Skip to main content
NCBI home page
EFSA Journal logoLink to EFSA Journal
. 2022 Dec 14;20(Suppl 2):e200907. doi: 10.2903/j.efsa.2022.e200907

Innovative in vitro approaches to toxicological investigations of mycotoxins effects

Arce López Beatriz 1,✉, Hymery Nolwenn 1
PMCID: PMC9749439  PMID: 36531276

Abstract

Among the potential contaminants, mycotoxins are of particular concern due to the importance in terms of food and feed safety. The difficulty in establishing a diagnosis for mycotoxicosis relies in the fact that the effects are subclinical, and that multicontamination by various toxins is the most common scenario. The co‐occurrence of these mycotoxins raises questions concerning both food safety and regulation. However, there is still limited knowledge on toxicity data on co‐exposure. The current technical report will describe the activities performed by the fellow in the LUBEM‐Brest University (France). In this context, the work programme offered by the hosting site consisted in vitro toxicological approaches to evaluate the toxicity of mycotoxin mixtures. The aim of this project was to assess human risk to the exposure of two main regulated mycotoxins (ochratoxin A and fumonisin B1) using different innovative cellular models (2D and 3D spheroids). In this framework, these mycotoxins were tested individually and as a combination on intestinal and hepatic cell lines alone or in co‐cultures. Overall, our results show the outstanding potential of using more predictive and realistic approaches for the risk assessment (RA) of mycotoxins. It is of high importance to pursue further toxicological characterisations and exposure evaluations for mycotoxins, in order to determine a more detailed RA. This will serve as a reference to understand multicontamination mechanism of mycotoxins at the cell level and help authority to revise regulation.

Keywords: Food contaminantsin vitro modelstoxicologymycotoxins

1. Introduction

Globally, throughout our lives we may be exposed to multiple chemicals from a variety of sources (OECD, 2018). Many microorganisms including bacteria, yeasts and fungi are associated with cereals but the latter are of great importance as some species are toxinogenic (CAST, 2003). Over the years, it has been estimated that the overall global contamination of agricultural products to be around 25%. However, although several mycotoxins are individually regulated (ochratoxins, fumonisins, etc.), mycotoxins levels are characterised by being higher than the European limits. Therefore, there are important economic impacts resulting from the application of the European legislation regarding these mycotoxins (Eskola et al., 2020).

In this context, detection and quantification of mycotoxins are particularly important due to their human and animal health associated risks (Habschied et al., 2021; Kępińska‐Pacelik and Biel, 2021). Recently, the European Food Safety Authority (EFSA) has undertaken to develop new methods to assess the risks associated with the complex issue of mixtures in the food chain and combined toxicity (EFSA Scientific Committee, 2019).

Presently, in vitro tests play a major role in obtaining information on toxicity mechanism with the perspective to be able to identify pathways of toxic responses by applying ‘omics’ techniques (Balmer et al., 2014). Physiology‐based toxicokinetic modelling is using data from in vitro studies to build up a model for a specific compound. Information from both areas is incorporated into the risk assessment to derive compound‐specific safety factors, which account for species differences and for the variability among the human population, including possible sensitive subpopulations. In link with the 3R concept, the question ‘Why animal testing for safety evaluations have not yet been replaced?’ can be raised (Smith et al., 2016).

1.1. Mycotoxins

Mycotoxins are secondary metabolites produced by fungi, mainly of the genera Aspergillus, Penicillium, Fusarium, Claviceps and Alternaria genera, which can contaminate food and reach humans food and reach humans, causing toxic effects and damaging their health (Marin et al., 2013). It is a group of heterogeneous compounds that differ in their structures, their biochemical and physicochemical characteristics and their toxicological properties (Alshannaq and Yu, 2017). Exposure to mycotoxins produces different toxic effects, called mycotoxicosis (Streit et al., 2012), which can result in an acute mycotoxicosis (due to ingestion of high levels of toxins in a short period of time) or chronic mycotoxicosis (due to ingestion of low levels of toxins in a long period of time) (Ostry et al., 2017; Claeys et al., 2020).

More than 300 mycotoxins are known, but only 40 compounds are regulated and monitored by national or international food regulation authorities, up to now (European Commission, 2006). This may lead to an underestimation of actual food and feed contamination patterns, as well as the actual health risk for both humans and animals. In addition, co‐exposure to multiple mycotoxins might generate additive, synergistic or antagonistic effects (Smith et al., 2016).

The International Agency for Research on Cancer (IARC) has studied some mycotoxins for their mycotoxins in terms of their potential carcinogenic risk. The studied mycotoxins for this project, ochratoxin A (OTA) and fumonisin B1 (FB1) (Figure 1), have been classified as ‘possible carcinogens to human’ (Group 2B) (IARC, 1987, 1993, 2012).

Figure 1.

Figure 1

Open in a new tab

Chemical structures of the studied mycotoxins: OTA (left); FB1 (right)

1.2. Cell culture

1.2.1. HepaRG cell line

The liver being the reference detoxification organ of the organism, hepatocyte cells represent one of the most suitable in vitro study models for xenobiotic metabolism and for toxicological studies. Indeed, the functions of the liver are mainly performed by hepatocytes which represent about 80% of the total cell population. In addition, the alteration of liver functions is one of the main effects observed on animals following exposure to high doses of mycotoxins (Pitt, 2000). However, more than 50% of the drugs inducing liver damage in human clinical trials are not hepatotoxic in animals, which highlights the importance of using human hepatocytes to more accurately assess drug toxicity, or other bioactive molecules, in vitro in humans (Olson et al., 2000). Nevertheless, as for all primary cells, primary human hepatocytes are phenotypically unstable, have a limited lifespan and a high interdrug variability. On the other hand, hepatocyte lines of tumour origin or obtained by oncogenic immortalisation, such as HepG2 and C3A cells, lack some important liver‐specific functions. In particular, these lines do not possess some major cytochromes of the P450 family involved in the metabolism of xenobiotics, and are therefore of limited interest for pharmaceutical and therapeutic studies (Guguen‐guillouzo and Guillouzo, 2010). In contrast, the human hepatocyte cell line HepaRG, obtained from a liver tumour from a patient with hepatocarcinoma and hepatitis C infection (Gripon et al., 2002), seems to be a good substitute for primary hepatocytes for toxicology studies. Indeed, this line possesses both the metabolic performance of primary human hepatocytes and the growth capacities of hepatocyte lines (Guillouzo et al., 2007). In particular, HepaRG cells express many liver‐specific functions such as the main cytochromes P450 and some nuclear receptors at levels comparable to those found in primary human hepatocytes.

Moreover, these cells can be maintained at confluence for several weeks while maintaining a stable metabolic activity, which makes them ideal model for the study of drug metabolism parameters as well as acute and chronic as well as the acute and chronic effects of xenobiotics on the human liver (Guillouzo et al., 2007; Anthérieu et al., 2012). On the other hand, the HepaRG cells have unique characteristics: when detached and reseeded at low density, they are able to dedifferentiate by reacquiring an undifferentiated morphology and dividing in a very active way to reach confluence quickly.

As a result, two types of cell populations morphologically different can be distinguished (Figure 2): colonies formed by clusters of granular epithelial cells resembling hepatocytes, surrounded by more flattened cells. These cells can be differentiated respectively (with the addition of DMSO in the culture medium) into more granular cells having one or two nuclei and closely resembling adult primary hepatocytes for the former, and into biliary epithelial cells for the latter. The two cell states (undifferentiated and differentiated) from this line can be used and compared in toxicological studies (Guillouzo et al., 2007). HepaRG cells thus represent an innovative model for this type of toxicological study.

Figure 2.

Figure 2

Open in a new tab

Morphology of HepaRG cells in different states: undifferentiated (left); differentiated (right). Scale bar: 100 μm

1.2.2. Caco‐2 cell line

The intestinal barrier plays an important role in protecting the body against ingested toxic substances. It also represents one of the major sites of exposure to agents because of its large area of exposure and its physiological role in the transfer of nutrients from the lumen to the blood. The gastrointestinal tract, in addition to its role in the absorption of xenobiotics, is also actively in their biotransformation (Vancamelbeke and Vermeire, 2017). The human tumour cell line Caco‐2 of intestinal origin, obtained from colorectal adenocarcinoma, is very commonly used in in vitro toxicology studies as a model to mimic the intestinal barrier (De Angelis and Turco, 2011).

Despite the fact that reproducibility problems have been reported in the literature making it difficult to compare results between laboratories, the Caco‐2 cell line has been widely used over the last 30 years in in vitro studies. These problems have been attributed to the intrinsic variability of the cells used in different laboratories, as well as to the different conditions related to the culture, such as the type of animal serum used, the supplements added to the culture medium, the number of passages and the origin of the clones (Natoli et al., 2012). In addition, these cells have the particularity of being able to differentiate spontaneously (Figure 3), leading to the formation of a monolayer of polarised intestinal cells expressing several morphological and functional characteristics of mature intestinal enterocytes (Sambuy et al., 2005).

Figure 3.

Figure 3

Open in a new tab

Morphology of Caco‐2 cells in different states: Undifferentiated (left); differentiated (right). Scale bar: 100 μm.

1.3. Innovative in vitro systems

1.3.1. Co‐culture models

In vitro cell culture models have proven to be extremely useful to understand the molecular mechanisms of transport, metabolism and toxicity of relevant molecules, such as mycotoxins. These systems allow to better mimic the in vivo situation by taking into account the interactions between different cell types, thus representing an interesting alternative to traditional systems (Smith et al., 2018).

However, one of their limitations is the need to culture individual types in isolation from other cells that are in constant and close physiological interaction in vivo. In an attempt to overcome these limitations, several interesting co‐culture models have recently been unveiled, combining intestinal cells with cells of different origin, such as neural, liver, pancreatic or monocytic cells. These models better reproduce the cross‐talk between tissues that occurs in vivo (Castell‐Auví et al., 2010a,b). Differentiated enterocytes in the small intestine are responsible for the transport and first metabolism of ingested molecules. After crossing the intestinal barrier, metabolites come into contact with local cytotypes such as intraepithelial lymphocytes, monocytes, fibroblasts and enteric neuronal cells, or are transported to the liver and other organs via the hepatic portal circulation and the lymphatic system.

For this purpose, it is necessary to study cytotoxicity on a co‐culture system to suggest the toxicological impact of mycotoxins on different cellular models. To reproduce this complex interaction in vitro, we used as an intestinal cell model the human adenocarcinoma cell line Caco‐2 that spontaneously differentiates in vitro, expressing several morphological and functional characteristics of mature small intestinal enterocytes (Sambuy et al., 2005) with HepaRG cells.

For these co‐culture experiments, Caco‐2 cells on inserts were transferred to culture plates containing HepaRG cells, as shown in Figure 4.

Figure 4.

Figure 4

Open in a new tab

Schematic representation of the in vitro co‐culture system

1.3.2. 3D models

Although co‐cultures overcome some of the drawbacks of classical 2D systems (in particular, intercellular communication), they are still far from accurately reproducing all cellular functions observed in a tissue. 3D in vitro culture systems seem to be relevant models to try to approximate in vivo conditions (Hoarau‐Véchot et al., 2018). In particular, spheroid systems have been developed that are now viable with high throughput (Kelm and Fussenegger, 2004). In such systems, the number of cells and thus the size of the spheroid can be adjusted (Fennema et al., 2013).

A study performed with HepaRG cells pointed out that 3D HepaRG spheroids had better functionality than conventional 2D HepaRG cultures and thus represented a good model for the study of drug toxicity, in particular for the study of drug metabolism (Mueller et al., 2014).

Therefore, there is a whole field of research possible today to mimic the in vivo context more precisely, and which could allow a more accurate assessment of the risk of mycotoxins to human and animal health.

2. Description of work programme

The fellow was hosted by the LUBEM (Laboratoire Universitaire de Biodiversité et Ecologie Microbienne), belonging to the Université de Bretagne Occidentale (UBO) in France. The hosting division has a long‐standing experience in the risk assessment of innovative topics related to moulds (e.g. fungal diversity in the agri‐food environment, predictive mycology, antifungal biopreservation), focuses mainly on secondary metabolites, in particular mycotoxins and their toxicological impact. Since 2022, the host laboratory team has been recognised as a participating institution of INRAE (National Research Institute for Agriculture, Food and the Environment).

In this field of research, the activities concern scientific and technological advances that can revolutionise the toxicological evaluation of these metabolites but also of various chemical compounds (e.g. pesticides). One of the main challenges in this field is the need to move away from animal testing and toward the use of in vitro methods, in agreement with the 3R concept. However, one of the main downfall of in vitro toxicological evaluation is the distance to the in vivo reality and thus the usability of the obtained data. In order to counteract this problem, we have developed 3D innovative new cellular models, like spheroids cell models, which are likely to have an impact on risk assessment procedures. Moreover, these models can also help to better take into account the reality of mycotoxin (or other compounds) exposure through multi‐contamination and/or chronic exposure studies. The proposed project is directly linked to these scientific questions and was split into two different phases to better answer the different aspects.

2.1. Aims

The lack of regulatory consideration of multi‐contaminations to mycotoxins is mainly due to the scarcity of toxicological data. Moreover, the effects of the combinations of mycotoxins on the cellular mechanisms are mechanisms are insufficiently known. In this context, the present work programme aimed to determine a 3D cell model relevant for in vitro toxicological evaluation using mycotoxin as an example.

More precisely, the aim was to study the cytotoxic effects of OTA and FB1 combinations and to identify the cellular mechanisms involved in the toxicity of these mixtures via toxicological approaches using different human cell lines (intestinal and hepatic) representative of the defence barriers of the organism and of the detoxification organ, privileged targets of mycotoxins.

The following specific tasks, articulated in two parts, arise from this general objective:

Task.1: Comparison of 2D and 3D hepatocyte models on mycotoxins toxicity.

Mycotoxins correspond to a critical food safety, in particular for cereal crops, posing significant health risk to humans and livestock. These fungal metabolites also have a significant economic impact that embraces yield losses, product recalls, costs linked to management, prevention and mitigation. Moreover, some are regulated at the European level and have been extensively studied in both in vivo and in vitro conditions. In this context, the goal of this task was to compare in vitro toxicological data obtained with the same cell line (human hepatoma) HepaRG but with two different experimental conditions, namely in 2D (classical cell culture) and 3D (spheroid). These models were used to compare the toxicological impact of two mycotoxins (OTA and FB1) in acute conditions.

Task.2: Comparison of 2D and 3D system cells to evaluate mycotoxins mixture effects.

As the reality of mycotoxin exposure corresponds to multi‐contamination, toxicity of mixtures at actual concentrations encountered in cereals was assessed using cell viability and compared to control (toxicity of each mycotoxin alone). Mycotoxin interactions were analysed by the isobologram‐combination index method and a toxicology predictive model was developed.

2.2. Activities and methodology of the project

The fellow was involved in the different steps of evaluation of mycotoxin in vitro toxicity from the beginning of the project. To gather contamination data, literature searches were included to further study about the topic and to select the tested mycotoxin concentrations.

In order to follow this work, the fellow was trained in cell culture methodology, data analysis and in vitro approaches. In this framework, to achieve the main goal of the project, toxicological effects of mycotoxins were assessed on different parameters to compare 2D and 3D approaches.

  • –

    Cell growth and viability effect were analysed in 2D and 3D system at the same time on intestinal cell line (Caco‐2), hepatocyte line (HepaRG). On cell lines, the proliferation was measured by common cytotoxicity tests based on the analysis of mitochondrial activity (MTS (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium assay) test or ATP assay for 3D models) after 48 h of exposure with mycotoxins alone or in cocktail.

  • –

    Barrier function. When intestinal epithelial cells (Caco‐2) are grown on inserts ‘transwell system’, they form a monolayer and differentiate into mature intestinal epithelium (expression of apical villi, formation of tight junctions, etc.). It is then possible to assess the integrity of this monolayer by measuring the transepithelial electrical resistance (TEER) established between the basal and the apical compartments (Bouhet, 2004). We investigated the effects of OTA and FB1 alone and in cocktail at no cytotoxic concentrations on the monolayer TEER during 48 h.

  • –

    For mycotoxin assay, the optimisation of mycotoxin extraction methods (suitable for cell lines and targeted mycotoxins) was carried out and their quantification was made by LC‐Q‐TOF in extra and intracellular systems.

According to the main Task 1, the following deliverables were obtained: results of cell viability study on each cell model exposed to mycotoxin alone or in cocktail, determination of interaction type (additive, synergic, potentiate or antagonist), results of mycotoxins effect on barrier function (Comparison of results in 2D and 3D systems) and quantification of OTA, FB1 in the co‐culture system in apical, basolateral and intracellular fraction after 48 h of exposure.

To better characterise the impact of combined mycotoxin exposure (Task 2): Human system cells were used to evaluate mycotoxins mixture effects after exposure. Comparison of cellular models and effects of OTA and FB1 on systems were performed.

2.3. In vitro cell culture systems for the assessment of toxicity of mycotoxins

Following an ethical approach aimed at reducing animal experimentation (European Commission, 2010), many toxicology studies are now conducted in vitro toxicology studies are now conducted on in vitro systems. In particular, since the introduction of the ‘3Rs’ rule (reduce, refine, replace), major efforts have been made to improve these have been made to improve these cell culture models so that they can replace animal experiments (Spielmann et al., 2008).

The use of an accurate in vitro model for toxicity studies should identify the cellular pathways and mechanisms affected while reflecting the in vivo situation (Committee on Toxicity Testing and Assessment of Environmental Agents and National Research Council, 2007). Such a system must also allow for long‐term functional culture of the cells. However, most of these studies are performed in classical 2D culture systems using a single type of cell, and therefore do not reflect the complexity of a 3D organ, leading to discrepancies between in vitro and in vivo experimental data (Antoni et al., 2015). In particular, this type of culture does not take into account some important factors that allow to accurately reproduce the physiology of cells and tissues, such as the communication between the cell and its matrix as well as the communication between cells, but also the consecutive exposure of several cell types during the ingestion of a toxin.

2.4. Additional scientific activities

Besides the specific activities in the risk assessment work programme (EU‐FORA training), the fellow participated in a full range of activities of the research unit, which is indeed a group young and committed, taking part in the exciting and numerous activities of the unit: contributed to teaching and learning conference (Master of Health Biology in Brest University).

In order to maximise knowledge transfer, the fellow was actively engaged through numerous events, including meetings and international conferences (The World Mycotoxin Forum and Mycotoxin Research) concerning mycotoxins, food contaminants, microbiology or toxicology, to present the results of these new innovative cell culture models. All these activities completed during the fellowship period are reported in Appendix A.

3. Conclusions

The work programme at the LUBEM (Laboratoire Universitaire de Biodiversité et Ecologie Microbienne)‐Université de Bretagne Occidentale (UBO) provided the fellow the opportunity to develop important skills within critical aspects of the risk assessment framework, mainly focused on toxicological aspects.

The fellowship programme, beyond the personal enrichment that an experience living abroad always brings, has provided the opportunity to gain expertise in different areas of toxicological risk assessment, especially with regard to cell culture, data analysis and toxicology predictive approaches. The fellow has also considered this a valuable experience to consolidate her background in terms of mycotoxin knowledge, especially in the context of impact on risk assessment.

Working at the LUBEM has been such an exciting opportunity to further develop research skills and complement education by learning new techniques in the field of metabolism and toxicological effects. The proposed work was a perfect fit with the experience and expectations of the fellow.

During this year, thanks to the participation in different scientific conferences and the EU‐FORA trainings (covering in‐depth all over the different areas of food safety risk assessment, risk management and risk communication), the fellow has gained autonomy in decision‐making, collaboration with other researchers and also a great capacity of interaction to communicate science. Attendance to these complementary ‘learning‐by‐doing’ courses has allowed the trainee to work in groups and to make quick decisions through the evaluation of high‐level scientific articles and case studies. The working methodology applied by EFSA in risk assessment and the integration in such a multidisciplinary research group has provided a great environment to build a strong network of professional and personal experiences, opening the door to future collaborations and maximising knowledge transfer.

The fellow and the supervisor have valued the EU‐FORA Fellowship programme as a positive professional experience to exchange and cooperate with advanced knowledge in a specific research area. In this framework, it is expected that the general risk assessment methodology applied for this particular project will be extended and applied by the fellow and the group in future positions, to expand knowledge and collaborations in the toxicological evaluation of mycotoxins.

3.1. Disclaimer

Detailed results obtained from the method development, sample analysis and risk assessment during the research study are not included in this report to avoid certain copyright claims, as the final deliverables and complete work will be subsequently published in other scientific journals.

Abbreviations

AGES

Austrian Agency for Health and Food Safety

BfR

German Federal Institute for Risk Assessment

EFET

Hellenic Food Authority

EU‐FORA

European Food Risk Assessment Fellowship Programme

FB1

fumonisin B1

IARC

International Agency for Research on Cancer

LC‐Q‐TOF

liquid chromatography coupled to quadrupole time‐of‐flight

LUBEM

Laboratoire Universitaire de Biodiversité et Ecologie Microbienne

MS

mass spectrometry

OTA

ochratoxin A

RA

risk assessment

TEER

transepithelial electrical resistance

UBO

Université de Bretagne Occidentale

WMF

World Mycotoxin Forum

Appendix A – Training activities

Table A.1: Training activities during the EU‐FORA Fellowship Programme

Type of event Event Contribution Location Date
Meeting Challenges in Public Health Protection in the 21st Century: New Methods, Omics and Novel Concepts in Toxicology Attendance Online 15–17 November 2021
Webinar ‘Mycotoxins in the food chain’ Attendance Online 7–8 October 2021
‘Toxicity and authenticity testing of foods with trace elemental and stable isotope analysis’ Attendance Online 4 November 2021
Conference Human Exposome WMF 2022 Oral Communication. ‘Biomonitoring and human exposure to multiple mycotoxins’ Online 12 October 2021
Virtual ICFC 2021 Oral Communication. ‘Exposure to mycotoxins in Spanish children through the analysis of their levels in plasma samples’ Online 27–28 September 2021
43rd Mycotoxin Workshop Toulouse Written Communication. ‘Toxicological effects of ochratoxin A and fumonisin B1 on human cells using innovative 2D and 3D in vitro models’ Toulouse (France) 30 May–1 June 2022
The World Mycotoxin Forum (WMF) 2022 Written Communication. ‘Innovative 2D and 3D in vitro models to evaluate toxicological effects of ochratoxin A and fumonisin B1 on human cells’ Parma (Italy) 16–18 May 2022
‘ONE – Health, Environment, Society – Conference 2022’ Attendance Online 21–24 June 2022
WMF 2022. Pre‐Conference on Analysis. Development and trends in (multi)mycotoxin detection Attendance Online 1 February 2022
WMF 2022. Pre‐Conference on Animal Health. Mycotoxins‐ongoing issues of animal health and productivity Attendance Online 30 November 2021
RAFA 2021 Attendance Online 3–4 November 2021

EU‐FORA

Training Courses

 Induction Training EFSA Training Online 30 August–17 September 2021
Module 1. Novel Foods (AGES–EFSA) Online 22–26 November 2021
Module 2. Risk Communication (BfR–EFSA) Online 21–25 March 2022
Module 3. Emerging Risks (EFET–EFSA) Athens (Greece) 6–10 June 2022
Module 4. Data Collection and Reporting (EFSA) Online 21–25 August 2022
Open in a new tab

Suggested citation: Beatriz A and Nolwenn H, 2022. Innovative in vitro approaches to toxicological investigations of mycotoxins effects. EFSA Journal 2022;20(S2):e200907, 12 pp. 10.2903/j.efsa.2022.e200907

Declarations of interest If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.

Acknowledgements This report is funded by EFSA as part of the EU‐FORA programme 2021–2022, in collaboration with the hosting site Université de Bretagne Occidentale ‐ (Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, LUBEM) (UBO) France that has provided the fellow with the necessary tools and equipment to develop this report. The authors would like to thank Dr. Monika Coton, professor at the Université de Bretagne Occidentale for their scientific support throughout the programme.

Approved: 31 August 2022

References

  1. Alshannaq A and Yu J‐H, 2017. Occurrence, toxicity, and analysis of major mycotoxins in food. International Journal of Environmental Research and Public Health, 14, 632. 10.3390/ijerph14060632 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anthérieu S, Chesné C, Li R, Guguen‐Guillouzo C and Guillouzo A, 2012. Optimization of the HepaRG cell model for drug metabolism and toxicity studies. Toxicology in Vitro, 26, 1278–1285. 10.1016/j.tiv.2012.05.008 [DOI] [PubMed] [Google Scholar]
  3. Antoni D, Burckel H, Josset E and Noel G, 2015. Three‐dimensional cell culture: a breakthrough in vivo. International Journal of Molecular Sciences, 16, 5517–5527. 10.3390/ijms16035517 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balmer NV, Dao T, Leist M, Vojnits K, Waldmann T, Zhao L and van Vliet E, 2014. In: Bal‐Price A and Jennings P (eds.). Vitro Toxicology Systems. 10.1007/978-1-4939-0521-8 [DOI] [Google Scholar]
  5. Bouhet S, 2004. The mycotoxin Fumonisin B1 alters the proliferation and the barrier function of porcine intestinal epithelial cells. Toxicological Sciences, 77, 165–171. 10.1093/toxsci/kfh006 [DOI] [PubMed] [Google Scholar]
  6. CAST , 2003. Mycotoxins: risks in plant, animal, and human systems. In Mycopathologia., 65, 43–45. 10.1007/BF00447172 [DOI] [Google Scholar]
  7. Castell‐Auví A, Cedó L, Pallarés V, Blay M, Pinent M, Motilva M and Ardévol A, 2010a. Development of a coculture system to evaluate the bioactivity of plant extracts on pancreatic β ‐cells. Planta Medica, 76, 1576–1581. 10.1055/s-0029-1241002 [DOI] [PubMed] [Google Scholar]
  8. Castell‐Auví A, Motilva MJ, Macià A, Torrell H, Bladé C, Pinent M and Ardévol A, 2010b. Organotypic co‐culture system to study plant extract bioactivity on hepatocytes. Food Chemistry, 122, 775–781. 10.1016/j.foodchem.2010.03.052 [DOI] [Google Scholar]
  9. Claeys L, Romano C, De Ruyck K, Wilson H, Fervers B, Korenjak M and Huybrechts I, 2020. Mycotoxin exposure and human cancer risk: a systematic review of epidemiological studies. Comprehensive Reviews in Food Science and Food Safety, 19, 1449–1464. 10.1111/1541-4337.12567 [DOI] [PubMed] [Google Scholar]
  10. Committee on Toxicity Testing and Assessment of Environmental Agents, & National Research Council . (2007). Toxicity testing in the 21st century ‐ NRC report.
  11. De Angelis I and Turco L, 2011. Caco‐2 cells as a model for intestinal absorption. Current Protocols in Toxicology, 47, 1–15. 10.1002/0471140856.tx2006s47 [DOI] [PubMed] [Google Scholar]
  12. EFSA Scientific Committee , More SJ, Hardy A, Bampidis V, Benford D, Hougaard Bennekou S, Bragard C and Hogstrand C, 2019. Guidance on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals. EFSA Journal 2019;17(3):5634, 45 pp. 10.2903/j.efsa.2019.5634 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eskola M, Kos G, Elliott CT, Hajšlová J, Mayar S and Krska R, 2020. Worldwide contamination of food‐crops with mycotoxins: validity of the widely cited ‘FAO estimate’ of 25%. Critical Reviews in Food Science and Nutrition, 60, 2773–2789. 10.1080/10408398.2019.1658570 [DOI] [PubMed] [Google Scholar]
  14. European Commission , 2006. Commission Recommendation 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union, 70, 12–34. [Google Scholar]
  15. European Commission , 2010. DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purpose. Official Journal of the European Union, 23, 33–79. [Google Scholar]
  16. Fennema E, Rivron N, Rouwkema J, van Blitterswijk C and de Boer J, 2013. Spheroid culture as a tool for creating 3D complex tissues. Trends in Biotechnology, 31(2), 108–115. 10.1016/j.tibtech.2012.12.003 [DOI] [PubMed] [Google Scholar]
  17. Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I and Guguen‐Guillouzo C, 2002. Infection of a human hepatoma cell line by hepatitis B virus. Proceedings of the National Academy of Sciences, 99(24), 15655–15660. 10.1073/pnas.232137699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Guguen‐guillouzo C and Guillouzo A, 2010. In: Maurel P (ed). General Review on In Vitro Hepatocyte Models and Their Applications. 10.1007/978-1-60761-688-7 [DOI] [PubMed] [Google Scholar]
  19. Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F and Guguen‐Guillouzo C, 2007. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chemico‐Biological Interactions, 168, 66–73. 10.1016/j.cbi.2006.12.003 [DOI] [PubMed] [Google Scholar]
  20. Habschied K, Kanižai Šarić G, Krstanović V and Mastanjević K, 2021. Mycotoxins—biomonitoring and human exposure. Toxins, 13, 113. 10.3390/toxins13020113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hoarau‐Véchot J, Rafii A, Touboul C and Pasquier J, 2018. Halfway between 2D and animal models: are 3D cultures the ideal tool to study cancer‐microenvironment interactions? International Journal of Molecular Sciences, 19, 181. 10.3390/ijms19010181 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. IARC (International Agency for Research on Cancer) , 1987. Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42 (Supplement 7). IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, 46. [PubMed] [Google Scholar]
  23. IARC (International Agency for Research on Cancer) , 1993. Some Naturally Occurring Substances: Food Items and Constituents. Heterocyclic Aromatic Amines and Mycotoxins, IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. 56 pp. [Google Scholar]
  24. IARC (International Agency for Research on Cancer) , 2012. Chemical agents and related occupations. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans / World Health Organization, International Agency for Research on Cancer, 100(Pt 3), 9–562. [PMC free article] [PubMed] [Google Scholar]
  25. Kelm JM and Fussenegger M, 2004. Microscale tissue engineering using gravity‐enforced cell assembly. Trends in Biotechnology, 22, 195–202. 10.1016/j.tibtech.2004.02.002 [DOI] [PubMed] [Google Scholar]
  26. Kępińska‐Pacelik J and Biel W, 2021. Mycotoxins—prevention, detection, impact on animal health. Processes, 9, 1–17. 10.3390/pr9112035 [DOI] [Google Scholar]
  27. Marin S, Ramos AJ, Cano‐Sancho G and Sanchis V, 2013. Mycotoxins: occurrence, toxicology, and exposure assessment. Food and Chemical Toxicology, 60, 218–237. 10.1016/j.fct.2013.07.047 [DOI] [PubMed] [Google Scholar]
  28. Mueller D, Krämer L, Hoffmann E, Klein S and Noor F, 2014. 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. Toxicology in Vitro, 28, 104–112. 10.1016/j.tiv.2013.06.024 [DOI] [PubMed] [Google Scholar]
  29. Natoli M, Leoni BD, D'Agnano I, Zucco F and Felsani A, 2012. Good Caco‐2 cell culture practices. Toxicology in Vitro, 26, 1243–1246. 10.1016/j.tiv.2012.03.009 [DOI] [PubMed] [Google Scholar]
  30. OECD (Organisation for Economic Co operation and Development) , 2018. Considerations for assessment of risk from the combined exposure to multiple chemicals. Series on Testing and Assessment, 296, 119. Aailable online: www.oecd.org/chemicalsafety/ [Google Scholar]
  31. Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G and Heller A, 2000. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regulatory Toxicology and Pharmacology, 32, 56–67. 10.1006/rtph.2000.1399 [DOI] [PubMed] [Google Scholar]
  32. Ostry V, Malir F, Toman J and Grosse Y, 2017. Mycotoxins as human carcinogens—the IARC Monographs classification. Mycotoxin Research, 33, 65–73. 10.1007/s12550-016-0265-7 [DOI] [PubMed] [Google Scholar]
  33. Pitt JI, 2000. Toxicogenic fungi and mycotoxins. In British Medical Bulletin, 56. 10.1201/9780203970553 [DOI] [PubMed] [Google Scholar]
  34. Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A and Zucco F, 2005. The Caco‐2 cell line as a model of the intestinal barrier: influence of cell and culture‐related factors on Caco‐2 cell functional characteristics. Cell Biology and Toxicology, 21, 1–26. 10.1007/s10565-005-0085-6 [DOI] [PubMed] [Google Scholar]
  35. Smith MC, Madec S, Coton E and Hymery N, 2016. Natural co‐occurrence of mycotoxins in foods and feeds and their in vitro combined toxicological effects. Toxins, 8, 94. 10.3390/toxins8040094 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smith M‐C, Gheux A, Coton M, Madec S, Hymery N and Coton E, 2018. In vitro co‐culture models to evaluate acute cytotoxicity of individual and combined mycotoxin exposures on Caco‐2, THP‐1 and HepaRG human cell lines. Chemico‐Biological Interactions, 281, 51–59. 10.1016/j.cbi.2017.12.004 [DOI] [PubMed] [Google Scholar]
  37. Spielmann H, Grune B, Liebsch M, Seiler A and Vogel R, 2008. Successful validation of in vitro methods in toxicology by ZEBET, the National Centre for Alternatives in Germany at the BfR (Federal Institute for Risk Assessment). Experimental and Toxicologic Pathology, 60, 225–233. 10.1016/j.etp.2008.01.012 [DOI] [PubMed] [Google Scholar]
  38. Streit E, Schatzmayr G, Tassis P, Tzika E, Marin D, Taranu I and Oswald IP, 2012. Current situation of mycotoxin contamination and co‐occurrence in animal feed—focus on Europe. Toxins, 4, 788–809. 10.3390/toxins4100788 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vancamelbeke M and Vermeire S, 2017. The intestinal barrier: a fundamental role in health and disease. Expert Review of Gastroenterology and Hepatology, 11, 821–834. 10.1080/17474124.2017.1343143 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from EFSA Journal are provided here courtesy of Wiley

RESOURCES