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Harmicines represent hybrid compounds composed of β-carboline alkaloid harmine and cinnamic acid derivatives (CADs). In this paper we report the synthesis of amide-type harmicines and the evaluation of their biological activity. N-harmicines 5a–f and O-harmicines 6a–h were prepared by a straightforward synthetic procedure, from harmine-based amines and CADs using standard coupling conditions, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA). Amide-type harmicines exerted remarkable activity against the erythrocytic stage of P. falciparum, in low submicromolar concentrations, which was significantly more pronounced compared to their antiplasmodial activity against the hepatic stages of P. berghei. Furthermore, a cytotoxicity assay against the human liver hepatocellular carcinoma cell line (HepG2) revealed favorable selectivity indices of the most active harmicines. Molecular dynamics simulations demonstrated the binding of ligands within the ATP binding site of PfHsp90, while the calculated binding free energies confirmed higher activity of N-harmicines 5 over their O-substituted analogues 6. Amino acids predominantly affecting the binding were identified, which provided guidelines for the further derivatization of the harmine framework towards more efficient agents.

Extracts of Rheum palmatum L., Rhamnus purshiana DC., Rhamnus frangula L., and Cassia senna L. are used in traditional medicine thanks to their beneficial properties. These species contain hydroxyanthracene derivatives, considered genotoxic and possibly related to colorectal cancer development. This research aimed to study, using a micronucleus assay in vitro, the genotoxic potential of Rheum palmatum L., Rhamnus purshiana DC., Rhamnus frangula L. (bark), and Cassia senna L. (leaves and fruits) extracts. The extracts were evaluated at different concentrations: from 0 to 2000 µg/mL for Rhamnus purshiana DC, from 0 to 2500 µg/mL for Rheum palmatum L. and Rhamnus frangula L., and from 0 to 5000 µg/mL for Cassia senna L. The cytokinesis-block proliferation index was calculated to analyse if the used concentrations showed cytotoxicity. The hydroxyanthracene content varied between 0.06% and 0.23% for aloe-emodin, and between 0.07% and 0.16% for emodin and rhein. No cytotoxic effect was detected at any of these concentrations. Micronucleus analyses showed a lack of genotoxicity for all the extracts tested. These results show that Rheum palmatum L., Rhamnus purshiana DC, Rhamnus frangula L., and Cassia senna L. extracts do not induce genotoxicity since no increase in micronuclei formation in human lymphocytes in vitro was detected.

Abstract Groundwater monitoring is the highest tier in the leaching assessment of plant protection products in the EU. The European Commission requested EFSA for a review by the PPR Panel of the scientific paper of Gimsing et al. (2019) on the design and conduct of groundwater monitoring studies. The Panel concludes that this paper provides many recommendations; however, specific guidance on how to design, conduct and evaluate groundwater monitoring studies for regulatory purposes is missing. The Panel notes that there is no agreed specific protection goal (SPG) at EU level. Also, the SPG has not yet been operationalised in an agreed exposure assessment goal (ExAG). The ExAG describes which groundwater needs to be protected, where and when. Because the design and interpretation of monitoring studies depends on the ExAG, development of harmonised guidance is not yet possible. The development of an agreed ExAG must therefore be given priority. A central question in the design and interpretation of groundwater monitoring studies is that of groundwater vulnerability. Applicants must demonstrate that the selected monitoring sites represent realistic worst‐case conditions as specified in the ExAG. Guidance and models are needed to support this step. A prerequisite for the regulatory use of monitoring data is the availability of complete data on the use history of the products containing the respective active substances. Applicants must further demonstrate that monitoring wells are hydrologically connected to the fields where the active substance has been applied. Modelling in combination with (pseudo)tracer experiments would be the preferred option. The Panel concludes that well‐conducted monitoring studies provide more realistic exposure assessments and can therefore overrule results from lower tier studies. Groundwater monitoring studies involve a high workload for both regulators and applicants. Standardised procedures and monitoring networks could help to reduce this workload.

Hydroxyanthracene derivatives are widely distributed in the plant kingdom, mainly in botanicals such as the Hypericum, Rheum, Rhamnus and Aloe genera. For centuries, plants containing hydroxyanthracene derivatives have been used as herbal remedies, mainly as laxatives. The root and underground stem (rhizome) are used to make medicine, primarily for digestive complaints including constipation, diarrhoea, heartburn, stomach pain, gastrointestinal bleeding, and preparation for certain gastrointestinal diagnostic procedures. The use of hydroxyanthracene-containing botanicals has raised the attention of European Food Safety Authority (EFSA) for the potential genotoxicity activity, that in 2018 concluded “[.] and that there is a safety concern for extracts containing hydroxyanthracene derivatives although uncertainty persists”. No genotoxic activity has been reported with other constituents such as rhein, physcion and chrysophanol. In the present study, Rhubarb ethanolic extract of ground rhubarb rhizome (hydroxyanthracene total content 1.39 %) was tested in the Ames Assay in Salmonella typhimurium and Escherichia coli, up to 5000 µg/plate and up to 5000 µg/mL in human lymphocytes Micronucleus Test (OECD 471 and 487 respectively) in vitro mutagenic and genotoxic effects. Under the experimental conditions used, the rhubarb rhizome extract showed no genotoxic activity.

Abstract Development of adverse outcome pathways (AOPs) for uterine adenocarcinoma can provide a practical tool to implement the EFSA‐ECHA Guidance (2018) for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. AOPs can give indications about the strength of the relationship between an adverse outcome (intended as a human health outcome) and chemicals (pesticides but not only) affecting the pathways. In this scientific opinion, the PPR Panel explored the development of AOPs for uterine adenocarcinoma. An evidence‐based approach methodology was applied, and literature reviews were produced using a structured framework assuring transparency, objectivity, and comprehensiveness. Several AOPs were developed; these converged to a common critical node, that is increased estradiol availability in the uterus followed by estrogen receptor activation in the endometrium; therefore, a putative AOP network was considered. An uncertainty analysis and a probabilistic quantification of the weight of evidence have been carried out via expert knowledge elicitation for each set of MIEs/KEs/KERs included in individual AOPs. The collected data on the AOP network were evaluated qualitatively, whereas a quantitative uncertainty analysis for weight of the AOP network certainty has not been performed. Recommendations are provided, including exploring further the uncertainties identified in the AOPs and putative AOP network; further methodological developments for quantifying the certainty of the KERs and of the overall AOPs and AOP network; and investigating of NAMs applications in the context of some of the MIEs/KEs currently part of the putative AOP network developed.

Abstract The EFSA Panel on Plant Protection Products and their Residues was requested by the European Commission, to conclude based upon available evidence if metabolites 3‐phenoxybenzoic acid (PBA) and 3‐(4′‐hydroxyphenoxy)benzoic acid (PBA(OH)), common to several pyrethroid compounds, have genotoxic properties, if they share the (neuro)toxicity profile of their parent compounds, and if evidence allows to conclude on their health‐based guidance values. Available body of evidence consisted of studies from regulatory dossiers submissions, as well as from public literature. In addition, the data gap for short‐term toxicity profile of PBA was addressed by read‐across. Assessment revealed that PBA and PBA(OH) do not raise a concern with respect to genotoxicity. As regards general toxicity, PBA and PBA(OH) have different qualitative (no neurotoxic mechanism) and quantitative (higher NOAELs) toxicity compared to the parent pyrethroid compounds. For both metabolites, acceptable daily intake (ADI) and acute reference dose (ARfD) values were derived at 0.1 mg/kg body weight (bw) per day and 1 mg/kg bw, respectively.

The toxicity of particulate matter (PM) is strictly associated with its physical-chemical characteristics, such as size or chemical composition. While these properties depend on the origin of the particles, the study of the toxicological profile of PM from single sources has rarely been highlighted. Hence, the focus of this research was to investigate the biological effects of PM from five relevant sources of atmospheric PM: diesel exhaust particles, coke dust, pellet ashes, incinerator ashes, and brake dust. Cytotoxicity, genotoxicity, oxidative, and inflammatory response were assessed in a bronchial cell line (BEAS-2B). BEAS-2B cells were exposed to different concentrations (25, 50, 100, and 150 μg/mL medium) of particles suspended in water. The exposure lasted 24 h for all the assays performed, except for reactive oxygen species, which were evaluated after 30 min, 1 h, and 4 h of treatment. The results showed a different action of the five types of PM. All the tested samples showed a genotoxic action on BEAS-2B, even in the absence of oxidative stress induction. Pellet ashes seemed to be the only ones able to induce oxidative stress by boosting the formation of reactive oxygen species, while brake dust resulted in the most cytotoxic. In conclusion, the study elucidated the differential response of bronchial cells to PM samples generated by different sources. The comparison could be a starting point for a regulatory intervention since it highlighted the toxic potential of each type of PM tested.

Aloe ferox Mill is widely used as a traditional herbal medicine for the treatment of a broad spectrum of illnesses given its laxative, anti-inflammatory, bitter tonic, anti-oxidant, antimicrobial and anti-cancer properties.Using the in vivo alkaline comet assay in animals (OECD 489), this study investigated the potential in vivo genotoxicity of dried Aloe ferox juice at dose levels of 500, 1000, and 2000 mg/kg/day in mice. Aloe ferox showed no genotoxic activity in preparations of single cells from the colon of the treated Hsd:ICR (CD-1) male mice. No statistically significant increase in DNA migration over the negative control was observed by analysis of variance for both comet parameters, tail moment and tail intensity, apart from the positive control ethyl methanesulphonate that induced clear and statistically significant increases in DNA migration parameters over the concurrent controls. The new reported scientific evidence unequivocally demonstrates that dried Aloe ferox juice containing hydroxyanthracene derivatives does not induce DNA damage in preparations of single cells from colon in in vivo comet genotoxicity studies. This suggests that the hyperplastic changes and mucosal hyperplasia observed after long-term administration of Aloe vera non-decolourised whole leaf extract may be attributed to an epigenetic effect of the material under investigation.

Abstract Flupyradifurone is a novel butenolide insecticide, first approved as an active substance for use in plant protection products by Commission Implementing Regulation (EU) 2015/2084. Following concerns that this substance may pose high risks to humans and the environment, the French authorities, in November 2020, asked the Commission to restrict its uses under Article 69 of Regulation (EC) No 1107/2009. To support this request, competent Authorities from France cited a series of literature papers investigating its hazards and/or exposure to humans and the environment. In addition, in June 2020, the Dutch Authorities notified the Commission, under Article 56 of Regulation (EC) No 1107/2009, of new information on flupyradifurone on the wild bee species Megachile rotundata. This notification is also referred to in the French notification on flupyradifurone. Consequently, the EFSA PPR Panel was mandated to quantify the likelihood of this body of evidence constituting proof of serious risks to humans or the environment. Therefore, the EFSA PPR Panel evaluated the likelihood of these studies indicating new or higher hazards and exposure to humans and the environment compared to previous EU assessments. A stepwise methodology was designed, including: (i) the initial screening; (ii) data extraction and critical appraisal based on the principles of OHAT/NTP; (iii) weight of evidence, including consideration of the previous EU assessments; (iv) uncertainty analysis, followed, whenever relevant, by an expert knowledge elicitation process. For the human health, only one study was considered relevant for the genotoxic potential of flupyradifurone in vitro. These data did not provide sufficient information to overrule the EU assessment, as in vivo studies already addressed the genotoxic potential of flupyradifurone. Environment: All available data investigated hazards in bee species. For honey bees, the likelihood of the new data indicating higher hazards than the previous EU assessment was considered low or moderate, with some uncertainties. However, among solitary bee species – which were not addressed in the previous EU assessment – there was evidence that Megachile rotundata may be disproportionately sensitive to flupyradifurone. This sensitivity, which may partially be explained by the low bodyweight of this species, was mechanistically linked to inadequate bodily metabolisation processes.

Abstract Acetamiprid is a pesticide active substance with insecticidal action currently under the third renewal (AIR3) of the Commission implementing regulation (EU) No 844/2012. Following concerns that this substance may pose high risks to humans and the environment, the French authorities asked the Commission to restrict its uses under Article 69 of Regulation (EC) No 1107/2009. To support this request, competent Authorities from France cited a series of literature papers investigating its hazards and/or exposure to humans and the environment. Consequently, the EFSA PPR Panel was mandated to advise on the likelihood that body of evidence would constitute proof of serious risks to humans or the environment. Therefore, the EFSA PPR Panel evaluated the likelihood of these studies indicating new or higher hazards and exposure to humans and the environment compared to previous EU assessments.A stepwise methodology was designed, including: (i) the initial screening; (ii) the data extraction and critical appraisal based on the principles of OHAT/NTP; (iii) the weight of evidence, including consideration of the previous EU assessments; (iv) the uncertainty analysis, followed, whenever relevant, by an expert knowledge elicitation process. For human health, no conclusive evidence of higher hazards compared to previous assessment was found for genotoxicity, developmental toxicity, neurotoxicity including developmental neurotoxicity and immunotoxicity. However, due to the lack of adequate assessment of the current data set, the PPR Panel recommends conducting an assessment of endocrine disrupting properties for acetamiprid in line with EFSA/ECHA guidance document for the identification of endocrine disruptors. For environment, no conclusive, robust evidence of higher hazards compared to the previous assessment was found for birds, aquatic organisms, bees and soil organisms. However, the potential of high inter‐species sensitivity of birds and bees towards acetamiprid requires further consideration.

Abstract 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.

Abstract The EFSA Panel on Plant Protection Products and their Residues (PPR) has developed, as a self‐task mandate (EFSA‐Q‐2019‐00100), two adverse outcome pathway (AOP)‐informed integrated approach to testing and assessment (IATA) case studies to answer a developmental neurotoxicity (DNT) hazard identification and characterisation problem formulation that could support the regulatory decisions for the pesticide active substances deltamethrin and flufenacet. The IATA were developed to assess the applicability of the DNT in vitro testing battery (IVB), designed to explore fundamental neurodevelopmental processes, in the regulatory risk assessment of pesticides. For this purpose, an evidence‐based‐approach methodology was applied: 1) systematic literature review and critical appraisal of all the evidence i.e. human observational studies, in vivo data from rodent models and new approach methodologies (NAMs, i.e. in vitro studies including high‐throughput testing from IVB and zebrafish studies from the literature) for both case studies; 2) a quantitative uncertainty analysis of all the evidence using expert knowledge elicitation (EKE) and a probabilistic approach; 3) integration of all the evidence using the AOP conceptual framework. This stepwise approach resulted in the postulation of an evidence‐based AOP network for one of the case studies. A probabilistic quantification of the weight of evidence (WoE) using Bayesian network analysis allowed the assessment and the quantification of the uncertainty in the postulated AOP. The approach taken allowed conclusions to be drawn with an acceptable level of certainty in DNT hazard identification and characterisation of deltamethrin and that flufenacet is not a developmental neurotoxicant, supporting the relevance of the mechanistic understanding. The case studies show the applicability of the DNT‐IVB for hazard identification and characterisation and illustrate the usefulness of an AOP‐informed IATA for regulatory decision making. The overall activity led to improved interpretation of human data by providing a plausible mechanistic link to adverse outcomes, which would support their contextualisation in the risk assessment process. This Scientific Opinion allows the PPR Panel to draft several recommendations for the implementation of the AOP‐informed IATA methodology and of the DNT‐IVB in the regulatory risk assessment of pesticides.

Abstract The European Commission requested EFSA to provide scientific advice on the translocation potential by Pseudomonas chlororaphis MA342 in plants after seed treatment of cereals and peas and, if applicable, for a revision of the assessment of the risk to humans by its metabolite 2,3‐deepoxy‐2,3‐didehydro‐rhizoxin (DDR) and this based on the evidence available in the dossier for renewal of the approval. The information from other P. chlororaphis strains than MA342 was taken into account with care, because the studies available in the dossier did not confirm the identity of the strain MA342 as belonging to the species P. chlororaphis. It has been concluded that there is a potential for translocation of P. chlororaphis MA342 to edible plant parts following seed treatment till an estimated concentration up to about 105 cfu/g and some exposure can be assumed by consumption of fresh commodities. Also, production of the metabolite DDR in the plant cannot be excluded. Regarding levels of DDR in the raw agricultural commodities, exposure estimates based on the limit of quantification (LOQ) for DDR in cereals cannot be further refined while there is no information on the levels of DDR in peas in the dossier. As regards genotoxicity, DDR induced chromosomal damage; however, it was not possible to conclude whether it is through an aneugenic or clastogenic mechanism. Hence, it is not possible to draw a reliable conclusion that DDR is producing an aneugenic effect nor to determine a threshold dose for aneugenicity. Thus, it is not possible to revise the human risk assessment as regards exposure to DDR. The concerns identified in the EFSA conclusion of 2017 remain.

Abstract The Panel received a mandate from the European Commission to assess the genotoxic potential of triazine amine based on available information submitted by the applicants. Available information includes experimental genotoxicity data on triazine amine, Quantitative Structure–Activity Relationship (QSAR) analysis and read across with structurally similar compounds. Based on the overall weight of evidence, the Panel, in agreement with the cross‐cutting Working Group Genotoxicity, concluded that there is no concern for the potential of triazine amine to induce gene mutations and clastogenicity; however, the potential to induce aneugenicity was not adequately investigated. For a conclusion, an in vitro micronucleus assay performed with triazine amine would be needed.

Rhus coriaria L. (sumac) is a small plant widely diffused in the Mediterranean region. Its fruit are often consumed as a spice but are also present in traditional medicine of several countries. Recently, interest in this plant has increased and many scientific works reported its beneficial effects including antioxidant and anti-inflammatory properties. Plant extracts can be successfully used against ultraviolet rays, which are able to reach and damage the human skin; however, sumac extracts were never applied to this usage. Thus, in this study, we used a macerated ethanol extract of Rhus coriaria L. dried fruit (mERC) to demonstrate its preventive role against the damage induced by ultraviolet-A rays (UV-A) on microvascular endothelial cells (HMEC-1). In vitro effects of the extract pre-treatment and UV-A exposure were evaluated in detail. The antioxidant capacity was assessed by reactive oxygen species (ROS) formation and cellular antioxidant activity measurement. Genoprotective effects of mERC were investigated as well. Our findings indicate that the extract acts as a cell cycle inhibitor or apoptosis inducer, according to the level of damage. The present work provides new insights into the usage of Rhus coriaria extracts against skin injuries.

Our research is aimed at devising and assessing a computational approach to evaluate the affinity of endocrine active substances (EASs) and their metabolites towards the ligand binding domain (LBD) of the androgen receptor (AR) in three distantly related species: human, rat, and zebrafish. We computed the affinity for all the selected molecules following a computational approach based on molecular modelling and docking. Three different classes of molecules with well-known endocrine activity (iprodione, procymidone, vinclozolin, and a selection of their metabolites) were evaluated. Our approach was demonstrated useful as the first step of chemical safety evaluation since ligand-target interaction is a necessary condition for exerting any biological effect. Moreover, a different sensitivity concerning AR LBD was computed for the tested species (rat being the least sensitive of the three). This evidence suggests that, in order not to over-/under-estimate the risks connected with the use of a chemical entity, further in vitro and/or in vivo tests should be carried out only after an accurate evaluation of the most suitable cellular system or animal species. The introduction of in silico approaches to evaluate hazard can accelerate discovery and innovation with a lower economic effort than with a fully wet strategy.