A prioritization strategy for functional alternatives to bisphenol A in food contact materials.

The use of bisphenol A (BPA), a substance of very high concern, is proposed to be banned in food contact materials (FCMs) in the European Union. To prevent regrettable substitution of BPA by alternatives with similar or unknown hazardous properties, it is of importance to gain the relevant toxicological information on potential BPA alternative substances and monitor them adequately. We created an inventory of over 300 substances mentioned as potential BPA alternatives in regulatory reports and scientific literature. This study presents a prioritization strategy to identify substances that may be used as an alternative to BPA in FCMs. We prioritized 20 potential BPA alternatives of which 10 are less familiar. We subsequently reviewed the available information on the 10 prioritized less familiar substances regarding hazard profiles and migration potential obtained from scientific literature and in silico screening tools to identify a possible risk of the substances. Major data gaps regarding the hazard profiles of the prioritized substances exist, although the scarce available data give some indications on the possible hazard for some of the substances (like bisphenol TMC, 4,4-dihydroxybenzophenone, and tetrachlorobisphenol A). In addition, very little is known about the actual use and exposure to these substances. More toxicological research and monitoring of these substances in FCMs are, therefore, required to avoid regrettable substitution of BPA in FCM.

Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Human Biomonitoring Guidance Values for Deoxynivalenol Derived under the European Human Biomonitoring Initiative (HBM4EU).

The mycotoxin deoxynivalenol (DON) was one of the priority substances in the European Joint Human Biomonitoring Initiative (HBM4EU) project. In this study, to better interpret the actual internal exposure of DON in the general population and safeguard public health, human biomonitoring guidance values of DON for the general population (HBM-GVGenPop) were derived. The HBM-GVGenPop of DON was based on either the total DON (DON and its glucuronides) or DON's main metabolite (DON-15-GlcA) levels in 24-h urine samples, resulting in a HBM-GVGenPop of 0.023 µg/mL for the total DON or a HBM-GVGenPop of 0.020 µg/mL for DON-15-GlcA. The use of 24-h urine samples is recommended based on the fact that DON and its metabolites have a short elimination half-life (T1/2), and 95% of the cumulative amount was excreted within 12 h after DON intake. The T1/2 for DON, DON-15-GlcA, and total DON were estimated to be 2.55 h, 2.95 h, and 2.95 h, respectively. Therefore, a 24-h urine sample reflects almost all of the DON exposure from the previous day, and this type of sample was considered for the derivation of a HBM-GVGenPop for DON.

Assessment of Mycotoxin Exposure and Associated Risk in Pregnant Dutch Women: The Human Biomonitoring Approach.

Mycotoxins are toxic secondary metabolites produced by various fungi that can contaminate food crops, which, in turn, may lead to human exposure. Chronic exposure to mycotoxins can cause adverse health effects including reproductive and developmental toxicity. Pregnant women and their foetuses present a vulnerable group for exposure to mycotoxins that can cross the placenta. Human biomonitoring of mycotoxins provides a real-life approach to estimate internal exposure. In this pilot study, 24-h urine samples from 36 pregnant Dutch women were analysed for aflatoxin M1 (AFM1), total deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), total zearalenone (ZEN), total α-zearalenol (α-ZEL), total ß-zearalenol (ß-ZEL) and total zearalanone (ZAN), where 'total' refers to mycotoxins and their conjugated forms. Serum samples from these women were analysed for fumonisin B1 (FB1) and ochratoxin A (OTA). All samples were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most prevalent mycotoxins were total DON, total ZEN and OTA, with a detection frequency of 100%. DOM-1, total α-ZEL and total ß-ZEL were detected but to a lesser extent, while AFM1, total ZAN and FB1 were undetected. Median concentrations were 4.75 µg total DON/L, 0.0350 µg DOM-1/L, 0.0413 µg total ZEN/L, 0.0379 µg total α-ZEL/L, 0.0189 µg total ß-ZEL/L, and 0.121 µg OTA/L. The calculated median concentration for total ZEN and its metabolites was 0.105 µg/L. Based on two separate risk assessment approaches, total DON exposure in this group was considered to be of low concern. Similarly, exposure to total ZEN and its metabolites in this group was of low concern. For OTA, the risk of non-neoplastic effects was of low concern based on exposure in this group, and the risk of neoplastic effects was of low concern in the majority of participants in this group. The findings of this pilot study confirm the presence of mycotoxins in the urine and serum of pregnant Dutch women, with total DON, total ZEN, and OTA most frequently detected. Exposure to all measured mycotoxins was considered to be of low concern in this group, except for exposure to OTA, which was of low concern for the majority of participants. The study's findings offer valuable insights but should be confirmed using a larger and more diverse sample of the Dutch general population.

Development of a Generic PBK Model for Human Biomonitoring with an Application to Deoxynivalenol.

Toxicokinetic modelling provides a powerful tool in relating internal human exposure (i.e., assessed through urinary biomarker levels) to external exposure. Chemical specific toxicokinetic models are available; however, this specificity prevents their application to similar contaminants or to other routes of exposure. For this reason, we investigated whether a generic physiological-based kinetic (PBK) model might be a suitable alternative for a biokinetic model of deoxynivalenol (DON). IndusChemFate (ICF) was selected as a generic PBK model, which could be fit for purpose. Being suited for simulating multiple routes of exposure, ICF has particularly been used to relate the inhalation and dermal exposure of industrial chemicals to their urinary excretion. For the first time, the ICF model was adapted as a generic model for the human biomonitoring of mycotoxins, thereby extending its applicability domain. For this purpose, chemical-specific data for DON and its metabolites were collected directly from the literature (distribution and metabolism) or indirectly (absorption and excretion) by fitting the ICF model to previously described urinary excretion data. The obtained results indicate that this generic model can be used to model the urinary excretion of DON and its glucuronidated metabolites following dietary exposure to DON. Additionally, the present study establishes the basis for further development of the model to include an inhalation exposure route alongside the oral exposure route.

Combined chronic dietary exposure to four nephrotoxic metals exceeds tolerable intake levels in the adult population of 10 European countries.

A mixture risk assessment (MRA) for four metals relevant to chronic kidney disease (CKD) was performed. Dietary exposure to cadmium or lead alone exceeded the respective reference values in the majority of the 10 European countries included in our study. When the dietary exposure to those metals and inorganic mercury and inorganic arsenic was combined following a classical or personalised modified reference point index (mRPI) approach, not only high exposure (95th percentile) estimates but also the mean exceeded the tolerable intake of the mixture in all countries studied. Cadmium and lead contributed most to the combined exposure, followed by inorganic arsenic and inorganic mercury. The use of conversion factors for inorganic arsenic and inorganic mercury from total arsenic and total mercury concentration data was a source of uncertainty. Other uncertainties were related to the use of different principles to derive reference points. Yet, MRA at the target organ level, as performed in our study, could be used as a way to efficiently prioritise assessment groups for higher-tier MRA. Since the combined exposure to the four metals exceeded the tolerable intake, we recommend a refined MRA based on a common, specific nephrotoxic effect and relative potency factors (RPFs) based on a similar effect size.

Combined Exposure to Multiple Mycotoxins: An Example of Using a Tiered Approach in a Mixture Risk Assessment.

Humans are exposed to mycotoxins on a regular basis. Exposure to a mixture of mycotoxins may, therefore, result in a combination of adverse effects, or trigger the same effects. This should be accounted for when assessing the combined risk of multiple mycotoxins. Here, we show the outcome of using different approaches in assessing the risks related to the combined exposure to mycotoxins. We performed a tiered approach using assessment groups with a common target organ (kidney, liver and haematologic system), or a common adverse effect (phenomenon) (reduced white blood cell count), to combine the exposure to mycotoxins. The combined exposure was calculated for the individuals in this assessment, using the Monte Carlo Risk Assessment (MCRA) tool. The risk related to this combined exposure was assessed using toxicological reference values, e.g., health based guidance values. We show that estimating the combined risk by adding the single compounds' risk distributions slightly overestimates the combined risk in the 95th percentile, as compared to combining the exposures at an individual level. We also show that relative potency factors can be used to refine the mixture risk assessment, as compared to ratios of toxicological reference values with different effect sizes and assessment factors.

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.

Providing Biological Plausibility for Exposure-Health Relationships for the Mycotoxins Deoxynivalenol (DON) and Fumonisin B1 (FB1) in Humans Using the AOP Framework.

Humans are chronically exposed to the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), as indicated by their widespread presence in foods and occasional exposure in the workplace. This exposure is confirmed by human biomonitoring (HBM) studies on (metabolites of) these mycotoxins in human matrices. We evaluated the exposure-health relationship of the mycotoxins in humans by reviewing the available literature. Since human studies did not allow the identification of unequivocal chronic health effects upon exposure to DON and FB1, the adverse outcome pathway (AOP) framework was used to structure additional mechanistic evidence from in vitro and animal studies on the identified adverse effects. In addition to a preliminary AOP for DON resulting in the adverse outcome (AO) 'reduced body weight gain', we developed a more elaborated AOP for FB1, from the molecular initiating event (MIE) 'inhibition of ceramide synthases' leading to the AO 'neural tube defects'. The mechanistic evidence from AOPs can be used to support the limited evidence from human studies, to focus FB1- and DON-related research in humans to identify related early biomarkers of effect. In order to establish additional human exposure-health relationships in the future, recommendations are given to maximize the information that can be obtained from HBM.

Citizens' Perception and Concerns on Chemical Exposures and Human Biomonitoring-Results from a Harmonized Qualitative Study in Seven European Countries.

Exposure to different chemicals is an inevitable part of our everyday lives. Within HBM4EU, focus group discussions were conducted to gather data on citizens' perceptions of chemical exposure and human biomonitoring. These discussions were hosted in Cyprus, Denmark, Hungary, Israel, Latvia, the Netherlands, and North Macedonia following a protocol developed in the first round of discussions. Results indicate the very high concern of European citizens regarding food safety and the environment. Focus group participants were well aware of potential uptake of chemicals through food consumption (e.g., preservatives, flavor enhancers, coloring agents, pesticides, fertilizers, metals), drinking water, or from polluted air and water. One of the positive aspects identified here, is the high interest of citizens in awareness and education on personal measures to control exposure. The promotion of personal behavioral changes requires active involvement of society (e.g., commuting habits, energy choices, waste disposal, dietary habits). Activities should focus on raising awareness of the general public, implementation of policy measures, and mainstreaming of related topics into the education system. Raising awareness of the general public may promote engagement of citizens, which in turn may empower them to put pressure on politicians to take effective actions. There is also a need for further research which might focus on the impact of country-specific situations and of the COVID-19 pandemic on the exposure of citizens to chemicals.

Modelling the Renal Excretion of the Mycotoxin Deoxynivalenol in Humans in an Everyday Situation.

The dietary exposure to the mycotoxin deoxynivalenol (DON) can be assessed by human biomonitoring (HBM). Here, we assessed the relation between dietary DON intake and the excretion of its major metabolite DON-15-glucuronide (DON15GlcA) through time, in an everyday situation. For 49 volunteers from the EuroMix biomonitoring study, the intake of DON from each meal was calculated and the excretion of DON and its metabolites was analyzed for each urine void collected separately throughout a 24-h period. The relation between DON and DON15GlcA was analyzed with a statistical model to assess the residence time and the excreted fraction of ingested DON as DON15GlcA (fabs_excr). Fabs_excr was treated as a random effect variable to address its heterogeneity in the population. The estimated time in which 97.5% of the ingested DON was excreted as DON15GlcA was 12.1 h, the elimination half-life was 4.0 h. Based on the estimated fabs_excr, the mean reversed dosimetry factor (RDF) of DON15GlcA was 2.28. This RDF can be used to calculate the amount of total DON intake in an everyday situation, based on the excreted amount of DON15GlcA. We show that urine samples collected over 24 h are the optimal design to study DON exposure by HBM.

Assessment of the combined nitrate and nitrite exposure from food and drinking water: application of uncertainty around the nitrate to nitrite conversion factor.

Dietary exposure to nitrate and nitrite occurs via three main sources; occurrence in (vegetable) foods, food additives in certain processed foods and contaminants in drinking water. While nitrate can be converted to nitrite in the human body, their risk assessment is usually based on single substance exposure in different regulatory frameworks. Here, we assessed the long-term combined exposure to nitrate and nitrite from food and drinking water. Dutch monitoring data (2012-2018) and EFSA data from 2017 were used for concentration data. These were combined with data from the Dutch food consumption survey (2012-2016) to assess exposure. A conversion factor (median 0.023; range 0.008-0.07) was used to express the nitrate exposure in nitrite equivalents which was added to the nitrite exposure. The uncertainty around the conversion factor was taken into account by using conversion factors randomly sampled from the abovementioned range. The combined dietary exposure was calculated for the Dutch population (1-79 years) with different exposure scenarios to address regional differences in nitrate and nitrite concentrations in drinking water. All scenarios resulted in a combined exposure above the acceptable daily intake for nitrite ion (70 µg/kg bw), with the mean exposure varying between 95-114 µg nitrite/kg bw/day in the different scenarios. Of all ages, the combined exposure was highest in children aged 1 year with an average of 250 µg nitrite/kg bw/day. Vegetables contributed most to the combined exposure in food in all scenarios, varying from 34%-41%. Food additive use contributed 8%-9% to the exposure and drinking water contributed 3%-19%. Our study is the first to perform a combined dietary exposure assessment of nitrate and nitrite while accounting for the uncertain conversion factor. Such a combined exposure assessment overarching different regulatory frameworks and using different scenarios for drinking water is a better instrument for protecting human health than single substance exposure.

Anti-tumor properties of methoxylated analogues of resveratrol in malignant MCF-7 but not in non-tumorigenic MCF-10A mammary epithelial cell lines.

Resveratrol is a plant-derived polyphenol that is known for its anti-inflammatory and anti-tumorigenic properties in in vitro and in vivo models. Recent studies show that some resveratrol analogues might be more potent anti-tumor agents, which may partly be attributed to their ability to activate the aryl hydrocarbon receptor (AHR). Here, the anti-tumorigenic properties of resveratrol and structural analogues oxyresveratrol, pinostilbene, pterostilbene and tetramethoxystilbene (TMS) were studied in vitro, using in the malignant human MCF-7 breast cancer cell line and non-tumorigenic breast epithelial cell line MCF-10A. Cell viability and migration assays showed that methoxylated analogues of resveratrol are more potent anti-tumorigenic compounds than resveratrol and its hydroxylated analogue oxyresveratrol, with 2,3',4,5'-tetramethoxy-trans-stilbene (TMS) being the most potent compound. TMS decreased MCF-7 tumor cell viability with 50% at 3.6 µM and inhibited migration with 37.5 ± 14.8% at 3 µM. In addition, TMS activated the AHR more potently (EC50 in a reporter gene assay 2.0 µM) and induced AHR-mediated induction of cytochrome P450 1A1 (CYP1A1) activity (EC50 value of 0.7 µM) more than resveratrol and the other analogues tested. Cell cycle analysis showed that TMS induced a shift in cell cycle status from the G1 to the G2/M phase causing a cell cycle arrest in the MCF-7 cells, while no effect of TMS was observed in the non-tumorigenic MCF-10A mammary epithelial cell line. Gene expression analysis showed that 3 µM TMS increased gene expression of CYP1A1 (289-fold), CYP1B1 (5-fold) and Nqo1 (2-fold), and decreased gene expression of IL-8 (3-fold) in MCF-7 cells. In MCF-10A cells, 10 µM TMS also increased gene expression of CYP1A1 (5-fold) and CYP1B1 (2-fold), but decreased gene expression of Nqo1 (1.4-fold) in contrast to MCF-7 cells. TMS displays more potent anti-tumorigenic properties and activates the AHR more effectively than resveratrol. In addition, this is the first study to show that TMS, but not resveratrol, selectively inhibits the cell cycle of breast tumor cells and not the non-tumorigenic cells. Our study provides more insight in the anti-tumor properties of the methoxylated analogues of resveratrol in breast cells in vitro.