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The conclusions of the European Food Safety Authority (EFSA) following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State Austria and co-rapporteur Member State Italy for the pesticide active substance folpet and of confirmatory data following the MRL review under Article 12 of Regulation (EC) No 396/2005. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012, as amended by Commission Implementing Regulation (EU) No 2018/1659. The conclusions were reached on the basis of the evaluation of the representative uses of folpet as a fungicide on barley, wheat and wine grape (field uses) and tomato (field and greenhouse uses). The reliable end points, appropriate for use in regulatory risk assessment and the confirmatory data, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are reported where identified.

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Accurate and highly sensitive analysis of folpet and captan was accomplished using liquid chromatography-triple quadrupole linear ion trap mass spectrometry (LC-QqQIT) with selective ion mode; mass filtering, collision, and trapping condition. Dimensional mass spectrometry (MS3) parameters were optimized for the residue detection of folpet and captan in six food commodities (apples, tomatoes, sweet pepper, wheat flour, sesame seeds, and fennel seeds). The sample preparation method was based on the known QuEChERS protocol, except a mixture of acetonitrile/acetone was used for the sample extraction from the sesame seeds. The robustness and reliability of the developed MS3 method were demonstrated by performing a full validation, according to SANTE/11312/2021, at 0.01-0.25 mg/kg. Recovery ranged from 83 to 118% with a relative standard deviation below 19% in all the tested commodities, and limits of quantifications (LOQs) were 0.01 mg/kg in apples and tomatoes; 0.03 mg/kg in sweet pepper; and 0.05 mg/kg in wheat flour, sesame seeds, and fennel seeds. Monitoring results showed that about 90% of apples contained captan residue, and in sweet pepper, concentrations of captan and folpet as high as 1.57 and 0.97 mg/kg were found, respectively. The novel developed MS3 method enabled more reliable identification of these commonly problematic fungicides at lower LOQs than previously reported methods.

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Folpet, a phthalimide fungicide, is an agrochemical used to prevent fungal diseases in several crops. The toxicity of folpet has been demonstrated in Cyprinus carpio, pigs, and the human respiratory system. However, despite the possibilities of ingestion of folpet through feed, detrimental influences of folpet on dairy cattle have not been documented. Thus, this study aimed to record the harmful effects of folpet on the bovine mammary system and milk production using mammary epithelial cells (MAC-T cells), which play an essential role in the maintenance of yield and quality of milk production. In this study, we first confirmed that folpet exhibited cytotoxicity against MAC-T cells in both 2D and 3D cultures. Folpet treatment caused apoptosis, dysregulated intracellular calcium levels, and mitochondrial membrane potential, leading to cell death. We further demonstrated the induction of oxidative stress upon folpet treatment by assessing reactive oxygen species (ROS) content and lipid peroxidation in MAC-T cells. ROS generation following folpet treatment induced activation of MAPK cascades, including ERK1/2, JNK, and p38 signaling. This is the first report highlighting the detrimental impacts of folpet on bovine mammary glands and, consequently, the dairy industry by elucidating intracellular mechanisms using MAC-T cells.

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Among pesticides, fungicides are the most extensively used ones in agriculture and their effects on fish health and indirectly human health are needed to evaluate. Folpet has been used for over 50 years as a fungicide across the world. The mechanism of action of folpet on non-target aquatic organisms is so poorly understood and there is no available information about potential acute toxicity of folpet and its mechanism of action in non-target aquatic organisms. With this motivation, two successive experiments were set up: first, 96 h-LC50 value of folpet for common carp (Cyprinus carpio) was determined; and second, effects of different sublethal concentrations (0, 0.025, 0.50, 0.1 and 0.15 mg/L) of folpet on hematological serum biochemical blood parameters, DNA damages, expression levels of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), and cortisol receptor (HSP70) genes were evaluated in this study. It was calculated that 0.199 mg/L of folpet was the 96 h-LC50 value. After the folpet exposure for 14 days, significant decreases in red blood cells, hemoglobin, hematocrit, serum total protein, and GST gene expression levels were observed while serum glucose, liver enzymes activities, and expression levels of CAT, SOD, GPx, HSP70 genes increased. Also, folpet induced a significant genotoxic effect on the blood cells regarding to DNA damages. Consequently, the results have shown the toxic effects of folpet even at the lowest concentration on common carp.

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The fungicide folpet is rapidly degraded into phthalimide (PI) during both thermal processing and analytical procedures in sample preparation; thus, its residue definition has been modified into the sum of itself and PI. Tea is one of the world's most popular nonalcoholic beverages, where folpet is not listed as an applicable pesticide. To demonstrate how serious false-positives and overestimation in dietary risk are caused by the application of a new residue definition, the residue pattern of PI in made tea and processed tea leaves, along with its transfer rate during tea brewing and corresponding dietary risk, were investigated in the present study. The results revealed that PI residue in tea ranged from <10 µg/kg to 180 µg/kg with a median value of 10 µg/kg, 7.3 % of which was over the maximum residue limit established by EU (100 µg/kg, expressed as folpet). The PI residue in green tea was obviously higher than that in black, dark and oolong tea. Simulated heating experiments revealed that PI can arise from improper heating of folpet-free fresh tea leaves, and thus green tea bears a higher risk for its manufacturing employing a comparatively higher temperature. The transfer rate of PI during tea brewing was 104 ± 14 %. Nevertheless, the risk of PI through drinking tea was negligible to humans depending on the risk quotient (RQ) value, which was significantly lower than 1.

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In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Probelte SA submitted a request to the competent national authority in Spain to modify the existing maximum residue levels (MRLs) for the active substance folpet in lettuces. The data submitted in support of the request were found to be sufficient to derive an MRL proposal for lettuces. Adequate analytical methods for enforcement are available to control the residues of folpet and phthalimide in high water content commodities at the validated LOQ values of 0.05 mg/kg for folpet and 0.01 mg/kg for phthalimide. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of folpet according to the reported agricultural practice is unlikely to present a risk to consumer health.

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Biomonitoring can be relevant for assessing pesticides exposure of residents living close to vineyards (LCTV). However, because xenobiotics are generally present at low levels in human biological matrices and the sources of pesticide exposure are multiple, several challenges need to be overcome to reliably assess exposure in residents LCTV. This includes particularly identifying the most appropriate exposure biomarkers, the biological matrices in which they should be measured, and analytical methods that are sufficiently sensitive and specific to quantify them. The aim of the present study was to develop a tiered approach to identify relevant biomarkers and matrices for assessing pesticide exposure in residents LCTV. We used samples from a biobank for 121 adults and children included in a national prevalence study conducted between 2014 and 2016 who lived near or far from vineyards. We analyzed five priority pesticides (folpet, mancozeb, tebuconazole, glyphosate, and copper) and their metabolites in urine and hair samples. We identified relevant biomarkers according to three criteria related to: i) the detection frequency of those pesticides and metabolites in urine and hair, ii) the difference in concentrations depending on residence proximity to vineyards and, iii) the influence of other environmental and occupational exposure sources on pesticide levels. This tiered approach helped us to identify three relevant metabolites (two metabolites of folpet and one of tebuconazole) that were quantified in urine, tended to be higher in residents LCTV than in controls, and were not significantly influenced by occupational, dietary, or household sources of pesticide exposure. Our approach also helped us to identify the most appropriate measurement strategies (biological matrices, analytical methods) to assess pesticide exposure in residents LCTV. The approach developed here was a prerequisite step for guiding a large-scale epidemiological study aimed at comprehensively measuring pesticides exposures in French residents LCTV with a view to developing appropriate prevention strategies.

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It is well documented that under some circumstances phthalimide, a known degradation product of the fungicide folpet, can be formed as an artifact during gas chromatographic analysis. This fact explains one phthalimide source, but does not explain a great number of positive findings in the group of dried plant commodities obtained with an artifact-free analysis. Therefore, in the framework of this study, herbal and tea plants were grown in a glasshouse under the best possible protection against external environmental influences and ensuring the exclusion of the use of folpet. It was demonstrated that relevant amounts of phthalimide are formed during the drying process as part of the routine production of tea and herbals and in the absence of folpet. In this context, the presence of the widespread environmental chemical phthalic anhydride and its impact was investigated. We conclude that phthalimide is no reliable indicator for the active use of folpet.

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Folpet, a fungicide, is utilized even in cosmetics and pharmaceuticals. The LD50 of folpet in mammals, birds, and fish is relatively high. Recently, several negative effects of folpet on the respiratory system and cornea have been reported. However, there is no study on the negative effects of folpet on maternal-fetus interactions. In the present study, we used porcine trophectoderm (pTr) cells and porcine luminal epithelial (pLE) cells to investigate the toxic effects of folpet during implantation. Folpet treatment decreased cell proliferation and promoted apoptosis with cell cycle arrest. In addition, the ERK, JNK, and AKT signal pathways were activated by folpet treatment. Folpet treatment induced calcium overload in pTr and pLE cells mediating antimigratory and antiadhesive effects in both cell lines. Co-treatment with calcium chelates decreased the anti-implantation effect of folpet. Overall, our results demonstrated potential reproductive toxicity of folpet in pig.

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Folpet is a phthalimide type of fungicide and has been used to control several crop diseases. Although it has adverse effects on the gastrointestinal tract, its mechanism and toxic effects on testis have not been demonstrated. In the present study, we elucidated the cytotoxic effect of folpet on the mouse Sertoli cell line, TM4. Our results revealed that folpet suppressed viability and proliferative capacity of TM4 cells and further inhibited 3D spheroid formation. Moreover, folpet impeded appropriate cell cycle progression and induced apoptotic cell death in TM4 cells. It disrupted the electrochemical gradient of mitochondria and calcium homeostasis in TM4 cells. Furthermore, endoplasmic reticulum stress-related proteins were activated in folpet-treated TM4 cells, and relative reactive oxygen species (ROS) production was also increased. N-acetylcysteine (NAC) treatment reinstated the folpet-induced ROS generation in TM4 cells. Additionally, NAC restored the proliferative capacity and reduced the apoptotic cells in folpet-treated TM4 cells. Collectively, we demonstrated that folpet causes ROS-mediated apoptotic cell death with mitochondrial dysfunction and calcium dysregulation in TM4 cells.

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In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Adama Agriculture B.V on behalf of Adama Makhteshim Ltd submitted a request to the competent national authority in France to modify the existing maximum residue levels (MRL) for the active substance folpet in barley, oat, rye and wheat. The data submitted in support of the request were found to be sufficient to derive MRL proposals for these commodities. Adequate analytical methods for enforcement are available to control the residues of folpet and phthalimide in dry commodities at the validated limit of quantification (LOQ) of 0.01 mg/kg per analyte. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of folpet according to the reported agricultural practices is unlikely to present a risk to consumer health.

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The quantitative determination of multiple pesticide residues in food is an iterative process given the frequent changes in monitoring specifications set by regulatory authorities, introduction of new pesticide active ingredients, variety of commodities encountered and advances in the capability of analytical instrumentation and software platforms. The method described here:•replaces our previous methodology [1] that was based on an ethyl acetate extraction [2], two different sample extract clean-up regimes depending on the commodity; either Gel Permeation Chromatography (GPC) or Solid Phase Extraction (SPE) and GC/MSMS analysis using cool on-column injection and permits higher throughput using the same QuEChERS extraction method used for LCMS/MS analysis [3]•uses PTV injection incorporating a deactivated (baffled) injection liner required to improve performance for 'difficult to analyse' pesticides e.g. captan, dichlofluanid, folpet, tolylfluanid.•has been validated for the quantitative determination of 113 different pesticides and their metabolites in a range of fruit and vegetables of high water content and high acid and high water content i.e. cabbage, lemon, pepper, plum and spinach and complies with requirements of European Commission guidance document on Analytical Quality Control and Method Validation Procedures for Pesticides Residues Analysis in food and feed - SANTE/12682/2019 [4].

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Serious difficulties in evaluating the fungicides captan and folpet by the usual chromatography systems coupled to mass spectrometry are well known. These compounds are highly prone to degradation due to different conditions into tetrahydrophthalimide (THPI) and phthalimide (PHI). Such an effect can be produced at different stages of the analytical procedure or during the growing crop, making their evaluation troublesome. As a consequence, the quantification of captan and folpet is typically performed through or together these metabolites. However, imide ring metabolites can be produced by other unknown sources, including other phthalimide derived pesticides enabling false positive results. For this reason, in the last decade, laboratories demand a robust method to quantify captan and folpet, that overcomes such a situation. In the present work, various operational parameters were optimized to ensure the no degradation of captan and folpet facilitated by supercritical fluid chromatography coupled to mass spectrometry (SFC-MS/MS). A direct comparison with reverse-phase LC-MS/MS and GC-MS/MS was conducted for comparative purposes. The representative commodities selected for this evaluation were pepper and tomato. Furthermore, possible oxidative degradation during the sample milling step was also evaluated and avoided by the application of crio-milling conditions and ascorbic acid addition. By the proposed procedure, captan and folpet were recovered in both matrices at the 84%-105% range and with an RSD below 8% at two concentration levels: 10 and 50 µg/kg. On the contrary, with GC-MS/MS, captan and folpet were not recovered, and, as a consequence, their evaluation was possible only by THPI and PI. In the case of LC-MS/MS a significant decrease in the sensitivity was observed compared to SFC-MS/MS. Other validation parameters evaluated were satisfactory. This new approach can assess the correct analysis of captan and folpet at low concentrations in fruits and vegetables.

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Small intestinal (SI) tumors are relatively uncommon outcomes in rodent cancer bioassays, and limited information regarding chemical-induced SI tumorigenesis has been reported in the published literature. Herein, we propose a cytotoxicity-mediated adverse outcome pathway (AOP) for SI tumors by leveraging extensive target species- and site-specific molecular, cellular, and histological mode of action (MOA) research for three reference chemicals, the fungicides captan and folpet and the transition metal hexavalent chromium (Cr(VI)). The gut barrier functions through highly efficient homeostatic regulation of SI epithelial cell sloughing, regenerative proliferation, and repair, which involves the replacement of up to 1011 cells per day. This dynamic turnover in the SI provides a unique local environment for a cytotoxicity mediated AOP/MOA. Upon entering the duodenum, cytotoxicity to the villous epithelium is the molecular initiating event, as indicated by crypt elongation, villous atrophy/blunting, and other morphologic changes. Over time, the regenerative capacity of the gut epithelium to compensate declines as epithelial loss accelerates, especially at higher exposures. The first key event (KE), sustained regenerative crypt proliferation/hyperplasia, requires sufficient durations, likely exceeding 6 or 12 months, due to extensive repair capacity, to create more opportunities for the second KE, spontaneous mutation/transformation, ultimately leading to proximal SI tumors. Per OECD guidance, biological plausibility, essentiality, and empirical support were assessed using modified Bradford Hill considerations. The weight-of-evidence also included a lack of induced mutations in the duodenum after up to 90 days of Cr(VI) or captan exposure. The extensive evidence for this AOP, along with the knowledge that human exposures are orders of magnitude below those associated with KEs in this AOP, supports its use for regulatory applications, including hazard identification and risk assessment.

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Wastewater-based epidemiology is an innovative approach to estimate a population's intentional and unintentional consumption of chemicals based on biomarker assays found in wastewater. This method can provide real-time objective information on the xenobiotics to which a population is directly or indirectly exposed. This approach has already been used to assess the population exposure to four classes of pesticides: organochlorines (chlordecone), triazines, organophosphates, and pyrethroids. This review aims to obtain the data (excretion rates) and characteristics (pesticide and metabolites stability, including in-sewer one) for other pesticides to broaden the scope of this new method. Excretion rates and stability descriptions for 14 pesticides, namely 2,4-D, aldrin, carbaryl, chlorobenzilate, dieldrin, diquat, ethion, glufosinate, glyphosate, folpet, malathion, parathion, penconazole, and tebuconazole, will be discussed in a practical framework.

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Carcinogenesis of the small intestine is rare in humans and rodents. Oral exposure to hexavalent chromium (Cr(VI)) and the fungicides captan and folpet induce intestinal carcinogenesis in mice. Previously (Toxicol Pathol. 330:48-52), we showed that B6C3F1 mice exposed to carcinogenic concentrations of Cr(VI), captan, or folpet for 28 days exhibited similar histopathological responses including villus enterocyte cytotoxicity and regenerative crypt epithelial hyperplasia. Herein, we analyze transcriptomic responses from formalin-fixed, paraffin-embedded duodenal sections from the aforementioned study. TempO-Seq technology and the S1500+ gene set were used to analyze transcription responses. Transcriptional responses were similar between all 3 agents; gene-level comparison identified 126/546 (23%) differentially expressed genes altered in the same direction, with a total of 25 upregulated pathways. These changes were related to cellular metabolism, stress, inflammatory/immune cell response, and cell proliferation, including upregulation in hypoxia inducible factor 1 (HIF-1) and activator protein 1 (AP1) signaling pathways, which have also been shown to be related to intestinal injury and angiogenesis/carcinogenesis. The similar molecular-, cellular-, and tissue-level changes induced by these 3 carcinogens can be informative for the development of an adverse outcome pathway for intestinal cancer.

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In this study, we propose an improved analytical method for the multiresidue analysis of captan (plus its metabolite, tetrahydrophthalimide), folpet (plus its metabolite, phthalimide), captafol, and iprodione in cereals using liquid chromatography tandem mass spectrometry (LC-MS/MS). As captan, captafol, and folpet are easily degraded during homogenisation and extraction, samples were comminuted with liquid nitrogen, and both QuEChERS and ethyl acetate-based extraction workflows provided a satisfactory method performance. The optimised LC-MS/MS procedure with electrospray ionisation did not degrade these compounds, and offered sufficient method selectivity by resolving and minimising co-eluting matrix-derived interferences. The method also resolved the problem of non-specific mass spectra that these compounds usually produce on GC-MS analysis involving electron ionisation. The method performance was satisfactory for all 6 compounds at 0.01 mg kg-1 and higher levels of fortification, and validated as per the SANTE/11813/2017 guidelines of analytical quality control in a wide range of cereals including rice, wheat, sorghum, and corn. The method provides special advantage of simultaneous analysis of captan, and folpet along with their metabolites (tetrahydrophthalimide, and phthalimide, respectively) in combination with captafol, and iprodione in a single chromatographic run. Although iprodione is known to degrade to 3,5-dichloroaniline, since this metabolite is not a part of the residue definition, it was not included in the scope of this method. As the method demonstrates satisfactory selectivity, sensitivity, accuracy, precision, and robustness in a wide range of cereal matrices, it is recommended for regulatory testing of these compounds in cereals.

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An improved liquid chromatography tandem mass spectrometry method is reported for the determination of residues of captan (+tetrahydrophthalimide), captafol, folpet (+phthalimide), and iprodione in fruits and vegetables. The optimized electrospray ionization parameters (high cone gas flow, and a low desolvation temperature) did not result in degradation of target compounds, rather they provided a significant advantage over the conventional GC-MS/MS methods, which lack sensitivity and repeatability. Strategies for minimizing losses in recovery of these compounds during sample preparation included cryogenic comminution, extraction with acidified ethyl acetate or acetonitrile, and dilution of the final extract with acidified water prior to LC-MS/MS analysis. The method performance complied with the SANTE/11813/2017 guidelines, with recoveries in the range of 70-120% at the LOQ of 0.01 mg/kg across the tested matrices at various pHs. The efficiency of the method was reflected in its precision (RSDs < 10%) for incurred residues.

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Two methods based on a modified QuEChERS sample preparation and either LC coupled to atmospheric pressure ionisation and high-resolution MS or GC coupled to electron ionisation and tripled quadrupole MS have been assessed for the quantification of folpet and phthalimide in tea and other dry herbal infusions. Both methods have been fully validated in green tea and further checked in black tea, verbena and rooibos, and they performed according to the SANTE/11813/2017 criteria at the target LOQ concentration level (50 µg/kg). These methods allow the accurate quantification of folpet in the selected matrices according to the new EU residue definition, which includes phthalimide. Phthalimide is the main metabolite and degradation product of folpet, although according to recent studies, it could be generated from different sources than folpet breakdown, such as food processing or analysis by GC.

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OBJECTIVE: Introduction: It is known that pesticides have both short-term and long-term effects of the action on the human body. Today, taking into account the growth rate of the agricultural crops protection means' market and the expansion of the range of pesticide mixtures and combined formulations, there is a need for a more in-depth study of its possible effects on the environment and the human body. Recently, a new fungicide containing a mixture of two active substances, benthiavalicarb isopropyl and folpet, was introduced for application in Ukraine. Considering the possible influence of both substances on the enzyme systems involved in the xenobiotic metabolism, potentiation of its carcinogenic action in the formulation can be expected. No genotoxic effect was revealed studying in vivo studies the mutagenic activity of both substances isolated. Therefore, both substances are epigenetic carcinogens with a promoter threshold mechanism of action. In this regard, the promoter action of these substances was studied by us in the mid-term test on a multi-organ model. The aim: The purpose of our work was an experimental study of the carcinogenic action of benthiavalicarb-isopropyl and folpet - substances with ascertained carcinogenic activity, under the conditions of its simultaneous influence on the organism of laboratory animals (rats and mice). PATIENTS AND METHODS: Materials and methods: Toxicological, toxicometric (weight of animals, absolute, relative mass of internal organs) histological, microscopic, histochemical, and statistical methods were used in the study. RESULTS: Results and conclusions: No combined action of folpet and benthiavalicarb-isopropyl on the proliferation of carcinogen-transformed hepatocytes and the formation of hyperplastic nodules expressing γ-glutamyltranspeptidases (γ-GTP) as markers of pre-tumor changes in hepatocarcinogenesis was revealed. This allows us to conclude that there is no modifying effect of the folpet on carcinogenicity.

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