Nanomaterials in Foods and Human Digestion: An Important Layer in the Assessment of Potential Toxic Effects.

The use of nanoparticles (e.g. titanium dioxide) in commercial food products to modify some properties, such as brightness and whiteness, increased in the last years and is nowadays widespread. Despite the inhalation of nanoparticles is already a topic of concern, the potential adverse health effects due to ingestion still present gaps of knowledge. In fact, gastrointestinal tract is the first interface between the body and the external environment and consequently could represent a target organ for compounds present in food, namely nanoparticles, that could exert toxic effects. The in vitro digestion models used to simulate the human digestion may contribute to fill these gaps. The applicability of the in vitro digestion methods is discussed concerning its potential use as a tool for addressing the toxicity of ingested nanomaterials or other food contaminants, mimicking the physiological processes.

Environmental exposures in young adults with declining kidney function in a population at risk of Mesoamerican nephropathy.

OBJECTIVES: There is an epidemic of Mesoamerican nephropathy (MeN) in Central America, where sugarcane production is prominent. Numerous causes are proposed, but to date limited evidence supports any one hypothesis. A nested case-control study using biosamples from a rural, community-based follow-up study of 350 young adults from Northwest Nicaragua at risk of MeN was conducted with the aim of characterising the associations between urinary concentrations of metals, pesticides and mycotoxins from samples collected in the first 6 months and decline in kidney function over 2 years. METHODS: Urine samples collected at baseline (pre-sugarcane harvest) and the first 6 month follow-up (post-sugarcane harvest) visit were tested. Twelve metals and metalloids (aluminium, total arsenic, cadmium, chromium, cobalt, copper, lead, manganese, mercury, selenium, silicon and strontium) were analysed by inductively coupled plasma-mass spectrometry. Twelve pesticides or their metabolites (2,4-dichlorophenoxyacetic acid, 3-phenoxybenzoic acid, 4-fluoro-3-phenoxybenzoic acid, chloro-3,3,3-trifluoro-1-propen-1-yl-2,2-dimethylcyclopropanecarboxylic acid, cis/trans 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, ethylenethiourea, glyphosate, 4-chloro-2-methylphenoxy acetic acid, 3-hydroxy-pyrimetanil, 5-hydroxytiabendazole, hydroxy-tebuconazole and 3,5,6-trichloro-2-pyridinol) and two mycotoxins (ochratoxin A (OTA) and citrinin (CIT)) were analysed by liquid chromatography coupled-mass spectrometry. Differences in the creatinine-corrected urinary concentrations of the measured exposures between outcome groups (participants with stable vs declining kidney function) were examined. RESULTS: Elevated levels of aluminium and total arsenic as well as metabolites of several pesticides were detected across the population. No differences were identified between the declining and stable groups in the levels of metals or pesticides tested. OTA and CIT were below the limit of detection. CONCLUSIONS: The tested metals, metalloids, pesticides and mycotoxins were not associated with loss of kidney function in participants at-risk of MeN.

Applicability of in vitro methods to study patulin bioaccessibility and its effects on intestinal membrane integrity.

In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, although the total amount of an ingested contaminant (external dose) does not always reflect the quantity available for the body (internal dose). In this study, two in vitro methods were applied to study bioaccessibility and intestinal membrane integrity of cells exposed to patulin, a mycotoxin with significant public health risk. Seven artificially contaminated fruit juices were assayed in the presence or absence of a standard meal, showing a significant difference for bioaccessibility values between contaminated samples alone (mean 27.65 ± 13.50%) and combinations with a standard meal (mean 7.89 ± 4.03%). Different concentrations of patulin (PAT) and cysteine (CYS) (protector agent) were assayed in Caco-2 cells monolayers. At 95 µM, PAT produced a marked decrease in transepithelial electrical resistance (TEER). This effect was significantly reduced when 400 µM and 4000 µM CYS was added to the cells. Combined use of in vitro digestion models with other techniques using intestinal cell lines, such as in vitro intestinal absorption models that use Caco-2 cells, may offer a more comprehensive model of what is occurring during digestion and absorption processes. The study of beneficial effects of protective agents would also be enhanced.

Challenges of the Application of In Vitro Digestion for Nanomaterials Safety Assessment.

Considering the increase in the production and use of nanomaterials (NM) in food/feed and food contact materials, novel strategies for efficient and sustainable hazard characterization, especially in the early stages of NM development, have been proposed. Some of these strategies encompass the utilization of in vitro simulated digestion prior to cytotoxic and genotoxic assessment. This entails exposing NM to fluids that replicate the three successive phases of digestion: oral, gastric, and intestinal. Subsequently, the resulting digestion products are added to models of intestinal cells to conduct toxicological assays, analyzing multiple endpoints. Nonetheless, exposure of intestinal cells to the digested products may induce cytotoxicity effects, thereby posing a challenge to this strategy. The aim of this work was to describe the challenges encountered with the in vitro digestion INFOGEST 2.0 protocol when using the digestion product in toxicological studies of NM, and the adjustments implemented to enable its use in subsequent in vitro biological assays with intestinal cell models. The adaptation of the digestion fluids, in particular the reduction of the final bile concentration, resulted in a reduced toxic impact of digestion products.

Maternal dietary exposure to mycotoxin aflatoxin B1 promotes intestinal immune alterations and microbiota modifications increasing infection susceptibility in mouse offspring.

Mycotoxins are secondary metabolites produced by fungi occurring in food that are toxic to animals and humans. Early-life mycotoxins exposure has been linked to diverse pathologies. However, how maternal exposure to mycotoxins impacts on the intestinal barrier function of progeny has not been explored. Here, exposure of pregnant and lactating C57Bl/6J female mice to aflatoxin B1 (AFB1; 400 µg/kg body weight/day; 3 times a week) in gelatine pellets, from embryonic day (E)11.5 until weaning (postnatal day 21), led to gut immunological changes in progeny. The results showed an overall increase of lymphocyte number in intestine, a reduction of expression of epithelial genes related to microbial defence, as well as a decrease in cytokine production by intestinal type 2 innate lymphoid cells (ILC2). While susceptibility to chemically induced colitis was not worsened, immune alterations were associated with changes in gut microbiota and with a higher vulnerability to infection by the protozoan Eimeria vermiformis at early-life. Together these results show that maternal dietary exposure to AFB1 can dampen intestinal barrier homeostasis in offspring decreasing their capability to tackle intestinal pathogens. These data provide insights to understand AFB1 potential harmfulness in early-life health in the context of intestinal infections.

Bioaccessibility data of potentially toxic elements in complementary foods for infants: A review.

The introduction of complementary foods (CFs) is a critical step in an infant's transition to solid foods, providing essential nutrients beyond breast milk. However, CFs may contain potentially toxic elements (PTEs), such as arsenic and cadmium that pose health risks to infants. In this context, understanding the bioaccessibility of PTEs is vital as it determines the fraction of a contaminant released from the food matrix and available for absorption in the gastrointestinal tract. Efforts have been made to standardize the assessment methodology for bioaccessibility, ensuring consistent and reliable data. Moreover, regulatory agencies have established guidelines for PTEs levels in food. However, important gaps still exist, which motivates many research opportunities on this topic.

A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

Harmonized human biomonitoring in European children, teenagers and adults: EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014-2021).

As one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants of three age groups: (i) 3,576 children aged 6-12 years, (ii) 3,117 teenagers aged 12-18 years and (iii) 4,102 young adults aged 20-39 years. The participants were recruited between 2014 and 2021 in 11-12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Σ (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability and will give leverage to national policy makers for the implementation of targeted measures.

How to use human biomonitoring in chemical risk assessment: Methodological aspects, recommendations, and lessons learned from HBM4EU.

One of the aims of the European Human Biomonitoring Initiative, HBM4EU, was to provide examples of and good practices for the effective use of human biomonitoring (HBM) data in human health risk assessment (RA). The need for such information is pressing, as previous research has indicated that regulatory risk assessors generally lack knowledge and experience of the use of HBM data in RA. By recognising this gap in expertise, as well as the added value of incorporating HBM data into RA, this paper aims to support the integration of HBM into regulatory RA. Based on the work of the HBM4EU, we provide examples of different approaches to including HBM in RA and in estimations of the environmental burden of disease (EBoD), the benefits and pitfalls involved, information on the important methodological aspects to consider, and recommendations on how to overcome obstacles. The examples are derived from RAs or EBoD estimations made under the HBM4EU for the following HBM4EU priority substances: acrylamide, o-toluidine of the aniline family, aprotic solvents, arsenic, bisphenols, cadmium, diisocyanates, flame retardants, hexavalent chromium [Cr(VI)], lead, mercury, mixture of per-/poly-fluorinated compounds, mixture of pesticides, mixture of phthalates, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), and the UV-filter benzophenone-3. Although the RA and EBoD work presented here is not intended to have direct regulatory implications, the results can be useful for raising awareness of possibly needed policy actions, as newly generated HBM data from HBM4EU on the current exposure of the EU population has been used in many RAs and EBoD estimations.

Mycotoxin Exposure during the First 1000 Days of Life and Its Impact on Children's Health: A Clinical Overview.

The first 1000 days of life are very sensitive to any event that alters health programming, and they represent a window for intervention to improve population health. Pregnant women, fetuses, and infants are particularly vulnerable to exposure to food contaminated with mycotoxins. This review aimed to gather data from the literature on mycotoxins exposure during intrauterine life and early childhood, and associated health risks, as assessed through human biomonitoring and mycotoxins occurrence in foods, in different continents. Maternal internal exposure to aflatoxins is associated with fetal growth restriction, while exposure to fumonisins increases the risk of offspring's neural tube defects. Mycotoxin contamination of breast milk is reported worldwide, but data on adverse effects of the lactational transfer of mycotoxins on infant health are lacking. Young children are exposed to mycotoxins through contaminated infant formulas and baby foods. Both external and internal exposure to aflatoxins and fumonisins in children are reported to be associated with growth impairment. In low-income settings, where other co-factors can affect growth, this association should be interpreted with caution. Further studies on human biomonitoring of mother-infant pairs and young children are needed to guide management strategies aiming to minimize mycotoxin exposure at critical developmental stages.

earlyMYCO: A Pilot Mother-Child Cohort Study to Assess Early-Life Exposure to Mycotoxins-Challenges and Lessons Learned.

Early-life exposure occurs during gestation through transfer to the fetus and later, during lactation. Recent monitoring data revealed that the Portuguese population is exposed to mycotoxins, including young children. This study aimed to develop a pilot study to assess the early-life exposure to mycotoxins through a mother-child cohort, and to identify the associated challenges. Participants were recruited during pregnancy (1st trimester) and followed-up in three moments of observation: 2nd trimester of pregnancy (mother), and 1st and 6th month of the child's life (mother and child), with the collection of biological samples and sociodemographic and food consumption data. The earlyMYCO pilot study enrolled 19 mother-child pairs. The analysis of biological samples from participants revealed the presence of 4 out of 15 and 5 out of 18 mycotoxins' biomarkers of exposure in urine and breast milk samples, respectively. The main aspects identified as contributors for the successful development of the cohort were the multidisciplinary and dedicated team members in healthcare units, reduced burden of participation, and the availability of healthcare units for the implementation of the fieldwork. Challenges faced, lessons learned, and suggestions were discussed as a contribution for the development of further studies in this area.

Adverse Outcome Pathways Associated with the Ingestion of Titanium Dioxide Nanoparticles-A Systematic Review.

Titanium dioxide nanoparticles (TiO2-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO2-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO2-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO2-NPs in food, announced by the European Food Safety Authority (EFSA).

Current Advances, Research Needs and Gaps in Mycotoxins Biomonitoring under the HBM4EU-Lessons Learned and Future Trends.

Mycotoxins are natural metabolites produced by fungi that contaminate food and feed worldwide. They can pose a threat to human and animal health, mainly causing chronic effects, e.g., immunotoxic and carcinogenic. Due to climate change, an increase in European population exposure to mycotoxins is expected to occur, raising public health concerns. This urges us to assess the current human exposure to mycotoxins in Europe to allow monitoring exposure and prevent future health impacts. The mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1) were considered as priority substances to be studied within the European Human Biomonitoring Initiative (HBM4EU) to generate knowledge on internal exposure and their potential health impacts. Several policy questions were addressed concerning hazard characterization, exposure and risk assessment. The present article presents the current advances attained under the HBM4EU, research needs and gaps. Overall, the knowledge on the European population risk from exposure to DON was improved by using new harmonised data and a newly derived reference value. In addition, mechanistic information on FB1 was, for the first time, organized into an adverse outcome pathway for a congenital anomaly. It is expected that this knowledge will support policy making and contribute to driving new Human Biomonitoring (HBM) studies on mycotoxin exposure in Europe.

The Interaction between Tribolium castaneum and Mycotoxigenic Aspergillus flavus in Maize Flour.

Tribolium castaneum is one of the most common insect pests of stored products. Its presence makes cereals more susceptible to the spread of the fungi Aspergillus flavus, which may produce mycotoxins. The aim of this work was to evaluate the influence of T. castaneum adults on the development of a mycotoxigenic A. flavus strain in maize flour as well as the influence of this fungus on the insects. Maize flour was exposed to T. castaneum, spores of A. flavus or to both. The results revealed an interaction between T. castaneum and A. flavus as the flour exposed to both organisms was totally colonized by the fungus whereas almost all the insects were killed. Aflatoxin B1 (AFB1) revealed a significantly higher concentration in the flour inoculated with both organisms (18.8 µg/kg), being lower when exposed only to A. flavus, suggesting that the presence of insects may trigger fungal development and enhance mycotoxin production. The ability of these organisms to thrive under the same conditions and the chemical compounds they release makes the interaction between them a subject of great importance to maintain the safety of stored maize. This is the first work evaluating the interaction between T. castaneum and A. flavus mycotoxin production.

Deoxynivalenol exposure assessment through a modelling approach of food intake and biomonitoring data - A contribution to the risk assessment of an enteropathogenic mycotoxin.

Deoxynivalenol (DON), an enteropathogenic mycotoxin produced by Fusarium species, is usually associated with adverse health outcomes such as gastrointestinal diseases and immunotoxicity. To estimate DON exposure of the Portuguese population at national level, a modelling approach, based on data from 94 Portuguese volunteers, was developed considering the inputs of the food consumption data generated within the National Food and Physical Activity Survey and the human biomonitoring data used to assess the exposure to DON. Ten models of association between DON urinary biomarkers and food items (pasta, cookies, biscuits, sweets, bread, rusks, nuts, oilseeds, beer, meat, milk) were established. Applying the most adequate model to the consumption data (n = 5811) of the general population, the exposure estimates of the Probable Daily Intake revealed that a fraction (0.1%) of the Portuguese population might exceed the Tolerable Daily Intake defined for DON. The analysis stratified by age revealed children (3.2%) and adolescents (6.0%) are more likely to exceed the Tolerable Daily Intake for DON. Although the unavoidable uncertainties, these results are important contributions to understand the exposure to this mycotoxin in Portugal, to assess the associated risk and the potential public health consequences.

Risk-Benefit Assessment of Cereal-Based Foods Consumed by Portuguese Children Aged 6 to 36 Months-A Case Study under the RiskBenefit4EU Project.

Cereal-based foods, including breakfast (BC) and infant cereals (IC), are among the first solid foods introduced to infants. BC and IC are sources of macro and micronutrients that have beneficial effects on health, but can also be sources of harmful chemical and microbiological contaminants and nutrients that may lead to adverse health effects at high consumption levels. This study was performed under the RiskBenefit4EU project with the aim of assessing the health impact associated with consumption of BC and IC by Portuguese children under 35 months. Adverse effects associated with the presence of aflatoxins, Bacillus cereus, sodium and free sugars were assessed against the benefits of fiber intake. We applied a risk-benefit assessment approach, and quantified the health impact of changes in consumption of BC and IC from current to various alternative consumption scenarios. Health impact was assessed in terms of disability-adjusted life years. Results showed that moving from the current consumption scenario to considered alternative scenarios results in a gain of healthy life years. Portuguese children can benefit from exchanging intake of IC to BC, if the BC consumed has an adequate nutritional profile in terms of fiber, sodium and free sugars, with levels of aflatoxins reduced as much as possible.

Analysis of the Characteristics and Cytotoxicity of Titanium Dioxide Nanomaterials Following Simulated In Vitro Digestion.

Several metallic nanomaterials (NMs), such as titanium dioxide nanomaterials (TiO2), present beneficial properties with a broad range of innovative applications. The human population is exposed to TiO2, particularly by ingestion, due to its increasing use as a food additive and inclusion in dietary supplements and food packaging materials. Whether this oral exposure may lead to adverse local or systemic outcomes has been the subject of research, but studies have generated contradictory results, reflecting differences in the physicochemical properties of the TiO2 studied, effects of the surrounding matrix, and modifications during digestion. This work aimed to investigate the toxic effects of three different TiO2 NMs (NM-103, NM-103 and NM-105) on the gastrointestinal tract cells, Caco-2 and HT29-MTX-E12, after the use of the standardized static INFOGEST 2.0 in vitro digestion method to mimic human digestion of TiO2, contributing to hazard assessment. The results show that, for one of the digested TiO2 NMs studied (NM-105), a more pronounced toxicity occurs after exposure of HT29-MTX-E12 intestinal cells, as compared to undigested NM, concomitantly with subtle changes in characteristics of the NM. Thus, the inclusion of the digestion simulation in the safety evaluation of ingested NMs through in vitro bioassays can better integrate the modifications that NMs suffer in the organism. It is expected that such an approach will reduce uncertainties in the hazard assessment of ingested NMs for human health.

Food Consumption Data as a Tool to Estimate Exposure to Mycoestrogens.

Zearalenone and alternariol are mycotoxins produced by Fusarium and Alternaria species, respectively, that present estrogenic activity and consequently are classified as endocrine disruptors. To estimate the exposure of the Portuguese population to these two mycotoxins at a national level, a modelling approach, based on data from 94 Portuguese volunteers, was developed considering as inputs: i) the food consumption data generated within the National Food and Physical Activity Survey; and ii) the human biomonitoring data used to assess the exposure to the referred mycotoxins. Six models of association between mycoestrogens urinary levels (zearalenone, total zearalenone and alternariol) and food items (meat, cheese, and fresh-cheese, breakfast cereals, sweets) were established. Applying the obtained models to the consumption data (n = 5811) of the general population, the median estimates of the probable daily intake revealed that a fraction of the Portuguese population might exceed the tolerable daily intake defined for zearalenone. A reference intake value for alternariol is still lacking, thus the characterization of risk due to the exposure to this mycotoxin was not possible to perform. Although the unavoidable uncertainties, these results are important contributions to understand the exposure to endocrine disruptors in Portugal and the potential Public Health consequences.

A multi-endpoint approach to the combined toxic effects of patulin and ochratoxin a in human intestinal cells.

Humans can be exposed to a complex and variable combination of mycotoxins. After ingestion, intestinal mucosa constitutes the first biological barrier that can be exposed to high concentrations of these toxins. The present study aimed to characterize the combined cytotoxicity, genotoxicity and impact on the gastrointestinal barrier integrity of patulin (PAT, 0.7 µM to 100 µM) and ochratoxin A (OTA, 1 µM to 200 µM) mixtures in Caco-2 cells. A dose-ratio deviation was verified for cytotoxicity, implying that OTA was mainly responsible for synergism when dominant in the mixture, while this pattern was changed to antagonism for the highest PAT concentrations. Genotoxicity (comet assay) results were compatible with an interactive DNA damaging effect at the highest PAT and OTA concentrations, not clearly mediated by the formation of oxidative DNA breaks. Regarding gastrointestinal barrier integrity, a potential synergism was attained at low levels of both mycotoxins, changing to antagonism at higher doses. The present results indicate that combined mycotoxins effects may arise at the intestinal level and should not be underestimated when evaluating their risk to human health.

Assessment of mycotoxin exposure and risk characterization using occurrence data in foods and urinary biomarkers in Brazil.

This study aimed to assess the exposure of Brazilian residents (N = 86) from rural areas to multiple mycotoxins and characterize the associated risk in two sampling periods (SP) (April-May and December/2016). Mycotoxins in food and urine samples were determined by liquid chromatography coupled to tandem mass spectrometry. Mean probable daily intake (PDI) values based on occurrence data in foods in both SP varied from 0.007 to 0.013, 0.069 to 1.002, 0.119 to 0.321 and 0.013-0.156 µg kg-1 body weight (bw) day-1 for aflatoxins (AFs), deoxynivalenol (DON), fumonisins (FBs) and zearalenone (ZEN), respectively. Mean PDI values based on urinary biomarkers were 0.001, 84.914, 0.031, 0.377 and 0.002 µg kg-1 bw day-1 for AFB1, DON, ochratoxin A (OTA), FB1 and ZEN, respectively. Hazard quotient (HQ) calculated using food data revealed a potential health concern for ZEN in 2nd SP. HQ > 1 based on urinary biomarkers were observed for DON in the two SP. Although OTA was not detected in any food sample, the HQ based on urinary OTA levels was >1 in the 1st SP. Margin of exposure values for AF from food and urine data in the 1st SP were below 10,000, indicating potential health risks.