Mycotoxins Beauvericin Research Articles

Nematicidal effect of Beauveria species and the mycotoxin beauvericin against pinewood nematode Bursaphelenchus xylophilus

Introduction and main objectiveBursaphelenchus xylophilus, commonly known as pine wood nematode (PWN), is considered one of the greatest threats to European and Asian pines. Regarding its management, most efforts have been directed toward control measures for the major vector (Monochamus spp.) and screening for genetic resistance in its hosts. However, an integrated pest management strategy which also implements pinewood nematode control is currently lacking. The aim of this study was to evaluate the nematicidal effect of two Beauveria species, a genus well known for its entomopathogenic activity.Summary methodologyFor this purpose, in vitro antagonism tests of fungi (Beauveria bassiana and B. pseudobassiana) and the mycotoxin beauvericin (C45H57N3O9) on B. xylophilus populations were conducted. Finally, the production of beauvericin in B. bassiana and B. pseudobassiana strains was quantified by high-performance liquid chromatography - mass spectrometry (HPLC-MS).Results and discussionBoth the B. bassiana and B. pseudobassiana fungal species and the mycotoxin beauvericin showed a clear nematicidal effect on B. xylophilus populations, substantially reducing their survival rate and even attaining 100% mortality in one case. HPLC-MS analysis confirmed and quantified the production of beauvericin by B. bassiana and demonstrated for the first-time beauvericin production in B. pseudobassiana.Final conclusionThese findings highlight the potential of Beauveria species and the mycotoxin beauvericin to be implemented in an integrated pest management strategy to control both nematode and vector.

Alterations in Daphnia magna exposed to enniatin B and beauvericin provide additional value as environmental indicators

Mycotoxins beauvericin (BEA) and enniatin B (ENN B) affect negatively several systems and demand more studies as the mechanisms are still unclear. The simultaneous presence of contaminants in the environment manifests consequences of exposure for both animals and flora. Daphnia magna is considered an ideal invertebrate to detect effects of toxic compounds and environmental alterations. In this study, the potential toxicity and the basic mechanism of BEA and ENN B individually and combined were studied in D. magna. Acute and delayed toxicity were evaluated, and transcript levels of genes involved in xenobiotic metabolism (mox, gst, abcb1, and abcc5), reproduction, and oxidative stress (vtg-SOD) were analyzed by qPCR. Though no acute toxicity was found, results revealed a spinning around and circular profile of swimming, a strong decrease of survival after 72 h for BEA and ENN B at 16 µM and 6.25 µM, respectively, while for BEA + ENN B [8 + 1.6] µM after 96 h. The amount of mycotoxin remaining in the media revealed that the higher the concentration assayed the higher the amount remaining in the media. Differential regulation of genes suggests that xenobiotic metabolism is affected denoting different effects on transcription for tested mycotoxins. The results provide new insights into the underlying risk assessment of BEA and ENN B not only through food for consumers but also for the environment.

Assessment of single-nucleotide variant discovery protocols in RNA-seq data from human cells exposed to mycotoxins

Food and feed contamination by nonlegislated mycotoxins beauvericin (BEA) and enniatin B (ENB) is a worldwide health concern in the present. The principal objective of this work is to assess some of the existing protocols to discover the single nucleotide variants (SNVs) in transcriptomic data obtained by RNA-seq from Jurkat cells in vitro samples individually exposed to BEA and ENB at three concentration levels (1.5, 3 and 5 µM). Moreover, previous transcriptomic results will be compared with new findings obtained using a different protocol. SNVs rs201003509 in BEA exposed cells and the rs36045790 in ENB were found in the differentially expressed genes in all doses compared to controls by means of the Genome Analysis Toolkit (GATK) Best Practices workflow. SNV-RNA-seq complementary pipeline did not show any SNV. Concerning gene expression, discrepant results were found for 1.5 µM BEA exposed cells compared with previous findings. However, 354 overlapped differentially expressed genes (DEGs) were identified in the three ENB concentrations used, with 147 matches with respect to the 245 DEGs found in the previous results. In conclusion, the two discovery SNVs protocols based on variant calling from RNA-seq used in this work displayed very different results and there were SNVs found manually not identified by any pipeline. Additionally, the new gene expression analysis reported comparable but non identical DEGs to the previous transcriptomic results obtained from these RNA-seq data.

Interaction of the Emerging Mycotoxins Beauvericin, Cyclopiazonic Acid, and Sterigmatocystin with Human Serum Albumin.

Beauvericin (BEA), cyclopiazonic acid (CPA), and sterigmatocystin (STC) are emerging mycotoxins. They appear as contaminants in food and animal feed, leading to economic losses and health risks. Human serum albumin (HSA) forms stable complexes with certain mycotoxins, including ochratoxins, alternariol, citrinin, and zearalenone. HSA binding can influence the toxicokinetics of xenobiotics, and albumin can also be considered and applied as a relatively cheap affinity protein. Therefore, we examined the potential interactions of BEA, CPA, and STC with HSA employing fluorescence spectroscopy, ultracentrifugation, ultrafiltration, and molecular modeling. Spectroscopic and ultracentrifugation studies demonstrated the formation of low-affinity BEA–HSA (Ka ≈ 103 L/mol) and moderately strong CPA–HSA and STC–HSA complexes (Ka ≈ 104 L/mol). In ultrafiltration experiments, CPA slightly displaced each site marker (warfarin, naproxen, and camptothecin) tested, while BEA and STC did not affect significantly the albumin binding of these drugs. Modeling studies suggest that CPA occupies Sudlow’s site I, while STC binds to the Heme site (FA1) on HSA. Considering the interactions of CPA with the site markers, the CPA–HSA interaction may have toxicological importance.

Mycotoxins in Maize Silage from China in 2019.

Animal feed (including forage and silage) can be contaminated with mycotoxins. Here, 200 maize silage samples from around China were collected in 2019 and analyzed for regulated mycotoxins, masked mycotoxins (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-glucoside), and emerging mycotoxins (beauvericin, enniatins, moniliformin, and alternariol). Deoxynivalenol and zearalenone were detected in 99.5% and 79.5% of the samples, respectively. Other regulated mycotoxins were detected in fewer samples. The highest deoxynivalenol and zearalenone concentrations were 3600 and 830 μg/kg, respectively. The most commonly detected masked mycotoxin was 15-acetyldeoxynivalenol, which was detected in 68.5% of the samples and had median and maximum concentrations of 61.3 and 410 μg/kg, respectively. The emerging mycotoxins beauvericin, alternariol, enniatin A, enniatin B1, and moniliformin were detected in 99.5%, 85%, 80.5%, 72.5%, and 44.5%, respectively, of the samples but at low concentrations (medians <25 μg/kg). The samples tended to contain multiple mycotoxins, e.g., the correlation coefficients for the relationships between the concentrations of beauvericin and deoxynivalenol, deoxynivalenol and zearalenone, and zearalenone and beauvericin were 1.0, 0.995, and 0.995, respectively. The results indicated that there needs to be more awareness of the presence of one or more masked and emerging mycotoxins in maize silage in China.

Emerging mycotoxins and reproductive effects in animals: A short review.

Emerging Fusarium mycotoxins beauvericin (BEA), enniatins (ENNs), and moniliformin (MON) are gaining increasing interest due to their wide presence especially in cereals and grain-based products. In vitro and in vivo studies indicate that Fusarium mycotoxins can be implicated in reproductive disorders in animals. Of these mycotoxins, BEA may affect reproductive functions, impairing the development of oocytes in pigs and sheep. Studies show dramatic inhibitory effects of BEA and ENNA on bovine granulosa cell steroidogenesis. ENNs also inhibit boar sperm motility and cause detrimental effects on embryos in mice and pigs. Although little data are reported on reproductive effects of MON, in vitro studies show inhibitory effects of MON on Chinese hamster ovary cells. The present review aims to summarize the reproductive toxicological effects of emerging Fusarium mycotoxins BEA, ENNs, and MON on embryo development, ovarian function, and testicular function of animals. In vitro and in vivo toxicological data are reported although additional studies are needed for proper risk assessment.

Identification and Production of Beauvericin by Fusarium subglutinansand F. sacchari from Sugarcane

HIGHLIGHTS Isolation two species of pathogens Fusarium from sugarcane. Identification the difference in morphological characterization between two species of Fusarium. Determination the ability of pathogens Fusarium to produce mycotoxin Beauvericin. Determination of the toxicity Beauvericin on the brine shrimp.

Mass Spectrometry-Based Metabolomics Reveals a Concurrent Action of Several Chemical Mechanisms in Arabidopsis-Fusarium oxysporum Compatible and Incompatible Interactions.

Fusarium oxysporum is a destructive root-infecting plant pathogen that causes significant yield losses in many economically important crop species. Hence, a deeper understanding of pathogen infection strategies is needed. With liquid chromatography-tandem mass spectrometry and gas chromatography-time of flight mass spectrometry platforms, we analyzed the metabolic changes in a time-course experiment with Arabidopsis accessions either resistant (Col-0) or susceptible (Ler-0) to isolates of Fusarium oxysporum forma specialis matthioli infection. We showed a concurrent effect of Fusarium-derived polyols and the mycotoxin beauvericin in the suppression of the immune response of susceptible hosts. A significant increase in oxidized glutathione in the resistant host was probably associated with effective reactive oxygen species-mediated resistance responses. Through a combination of targeted and untargeted metabolomics, we demonstrated the concurrent action of several Arabidopsis defense systems as well as the concurrent action of several virulence systems in the fungal attack of susceptible Arabidopsis.

Beauvericin and Enniatins: In Vitro Intestinal Effects.

Food and feed contamination by emerging mycotoxins beauvericin and enniatins is a worldwide health problem and a matter of great concern nowadays, and data on their toxicological behavior are still scarce. As ingestion is the major route of exposure to mycotoxins in food and feed, the gastrointestinal tract represents the first barrier encountered by these natural contaminants and the first structure that could be affected by their potential detrimental effects. In order to perform a complete and reliable toxicological evaluation, this fundamental site cannot be disregarded. Several in vitro intestinal models able to recreate the different traits of the intestinal environment have been applied to investigate the various aspects related to the intestinal toxicity of emerging mycotoxins. This review aims to depict an overall and comprehensive representation of the in vitro intestinal effects of beauvericin and enniatins in humans from a species-specific perspective. Moreover, information on the occurrence in food and feed and notions on the regulatory aspects will be provided.

The mycotoxin beauvericin impairs development, fertility and life span in the nematode Caenorhabditis elegans accompanied by increased germ cell apoptosis and lipofuscin accumulation

Beauvericin is an ubiquitous mycotoxin with relevant occurrence in food and feed. It causes a high toxicity in several cell lines, but its general mechanism of action is not fully understood and only limited in vivo studies have been performed. We used Caenorhabditis elegans as a model organism to investigate effects of beauvericin. The mycotoxin displays a moderate acute toxicity at 100 μM; at this concentration also reproductive toxicity occurred (reduction of total progeny to 32.1 %), developmental toxicity was detectable at 250 μM. However, even lower concentrations were capable to reduce stress resistance and life span of the nematode: A significant reduction was detected at 10 μM beauvericin (decrease in mean survival time of 4.3 % and reduction in life span of 12.9 %). An increase in lipofuscin fluorescence was demonstrated starting at 10 μM suggesting oxidative stress as a mechanism of beauvericin toxicity. Beauvericin (100 μM) increases the number of apoptotic germ cells comparable to the positive control UV-C (400 J/m2). Conclusion: Low concentrations of beauvericin are capable to cause adverse effects in C. elegans, which may be relevant for hazard identification of this compound.

A Novel and Potentially Multifaceted Dehydroascorbate Reductase Increasing the Antioxidant Systems is Induced by Beauvericin in Tomato

Dehydroascorbate reductases (DHARs) are important enzymes that reconvert the dehydroascorbic acid (DHA) into ascorbic acid (ASC). They are involved in the plant response to oxidative stress, such as that induced by the mycotoxin beauvericin (BEA). Tomato plants were treated with 50 µM of BEA; the main antioxidant compounds and enzymes were evaluated. DHARs were analyzed in the presence of different electron donors by native and denaturing electrophoresis as well as by western blot and mass spectrometry to identify a novel induced protein with DHAR activity. Kinetic parameters for dehydroascorbate (DHA) and glutathione (GSH) were also determined. The novel DHAR was induced after BEA treatment. It was GSH-dependent and possessed lower affinity to DHA and GSH than the classical DHARs. Interestingly, the mass spectrometry analysis of the main band appearing on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a chloroplast sedoheptulose 1,7-bisphosphatase, a key enzyme of the Calvin cycle, and a chloroplast mRNA-binding protein, suggesting that the DHA reducing capacity could be a side activity or the novel DHAR could be part of a protein complex. These results shed new light on the ascorbate-glutathione regulation network under oxidative stress and may represent a new way to increase the plant antioxidant defense system, plant nutraceutical value, and the health benefits of plant consumption.

Molecular crosstalk between the endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum – Modulation of lipoxygenase activity and beauvericin production during the interaction

Plants comprise many asymptomatic fungal endophytes with potential roles of plant protection against abiotic and biotic stresses. Endophytes communicate with their host plant, with other endophytes and with invading pathogens but their language remains largely unknown. This work aims at understanding the chemical communication and physiological interactions between the fungal endophyte Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum. Oxylipins, common means of communication, such as 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), had been shown in our earlier studies to be overproduced during dual culture between the two fungal antagonists. On the other hand, the mycotoxin beauvericin was reduced in the interaction zone. The present work addresses the mechanisms underlying these changes. Hydroperoxy oxylipins are produced by lipoxygenases and P. variabile contains two lipoxygenase genes (pvlox1 and pvlox2), whereas pvlox2, but not pvlox1, is specifically up regulated during the interaction and none of the F. oxysporum lox genes vary. Heterologous expression of pvlox2 in yeast shows that the corresponding enzyme PVLOX2 produces 13-HPODE and, therefore, is most likely at the origin of the overproduced 13-HPODE during the interaction. Compellingly, beauvericin synthase gene beas expression is induced and beauvericin amounts increase in F. oxysporum mycelium when in contact with P. variabile. 13-HPODE, however, does not affect beas gene expression. Beauvericin, indeed, inhibits P. variabile growth, which counteracts and biotransforms the mycotoxin leading to reduced amounts in the interaction zone which allows further expansion of the endophyte. In order to study the interaction between the protagonists in planta, we set up an in vitro tripartite interaction assay, including the model plant Arabidopsis thaliana. F. oxysporum rapidly kills A. thaliana plants, whereas P. variabile provides up to 85% reduction of plant death if present before pathogen attack. Future studies will shed light on the protection mechanisms and the role of oxylipins and beauvericin degradation herein with the long-term aim of using endophytes in plant protection.

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep.

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus-oocyte-complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase-mediated dUTP nick-End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short-term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long-term carry-over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late-stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3-0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.

Counteracting in Vitro Toxicity of the Ionophoric Mycotoxin Beauvericin-Synthetic Receptors to the Rescue.

Beauvericin (BEA) and enniatins are toxic ionophoric cyclodepsipeptides that mainly occur in grains. As such, their presence in food commodities poses a concern for public health. To date, despite recent European Food Safety Authority emphasis on the need for more data to evaluate long-term toxicity effects, no suitable affinity reagents are available to detect the presence of BEA and derivatives in food samples. We here report on the synthesis of a small library of artificial receptors with varying cavity sizes and different hydrophobic building blocks. Immobilization of one of the receptors on solid support resulted in a strong retention of beauvericin, thus revealing promising properties as solid-phase extraction material for sample pretreatment. Furthermore, treatment of HepG2 cells with the most promising receptor markedly reduced beauvericin-induced cytotoxicity, hinting toward the possibility of using synthetic receptors as antidotes against ionophoric toxins.

Metabolic profiling of Fusarium oxysporum f. sp. conglutinans race 2 in dual cultures with biocontrol agents Bacillus amyloliquefaciens, Pseudomonas aeruginosa, and Trichoderma harzianum

There are increasing efforts to identify biocontrol-active microbial metabolites in order to improve strategies for biocontrol of phytopathogens. In this work, Fusarium oxysporum f. sp. conglutinans was confronted with three different biocontrol agents: Trichoderma harzianum, Bacillus amyloliquefaciens, and Pseudomonas aeruginosa in dual culture bioassays. Metabolites produced during the microbial interactions were screened by a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). T. harzianum exhibited the strongest inhibition of growth of F. oxysporum resulting in overlay of the pathogen colony with its mycelium. Recorded metabolite profiles suggested a direct attack of F. oxysporum mycelium by T. harzianum and B. amyloliquefaciens by means of membrane-attacking peptaibols and a set of antimicrobial lipopeptides and siderophores, respectively. The direct mode of the biocontrol activity of T. harzianum and B. amyloliquefaciens corresponded to their ability to suppress F. oxysporum production of mycotoxin beauvericin suggesting that this ability is not specific only for Trichoderma species. In the case of P. aeruginosa, siderophores pyoverdine E/D and two rhamnolipids were produced as major bacterial metabolites; the rhamnolipid production was blocked by F. oxysporum. The results showed that this type of biocontrol activity was the least effective against F. oxysporum. The effective application of MALDI-MS profiling to the screening of nonvolatile microbial metabolites produced during the interaction of the phytopathogen and the biocontrol microorganisms was demonstrated.

Cytotoxicity against fish and mammalian cell lines and endocrine activity of the mycotoxins beauvericin, deoxynivalenol and ochratoxin-A

The toxicity of mycotoxins is well recognized in mammals, but their effects on fish have received less attention. Moreover, in the last years several studies have reported that some mycotoxins may act as endocrine disruptors. The aim of this study was to determine the cytotoxic effects and endocrine activities of three mycotoxins: beauvericin, deoxynivalenol and ochratoxin-A. Cytotoxicity in two fish hepatoma and one mammalian hepatoma cell lines was determined by the AlamarBlue, Neutral Red Uptake and CFDA-AM assays. For the assessment of androgenic, estrogenic and thyroidal agonistic/antagonistic effects three cell lines stably expressing luciferase as reporter gene under the control of hormone receptors were used. Results showed that both fish and mammalian cell lines were very sensitive to the mycotoxins tested. OTA was the least toxic mycotoxin and DON and BEA showed similar acute toxicity. None of the three mycotoxins tested presented agonistic effects at the receptors studied, but all of them showed strong antagonistic effect at the thyroid receptor. BEA showed a weak antagonistic effect at the androgen receptor and OTA produced a biphasic dose-response curve at the estrogen receptor. The data obtained in this work are of high interest for aquaculture industries and for regulators.

First report of Fusarium foetens as a mycotoxin producer

Fusarium foetens, a pathogen of Begonia plants, has been recently described as a new fungal species. This Fusarium species causes a destructive vascular wilt disease which leads to the death of the plant. Moreover, Fusarium species are known to produce a huge variety of secondary metabolites such as mycotoxins and phytotoxins. Here, we studied the toxicogenic profile of one F. foetens strain, isolated from maize, employing two methods based on the use of ultra-performance liquid chromatography coupled to mass spectrometry-ion trap-time of flight detection. The mycotoxins beauvericin and fusaric acid were detected in a pure culture of F. foetens. In addition, four fusaric acid analogs (10,11-dihidroxyfusaric acid, hydroxyfusaric acid, dehydrofusaric acid, and a hydroxylated unsaturated fusaric acid analog) were tentatively identified on the basis of their accurate mass and fragmentation patterns. Therefore, these preliminary data indicate that F. foetens isolated from maize is able to produce Fusarium mycotoxins including beauvericin and fusaric acid.

Comparative in vitro cytotoxicity of the emerging Fusarium mycotoxins beauvericin and enniatins to porcine intestinal epithelial cells

The emerging Fusarium mycotoxins beauvericin (BEA) and enniatin (ENN) A, ENN A1, ENN B and ENN B1 gain increasing interest due to their highly prevalent contamination of cereals and cereal products. After oral intake, the gastro-intestinal tract is the first possible site of interaction. In the present in vitro study, the relative cytotoxicity of these mycotoxins towards proliferating and differentiated intestinal porcine epithelial cells of the jejunum (IPEC-J2) was evaluated using flow cytometric viability analysis. IPEC-J2 cells showed the highest sensitivity to BEA and ENN A. In proliferating cells, incubation for 24h with 10 μM BEA caused complete disruption, while the viability percentage declined to 32% after 24h of incubation with 10 μM ENN A. ENN A1 and ENN B1 were less cytotoxic with 87% and 93% viable cells after 24h of incubation with 10 μM ENN A1 and B1, respectively. ENN B was the least cytotoxic since incubation at concentrations up to 100 μM resulted in 83% viable proliferating cells. The same trend was observed for differentiated cells. The limited in vitro cytotoxic effect of ENN B on intestinal cells corroborates previous in vivo findings in broiler chicken in which dietary ENN B had minimal effect on intestinal morphometry.

Impact of fungicides and weather on cyclodepsipeptide-producing Fusarium spp. and beauvericin and enniatin levels in wheat grains.

Fusarium head blight (FHB) is a well-known disease of wheat caused by a complex of Fusarium species. In this research, an extensive study on the occurrence of the emerging Fusarium cyclodepsipeptide mycotoxins beauvericin and enniatins was conducted in Belgian wheat grains harvested in 2015 and 2016. To assess the link between Fusarium species and their mycotoxin production, ultra-performance liquid chromatography-tandem mass spectrometry was used to quantify the cyclodepsipeptide mycotoxins, while quantitative polymerase chain reaction was applied to quantify the presence of Fusarium species. It was shown that enniatins were mainly associated with the presence of F. avenaceum, while beauvericin, despite its low incidence, correlated significantly with F. poae. The application of fungicides resulted in a species shift and in the occurring mycotoxins. Concerning the effect of weather conditions, it was seen that levels of enniatins were positively correlated with the rainfall in May and June, while a negative correlation was observed with rainfall in the first half of July. Our study provides new insights into the occurrence of the emerging cyclodepsipeptide mycotoxins in an agro-ecosystem in which fungicides are the main control measure against FHB. It seems that beauvericin and enniatin levels are affected by different parameters and behave differently upon application of fungicides. © 2018 Society of Chemical Industry.

In vivo toxicity and genotoxicity of beauvericin and enniatins. Combined approach to study in vivo toxicity and genotoxicity of mycotoxins beauvericin (BEA) and enniatin B (ENNB)

Beauvericin (BEA) and Enniatins (ENN) are mycotoxins produced by Fusarium fungi detected in food and feed; there are insufficient data to establish their reference values. To evaluate BEA and ENN oral toxicity, an integrated approach was applied. Among ENN, Enniatin B (ENNB) was selected as test substance. The approach is composed by: i) in vitro and acute in vivo genotoxicity tests; ii) a repeated‐dose oral toxicity study focused on genotoxic, immune, endocrine, nervous endpoints and the reproductive/developmental toxicity screening. For BEA, all the genotoxicity endpoints yielded negative results excluding Comet assay in duodenum and kidney after repeated doses. BEA immunotoxicity was observed in female mice, concentrated in number and functional activity of effector T cells in the spleen. Based on the repeated‐dose BEA study, the No Observed Adverse Effect Level (NOAEL) for female mice is 1 mg/kg b.w. per day (increased thyroid pycnotic nuclei and endometrial hyperplasia). In males, the NOAEL is 0.1 mg/kg b.w. per day (reduced colloid and altered T4 serum levels). Maternal NOAEL is 0.1 mg/kg b.w. per day (increased thymus weight), developmental NOAEL is 10 mg/kg b.w. per day. For ENNB, the results support a genotoxic effect in bone marrow and liver cells after acute treatment, but not after repeated exposure. Immunotoxic ENNB effects were observed in both genders, suggestive of a suppressive/inhibiting activity particularly evident in males. Based on the repeated‐dose ENNB study, the NOAEL for females is 0.18 mg/kg b.w. per day (histomorphometrical effects on thymus, uterus and spleen). In male mice, the NOAEL is 1.8 mg/kg b.w. per day (enterocyte vacuolization in duodenum and increased Reactive Oxygen Species and reduced Glutathione brain levels). The maternal NOAEL is 1.8 mg/kg b.w. per day (decreased white pulp area and increased red/white pulp area ratio in spleen), developmental NOAEL is 18 mg/kg b.w. per day.