Ochratoxin A: 50 Years of Research

1Ochratoxin A (OTA) is one of the most important and deleterious mycotoxins.

2OTA was isolated and chemically characterized in 1965. OTA was discovered in South Africa as a toxic metabolite of Aspergillus ochraceus in a corn meal that was intentionally inoculated with this microfungus. Further research has shown that OTA is nephrotoxic, hepatotoxic, embryotoxic, teratogenic, neurotoxic, immunotoxic, genotoxic, and carcinogenic in many species with species and sex-related differences. The International Agency for Research on Cancer classified OTA as a possible human carcinogen (group 2B) in 1993 based on a great amount of evidence of its carcinogenity discovered in several animal studies. The susceptibility to cancer is species- and sex-dependent. Frequent exposure of animals or humans to OTA may cause a range of health problems. In particular, OTA could be a threat of cancer for humans. It will be shown further in this article that OTA acts as a nephrotoxin and an urothelial carcinogen as a result of both the oxidative stress and direct genotoxic mechanisms. Strikingly, chronic exposure to low OTA doses could be even more damaging than acute exposure to a high dose. Humans are normally exposed to OTA—as they are to other mycotoxins—through several routes, dietary intake being the most prominent. Dermal contact or inhalation exposures are of a minor importance with respect to the general population, although, occasionally, these routes may also play a role.

3In this paper, we attempt to review the data on OTA research from its discovery. The principal milestones in OTA research in 1965–1990, 1991–2000, and 2000–2015 are summarized in Figure 1, Figure 2 and Figure 3.

4Aspergillus ochraceus was the first producer of OTA ever identified. OTA was first discovered in corn meal intentionally inoculated with Aspergillus ochraceus. Then, in a survey on OTA occurrence, producing strains isolated from feedstuffs, 2/19 isolates of Aspergillus niger var. niger were able to produce OTA in medium containing 2% yeast extract and 15% of sucrose broth, and in maize cultures. This was the first report on the production of OTA by Aspergillus niger. Furthermore, Teren et al. (1996) tested 157 strains belonging to Aspergillus section Nigri for OTA production. OTA was also detected in the culture filtrates of 5/12 Aspergillus carbonarius strains and 3/100 isolates in the A. niger aggregate (A. foetidus and A. niger). OTA-producing Aspergillus species, A. carbonarius (and the closely related A. niger which produces OTA more rarely), grow well at high temperatures and produce pigmented hyphae and spores, making these species resistant to UV light. Consequently, A. carbonarius is commonly found in grapes and similar fruits that mature in sunlight and at high temperatures. The ability of Aspergillus tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes were described for the first time in 2005. New OTA-producing species of Aspergillus section, Circumdati A. westerdijkiae and A. steynii isolated from coffee, were discovered in 2004. Moreover, Samson et al. (2004) found new OTA-producing species in Aspergillus section Nigri—Aspergillus lacticoffeatus and A. sclerotioniger—which were also isolated from coffee.

5In 1969, Walbeek et al. isolated OTA from Penicillium viridicatum. Due to considerable revisions in taxonomy, particularly within the genus Penicillium, and ensuing difficulties in correct assignation, this identity has changed over the years. Several authors have drawn attention to the fact that isolates of Penicillium viridicatum as defined at that time could be now divided into three groups depending on their various properties, including growth rates, mycotoxin production, and source. Penicillium viridicatum isolates from group I grow rapidly, and they are first bright yellow green and turn forest green with age. They are mostly isolated from moldy grain but have not been found to produce either OTA or citrinin (CIT). P. viridicatum isolates from group II grow slowly and are yellow green both at maturity and in age. They are isolated from various plant sources, and produce both OTA and citrinin. P. viridicatum isolates from group III grow moderately quickly and turn brown with age. They come from meat or meatpacking plants in Europe. These latter isolates produce OTA when freshly isolated, but have not been found to produce citrinin. The taxonomy of P. viridicatum and P. verrucosum has been reviewed to clarify the conflict relating to the three P. viridicatum groups as laid down by Ciegler et al. (1973). It has been concluded that P. viridicatum group II corresponds to P. verrucosum and not to P. viridicatum, as indicated by Pitt (1979). Among species in subgenus Penicillium, only P. verrucosum is known to produce OTA. The main food habitat for P. verrucosum appears to be cereals growing in cool temperate zones, ranging across Northern and Central Europe and Canada. In 2001, Penicillium nordicum was determined and confirmed as the second OTA-producing Penicillium species along with P. verrucosum. Despite their shared ability to produce OTA, Larsen et al. (2001) claimed that the two species differ in several ways. P. nordicum and P. verrucosum occupy different ecological niches. OTA-producing isolates originating from plant-derived material are almost always contaminated by P. verrucosum, whereas OTA producers in meat or cheese are derived from P. nordicum. Under many laboratory conditions, P. nordicum produces more OTA than P. verrucosum isolates, and lack to produce citrinin.

6Table 1 and Table 2 provide an overview of the current identity of microfungi Aspergillus and Penicillium species, which are capable of producing OTA in foodstuffs.

7CAS name (Chemical Abstracts Services) Registry No.: 303-47-9.

8Chemical Abstracts: L-Phenylalanine, N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1-H-2-benzopyran-7-yl)carbonyl]-,(R)-.

9IUPAC name: (N-[[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl] carbonyl]-3-phenyl-l-alanine).

10Other name: (−)-N-[(5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl) carbonyl]-3-phenylalanine.

11Summary formula: C20H18 O6ClN.

12OTA consists of a para-chlorophenolic moiety containing a dihydroiso-coumarin group that is amide-linked to L–phenylalanine. See Figure 4 and Table 3 for structure of the OTA derivatives.

13Molecular Weight: 403.8.

14Chemical and physical properties of OTA were comprehensively described by Budavari (1989) and IARC (1993), its melting point was determined by van der Merwe et al. (1965) and Kuiper-Goodman and Scott (1989), and its optical rotation by Pohland et al. (1982). Spectroscopic data on OTA (as ultraviolet, infrared, mass spectral and proton nuclear magnetic resonance data) were reported by van der Merwe et al. (1965) and Pohland et al. (1982), OTA solubility (e.g., in chloroform, ethanol, methanol, xylene) by WHO (1990), and its stability (partial degradation under normal cooking conditions) by Müller (1982). OTA degradation was performed by treatment with an excess of sodium hypochlorite solution. Physico-chemical properties of OTA and the progress in their knowledge have been recently reviewed in great detail by Khoury and Atoui (2010). OTA is a weak acid with two pka (4 and 7).

15Table 3 described several derivatives occurring naturally or formed in the body after biotransformation. Some are hydroxylated, others lack phenylalanine moiety or are conjugated (e.g., with glutathione, glucuronic acid, sulfate, or pentose).

16The most recently discovered ones include a decarboxylated hydroquinone derivative, DC-OTHQ (often linked to glutathione).

17During coffee roasting (at 225 °C), 2′-DC-OTA and 2′R-OTA, two products of thermal degradation of OTA, were identified. Ochratoxin α amide, which is formed at high temperatures during coffee roasting, was discovered. This represents another product of thermal degradation of OTA.

18Principal methods developed for OTA determination in biological materials are summarized in Table 4.

19In fact, more sensitive analytical methods or new methods for determining OTA and ochratoxins in biological materials are being developed consecutively toward the sophisticated development of instrumentation and analytical techniques but also toward the improvement of laboratory analytical methods. The most used and traditional analytical techniques include thin-layer chromatography, HPLC, and ELISA. Therefore, in the present article, the analytical techniques are divided into traditional ones, and the others.

20Generally, all chemical methods for the analysis of OTA consist of several steps (extraction, clean-up, separation, detection, quantification, and confirmation of identity). Conventional sample extraction and clean-up are usually achieved by liquid extraction for OTA determination in kidneys of swine. More recently, solid-phase extractions (SPE) notably for OTA determination in animal feed and immunoaffinity columns (IAC) (/homemade of IAC/; immunoaffinity cartridges commercially available) have become popular. At present, different kinds of cartridges are commercially available for clean-up and pre-concentration, including IAC and molecular imprinted polymers (MIPs) cartridges, composed by anti-OTA antibodies and three-dimensional network specific for the target molecule. In this case, OTA passed through cartridges (e.g., Mycosep™ or Mycospin™). It is based on adsorption and the ion-exchange process. The use of immunoaffinity chromatography in the clean-up step improves mycotoxin analysis and has a number of advantages: clean extracts, precision and accuracy, rapidity, and reduction of the use of dangerous solvents. The main advantages of these columns are the specific binding of OTA onto the antibody and the near-complete removal of matrix interference. Nevertheless, in the case of OTA, underestimation can be observed if extraction is done in an alkaline condition, because OTA is converted into open-ring OTA (OP-OA) and no longer recognized by antibodies.

21The confirmation of OTA presence in biological materials is very important in order to guarantee quality of analytical results. Hult and Gatenbeck (1976) presented the OTA confirmation with carboxypeptidase A, as did Hunt et al. (1980) with boron trifuoride methanol and Studer-Rohr et al. (1995) with diazomethane. Quality assurance of analytical results (a laboratory accreditation, participation in proficiency testing, and the use of certified reference materials) according to the past norm EN 45001 (1989) and the recent norm which is in force EN ISO/IEC 17025 (2005) is very important for the purposes of OTA determination in biological materials.

22Many analytical methods for the determination of OTA have been developed over time, and most of them involve the use of thin-layer chromatography (TLC) and, predominantly, high-performance liquid chromatography (HPLC) with fluorescence detection (FLD). Subsequently, OTA is identified and detected by LC-MS, LC-MS/MS, aptamers, ELISA, and immunosensing methods. However, the technique most commonly used is based on liquid chromatography (LC) coupled with a fluorometric detector for highly sensitive detection signal. It is known that, due to natural OTA fluorescence, OTA is generally determined by chromatographic techniques.

23The other methods for the OTA determination used include gas chromatography–mass spectrometry (GC-MS), fluorometric kits (the immunoaffinity columns coupled with a fluorometer), fluorescence polarization immunoassay (PFIA), isotope dilution, and a radioimmunoassay (RIA); however, due to health hazards of radiolabeled compounds and specialized waste disposal, RIA has not been in use for a long time. More recent methods for OTA determination are inductively coupled plasma mass spectrometry ICP-MS, and capillary electrophoresis techniques: capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), micellar electrokinetic capillary chromatography/MEKC/, molecular imprint polymers (MIPs), biosensors, and aptamers (single-stranded oligonucleotides (DNA or RNA) selected in vitro to bind with high affinity and specificity to molecular targets). The applications of aptamers are known and developed, e.g., in chromatography, capillary electrophoresis, mass spectrometry, and biosensors.

24In 1969, Shotwell et al. with colleagues from the U.S. Department of Agriculture (USDA) published the very first piece of information about the amount of OTA in a maize sample at levels from 110 to 150 ng/g. In 1970, Scott et al. from Health Canada published data on OTA in moldy cereals, beans, and peanuts. OTA concentrations in wheat, oats, barley, and rye (62.0% positive samples) ranged from 30 to 27.000 ng/g. The occurrence of OTA in pig kidney was first mentioned by Hald and Krogh in 1972 and by Hunt et al. in 1979. Since that time, more than 90 kinds of foodstuffs of both plant and animal origins, including milk, have been found to contribute to the OTA dietary exposure.

25As for foodstuffs of plant origin, OTA occurs in cereal products, olives, beans, beer, wine, coffee, cocoa products, raisins, figs, licorice, pulses, pumpkin seeds, and tea. In general, the average concentration of OTA is reported to range from 0.1 to 100 ng/g. OTA concentration in black pepper, cayenne pepper, caraway, cardamom, coriander, chili powder, curcuma, and dried red pepper ranges from 1 to 100 ng/g. Feedstuffs of plant origin—those made of wheat, oats, barley, rye, maize, rice, millet, sorghum, soybean, horse bean, peas, bean, broad bean, alfalfa, sunflower or pumpkin seeds, coconut, peanut cake, and hay/silage—also contain from 1 to 100 ng/g of OTA.

26In foodstuffs of animal origin, e.g., in pork blood products, edible offal, pork meat, chicken meat and offal, and dry-cured ham, the levels of OTA range from 0.1 to 1 ng/g. The same amounts are measured in feedstuffs of animal origin, e.g., in pork kidney and liver, pork meat, chicken liver, and viscera, and in mechanically separated chicken used as ingredients in pet food for cats and dogs.

27Table 5, Table 6 and Table 7 summarize the recent data related to OTA in foodstuffs obtained from the EU Rapid Alert System for Food and Feed (RASFF). The RASFF dealt with OTA in 175 cases in 2000–2015.

28OTA has been found to cause porcine and poultry nephropathy. OTA is implicated in the pathogenesis of some renal diseases including Balkan endemic nephropathy (BEN), kidney tumors occurring in certain endemic regions of the Balkan Peninsula, and chronic interstitial nephropathy (CIN) occurring in Tunisia and other North African countries.

29Kidney lesions have been observed on proximal tubules. The epithelial cells were damaged, for example, membrane integrity was lost, and the size and the density of the brush border were reduced. The chromatin was condensed, and the nuclear envelope disappeared. The histologic picture shows an enlargement of tubular membrane and an apparition of collagen fibers.

30At the beginning, the BEN disease is characterized by a modification to epithelial cells without any change in the size of the organ. After chronic exposure, kidneys are reduced and interstitial fibrosis is the most important picture. At the end stage, impairment of renal function leads to enzymuria (e.g., gamma glutamyl transferase, alkaline phosphatase, lactate dehydrogenase), polyuria accompanied with red tongue, thirst, and bitter taste. Neither edema nor hypertension can be observed. Other symptoms such as headaches, lumbar pain, asthenia, and anemia (iron deficiency) were recorded. Several biochemical parameters changed including glycosuria, proteinuria (0.15–0.5 g/ 24 h), alkalinization of urine, elevated serum creatinine, and an increase in immunoglobulin M (IgM) and immunoglobulin E (IgE).

31Data on OTA nephrotoxicity are summarized in Table 8.

32Data on OTA carcinogenicity are summarized in Table 9.

33In 1976 and 1983, IARC first evaluated the carcinogenic risk that OTA poses toHuman. No report on cases of cancer or epidemiological studies were available at that time and, in the absence of adequate epidemiological data, no evaluation of the carcinogenicity of OTA with respect to Humans could have been made. In 1987, the IARC reclassified OTA into Group 3 (not classifiable for its carcinogenicity to humans). Based on a great amount of evidence of OTA carcinogenicity revealed in new animal studies, it was again reclassified into Group 2B (possibly carcinogenic to humans) in 1993. At present, new information regarding genotoxicity of OTA (formation of OTA-DNA adducts), its role in oxidative stress, and the identification of epigenetic factors involved in OTA carcinogenesis—should they indeed provide strong evidence that OTA carcinogenicity is mediated by a mechanism that also occurs in humans—could lead to another reclassification of OTA. In the light of recently available data, it does not seem inappropriate to upgrade its carcinogenicity from Group 2B (possibly carcinogenic to humans) to at least Group 2A (probably carcinogenic to humans) or, in our opinion, even to Group 1 (carcinogenic to humans).

34Biomonitoring of OTA provides the best approach to assess the human exposure to OTA from any source and through any route. The first studies reporting the presence of OTA in human blood were carried out in the Balkans in the 1970s. The exposure of the human population to OTA and other ochratoxins represents a worldwide problem. Baldwin et al. (2011) reviewed biomarker researches for the most important mycotoxins and defined biomarkers. Recently, a biomarker of exposure has been defined to be a biological measure which is correlated with the quantity of the xenobiotic ingested; resulting in the improved exposure classification in comparison with more traditional approaches. OTA in milk (non-invasive sampling), OTA in blood serum (invasive sampling), OTA in urine (non-invasive sampling), and OTA in human kidneys (sampling post-mortem or after nephroctomia) are qualified as biomarker of exposure to OTA. Soto et al. (2015) have recently used several biomarkers for evaluating the OTA exposure. The values of OTA detected in potential biomarkers of exposure for blood, breast milk, and urine ranged from 0.15 to 18.0, from 0.002 to 13.1, and from 0.013 to 0.2 ng/mL, respectively. The calculated EDI for OTA in plasma ranged from 0.15 to 26 ng/kg bw/day and has turned out to be higher than that obtained in urine (0.017 to 0.4 ng/kg bw/day). All these values have been correlated with the range of EDI for OTA calculated from food products: 0.0001–25.2 ng/kg bw/day.

35In 1979, OTA determination in human whole blood and serum was developed. In the past several decades, OTA has been detected in human blood samples on a worldwide scale. Scott (2005) has described OTA in blood serum as a uniquely useful biomarker of OTA exposure due to its high-affinity binding to serum albumin or to other small proteins, which should result in higher serum OTA levels and long persistence of OTA in blood serum. OTA blood amounts will integrate exposure over longer periods. The use of serum or plasma has been described as more suitable matrices in comparison to whole blood. Generally, the determination of OTA in blood samples remains the basic method of how to monitor human exposure to OTA, which is ubiquitous in human blood serum/plasma and indicates continuous exposure to the toxin, originating mainly from food intake.

36Table 10 describes some of the most notable findings of OTA in blood on a worldwide scale.

37Advantages arising from monitoring OTA in the blood of healthy persons consist mainly in relatively high OTA levels found compared with OTA determinations in urine. OTA blood determination will integrate exposure over longer periods, while biomarker analysis in urine apparently better reflects day–to-day variations in the exposure of adults and infants.

38Urine is a major excretion route for both OTA and OTα (5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydroisochromene-7-carboxylic acid; formula, see Table 3) in humans. OTA can be found in urine several days after OTA ingestion. The elimination of OTA through human urine has been reported to be low (mean value between 20 and 80 ng/day) and independent of the dose ingested. The OTA uptake has been described as dependent on the free OTA concentration, which is severely limited by the binding of OTA to serum albumin. Thus, the relationship between OTA in urine and OTA intake remains a complex issue as in the case of OTA in blood.

39The first study measuringing OTA in urine in Europe was conducted by Mac Donald et al. (2001) in the UK. In this study, OTA was found in 46 urine samples (92%) collected over 24 h from 50 volunteers (healthy individuals from the UK). OTA concentrations ranged from <10 to 58 ng/L, and the mean value was about 21 ng/L. This study demonstrated a strong correlation between OTA concentrations in urine and its dietary intake. The second study in Europe was conducted in Bulgaria by Castegnaro et al. (1991). A total of 152 urine samples collected from patients with BEN (Balkan endemic nephropathy) or urinary tract tumors (UTT) and from the control families were analyzed, and OTA was detected in about 33% of the samples of urine (more often in endemic villages than in nonendemic ones) in the range 5–604 ng/L and in healthy people in the range 5–43 ng/L (LOQ = 5 ng/L). In Europe, another one-month follow-up study of OTA in urine samples after a 24-h collection of urine from the inhabitants with BEN in Bulgaria (from 16 healthy volunteers from two villages located in the Vratza district with a high risk area for BEN; 5 of Gorno Pestehne, 11 of Beli Izvor) was conducted by Petkova-Bocharova et al. (2003). 98% of samples were positive and contained OTA in the range 10–1910 ng/L. The OTA mean value in Gorno Pestehne was 50.8 ng/L, and in Beli Izvor it was 168.6 ng/L. In a Czech study carried out in 2010, OTA was measured in a total of 236 samples of urine collected from healthy persons within a 24-h cycle (males/females, 45–60 years old, two samples per person from non-consecutive days, with at least a 14-day time difference). A total of 185 samples (78%) of these 236 samples were positive, with a limit of quantification (LOQ) of 2.0 ng/L, a mean of 7.32 ng/L, and a median of 4.47 ng/L. These data signalize the real exposure of the given population group to OTA, with a higher percentage of positive urine samples in men (92%) than women (65%).

40OTA was usually determined in morning urine (not 24-h urine) in these countries (see also Table 11). However, in exposure studies, it is recommended that urine is collected over 24 h—representative of the excretion throughout a day.

41The multibiomarker methods have been applied in several pilot studies to prove their applicability and to estimate mycotoxin exposure in the populations/individuals tested. The application of these methods resulted in advanced data on exposure patterns and revealed new findings on co-exposure to the mycotoxin combinations. In addition, it must be mentioned that urinary excretion mainly reflects the recent mycotoxin intake, whereas measurements in plasma/serum are more likely to reflect the long-term exposure. As a result of the advent of the latest generation of high-performance LC-MS/MS instruments, a clear trend toward the development and application of multianalyte methods in mycotoxin biomarker research can be observed. Warth et al. (2012) injected samples directly into the LC-MS/MS system to facilitate the quantification of 15 analytes. A method developed by Ediage et al. (2012) covered seven mycotoxins and several important conjugation and breakdown products (for a total of 18 analytes). In this study, OTA, OTα, and 4-OH OTA were measured in human volunteers. However, none of the target metabolites of OTA such as OTα or 4-OH OTA were confirmed in another study performed with urine samples in Cameroon, but the data correlate with similar findings reported for a Korean population. According to Munoz et al. (2010a), interindividual variability in the detoxification of OTA in human urine may account for the observed variations in urinary OTα, and the possibility cannot be excluded that a low rate of OTA detoxification is a characteristic of some human populations. The highest concentration of OTA reported so far in human urine was detected in Sierra Leone with a range of 70–148,000 ng/L, but no mean was reported. Table 11 summarizes the OTA detection in human morning urine around the world. Last but not least, in dietary studies carried out in Serbia, in addition to OTA, several OTA derivatives have been detected in urine (and in blood). A clear difference between men and women has been observed.

42As OTA is also excreted via human milk, breastfed children including babies are exposed to OTA as well. Nevertheless, OTA amounts in milk are reported to be much lower than concentrations of OTA in blood (down to 10 times). In Italy, OTA was detected in milk from healthy women with varying daily diets in different geographical regions. The relationship between OTA contamination of human milk and its dietary intake was examined, and it was confirmed that OTA occurrence in human milk was likely associated with maternal dietary habits. The strongest associations were observed with foodstuffs of plant origin and, to a lesser extent, with foodstuffs of animal origin.

43Table 12 summarizes data on OTA presence in human milk worldwide.

44In some countries, e.g., Egypt, Turkey, and Sierra Leone, OTA milk concentrations were found to be more than 100-fold higher in comparison with Europe (see Table 12). It can be concluded that, despite the fact OTA concentrations in milk compared with blood are much lower, OTA contamination of human breast milk presents a potentially serious health hazard.

45OTA presence in human tissues seems to be direct and definite proof of human exposure to OTA, although practicability of such measurements “in vivo” is obviously limited. Taking OTA’s nephrotoxicity in mind, in particular, there are not many studies available that have attempted to determine OTA in human kidneys. Several studies have been carried on the content of OTA in human kidneys, e.g., in Germany, in the Czech Republic in 30 samples of kidney (40% positive/detectable/samples; OTA ranged from 0.1 to 0.2 ng/g; mean 0.07 ng/g; results of OTA < 0.1 ng/g (LOQ) given as 1/2 limit of quantification = 0.05 ng/g), and in Poland in 19 samples of kidney (78.9% positive/detectable; OTA ranged from 0.15 to 0.39 ng/g with mean 0.26 ng/g). Several human kidneys samples (60) obtained from patients suffering from kidney (or urinary bladder) cancer from Bulgaria (8 samples), Serbia (10 samples), Croatia (16 samples), and France (18) have been analyzed up to now. Not only was OTA detected but also OTA derivatives such as OTHQ, OTHQ-GSH, 4-OH OTA, and OTB. Interestingly, DNA adducts were detected, and the nature of the DNA was in relation to the OTA derivatives. In Croatia, the DNA adducts profile of a farmer was similar to the profile of the pigs and poultry from his farm. It has been observed that the exposure has been higher in rural regions, and co-exposure to CIT and/or FB has been systematic.

46Due to its toxic properties, OTA is subject to legal regulation both on national and international levels. The toxicity of OTA became more or less evident by the end of the 1970s. A real debate on whether OTA in food and feed shall be regulated on a national or international level does not seem to predate the 1990s. This circumstance contrasts with the case of other mycotoxins, in particular, the aflatoxins (in the USA, the first limits for aflatoxins were established as early as the 1960s; soon after their discovery, the European Communities followed in the 1970s).

47For OTA, in 1991, van Egmond estimated that in 60 countries where some legal regulations with respect to mycotoxins existed, only 11 set limits on OTA (Brazil, Czechoslovakia, Denmark, France, Greece, Hungary, Israel, The Netherlands, Romania, Sweden, and the United Kingdom). In 2003, when a worldwide survey on legal regulation of mycotoxins was conducted by the FAO in cooperation with the Dutch Foreign Service, the number of countries with legal limits on OTA in food and feed grew to 37 (compared to more than 76 countries with legal limits for aflatoxins). No such large-scale survey has been reported ever since. However, it may be assumed that the number of countries where OTA presence in food and feed is subject to legal regulation is not lower now than it was in 2003 (see Figure 5). This assumption can be based on two major arguments. Firstly, since 2003, research has provided new data on OTA’s harmful effects to human and animal health. Secondly, due to the globalization of food and feed markets, discussion on how to tackle the health hazards linked to OTA (and other mycotoxins) has intensified on an international level and has had repercussions back on the national level. By way of example, China seems to have recently established limits on OTA in both food and feed.

48Membership of States in international or regional organizations may also contribute to adoption of legal regulations on OTA. For the time being, the binding maximum limits on OTA appear to exist only in the European Union (EU) (see infra). On the global level, debate on the feasibility of establishing the maximum limits on OTA has taken place at the Codex Alimentarius Commission (CAC), the joint intergovernmental body established by the FAO and WHO responsible for implementing the Joint FAO/WHO Food Standards Programme. After the Joint Food and Agricultural Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA), an expert body which provides scientific advice to the CAC repeatedly dealt with OTA in 1991, 1995, 2001, and 2007, the maximum limit of 5 µg/kg with respect to wheat, barley, and rye has been recently established under the Codex General Standard for Contaminants and Toxins in Food and Feed. In addition, there are four codes of practice that aim at the prevention and reduction of OTA contamination in cereals, wine, coffee, and adopted between 2007 and 2014. Although the Codex Alimentarius standards are not per se binding, their importance stems especially from the fact the World Trade Organization (WTO) considers the measures taken by its Member States in conformity with the Codex Alimentarius standards to be science-based, appropriate, and nondiscriminatory under the WTO Agreement on the Application of Sanitary and Phytosanitary Measures signed in 1994 and thus does not treat them as breaches of world trade rules.

49As far as the existing limits on OTA are concerned, those of the EU are generally assessed to be the most comprehensive and detailed.

50As for the limits on OTA in food, these were first established on the EU level by the Commission Regulation (EC) No 472/2002 of 12 March 2002 amending Regulation (EC) No 466/2001 setting maximum levels for certain contaminants in foodstuffs (see Table 13). As the Regulation No 466/2001 was repeatedly amended, in 2006, it was replaced by completely a new act, Commission Regulation (EC) No 1881/2006 of 19 December 2006, setting maximum levels for certain contaminants in foodstuffs. The adoption of Regulation No 1881/2006 was based on the scientific opinion of the Scientific Panel on contaminants in the Food Chain of the EFSA adopted on 4 April 2006, which updated the earlier opinion of the Scientific Committee on Food on OTA adopted on 17 September 1998.

51In the EU, the Regulation 1881/2006 remains in force today, although it has been amended nearly 26 times. As of February 2016, the Regulation No 1881/2006 sets the maximum limits on OTA not only in cereals (both in the unprocessed cereals and cereal products) but in a wide variety of other food commodities as well (see Table 14). These limits are legally binding on all 28 EU Member States, which are obliged to apply these rules in full.

52Apart from setting binding limits on OTA in food, since 2002, the EU has also unified the methods of sampling and analysis for purposes of the official control of the levels of mycotoxins in foodstuffs performed by the authorities of the Member States (first by the Commission Directive 2002/26/EC of 13 March 2002, later replaced by the Commission Regulation (EC) No 401/2006 of 23 February 2006 which remains in force today).

53As for OTA in feed, however, up to now, only a non-binding recommendation exists with respect to cereal feed, and feed for pigs and poultry on the EU level (Commission Recommendation 2006/576/EC of 17 August 2006 on the presence of deoxynivalenol, zearalenone, OTA, T-2 and HT-2, and fumonisins in products intended for animal feeding). For details, see Table 15.

54There are, however, approaches to legal regulation of OTA other than establishing and enforcing the binding maximum limits on OTA in food and feed commodities as in the EU. Most notably, no binding limits on OTA in food or feed exist in the USA. Even more strikingly, no advisory or regulatory action limits have been established by the US authorities. Instead, the US Food and Drug Administration (FDA), acting under the Federal Food, Drug and Cosmetic Act (FFDCA), has instead consistently relied on laying down good agricultural and manufacturing practices and on requiring the implementation of food safety plans in food industry undertakings. In extension, the FDA monitors the compliance with these practices and the presence of OTA in domestic and imported foods (Food Compliance Programme No 7307.001 entitled “Mycotoxins in Domestic and Imported Foods”). An approach analogous to that of the USA has been adopted by a range of other countries such as Australia, Canada, and Japan.

55For some authors, the US approach to regulating mycotoxins including OTA is clearly preferable because it is seen as an option that might “diffuse trade frictions, and at the same time help reduce economic losses from mycotoxin contamination and divergent standards”. The truth is that the US approach seems to exert a non-negligible influence on the international level, e.g., within the CAC, which has, as mentioned above, adopted four codes of good practice with the aim of reducing the OTA occurrence in several food commodities that are commercially important.

56To sum up, 50 years after the discovery of OTA, differences in how to legally regulate mycotoxins including OTA are still marked. However, even in an era when further liberalization of world trade is envisaged (e.g., a project of the Transatlantic Trade and Investment Partnership and the TTIP between the USA and the EU), due to economic and political controversies linked to the existing policies on mycotoxins, it cannot be expected that some harmonized approach to legally regulating mycotoxins including OTA will be easily established on a global level.

57OTA is ubiquitously found all over the world in many foodstuffs and feedstuffs. OTA is recognized for its nephrotoxicity and, to date has been identified as one of the most potent renal carcinogens in rodents ever studied by the National Cancer Institute/National Toxicological Program (NCI/NTP). OTA is deleterious for the pig and poultry industries. For human beings, many authors consider it to be the main contributor in the pathogenesis of Balkan endemic nephropathy and some nephropathies in other parts of the world.

58The development of effective strategies alleviating OTA-induced toxicity is very complex because the mechanism of action of OTA is still unclear. The toxic effect is the result of many effects such as the inhibition of protein synthesis, direct genotoxicity, and cell cycle arrest. Inhibition of OTA uptake and stimulation of OTA elimination of the body preventing OTA accumulation will be promising approaches.

59Since its discovery in 1965, numerous studies have been performed with respect to OTA, which have permitted the establishment of different mechanisms for OTA nephrotoxicity and carcinogenicity (summarized in Figure 6 and Figure 7). The mechanisms leading to OTA nephrotoxicity, its hepatotoxicity and immunotoxicity, can be linked to inhibition of protein synthesis, lipoperoxidation, and modulation of MAP kinase cascade (Figure 6), whereas its carcinogenicity arises after the metabolic activation of OTA in a way similar to pentachlorophenol derivatives (Figure 7).

60OTA forms covalent DNA adducts through radical and benzoquinone intermediates. The OTHQ metabolite of OTA can undergo an autoxidative process to generate the quinone electrophile OTQ that reacts with DNA. In addition, the formation of OTQ or phenoxy and aryl radicals can lead to increased ROS production that causes cytotoxicity. Radical species generate a C-bound C8-dG adduct, while benzetheno-type DNA adducts are expected from the quinone electrophile. The quinone-type adducts form faster in cells and stem from P450 activation of OTA. The C-bound C8-OTA adduct forms at a slower rate and is predicted to stem from reductive dehalogenation of OTA (via GSH and cyclooxygenase or lipoxygenase). The C5-Cl atom is critical for DNA adduction (genotoxicity) but not for cytotoxicity (OTB is cytotoxic but not genotoxic) (Figure 7).

61Several quinone derivatives have been isolated from blood and urine and also in human or animal tissues exposed to OTA. The OTB-dG adduct is consistently found by 32P-postlabeling in kidney DNA from OTA-treated rats, pigs, and humans. These metabolites and this adduct could serve as biomarker for OTA exposure.

62Increases in carcinogenicity and genotoxicity during co-exposure with citrinin (CIT), fumonisin (FB), or both can be explained by both factors. FB and CIT induce COX2, thus favoring the biotransformation of OTA into a genotoxic compound. Moreover, the quinone methide structure of CIT could easily explain the generation of DNA adduct. It may be capable of oxidizing OTA into the phenoxyl radical to promote C-C8 adduct formation. The new findings on OTA mutagenicity favor direct genotoxicity and rule out oxidative DNA damage as a contributor to the induction of deletion mutations or renal carcinogenesis. Therefore, further research should focus on co-exposure.

63Altogether, OTA is a complete carcinogen, active since the earliest stage of life. Intake evaluation based on real analysis shows that the daily intake was three times greater than the virtual safety dose of 4 ng/kg bw/day—against carcinogenicity (intake per day 648 ng/60 kg adult).

64Maternal-fetal risk assessment of OTA during pregnancy was conducted using the benchmark dose approach for genotoxic carcinogens. Considering the sensitivity of a fetus, risk reduction is a high priority. It is essential to keep exposure to OTA as low as possible in women, notably during pregnancy.

65Among the professional community, it is agreed that OTA is one of the five most agriculturally important mycotoxins; therefore, continued attention must be paid to research on ochratoxins and OTA in order to elucidate their metabolism, genotoxicity, and mechanism of action for renal carcinogenicity, with the ultimate aim of protecting public health and preventing economic losses.