As the term “masked mycotoxins” encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, “modified mycotoxins” into “biologically modified” and “chemically modified” with all variations of metabolites of the former and dividing the latter into “thermally formed” and “non-thermally formed” ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term “modified mycotoxins” should be used in the future and the term “masked mycotoxins” to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.

The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumor formation is unknown and conflicting results have been obtained regarding the potential of OTA to bind to DNA. OTA is poorly metabolized, and no reactive intermediates capable of interacting with DNA have been detected in vitro or in vivo. Recently, a hydroquinone/quinone redox couple and a carbon-bonded OTA-deoxyguanosine (OTA-dG) adduct formed by electrochemical oxidation and photoreaction of OTA have been reported and suggested to be involved in OTA carcinogenicity. This study was designed to characterize the role of DNA binding and to determine if formation of these derivatives occurs in vivo and in relevant activation systems in vitro using specific and sensitive methods. Horseradish peroxidase activation of OTA and its dechlorinated analogue ochratoxin B (OTB) yielded ochratoxin A-hydroquinone (OTHQ), but the postulated OTA-dG adduct was not detectable using LC-MS/MS. In support of this, no OTA-related DNA adducts were observed by 32P-postlabeling. In vivo, only traces of OTHQ were found in the urine of male F344 rats treated with high doses of OTA (2 mg/kg body wt) for 2 weeks, suggesting that this metabolite is not formed to a relevant extent. In agreement with the in vitro data, OTA-dG was not detected by LC-MS/MS in liver and kidney DNA extracted from treated animals. In addition, DNA binding of OTA and OTB was assessed in male rats given a single dose of 14C-OTA or 14C-OTB using accelerator mass spectrometry, a highly sensitive method for quantifying extremely low concentrations of radiocarbon. The 14C content in liver and kidney DNA from treated animals was not significantly different from controls, indicating that OTA does not form covalent DNA adducts in high yields. In summary, the results presented here demonstrate that DNA binding of OTA is not detectable with sensitive analytical methods and is unlikely to represent a mechanism for OTA-induced tumor formation.

The European Commission asked EFSA to update their 2006 opinion on ochratoxin A (OTA) in food. OTA is produced by fungi of the genus Aspergillus and Penicillium and found as a contaminant in various foods. OTA causes kidney toxicity in different animal species and kidney tumours in rodents. OTA is genotoxic both in vitro and in vivo; however, the mechanisms of genotoxicity are unclear. Direct and indirect genotoxic and non‐genotoxic modes of action might each contribute to tumour formation. Since recent studies have raised uncertainty regarding the mode of action for kidney carcinogenicity, it is inappropriate to establish a health‐based guidance value (HBGV) and a margin of exposure (MOE) approach was applied. For the characterisation of non‐neoplastic effects, a BMDL10 of 4.73 μg/kg body weight (bw) per day was calculated from kidney lesions observed in pigs. For characterisation of neoplastic effects, a BMDL10 of 14.5 μg/kg bw per day was calculated from kidney tumours seen in rats. The estimation of chronic dietary exposure resulted in mean and 95th percentile levels ranging from 0.6 to 17.8 and from 2.4 to 51.7 ng/kg bw per day, respectively. Median OTA exposures in breastfed infants ranged from 1.7 to 2.6 ng/kg bw per day, 95th percentile exposures from 5.6 to 8.5 ng/kg bw per day in average/high breast milk consuming infants, respectively. Comparison of exposures with the BMDL10 based on the non‐neoplastic endpoint resulted in MOEs of more than 200 in most consumer groups, indicating a low health concern with the exception of MOEs for high consumers in the younger age groups, indicating a possible health concern. When compared with the BMDL10 based on the neoplastic endpoint, MOEs were lower than 10,000 for almost all exposure scenarios, including breastfed infants. This would indicate a possible health concern if genotoxicity is direct. Uncertainty in this assessment is high and risk may be overestimated.

No abstract

Ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rats, but the mechanism of OTA tumorigenicity is unknown. Ochratoxin A has been shown to be negative in many genetic toxicology test in vitro. However, the potential of OTA to induce genotoxic effects has not been investigated in male rats, the most sensitive species for OTA-induced tumor formation. In this study, male F344 rats were repeatedly administered OTA (0, 250, 500, 1000, and 2000 microg/kg of body wt) or the non-chlorinated analogue ochratoxin B (OTB; 2000 microg/kg of body wt) for 2 weeks (5 days/week), and DNA breakage was analyzed in target and nontarget tissues using the comet assay both in the absence and presence of formamidopyrimidine-DNA (Fpg) glycosylase. Potential DNA-adduct formation was also analyzed in the target organ kidney by 32P-postlabeling using two different solvent systems. DNA-strand breaks were evident in liver, kidney, and spleen of animals treated with OTA, and a similar degree of DNA damage was observed in rats treated with OTB, despite the lower toxicity of OTB. Moreover, the presence of DNA damage did not correlate with histopathological alterations, which were evident in the kidney but not in the liver. In liver and kidney, the extent of DNA damage was further enhanced in the presence of Fpg glycosylase, which is known to convert oxidative DNA damage into strand breaks, suggesting the presence of oxidative DNA damage. Oxidative DNA damage as a mechanism of OTA-dependent DNA damage is consistent with the absence of lipophilic DNA adducts as assessed by 32P-postlabeling analysis. No spots indicative of OTA-related DNA adducts were observed in kidney DNA extracted from OTA-treated animals by 32P-postlabeling analysis, despite the use of synthetic standard for postulated adducts. A small, but not significant, increase in the incidence of chromosomal aberrations (essentially chromatid and chromosome-type deletions) was observed in splenocytes from rats treated with OTA in vivo and subsequently cultured in vitro to express chromosomal damage. These aberrations are also compatible with oxidative DNA lesions since they are not typically caused by chemical carcinogens which form covalent DNA adducts. Together, with the lack of evidence for formation of lipophilic DNA adducts as assessed by postlabeling, these data suggest that OTA may cause genetic damage in both target and nontarget tissues independent of direct covalent binding to DNA.