Portuguese children exposure to multiple mycotoxins through food consumption: toward a holistic approach for multiple mycotoxins risk assessment

Humans can be exposed to multiple chemicals at once from a variety of sources, and human risk assessment of multiple chemicals poses several challenges to scientists, risk assessors and risk managers. Ingestion of food is considered a major route of exposure to many contaminants, namely mycotoxins, especially for vulnerable population groups, as children. A lack of sufficient data regarding mycotoxins children risk assessment, could contribute to an inaccuracy of the estimated risk. Efforts must be undertaken to develop initiatives that promote a broad overview of multiple mycotoxins risk assessment. The present work, developed within the MYCOMIX project, aims to assess the risk associated to the exposure of Portuguese children (< 3 years old) to multiple mycotoxins through consumption of foods primarily marketed for this age group. A holistic approach was developed applying deterministic and probabilistic tools to the calculation of mycotoxin daily intake values, integrating children food consumption (3-days food diary), mycotoxins occurrence (HPLC-UV, HPLC-FD, LC-MS/MS and GC-MS), bioaccessibility (standardized in vitro digestion model) and toxicological data (in vitro evaluation of cytotoxicity, genotoxicity and intestinal impact). A case study concerning Portuguese children exposure to patulin (PAT) and ochratoxin A (OTA), two mycotoxins co-occurring in processed cereal-based foods (PCBF) marketed in Portugal, was developed. Main results showed that there is low concern from a public health point of view relatively to PAT and OTA Portuguese children exposure through consumption of PCBF, considering the estimated daily intakes of these two mycotoxins (worst case scenarios, 22.930 ng/kg bw/day and 0.402 ng/kg bw/day, for PAT and OTA, respectively), their bioaccessibility and toxicology results. However, the present case study only concerns the risk associated with the consumption of PCBF and child diet include several other foods. The present work underlines the need to adopt a holistic approach for multiple mycotoxins risk assessment integrating data from exposure, bioacessibility and toxicity domains in order to contribute to a more accurate risk assessment.

Risk assessment of exposure to multiple mycotoxins in food

Moulds may produce a diversity of toxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins and others. Although toxicological, environmental and epidemiological studies have addressed the problem of these toxins one by one, more than one mycotoxin are found usually in the same contaminated food. Risk assessment for humans potentially exposed to multimycotoxins suffers very much from the lack of adequate food consumption data. Furthermore, for a given mycotoxin, synergism and antagonism with other mycotoxins, found in the same food commodities, are not taken into account. Aflatoxin B1 and ochratoxin A belong to the most frequently occurring mycotoxins. This has repeatedly been demonstrated, however, normally, the risk resulting from their simultaneous occurrence is not considered. A descriptive study was developed to monitor air fungal contamination in one hospital food unit.

Is there a health risk concerning the children´s consumption of cereal-based products? A cumulative mycotoxin risk assessment approach

Historically, the health risk of mycotoxins had been evaluated on the basis of single-chemical and single-exposure pathway scenarios. However, the co-contamination of foodstuffs with these compounds is being reported at an increasing rate and a multiple-exposure scenario for humans and vulnerable population groups as children is urgently needed. Cereals are among the first solid foods eaten by child and thus constitute an important food group of their diet. Few data are available relatively to early stages child´s exposure to mycotoxins through consumption of cereal-based foods. The present study aims to perform the cumulative risk assessment of mycotoxins present in a set of cereal-based foods including breakfast cereals (BC), processed cereal-based foods (PCBF) and biscuits (BT), consumed by children (1 to 3 years old, n=75) from Lisbon region, Portugal. Children food consumption and occurrence of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) in cereal-based foods were combined to estimate the mycotoxin daily intake, using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) and aflatoxin daily exposure. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (HQ, ratio between exposure and a reference dose). The concentration addition (CA) concept was used for the cumulative risk assessment of multiple mycotoxins. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. Main results revealed a significant health concern related to aflatoxins and especially aflatoxin M1 exposure according to the MoET and MoE values (below 10000), respectively. HQ and HI values for the remaining mycotoxins were below 1, revealing a low concern from a public health point of view. These are the first results on cumulative risk assessment of multiple mycotoxins present in cereal-based foods consumed by children. Considering the present results, more research studies are needed to provide the governmental regulatory bodies with data to develop an approach that contemplate the human exposure and, particularly, children, to multiple mycotoxins in food. The last issue is particularly important considering the potential synergistic effects that could occur between mycotoxins and its potential impact on human and, mainly, children health.

Combined exposure to multiple mycotoxins in food: are we ready for risk assessment and risk management?

It is nowadays recognized that the risk of human co-exposure to multiple mycotoxins is real. In the last years, a number of studies have approached the issue of co-exposure and the best way to develop a more precise and realistic assessment. Likewise, the growing concern about the combined effects of mycotoxins and their potential impact on human health has been reflected by the increasing number of toxicological studies on the combined toxicity of these compounds. Nevertheless, risk assessment of these toxins, still follows the conventional paradigm of single exposure and single effects, incorporating only the possibility of additivity but not taking into account the complex dynamics associated to interactions between different mycotoxins or between mycotoxins and other food contaminants. Considering that risk assessment is intimately related to the establishment of regulatory guidelines, once the risk assessment is completed, an effort to reduce or manage the risk should be followed to protect public health. Risk assessment of combined human exposure to multiple mycotoxins thus poses several challenges to scientists, risk assessors and risk managers and opens new avenues for research. This presentation aims to give an overview of the different challenges posed by the likelihood of human co-exposure to mycotoxins and the possibility of interactive effects occurring after absorption, towards knowledge generation to support a more accurate human risk assessment and risk management. For this purpose, a physiologically-based framework that includes knowledge on the bioaccessibility, toxicokinetics and toxicodynamics of multiple toxins is proposed. Regarding exposure assessment, the need of harmonized food consumption data, availability of multianalyte methods for mycotoxin quantification, management of left-censored data and use of probabilistic models will be highlight, in order to develop a more precise and realistic exposure assessment. On the other hand, the application of predictive mathematical models to estimate mycotoxins’ combined effects from in vitro toxicity studies will be also discussed. Results from a recent Portuguese project aimed at exploring the toxic effects of mixtures of mycotoxins in infant foods and their potential health impact will be presented as a case study, illustrating the different aspects of risk assessment highlighted in this presentation. Further studies on hazard and exposure assessment of multiple mycotoxins, using harmonized approaches and methodologies, will be crucial towards an improvement in data quality and contributing to holistic risk assessment and risk management strategies for multiple mycotoxins in foodstuffs.

Contribute of the MYCOMIX project and future perspectives for Human Biomonitoring and health risk assessment in Portugal

There is a growing concern within public health about mycotoxin involvement in human diseases, namely those related to children. The MycoMix project (2012-2015), funded by the Portuguese Foundation for Science and Technology, gathered a multidisciplinary team aiming at answering several questions: 1) Are Portuguese children exposed daily to one or several mycotoxins through food? 2) Can this co-exposure affect children´s health? and 3) Are there interaction effect between mycotoxins? Mycomix results revealed that Portuguese children (< 3 years old, n=103) are exposed to multiple mycotoxins through food consumption. Cumulative risk assessment results revealed a potential health concern for the high percentiles of intake, specially for aflatoxins which are carcinogenic compounds. This fact assumes particular importance considering the interactive effects found in in vitro bioassays. These results highlight the need for a more accurate approach to assess the human exposure to mycotoxins6. Within the Mycomix project the assessment of mycotoxin exposure was based on calculations combining mycotoxin data in food with population data on food consumption. This approach does not consider some aspects as the inter-individual metabolism variation, the exposure through sources other than food and the heterogeneous distribution of mycotoxins in food. Exposure assessment of mycotoxins in Portuguese population through biomarkers is still missing and further studies are urgent to be developed. The European Human Biomonitoring Initiative (EHBMI), a proposal within the European Joint Programme, aims to advance the understanding of the extent of exposure to environmental chemicals across Europe and the impact on human health, by gathering national expertise in human biomonitoring domain. At national level Mycomix project uncovered the potential health risk of exposure of Portuguese children to multiple mycotoxins. The risk assessment expertise acquired within Mycomix, namely in analysis and toxicology of chemical mixtures, will be brought together as a contribute to EHBMI objectives.

Application of mathematical models to mycotoxins children risk assessment: a case study of Portuguese children exposure to co-occurring mycotoxins in processed cereal-based foods

People, animals and the environment can be exposed to multiple chemicals at once from a variety of sources, but current risk assessment is usually carried out based on one chemical substance at a time. In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, namely mycotoxins, a wide group of fungal secondary metabolites that are known to potentially cause toxicity and carcinogenic outcomes. Mycotoxins are commonly found in a variety of foods including those intended for consumption by infants and young children and have been found in processed cereal-based foods available in the Portuguese market. The use of mathematical models, including probabilistic approaches using Monte Carlo simulations, constitutes a prominent issue in human health risk assessment in general and in mycotoxins exposure assessment in particular. The present study aims to characterize, for the first time, the risk associated with the exposure of Portuguese children to single and multiple mycotoxins present in processed cereal-based foods (CBF). Portuguese children (0-3 years old) food consumption data (n=103) were collected using a 3 days food diary. Contamination data concerned the quantification of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) were evaluated in 20 CBF samples marketed in 2014 and 2015 in Lisbon; samples were analyzed by HPLC-FLD, LC-MS/MS and GC-MS. Daily exposure of children to mycotoxins was performed using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) to the aflatoxin exposure. The magnitude of the MoE gives an indication of the risk level. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (ratio between exposure and a reference dose, HQ). For the cumulative risk assessment of multiple mycotoxins, the concentration addition (CA) concept was used. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. 71% of CBF analyzed samples were contaminated with mycotoxins (with values below the legal limits) and approximately 56% of the studied children consumed CBF at least once in these 3 days. Preliminary results showed that children exposure to single mycotoxins present in CBF were below the TDI. Aflatoxins MoE and MoET revealed a reduced potential risk by exposure through consumption of CBF (with values around 10000 or more). HQ and HI values for the remaining mycotoxins were below 1. Children are a particularly vulnerable population group to food contaminants and the present results point out an urgent need to establish legal limits and control strategies regarding the presence of multiple mycotoxins in children foods in order to protect their health. The development of packaging materials with antifungal properties is a possible solution to control the growth of moulds and consequently to reduce mycotoxin production, contributing to guarantee the quality and safety of foods intended for children consumption.