) is a critical endpoint for hazard characterization of chemicals and is assessed to derive safe levels of exposure for human health. Here we present the first attempt to model simultaneously no-observed-(adverse)-effect level (NO(A)EL) and lowest-observed-(adverse)-effect level (LO(A)EL). Classification and regression models were derived based on rat sub-chronic repeated dose toxicity data for 327 compounds from the Fraunhofer RepDose database. Multi-category classification models were built for both NO(A)EL and LO(A)EL though a consensus of statistics- and fragment-based algorithms, while regression models were based on quantitative relationships between the endpoints and SMILES-based attributes. NO(A)EL and LO(A)EL models were integrated, and predictions were compared to exclude inconsistent values. This strategy improved the performance of single models, leading to R2 greater than 0.70, root-mean-square error (RMSE) lower than 0.60 (for regression models), and accuracy of 0.61-0.73 (for classification models) on the validation set, based on the endpoint and the threshold applied for selecting predictions. This study confirms the effectiveness of the modeling strategy presented here for assessing RDT of chemicals using in silico models.

Acetylcholinesterase inhibition in electric eel and human donor blood: an in vitro approach to investigate interspecies differences and human variability in toxicodynamics.

In chemical risk assessment, default uncertainty factors are used to account for interspecies and interindividual differences, and differences in toxicokinetics and toxicodynamics herein. However, these default factors come with little scientific support. Therefore, our aim was to develop an in vitro method, using acetylcholinesterase (AChE) inhibition as a proof of principle, to assess both interspecies and interindividual differences in toxicodynamics. Electric eel enzyme and human blood of 20 different donors (12 men/8 women) were exposed to eight different compounds (chlorpyrifos, chlorpyrifos-oxon, phosmet, phosmet-oxon, diazinon, diazinon-oxon, pirimicarb, rivastigmine) and inhibition of AChE was measured using the Ellman method. The organophosphate parent compounds, chlorpyrifos, phosmet and diazinon, did not show inhibition of AChE. All other compounds showed concentration-dependent inhibition of AChE, with IC50s in human blood ranging from 0.2-29 µM and IC20s ranging from 0.1-18 µM, indicating that AChE is inhibited at concentrations relevant to the in vivo human situation. The oxon analogues were more potent inhibitors of electric eel AChE compared to human AChE. The opposite was true for carbamates, pointing towards interspecies differences for AChE inhibition. Human interindividual variability was low and ranged from 5-25%, depending on the concentration. This study provides a reliable in vitro method for assessing human variability in AChE toxicodynamics. The data suggest that the default uncertainty factor of ~ 3.16 may overestimate human variability for this toxicity endpoint, implying that specific toxicodynamic-related adjustment factors can support quantitative in vitro to in vivo extrapolations that link kinetic and dynamic data to improve chemical risk assessment.

Investigating the interaction between melamine and cyanuric acid using a Physiologically-Based Toxicokinetic model in rainbow trout.

Following outbreaks of feed and food adulterations with a melamine and cyanuric acid mixture in 2007 and melamine in 2008 respectively, the kinetics and toxicodynamics of the mixture have been investigated particularly in sensitive species such as the rainbow trout. Tissue concentrations and intensity of the adverse effect, melamine-cyanurate crystal formation in kidney, were reported in similar experimental conditions. Here, a recent PBTK model for rainbow trout has been applied to model the kinetics of both single compounds based on residue levels in tissues. Both PBTK models for the single compounds were combined and a model of crystal formation for the mixture melamine-cyanuric acid was also added to predict the intensity of crystal formation under the assumptions that crystals formed either in urine or in kidney tissue. Modelling the kinetics of melamine and cyanuric acid provided a better understanding and prediction of intensity of crystal formation in case of sequential exposures with varying intensity or co-exposure. This study demonstrates, for the first time, how fish PBTK models can play a key role in the understanding and prediction of toxicokinetics and toxicodynamics of mixtures. This study also illustrates how adverse effects may potentially occur even when the compounds are not administered together as a mixture.

The Yin-Yang of CYP3A4: a Bayesian meta-analysis to quantify inhibition and induction of CYP3A4 metabolism in humans and refine uncertainty factors for mixture risk assessment.

Quantifying differences in pharmacokinetics (PK) and toxicokinetics (TK) provides a science-based approach to refine uncertainty factors (UFs) for chemical risk assessment. Cytochrome P450 (CYP) 3A4-the major hepatic and intestinal human CYP-and the P-glycoprotein (Pgp) transporter share a vast range of common substrates for which PK may be modulated through inhibition or induction in the presence of grapefruit juice (GFJ) or St. John's wort (SJW), respectively. Here, an extensive literature search was performed on PK interactions for CYP3A4 and Pgp substrates after oral co-exposure to GFJ and SJW. Relevant data from 109 publications, extracted for both markers of acute (Cmax) and chronic [clearance and area under the plasma concentration-time curve (AUC)] exposure, were computed into a Bayesian hierarchical meta-analysis model. Bioavailability (F) and substrate fraction metabolised by CYP3A4 (Fm) were identified as the variables exhibiting the highest impact on the magnitude of interaction. The Bayesian meta-regression model developed provided good predictions for magnitudes of inhibition (maximum 5.3-fold with GFJ) and induction (maximum 2.3-fold with SJW). Integration of CYP3A4 variability, F, Fm and magnitude of interaction provided the basis to derive a range of CYP3A4 and Pgp-related UFs. Such CYP3A4 and Pgp-related UFs can be derived in the absence of human data using in vitro TK evidence for CYP3A4/Pgp inhibition or induction as conservative in silico options. The future development of quantitative in vitro-in vivo extrapolation models for mixture risk assessment is discussed with particular attention to integrating human in vitro and in vivo P/TK data on interactions with pathway-related variability.

A Guide to the Clinical Management of Vipera Snakebite in Italy.

The genus Vipera encompasses most species of medically significant venomous snakes of Europe, with Italy harbouring four of them. Envenomation by European vipers can result in severe consequences, but underreporting and the absence of standardised clinical protocols hinder effective snakebite management. This study provides an updated, detailed set of guidelines for the management and treatment of Vipera snakebite tailored for Italian clinicians. It includes taxonomic keys for snake identification, insights into viper venom composition, and recommendations for clinical management. Emphasis is placed on quick and reliable identification of medically relevant snake species, along with appropriate first aid measures. Criteria for antivenom administration are outlined, as well as indications on managing potential side effects. While the protocol is specific to Italy, its methodology can potentially be adapted for other European countries, depending on local resources. The promotion of comprehensive data collection and collaboration among Poison Control Centres is advocated to optimise envenomation management protocols and improve the reporting of epidemiological data concerning snakebite at the country level.

Evaluation of the ecological risk of pesticide residues from the European LUCAS Soil monitoring 2018 survey.

The 2018 LUCAS (Land Use and Coverage Area frame Survey) Soil Pesticides survey provides a European Union (EU)-scale assessment of 118 pesticide residues in more than 3473 soil sites. This study responds to the policy need to develop risk-based indicators for pesticides in the environment. Two mixture risk indicators are presented for soil based, respectively, on the lowest and the median of available No Observed Effect Concentration (NOECsoil,min and NOECsoil,50) from publicly available toxicity datasets. Two further indicators were developed based on the corresponding equilibrium concentration in the aqueous phase and aquatic toxicity data, which are available as species sensitivity distributions. Pesticides were quantified in 74.5% of the sites. The mixture risk indicator based on the NOECsoil,min exceeds 1 in 14% of the sites and 0.1 in 23%. The insecticides imidacloprid and chlorpyrifos and the fungicide epoxiconazole are the largest contributors to the overall risk. At each site, one or a few substances drive mixture risk. Modes of actions most likely associated with mixture effects include modulation of acetylcholine metabolism (neonicotinoids and organophosphate substances) and sterol biosynthesis inhibition (triazole fungicides). Several pesticides driving the risk have been phased out since 2018. Following LUCAS surveys will determine the effectiveness of substance-specific risk management and the overall progress toward risk reduction targets established by EU and UN policies. Newly generated data and knowledge will stimulate needed future research on pesticides, soil health, and biodiversity protection. Integr Environ Assess Manag 2024;00:1-15. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Recent advances in the risk assessment of melamine and cyanuric acid in animal feed.

Melamine can be present at low levels in food and feed mostly from its legal use as a food contact material in laminates and plastics, as a trace contaminant in nitrogen supplements used in animal feeds, and as a metabolite of the pesticide cyromazine. The mechanism of toxicity of melamine involves dose-dependent formation of crystals with either endogenous uric acid or a structural analogue of melamine, cyanuric acid, in renal tubules resulting in potential acute kidney failure. Co-exposure to melamine and cyanuric acid in livestock, fish, pets and laboratory animals shows higher toxicity compared with melamine or cyanuric acid alone. Evidence for crystal formation between melamine and other structural analogs i.e. ammelide and ammeline is limited. Illegal pet food adulterations with melamine and cyanuric acid and adulteration of milk with melamine resulted in melamine-cyanuric acid crystals, kidney damage and deaths of cats and dogs and melamine-uric acid stones, hospitalisation and deaths of children in China respectively. Following these incidents, the tolerable daily intake for melamine was re-evaluated by the U.S. Food and Drug Administration, the World Health Organisation, and the Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA). This review provides an overview of toxicology, the adulteration incidents and risk assessments for melamine and its structural analogues. Particular focus is given to the recent EFSA risk assessment addressing impacts on animal and human health of background levels of melamine and structural analogues in animal feed. Recent research and future directions are discussed.

Nitrite in feed: from animal health to human health.

Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance. Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies differences in sensitivity to nitrite intoxication principally result from physiological and anatomical differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case histories reported in the literature no-observed-effect levels of 3.3mg/kg body weight (b.w.) per day for nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for pigs and cattle, respectively. Recognising that the bulkiness of animal feed limits their consumption, these margins in conjunction with good agricultural practise were considered satisfactory for the protection of livestock health. A human health risk assessment was also carried out taking into account all direct and indirect sources of nitrite from the human diet, including carry-over of nitrite in animal-based products such as milk, eggs and meat products. Human exposure was then compared with the acceptable daily intake (ADI) for nitrite of 0-0.07 mg/kg b.w. per day. Overall, the low levels of nitrite in fresh animal products represented only 2.9% of the total daily dietary exposure and thus were not considered to raise concerns for human health. It is concluded that the potential health risk to animals from the consumption of feed or to man from eating fresh animal products containing nitrite, is very low.

An in silico insight on the mechanistic aspects of gelsenicine toxicity: A reverse screening study pointing to the possible involvement of acetylcholine binding receptor.

Gelsedine-type alkaloids are highly toxic plant secondary metabolites produced by shrubs belonging to the Gelsemium genus. Gelsenicine is one of the most concerning gelsedine-type alkaloids with a lethal dose lower than 1 mg/Kg in mice. Several reported episodes of poisoning in livestock and fatality cases in humans due to the usage of Gelsemium plants extracts were reported. Also, gelsedine-type alkaloids were found in honey constituting a potential food safety issue. However, their toxicological understanding is scarce and the molecular mechanism underpinning their toxicity needs further investigations. In this context, an in silico approach based on reverse screening, docking and molecular dynamics successfully identified a possible gelsenicine biological target shedding light on its toxicodynamics. In line with the available crystallographic data, it emerged gelsenicine could target the acetylcholine binding protein possibly acting as a partial agonist against α7 nicotinic acetylcholine receptor (AChR). Overall, these results agreed with evidence previously reported and prioritized AChR for further dedicated analysis.

A Computational Inter-Species Study on Safrole Phase I Metabolism-Dependent Bioactivation: A Mechanistic Insight into the Study of Possible Differences among Species.

Safrole, a 162.2 Da natural compound belonging to the alkenylbenzenes class, is classified as a possible carcinogen to humans by IARC (group IIB) and has proven to be genotoxic and carcinogenic to rodents. Despite its use as a food or feed additive, it is forbidden in many countries due to its documented toxicity; yet, it is still broadly present within food and feed and is particularly abundant in spices, herbs and essential oils. Specifically, safrole may exert its toxicity upon bioactivation to its proximate carcinogen 1'-hydroxy-safrole via specific members of the cytochrome P450 protein family with a certain inter/intra-species variability. To investigate this variability, an in-silico workflow based on molecular modelling, docking and molecular dynamics has been successfully applied. This work highlighted the mechanistic basis underpinning differences among humans, cats, chickens, goats, sheep, dogs, mice, pigs, rats and rabbits. The chosen metric to estimate the likeliness of formation of 1'-hydroxy-safrole by the species-specific cytochrome P450 under investigation allowed for the provision of a knowledge-based ground to rationally design and prioritise further experiments and deepen the current understanding of alkenylbenzenes bioactivation and CYPs mechanics. Both are crucial for a more informed framework of analysis for safrole toxicity.

A computational study on the biotransformation of alkenylbenzenes by a selection of CYPs: Reflections on their possible bioactivation.

Alkenylbenzenes are aromatic compounds found in several vegetable foods that can cause genotoxicity upon bioactivation by members of the cytochrome P450 (CYP) family, forming 1'-hydroxy metabolites. These intermediates act as proximate carcinogens and can be further converted into reactive 1'-sulfooxy metabolites, which are the ultimate carcinogens responsible for genotoxicity. Safrole, a member of this class, has been banned as a food or feed additive in many countries based on its genotoxicity and carcinogenicity. However, it can still enter the food and feed chain. There is limited information about the toxicity of other alkenylbenzenes that may be present in safrole-containing foods, such as myristicin, apiole, and dillapiole. In vitro studies showed safrole as mainly bioactivated by CYP2A6 to form its proximate carcinogen, while for myristicin this is mainly done by CYP1A1. However, it is not known whether CYP1A1 and CYP2A6 can activate apiole and dillapiole. The present study uses an in silico pipeline to investigate this knowledge gap and determine whether CYP1A1 and CYP2A6 may play a role in the bioactivation of these alkenylbenzenes. The study found that the bioactivation of apiole and dillapiole by CYP1A1 and CYP2A6 is limited, possibly indicating that these compounds may have limited toxicity, while describing a possible role of CYP1A1 in the bioactivation of safrole. The study expands the current understanding of safrole toxicity and bioactivation and helps understand the mechanisms of CYPs involved in the bioactivation of alkenylbenzenes. This information is essential for a more informed analysis of alkenylbenzenes toxicity and risk assessment.

Exploring Dimensionality Reduction Techniques for Deep Learning Driven QSAR Models of Mutagenicity.

Dimensionality reduction techniques are crucial for enabling deep learning driven quantitative structure-activity relationship (QSAR) models to navigate higher dimensional toxicological spaces, however the use of specific techniques is often arbitrary and poorly explored. Six dimensionality techniques (both linear and non-linear) were hence applied to a higher dimensionality mutagenicity dataset and compared in their ability to power a simple deep learning driven QSAR model, following grid searches for optimal hyperparameter values. It was found that comparatively simpler linear techniques, such as principal component analysis (PCA), were sufficient for enabling optimal QSAR model performances, which indicated that the original dataset was at least approximately linearly separable (in accordance with Cover's theorem). However certain non-linear techniques such as kernel PCA and autoencoders performed at closely comparable levels, while (especially in the case of autoencoders) being more widely applicable to potentially non-linearly separable datasets. Analysis of the chemical space, in terms of XLogP and molecular weight, uncovered that the vast majority of testing data occurred within the defined applicability domain, as well as that certain regions were measurably more problematic and antagonised performances. It was however indicated that certain dimensionality reduction techniques were able to facilitate uniquely beneficial navigations of the chemical space.

The Endangered Sardinian Grass Snake: Distribution Update, Bioclimatic Niche Modelling, Dorsal Pattern Characterisation, and Literature Review.

The Sardinian grass snake, Natrix helvetica cetti, is an endangered endemic snake subspecies with a restricted and highly fragmented geographic distribution. Information on its ecology and detailed geographic distribution are scarce and may negatively impact on its conservation status. Therefore, a literature review on its taxonomy, morphology, ecology, and conservation is presented here. Moreover, field records from the authors, citizen science and the existing literature provide an updated geographic distribution highlighting its presence within 13 new and 7 historic 10 × 10 km cells. Bioclimatic niche modelling was then applied to explore patterns of habitat suitability and phenotypic variation within N. h. cetti. The geographic distribution of the species was found to be positively correlated with altitude and precipitation values, whereas temperature showed a negative correlation. Taken together, these outcomes may explain the snake's presence, particularly in eastern Sardinia. In addition, analysis of distribution overlap with the competing viperine snake (N. maura) and the urodeles as possible overlooked trophic resources (Speleomantes spp. and Euproctus platycephalus) showed overlaps of 66% and 79%, respectively. Finally, geographical or bioclimatic correlations did not explain phenotypic variation patterns observed in this highly polymorphic taxon. Perspectives on future research to investigate N. h. cetti's decline and support effective conservation measures are discussed.

The use of new approach methodologies for the environmental risk assessment of food and feed chemicals.

New Approach Methodologies (NAMs) provide tools for supporting both human and environmental risk assessment (HRA and ERA). This short review provides recent insights regarding the use of NAMs in ERA of food and feed chemicals. We highlight the usefulness of tiered methods supporting weight-of-evidence approaches in relation to problem formulation (i.e., data availability, time, and resource availability). In silico models, including quantitative structure activity relationship models, support filling data gaps when no chemical property or ecotoxicological data are available, and biologically-based models (e.g., toxicokinetic-toxicodynamic models, dynamic energy models, physiologically-based models and species sensitivity distributions) are applicable in more data rich situations, including landscape-based modelling approaches. Particular attention is given to provide practical examples to apply the approaches described in real-world settings. We conclude with future perspectives, with regards to the need for addressing complex challenges such as chemical mixtures and multiple stressors in a wide range of organisms and ecosystems.

Pilot survey reveals ophidiomycosis in dice snakes Natrix tessellata from Lake Garda, Italy.

Ophidiomycosis is an emerging infectious disease caused by the fungus Ophidiomyces ophidiicola (Oo). To date, Oo presence or associated disease condition has been recorded in wild and/or captive snakes from North America, Europe, Asia and Australia, but the data is still scarce outside the Nearctic. Although Italy is a country with a high snake biodiversity in the European panorama, and animals with clinical signs compatible with Oo infection have been documented, to date no investigations have reported the disease in the wild. Therefore, a pilot survey for the Italian territory was performed in conjunction with setting up a complete diagnostic workflow including SYBR Green-based real-time PCR assay for the detection of Oo genomic and mitochondrial DNA combined with histopathology of scale clips. Oo presence was investigated in 17 wild snake specimens from four different species. Four snakes were sampled in a targeted location where the mycosis was suspected via citizen science communications (i.e. North of the Lake Garda), whereas other ophidians were collected following opportunistic sampling. Oo genomic and mitochondrial DNA were detected and sequenced from all four Lake Garda Natrix tessellata, including three juveniles with macroscopic signs such as discolouration and skin crusts. From histopathological examination of scale clips, the three young positive individuals exhibited ulceration, inflammation and intralesional hyphae consistent with Oo infection, and two of them also showed the presence of arthroconidial tufts and solitary cylindrical arthrospores, allowing "Ophidiomycosis and Oo shedder" categorisation. For the remaining snake samples, the real-time PCR tested negative for Oo. This pilot survey permitted to localise for the first time Oo infection in free-ranging ophidians from Italy. Ophidiomycosis from Lake Garda highlights the need to increase sampling efforts in this area as well as in other northern Italian lakes to assess the occurrence of the pathogen, possible risk factors of the infection, its impact on host population fitness and the disease ecology of Oo in European snakes.

Risk Assessment of Combined Exposure to Multiple Chemicals at the European Food Safety Authority: Principles, Guidance Documents, Applications and Future Challenges.

Human health and animal health risk assessment of combined exposure to multiple chemicals use the same steps as single-substance risk assessment, namely problem formulation, exposure assessment, hazard assessment and risk characterisation. The main unique feature of combined RA is the assessment of combined exposure, toxicity and risk. Recently, the Scientific Committee of the European Food Safety Authority (EFSA) published two relevant guidance documents. The first one "Harmonised methodologies for the human health, animal health and ecological risk assessment of combined exposure to multiple chemicals" provides principles and explores methodologies for all steps of risk assessment together with a reporting table. This guidance supports also the default assumption that dose addition is applied for combined toxicity of the chemicals unless evidence for response addition or interactions (antagonism or synergism) is available. The second guidance document provides an account of the scientific criteria to group chemicals in assessment groups using hazard-driven criteria and prioritisation methods, i.e., exposure-driven and risk-based approaches. This manuscript describes such principles, provides a brief description of EFSA's guidance documents, examples of applications in the human health and animal health area and concludes with a discussion on future challenges in this field.