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OCHRATOXIN A

First draft prepared by

Diane Benford1, Catherine Boyle1, Wolfgang Dekant2, Radovan Fuchs3, David W. Gaylor4, Gordon Hard5, Douglas B. McGregor6, John I. Pitt7, Radovan Plestina3, Gordon Shephard8, Michelle Solfrizzo9, Philippe J.P.Verger10, Ronald Walker11

1 Food Standards Agency, London, United Kingdom

2 University of Würzburg, Würzburg, Germany

3 Institute for Medical Research and Occupational Health, Zagreb, Croatia

4 Science International, Little Rock, Arkansas, USA

5American Health Foundation, Valhalla, New York, USA

6Lyon, France

7Food Science Australia, North Ryde, New South Wales, Australia

8Medical Research Council, Tygerberg, South Africa

9Consiglio Nazionale delle Ricerche, Bari, Italy

10Institut National de la Recherche Agronomique, Paris, France

11University of Surrey, Guildford, United Kingdom

Explanation

Biological data

Biochemical aspects

Absorption, distribution, and excretion

Absorption

Distribution

Excretion

Biotransformation

Effects on enzymes and other biochemical parameters

Toxicological studies

Acute toxicity

Short-term studies of toxicity

Long-term studies of toxicity and carcinogenicity

Genotoxicity

Reproductive toxicity

Special studies

Covalent binding to nucleic acids and/or proteins

Immunotoxicity

Neurotoxicity

Nephrotoxicity

Mechanism of tumorigenesis

Mechanisms of cytotoxicity

Effects on the male reproductive system

Observations in domestic animals and veterinary toxicology

Observations in humans

Biomarkers of exposure

Biomarkers of effect

Epidemiological studies

Analytical methods

Screening tests

Conclusions

Effects of processing

Levels and patterns of contamination of food commodities

Ochratoxin A-producing fungi

Aspergillus species that produce ochratoxin A

Penicillium species that produce ochratoxin A

Physiology and ecology of fungi that produce ochratoxin A

Results of surveys

Distribution

Annual variation

Food consumption and dietary intake assessments

National and regional estimates of intake

Occurrence of achratoxin A in foods

Consumption of potentially contaminated foods

Biomarkers of exposure

Assessment of intake at the international level

Impact of alternative maximum limits on intake

Prevention and control of ochratoxin A production

By Aspergillus ochraeus

By Aspergillus carbonarius

By Penicillium verrucosum

Comments

Evaluation

References

Appendix A. Results of surveys

1. EXPLANATION

Ochratoxin A was evaluated by the Committee at its thirty-seventh meeting (Annex 1, reference 94), when it established a provisional tolerable weekly intake (PTWI) of 112 ng/kg bw, on the basis of deterioration of renal function in pigs, for which the lowest-observed-effect level (LOEL) was 0.008 mg/kg bw per day, and a safety factor of 500. At that time, the Committee recommended that further studies be conducted to elucidate the role of ochratoxin A (and other mycotoxins) in causing nephropathy in pigs and humans, the mechanisms of induction of tumours, and the role of phenylalanine in antagonizing the adverse effects of ochratoxin A. (The present Committee noted that the adverse effects noted at the thirty-seventh meeting consisted of nephrotoxicity.) Ochratoxin A was re-evaluated by the Committee at its forty-fourth meeting (Annex 1, reference 116), when it considered toxicological data that had become available since the previous evaluation, including studies on the epidemiology of nephropathy, on genotoxicity and on experimental nephrotoxicity. At that meeting, the Committee reconfirmed the PTWI, rounding it to 100 ng/kg bw, and reiterated its request for further studies on ochratoxin A.

The Codex Committee on Food Additives and Contaminants at its Thirty-first Session requested the Expert Committee to perform a risk assessment of the consequences of establishing a maximum level of 5 or 20 ΅g/kg in cereals and cereal products.

Ochratoxin A is produced by a single Penicillium species, P. verrucosum, by Aspergillus ochraceus and several related Aspergillus species, and by A. carbonarius, with a small percentage of isolates of the closely related A. niger. These three groups of species differ in their ecological niches, in the commodities affected, and in the frequency of their occurrence in different geographical regions. P. verrucosum grows only at temperatures below 30 °C and at water activity as low as 0.8. It is therefore found only in cool temperate regions; it is the source of ochratoxin A in cereals and cereal products in Canada and Europe. As cereals are widely used in animal feeds in Europe, and ochratoxin A is relatively stable in vivo, this mycotoxin is also found in some animal products in that region, especially in pig kidney and liver. As P. verrucosum does not occur in the tropics and subtropics, cereals from these regions are unlikely to contain ochratoxin A from this source. A. ochraceus grows at moderate temperatures and at a water activity above 0.8. It is found sporadically in a wide range of stored food commodities, including cereals, but is seldom the cause of substantial concentrations of ochratoxin A. It may also infect coffee beans during sun-drying and is a source of ochratoxin A in green coffee beans. A. carbonarius grows at high temperatures and is associated with maturing fruits, especially grapes. Because of its black spores, it is highly resistant to sunlight and survives sun-drying. It is the source of ochratoxin A in fresh grapes, dried vine fruits, and wine; it is also one source of ochratoxin A in coffee.

The Committee considered several new studies that had become available since the previous evaluation of ochratoxin A. These included further studies of absorption, distribution (including secretion into the milk of experimental animals), metabolism, and excretion; biochemical studies; toxicological studies on genotoxicity, immunotoxicity, neurotoxicity, embryotoxicity, and hepatotoxicity; and studies on the mechanisms of cytotoxicity and nephrotoxicity. The results of epidemiological studies were also reviewed. New data from surveys of food commodities for ochratoxin A and of food consumption were also considered, and intakes were estimated for various countries and regions of the world.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.1.1 Absorption, distribution, and excretion

(a) Absorption

It has been suggested that, in most species, ochratoxin A is absorbed from the stomach as a result of its acidic properties (pKa = 7.1) (Galtier, 1978; Roth et al., 1988). In studies of animals with ligated gastrointestinal loops, however, the small intestine was found to be the major site of absorption, with maximal absorption from the proximal jejunum. Absorption from the jejunum can take place against a concentration gradient and depends on the pH at the mucosal surface of the jejunum. Ochratoxin A that is so transferred is lipid-soluble and non-ionized (Kumagai & Aibara, 1982; Kumagai, 1988).

The results of studies in which a low dose of [3H]ochratoxin A was given by intubation to mice were interpreted by the authors as indicating rapid absorption from the stomach, but they could also be interpreted as showing that intestinal absorption is the major route, with rapid transit from the stomach to the intestine. Secondary peaks of ochratoxin A found in the intestinal contents and serum may have been a consequence of enterohepatic circulation, since the biliary excretion of this toxin is very efficient (Fuchs et al., 1988a; Roth et al., 1988).

The overall percentage of ochratoxin A absorbed was 66% in pigs, 56% in rats, 56% in rabbits, and 40% in chickens (Suzuki et al., 1977; Galtier et al., 1981).

In male Wistar rats that received a single intratracheal dose of crystalline ochratoxin A (purity unknown) at 50 ng/g bw, absorption from the lungs was found to be very efficient, the bioavailability being calculated as 98%. The biological half-life of ochratoxin A was estimated to be 127 h. The toxicokinetics of the toxin when given intratracheally, orally, or intravenously were comparable (Breitholtz-Emanuelsson et al., 1995).

Phenylalanine given to mice by gavage with ochratoxin A in a 10:1 molar ratio appeared to increase the absorption of ochratoxin A from the stomach and intestine and to increase gastrointestinal transit. This resulted in an eightfold higher concentration of ochratoxin A in serum and and a fourfold higher concentration in liver during the next 12 h (Roth et al., 1988).

b) Distribution

The bioavailability of ochratoxin A, estimated from a comparison of the maximal serum concentration after oral and intravenous administration, was very low in fish but 44 and 97% for two mammalian species investigated (Hagelberg et al., 1989). Once it reaches the blood, ochratoxin A bound readily to serum albumin (Galtier et al., 1980) and other macromolecules (Hult & Fuchs, 1986). Erythrocytes contained only traces (Galtier, 1978).

The association constant for the binding of ochratoxin A to serum albumin was 7.1 Χ 104 per mol for pigs, 5.1 Χ 104 per mol for chickens, and 4.0 Χ 104 per mol for rats (Galtier et al., 1981). The fraction of ochratoxin A bound to serum albumin and other macromolecules constitutes a mobile reserve of mycotoxin that can be made available for release to the tissues for a long time (Galtier, 1978; Hult et al., 1982). Studies with albumin-deficient rats showed that the main effect of ochratoxin A binding to serum albumin is to retard its elimination by limiting its transfer from the bloodstream to hepatic and renal cells (Kumagai, 1985).

In studies of the stability of ochratoxin A bound to porcine albumin, it was displaced by the acidic drug phenylbutazone, so that more free toxin was available. In male rats, ochratoxin A was more toxic in the presence of phenylbutazone, with a significant decrease in the LD50 value from 33 to 21 mg/kg bw (Galtier et al., 1980).

Ochratoxin A had strong affinity for an unidentified serum macromolecule (relative molecular mass, 20 000), with association constants of 2.3 Χ 1010 per mol in human serum and 0.59 Χ 1010 per mol in porcine serum. The specific binding of this macromolecule was saturated at concentrations of ochratoxin A of 10–20 ng/ml serum. Significant amounts of serum albumin were bound at higher concentrations of ochratoxin A, with saturation above several hundred micrograms per millilitre of serum (Stojkovic et al., 1984; Hult & Fuchs, 1986).

The fraction of ochratoxin A that remained unbound to two identified plasma proteins was 0.02% in humans and rats, 0.08% in monkeys, 0.1% in mice and pigs, and 22% in fish (Hagelberg et al., 1989).

Once ochratoxin A has been absorbed, the concentrations of the toxin and its metabolites in tissues and plasma residues depend on the length of feeding, the dose, whether the ochratoxin A is naturally occurring or crystalline, the route, the degree of serum binding, the half-life of ochratoxin A, and the duration on an ochratoxin A-free diet before sacrifice. These factors are important in assessing the natural occurrence of residues in animal tissues (Kuiper-Goodman & Scott, 1989).

After a single oral dose, the maximum serum concentrations of ochratoxin A were found within 10–48 h in pigs and rats (Suzuki et al., 1977; Galtier, 1978; Galtier et al., 1981; Mortensen et al., 1983a), at 2–4 h in ruminant calves (Sreemannarayana et al., 1988), after 1 h in rabbits, and after 0.33 h in chickens (Galtier et al., 1981). Maximum concentrations in tissues were found within 48 h in rats.

Wide species differences have been reported in the serum half-life of ochratoxin A. The half-life after oral administration was found to be 510 h in Macaca mulata monkeys (Hagelberg et al., 1989), 72–120 h in pigs (Galtier et al., 1981; Mortensen et al., 1983a), 77 h in pre-ruminant calves (Sreemannarayana et al., 1988), 55–120 h in rats (Galtier et al., 1979; Ballinger et al., 1986; Hagelberg et al., 1989), 6.7 h in quail (Hagelberg et al., 1989), and 4.1 h in chickens (Galtier et al., 1981). In those species tested, the serum half-time was longer after intravenous administration (Hagelberg et al., 1989), perhaps due in part to differences in absorption (Galtier et al., 1981), differences in peak plasma concentrations (see above), and species differences in the degree of binding to serum macromolecules, including albumin.

The rate of disappearance of ochratoxin A was slower from blood than from kidney, liver, and other tissues in pigs (Hult et al., 1979).

Whole-body autoradiography of mice after a single intravenous dose of [14C]ochratoxin A at approximately 200 ΅g/kg bw showed that the toxin persisted for > 4 days in the blood, interpreted as showing that the toxin is present mainly in bound form at this dose (Fuchs et al., 1988a). In a similar experiment in rats, the distribution after 24 h was greatest in lung and, in decreasing order, in adrenal medulla, skin, liver, myocardium, kidney, salivary gland, adrenal cortex, muscle, gastric mucosa, and bone marrow (Breitholtz-Emanuelsson et al., 1992). The tissue distribution in pigs, rats, chickens, and goats generally followed the order kidney > liver > muscle > fat (Harwig et al., 1983) or kidney > muscle > liver > fat (Mortensen et al., 1983a; Madsen et al., 1982).

In hens fed ochratoxin A, none was found in eggs (Krogh et al., 1976). In another study, it was found in eggs when the birds were fed 10 mg/kg bw (Juszkiewicz et al., 1982). A study of the tissue distribution of [14C]ochratoxin A in laying Japanese quail showed specific retention of unidentified radiolabel as a ring-shaped deposition in eggs, indicating that the toxin could be deposited over a short period (Fuchs et al., 1988b). Egg-laying Japanese quail were given a single oral dose of 0, 1, 5, or 20 mg/kg bw. The concentrations of ochratoxin A in abdominal yolk of birds 6 h later were 13 ΅g/kg in those given 5 mg/kg bw and and 34 ΅g/kg in those given 20 mg/kg bw. The toxin was still present in abdominal yolks 4 days after administration, and the mean concentration was 10-fold higher than in whole eggs. No ochratoxin A was found in eggs of birds given 1 mg/kg bw (Piskorska-Pliszczynska & Juszkiewicz, 1990).

Lactating rats, treated orally with a single dose of ochratoxin A up to 250 ΅g/kg bw excreted the toxin in their milk. The milk:blood concentration ratio was 0.4 at 24 h and 0.7 at 72 h. A linear relationship was found between the concentration of ochratoxin A in the dam’s milk and that in the blood and kidneys of pups at 72 h. The pup milk:blood concentration ratio was approximately 1.7. At 72 h, the suckling pups had higher concentrations of ochratoxin A than their dams in both blood and kidney (Breitholtz-Emanuelsson et al., 1993a).

Whole-body autoradiography after intravenous administration of high doses of [14C]ochratoxin A showed that it could cross the placenta more readily when given on days 8 and 9 than day 10 of gestation, with radiolabel appearing within 20 min in the uterine wall, placenta, and fetal tissues. Ochratoxin A given to mice on day 17 of gestation resulted in very little radiolabel in fetuses (Appelgren & Arora, 1983a,b).

Differences in fetal uptake of ochratoxin A at different times during gestation were suggested to be due to differences in the placenta, which was considered to be completely developed by day 9 of gestation. After intraperitoneal injection of ochratoxin A on day 11 or 13 of gestation, residues appeared more slowly and reached maximum values 30–48 h after dosing. The concentrations in the placenta were high 2–6 h after injection and then decreased more slowly than from other tissues. The serum half-life of ochratoxin A was 29 h at day 11 and 24 h at day 13 of gestation. The authors considered the embryo to be a ‘deep compartment’ (Fukui et al., 1987).

A group of 39 female Sprague-Dawley rats received ochratoxin A orally at 50 ΅g/kg bw five times a week for 2 weeks before mating, during gestation, and then 7 days a week during lactation. Pups from ochratoxin A-treated dams were cross-fostered at birth to control dams and vice versa. Treatment did not affect maternal body weight nor alter the birthweight or development of pups. The concentrations of ochratoxin A in the blood and kidney of exposed pups were three to four times higher than those in the dams. No differences in weight gain or in body or kidney weight were seen between pups exposed in utero, via lactation, or both. The transfer of ochratoxin A to milk was very efficient (60% of the blood concentration at 8 weeks). The highest blood and kidney concentrations were found in offspring exposed in utero and via milk, but the most significant exposure was via milk (Hallén et al., 1998).

After subcutaneous administration of [3H]ochratoxin A to rats on day 12 of gestation, fetal uptake was delayed, with maximum concentrations 48–72 h after dosing, representing about 0.1% of the dose administered (Ballinger et al., 1986).

Four lactating Blanc de Termonde rabbits received ochratoxin A from feed naturally contaminated at 190 ng/g, equivalent to 16 ΅g/kg bw, on days 3–19 of lactation. The toxin was effectively transported from blood to milk and subsequently to the offspring. Higher concentrations were found in maternal plasma than in milk, and a linear relationship was found between the concentrations in milk and plasma of offspring. The plasma:kidney concentrations were much higher in offspring than in adults, perhaps due to slower detoxication in the former (Ferrufino-Guardia et al., 2000).

Ochratoxin A given at 0.38 mg/kg bw to pregnant sows on days 21–28 of pregnancy did not cross the placenta (Patterson et al., 1976). Similarly, no residues were found in piglets of sows fed diets containing ochratoxin A at 7–16 ΅g/kg bw per day throughout gestation (Mortensen et al., 1983b). In a more recent study, however, ochratoxin A was transmitted to six piglets in utero when the sow was fed naturally contaminated feed; the blood concentrations in the newborn piglets were 0.075–0.12 ng/ml, whereas that in the sow was 0.20 ng/ml (Barnikol & Thalmann, 1988).

(c) Excretion

Both biliary excretion and glomerular filtration play important roles in the plasma clearance of ochratoxin A in rats. This is related to its relative molecular mass of 403.8, since both pathways are used in this species for substances with relative molecular masses between 350 and 450. Thus, in rats, both the urinary and faecal excretory routes are important, the relative contribution of each depending on factors such as route of administration and dose (Kuiper-Goodman & Scott, 1989).

In all species, the relative contribution of each excretory route is also influenced by the degree of serum macromolecular binding and differences in the degree of enterohepatic recirculation of ochratoxin A (Hagelberg et al., 1989).

In rats, the major excretory products were ochratoxin alpha (in urine and faeces), ochratoxin A, and the 4R-OH-ochratoxin A epimer. In urine, these represented 25–27%, 6%, and 1–1.5% of the administered dose, respectively (Storen et al., 1982a).

Up to 33% of the radiolabel on an orally administered dose of ochratoxin A was excreted into the bile of rats up to 6 h after dosing; only trace amounts of ochratoxin alpha were detected in the bile (Suzuki et al., 1977).

Biliary excretion of ochratoxin A was increased and urinary excretion of ochratoxin A and ochratoxin alpha was decreased in mice pretreated with phenobarbital (Moroi et al., 1985).

When ochratoxin A was administered to rats intraperitoneally, only traces of ochratoxin A and ochratoxin alpha were identified in faeces, whereas after oral administration 12% ochratoxin A and 9% ochratoxin alpha were found in faeces (Storen et al., 1982a).

In pre-ruminant and ruminant calves, 85–90% of orally administered ochratoxin A was excreted as ochratoxin alpha, most of it in the urine (Sreemannarayana et al., 1988).

2.1.2 Biotransformation

Ochratoxin A is hydrolysed to the non-toxic ochratoxin alpha at various sites. In rats, detoxication by hydrolysis to ochratoxin alpha is a function of the bacterial microflora of the caecum (Galtier, 1978). The enzymes responsible for hydrolysis to ochratoxin alpha in cows and rodents are carboxypeptidase A and chymotrypsin (Pitout, 1969a,b; Pitout & Nel, 1969). Other mycotoxins such as penicilloic acid inhibit this reaction (Parker et al., 1982). Inhibition of the flora of the lower gastrointestinal tract of rats by neomycin reduced hydrolysis of ochratoxin A to ochratoxin alpha and increased the blood concentration of ochratoxin A (Madhyastha et al., 1992).

Studies with rat tissue homogenate showed that the duodenum, ileum, and pancreas also have a high capacity to carry out this reaction, whereas the activity in the liver and kidney was low (Suzuki et al., 1977). It was non-existent in rat hepatocytes (Hansen et al., 1982) and rabbit and rat liver (Kanisawa et al., 1979; Stormer et al., 1983).

In rats given [14C]ochratoxin A, most of the radiolabel was attached to ochratoxin A, indicating that efficient metabolism of this toxin is lacking in most tissues other than the intestine (Galtier et al., 1979).

Incubation of the contents of the four stomachs of cows indicated effective hydrolysis of ochratoxin A to ochratoxin alpha by the ruminant protozoa. Assuming a similar reaction velocity in vivo, it was estimated that up to 12 mg/kg of feed could be degraded (Hult et al., 1976; Pettersson et al., 1982), so that this species is assumed to be relatively resistant to the effects of ochratoxin A in feed. Sheep also have a good capacity to detoxify ochratoxin A before it reaches the blood (Kiessling et al., 1984).

Studies in mice suggest that ochratoxin A circulates from the liver into the bile and into the intestine, where it is hydrolysed to ochratoxin alpha (Moroi et al., 1985).

About 25–27% of ochratoxin A, given either intraperitoneally or orally to rats, was present as ochratoxin alpha in the urine. Its presence in the urine can be explained by reabsorption from the intestine (Storen et al., 1982a). A similar mechanism of intestinal reabsorption of ochratoxin alpha has been suggested to occur in ruminant calves (Sreemannarayana et al., 1988).

Other minor urinary metabolites of ochratoxin A are 4-OH (4R-and 4S) epimers produced in rat and rabbit liver (Størmer et al., 1981) and rat kidney (Stein et al., 1985) by the action of cytochromes P450 (CYPs; Størmer et al., 1981, 1983). The 4R-OH epimer, which is considered less toxic than ochratoxin A, is the main one formed in human and rat liver microsomal systems (Størmer et al., 1981), whereas the 4S-OH epimer is more prevalent in pig liver microsomes. No data were available on its toxicity (Moroi et al., 1985).

The biotransformation of ochratoxin A has also been studied in various microsomal preparations and in recombinant human and rat CYP preparations (Gautier et al., 2001; Zepnik et al., 2001). Incubation of ochratoxin A with liver microsomes from rats and mice produced 4R- and 4S-hydroxyochratoxin A, but at very low rates, whereas oxidation of ochratoxin A was not observed in kidney microsomes from these species. 4R-Hydroxyochratoxin A was also formed at low rates by recombinant human CYP 3A4 (both studies), CYP 1A1 and CYP 2C9-1 (both in single studies), while conflicting results were obtained with CYP1A2. Oxidation was not observed with recombinant human CYP 2E1 or rat CYP 1A2 or the male rat-specific CYP 2C11 (all in one study). Prostaglandin H-synthase produced small amounts of a non-polar product.

The 10-OH derivative was formed from ochratoxin A in a rabbit liver microsomal system (Størmer et al., 1983). Ochratoxin C, a metabolite of ochratoxin A produced in rumenal fluid, is as toxic as ochratoxin A (cited by Galtier et al., 1981). Ochratoxin B, a dechloro derivative of ochratoxin A, may occur with ochratoxin A in cereal products. In rats, it is less toxic than ochratoxin A and is metabolized to 4-OH-ochratoxin B and ochratoxin beta (Størmer et al., 1985).

Ochratoxin B was not antagonistic to ochratoxin A with respect to effects on the formation of phenylalanyl-tRNA and protein synthesis (Roth et al., 1989).

Many researchers consider that the toxicity of ochratoxin A is due to one of its metabolites. The studies cited above indicate, however, that, in rats, ochratoxin A itself, rather than one of its metabolites, is the active toxic agent, since the known metabolites are less toxic than or as toxic as ochratoxin A. This conclusion concurs with findings in mice, in which the LD50 of ochratoxin A increased by 1.5- to 2-fold after the animals were fed phenobarbital at 500 mg/kg of diet for 1 week before oral or intraperitoneal administration (Moroi et al., 1985).

Similarly, pretreatment with sodium phenobarbital at 80 mg/kg bw per day by gavage for 5 days or 3-methylcholanthrene at 20 mg/kg bw per day by gavage for 2 days resulted in increased LD50 values for ochratoxin A. With phenobarbital, the difference was smaller 144 h after dosing with ochratoxin A than at 48 h. Administration of piperonyl butoxide, an inhibitor of microsomal monooxygenases, decreased the 144-h LD50 of ochratoxin A from 40 to 19 mg/kg bw (Chakor et al., 1988). In contrast, preliminary studies in mice showed that simultaneous feeding of phenobarbital slightly increased the incidence of liver tumours seen with ochratoxin A alone, and that the mice developed large, multiple hepatomas (Suzuki et al., 1986).

Few data are available on the metabolic disposition of ochratoxin A in humans. It has been suggested that it has a long serum half-life, on the basis of its strong binding to human serum macromolecules (Bauer & Gareis, 1987; Hagelberg et al., 1989).

2.1.3 Effects on enzymes and other biochemical parameters

The activities of glycolytic enzymes were reduced, whereas those of gluconeo-genic enzymes were increased. The diabetogenic effect of ochratoxin A was thought to be due to inhibited synthesis and/or release of insulin from pancreatic cells, thereby suppressing glycolysis and glycogenesis and enhancing gluconeogenesis and glycogenolysis (Subramanian et al., 1989).

Calcium homeostasis was studied in rats treated intraperitoneally with ochratoxin A at a single dose of 10 mg/kg bw or multiple doses of 0.5–2 mg/kg bw per day. An increase in renal endoplasmic reticulum calcium pump activity was observed, suggesting an association with ochratoxin A-induced renal cytotoxicity (Rahimtula & Chong, 1991).

Studies with pig renal cortical explants indicated that inhibition of the biosynthesis of macromolecules (protein, RNA and DNA) by ochratoxin A was not due to impairment of cellular respiration (Braunberg et al., 1992).

The biochemistry and molecular aspects of the action of ochratoxin A in both prokaryotes and eukaryotes have been reviewed (Röschenthaler et al., 1984). The findings are inconsistent, owing to differences and limitations in experimental models and procedures as well as interfering factors, especially in more complex organisms. In prokaryotes (Konrad & Röschenthaler, 1977), eukaryotic microorganisms (Creppy et al., 1979a), mammalian cells (Creppy et al., 1980a, 1983a), and experimental animals in vivo (Creppy et al., 1980b, 1984), the primary effect of ochratoxin A is inhibition of protein synthesis; secondarily, RNA and DNA synthesis may be inhibited.

The inhibition of protein synthesis is specific and occurs at the post-transcriptional level, ochratoxin A having a direct effect on the translation step in protein synthesis. This involves competitive inhibition of phenylalanine-tRNAPhe synthetase, so that amino-acylation and peptide elongation are stopped. This reaction is fundamental for all living organisms. In yeast, the first part of this reaction, phenylalanine-dependent pyrophosphate exchange, was inhibited five times more than transfer to tRNA, the second part. In this reaction ochratoxin A may be regarded as an analogue of phenylalanine, and in cell cultures the competitive inhibition could be reversed by an increase in phenylalanine concentration (Creppy et al., 1979a). Similarly, in mice, the lethality of a single dose of 0.8 mg of ochratoxin A injected intraperitoneally was completely prevented by simultaneous injection of 1 mg of phenylalanine (Creppy et al., 1980b).

In yeast, the effect on protein synthesis of the rR-OH-ochratoxin A epimer was similar to that of ochratoxin A, but ochratoxin alpha, which lacks the phenylalanine moiety, had no effect (Creppy et al., 1983a). Analogues of ochratoxin A in which phenylalanine has been replaced by other amino acids, such as tyrosine, inhibit the respective amino acid-specific tRNA synthetases similarly (Creppy et al., 1983b).

The binding affinity of phenylalanine-tRNAPhe synthetase for ochratoxin A is weaker than for phenylalanine and ranges from 1/300 in yeast (KM = 1.3 mmol/L for ochratoxin A; 3.3 ΅mol/L for phenylalanine) (Creppy et al., 1983a) to 1/20 in rat liver (Km = 0.28 mmol/L for ochratoxin A; 6 ΅mol/L for phenylalanine) (Röschenthaler et al., 1984). Despite these differences in binding affinity, the inhibition of phenylalanine-tRNAPhe by ochratoxin A is very effective, since the toxin is more readily concentrated by cells than phenylalanine. The concentration of ochratoxin A inside hepatoma cells was 200- to 300-fold that in the medium (Creppy et al., 1983a).

A dose-related inhibition of protein synthesis was found in mice given ochratoxin A intraperitoneally at a dose > 1 mg/kg bw. The degree of inhibition of protein synthesis 5 h after administration of ochratoxin A at 1 mg/kg bw was 26% in liver, 68% in kidney, and 75% in spleen as compared with controls (Creppy et al., 1984).

Ochratoxin A may also act on other enzymes that use phenylalanine as a substrate, although no direct effect on the activity of other isolated enzyme systems has been demonstrated (Röschenthaler et al., 1984). In kidney slices from rats 2 days after they had been fed ochratoxin A at 2 mg/kg bw, the activity of renal phosphoenolpyruvate carboxykinase, a key enzyme in the gluconeogenic pathway, was lowered by 50% (Meisner & Krogh, 1986). The inhibition was due indirectly to specific degradation of the mRNA coding for this enzyme. A similar effect was not seen in rat liver (Meisner et al., 1983).

The effect of ochratoxin A on phenylalanine metabolism was studied in isolated hepatocytes and in liver homogenates from male rats treated in vivo. Both the hydroxylation of phenylalanine to tyrosine and the subsequent metabolism of tyrosine, as measured by homogenate oxidation, were inhibited when ochratoxin A at a concentration of 0.12–1.4 mmol/L was incubated with isolated hepatocytes (Creppy et al., 1990).

Ochratoxin A enhanced NADPH- or ascorbate-dependent lipid peroxidation in rat liver microsomes and NADPH-dependent lipid peroxidation in kidney microsomes in vitro, as measured by malondialdehyde formation or oxygen uptake. It was suggested that ochratoxin A stimulates lipid peroxidation by complexing Fe3+ and facilitating its reduction. Subsequent to oxygen binding, an iron–oxygen complex initiates lipid peroxidation. Cytochrome P450, free active oxygen species, and free hydroxy radicals do not appear to be involved in Fe3+–ochratoxin A- stimulated lipid peroxidation. Oral administration of ochratoxin A at 6 mg/kg bw to rats appeared to increase lipid peroxidation in vivo, causing a sevenfold increase in ethane exhalation (Rahimtula et al., 1988; Omar et al., 1990).

In pig renal cortical tissue, ochratoxin A and citrinin added singly or in combination at a concentration of 10–6 or 10–3 mol/L did not elicit consistent or strong synergistic effects, as measured by transport of tetraethylammonium and paraaminohippurate ions, or protein synthesis measured with [3H]leucine (Braunberg et al., 1994).

The effects of superoxide dismutase and catalase on ochratoxin A-induced nephrotoxicity were studied. Superoxide removes oxygen by converting it to hydrogen peroxide; this enzyme works in conjunction with catalase, which removes hydrogen peroxide within cells. Rats were given 20 mg/kg bw of each enzyme by subcutaneous injection every 48 h, 1 h before gavage with ochratoxin A at 290 ΅g/kg bw every 48 h, for 3 weeks. Superoxide dismutase and catalase prevented most of the nephrotoxic effects induced by ochratoxin A, observed as enzymuria, proteinuria, and creatinaemia, and increased the urinary excretion of ochratoxin A. The results indicated that superoxide radicals and hydrogen peroxide are likely to be involved in the nephrotoxic effects of ochratoxin A in vivo (Baudrimont et al., 1994).

After short-term administration of ochratoxin A to rats, the renal proximal tubule did not appear to be the main target for nephrotoxicity, although decreased capacity to eliminate the toxin may result in a self-enhancing effect (Gekle & Silbernagl, 1994). The main renal effect of ochratoxin A in rats was found in the ‘postproximal’ nephron, as measured by a reduced glomerular filtration rate, increased fractional water, Na+, K+, and Cl– excretion, and increased dependence of osmol clearance on urine flow. In addition, ochratoxin A blocked membrane anion conductance in canine kidney cells in vitro (Gekle et al., 1993).

2.2 Toxicological studies

2.2.1 Acute toxicity

The LD50 values found in various species treated by various routes are shown in Table 1. Dogs and pigs were the most sensitive species and rats and mice the least sensitive. Simultaneous oral administration of phenylalanine at 100 mg/kg bw to mice increased the oral LD50 from 46 mg/kg bw to 71 mg/kg bw (Moroi et al., 1985). As is the case with many xenobiotics, neonatal rats were considerably more susceptible than adults.

Table 1. LD50 values for ochratoxin A in various species

Species

LD50 (mg/kg bw)

Oral

Intraperitoneal

Intravenous

Mouse

46–58

22–40

26–34

Rat

20–30

13

13

Rat neonate

3.9

 

 

Dog

0.2

 

 

Pig

1

 

 

Chicken

3.3

 

 

Based on a literature compilation by Harwig et al. (1983)

Histopathological and electron microscopic studies were conducted with groups of 10 male Long-Evans and Sprague-Dawley rats given benzene-free ochratoxin A at a single dose of 0, 17, or 22 mg/kg bw in 0.1 mol/L sodium bicarbonate by gavage and examined for up to 48 h afterwards. The earliest changes were multifocal haemorrhages in many organs and fibrin thrombi in the spleen, the choroid plexus of the brain, liver, kidney and heart, suggesting disseminated intravascular coagulation. The effect was postulated by the authors to be due to activation of extrinsic and intrinsic systems of coagulation. Other changes were hepatic and lymphoid necrosis, enteritis with villous atrophy, affecting the jejunum most severely, and nephrosis. The myocardial changes were considered to be related to shock and subsequent ischaemic injuries (Albassam et al., 1987).

2.2.2 Short-term studies of toxicity

Ochratoxin A had nephrotoxic effects in all monogastric mammalian species tested so far (Kuiper-Goodman & Scott, 1989). The results of short-term studies with this toxin are shown in Table 2.

Table 2. Results of short-term studies of the toxicity of ochratoxin A

Species, strain, sex, age

No.

Route

Dose
(mg/kg bw per day [mg/kg of diet])

Time
(days)

NOEL
(mg/kg bw per day)

Effects

Reference

Rat, Wistar, male, weanling

10

Diet

0.24–2.4 [2.4–24]

14

~0.48

Growth retardation

Munro et al. (1974)

~0.48

Increased serum blood urea nitrogen

~0.96

Increased kidney weight

< 0.24

Decreased urine volume

< 0.24

Renal lesions

Rat Wistar, male, female, weanling

15

Diet

0.015–0.37 [0.2–5]

90

~0.075

Reduced weight gain

Munro et al. (1974)

~0.016

Reduced kidney weight; no change in blood urea nitrogen, urinary or haematological parameters

0.37

Desquamation; increase in smooth endoplasmic reticulum, changes in rough endoplasmic reticulum, basement membrane thickening of proximal convoluted tubule cells; increased eosinophilia and karyomegaly in proximal convoluted tubule cells

Rat, Wistar, male, adult

5

Gavage

5

3

< 5

Reduced para-amino hippuric acid clearance, basement membrane thickening

Suzuki et al. (1975)

Rat, Wistar, male, adult

10

Gavate

0.5-2

10

1

Increased blood urea nitrogen

Haley & Galtier (1977)

< 0.5

Increased urine volume

Rat, Sprague-Dawley and Wistar, male, female adult

4-6

intraperi-toneal

0.75, 2

5-7

< 0.75

Decreased body weight, increased urine flow; decreased urine osmolality; increased urinary protein; increased urinary glucose; impaired urinary transport of organic substances; Sprague-Dawley more sensitive than Wistar, females less sensitive than males

Berndt & Hayes (1979)

Rat, Wistar, male, adult

14

Gavage

4

4-10

< 4

Decreased factors II, VI, X; decreased plasma fibrinogen, decreased thrombocyte, megakaryocyte counts

Gaultier et al. (1979)

Rat, Wistar, male, adult

9

Gavage

4

10

< 4

Hypothermia, cachexia, tremors, diarrhoea

Galtier et al. (1980)

Rat, Wistar, male, adult

3

Gavage

0.14-2

56-84

< 0.14

Decreased kidney enzyme activity; increased urinary enzyme activity

Kane et al. (1986a)

Rat, Fischer 344/N, male, female, weanling

5

Gavage

1–6

16 (12 doses)

1

Increased relative kidney, heart, and brain weight; thymus atrophy; forestomach necrosis; adrenal haemorrhage

National Toxicology Program (1989)

< 1

Bone-marrow hypoplasia

< 1

Renal nephropathy

Rat, Fischer 344/N, male, female, weanling

10

Gavage

0.06–1

91

0.12, males

Growth retardation

National Toxicology Program (1989)

0.12, males

Reduced relative kidney weight

0.06

Kidney tubular necrosis

< 0.06

Karyomegaly

Dog, beagle, male, young

3–6

Capsule

0.1–0.2

14

0.2

No change in kidney function

Kitchen et al. (1977a,b,c)

< 0.1

Renal tubular necrosis

< 0.1

Proximal tubule changes; thymus, lymphoid necrosis

Pig, female, 8–12 weeks

3–6

Diet

0.008. 0.04, 0.2
[0.2 1, 5]

5–90

< 0.008

Renal enzyme changes; changes in renal function

Elling (1979a); Krogh et al. (1988)

(a) Rats

Groups of 10 male weanling Wistar rats were fed semi-purified diets containing ochratoxin A at a concentration of 0, 2.4, 4.8 9.6, or 24 mg/kg, equivalent to 0, 0.24, 0.48, 0.96, and 2.4 mg/kg bw per day, for 14 days. At the two higher doses, growth retardation, reduced food consumption, and increased serum urea nitrogen were seen. At the highest dose, the relative kidney weight was increased. Renal lesions, involving degenerative changes in the entire tubular system, and a decrease in urine volume were seen at all doses. Increased eosinophilia and karyomegaly in cells of the proximal convoluted tubules were noted at all doses (Munro et al., 1974).

Semi-purified diets containing ochratoxin A at 0, 0.2, 1, or 5 mg/kg, equivalent to 0, 0.015, 0.075, or 0.37 mg/kg bw per day, were fed to groups of 15 weanling Wistar rats of each sex for 90 days. At that time, eight animals from each group were killed, and the remaining rats were fed control diet for an additional 90 days. No changes in blood urea nitrogen or urinary or haematological parameters were seen at any dose. After 90 days at the two higher dietary concentrations, the relative kidney weights were reduced in animals of each sex; these had returned to control values after the 90-day recovery period, except in males at the highest dose. Dose-related changes in morphological appearance were seen after 90 days of treatment at doses > 0.2 mg/kg of diet and involved karyomegaly and increased eosinophilia in cells of the proximal convoluted tubules. The authors considered the latter change to be a phenomenon of ageing which had been accelerated by administration of ochratoxin A. Desquamation of proximal tubular cells, autolysis, changes in the rough and smooth endoplasmic reticulum, and tubular basement membrane thickening up to 4 ΅m were noted at the highest dose. In animals at the highest dose that were subsequently given control diet for 90 days, the karyomegaly and tubular basement membrane thickening persisted, but otherwise the kidneys appeared normal (Munro et al., 1974).

Similar effects were seen when ochratoxin A was administered to groups of four to six adult Sprague-Dawley and Wistar rats by intraperitoneal injection for 5–7 days at a dose of 0, 0.75, or 2 mg/kg bw per day. Decreased body weight, increased urine flow, increased urinary protein, increased urinary glucose, and impaired urinary transport of organic substances were seen at all doses. Sprague-Dawley rats were more sensitive than Wistar rats, and males were more sensitive than females. It was suggested that the increased urinary protein indicated interference with protein reabsorption by cells of the convoluted tubules (Berndt & Hayes, 1979).

Ochratoxin A was administered by gavage in maize oil to groups of five weanling male and female Fischer 344/N rats at a dose of 0, 1, 4, or 16 mg/kg bw per day on 5 days per week for a total of 12 doses over 16 days. All rats that received the highest dose had diarrhoea and nasal discharge and died before the end of the study. Increased relative weights of kidneys, heart, and brain, thymus atrophy, forestomach necrosis and/or hyperplasia, and haemorrhage of adrenal glands were seen at the two higher doses. Bone-marrow hypoplasia and nephropathy were seen at all doses, involving renal tubular degenerative and regenerative changes (National Toxicology Program, 1989).

Ochratoxin A was administered by gavage in maize oil to groups of 10 male and female weanling Fischer 344/N rats at a dose of 0, 0.06, 0.12, 0.25, 0.5, or 1 mg/kg bw per day for 5 days/week for 91 days. Growth retardation and a reduced relative kidney weight were seen in males at the two higher doses. The NOEL for renal tubular necrosis was 0.062 mg/kg bw, but karyomegaly of dose-related severity was observed in the proximal tubules at all doses. Milder renal changes consisting of tubular atrophy were seen at lower doses (National Toxicology Program, 1989).

Groups of 15 weanling rats were given ochratoxin A in 0.1 mol/L sodium bicarbonate at a dose of 0 or 100 ΅g/rat (equivalent to 1.25 mg/kg bw per day) by gavage for 8 weeks. Blood samples from fasted treated rats contained about twice the amount of glucose as those of controls. In a glucose tolerance test, the insulin concentration did not reach that in control rats. Total carbohydrate and glycogen concentrations in liver of treated rats were reduced, as seen earlier (Suzuki et al., 1975; T. Kuiper-Goodman, personal observation).

(b) Dogs

Groups of three to six young beagle dogs were given ochratoxin A by capsule at a dose of 0, 0.1, or 0.2 mg/kg bw per day for 14 days. No changes were observed in renal function, but tubular necrosis and ultrastructural changes in the proximal tubules were observed at all doses. Necrosis of lymphoid tissues of the thymus and tonsils was also seen at all doses (Kitchen et al., 1977a,b,c).

(c) Pigs

In a series of experiments, groups of three to six sows were given feed containing ochratoxin A at a concentration of 0, 0.2, 1, or 5 mg/kg, equivalent to 0, 0.008, 0.04, and 0.2 mg/kg bw per day, for periods of 5 days, 8 or 12 weeks, or up to 2 years. Decreased renal function, nephropathy, and reduced renal enzyme activity were reported. Progressive nephropathy but no renal failure was seen in female pigs given feed containing 1 mg/kg for 2 years; no results were reported for male pigs (Krogh & Elling, 1977; Elling, 1979a,b, 1983; Elling et al., 1985; Krogh et al., 1988).

(d) Chickens

In groups of 10 broiler chicken given ochratoxin A at a dietary concentration of 4 mg/kg for 2 months, the mortality rate was 42%. When the feed was supplemented with 0.8 or 2.4% L-phenylalanine, the mortality rate decreased to 12 and 15%, respectively (Gibson et al., 1990).

2.2.3 Long-term studies of toxicity and carcinogenicity

Mice

Diets containing ochratoxin A at 0 or 40 mg/kg, equivalent to 5.6 mg/kg bw per day, were fed to groups of adult male 10 ddY mice for 44 weeks, followed by 5 weeks of basal diet. Of the nine surviving treated mice, five had hepatic-cell tumours, nine had renal cystic adenomas, and two had solid renal-cell tumours (terms used by the authors). No hepatic or renal tumours were observed in control mice, and no data on the incidence of these tumours in other control groups of this strain of mice were presented. It was not clear indicated whether the liver tumours were benign or malignant (Kanisawa & Suzuki, 1978).

In a second study from the same laboratory, diets containing ochratoxin A at 0 or 25 mg/kg, equivalent to 3.5 mg/kg bw per day, were fed to groups of 20 6-week-old male DDD mice for 70 weeks. All 20 surviving treated mice had renal cystic adenomas, six had solid renal tumours, and eight had hepatic-cell tumours. One of the 17 control mice had a hepatic-cell tumour (Kanisawa, 1984).

In a third study from the same laboratory, the mice were not exposed for life but for 70 weeks. Diets containing ochratoxin A at 0 or 50 mg/kg, equivalent to 7 mg/kg bw per day, were fed to groups of 16 adult male ddY mice for 5–30 weeks, followed by control diet for 40–65 weeks. No renal or liver tumours were observed in control mice or in mice fed ochratoxin A for ­ 10 weeks. The incidences of renal-cell tumours were 3/15, 1/14, 2/15, and 4/17 after 15, 20, 25, and 30 weeks on the ochratoxin A-containing diet, respectively. The incidence of renal cystic adenomas was not indicated. A significant increase in the incidence of liver tumours was observed after mice had been fed ochratoxin A for 25 weeks (5/15) or 30 weeks (6/17). These results indicated that the renal and liver tumours persisted through subsequent feeding of control diet (Kanisawa, 1984).

In these studies, two types of renal tumour were distinguished by the authors: papillary cyst adenomas (benign) and solid renal-cell tumours, which contained atypical cells, displayed infiltrative growth, and were interpreted by the Committee as malignant. Preneoplastic renal lesions were frequent and multiple and consisted of distended tubules with atypical epithelial cells. No metastases attributable to the kidney or liver tumours were found.

Diets containing ochratoxin A at a concentration of 0, 1, or 40 mg/kg were fed to groups of 50 weanling B6C3F1 mice of each sex for 24 months. The test compound contained about 84% ochratoxin A, 7% ochratoxin B, and 9% benzene. Dead and moribund mice were identified daily. The mice were examined and weighed, and their food consumption was recorded weekly for the first 4 weeks, then monthly. Animals at the high dose showed decreased body weights, by 25% for females and 33% for males, indicating that the maximum tolerated dose was exceeded, although no other signs of toxicity were observed. Nephropathy, characterized by cystic dilatation of renal tubules often with hyperplasia of the lining epithelium, was seen only in mice fed diets containing the highest concentration and was more severe in males than in females. No nephropathy was found in males or females given a control diet or the lower concentration of ochratoxin A. Benign and malignant renal tumours were seen only in male mice fed diets containing the high concentration, at incidences of 53% and 29%, respectively (combined incidence, 63%). No metastases from the renal tumours were found.

When the combined incidence of hepatocellular adenomas and carcinomas in treated mice was compared with that in concurrent controls, the increase was statistically significant in both male and female mice given the high dose; however, the 20% incidence in males was within the range of past controls of 0–22% for this strain of mouse, but the 14% incidence in females was greater than the incidence of 0–3.9% in previous controls (Ward et al., 1979). The authors noted that the ochratoxin A used in their study contained 9% benzene, a proven carcinogen, and thus the possibility of synergism must be considered. The presence of renal tumours in males did not decrease their survival rate. In fact, the survival rates of males at 18 months were 75% in the controls and 65% among those at 1 mg/kg of diet, compared with 98% for those at 40 mg/kg of diet, owing to a high incidence of fatal obstructive urinary-tract disease among the controls and low-dose mice, with onset as early as 4 months (Bendele et al., 1985a). It was suggested that the apparent protective effect of ochratoxin A at 40 mg/kg of diet was due to inhibition of the growth of gram-positive bacteria and to the induction of polyuria as a result of renal proximal tubule damage (Bendele & Carlton, 1986). Group caging and fighting-related lesions of the prepuce and penis may have contributed to the chronic uropathy (Rao, 1987).

Rats

Groups of 80 male and female Fischer 344/N rats were given ochratoxin A by gavage in maize oil at a concentration of 0, 21, 70, or 210 ΅g/kg bw per day, 5 days/week for 9 months, 15 months, or 103 weeks. The rats were observed twice daily, and body weights and food consumption were recorded weekly for the first 13 weeks and then monthly. Feed and water were available ad libitum. Groups of 15 rats of each sex were killed after 9 and 15 months. The body weight of rats at the highest dose was decreased by 4–7% between 18 and 77 weeks for male rats and between 6 and 89 weeks for female rats. No compound-related clinical signs were seen, and the results of haematological and serum chemical analyses showed no effects of biological significance. Urinary analysis indicated a mild to moderate change in the ability to concentrate urine, with no other change in renal function.

The incidences of renal adenomas in males were 1/50, 1/51, 6/51, and 10/50 and those of renal carcinomas were 0/50, 0/51, 16/51, and 30/50, in the four groups, respectively. The combined incidences of renal tubule-cell adenomas and carcinomas were 20/51 and 36/50 at the two higher doses. At the highest dose, many of the renal adenomas and carcinomas were multiple or bilateral. There was a dose-related increase in the number of males found dead or moribund before the terminal sacrifice (7, 19, 23, and 26, respectively, at 0, 21, 70, and 210 ΅g/kg bw per day). The decreased survival rates among rats at the two higher doses were attributed by the authors to the presence of kidney tumours, since 15/23 and 18/26 rats that died at these two doses had kidney tumours. In addition. a larger proportion of animals that died before the terminal sacrifice had carcinomas that had become metastatic (3/8 and 11/15 at the intermediate and high doses, respectively) than of animals killed at terminal sacrifice (0/7 and 3/15 at the intermediate and high doses, respectively). In male rats given the low dose of ochratoxin A, only one kidney tumour was present, although the decrease in survival was similar to that of rats at the two higher doses. The reduced survival of this group must therefore be attributed to a non-neoplastic treatment-related effect. In females, the combined incidences of renal adenomas and carcinomas were 0/50, 0/51, 2/50, and 8/50 at 0, 21, 70, and 210 ΅g/kg bw per day, respectively. The significance of the ochratoxin A-induced renal carcinomas in rats is increased by the high frequency of metastases, attributed to renal-cell carcinomas, mainly in the lungs and lymph nodes. Females at the high dose also had a greater multiplicity of fibroadenomas in the mammary gland (14/50) than controls and rats at lower doses (4–5/50).

The non-neoplastic lesions involved mainly the kidney. Chronic diffuse nephropathy, common to old rats, was seen at about the same incidence in all groups, but the extent and grade were not reported. Karyomegaly or karyocytomegaly (large kidney epithelial cells with giant polyploid nuclei and prominent nucleoli) was seen in all males and females at the two higher doses, and it was the most consistent finding in these groups at the the 9- and 15-month interim sacrifices as well as in a preliminary 13-week study (National Toxicology Program, 1989).

In reviewing these data at its forty-fourth meeting, the Committee noted that renal carcinomas were found in 16/51 male rats at 70 ΅g/kg bw per day and in 30/50 at 210 ΅g/kg bw per day; no carcinomas were found at the lower doses. In female rats, renal carcinomas were less common, with 0/50, 1/50, and 3/50 animals showing carcinomas at the low, intermediate, and high doses, respectively. Renal adenomas were found in all groups of male rats, increasing in frequency with increasing dose. In the female rats, renal adenomas were found only at the two higher doses. Fibroadenomas in the mammary gland were found in 45–56% of treated females, a significantly higher percentage than in the control group (Annex 1, reference 117).

The slides of the kidneys from the National Toxicology Program study were reviewed subsequently (Hard, 2000). The review confirmed that the site of injury was the straight segment of proximal tubule S3 in the outer stripe of the outer medulla. In the 2-year bioassay, the lesion consisted of contraction and disorganization of the normal linear pattern of the S3 tubules due to marked development of karyomegaly and cytomegaly. This change showed a clear dose–response relationship in both males and females. The 16-day and 13-week studies showed that this response was preceded by focal tubule basophilia involving mainly the outer stripe of the outer medulla, associated with single-cell death, increased mitotic activity, and some simple tubule hyperplasia. Other non-neoplastic lesions involving only the outer stripe of the outer medulla in the 2-year bioassay were dilated atypical tubules, chromophobic tubules, and cystic tubules, the latter being more prominent in females than in males. The review also confirmed that low (microgram) concentrations of ochratoxin A induced a high incidence of renal tubule tumours (74% in males at the high dose), with carcinomas predominating over adenomas. The carcinomas had a relatively rapid onset, progressing with malignant and aggressive behaviour, some tumours showing a tendency towards an uncommon anaplastic phenotype. There was a relatively high incidence of metastasis, and some tumours were undoubtedly the cause of death. These various features of the ochratoxin A-induced tumours distinguish them from the kidney tumours induced by model non-genotoxic renal carcinogens such as d-limonene and chloroform. However, the tendency towards anaplasia and their aggressive nature were reminiscent of renal tubule tumours induced by fumonisin B1. Renal tumour development was clearly related to the site of ochratoxin A-induced tubule damage, in that preneoplastic atypical tubule hyperplasia, adenomas, and very early carcinomas developed within the outer stripe of the outer medulla. However, a mode of action of sustained cytotoxicity and compensatory cell regeneration coupled with simple tubule hyperplasia, although a possibility, could not be established within the limits of conventional histology alone. Nevertheless, the very high incidence of renal neoplasms, their relatively rapid onset and highly malignant behaviour, coupled with a tendency towards an aggressive anaplastic phenotype and their contribution to death all favour a conclusion that ochratoxin A-induced renal tumour development occurs via DNA reactivity.

The Committee noted that the long-term effects were preceded by evidence of renal toxicity in the 16-day and 13-week studies. It is unclear whether the malignancy and aggressive nature of the tumours is a secure indication that the mechanism of induction is via DNA reactivity. The analogy with tumours induced by fumonisin B1 is not evidence of a genotoxic mechanism, since it has been postulated that the mechanism by which fumonisins induce tumours may be indirect, involving altered sphingolipid metabolism.

2.2.4 Genotoxicity

The results of studies of genotoxicity with ochratoxin A are summarized in Table 3.

Table 3. Results of assays for genotoxicity with ochratoxin A

Test system

Test object

Concentration

Results

Reference

In vitro

Reverse mutation

S. typhimurium TA 98, TA100, TA1535, TA1537, TA1538

0.4–400 ΅g/plate

Negative (highly variable TA100 controls, not tested to cytotoxicity)

Wehner et al. (1978); Kuczuk et al. (1978)

Reverse mutation

S. typhimurium TA100, TA1538

~ 200 ΅g/plate

Negative with mouse and rat liver activation

Bartsch et al. (1980)

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

50–600 ΅g/plate

Negative

Bendele et al. (1985b)

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538, G46, G3076, D3052

0.1–100 ΅g/ml

Negative al. (1985b)

Bendele et

Reverse mutation

S. typhimurium TA1538

0.1–500 ΅g/plate (mixture of ochratoxin A:ochratoxin B, 17)

Positive > 100 ΅g/plate

Kuczuk et al. (1978)

Reverse mutation

S. typhimurium TA97, TA98, TA100, TA1535

1–100 ΅g/plate

Negative with hamster or rat liver activation

National Toxicology Program (1989)

Reverse mutation

S. typhimurium TA98, TA1535, TA1538

0–1200 ΅g/plate

Positive only after activation by mouse kidney microsomes

Obrecht-Pflumio et al. (1999)

Reverse mutation

S. typhimurium TA100, TA2638

0–200 ΅g/plate

Negative in preincubation assay with mouse liver and kidney, and isolated enzyme activation systems

Zepnik et al. (2001)

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

 

Positive after activation by medium derived from hepatocytes exposed to ochratoxin A

Hennig et al. (1991)

Gene mutation

S. cerevisiae D3

75, 200 ΅g/plate

 

Kuczuk et al. (1978)

Gene mutation

B. subtilis rec

20–100 ΅g/disc

Negative

Ueno & Kubota (1976)

DNA repair

E. coli, SOS assay

1–2 mg/100 ΅l

Negative

Reiss (1986); Auffray & Boutibonnes (1986)

DNA repair

E. coli WP2

Gradient plate, not stated

Negative

Bendele et al. (1985b)

Forward gene mutation

Mouse lymphoma cells, Tk locus

0.1–13 ΅g/ml

Negative (>12 ΅g/ml cytotoxic)

Bendele et al. (1985b)

Gene mutation

C3H mouse mammary cells

5–10 ΅g/ml

Negative (10 ΅g/ml cytotoxic)

Umeda et al. (1977)

Gene mutation (lacZ on shuttle vector)

NIH 3T3 cells transfected with human cytochrome P450

25 ΅g/ml

Positive

De Groene et al. (1996)

Unscheduled DNA synthesis

Fischer 344 rat primary hepatocytes

0.000025–500 ΅g/ml (2 lots tested at 15 doses)

Negative (> 0.05 ΅g/ml cytotoxic)

Bendele et al. (1985b)

Unscheduled DNA synthesis

ACI rat primary hepatocytes

0.4, 4 ΅g/ml

Weakly positive at 0.4, cytotoxic at 4.0 ΅g/ml

Mori et al. (1984)

Unscheduled DNA synthesis

C3H mouse primary hepatocytes

4, 40 ΅g/ml

Weakly positive at 4.0, cytotoxic at 40 ΅g/ml

Mori et al. (1984)

Unscheduled DNA synthesis

Rat hepatocytes; porcine urinary bladder epitheilial cells

250 nmol/L–1 ΅mol/L

Positive

Dorrenhaus & Follmann (1997)

Unscheduled DNA synthesis

Cultured human urothelial cells

0.005–0.05 ΅mol/L

Positive

Flieger et al. (1998)

Unscheduled DNA synthesis

Primary human urothelial cells

10–2000 nmol/L

Positive

Dorrenhaus et al. (2000)

DNA strand break, alkaline elution

Chinese hamster ovary cells; rat fibroblasts

200 ΅g/ml

Positive (1.2 strand breaks/109 Da)

Stetina & Votava (1986)

DNA damage

Mouse spleen, phytohaemagglutinin-stimulated

1–10 ΅g/ml

Positive (dose-related)

Creppy et al. (1985)

DNA damage, 32P-post-labelling assay

Mouse kidney, liver, spleen

0.6, 1.2, 2.5 mg/kg bw

Positive (‘adducts’ not shown to contain bound ochratoxin A)

Pfohl-Leszkowicz et al. (1991)

DNA binding

Rat kidney, liver, seminal vesicle; mouse kidney

100 ΅mol/L incubated with S9 protein

Negative

Gautier et al. (2001)

Sister chromatid exchange

Human peripheral blood lymphocytes

5–10 ΅g/ml

Negative (mitotic inhibition at 10 ΅g/ml)

Cooray (1984)

Sister chromatid exchange

Chinese hamster ovary cells, 26 h with ochratoxin A

0.5–5 ΅g/ml

Negative

National Toxicology Program (1989)

Sister chromatid exchange

Chinese hamster ovary cells, 2 h with ochratoxin A

5–160 ΅g/ml

Positive (frequency ­ 37% above control, weak dose–response relationship)

National Toxicology Program (1989)

Sister chromatid exchange

Human lymphocytes

 

Positive

Hennig et al. (1991)

Chromosomal aberration

Chinese hamster ovary cells, 8–10 h with ochratoxin A

30–160 ΅g/ml

Negative

National Toxicology Program (1989)

2 h with ochratoxin

100–300 ΅g/ml

Negative

Chromosomal aberration

Human lymphocytes, 48 h with ochratoxin A

4.5 ΅g/ml

Positive (4.5–5-fold increase)

Manolova et al. (1990)

Micronucleus formation

Ovine seminal vesicle cell cultures

12–30 ΅mol/L

Positivea

Degen et al. (1997)

Micronucleus formation

Syrian hamster embryo fibroblasts

 

Positiveb

Dopp et al. (1999)

In vivo

Chromosomal aberration

Mouse

1 ΅g/kg bw per day in diet, 45 days

Positive (ameliorated by 10 mg/kg bw ascorbic acid)

Bose & Sinha (1994)

Chromosomal aberration

Mouse

1 ΅g/kg bw per day in diet, 14 days

Positive (ameliorated by 130 IU vitamin A/kg bw)

Kumari & Sinha (1994)

Sister chromatid exchange

Chinese hamster bone marrow

25–400 mg/kg bw by gavage

Negative (> 100 mg/kg bw cytotoxic)

Bendele et al. (1985b)

DNA damage (single-strand breaks)

BALB/c mouse

2500 ΅g/kg bw intraperitoneally

 

Creppy et al. (1985)

Spleen 4,16, 24 h after treatment

Positive (max. response at 24 h)

Kidney 24, 48 h after treatment

Positive (max. response at 24 h)

Liver 24, 48, 72 h after treatment

Positive (max. resposne at 48 h; recovery at 72 h)

DNA damage

Wistar rat kidney, liver

290 ΅g/kg bw by gavage every 48 h for 6 or 12 weeks

Positive, no recovery between treatments

Kane et al. (1986b)

a No inhibition by indomethacin, suggesting absence of activation by prostaglandin H synthase

b Clastogenic effects due to changes in intracellular calcium

(a) DNA adducts

Almost all the available studies in which DNA adducts were detected by 32P-postlabelling after exposure to ochratoxin A were from one laboratory (Pfohl-Leszkowicz et al., 1991, 1993; Grosse et al., 1995, 1997; Castegnaro et al., 1998; Pfohl-Leszkowicz et al., 1998). All showed positive results in rats and mice given 0.4–2.5 mg/kg bw for 1–16 days or even up to 2 years. The number of adducts ranged from 1 to 200/109 nucleotides in kidney DNA. However, the nonspecific postlabelling technique used may have resulted in adducts that did not contain an ochrotoxin A or ochratoxin A metabolite moiety. At least some of the adducts might have been due to ochratoxin A-induced cytotoxic effects that generate reactive oxygen species. Thus, Grosse et al. (1997) found that prior treatment of rats with superoxide dismutase or catalase, ascorbic acid, or alpha-tocopherol significantly decreased the number of adducts.

Indications that oxidative damage to DNA is not the only source of the presumed adducts are provided by the results of experiments in vitro with purified DNA and mononucleotides incubated with kidney or liver microsomes from mouse and rabbit, ochratoxin A, and either NADPH or arachidonic acid as cofactors (Obrecht-Pflumio & Dirheimer, 2000). Presumed adducts were obtained in all cases but particularly with mouse and rabbit kidney microsomes and arachidonic acid as the cofactor. Liver microsomes were much less active. With NADPH as the cofactor with mouse kidney microsomal enzymes, the adduct level was only 44% that obtained with arachidonic acid. When dAMP, dGMP, dTMP, and dCMP were used as substrates, three adducts were formed with dGMP, mouse kidney microsomes, and either cofactor. However, only one of these adducts was common to the two cofactors. Inhibition of lipid peroxidation and the generation of hydroxyl radicals with desferrioxamine B methanesulfonate did not change the adduct profile. The major adduct obtained with dGMP co-chromatographed with the major adduct obtained with purified DNA. No adducts were obtained with the other three mononucleotides.

In contrast to these results with 32P-postlabelling methods, Schlatter et al. (1996) and Rasonyi (1995) reported that the level of covalent binding of [3H]ochratoxin A to DNA was below the limit of detection (LOD) of scintillation counting in kidney and liver (< 1.3/1010 and 5.6/1011 DNA bases, respectively). In addition, Gautier et al. (2001), using scintillation counting, did not find covalent binding of [3H]ochratoxin A to the DNA of the kidneys of male Fischer 344 rats dosed by gavage 24 h earlier. The LOD was 2.7 adducts/109 purified DNA bases. The authors also used a 32P-postlabelling method with these rat kidney DNA samples and found adducts at levels ranging from 31 to 71/109 DNA bases 24 h after dosing, compared with 6–24/109 DNA bases in untreated controls. Since the adducts occurred at a level 3–17 times higher than the detection limit for scintillation counting and there was no evidence of tritium exchange, most, if not all, of the adducts observed by the 32P-postlabelling method would not have contained an ochratoxin A moiety.

Furthermore, no adducts were found (detection limit, 20 adducts/109 DNA bases) by scintillation counting when DNA and [3H]ochratoxin A were incubated in the presence of male rat kidney microsomes with NADPH, mouse kidney microsomes with NADPH, rat seminal vesicle microsomes with arachidonic acid, or horseradish peroxidase with hydrogen peroxide.

(b) DNA damage and repair

There was no evidence of DNA repair as a result of possible DNA damage in bacteria, whereas DNA single-strand breaks were consistently induced in cultured mammalian cells. DNA single-strand breaks were also observed in vivo in spleen, liver, and kidney cells of mice after intraperitoneal injection of ochratoxin A. DNA repair, manifested as unscheduled DNA synthesis, was observed in most studies with primary cultures of rat and mouse hepatocytes, porcine epithelial cells from bladder, and human urothelial cells.

(c) Gene mutation

Most tests for gene mutation induction in bacteria showed no effect of exposure to ochratoxin A. Two studies showed positive results. One was in S. typhimurium strains TA1535 and TA1538 treated in the presence of mouse kidney microsomes (Obrecht-Pflumio et al., 1999), while the other was in S. typhimurium strains TA1535, TA1538, and TA100 treated with the culture medium of rat hepatocytes exposed to ochratoxin A (Hennig et al., 1991). Both papers described preliminary results that required further investigation before they could be readily accepted. It should be noted, however, that Hennig et al. (1991) obtained negative results with the same bacterial strains when rat liver microsomes were used as the exogenous metabolic activation system. This portion of the results has been confirmed in independent studies in other laboratories.

Gene mutations were not induced in the yeast Saccharomyces cerevisiae D3 (Kuczuk et al., 1978). In mammalian cells, gene mutations were not induced in two studies, while positive results were observed in a third. The last study was performed with NIH 3T3 cells transfected with a human CYP gene (De Groene et al., 1996) at a concentration of 25 ΅g/ml. In the studies with negative results, concentrations of 10 ΅g/ml (C3H mouse mammary cells) and > 12 ΅g/ml (mouse lymphoma L5178Y cells) were cytotoxic. The positive result therefore requires confirmation. No studies of mutation in vivo have been reported.

(d) Chromosomal aberrations

Sister chromatid exchange was induced in two of four studies in vitro but not in a single study in vivo after gavage of a range of doses that included cytotoxic doses.

Chromosomal aberrations were not induced in Chinese hamster ovary cells (National Toxicology Program, 1989) but were induced in cultured human lymphocytes (Manolova et al., 1990), and micronuclei were induced in ovine seminal vesicle cells and Syrian hamster embryo fibroblasts. In vivo, chromosomal aberrations were induced in mouse cells, an effect that was reduced by treatment of the mice with either ascorbic acid (by gavage) or vitamin A (in the diet). These protective effects are consistent with the observation that the formation of 32P-postlabelling spots was prevented in some studies in which mice were treated with ochratoxin A (Grosse et al., 1997).

2.2.5 Reproductive toxicity

No adequate studies on the reproductive toxicity of ochratoxin A were available for review. Several studies of effects on developmental toxicity are summarized.

(a) Mice

Groups of 4–26 pregnant CBA mice were given a single dose of ochratoxin A in maize oil by gavage at 0, 1, 2, or 4 mg/kg bw on day 8 or 9 of gestation (day of vaginal plug considered to be day 1 after conception) or 4 mg/kg bw per day 2 days before mating and on days 2, 4, 6, 7, 10, and 14 of gestation, and observed until day 19. At this time, the numbers of viable and dead fetuses and the number of resorption sites were determined, and fetuses were weighed and examined for morphological changes. No mention was made of maternal toxicity. Prenatal survival was decreased in groups that had received 4 mg/kg bw on days 7 (24% deaths), 8 (17% deaths), and 9 (22% deaths) of gestation. Overt craniofacial anomalies were seen only after treatment on day 8 or 9; their incidence, multiplicity, and severity increased with increasing dose, the peak effect being seen on day 9. The incidences of malformed pups among surviving pups were 0%, 0%, 8.1%, and 16% of mice given ochratoxin A at 0, 1, 2, or 4 mg/kg bw on day 8 of gestation, and 0%, 29%, 42%, and 91% of mice given the same doses on day 9 of gestation. The mean number of malformations per fetus was 0.3 and 2.3 on days 8 and 9 at 4 mg/kg bw, and 1.7 and 3.9 in animals given 8 mg/kg bw in a separate study. The central nervous system, the eye, and the axial skeleton were the main systems affected. The most important malformations were those affecting the craniofacial structures, including aplasia and dysplasia of the upper facial structures, such as exencephaly, microcephaly, blunt jaws, anophthalmia, microphthalmia, and median cleft face. In animals treated on day 9 of gestation at 4 mg/kg bw, the incidences of the various major anomalies were exencephaly, 89%; anophthalmia, 45%; microphthalmia, 27%; open eyelids, 16%; agenesis of external nares, 21%; cleft lip, 7.1%; median cleft face, 8.9%; and malformed jaws or short maxilla with protruding tongue, 41%. The craniofacial anomalies were considered by the authors to have arisen from failure of closure of the neurocranium, resulting in abnormal configuration, position, and size of the bones of the base and lateral walls of the skull (Arora & Frölen, 1981).

The effects of protein deprivation on the teratogenic effects of ochratoxin A were studied in groups of 10–13 CD-1 mice, maintained on diets providing 26% (control), 16%, 8%, and 4% purified protein (casein), after mating and throughout gestation. A single dose of ochratoxin A in 0.1 N sodium bicarbonate was administered by gavage at a dose of 0, 2, or 3 mg/kg bw on day 8 of gestation (vaginal plug considered to be day 1), and the mice were killed on day 18 of gestation for examination. The dams were monitored twice daily, and food consumption was monitored. Diets and water were available ad libitum.

Ochratoxin A treatment did not affect maternal food consumption, but maternal deaths were significantly more frequent in the group receiving ochratoxin A at 3 mg/kg bw and 26% protein (five deaths), in that given the same dose and 4% protein (four deaths), and in that given 2 mg/kg bw and 4% protein (nine deaths), with no deaths in the untreated groups. The percentages of litters with grossly malformed fetuses and the percentages of malformed fetuses (in brackets) for each of the four diets (26, 16, 8, and 4% protein, respectively) were 58 (25), 50 (17), 75 (45), and 100 (81) at 3 mg/kg bw; 25 (5), 50 (21), 30 (13), and 100 (78) at 2 mg/kg bw; and 0 (0), 0 (0), 18 (3), and 31 (9.8) without ochratoxin A. The fetal weights were reduced as a result of treatment and protein deprivation. Cranofacial malformations were the commonest abnormality, but at lower protein concentrations gross malformations affecting the limbs and tail were also seen (Singh & Hood, 1985).

In microcephalic mice derived from females treated intraperitoneally with ochratoxin A at 3 mg/kg bw on day 10 of gestation, a quantitative assessment of neurons and synapses at 6 weeks of age showed that the somatosensory cortices of treated mice had fewer synapses per neuron than those of controls, indicating reduced dendritic growth (Fukui et al., 1992).

(b) Rats

Five groups of 12–20 pregnant Wistar rats were given ochratoxin A at a total dose of 5 mg/kg bw in 0.16 mol/L sodium bicarbonate by gavage, as follows: a single dose of 2.5 mg/kg bw on each of days 8 and 9 of gestation (vaginal plug considered to be day 1), a dose of 1.2 mg/kg bw on each of days 8–11 of gestation, a dose of 0.83 mg/kg bw on each of days 8–13 of gestation, or a dose of 0.63 mg/kg bw on each of days 8–15 of gestation. A control group was given the vehicle only. In a similar way, three groups of 20 rats were given ochratoxin A at a single dose of 2.5 mg/kg bw on each of days 8 and 9 of gestation or a dose of 1.7 mg/kg bw on each of days 8–10 of gestation. The rats were killed on day 20 of gestation. No significant difference was seen in the number of implantations per female in the various groups. Females that had received the same total amount of ochratoxin A but divided into fewer single doses and early in gestation were most affected. There was a dose-related increase in the number of resorptions per female and decreases in the mean number of fetuses per female, mean fetal weight, and mean placental weight. A high dose-related incidence of fetal haemorrhages (seen at 2, 2.5, and 4 times the 1.2 mg/kg dose) and coelosome with or without oedema were considered to be teratogenic responses (Moré & Galtier, 1974).

In a study from the same laboratory, a similar protocol for administration of ochratoxin A was used, but the rats were observed until 82 days after birth. Dose-related decreases in the mean number of newborn rats, the mean number of rats alive at 4 days, and the viability index were seen, but not in the lactation index. In the group given 2.5 mg/kg bw twice, the mean body weights of male and female offspring at 82 days were reduced by 12 and 8%, respectively. Hydrocephalus was observed on day 15 after birth in 26% of the male offspring at that dose, and 40% of these animals died by 20 days after birth. A second generation was bred to examine residual maternal or paternal effects, without further administration of ochratoxin A. No differences in reproductive parameters were noted, and few details were given (Moré & Galtier, 1975).

A dose of 0.5 mg/kg bw given by gavage to rats on days 11–14 of gestation caused learning deficits in pups tested for 26 weeks (Kihara et al., 1984).

Oral administration of ochratoxin A to pregnant rats at 1 mg/kg bw per day on days 6–15 of gestation resulted in decreased fetal weight and increased numbers of resorptions but no overt adverse effects on the dams. Skeletal and/or lung malformations were reported in up to 20% of the fetuses; the incidence of renal malformations was 40%. Concurrent administration of methionine at 43 mg/kg bw protected against these adverse effects (Abdel-Wahhab et al., 1999).

Other studies on the teratogenicity of ochratoxin A in mice and rats treated intraperitoneally or subcutaneously were reviewed by Kuiper-Goodman & Scott (1989).

(c) Chickens

The embryotoxic potential of ochratoxin A was tested in chicks by injecting hens’ eggs on day 3 and incubating them until day 13 or 18, when visible abnormalities, weight, and length of chicks were recorded. A dose-related increase in the mortality rate was seen after injection of 1–2 ΅g of ochratoxin A. An increased frequency of abnormalities was seen in one of the two reported experiments (Edrington et al., 1995). The Committee noted that this is not a validated method, and the results could not be used in risk assessment.

(d) In vitro

Prechondrogenic mesenchymal cells from the limb buds of 4-day-old chick embryos were cultured with ochratoxin A for 6 days. Ochratoxin A inhibited the accumulation of cartilage proteoglycans and general protein synthesis in a dose-related manner (Wiger & Stormer, 1990).

Rat embryos explanted on day 10 of gestation were cultured in a medium containing ochratoxin A at concentrations up to 300 ΅g/ml. Dose-dependent reductions in the protein and DNA content of the embryos were seen. The malformations induced included hypoplasia of the telencephalon, stunted limb bud development, and decreased size of mandibular and maxillary bones. Cellular necrosis of mesodermal and neuroectodermal structures was observed (Mayura et al., 1989).

2.2.6 Special studies

(a) Covalent binding to nucleic acids and/or proteins

Preliminary observations indicated no specific binding of ochratoxin A to macromolecules in porcine kidney cytosol (Stojkovic et al., 1984).

Subcellular fractions of a number of kidney-derived cell lines and rat intestine, liver, spleen, kidney, and plasma were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then incubated with ochratoxin A coupled with horseradish peroxidase in order to locate the ochratoxin A-binding proteins. The toxin was shown to bind to virtually all rat blood serum proteins and to some proteins in rat intestine, liver, spleen, and kidney, particularly at 60, 40, and 27 kDa. Binding of ochratoxin A to the 60- and 27-kDa proteins, but not the 40-kDa protein, was inhibited by phenylalanine and aspartame in liver but not in the other organs. The binding of ochratoxin A to cytosolic or organelle proteins was comparable in all the kidney cell lines, which were derived from various species and from various regions of the kidney. Phenylalanine and aspartame had no effect on the binding. The authors concluded that ochratoxin A can bind to several cellular proteins, and that this accounts for its accumulation in cells, but that the results do not explain the protective effects of phenylalanine and aspartame described previously (Schwerdt et al., 1999a).

(b) Immunotoxicity

Mice

The size of the mouse thymus was reduced to 33% that of controls after four intraperitoneal injections of ochratoxin A at 20 mg/kg bw on alternate days, a dose which caused minimal nephrotoxicity. Bone marrow depression was shown as dose-related, significantly (p < 0.01) decreased marrow cellularity, including a reduction in bone marrow macrophage–granulocyte progenitors, a decreased number of haematopoietic stem cells and a significant decrease in erythropoiesis as measured by 59Fe uptake; increased phagocytosis by macrophages was also observed (Boorman et al., 1984).

Residual damage was seen 3 weeks after exposure as increased sensitivity to radiation, even though bone-marrow cellularity and the peripheral blood count had returned to normal (Hong et al., 1988; National Toxicology Program, 1989).

Ochratoxin A administered to 8–10-week-old Swiss mice at 5 mg/kg bw per day by intraperitoneal injection for 50 days reduced the antibody response to Brucella abortis, a cell-mediated immune response. This was postulated to be due to suppression of immunoglobulin (Ig)M synthesis. The same treatment also reduced mitogen (concanavalin A)-induced blast formation in lymphocytes derived from mouse spleen (Prior & Sisodia, 1982).

Groups of eight female BALB/c mice were fed diets containing ochratoxin A at a concentration of 6, 250, or 2600 ΅g/kg for 28 or 90 days, equivalent to 1, 40, and 400 ΅g/kg bw per day. Treatment did not cause changes in body or lymphoid organ weights. Kidney weights were reduced at the two higher doses at 28 days and at the highest dose at 90 days. The concentrations of ochratoxin A in kidney were clearly dose-related. No differences in leukocyte count were observed, but a significant reduction in the number of spleen cells (by about 20%) was observed at the highest dose after 90 days. No changes were observed in blood or thymic T lymphocytes at 28 days, but a decreased proportion of mature CD4+ and CD8+ cells was seen with a corresponding increase in the immature double-positive sub-population at the two higher doses after 90 days. After 28 days, the primary (humoral) antibody response to sheep red blood cells was significantly suppressed in a dose-dependent manner at the two higher doses. The antibody response to another T-cell-dependent antigen (viral antigen PR8) was not affected, suggesting that exposure to ochratoxin A alters certain immune functions in mice, and, as previously demonstrated, the spleen may be the most sensitive immune tissue to ochratoxin A. Differences in the proportions of mature and immature CD4+ and CD8+ populations suggest that ochratoxin A may affect late-stage differentiation of T cells (Thuvander et al., 1995).

Female BALB/c mice were given diets containing ochratoxin A to provide a calculated average dietary intake of 5–30 ΅g/kg bw per day for 2 weeks before mating. At birth, the pups were cross-fostered to unexposed dams. Exposed and control pups were killed at 14 and 28 days of age. Ochratoxin A did not effect the reproductive outcome or body weight of pups. No differences in spleen or thymus weight or cell numbers were observed on day 14, but significant increases were seen in both thymus weights (by 20%) and cell number (by 67%) in the offspring of dams at the high dose on day 28. Although the percentages of splenic CD4+ and CD8+ cells were decreased in pups at the high dose, there were no alterations in absolute numbers. No significant differences were observed in the proliferative responses of splenic or thymic lymphocytes to mitogens nor in the production of interleukin-2 in concanavalin A-stimulated cell cultures. No significant differences in the humoral antibody response to sheep red blood cells or viral antigen PR8 were found. Natural killer cell activity on day 28 was not affected by prenatal exposure to ochratoxin A. Thus, the treatment did not suppress immune function but altered the absolute and relative numbers of lymphocyte subpopulations in lymphoid organs (Thuvander et al., 1996a).

Groups of 10 Han-NMRI mice (sex not specified) received commercial (Serva) or ‘raw’ ochratoxin A at a dose of 1, 3, or 6 mg/kg bw per day by intraperitoneal injection for 8–17 days and were then monitored for up to 20 days. Animals receiving ‘raw’ ochratoxin A at 3 mg/kg bw per day had a significantly lower body weight than controls on days 5–17; however, this correlated with a reduction in feed consumption. No significant change in body weight was noted in the groups receiving crystalline ochratoxin A. The total leukocyte count was unchanged in all groups; however, lymphopenia, neutrophilia, and eosinophilia were observed at 3 and 6 mg/kg bw per day. The blood IgM titre was suppressed at these doses in a dose-dependent manner. The authors concluded that ochratoxin A has a nonselective suppressive effect on various immune reactions, but the paper contains inadequate detail to verify their conclusion (Müller et al., 1995).

Rats

Bone-marrow hypocellularity and a reduced thymic size were also seen in Fischer rats given ochratoxin A at 1 or 4 mg/kg bw per day by gavage for 16 days (National Toxicology Program, 1989).

Necrosis of germinal centres in the spleen and lymph nodes was seen in Wistar rats given a single dose of ochratoxin A at 5–50 mg/kg bw (Kanisaw et al., 1977) and in dogs given ochratoxin A by capsule at doses of 0.1–0.2 mg/kg bw per day for 14 days (Kitchen et al., 1977c).

The effects of ochratoxin A on the bone marrow and lymphatic cell population may reflect the sensitivity of these cells to the inhibition of protein synthesis induced by ochratoxin A. These effects on the structural components of the immune system indicated that ochratoxin A is likely to have an effect on immune function.

The immunotoxic effects of perinatal exposure to ochratoxin A were investigated in the offspring of Sprague-Dawley rats treated singly or repeatedly. In a short-term study, dams received a single oral dose of 10, 50, or 250 ΅g/kg bw on day 11 of lactation, and the pups were examined on day 14. Dose-dependent uptake of ochratoxin A was observed in both dams and pups. The toxin did not induce consistent changes in the weights of the lymphoid organs of pups. A small but significant increase in the number of thymocytes was observed in offspring of dams dosed at 50 ΅g/kg bw, but it was not dose-dependent. A small but significant decrease in the proliferative response of splenocytes to T-cell mitogen lipopolysaccharide was seen in pups of dams given 250 ΅g/kg bw. In contrast, exposure to 10–50 ΅g/kg bw per day resulted in significant increases in the proliferative responses of both splenocytes and thymocytes of pups to concanavalin A. This was not seen at the higher dose. The authors proposed that short-term exposure of suckling pups via the milk stimulates the immune response, measured as proliferation of lymphocytes in response to concanavalin A and lipopolysaccharide (Thuvander et al., 1996b).

In a long-term study, dams received repeated oral doses of ochratoxin A at 50 ΅g/kg bw on 5 days/week for 2 weeks before mating, during gestation, and then 7 days/week until weaning. At parturition, the number of pups was reduced to eight per litter and they were cross-fostered to produce groups of prenatally, postnatally, and pre- and postnatally exposed pups. The highest blood concentrations of ochratoxin A were detected in pups exposed both pre- and postnatally, but exposure via the milk appeared to account for most of the content. Long-term exposure to 50 ΅g/kg bw per day did not induce any consistent changes in body or lymphoid organ weights of pups, but prenatal exposure suppressed the lymphocyte response to both B- and T-cell mitogens at 14 days of age. The background proliferation of unstimulated cells was significantly suppressed in cultures from prenatally exposed pups. These effects were not observed in pups exposed during lactation, although the blood concentrations were higher in pups exposed postnatally. Prenatally exposed pups showed a significantly lower primary antibody response to PR8 viral antigen (± 0.36). No significant difference in the natural killer cell activity of splenocytes was measured in exposed pups at 13 weeks of age. The authors concluded that long-term prenatal exposure to ochratoxin A, but not postnatal exposure via milk, may cause immunosuppression; however, short-term postnatal exposure may stimulate proliferation of lymphocytes in response to mitogens (Thuvander et al., 1996b).

The Committee noted that no details were given about the ochratoxin A used. These authors previously used commercial ochratoxin A, but in this paper they quoted a 1984 reference for details of how the ochratoxin A was produced. The size of the groups was not given, but they seem to have consisted of four to five dams.

Pigs

Groups of six weanling hybrid pigs received either pure or crude ochratoxin A at doses of 7–50 ΅g/kg bw per day by subcutaneous injection for 19–39 days. The animals were immunized 8 days after ochratoxin A challenge with Pasteurella by inhalation. The authors stated that ochratoxin A had no effect on body-weight gain and that the serum concentrations were dose-dependent. The concentrations were reported to be lower after administration of crude ochratoxin A than pure material. A reduction in relative lymphocyte count and increases in total leukocyte, relative neutrophil, and eosinophil counts were seen. Crude toxin had a greater effect than pure toxin. Ochratoxin A decreased the phagocytosis index of individual cells and decreased expression of SWC1 (a lymphocyte cell surface marker) but did not change lymphocyte proliferation (Müller et al., 1999).

The Committee noted that many of the results were conflicting and the study was inadequately reported.

Chickens

In chickens fed diets containing ochratoxin A at a concentration of 2–4 mg/kg for 20 days, the lymphoid cell population of immune organs was decreased (Dwivedi & Burns, 1984a).

Several studies have shown that ochratoxin A affects both humoral and cell-mediated immunity. In chickens fed a diet containing ochratoxin A at 5 mg/kg for 56 days, the contents of alpha1-, alpha2-, beta-, and gamma-globulins in plasma were reduced (Rupic et al., 1978).

In chickens fed diets containing ochratoxin A at a concentration of 2–4 mg/kg for 20 days, immunoglobulin (Ig)G, IgA, and IgM in lymphoid tissues and serum were depressed (Dwivedi & Burns, 1984b), and complement activity was slightly affected in birds fed at diets containing 2 mg/kg for 5–6 weeks (Campbell et al., 1983).

Ochratoxin A also reduced IgG and increased IgM in the bursa of Fabricius in chick embryos that had been injected with 2.5 ΅g of the toxin on day 13. This did not affect their immunocompetence, however, as seen after challenge of the hatched chickens with E. coli at 1, 2, and 4 weeks of age, indicating that the effect on immunoglobulins may have been transient (Harvey et al., 1987).

Immunosuppression was observed in chickens fed diets containing ochratoxin A at 0.5 or 2 mg/kg for 21 days. When compared with controls, the treated animals had reduced total serum protein, lymphocyte counts, and weights of the thymus, bursa of Fabricius, and spleen (Singh et al., 1990).

In vitro

The effects of ochratoxin A on T-cell activation were investigated in purified (> 95%) human lymphocytes cultured in medium containing 1% bovine serum albumin. Intracellular free Ca2+ and activation of protein kinase C were measured as indicators of the early stages of activation; the effect on phytohaemagglutinin-induced proliferation was measured as a late event mediated by expression of functional interleukin-2 receptors. The early-stage events were not inhibited by ochratoxin A at a concentration of 12 ΅mol/L. In contrast, incubation of ochratoxin A with phytohaemagglutinin-stimulated lymphocytes resulted in inhibition of DNA synthesis at concentrations > 6.4 ΅mol/L. Protein synthesis in resting lymphocytes was markedly inhibited by 12 ΅mol/L but to a lesser extent in phytohaemagglutinin-stimulated lymphocytes. The authors concluded that ochratoxin A can block DNA synthesis at a late stage in lymphocyte activation and that this effect may be partially mediated by inhibition of protein synthesis (Størmer & Lea, 1995).

Ochratoxin A also inhibited the proliferative response of bovine peripheral blood mononuclear cells cultured in 10% fetal calf serum. The ID50 value varied from 0.1 to 4 ΅g/ml, depending on the mitogen used to stimulate the cells and the incubation time. The authors considered these results indicative of immunosuppressive potential (Charoenpornsook et al., 1998).

(c) Neurotoxicity

Rats

Three male Wistar rats received 1 nmol (about 400 ng) of ochratoxin A by intracerebral administration and four received a diet containing 290 ΅g/kg by oral gavage for 8 days. The animals were killed 24 h after dosing. Although ochratoxin A was detected in areas of the central nervous system after intracerebral injection, it was not detected in the periphery or blood, kidney, or urine, indicating that it is transferred poorly or not at all from the spinal fluid to blood, kidney, or urine. After administration in the diet, the ventral mesencephalon, hippocampus, striatum, and cerebellum were the main targets of cytotoxicity in rat brain (Belmadani et al., 1998a)

Four male Wistar rats received ochratoxin A at 290 ΅g/kg bw orally every 48 h for 1–6 weeks. The treated animals had a slight reduction in body weight after 4 weeks, but feed and water consumption were not significantly different from those of controls. Ochratoxin A accumulated in the brain in a linear time-dependent manner, to reach about 100 ng/g of brain after 6 weeks. The toxin was shown to change the concentrations of the amino acids tyrosine and phenanthrene and to damage tissues in the hippocampus (Belmadani et al., 1998b)

Ten adult female Fischer rats received ochratoxin A at 120 ΅g/kg bw per day by oral gavage for 10, 20, or 35 days. Treatment altered the activity of all enzymes tested. Significant increases in gamma-glutamyl transferase activity were observed in the three brain regions examined. The changes in the other enzyme activities were regionally selective, but most of the activities had returned to control levels by day 35 of dosing (Zanic-Grubisic et al., 1996).

(b) In vitro

The neurotoxicity of ochratoxin A has been investigated in nerve tissue cell cultures (embryonic chick neural retina and brain) and cultured meningeal fibroblasts. The cells were incubated with ochratoxin A in serum-free medium for 8 days. The median inhibitory concentration (IC50) for a number of parameters of cytotoxicity (cellular protein, 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide [MTT] reduction, neutral red uptake) were found to be about 170 nmol/L in all three culture systems, indicating that ochratoxin A did not have cell-specific effects. Ochratoxin B and the heat-induced 3S-epimer of ochratoxin A induced comparable effects at 19- and 10-fold higher concentrations, respectively (Bruinink et al., 1997).

In a study with a similar protocol, markers of neuritic outgrowth and differentiation (NF68 and 160 kDa, MAP2 and MAP5) were affected at significantly lower concentrations than the markers of cytotoxicity. Although the presentation of the data is unclear, the IC50 values for the most sensitive parameters appeared to be 20–50 nmol/L for the embryonic brain and neural retinal cultures. Binding of ochratoxin A to bovine serum albumin resulted in significantly decreased potency (IC50 values increased by 15–30-fold). Differences were noted between serum-free primary cultures and the cell lines. In these culture systems, phenylalanine did not decrease the effects of ochratoxin A and in contrast appeared to cause a concentration-related decrease in the IC50 [no statistical analysis presented]. The authors concluded that ochratoxin A specifically affected neurite formation and that its toxicity was decreased by protein binding but not by phenylalanine (Bruinink & Sidler, 1997).

These authors also investigated whether the effects of ochratoxin A could be attributed to its isocoumarin structure, by comparing the toxicity of ochratoxin A with that of ochratoxin alpha and ochracin in serum-free embryonic chick brain cultures. Ochratoxin A decreased the end-points at concentrations > 15 nmol/L, with a greater effect on neurite outgrowth (neurofilament 68 kD). Ochratoxin alpha and ochracin had minimal effects at concentrations up to 1 mmol/L. The isocoumarin structure was therefore considered not to be responsible for the toxicity of ochratoxin A in this brain cell culture model (Bruinink et al., 1998).

The regional selectivity of ochratoxin A was investigated in primary cultures of neurons and astrocytes isolated from embryonic or newborn rat brain ventral mesencephalon and cerebellum. The cultures were exposed to ochratoxin A in a medium containing 10% fetal calf serum for 46 h, before measurement of DNA and protein synthesis, lactate dehydrogenase leakage, and lipid peroxidation. Ochratoxin A inhibited protein and DNA synthesis in all cell types, with IC50 values ranging from 14 to 69 ΅mol/L. Neuronal cells were more sensitive than astrocytes, and the cells of the ventral mesencephalon were more sensitive than those of the cerebellum. Increases in lactate dehydrogenase leakage and lipid peroxidation were also seen, but the sensitivity of the cell types did not mirror that for DNA and protein synthesis. The authors concluded that ochratoxin A is neurotoxic and may affect particular structures of the brain (Bruinink et al., 1998).

(d) Nephrotoxicity

In vivo

Renal function and morphology are greatly affected at high doses of ochratoxin A, as indicated by increased kidney weight, urine volume, blood urea nitrogen (Hatey & Galtier, 1977), urinary glucose, and proteinuria (Berndt & Hayes, 1979). The last two findings indicate that the site of reabsorption, i.e. the proximal convoluted tubules, is damaged. The NOELs for changes in renal function depend on the species and on the parameter tested. At low doses of ochratoxin A, no increase in blood urea nitrogen, creatinine, or glucose was found in the urine of male or female rats given 210 ΅g/kg bw per day by gavage for 6–12 months, but a mild to moderate decrease in the ability to concentrate urine was seen. The NOEL for this effect was 70 ΅g/kg bw per day for male rats and 21 ΅g/kg bw per day for female rats (National Toxicology Program, 1989).

Various groups of investigators have shown that this specific nephrotoxic effect is due to an ochratoxin A-induced defect of the organic anion transport mechanism located on the brush border of the proximal convoluted tubule cells and basolateral membranes (Endou et al., 1986; Sokol et al., 1988). The organic ion transport system is also the mechanism by which ochratoxin A enters proximal tubular cells (Friis et al., 1988; Sokol et al., 1988).

The middle (S2) and terminal (S3) segments of the proximal tubule of isolated nephron segments were found to be the most sensitive to the toxic effects of ochratoxin A (0.05 mmol/L), as shown by a significant decrease in cellular ATP and a dose-related decrease in mitochondrial ATP content (Jung & Endou, 1989).

Several investigators have measured the effect of ochratoxin A on the release of enzymes from the kidney into the urine. Changes in enzyme and protein patterns can be used to distinguish different types of renal injury (Stonard et al., 1987).

Subcutaneous doses of ochratoxin A at 10 mg/kg bw for 5 days decreased the activity of muramidase and then decreased the activities of lactate dehydrogenase, alkaline phosphatase, glutamate dehydrogenase, and acid phosphatase in the kidney (Ngaha, 1985). The activities of alanine peptidase, leucine amino peptidase, and alkaline phosphatase were decreased by 60%, 50%, and 35%, respectively in isolated kidney tubules in the presence of 0.1 mmol/L ochratoxin A (Endou et al., 1986).

In male rats given ochratoxin A at 0.1–2 mg/kg bw per day orally for 2–5 days, the phosphoenolpyruvate carboxykinase activity decreased by 50–70% at the highest dose (Meisner et al., 1983; Meisner & Krogh, 1986). The minimum effect level was 0.1 mg/kg bw per day (Meisner & Polsinelli, 1986); at 2 mg/kg bw per day, enzymes such as pyruvate carboxylase, malate dehydrogenase, hexokinase, and gamma-glutamyl transpeptidase were not affected (Meisner & Selanik, 1979).

In rats given ochratoxin A by gavage at a dose of 0.14 mg/kg bw every 48 h (equivalent to about 2 mg/kg diet) for 8–12 weeks, the activities of lactate dehydrogenase, alkaline phosphatase, leucine aminopeptidase, and gamma-glutamyl transferase decreased significantly. The last three enzymes are located in the brush border of the proximal convoluted tubules, indicating damage at that site. Concomitantly with the decrease of enzyme activity in the kidney, these enzymes appeared in the urine. A late event was a urinary increase in the activity of N-acetyl beta-D-glucosidase, a lysosomal enzyme. The activity of this enzyme in the kidney was not affected (Kane et al., 1986a). The late appearance of this enzyme may indicate active regeneration and exfoliation of necrotic proximal convoluted tubular cells, releasing lysosomal enzymes (Stonard et al., 1987). In this study, para-aminohippurate clearance was reduced initially by 56% at 2 weeks and 8% at 12 weeks of dosing, indicating damage followed by regeneration.

Pigs are very sensitive to the effect of ochratoxin A on renal enzyme activity. In the kidneys of pigs fed diets containing ochratoxin A at 0.2–1 mg/kg, equivalent to 0.008–0.041 mg/kg bw per day, a dose-related decrease in the activity of phosphoenolpyruvate carboxykinase and gamma-glutamyl transpeptidase was accompa-nied by a dose-related decrease in renal function, as indicated by a reduction in the maximal tubular excretion of para-aminohippurate per clearance of inulin and an increase in glucose excretion. Only cytosolic, and not mitochondrial, phosphoenol-pyruvate carboxykinase activity was inhibited (Meisner & Krogh, 1986; Krogh et al., 1988).

Subcutaneous administration of superoxide dismutase and catalase together was found to offset the nephrotoxic effects of ochratoxin A, leading to the suggestion that superoxide radicals and hydrogen peroxide are likely to be involved in the nephrotoxic effects of ochratoxin A in vivo (Baudrimont et al., 1994).

Male Wistar rats weighing 70–150 g were given ochratoxin A in order to determine its effects on the pH in the vasa recta of the renal papilla after a single intravenous injection pf 3 ΅mol/kg bw or six intraperitoneal injections of 1.2 ΅mol/kg bw per day. Both regimes increased the pH in the descending and ascending vasa recta, with no significant difference between the renal arterial and aortic pH values, in serum and urine osmolality, or in urinary flow rate. An increased pH over that before treatment was detected in the collecting ducts of individual animals after an intravenous dose, but the differences between groups were not significant. Treated rats had a lower body-weight gain (19%) than controls (34%) during the 6-day intraperitoneal treatment. The authors suggested that ochratoxin A upsets pH homeostasis in the interstitium of the renal papilla, leading to alkalinization, in addition to impairment of urinary acidification (Kuramochi et al., 1997a).

In a subsequent study in which male Wistar rats were treated intravenously with ochratoxin A at 3 ΅mol/kg bw, the pH in the proximal tubule, distal tubule, and collecting ducts and the descending and ascending vasa recta was increased. The concentration of bicarbonate ion increased significantly in the proximal tubule and collecting ducts of treated animals, but there were no significant differences between treated and control animals in any of the parameters in aortic blood. No significant differences were observed in pCO2 in any region, and there were no significant differences in serum or urine osmolality or urinary flow rate. The authors concluded that ochratoxin A increased the pH and bicarbonate ion concentration in the tubular fluid or vasa recta but did not alter pCO2. They hypothesized that this disturbance in pH homeostasis could contribute to alterations in acid–base status and hence to the nephrotoxicity of ochratoxin A (Kuramochi et al., 1997b).

Groups of Wistar rats (sex not specified) were given ochratoxin A at 0, 0.4, or 0.8 mg/kg bw intraperitoneally every 72 h for 90 days in order to investigate the relationship between the pathogenesis of nephropathy and the genotoxic and carcinogenic effects of ochratoxin A. Treatment resulted in significant, dose-related decreases in relative kidney weight (80 and 77% of control), average kidney length (83 and 78% of control), and creatinine clearance (90 and 76% of control) at 0.4 and 0.8 mg/kg bw, respectively. Severe renal atrophy was reported in animals at both doses, but these were not dose-related. Dose-dependent concentrations of ochratoxin A were found in the blood (920 and 1900 ng/ml) and kidney (30 and 170 ng/g) at the two doses, respectively. The urinary concentration was similar at both doses, consistent with the low urinary elimination of ochratoxin A. Histological examination of the kidneys of rats given 0.8 mg/kg bw every 72 h for 30 days showed giant karyomegalic tubule cells with limited degeneration of interstitial tissue and fewer apoptotic bodies in the tubule epithelium than in the kidneys of control animals. The authors also found abnormal mitoses after 30 days of dosing and suggested that regeneration would have occurred if treatment had been stopped (Maaroufi et al., 1999).

Administration of ochratoxin A at 1 or 3 mg/kg of diet and cholestyramine at 1 or 5% of diet for up to 14 days decreased the plasma concentrations of ochratoxin A and the urinary and biliary excretion of this toxin and its metabolites. Increased concentrations of ochratoxin A were found in the faeces. The authors suggested that cholestyramine may have decreased the absorption of ochratoxin A, either by interfering with bile acid secretion or by direct binding (Kerkadi et al., 1998).

These authors subsequently confirmed that cholestyramine can bind ochratoxin A and bile salts in vitro and that depletion of bile salts by interruption of enterohepatic circulation in rats resulted in decreased plasma concentrations of ochratoxin A (Kerkadi et al., 1999).

In vitro

When ochratoxin A was added to isolated rat renal proximal tubules in suspension, mitochondrial dysfunction was seen as an early event in the process of nephrotoxicity. Mitochondrial impairment apparently occurred at sites I and II of the respiratory chain. Although lipid oxidation occurred before cell death, it did not seem to be responsible for the toxic effect (Aleo et al., 1991).

The effects of ochratoxin A on cell growth, cell viability and transepithelial transport were investigated in male Wistar rat proximal tubule cells in serum-free primary culture. Biphasic effects were reported, depending on the concentration of ochratoxin A (0.1–10 ΅mol/L) and the incubation time (24–72 h). The authors considered that these effects could be explained by accelerated cell growth, followed by decreased DNA synthesis due to increased cell density. Addition of albumin or acidification of the culture medium reduced the effects of ochratoxin A in a concentration-dependent manner, whereas alkalinization to pH 7.7 had little effect. Transepithelial electrolyte transport was disrupted at 10 ΅mol/L but not at 0.1 ΅mol/L. The authors concluded that ochratoxin A at physiological (nanomolar) concentrations can stimulate proliferation of proximal tubule cells without exerting toxic effects or reducing cell viability, and that this effect may be mediated by ochratoxin A-induced changes of cellular pH homeostasis (Gekle et al., 1995).

Transport of ochratoxin A by the kidney-specific organic anion transporter 1 (ochratoxin AT1) was investigated in Xenopus oocytes, which transiently express ochratoxin AT1, and cultured S3 cells, with stable expression of ochratoxin AT1, in 5% fetal calf serum. Oocytes with ochratoxin AT1 took up significantly more ochratoxin A (4 ΅mol/L) than oocytes transfected with the vector alone. para-Aminohippurate, probenecid, prioxicam, octanoate, and citronin, which have been reported previously to inhibit the nephrotoxicity of ochratoxin A, inhibited uptake. Uptake of the compound was also greater in ochratoxin AT1-expressing S3 cells than in the mock-transfected parent cells. Cell proliferation was significantly decreased by ochratoxin A at 2 and 10 ΅mol/L, and their viability was decreased by 10 ΅mol/L, in ochratoxin AT1-expressing S3 cells but not in the mock-transfected cells. Incubation with para-aminohippurate suppressed the effects of ochratoxin A. The authors concluded that ochratoxin AT1 plays a pivotal role in the nephrotoxicity of mycotoxins (Tsuda et al., 1999).

The transport of ochratoxin A across the renal peritubular membrane was studied in suspensions of freshly isolated rabbit renal proximal tubules, in order to investigate whether the accumulation of ochratoxin A in proximal tubule cells is involved in its nephrotoxicity. The accumulation and kinetics were determined by fluorescence, which correlates linearly with the concentration of ochratoxin A over a range of 1–70 nmol/L. Accumulation of 10 ΅mol/L was approximately linear for 60 s and approached steady state after 5 min. The uptake was almost completely blocked by 2 mmol/L probenecid, which inhibits the organic anion transport pathway. para-Aminohippurate, which is the prototypic substrate for the peritubular organic anion transporter, also inhibited ochratoxin A uptake, but only by 40–50% at a concentration of 2.5 mmol/L, indicating that uptake occurs by a mechanism in addition to the organic anion transporter. Use of other inhibitors indicated that phenylalanine was not involved, but that a fatty acid transporter may also contribute to uptake of ochratoxin A. The overall results suggest that the peritubular membrane is a significant site for accumulation of ochratoxin A (Groves et al., 1998).

The potential of ochratoxin A to induce apoptosis was examined in human proximal tubule-derived cells (IHKE cells) and compared with that in renal-cell lines derived from opossum proximal tubule (OK cells) and from canine renal collecting duct (MDCK-C11 and MDCK-C7 cells), cultured in 19% fetal calf serum. Ochratoxin A induced a time- and concentration-dependent increase in caspase 3 activity in IHKE cells. Significant increases were seen at 5 nmol/L and a 7-day incubation and at 10 nmol/L with a 24 or 72-h incubation. DNA fragmentation and chromatin condensation confirmed the occurrence of apoptosis at concentrations of 30 and 100 nmol/L. The free-radical scavenger N-acetylcysteine and the intracellular calcium chelator BAPTA-AM, had no effect on ochratoxin A-induced caspase 3 activation, indicating that the mechanism did not involve free-radical production or disturbed calcium homeostasis. IKHE cells were more sensitive to low concentrations of ochratoxin A than MDCK-C11, MDCK-C7, or OK cells. The authors concluded that low concentrations of ochratoxin A led to caspase 3 activation and subsequently apoptosis in cultured human proximal tubule cells but that the mechanism was unclear (Schwerdt et al., 1999b).

The ability of ochratoxin A to activate c-Jun N-terminal kinase has also been investigated in two clones of the MDCK kidney-derived cell line (C7 resembling principal cells and C11 resembling intercalated cells), cultured in 10% fetal calf serum. Incubation with ochratoxin A for 8 h at 10 nmol/L, 100 nmol/L, or 1 ΅mol/L resulted in stimulation of c-Jun N-terminal kinase 1 in MDCK-C7 cells but not in MDCK-C11 cells. Apoptosis, as measured by caspase 3 activity and DNA fragmentation, was observed in MDCK-C7 cells treated with ochratoxin A at 100 nmol/L, which did not cause necrosis as measured by leakage of the cytosolic enzyme lactate dehydrogenase. MDCK-C11 cells were less responsive than MDCK-C7 cells, with a smaller increase in caspase 3 activity at 300 nmol/L ochratoxin A. Lactate dehydrogenase leakage was proportional to DNA fragmentation, indicating that ochratoxin A primarily caused necrosis in MDCK-11 cells. Ochratoxin A at 0.1–0.5 ΅mol/L also potentiated the pro-apoptotic action of tumour necrosis factor-alpha in a concentration-dependent manner, with a greater effect in MDCK-C7 cells than in MDCK-C11 cells. The cell specificity demonstrated in this study indicates that c-Jun N-terminal kinase signalling pathways may play a role in ochratoxin A-induced apoptosis. The authors suggested that this may explain some of the changes in renal function and teratogenicity induced by the toxin (Gekle et al., 2000).

Ochratoxin A inhibited protein synthesis and caused leakage of cytosolic enzymes in Vero monkey kidney cells cultured in the presence of 5% newborn calf serum. Incubation with ochratoxin A for 24 h in the presence of aspartame at 250 ΅mol/L increased the IC50 for inhibition of protein synthesis from 14 ΅mol/L to 22 ΅mol/L. An increase to 34 ΅mol/L was seen when the cells were incubated with the same concentration of aspartame for 24 h before addition of ochratoxin A. Aspartame was also shown to prevent binding of ochratoxin A to plasma proteins and to displace ochratoxin A already bound to plasma proteins. The authors concluded that aspartame could decrease the toxicity of ochratoxin A by affecting binding to plasma proteins as well as by preventing inhibition of protein synthesis (Baudrimont et al., 1997). The Committee noted that these effects required high concentrations of aspartame.

(e) Mechanism of tumorigenesis

The mechanisms of tumour induction in rodent kidney by ochratoxin A have been addressed in many studies, including investigations of the role of biotransformation and bioactivation and the formation of ochratoxin A-derived nucleic acid derivatives in target and non-target organs for toxicity. The results diverge, as do those of the studies on mutagenicity. Although no definite mechanism for the carcinogenicity of ochratoxin A to rodent kidney has been described, non-genotoxic events make a major contribution to the induction and progression of ochratoxin A-derived renal tumours.

Several studies have addressed the biotransformation of ochratoxin A and its role in its toxicity. Biotransformation has been postulated to be involved in the DNA binding and renal tumorigenicity of ochratoxin A, and a variety of CYPs, peroxidases, and glutathione S-transferases have been suggested to catalyse the transformation of ochratoxin A to reactive intermediates (Hietanen et al., 1991; Hennig et al., 1991; Würgler et al., 1991; Malaveille et al., 1994; Fink-Gremmels et al., 1995; Obrecht-Pflumio et al., 1996; Grosse et al., 1997; Pfohl-Leszkowicz et al., 1998; Obrecht-Pflumio et al., 1999; El Adlouni et al., 2000). However, none of these studies assessed the capacity of the respective enzymes to transform ochratoxin A to metabolites or suggested the structure(s) of a reactive metabolite (Castegnaro et al., 1998). Most studies assessed potentially relevant end-points in the toxicity of ochratoxin A and their modulation by changes in xenobiotic-metabolizing enzyme activities. Because of these limitations, no conclusions can be drawn about the mechanisms of ochratoxin A-induced tumour formation in rat kidney.

The possible biotransformation reactions of ochratoxin A have been postulated on the basis of rigorous analytical chemistry. Formation of an ochratoxin A-derived reactive quinone was suggested (Gillman et al., 1999), but this metabolite was formed only by a chemical system that mimics the CYP system. The ochratoxin A-derived reactive quinone was not detected by the use of isolated enzymes and microsomes with high activity for specific CYPs, and only 4R- and 4S-hydroxy-ochratoxin A were formed at very low yields (Gautier et al., 2001; Zepnik et al., 2001). Subcellular fractions rich in prostaglandin synthase activity or purified CYP enzymes also did not catalyse the formation of reactive ochratoxin A metabolites (Gautier et al., 2001).

The known mechanisms of formation of ochratoxin A metabolites (insertion of an oxygen into a carbon–hydrogen bond) do not suggest formation of reactive and toxic intermediates. The lack of involvement of CYP-mediated oxidation in the toxicity of ochratoxin A is supported by the observation that increasing the rates of biotransformation of the toxin by induction of CYP decreases its renal toxicity (Omar et al., 1996), and the observation of typical toxic effects of ochratoxin A in cell systems with very low or no CYP activity (Seegers et al., 1994; Hoehler et al., 1996; Xiao et al., 1996; Dopp et al., 1999). The formation of ochratoxin A-derived radicals capable of interacting with macromolecules is also not indicated. In contrast, the electron spin resonance spectra suggest the formation of hydroxy radicals (Hoehler et al., 1996, 1997).

Formation of DNA adducts has also been postulated as an important event in the tumorigenicity of ochratoxin A. The formation of spots interpreted as ochratoxin A-derived DNA adducts was observed in target tissues in rodents by the very sensitive 32P-postlabelling assay. The nature of the DNA damage and/or mutations caused by ochratoxin A is unknown (Pfohl-Leszkowicz et al., 1991; Würgler et al., 1991; Grosse et al., 1995, 1997; Obrecht-Pflumio & Dirheimer, 2000). The end-points in many of the studies on the mechanisms of tumorigenicity of ochratoxin A was the possible formation of DNA adducts (spots by 32P-postlabelling). However, a role of DNA binding of ochratoxin A is not supported by the results of studies of biotransformation cited above or of experiments to investigate the binding of radiolabelled ochratoxin A to nucleic acids (Gautier et al., 2001). Studies of DNA binding with [3H]ochratoxin A revealed no binding of ‘metabolically activated’ ochratoxin A to calf thymus DNA in vitro or to DNA from rat liver or kidney in vivo. The sensitivity of these experiments was similar to that of the postlabelling studies. Lack of DNA binding of ochratoxin A or its metabolites was observed in vivo after administration of a single dose of [3H]ochratoxin A (Rasonyi, 1995).

In summary, these data cast doubt on the hypothesis that ochratoxin A causes renal tumours by covalent binding of reactive intermediates to DNA. The hypothesis that DNA damage induced by ochratoxin A is due to oxidative stress represents an alternative explanation for the discrepant data and is more consistent with the observations. Several experimental observations support this hypothesis. An unusually large number of DNA adducts (up to 30 individual adducts) was formed from ochratoxin A in low yields in various experimental systems (Castegnaro et al., 1998; Pfohl-Leszkowicz et al., 1998). Patterns of modifications similar to those observed with ochratoxin A by postlabelling were observed in kidney DNA of rodents exposed to iron(III) nitrilotriacetate (Randerath et al., 1995), a renal carcinogen that acts through oxidative stress, or in DNA exposed to hydrogen peroxide (Randerath et al., 1996). Some of these results are consistent with a major role of oxidative stress in the toxicity of ochratoxin A. For example, antioxidants prevent the induction of DNA damage by ochratoxin A in mice (Grosse et al., 1997).

Induction of renal toxicity, oxidative stress due to mitochondrial dysfunction, and persistent cell proliferation represent an alternative mechanism for the renal carcinogenicity of ochratoxin A. The toxin is known to induce oxidative stress (Aleo et al., 1991) and the formation of hydrogen peroxides (Omar et al., 1990). In addition, mechanisms linked to long-term renal toxicity and oxidative stress are known to play an important role in tumour induction in rat kidney (Swenberg & Maronpot, 1991; Dietrich & Swenberg, 1993; Hard, 1998). Several non-genotoxic chemicals that do not undergo bioactivation reactions induce renal tumours in rodents. For example, DNA damage and cellular toxicity mediated by oxidative stress seem to be involved in the renal carcinogenicity of iron(III) nitrilotriacetate and potassium bromate in rodents. These compounds are potent renal carcinogens and induce renal tumours in rodents in high yields after short exposure (Li et al., 1987; Wolf et al., 1998). Sex differences in tumour incidences are also seen with these compounds. For example, as seen with ochratoxin A, male rats are more susceptible to renal tumour induction by potassium bromate (Kurokawa et al., 1983, 1990; Umemura et al., 1998).

(f) Mechanisms of cytotoxicity

Ochratoxin A induced apoptosis in the HL-60 human promyelotic leukaemia cell line as seen by a DNA fragmentation technique and ultrastructural observation. Incubation for 24 h with ochratoxin A at a concentration of 3–4 ΅g/ml resulted in both apoptosis and cytotoxicity, as measured by MTT reduction (Ueno et al., 1995).

A brief report on the possible etiology of Balkan endemic nephropathy noted that apoptosis was not observed in kidneys of rats given ochratoxin A in the diet at 0.8 mg/rat per day for 5 days, which was sufficient to cause extensive necrosis (Mantle et al., 1998). No other details were available.

The toxicity of ochratoxin A, three natural analogues, and 10 synthetic analogues was compared in vitro and in vivo in order to identify the active moiety of the ochratoxin A structure. The studies in vivo involved intraperitoneal injection of mice and intravenous injection of rats, with lethality as the end-point. The hydroxyl, carboxyl, chlorine, and lactone groups of ochratoxin A affected its bactericidal activity (Bacillus brevis), its cytotoxicity to HeLa cells, and its toxicity to mice and rats. Its biological reactivity may be partly associated with the lactone carbonyl group of the isocoumarin moiety. There appeared to be no direct relationship between toxicity and the extent of iron chelation. In addition, formation of a previously undescribed ring-opened metabolite of ochratoxin A was detected in the bile but not in the blood or urine of rats after instillation of 100 ΅g of ochratoxin A into the carotid artery (Xiao et al., 1996).

The Committee noted that these studies are not helpful for risk assessment, because high doses were given by injection and lethality was the only end-point. Furthermore, the studies were inadequately reported.

(g) Effects on the male reproductive system

Ochratoxin A inhibited testosterone secretion in isolated testicular interstitial cells of gerbils (Fenske & Fink-Gremmels, 1990).

Male rats treated by gavage with ochratoxin A at 290 ΅g/kg bw every second day for up to 8 weeks showed a twofold increase in the testicular content of testosterone and accumulation of premeiotic germinal cells, as measured by increases in alpha-amylase, alkaline phosphatase, and gamma-glutamyl transpeptidase activities in testis homogenate. All of these effects were indicative of a disturbance of spermatogenesis (Gharbi et al., 1993).

2.3 Observations in domestic animals and veterinary toxicology

2.3.1 Chickens

Groups of 20 Peterson x Hubbard broiler chickens were fed diets containing ochratoxin A alone at 0 or 2.5 mg/kg of diet or in combination with cyclopiazonic acid for 3 weeks. A significant reduction in body-weight gain was seen by the second week of feeding and was still present at the third week (by 19%). The relative kidney weight was increased in the group given ochratoxin A, and significant increases in serum uric acid and triglycerides but decreased total protein, albumin, and cholesterol were seen (Gentles et al., 1999).

2.3.2 Pigs

Commercial ochratoxin A was administered orally at a dose of at 20 or 40 ΅g/day for 5 weeks to groups of two sexually mature male Hungarian large white and Dutch Landrace boars weighing 250 kg. Ochratoxin A was detected in serum and seminal plasma of both groups (Solti et al., 1996).

2.4 Observations in humans

2.4.1 Biomarkers of exposure

Ochratoxin A has a half-life of about 35 days in humans (Bauer & Gareis, 1987; Hagelberg et al., 1989; Studer-Rohr et al., 1995), and the blood concentrations are considered to represent a convenient biomarker of exposure during recent weeks. This biomarker has been used extensively in epidemiological studies (see below). Similar estimates of exposure have been derived from dietary surveys and from blood analyses, suggesting that the latter is a reliable biomarker.

2.4.2 Biomarkers of effects

The nephrotoxic effect of ochratoxin A is detectable by urinary analysis, but this is a relatively non-specific effect and late in onset. Anaemia is an early manifestation but is also non-specific, and early diagnosis is difficult.

2.4.3 Epidemiological studies

Since ochratoxin A was suggested to be a possible determinant of endemic nephropathy, considerable efforts have been made to determine a correlation between human exposure to this toxin and the incidence of the disease. Endemic nephropathy is a fatal human renal disease, recognized as a specific entity and affecting predominantly rural populations in limited areas of the central Balkan peninsula. So far, the disease has been reported in Bosnia and Herzegovina, Bulgaria, Croatia, Romania, and Yugoslavia (Serbia). The disease was first recognized in the 1950s (Tancev et al., 1956), but there is evidence that it occurred even earlier (Belicza et al., 1979).

The disease starts without an acute episode. Onset is common between the ages of 30 and 50, although there have been reports of patients aged 10–19 (Stoyanov et al., 1978). Its progress is very slow, and after development of nonspecific signs and symptoms there is atypical manifestation of renal impairment (Radonic et al., 1966). The effect on the primary tubules is characterized by a decrease in tubular transport and becomes evident through proteinuria. As a rule, the proteinuria is very mild and is accompanied by the characteristic presence of low-relative-molecular-mass proteins (Hall & Vasiljevic, 1973). Anaemia of the normochromic type is among the first signs of the disease and precedes clinical manifestation of renal impairment (Radonic et al., 1966). The ultrasonic appearance of the kidney is normal at the early stage of the disease, but it becomes smaller as the disease progresses (Borso, 1996). Since there are neither characteristic clinical data nor pathognomonic laboratory indicators, the early diagnosis of endemic nephropathy is difficult and relies on repeated findings of proteinuria, creatininaemia, anaemia, and a family history of the disease.

The prevalence rate of the disease is reported to be 2–10%. In the endemic area of Croatia, a systematic field survey of cases between 1975 and 1990 revealed a prevalence of 0.5–4.4%. The average specific mortality (based on official statistics and documented cases) during the period 1957–84 was 1.5/1000 per year, although some studies have shown that the mortality rate is actually more than twice as high. The disease affects more women than men, and women die more frequently from endemic nephropathy (Ceovic et al., 1992).

A remarkable reduction in the size of the kidney is seen post mortem: in one extreme case, one organ weighed only 20 g. In almost all advanced cases, a characteristic pale dirty yellow discolouration of the skin was common, with a peculiar yellowish colouration of the adipose tissue. The shrinking is progressive, and the organs can be normal or rather small in the early stages of the disease. The kidneys are pale grey and hard to cut (Vukelic et al., 1991). Pathomorphologically, the disease can be described as interstitial, bilateral, non-inflammatory, and non-obstructive nephropathy with heavy damage to the tubular epithelium and extensive interstitial fibrosis starting in the cortex (Vukelic et al., 1992).

The reported incidence of epithelial tumours of the upper urinary tract is much higher in endemic than in non-endemic areas (Chernozemsky et al., 1977; Nicolov et al., 1978; Ceovic & Miletic-Medved, 1996). In the endemic region of Croatia, the prevalence of tumours of the pyelon and ureter is 11 times that in the non-endemic area (Vukelic et al., 1987). Of the malignant tumours, transitional-cell carcinomas were the most frequent (95%); squamous-cell carcinomas were seen in only 5% of cases. Generally, the differences in urothelial tumours between endemic and non-endemic regions include the following: the incidence of tumours is higher in the endemic region, and they affect younger people and women more frequently; the renal pelvis and urethra are the usual sites of tumours in the endemic region, whereas in non-endemic regions the most frequent site is the urinary bladder (Vukelic & Sostaric, 1991). A study of 766 patients treated at the Belgrade Department of Urology for upper urinary tract tumours in 1970–97 showed that the incidence of these tumours was 68% in patients from endemic and probably endemic regions and 32% in patients from non-endemic regions in Yugoslavia (Serbia). The tumours were more frequent in women. A much higher incidence of bilateral tumours was reported in patients from the endemic region (13%) than from non-endemic regions (2%) (Djokic et al., 1999; Table 4).

Table 4. Incidence, by anatomical location, of urothelial tumours among inhabitants of areas endemic and non-endemic for endemic nephropathy

Anatomical location

Endemic area

Non-endemic area

(10 094 inhabitants)

(96 306 inhabitants)

No.

%

No:

%

Pyelon

29

0.286

20

0.021

Ureter

9

0.089

13

0.013

Urinary bladder

23

0.23

86

0.089

Combination
(pyelon–ureter)

6

0.059

7

0.007

Total

67

0.66

126

0.13

From Vukelic et al. (1992)

Striking similarities between the changes in the renal structure and function found in endemic nephropathy and in ochratoxin A-induced porcine nephropathy suggested a common causal relationship (Krogh, 1974). Epidemiological similarities, in particular the endemic occurrence (Krogh, 1976), support the hypothesis that ochratoxin A is a causative agent of endemic nephropathy in humans.

Although ochratoxin A has been found as a contaminant of food and feed all over the world (Krogh, 1992), food samples collected in the endemic areas showed higher contamination. In 1979 in the endemic region of Croatia, ochratoxin A was found in 9.4% of food samples. In a 5-year study in Bulgaria in which 524 food samples from endemic and control villages were analysed, the frequency of positive samples from endemic villages was several times higher than that from non-endemic villages (Pavlovic et al., 1979).

Ochratoxin A was first detected in humans in blood samples from inhabitants of endemic villages (Hult et al., 1982), at a much higher concentration than in non-endemic villages. The prevalence rate was 17% in endemic and 6.0% in non-endemic villages, and similar rates were found in blood samples from endemic (18%) and non-endemic (7.7%) areas in Bulgaria (Petkova-Bocharova et al., 1988).

Low blood concentrations of ochratoxin A have been found in countries where endemic nephropathy has not been detected, such as Canada, the Czech Republic, Egypt, France, Germany, Italy, Sweden, Switzerland, and Tunisia (Bauer & Gareis, 1987; Hadlok, 1993; Breitholtz-Emanuelsson et al., 1994; Zimmerli & Dick, 1995; Malir et al., 1998; Wafa et al., 1998). Some regional differences in exposure to ochratoxin A have been found (Breitholtz et al., 1991; Creppy et al., 1993; Maaroufi et al., 1995a; Scott et al., 1998), and in Croatia in a study of blood from donors in five major cities (Peraica et al., 1999). The mean concentration in the 250 samples was 0.39 ng/ml of plasma, and 59% of samples contained the toxin (detection limit, 0.2 ng/ml). The highest frequency of positive samples (100%), the highest mean ochratoxin A concentration (0.68 ng/ml), and the largest number of samples with a concentration > 1.0 ng/ml (18%) were found in a city relatively near the endemic region. The concentrations reported in blood from healthy persons are shown in Table 5.

Table 5. Occurrence of ochratoxin A in blood samples from healthy persons

Country

Period of collection

Positive/analysed

Concentration (ng/ml)

Reference

No.

%

Mean

Range

Bulgaria

1984–90

9/125

7

 

1.0–10

Petkova-Bocharova et al. (1991)

Canada

1994

144/144

100

0.88

0.29–2.4

Scott et al. (1998)

Czechoslovakia

1990

35/143

24

0.14

0.1–1.3

Fukal & Reisnerova (1990)

Czech Republic

1994

734/809

91

0.23

0.1–14

Malir et al. (1998)

1995

404/413

98

0.24

0.1–1.9

Croatia

1997

148/249

59

0.39

0.2–16

Peraica et al., (1999)

Denmark

1986

 

 

1.5

0.1–9.7

Hald (1991)

1987

 

 

2.3

0.1–9.4

1988

 

 

1.6

0.1–13

France

1991–92

 

 

 

 

Creppy et al. (1993)

Alsace

 

97/500

19

 

0.1–12

Aquitaine

 

385/2055

19

 

0.1–160

Rhone-Alpe

 

75/515

15

 

0.1–4.3

Germany

1977

84/165

51

0.79

0.1–14

Bauer & Gareis (1987)

1985

89/141/

63

0.42

0.1–1.8

1988

142/208

68

0.75

0.1–8.4

Hadlok (1993)

Hungary

1995

291/355

82

 

0.2–10

Solti et al. (1997)

1997

213/277

77

 

0.1–1.4

Tapai et al. (1997)

Italy

1992

65/65

100

0.53

0.1–2.0

Breitholtz-Emanuelsson et al. (1994)

Japan, Tokyo

1992–96

156/184

85

0.068

0.004–0.28

Ueno et al. (1998)

Poland

1983–84

25/397

6

0.21

1.0–13

Golinski (1987)

1984–85

52/668

8

0.31

1.0–40

Sierra Leone

1996

12/36

33a

 

1.5–18

Jonsyn (1996)

Spain

1996–98

40/75

53

0.71

0.5–4.0

Jimenez et al. (1998)

Switzerland

1992–93

 

 

 

 

Zimmerli & Dick (1995)

North of Alps

251/252

100

 

0.06–2.1

South of Alps

116/116

100

 

0.11–6.0

Sweden

1989

 

 

 

 

Breitholtz et al. (1991)

Visby

 

29/99

29

0.26

0.3–7.0

Uppsala

 

3/99

3

0.02

0.3–0.8

Ostersund

 

6/99

6

0.03

0.3–0.8

 

1990–91

39/39

100

0.17

0.09–0.94

Breitholtz-Emanuelsson et al. (1993b)

Tunisia

1993–95

73/140

52

1.2

0.1–8.8

Maaroufi et al. (1995a

From Peraica et al. (1999). Mean concentration calculated for all samples, range given only for positive samples

a Non-breastfed infants up to 5 years of age

Ochratoxin A has been found in human milk. Nine of 50 samples of milk from women in various regions of Italy contained the toxin, at concentrations of 1.2–6.6 ng/ml of milk (Micco et al., 1991).

3. ANALYTICAL METHODS

Ochratoxin A has been found in many commodities, including cereals, cereal products, coffee, grapes, dried vine fruit, grape juice, wine, cocoa and chocolate, beer, meat, pork products, pulses, milk and milk products, and spices. Several published analytical methods for the determination of ochratoxin A in maize, barley, wheat, wheat bran, wheat wholemeal, rye, wine, beer, and roasted coffee have been formally validated in collaborative studies. The methods are based on liquid chromatography (LC) with fluorescence detection, include a solid-phase extraction clean-up step with reversed-phase C18, silica gel 60, or immunoaffinity columns, and can guarantee detection of < 0.5 ΅g/kg. These methods have also been used successfully to analyse a number of other cereals, cereal products, and dried fruit.

The first LC method for determining ochratoxin A in maize and barley was validated in a collaborative study with materials spiked with ochratoxin A in the range of 10–50 ng/g. Ochratoxin A was extracted from grains with chloroform:aqueous phosphoric acid and isolated by liquid–liquid partitioning into aqueous bicarbonate solution that had been cleaned-up on a C18 (solid-phase extraction) cartridge. Identification and quantification were performed by reversed-phase LC with fluorescence detection. The identity of ochratoxin A in samples that contained it was confirmed by methyl ester derivatization followed by LC analysis (Nesheim et al., 1992). The performance characteristics achieved in an international collaborative study involving 16 laboratories are shown in Table 6. The method, which is quantitative for ochratoxin A at concentrations > 10 ΅g/kg in maize and barley, has been accepted as final-action AOAC International Official Method 991.44.

Table 6. Results of collaborative study for determination of ochratoxin A in maize and barley

Matrix

Mean
(ng/g)

No. acceptable results

Mean recovery
(%)

RSDr
(%)

RSDR
(%)

Maize

0.8

15

–

–

–

8.2

15

82

–

21

16

15

82

20

28

40

14

77

–

32

Barley

0.8

15

–

–

–

7.4

15

74

–

27

14

14

72

7.9

26

3

15

74

–

28

RSDr, relative standard deviation for repeatability; RSDR, relative standard deviation for reproducibility

This method was successively validated for other cereals and at lower ochratoxin A concentrations (Larsson & Moeller, 1996). Spiked and naturally contaminated barley, wheat bran, and rye containing ochratoxin A at concentrations of 2–9 ΅g/kg were used. The performance characteristics achieved in the international collaborative study involving 12 laboratories are shown in Table 7. The European Committee for Standardisation (CEN, technical committee 275/WG5 ‘Biotoxins’), which uses specific criteria to select methods, has adopted this method as CEN standard (EN ISO 15141-2) for determination of ochratoxin A in barley, maize, and wheat bran.

Table 7. Results of collaborative study for determination of ochratoxin A in rye, barley, and wheat bran

Matrix

Mean
(ng/g)

No. acceptable
results

Mean recovery
(%)

RSDr
(%)

RSDR
(%)

Rye

2.8

12

64

22

29

4.8

12

65

16

23

Barley

2.9a

12

–

17

22

3.0a

12

–

15

23

Wheat bran

3.8

12

70

21

24

4.5

12

68

17

26

For abbreviations, see Table 6.

a Naturally contaminated sample

The second LC method, adopted as CEN standard (EN ISO 15141-1) for determination of ochratoxin A in cereals and cereal products, was validated in a collaborative study on wheat wholemeal containing ochratoxin A at 0.4 or 1.2 ΅g/kg (Majerus et al., 1994). Ochratoxin A was extracted from grains with toluene after addition of hydrochloric acid and magnesium chloride solution. The filtered extract was cleaned-up on a mini-silica gel column, and ochratoxin A was determined by reversed-phase LC with fluorescence detection. The performance characteristics achieved in the collaborative study involving 13 laboratories according to ISO 5725: 1986 are shown in Table 8. Laboratory experience has shown that this method is also applicable to cereals, dried fruits, oilseeds, pulses, wine, beer, fruit juices, and raw coffee (Jiao et al., 1992; Majerus et al., 1993; Jiao et al., 1994).

During the past few years, the use of antibody-based immunoaffinity columns in the clean-up step has improved the analysis of ochratoxin A. Two methods based on immunoaffinity clean-up for determination of ochratoxin A in barley and roasted coffee have been developed and validated in collaborative studies under the auspices of the European Commission, Standard and Measurement Testing programme (Entwisle et al., 2000a,b). Ochratoxin A was extracted from barley with acetonitrile: water solution, and the filtered sample extract was diluted with phosphate-buffered saline that had been cleaned-up by passage through an immunoaffinity column. For the determination of ochratoxin A in roasted coffee, phenyl silane solid-phase extraction clean-up before the immunoaffinity column stage was introduced to avoid any deleterious effects of caffeine on the immunoaffinity columns (Koch et al., 1996; Entwisle et al., 2000b). In both these methods, ochratoxin A was identified and quantified by reversed-phase LC with fluorescence detection. Spiked and naturally contaminated samples containing ochratoxin A at 1.2–3.7 ΅g/kg were used in the collaborative study. These two methods have been accepted by AOAC International as first-action methods and are being considered for adoption by the CEN as standards. The performance characteristics achieved in the collaborative studies involving 15 laboratories are shown in Tables 9 and 10 for barley and roasted coffee, respectively.

Table 8. Results of collaborative study for determination of ochratoxin A in wholemeal wheat

Matrix

Mean
(ng/g)

No. acceptable results

Mean recovery
(%)

RSDr
(%)

RSDR
(%)

Wheat,

0.41

13

80

15

26

wholemeal

1.2

13

80

20

32

For abbreviations, see Table 6.

Table 9. Results of collaborative study for determination of ochratoxin A in barley

Matrix

Mean
(ng/g)

No. of acceptable results

Mean recovery
(%)

RSDr
(%)

RSDR
(%)

Barley

0.1a

14

–

26

72

1.3b

15

–

24

33

3.0b

14

–

12

17

4.5b

12

–

14

15

3.7

12

93

4

12

For abbreviations, see Table 6.

a Blank sample

b Naturally conataminated sample

Table 10. Results of collaborative study for determination of ochratoxin A in roasted coffee

Matrix

Mean
(ng/g)

No. of acceptable results

Mean recovery
(%)

RSDr
(%)

RSDR
(%)

Roasted coffee

0.1a

13

–

27

71

1.2a

14

–

22

26

2.6a

15

–

11

15

5.4a

12

–

2

14

3.5

13

85

6

13

For abbreviations, see Table 6.

a Naturally contaminated sample

The occurrence of ochratoxin A in wine, especially red wine, was first reported by Zimmerli and Dick (1995). The analytical method they used involved extraction of ochratoxin A by liquid–liquid partitioning with chloroform, clean-up on an immunoaffinity column, and determination by reversed-phase LC with fluorescence detection. A more accurate and precise method has since been developed for determination of ochratoxin A in red, rosé, and white wine (Visconti et al., 1999) and beer (Visconti et al., 2000a). After simple dilution with water containing polyethylene glycol and NaHCO3, wine or beer samples were cleaned-up on immunoaffinity columns and analysed by reversed-phase LC with fluorescence detection. The method was validated in a collaborative study. It has been adopted as a First Action Method by AOAC International and is being considered for adoption by the CEN as a standard (Visconti et al., 2000b). The performance characteristic obtained in the collaborative study are reported in Table 11.

Table 11. Results of collaborative study for determination of ochratoxin A in wine and beer

Matrix

Mean
(ng/g)

No. of acceptable results

Mean recovery
(%)

RSDr
(%)

RSDR
(%)

White wine

< 0.01a

–

–

–

–

0.10

13

100

10

14

1.0

14

91

6.6

14

1.8

14

88

8.5

13

0.28b

15

–

11

15

Red wine

< 0.01a

–

–

–

–

0.19

12

93

5.5

9.9

0.81

14

90

9.9

12

2.5

15

85

8.9

13

1.7b

14

–

11

13

Beer

< 0.01a

–

–

–

–

0.19

13

95

10

18

0.70

15

87

7.2

18

1.4

13

94

4.6

16

0.069b

14

–

19

20

For abbreviations, see Table 6.

a Blank sample

b Naturally contaminated sample

The European Commission, Measurement and Testing Programme sponsored projects to improve the method and to prepare certified reference materials for determination of ochratoxin A in wheat and pig kidney (Hald et al., 1993; Wood et al., 1996; Entwisle et al., 1996; Wood et al., 1997; Williams et al., 1998). The study in wheat involved 26 European laboratories and was conducted in three phases: a first comparison of procedures, a second comparison of procedures, and certification of two reference materials. Various procedures were compared in the first step, including chloroform, methanol, toluene, or ethyl acetate for extraction, and silica, reversed-phase, or immunoaffinity columns for clean-up. LC was used for deter-mination in all laboratories except one, where thin-layer chromatography TLC) was used (Hald et al., 1993). In the second comparison of procedures, all laboratories used high-performance liquid chromatography (HPLC) for quantification of ochratoxin A, whereas acetonitrile, chloroform, dichloromethane, ethyl acetate, methanol, and toluene were used as extraction solvents. Various clean-up procedures were used, including silica, reversed-phase, diatomaceous earth, and immunoaffinity columns and liquid–liquid partitioning. Fifteen of 26 participants had results that fulfilled the agreed acceptance criteria and therefore participated in the certification study (Wood et al., 1996). The results of nine of these 15 laboratories were accepted for certification of the two wheat reference materials. The methods used by these nine laboratories for setting the certification value of ochratoxin A in blank and naturally contaminated wheat flour are summarized in Table 12. The two certified reference materials are useful for ensuring the quality of analyses and can be used to prepare in-house reference materials easily and inexpensively. Certified reference materials for mycotoxins, including ochratoxin A, are available from the European Union Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium.

Table 12. Methods used by laboratories accepted for setting the certification value for ochratoxin A in wheat flour

Extraction

No. of laboratories

Clean-up

25-g test portion

4

Silica column (1 laboratory)

125 ml CHCl3

 

C18 column (2 laboratories)

12.5 ml 0.1 mol/L H3PO4

 

Liquid–liquid defatting (1 laboratory)

25-g test portion

3

Silica column

125 ml toluene

 

 

30 ml 2 mol/L HCl

 

 

25 ml 0.4 mol/L MgCl2

 

 

25-g test portion

1

Immunoaffinity column

24 ml acetonitrile

 

 

16 ml phosphate-buffered saline

 

 

25-g test portion

1

C18 column

80 ml ethyl acetate

 

 

8 ml 5% acetic acid

 

 

All laboratories used liquid chromatography with reversed-phase C18 and fluorescence detection

Two comparative studies of methods for the analysis of ochratoxin A in freeze-dried pig kidney were performed in order to determine the feasibility of preparing certified reference materials. In the first study, almost all of the 20 European laboratories reported lower than usual recoveries for the freeze-dried material (Entwisle et al., 1996). The methods used were similar to those used in the comparative studies for ochratoxin A in wheat (see above). The results of the second comparative study of methods for the analysis of ochratoxin A in freeze-dried pig kidney indicated the following: the reconstitution of freeze-dried material is crucial, as small lumps of powdered material may be formed; in recovery experiments, the spiking solution must be added to reconstituted material and not to powdered material in order to avoid formation of small lumps; immunoaffinity clean-up resulted in clearer extracts and chromatograms than reversed-phase or silica gel columns or liquid–liquid partitioning; the use of 1 mol/L instead of 0.1 mol/L phosphoric acid in the extraction step did not improve the recovery of ochratoxin A (Williams et al., 1998).

3.1 Screening tests

Screening methods based on TLC are available, and one has been collaboratively validated (Nesheim et al., 1973). These methods are used in only a few laboratories since they do not provide an adequate limit of quantification (LOQ). Enzyme-linked immunoabsorbent assays (ELISAs) have been developed for the detection of ochratoxin A in pig kidney, animal and human sera, cereals, and mixed feed. The results obtained with these methods require confirmation since the antibodies produced often show cross-reactivity to compounds similar to ochratoxin A. These methods were not used in the surveys considered by the Committee.

3.2 Conclusions

Because of the large number of commodities contaminated with ochratoxin A, several LC analytical methods have been proposed. Validated analytical methods are available for accurate and precise determination of ochratoxin A in maize, barley, rye, wheat, wheat bran, wheat wholemeal, roasted coffee, wine, and beer. The introduction of immunoaffinity columns has improved analytical methods for ochratoxin A. Use of these columns reduces the need for dangerous solvents, drastically improves the clean-up of extracts, improves detection, and simplifies sample preparation and clean-up.

The analytical methods used for the determination of ochratoxin A in foods are summarized in Table 13.

Table 13 (a). Analytical methods used to determine ochratoxin A in foods

Reference

Commodity

Test portion
(g)

Extraction

Clean-up

Quantification

Wolff et al. (2000)

Wine, juices, oil, vinegar

5 (ml)

Diluted with 1 ml PBS

Immunoaffinity column

HPLC (RP18)/ fluorescence detection

Wolff et al. (2000)

Meat and meat products

25

HCl–MgCl2 solution + CHCl3

Liquid–liquid partitioning with NaHCO3 + immunoaffinity

HPLC (RP18)/
fluorescence detection column

Wolff et al. (2000)

Cereals

40

30 ml HCl–MgCl2 + 125 ml toluene

SiO2 column Sep pak

HPLC (RP18)/
fluorescence detection

Wolff et al. (2000)

Beer

5 (ml)

Diluted with 1 ml PBS

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Wolff et al. (2000)

Coffee, tea

25

500 ml H2O–NaHCO3

Dilution with PBS and clean-up on immunoaffinity column

HPLC (RP18)/
fluorescence detection

Wolff et al. (2000)

Milk products, sweets, oilseeds

50

200 ml acetonitrile: water (6:4) 2-min blending

Dilution with PBS and clean-up on immunoaffinity column

HPLC (RP18)/
fluorescence detection

Langseth et al. (1989); Langseth (1999)

Cereals

25

125 ml CHCl3 + 12.5 ml 0.1 mol/L H3PO4; shaking 60 min

Silica Sep-pack cartridge or immunoaffinity column

HPLC (RP18)/
fluorescence detection

Larsson & Moeller (1996)

Wheat, barley, rye

50

25 ml 0.1 mol/L H3PO4 + 250 ml CHCl3 + 10 g diatomaceus earth; 3-min blending

Liquid–liquid partitioning with 3% NaHCO3 + C18 (Sep-pack)

HPLC (RP18)/
fluorescence detection

Soares et al. (1985)

Corn, peanuts, beans, rice, cassava

50

270 ml MeOH + 30 ml 4% KCl; 5 min blending

Clarification with (NH4)2SO4 and Hyflo Super-Cel; dilution with water and liquid-liquid partitioning with CHCl3

Visual TLC (20 x 20 cm silica gel 60)

Trucksess et al. (1999)

Wheat, barley, coffee

25

100 ml MeOH:1% NaHCO3 (7+3); 3-min blending

Dilution with PBS and clean-up on Immunoaffinity

HPLC (RP18)/
fluorescence detection column

Solfrizzo et al. (1998)

Wheat, oats

10

60 ml CHCl3 + 5 ml 0.1 mol/L H3PO4 30 min shaking

Dried extract dissolved in HPLC mobile phase and defatted with n-

HPLC (RP18)/
fluorescence detection hexane by liquid–liquid partitioning

Jorgensen et al. (1996)

Wheat, rye, barley, oats, bran, pork, poultry meat and liver

50

250 ml CH2Cl2:EtOH (4+1) + 25 ml 0.1 mol/L H3PO4 30-min shaking

Liquid–liquid partitioning with 0.35 mol/L NaHCO3+EtOH (5+2) and CH2Cl2

HPLC (RP18)/
fluorescence detection

Jorgensen (1998)

Beer

150 (ml)

Beer degassed for 60 min

Degassed beer passed on immunoaffinity column

HPLC (RP18)/
fluorescence detection

Jorgensen (1998)

Roasted coffee

50

200 ml 1% m/m NaHCO3; 2-min blending

Dilution with PBS and clean-up on immunoaffinity column

HPLC (RP18)/
fluorescence detection

Jorgensen (1998)

Pulses

50

200 ml acetonitrile: water (6:4); 2-min blending

Dilution with PBS and clean-up on immunoaffinity column

HPLC (RP18)/
fluorescence detection

Patel et al. (1996)

Cereals, oils, nuts, seeds, herbs, pickles, canned food

25

125 ml CHCl3 + 12.5 ml 0.1 mol/L H3PO4 30-min shaking

Silica Sep-pack cartridge

HPLC (RP18)/
fluorescence detection

Maaroufi et al. (1995)

Olives, cereals and derived foods, vegetables

100

120 ml CHCl3+HCl (100+1); shaking overnight

Preparative TLC

HPLC (RP18)/
fluorescence detection0

Sharman et al. (1992)

Cereal products

10

40 ml PBS:MeOH (50:50); 3–5-min blending

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Sharman et al. (1992)

Animal products

10

100 ml CHCl3+ 0.6 g 85% H3PO4 3–5-min blending

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Rao (2000)

Foods

NR

Acetonitrile:water or MeOH:water

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

MAFF (1999a)

Cereals

25

CHCl3 + 0.1 mol/L H3PO4

Silica Sep-pack cartridge

HPLC (RP18)/
fluorescence detection

Howell & Taylor (1981; modified)

Maize

25

250 ml CHCl3 + 25 ml 0.1 mol/L H3PO4 30-min shaking

Silica Sep-Pak cartridge

HPLC (RP18)/
fluorescence detection

Pineiro & Giribone (1994)

Various foods and feeds

NR

NR

NR

TLC

MAFF (1999b)

Dried fruit, chocolate, cocoa, pulses

NR

NR

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

MAFF (1999b)

Wine, grape juice

NR

NR

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

MAFF (1999c)

Total diet

NR

CHCl3:H3PO4

Liquid–liquid partitioning with NaHCO3 solution and clean-up on immunoaffinity column

HPLC (RP18)/
fluorescence detection

MAFF (1996b)

Green coffee beans

50

25 ml 0.1 mol/L H3PO4 + 250 ml CHCl3 + 10 g diatomaceus earth; 3-min blending

Liquid–liquid partii- tioning with 3% NaHCO3, immunoaffinity and C18 (Sep-pack)

HPLC (RP18)/
fluorescence detection

Stegen et al. (1997)

Coffee products

Cooperative study with 9 laboratories using different methods

HPLC (RP18)/
fluorescence

Nesheim et al. (1973)

Barley

50

25 ml 0.1 mol/L H3PO4 + 250 ml CHCl3 + 10 g

Liquid–liquid partitioning

TLC sprayed with ammonia and scanned with fluoridensitometer

Scott et al. (1991)

Meat, kidney, liver

25

100 ml CHCl3 + 50 ml 2 nmol/L NaCl + 50 ml 0.5 mol/L H3PO4 60-min shaking

Silica gel column

HPLC (RP18)/
fluorescence detection

Patel et al. (1997)

Roasted and soluble coffee

25

125 ml CHCl3 + 12.5 ml 0.1 mol/L H3PO4; 30-min shaking

Silica Sep-Pak cartridge + immunoaffinity column

HPLC (RP18)/
fluorescence detection

Burdaspal & Legarda (2000)

Baby food

25

100 ml 0.5 mol/L H3PO4 2 mol/L NaCl + 50 ml tert-butyl methyl ether; 2-min blending

Liquid–liquid partitioning + immunoaffinity clean-up

HPLC (RP18)/
enhanced fluorescence detection

Zimmerli & Dick (1995, 1996)

Wine, grape juice

5 (ml)

Dilution with H3PO4: 2 mol/L NaCl (33.7: 966.3), extraction with 5 ml CHCl3 1-min vortex

immunoaffinity column

HPLC (RP18)/
enhanced fluorescence detection

Burdaspal & Legarda (1998a)

Beer

5

Dilution with 1 ml 1% NaHCO3 15% NaCl

Immunoaffinity column

HPLC (RP18)/
enhanced fluorescence detection

Ueno (1998)

Coffee

0.5

8 ml 1% Na2CO3 and diluted with PBS

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Canned coffee

5 (ml)

Filtration

Wine and beer

5 (ml)

Mixed with 1 ml 2.5% Na2CO3 1.5% NaCl

Visconti et al. (1999, 2000a)

Wine, beer

10 (ml) 10 ml 1%

Dilution with PEG and 5% NaHCO3

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Ottender & Majerus (2000)

Wine

25 (ml)

Dilution with 25 ml PBS and pH 7.0–7.5

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Koch et al. (1996)

Roasted coffee

15

150 ml MeOH + 3% NaHCO3 (1+1, v/v) 30-min shaking column

Double clean-up: phenyl silane column and immunoaffinity

HPLC (RP18)/
fluorescence detection

Majerus et al. (1994)

Wheat wholemeal, cereals, dried fruits, pulses, wine, beer, fruit juice, raw coffee

20

30 ml HCl+ 50 ml 0.4 mol/L MgCl2; extracted with 100 ml toluene by shaking 60 min

Silica gel column

HPLC (RP18)/
fluorescence detection

Leoni et al. (2000)

Instant and roasted coffee

10

200 ml 1% NaHCO3 2-min blending

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Sizoo & van Egmond

Wheat and wheat products

25

CH3CN/water

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Inspectorate of Health Protection

Wheat and wheat products

20

MeOH/water or CH3CN/water

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Inspectorate for Health Protection

Wine

5 (ml)

Dilution with water

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Inspectorate for Health Protection

Roasted coffee

10

MeOH/3% NaHCO3

Phenyl silane column + immunoaffinity column

HPLC (RP18)/
fluorescence detection

Inspectorate for Health Protection

Paprika, pepper, nuts

20

MeOH/water or CH3CN/water

Immunoaffinity column

HPLC (RP18)/
fluorescence detection

Entwisle et al. (2000b)

Roasted coffee

15

MeOH/3% NaHCO3

Phenyl silane column + immunoaffinity column

HPLC (RP18)/
fluorescence detection

 

Table 13 (b). Analytical methods used to determine ochratoxin A in foods

Reference

Commodity

Test portion
(g)

LOD/LOQ
(΅g/kg)

Accuracy

Spiking
(΅g/kg)

Recovery
(%)

RSDr
(%)

RSDR
(%)

Wolff et al. (2000)

Wine, juices, oil, vinegar

5 (ml)

0.01/

 

 

 

 

Wolff et al. (2000)

Meat and meat products

25

0.01/

0.05 –5.0

34–104

 

 

Wolff et al. (2000)

Cereals

40

0.01/

 

 

 

 

Wolff et al. (2000)

Beer

5 (ml)

 

0.01/

 

 

 

Wolff et al. (2000)

Coffee, tea

25

 

0.3/

 

 

 

Wolff et al. (2000)

Milk products, sweets, oilseeds

50

 

0.01/

 

 

 

Langseth et al. (1989); Langseth (1999)

Cereals

25

0.01/

10

80–114

 

 

Larsson & Moeller (1996)

Wheat, barley, rye

50

0.1/

4.96

72

 

 

5.95

69

17–22 (5–7 ΅g/kg)

24–28 (5–7 ΅g/kg)

7.44

66

Soares et al. (1985)

Corn, peanuts, beans, rice, cassava

50

10/

10–400

86–125

0–24

 

Trucksess et al. (1999)

Wheat, barley, coffee

25

0.03/

1–4

71–96

2–17

 

Solfrizzo et al. (1998)

Wheat, oats

10

/0.8

1–100

82–104

3–7

 

Jorgensen et al. (1996)

Wheat, rye, barley, oats, bran, pork, poultry meat and liver

50

0.05/

5

80-90

<15

 

0.5–4 (meat, liver)

60–121

Jorgensen (1998)

Beer

150 (ml)

 

0.001/

0.3

66–113

 

Jorgensen (1998)

Roasted coffee

50

 

0.1/

5

59–83

 

Jorgensen (1998)

Pulses

50

 

0.1/

5

78–103

 

Patel et al. (1996)

Cereals, oils, nuts, seeds, herbs, pickles, canned food

25

 

0.1/

10

90

 

Maaroufi et al. (1995)

Olives, cereals and derived foods, vegetables

100

0.1/

10–100

60–85

 

 

Sharman et al. (1992)

Cereal products

10

0.2/

10

74

4

 

Sharman et al. (1992)

Animal products

10

0.2/

10

74–79

3–6

 

Rao (2000)

Foods

NR

0.2/0.5

1–5

70–126

8–13

 

MAFF (1999a)

Cereals

25

0.1/0.2

2

71–104

 

 

Howell & Taylor (1981; modified)

Maize

25

0.1/0.2

2

90–98

5

 

Pineiro & Giribone (1994)

Various foods and feeds

NR

50/

 

 

 

(

MAFF (1999b)

Dried fruit, chocolate, cocoa, pulses

NR

0.1/0.2

 

79–92

1.7–11.1

 

MAFF (1999b)

Wine, grape juice

NR

0.01/0.02

 

79–92

5.9–7.0

 

MAFF (1999c)

Total diet

NR

 

/0.002

 

89

16.8

MAFF (1996b)

Green coffee beans

50

 

/0.26

 

73.3

18

Stegen et al. (1997)

Coffee products

Cooperative study with 9 laboratories using different methods

0.2–1/

 

 

20

42 (CV)

Nesheim et al. (1973)

Barley

50

2/

 

 

 

 

Scott et al. (1991)

Meat, kidney, liver

25

0.5/

1–10

93–106

9–12

 

Patel et al. (1997)

Roasted and soluble coffee

25

0.1/

2

91

5

 

Burdaspal & Legarda (2000)

Baby food

25

/0.008

< 1

91–109

18

 

Zimmerli & Dick (1995, 1996)

Wine, grape juice

5 (ml)

0.003/0.005

0.055

74–91

6

 

Burdaspal & Legarda (1998a)

Beer

5

0.004/

0.114

100

2.4

 

Ueno (1998)

Coffee

0.5

0.003–0.06/

NR

80–90

 

 

Canned coffee

5 (ml)

Wine and beer

5 (ml)

Visconti et al. (1999, 2000a)

Wine, beer

10 (ml) 10 ml 1%

0.01/

0.1–3.0

85–102

5–19

10–20

Ottender & Majerus (2000)

Wine

25 (ml)

0.01/

 

 

8.4

 

Koch et al. (1996)

Roasted coffee

15

0.1/

6

87–94

 

 

Majerus et al. (1994)

Wheat wholemeal, cereals, dried fruits, pulses, wine, beer, fruit juice, raw coffee

20

0.1/

0.41

80

15

26

1.2

80

20

32

 

Leoni et al. (2000)

Instant and roasted coffee

10

0.2/

10

77–100

 

 

Sizoo & van Egmond

Wheat and wheat products

25

0.3/1

5

99

6

 

Inspectorate of Health Protection

Wheat and wheat products

20

0.1/0.25

0.51–54

86–106

8

NA

Inspectorate for Health Protection

Wine

5 (ml)

0.05/0.1

0.33

83–93

9

NA

Inspectorate for Health Protection

Roasted coffee

10

0.13/0.25

0.2–1.0

76–104

33

NA

Inspectorate for Health Protection

Paprika, pepper, nuts

20

0.13/0.25

NA

> 70

NA

NA

Entwisle et al. (2000b)

Roasted coffee

15

0.2/

4

65–97

2.0–22

14–26

 

CV, coefficient of variation; HPLC, high-performance liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MAFF, Ministry of Agriculture, Fisheries and Food (United Kingdom); RSDr, relative standard deviation for repeatability; RSDR, relative standard deviation for reproducibility; NR, not reported; PBS, phosphate-buffered saline; PEG, polyethylene glycol; TLC, thin-layer chromatography

4. EFFECTS OF PROCESSING

The main foods in which the effects of processing have been studied are cereals and coffee, although a little work has been carried out on other foods as well.

4.1 Cereals

In flour manufacture, some parts of the wheat grain are removed, possibly reducing the concentrations of ochratoxin A in flour and subsequent products. This was investigated by Osborne et al. (1996), who allowed growth of a toxin-producing strain of P. verrucosum in two samples of clean hard and soft wheat and then produced both white and wholemeal flour and bread from them. The process greatly influenced the final concentration of ochratoxin A in the bread (Table 14). The scouring process used by Osborne et al. (1996) resulted in a reduction of more than 50% in the concentration of ochratoxin A, but this process is not standard milling practice. Milling hard wheat to produce white flour resulted in an approximately 65% reduction, and a further 10% decrease occurred during baking. The reduction in soft wheat was much smaller; explanations were offered, but these were somewhat academic because bread is usually made from hard wheat. Wholemeal flour and bread showed much smaller reductions in the concentration of ochratoxin A during processing, as might be expected, because less of the grain is discarded. The loss during milling was only 10%, with a further 4% loss during baking (Table 14).

Table 14. Effect of flour and bread manufacture on the concentration of ochratoxin A (fg/kg) in wheat

Wheat type

Initial concentration

Cleaning process

After cleaning

White flour

White bread

Wholemeal flour

Wholemeal bread

Hard

618

Clean

624 (0)

209 (66)

140 (77)

555 (10)

531 (14)

Clean and scour

256 (59)

60 (90)

72 (88)

127 (80)

226 (63)

Soft

643

Clean

473 (27)

389 (40)

224 (65)

553 (9)

476 (26)

Clean and scour

179 (72)

111 (83)

94 (85)

189 (71)

160 (75)

From Osborne et al. (1996). Percentage reductions are given in parentheses. All values are from duplicate experiments, with generally good agreement.

In a study of the stability of ochratoxin A in cereals during heating, a toxigenic strain of A. ochraceus on wheat, and dry and moistened samples were heated at several temperatures and times and the level of destruction recorded. From those figures, the times to 50% destruction of ochratoxin A under various heating conditions were calculated (Table 15; Boudra et al., 1995).

Table 15. Time to reduce ochratoxin A content in wheat by 50% under various conditions

Moisture content of wheat

Temperature of heating (°C)

Time to 50% destruction of ochratoxin A (min)

Dry wheat

100

700

150

200

200

12

250

6

Wet wheat

100

140

150

60

200

19

From Boudra et al. (1995). The wheat samples had been ground; the dry wheat had been dried in a vacuum oven, the wet wheat was 50:50 dry wheat and water.

A reduction in ochratoxin A concentrations resulting from manufacture of retail products from contaminated raw materials was reported by the Ministry of Agriculture, Fisheries and Food (1996a). The processes involved in making breakfast cereals and biscuits caused substantial reductions in the ochratoxin A content; however manufacture of egg noodles and pasta caused little or no reduction (Table 16).

Table 16. Reduction in concentrations of ochratoxin A during food manufacture

Raw material

Ochratoxin
A (fg/kg)

Rate of incorporation of raw material into food

Retail food

Ochratoxin
A (fg/kg)

Reduction (%) (allowing for incorporation rate)

Breakfast cereal grain

0.8

43

Breakfast cereal

< 0.2

> 50

Breakfast cereal grain

0.8

90

Breakfast cereal

< 0.2

> 76

Biscuit flour

6.4

40

Biscuits

0.4

84

Durum wheat flour

1.4

82.5

Egg noodles

1.0

13

Durum wheat flour

0.9

82.5

Egg noodles

0.8

0

Durum wheat flour

1.6

82.5

Egg noodles

1.3

2

Semolina

0.8

100

Pasta (dried)

0.6

25

Semolina

0.8

100

Pasta (dried)

1.0

0

Modified from Ministry of Agriculture, Fisheries and Food (1996a)

4.2 Coffee

The perception that coffee may be an important source of ochratoxin A led to much work on its formation, destruction during roasting, and presence in brewed coffee. It now seems clear, however, that coffee is not a major source of ochratoxin A in the normal diet (Ministry of Agriculture, Fisheries and Food, 1995).

Reductios in the concentration of ochratoxin A during cleaning and roasting of coffee have been studied, with variable results. Wilkens & Jörissen (1999) showed that cyclone cleaning of green coffee beans had little effect: although the ochratoxin A concentration in the discarded fraction was high, the dust comprised < 1% of the weight of the cleaned coffee. Sorting with colour sorters resulted in some reduction, and steaming caused a mean 25% reduction. Decaffeination is an effective process, resulting in 92% reduction (Heilmann et al., 1999).

Several reports on the effect of roasting on the concentration of ochratoxin A are summarized in Table 17. The reported reductions vary, but a major factor in such variation is that the natural concentration of ochratoxin A in coffee destined for retail markets, i.e. in coffee of sufficient quality for human consumption, seldom exceeds 10 ΅g/kg. Reports based on samples of such coffee usually showed high rates of reduction, e.g. 50–90% (Micco et al., 1989), 30–90% (Wilkens & Jörissen, 1999), and 81% (Blanc et al., 1998). Much less reduction (0–22%) was reported by Studer-Rohr et al. (1995); however, they studied green coffee beans that were naturally spoiled or on which a culture of A. ochraceus had been grown in the laboratory, creating artificially high concentrations (90–1300 ΅g/kg). It is unlikely that such high concentrations are relevant to the conditions found in commercial beans or that degradation of such high concentrations by natural chemical reactions during heating would resemble those that occur in the minute traces of ochratoxin A normally found in coffee beans.

Table 17. Reduction in concentrations of ochratoxin A during roasting of coffee

Sample origin

Method of contamination

Ochratoxin A concentration (fg/kg)

Reduction
(%)

Reference

In green coffee

After roasting

Zaire

Natural

8.6

0.2

98

Micco et al. (1989)

Conillon

Natural

4.0

0.3

92

Micco et al. (1989)

Santos

Spiked

46

6.1

87

Micco et al. (1989)

Costa Rica

Spiked

42

20

49

Micco et al. (1989)

Brazil/Ivory Coast

Spiked

47

11

80

Micco et al. (1989)

Thailand
Robusta

Natural

7.3

1.4

81

Blanc et al. (1998)

Commercial

Spiked

780

890

0

Studer-Rohr et al. (1995)

 

Spiked

1300

1200

11

Studer-Rohr et al. (1995)

 

Natural (spoiled)

360

280

22

Studer-Rohr et al. (1995)

 

Natural (spoiled)

140

121

16

Studer-Rohr et al. (1995)

 

Natural (spoiled)

92

92

0

Studer-Rohr et al. (1995)

Unknown

Natural

0.90

0.63

30

Wilkens & Jörissen (1999)

 

Natural

9.9

2.1

79

Wilkens & Jörissen (1999)

 

Natural

18

1.9

89

Wilkens % Jörissen (1999)

Ivory Coast
Robusta

Natural

4.9

1.5

69

Stegen et al. (2001)

5. LEVELS AND PATTERNS OF CONTAMINATION
OF FOOD COMMODITIES

5.1 Ochratoxin A-producing fungi

Ochratoxin A was originally described as a metabolite of Aspergillus ochraceus in laboratory experiments (van der Merwe et al., 1965). It was subsequently reported in several related Aspergillus species (Ciegler, 1972; Hesseltine et al., 1972), but the first report of its natural occurrence, and its potential importance, was in a different source, a Penicillium species (Scott et al., 1970; Krogh et al., 1973). Recently, A. carbonarius was identified as a third major source, with a low percentage of isolates of the closely related species A. niger (Abarca et al., 1994; Téren et al., 1996). It is now clear that ochratoxin A is produced by a single Penicillium species, P. verrucosum, and a rather remarkable range of Aspergillus species. The following sections deal with these species in more detail.

5.1.1 Aspergillus species that produce ochratoxin A

The latest information (Frisvad & Samson, 2000 and unpublished data) indicates that ochratoxin A is produced by only a few species related to A. ochraceus, all of which are classified in Aspergillus subgenus Circumdati section Circumdati. Apart from A. ochraceus, this group of ochratoxin A producers includes two ascosporic fungi, Neopetromyces muricatus (asexual state A. muricatus) and Petromyces alliaceus (asexual state A. alliaceus) plus Aspergillus sclerotiorum and A. sulphureus. N. muricatus is the correct name for isolates producing ochratoxin A that were previously identified as A. melleus. Both N. muricatus and P. alliaceus are uncommon species. P. sclerotiorum isolates make this toxin only rarely, and although isolates of A. suphureus are usually producers, this is a rare species (J.C. Frisvad, personal communication). Apart from A. ochraceus, all of these species are very uncommon in foods and are not known to cause food spoilage. Hence, the only species of any importance for ochratoxin A production in the Aspergillus section Circumdati is A. ochraceus.

A. carbonarius has been recognized as a source of ochratoxin A only recently (Horie, 1995; Téren et al., 1996; Wicklow et al., 1996). It is now known that most if not all isolates of A. carbonarius are producers when grown in pure culture (Heenan et al., 1998; Taniwaki et al., 1999; J.I. Pitt and colleagues, unpublished), although the extent of production is variable. The closely related species A. niger has also been reported reliably as a producer (Ueno et al., 1991; Abarca et al., 1994; Heenan et al., 1998; Taniwaki et al., 1999; J.I. Pitt and colleagues, unpublished). All reports agree, however, that ochratoxin A production by A. niger is very uncommon, being formed under pure culture conditions by only 1–2% of isolates. A. carbonarius and A. niger are classified in Aspergillus subgenus Circumdati section Nigri.

A number of other species of Aspergillus have been reported to produce ochratoxin A, but in the opinion of Dr J.C. Frisvad, Department of Biotechnology, Technical University of Denmark, an authority on Aspergillus secondary metabolism, none of these has been substantiated (personal communication to J.I. Pitt, 2000).

5.1.2 Penicillium species that produce ochratoxin A

Soon after the isolation of ochratoxin A from A. ochraceus, the formation of ochratoxin A by a Penicillium species, P. viridicatum, was reported (van Walbeek et al., 1969), and its natural occurrence was confirmed (Krogh et al., 1973). The view that P. viridicatum was a major source of ochratoxin A in foods and feeds in some parts of the world was accepted for more than a decade. The species involved was later correctly identified as P. verrucosum (Pitt, 1987), as confirmed by others (Frisvad, 1989; Frisvad & Filtenborg, 1989). Although a number of more recent reports refer to ochratoxin A production by other, often unspecified, Penicillium species, this is known to be erroneous (Frisvad 1989; Frisvad & Filtenborg, 1989; Pitt & Hocking, 1997). It is now clear that P. verrucosum is the only Penicillium species that has been shown to produce ochratoxin A. It is classified in Penicillium subgenus Penicillium section Penicillium, along with many other mycotoxin-producing species.

5.1.3 Physiology and ecology of fungi that produce ochratoxin A

Each of the three major producers of ochratoxin A, P. verrucosum, A. ochraceus, and A. carbonarius, has a quite different physiology and consequently quite different ecological habitat. To understand the kinds of foods in which ochratoxin A occurs and to predict the potential for its formation, it is necessary to understand the physiology and ecology of these species and the differences between them.

(a) Penicillium verrucosum

P. verrucosum is a slowly growing species under any conditions but is capable of growth at low water activity (aw) (down to 0.80) and at low temperature (range, 0–31 oC; optimum, 20 °C) (Pitt & Hocking, 1997).

A notable feature of the ecology of P. verrucosum is that it grows only at lower temperatures. This results in a distribution which is apparently confined to cool temperate regions. Its major food habitat is cereal crops grown in cool temperate climates, ranging across northern and central Europe and Canada. It also occurs in European meat products and in cheese. It appears to be uncommon, indeed almost unknown, in warm climates or in other kinds of foods. The occurrence of this species in European cereals has two consequences: ochratoxin A is present in many kinds of European cereal products, especially bread and flour-based foods, and in animals that eat cereals as a major dietary component. Ochratoxin A was detected in Danish pig meat 25 years ago (Krogh et al., 1973), and its implications for human and animal health were recognized at the same time. As bread, other cereal products, and pig meats are major components of the European diet, the further consequence is that most Europeans who have been tested had appreciable concentrations of ochratoxin A in their blood (Hald, 1991; Petkova-Bocharova & Castegnaro, 1991; Breitholtz-Emanuelsson et al., 1993b; Zimmerli & Dick, 1995; Burdaspal & Legarda, 1998; Jiménez et al., 1998b; Scott et al., 1998). There is no doubt that this results from the growth of P. verrucosum in cereals.

(b) Aspergillus ochraceus

A. ochraceus can be described as a mesophilic xerophile. Growth occurs between 8 and 37 °C, with the optimum at 24–31 °C. Optimal conditions for growth are 0.95–0.99 aw, while the lower limit for growth is 0.79 aw on media containing sugars and down to 0.81 aw on media based on NaCl. A. ochraceus grows slowly at pH 2.2 and well between pH 3 and 10 (Pitt & Hocking, 1997).

A. ochraceus has been isolated from a wide range of food products but is more common in dried and stored foods than elsewhere. Stored foods from which it has been isolated include smoked and salted dried fish, dried beans, biltong, soya beans, chickpeas, rapeseed, pepper, dried fruit, and sesame seeds. Nuts are also a major source, especially pecans and pistachios, and also peanuts, hazelnuts, and walnuts. It has been reported infrequently in cereals and cereal products, including rice, barley, maize, wheat, flour, and bran. A. ochraceus has also been reported in cheese, spices, black olives, cassava, and processed meats. However, this species rarely causes spoilage and is often found in foods only at low concentrations; its presence is therefore not a good indicator of significant mycotoxin production (Pitt & Hocking, 1997).

Several workers have detected A. ochraceus in green coffee beans (Levi et al., 1974; Cantafora et al., 1983; Tsubouchi et al., 1984; Micco et al., 1989; Studer-Rohr et al., 1994), and this species appears to be one source of ochratoxin A in coffee (Taniwaki et al., 1999).

A. ochraceus has been isolated from a variety of South-East Asian commodities, including maize, peanuts, soya beans and other beans, cashews, and sorghum. Its presence or absence in any sample was probably related to the length of storage rather than to geographical location or other factors (Pitt et al., 1993, 1994, 1998).

As noted above, a few species closely related to A. ochraceus can produce ochratoxin A. Little is known about the physiology and ecology of any of these species, but what information there is suggests that their important features are similar to those of A. ochraceus. The occurrence of any of them in foods or food commodities is very rare, however.

(c) Aspergillus carbonarius

Relatively little is known about the third major ochratoxin A producer, A. carbonarius. The ability of this species to produce ochratoxin A was reported only recently (Horie, 1995; Téren et al., 1996; Varga et al., 1996; Heenan et al., 1998). It resembles A. niger in many features, and indeed the two species are very closely related. A. carbonarius differs from A. niger most notably in the production of larger spores, although other minor morphological differences exist. The available information on its physiology indicates a broad similarity to A. niger. However, preliminary studies indicate that A. carbonarius grows at rather lower temperatures than A. niger, with a maximum around 40 °C and optimal conditions about 32–35 °C. The ability to grow at reduced aw is also more restricted: germination occurs down to 0.82 aw at 25 and 30 oC. Unlike A. niger, A. carbonarius failed to germinate at 0.82 aw and 37 °C (S.-L. Leong & J.I. Pitt, unpublished data).

A. carbonarius appears to be less common than A. niger, but many surveys of Aspergilli in foods have probably not differentiated the two species, calling all black Aspergilli A. niger.

As a result of the high resistance of the black Aspergilli, which include A. niger, A. carbonarius, and A. japonicus, to sunlight and ultra-violet light, a major habitat of these species is dried vine fruits, which in most producing countries are dried in the sun without preservatives. The incidence of the black Aspergilli in grapes at harvest and during drying has been studied in the major grape-growing region surrounding Mildura, Victoria, Australia, which is an irrigated area with a hot (35–42 °C) climate during the harvest season. The three major black species, A. niger, A. carbonarius, and A. japonicus, were all very common. The percentage of each species varied from season to season, presumably due to seasonal differences in climatic factors, especially average temperatures and rainfall patterns (S.-L. Leong & J.I. Pitt, unpublished data).

In that study, 470 A. niger isolates, 200 of A. japonicus, and 245 of A. carbonarius were isolated and identified. All were subsequently assayed for ochratoxin A production. The techniques included examination under ultra-violet light after growth on coconut cream agar (Heenan et al., 1998), TLC from agar plugs (Filtenborg & Frisvad, 1980), and, on some isolates, growth in culture, extraction, and HPLC. A. japonicus did not make ochratoxin A, A. niger made it rarely and at low concentrations, but all isolates of A. carbonarius were capable of producing ochratoxin A. These findings indicate that it is extremely likely that A. carbonarius is the major source of ochratoxin A in grapes and grape products, including table grapes, wines, and dried vine fruits.

(d) Aspergillus niger

A detailed account of the ecology of A. niger is included here on the basis that A. carbonarius probably occurs in most habitats in which A. niger has been found. However, this must be regarded as a hypothesis, not a factual statement, at present.

Like many Aspergilli, A. niger grows optimally at relatively high temperatures, with minimal growth at 6–8 °C, maximal growth at 45–47 °C, and optimal conditions of 35–37 °C. A. niger is a xerophile with germination reported at 0.77 aw at 35 °C. The growth rates vary only slightly on media based on sugars, NaCl, or glycerol or at pH 4.0 and 6.5, and at various water activities. Thus, the growth of A. niger appears to be little affected by food type. A. niger can grow down to pH 2.0 at high aw (Pitt & Hocking, 1997).

Among the fungi most commonly reported in foods, A. niger is prevalent in warmer climates, both in field situations and stored foods. It is by far the commonest Aspergillus species responsible for post-harvest decay of fresh fruit, including apples, pears, peaches, citrus, grapes, figs, strawberries, mangoes, tomatoes, and melons and some vegetables, especially onions, garlic, and yams (Snowdon, 1990, 1991). Most of these diseases are sporadic and of minor significance. A. niger sometimes causes kernel rot in cashews and can cause thread mould spoilage of cheese (Hocking & Faedo, 1992).

A. niger is among the commonest fungi isolated from nuts, especially peanuts, and has also been reported in pecans, pistachios, hazelnuts, walnuts, coconut, and copra. Cereals and oilseeds are also sources, especially maize and also barley, soya beans, canola, sorghum, stored and parboiled rice, and dried beans (Pitt & Hocking, 1997).

A. niger has commonly been isolated from South-East Asian foods (Pitt et al., 1993, 1994, 1998). The heaviest contamination was found in peanuts, maize, cashews, copra, pepper, and spices from Indonesia, the Philippines, and Thailand and in kemiri nuts from Indonesia.

In terms of mycotoxin production, A. niger is usually regarded as a benign fungus and has been widely used in food processing. It is categorized as ‘generally regarded as safe’ by the Government of the USA. However, 2 of 19 A. niger isolates were reported to produce ochratoxin A by Abarca et al. (1994) and 2 of 115 by Heenan et al. (1998). Ochratoxin A production by A. niger in commercially grown crops appears possible but is probably uncommon.

5.2 Results of surveys

The results of surveys for ochratoxin A are shown in Appendix A. As contamination with ochratoxin-producing fungi is widespread, numerous commodities have been analysed, including cereal and cereal products, green and roasted coffee, dried fruits, wine, grape juice, cocoa and chocolate, herbs and spices, canned foods, oils, olive, pulses, chickpeas, lentils, soya products, sweets, milk and milk products, meat, kidney, liver, beer, tea, vinegar, mustard, baby food, and house dust.

Most of the information for the past 5 years was taken from the literature and one Internet site. Other data were submitted to the Committee for the current meeting. The natural occurrence of ochratoxin A before 1995 was reviewed by WHO (1990) and the Commission of the European Union (1997).

The total number of samples for which data are shown in Appendix A was 23 167, with 85% from Europe (Croatia, Denmark, Finland, France, Germany, Italy, the Netherlands, Norway, Spain, Sweden, Switzerland, and the United Kingdom), 7% from South America (Brazil and Uruguay), 6% from North America (Canada and the USA), 1% from Africa (Sierra Leone and Tunisia), and 1% from Asia (Dubai and Japan).

When mean values and 90th percentiles were not available, they were calculated from a single datum, if available, assuming 0 for those samples containing no detectable toxin. The parent reference (P), analytical method (A) and sampling method used (S) are shown for each entry. When available, details of the sampling procedure are reported. The analytical methods cited in Appendix A are described in Table 13.

Adequate sampling procedures should be used for future surveys of ochratoxin A in cereals and cereal products. For example, 10 of 22 submitted papers giving data on cereals described the sampling procedure, whereas no description was reported in the remaining 12 papers. When the collected sample was small, the number of total samples was adequately reduced in order to limit the impact of occasionally high values on the weighted mean concentration (Jorgensen et al., 1996; Solfrizzo et al., 1998; Jurcevic et al., 1999). No sampling plans for the determination of ochratoxin A in foods have been published, and details of sampling variation have not been reported.

Most of the samples reported in Appendix A (95%) were analysed by LC with fluorescence detection, and the LOD or LOQ was usually < 0.5 ΅g/kg. The remaining 5% of samples were analysed by TLC with limits of detection or quantification > 5 ΅g/kg. These data were not used to estimate intake as the analytical method used did not provide an LOQ down to 5 ΅g/kg, the lower level that the Committee was requested to evaluate by the Codex Committee on Food Additives and Contaminants. In fact, only one of the 1225 samples analysed by TLC, a sample of wheat, was found to be contaminated with 40 ΅g/kg of ochratoxin A (Pineiro & Giribone, 1994; Furlong et al., 1995a; Pineiro et al., 1996). When a more sensitive method was used (LOD = 0.2 ΅g/kg), most of the samples (78%) from the same geographical area were contaminated with ochratoxin A (Leoni et al., 2000).

The incidence of contaminated samples varied by commodity, and the incidence was higher in the same commodity when analytical methods with lower LOQs were used. For example, a survey of 300 samples of pig kidney in Denmark showed a high incidence of contamination (79%), and five samples were contaminated at > 5 ΅g/kg (Petersen, 2000). Similar results were obtained in an analysis of 61 samples of pig kidney collected in Germany (Wolff et al., 2000), whereas a low incidence of positive samples (6%) was found among 1010 samples collected in France (Dragacci et al., 1999). The different analytical methods used in these studies, with different LOQs (0.06 ΅g/kg and 0.5 ΅g/kg), could explain the discrepant results.

The concentrations of ochratoxin A in the various commodities were highly variable; 1.4% and 0.6% of all samples contained more than 5 ΅g/kg and 20 ΅g/kg, respectively. Within one type of cereal, 1.2% and 0.3% of samples contained more than 5 ΅g/kg and 20 ΅g/kg, respectively. Within cereal products, 0.3% and 0.05% of samples contained more than 5 ΅g/kg and 20 ΅g/kg, respectively. For this calculation, the cereal product samples from Tunisia were not considered, as they were selected samples from families of patients with nephropathy (Maaroufi et al., 1995b).

The weighted mean concentrations of ochratoxin A in cereals, cereal products, cocoa and chocolate, coffee, dried vine fruit, grape juice, pig kidney, other products of animal origin, and wine are shown in Table 18. Samples for which the mean concentration of ochratoxin A was lacking were not considered. High concentrations (­ 50 ΅g/kg) were also reported in herbs and spices (Patel et al., 1996; Rao, 2000) as well as in house dust (< 1600 ΅g/kg), confirming the widespread presence of ochratoxin A (Richard et al., 1999). Low concentrations were found in all 50 samples of the total diet analysed in the United Kingdom by a very sensitive method capable of detecting down to 0.002 ΅g/kg (Ministry of Agriculture, Fisheries and Food, 1999b).

Table 18. Weighted mean concentrations of ochratoxin A in commodities evaluated

Commodity

No. of samples

Weighted mean concentration (΅g/kg)

Beer

660

0.025

Cereals, all

2700

0.94

Barley

350

0.53

Maize

95

7.5

Oats

280

0.44

Rice

45

0.06

Rye

790

1.2

Wheat

1200

0.38

Cereal products

1500

0.19

Cocoa and chocolate

270

0.18

Coffee, green and roasted

1900

0.86

Green

130

1.0

Roasted

1700

0.76

Instant

290

1.4

Dried vine fruit

860

2.3

Grape juice

68

0.44

Pig kidney

380

0.12

Products of animal origin
(liver, meat, sausages)

810

0.052

Wine, all

1800

0.32

Red

1300

0.4

White

260

0.1

5.3 Distribution

As most of the samples (85%) represented the European diet, it is difficult to evaluate the geographical distribution of ochratoxin A. The data indicate that it occurs in coffee regardless of the geographical origin of the samples. For example, high percentages of contaminated samples were found in all countries and regions where coffee has been analysed, i.e. Brazil, Canada, Dubai, Europe, including eastern Europe, Japan, and the USA (Pittet et al., 1996; Stegen et al., 1997; Ueno, 1998; Trucksess et al., 1999; Government of Canada; Leoni et al., 2000; Rao, 2000; Wolff et al., 2000). There is clear evidence for the occurrence of ochratoxin A in cereals and cereal products in the European diet, but little information was available for other diets. The limited data indicate that ochratoxin A is also found in cereals produced elsewhere; in particular, contamination was found in 1 of 10 wheat samples and 3 of 28 mixed cereal samples in Dubai (Rao, 2000) and 56 of 383 wheat samples and 11 of 103 barley samples in the USA (Trucksess et al., 1999).

Ochratoxin A was found in 13 of 25 oat samples and 8 of 22 rye samples imported into Denmark (Jorgensen et al., 1996), 15 of 108 wheat samples and 12 of 41 rye samples imported into Norway (Langseth, 1999), and 14 of 139 maize samples imported into the United Kingdom (Scudamore & Patel, 2000). The data from Brazil and Uruguay could not be used as they had been obtained by analytical methods with high LODs, ranging from 5 ΅g/kg to 50 ΅g/kg, which are inadequate to detect and measure ochratoxin A at 0.94 ΅g/kg and 0.19 ΅g/kg, the weighted mean concen-trations found in Europe and the USA in cereals and cereal products, respectively (Pineiro & Giribone, 1994; Furlong et al., 1995a,b; Soares & Furlani, 1996; Pineiro et al., 1996).

Indirect evidence for the occurrence of ochratoxin A in foodstuffs in Africa is provided by the high incidence of contaminated samples (35%) and the high mean ochratoxin A concentration (7.9 ΅g/kg) found in breast milk collected in Sierra Leone, where some infants were exposed to concentrations that far exceeded the permissible levels in animal feeds in developed countries (Jonsyn et al., 1995). These high concentrations indicate considerable human exposure to this toxin from food; however, no surveys have been reported from that area. More surveys are needed in regions of the world other than Europe in order that intake in those regions can be assessed.

An unexpectedly high mean concentration of ochratoxin A, < 33 000 ΅g/kg, was reported in foods (wheat, barley, mixed cereals, dried vegetables, and olives) collected in Tunisia (Maaroufi et al., 1995b). One of the authors (E. Creppy) of the paper was contacted in order to check whether results had erroneously been reported as ng/kg instead of ΅g/kg. The response confirmed the concentrations reported in the paper, and the information was provided that the samples had been taken from members of families with one or more patient with nephropathy. These results should be confirmed in further studies with validated methods for determination of ochratoxin A, and they were not considered for estimating intake.

5.4 Annual variation

Data on annual variations in contamination with ochratoxin A were available for wine (Pietri, 2000), wheat, barley (Scudamore, 1999), and maize (Jurcevic et al., 1999). Higher incidences of contaminated samples and higher mean concentrations of ochratoxin A were found in maize collected in Croatia in 1997 (35%, 57 ΅g/kg) than in 1996 (10%, 38 ΅g/kg) (Jurcevic et al., 1999). A survey of wheat and barley was carried out in the United Kingdom during the crop year 1993–94. In 1993, only two of 611 cereal samples (0.3%) were contaminated, each containing 15 ΅g/kg. In 1994, the incidence was much higher, with 22 of 450 samples (9%) containing ochratoxin A at concentrations ranging from 1 to 32 ΅g/kg (Scudamore, 1999). Limited data from Italy for red wine (169 samples) showed no substantial variation of incidence over the years 1996–99, ranging from 70% to100%, whereas the mean concentration was 0.54–0.76 ΅g/kg in 1996–98 (54 samples) and 2.1 ΅g/kg in 1999 (115 samples) (Pietri, 2000).

6. FOOD CONSUMPTION AND DIETARY INTAKE ASSESSMENTS

6.1 National and regional estimates of intake

Ochratoxin A was first encountered as a natural contaminant in maize and to a lesser extent in some beans, including coffee and cocoa. Residues of ochratoxin A are not generally found in ruminants, because the toxin is cleaved in the rumen by protozoan and bacterial enzymes. Residues have been detected in a number of tissues of non-ruminant food animals, such as pigs, and in the muscle of hens and chickens but not in eggs.

Three types of data were submitted, the first on the occurrence of ochratoxin A in foods, the second on the intake of potentially contaminated foods, and the last on biomarkers of the exposure for use in epidemiological studies.

6.1.1 Occurrence of ochratoxin A in foods

Data were submitted by 13 countries: Argentina, Brazil, Canada, China, Denmark, France, Germany, Italy, Norway, Sweden, the United Kingdom, Uruguay, and the USA. Additional data were submitted by the Commission of the European Union and the Coffee Science Information Centre. Most of the data referred to cereals and cereal products, of which a total of 7877 samples were analysed. More than 2000 analyses were provided by Germany, pooled into six categories; 485 were from the USA, 212 from Sweden, 117 from Canada, and 75 from France. More than 2500 analyses were provided by the Commission of the European Union on the contamination of cereals by ochratoxin A in Denmark, Italy, The Netherlands, Spain, and the United Kingdom.

Brazil submitted information about ochratoxin A contamination in the electronic submission format of GEMS/Food. The data related to peanuts, grapes, beans, coffee, and maize, with a total number of 806 samples. The results for most of the samples (624) were, however, expressed as a percentage of positive values or as a range.

Canada submitted the results of surveys conducted between 1997 and 1999 on ochratoxin A in coffee and cereals, in a total of 101 samples of coffee and 117 samples of cereals and cereal products.

China submitted analytical data for 1989–91. The LOQ was very high (10 ΅g/kg), and none of the samples had concentrations that exceeded this value.

A sample of kidney and a sample of muscle were taken from 300 pigs from all parts of Denmark in 1999 and were analysed for their content of ochratoxin A. The analyses were performed at the Division of Chemical Contaminants, Danish Veterinary and Food Administration.

France provided results on the occurrence of ochratoxin A in pork offal (1011 samples) and in various fruits and vegetables (333 samples). The mean, the median, and the distribution of the contamination in intervals were provided for each food category.

Germany provided information on a total of 6476 samples of various foods analysed between 1995 and 1998. The data were very detailed, giving the number of samples, the LOQ of the method of analysis, the number of samples with concentrations above the LOQ, the percentage of samples with amounts below the LOQ, and the range of ochratoxin A concentrations for each food category, and, in a majority of cases, the mean, median, and 90th percentile of the distribution of contamination.

Italy provided data on the occurrence of ochratoxin A in 280 samples of wine.

Norway provided results for more than 1000 samples of cereals and cereal products.

Sweden submitted information on ochratoxin A contamination in the GEMS/Food electronic submission format. The data were for wheat, oat, rye, beans, peas, and coffee (soluble, green, and roasted). Analytical results were also provided for each sample of wheat, pulses, and coffee, making it possible to calculale the standard deviation of the distribution.

More than 2000 analytical results for various commodities were submitted by the United Kingdom.

Uruguay submitted the abstracts of scientific publications presented during the last meeting of AOAC/IUPAC (Guaruja, May 2000). Only one publication was relevant to the occurrence of ochratoxin A and gave the percentage of positive values in 600 samples of maize collected between 1991 and 1998 in Argentina, Brazil, and Paraguay.

The USA submitted data from five laboratories, reflecting the concentrations of ochratoxin A in coffee, raisins, wheat, and barley. Some of the results for green coffee (180 samples) and soluble coffee (23 samples) were expressed as percentages of positive values.

The Commission of the European Union submitted an assessment of the dietary intake of ochratoxin A by the populations of Member States of the European Union, for which data on occurrence were collected from the 12 Member States and Norway. ‘Best estimates’ were made of the mean concentrations in foods and food groups, and these are included in this monograph except for countries that provided more recent and more accurate data directly to the Committee, i.e. France, Germany, and Sweden.

The Coffee Science Information Centre provided information on total exposure to ochratoxin A contained in the position paper of the Codex Alimentarius Commission (CX/FAC 99/14), the report of a survey by the Ministry of Agriculture, Fisheries and Food of the United Kingdom of human exposure to ochratoxin A, and several published papers on the occurrence of ochratoxin A in coffee in European Member States and the USA (Patel et al., 1997; Stegen et al., 1997; Burdaspal & Legarda, 1998b; Jorgensen, 1998; Trucksess et al., 1999).

6.1.2 Consumption of potentially contaminated foods

National food consumption data were provided by France, Germany, Sweden, and the Commission of the European Union.

The distribution of consumption of foods in the relevant food categories was provided on the basis of individual data. A report in which data on food intake were compared with data on contamination by ochratoxin A indicated the distribution of intake and the probability of high intake.

The German approach consisted of combining the median and the 90th percentile of the distribution of contamination with various portion sizes (small, medium, and large) obtained in 3-day and 4-week studies of mean intake. The basis of the intake studies was not provided. Surprisingly, the intakes in the longer study were higher than those obtained in the shorter study.

A Swedish report provided an estimate of the intake of ochratoxin A from various foods and was based on data on food consumption from their study of dietary habits and nutrient intake in Sweden (Becher, 1992; Swedish National Food Administration, 1994).

Data on consumption were submitted in two reports from the European Union Scientific Cooperation (SCOOP) programme: from Task Group 3.2.2 (Commission of the European Union, 1997) and from Task Group 4.1 (Commission of the European Union, 1996). These reports provided a compilation of the mean intakes of various food categories across the European Union.

6.1.3 Biomarkers of exposure

The results of intake assessments that included biomarkers were provided by Sweden and the Commission of the European Union. The results of a study in the United Kingdom, available on the Internet, were submitted by the Coffee Science Information Centre.

Sweden submitted a publication on the concentrations of ochratoxin A in blood from Norwegian and Swedish donors (Thuvander et al., 2000). The mean concentration of ochratoxin A in plasma ranged from 0.17 to 0.21 ng/ml, and the 95th percentile concentration was 0.38–0.57 ng/ml.

The Ministry of Agriculture, Fisheries and Food in the United Kingdom conducted a survey of intake of ochratoxin A in which samples of duplicate diets, plasma, and urine were collected each week from 50 volunteers living in one area of the United Kingdom. A statistical analysis of the results indicated a stronger correlation between the urinary concentration of ochratoxin A and the level of consumption than with the plasma concentration.

SCOOP report 3.2.2 contains data on the occurrence of ochratoxin A in plasma and milk collected from healthy persons between 1977 and 1994 in Denmark, France, Germany, Italy, and Sweden.. These data showed a mean concentration of ochratoxin A in plasma of 1.8 ng/ml in Denmark (1986–88), 0.4 ng/ml in France (1993), 0.45 ng/ml in Germany (1977–94), 0.53 ng/ml in Italy (1992), and 0.18 ng/ml in Sweden (1994).

6.2 Assessment of intake at the international level

Intake at the international level was assessed from data on mean consumption combined with the weighted mean of contamination. As ochratoxin A occurs mainly in Europe, data on food consumption in Europe obtained from the GEMS/Food programme were considered the most relevant for risk assessment. As no information was available on coffee, beer, wine, or fruit juices in the GEMS/Food database, the mean intakes of the food categories in Europe were obtained from a report of the Commission of the European Union (1997), and those for coffee from a published paper (Jorgensen, 1998).

The available data on levels of contamination were aggregated as per the recommendations of a FAO/WHO workshop (Geneva, June 2000). The national results were presented in an aggregated format, so that one figure could represent the mean of a large number of individual samples. The first step therefore consisted of weighting each result as a function of the number of samples it represented. Each result was then multiplied by the number of individual samples in the original survey. The sum was then divided by the total number of individual samples. The result of this operation provided a weighted mean level of contamination for the food category considered. Some data were not used because they were expressed only in terms of the presence or absence of ochratoxin A, with no quantification of the mean level of contamination. As it was not possible to identify analytical results for targeted samples, all the data were considered to be representative of the total contamination of foodstuffs.

Each weighted mean can therefore be multiplied by the mean consumption of the corresponding food category to derive the contribution of that food category to human intake. The results are presented in Table 19. With this approach, the mean total intake of ochratoxin A was about 45 ng/kg bw per week, assuming a body weight of 60 kg.

Table 19. Classification of food categories as a function of their relative contribution to human exposure

Food category

Contamination
(΅g/kg)

Intake

g

΅g/person per week

ng/kg bw per week

Cerealsa

0.94

230

1.5

25

Beerb

0.023

260

0.04

0.69

Winec

0.32

240

0.54

8.9

Grape juiced

0.39

69

0.19

3.1

Tea

0.3

2.3

0.00

0.08

Cocoa

0.55

6.3

0.02

0.40

Pork

0.17

76

0.09

1.5

Poultry

0.041

53

0.02

0.25

Dried fruits

2.2

2.3

0.03

0.58

Pulses

0.19

25

0.03

0.55

Roasted coffeee

0.76

24

0.13

2.1

a From GEMS/Food database, data for the Far Eastern diet

b From SCOOP report 4.1, data for Norway

c From SCOOP report 4.1, data for the United Kingdom

d From SCOOP report 4.1, data for Portugal

e From Jorgensen (1998)

From these calculations, various food categories could be classified as a function of their potential impact in terms of public health. Cereals and wine contributed about 25 and 10 ng/kg bw per week, respectively, to average intake, whereas grape juice and coffee each contributed 2–3 ng/kg bw per week. Other food products (dried fruits, beer, tea, milk, cocoa, poultry, and pulses) contributed < 1 ng/kg bw per week. Most of the results submitted for pig meat and products were for pig liver and kidney, whereas the figure for food consumption was based on pig meat: the resulting estimate of 1.5 ng/kg bw per week can therefore be considered a gross overestimate of intake.

The data on ‘cereal products’ included several foods manufactured from cereals. In these products, the reported mean levels of contamination were about one-fifth those in the raw material. However, these data were not adequate for use in the intake assessment.

The second step, relating to contamination of the most relevant food categories for human intake (i.e. cereals, wine, grape juice, and coffee), consisted of simulating a worldwide distribution of ochratoxin A on the basis of several assumptions (WHO, 2000b). The first concerns the form of the distribution curve. It is generally considered that food contaminants follow a log-normal distribution. In order to construct a global distribution curve assuming log-normality, the mean and the standard deviation must be determined.

The results are presented in Table 20 and in Figures 1–4.

Table 20. Distribution of contamination of major food categories with ochratoxin A

Food category

No. of samplees

Weighted mean

Standard deviation (ln)

Cereals

2714

0.94

1.23

Cereal products

1536

0.19

1.96

Wine

1834

0.32

1.26

Grape juice

68

0.44

0.48

Green coffee

127

1.02

6.12

Roasted coffee

1726

0.76

0.29

Grapes

857

2.29

0.79

Figure 1
Figure 2
Figure 3
Figure 4

6.3 Impact of alternative maximum limits on intake

In order to assess intake from cereals, a probabilistic approach was used, in which a simulated distribution of contamination and the distribution of cereal consumption in France were used.

For cereal consumption, the results of a survey of food consumption by 1161 individuals were used, with individual body weights, assuming that 100% were consumers with a mean consumption of 28 g/week per kg bw and consumption at the 95th percentile of 61 g/week per kg bw.

For contamination of cereals, assuming that the distribution is log-normal, the model included the mean and the standard deviation and, for the specific task, the minimum and the maximum values. The maximum value was assumed to be 0 in all situations. Three simulations were made, the first with the maximum observed value (121 ΅g/kg), the second with the higher proposed maximum limit (20 ΅g/kg), and the third with the lower proposed maximum limit (5 ΅g/kg).

A probabilistic approach with a Monte-Carlo simulation made it possible to assess the intake of ochratoxin A by the population. The software used was @risk, and the equation for constructing the curves for contamination was riskTlognorm (mean, SD, min, max).

This simulation, which was considered to be realistic for European-type diets, showed that the intake of ochratoxin A by consumers of cereals at the 95th percentile would be 92 ng/kg bw per week. Use of the proposed maximum limit of 5 ΅g/kg, as opposed to 20 ΅g/kg, would have a statistically significant effect on intake only for consumers of cereals above the 95th percentile. However, the difference would be very small (84 vs 92 ng/kg of body weight per week) in view of the distribution of the level of contamination indicated by the available data.

7. PREVENTION AND CONTROL OF OCHRATOXIN A PRODUCTION

Control of ochratoxin A in foods depends on the kind of crop and its geographical location, as those two factors are primarily responsible for determining which of the three major ochratoxin A-producing fungi is likely to grow and produce toxin. Moreover, as experience with other mycotoxins has shown, the likelihood and extent of toxin production by any particular species is greatly influenced by whether the fungus concerned has a particular affinity for a specific crop, i.e. can invade and grow in a crop before or during harvesting and drying. As these factors, and the physiology of the three species, differ, the control of ochratoxin A production by each species is considered separately.

7.1 By Aspergillus ochraceus

As stated above, A. ochraceus occurs primarily in stored foods, and no association with plants before harvest is known. Its control therefore consists mainly of the standard methods used for controlling the growth of any fungus in dried foods.

7.1.1 Grains

The main commodities in which A. ochraceus may produce ochratoxin A are stored grains. The traditional way of avoiding microbial growth in grains is to dry them thoroughly and to keep them dry. Adequate ventilation in storage bins will remove moisture, prevent condensation, lower and equilibrate temperatures, and prevent heating. Over the past 20 years, bag stacks and manual handling of grain have given way to bulk handling and storage, with great improvements in the control of insect and fungal damage, even in some tropical areas (Champ & Highley, 1988). Drying techniques have made great advances, with sophisticated computer control of drying rates and temperatures now in use, at least in developed countries. Control of insect and fungal damage in grain stores is of particular importance in the tropics, where most grains are stored in sacks in warehouses unsuitable for sealing and fumigation. Recent approaches to sealing stacks of bags in such stores, fumigating and then maintaining the sealed stacks under controlled atmospheres have shown the potential to greatly reduce grain losses (Annis, 1990a; Graver, 1990).

The moisture content of grains must be reduced to below 0.8 aw (17–19% moisture; Iglesias & Chirife, 1982) to prevent ochratoxin A formation by A. ochraceus.

Modern approaches to grain storage rely on fumigation and sealed storage under controlled atmospheres, especially in tropical and subtropical regions where insect damage is a major problem (Champ et al., 1990). Fumigants, highly toxic gases or vapours added to grain stores specifically to kill insects, in some cases also achieve fungal destruction. Fumigants are usually used as a rapid method for killing insects and are subsequently removed by ventilation. A variety of gases have been used as fumigants, either singly or in combination, including ethylene dichloride, carbon tetrachloride, carbon disulfide, ethylene dibromide, chloropicrin, hydrogen cyanide, ethylene oxide, methyl chloride, methyl bromide, and phosphine. For various reasons, only methyl bromide and phosphine are in widespread use (Annis, 1990b). The concentrations recommended for use are given in Table 21. Environmental considerations are resulting in the phasing out of methyl bromide, and the search for alternative fumigants continues.

Table 21. Suggested target doses for gaseous treatments of grain at 25 °C

Gas

Time (days)a

Concentrationb

Concentration x time

Carbon dioxide

15

> 35%

–

Oxygen

20

< 1%

–

Phosphine

7

100 mg/m3

–

Methyl bromide

1–2

–

150 g h/m3

Hydrogen cyanide

1

Not well defined

–

From Annis (1990b)

a In cases of slow gas introduction or poor gas distribution, longer exposure may be necessary.

b Minimum concentration achieved at end of exposure

Controlled atmospheres may be used for grain storage. This technique relies on continuous application of atmospheres low in O2 or with high CO2 concentrations. The recommended approach is to add such gas mixtures to sealed storage and maintain the grain in totally sealed systems. Where this is not practicable, continuous flow of such gas mixtures may be possible (Annis, 1990b). The recommended O2 and CO2 concentrations are given in Table 21.

Fumigation and controlled atmospheres help control mould growth on grains by directly destroying spores, by inhibiting growth, or by killing insects that damage kernels. Fumigants that merely destroy insects have no lasting effect on mould growth (Vandegraft et al., 1973); however, methyl bromide destroys fungi as well as insects (Majumder, 1974), and phosphine has some fungicidal properties (Hocking & Banks, 1993). Modified atmospheres that control insects may have a substantial effect in controlling fungi as well (Hocking, 1990).

Many investigators have suggested using heat or chlorine to destroy micro-organisms in grains; however, this technique has had little use, with the recognition that such remedial processes do not destroy mycotoxins and are not a substitute for clean grain. In the same way, ionizing radiation at 2–3 kGy destroys moulds that ordinarily spoil rice (Iizuka & Ito, 1968; Ito et al., 1971), but this process remains illegal or unacceptable to consumers in many countries.

7.1.2 Coffee

Control of ochratoxin A production in coffee by either A. ochraceus or A. carbonarius is similar, but the control measures used are different from those for other commodities. Although some research is still required, it appears that ochratoxin A can be controlled in coffee by good manufacturing practice. Studies in Brazil by Taniwaki et al. (1999) and Taniwaki and Pitt (unpublished) have shown that mould growth and ochratoxin A production occur only during drying of green coffee beans, and that if drying is rapid and effective ochratoxin A will not be produced. Good sun-drying or a combination of sun-drying and mechanical dehydration provide effective control. No evidence has been found that either A. ochraceus or A. carbonarius invades coffee beans before harvest or has an association with the coffee tree.

The formation of ochratoxin A during drying of coffee was studied in Thailand by Bucheli et al. (2000), who showed that the toxin was normally produced during sun-drying of coffee in that country, that overripe cherries were more susceptible than green ones, and that defects, especially the inclusion of husks, were the most important source of ochratoxin A contamination. They agreed with Taniwaki et al. (1999), that better quality raw material, appropriate drying and dehulling procedures, and reduction of defects can substantially reduce the concentration of ochratoxin A in green coffee.

The formation of ochratoxin A did not increase during storage for 18 months in Thailand, even with bag storage at high humidity (Bucheli et al., 1998). However, as the initial aw of the beans stored in bags was 0.72 and did not exceed 0.75 even in the rainy season, these results are not surprising.

The suggestion by Mantle (1998) that ochratoxin A in coffee beans may result from uptake of ochratoxin A in soil by the roots of the coffee tree and then translocation is conjectural at best.

7.2 By Aspergillus carbonarius

The available evidence indicates that A. carbonarius and A. niger are not pathogens on fruit, but saprophytes, and hence cannot gain entry to sound fruit. However, damage to fruit by any means, mechanical, chemical, or by disease microorganisms, may allow entry into fruit tissue, where the low pH, high sugar, and often warm temperature provide an ideal habitat for these species. This is especially true of grapes, which have very tough skins. When the skins are intact, they are resistant to attacks by these fungi, but ideal growth conditions prevail once the skin is disrupted. Control of growth of these species in grapes before harvest therefore relies on:

•

control of pathogenic fungi, especially Rhizopus stolonifer, Botrytis cinerea, and powdery mildews such as Erysiphe species;

•

control of mechanical damage, from pruning, leaf reduction and, for dried fruit, harvesting equipment; and

•

control of splitting due to rain just before harvest.

Growth of fungal pathogens is especially difficult to control on cultivars that have tight bunches or excessive leaf cover over bunches, or under rainy or misty conditions at harvest time. Control of Rhizopus stolonifer, Botrytis cinerea, and Erysiphe species relies primarily on vineyard hygiene, with removal of diseased plant tissue, thinning of tight bunches, and removal of excessively tight leaf clusters (Snowdon, 1990; Emmett et al., 1992). Fungicides are sometimes used against pathogenic fungi, but the effectiveness of such treatments is variable and depends on seasonal and geographic factors (Nair et al., 1987; Snowdon, 1990).

Control of mechanical damage relies on good farm management. In many countries, the importance of minimizing damage to grapes probably still needs emphasis. This is a serious problem when grapes are to be dried, as mechanical damage at harvest is difficult to avoid and the length of the drying process (2 weeks or more) provides ample time for growth of A. carbonarius and ochratoxin A production. This is much less of a problem with wine grapes, which are usually crushed within a few hours of picking, and it is a reasonable assumption that the rapid establishment of anaerobic conditions prevents further growth of A. carbonarius or ochratoxin A production.

Some cultivars, especially the sultana grapes favoured for drying, are susceptible to rain damage during the week preceding harvest, when turgor pressure inside the fruit is high and the skins often inflexible. Splitting around the neck of the grape below the stem provides an ideal environment for invasion by A. carbonarius. Control is very difficult. The only useful recommendation is to cut vines or bunches and commence drying as quickly as possible after rain, if damage is seen as a possibility.

Little has been published specifically about preharvest and postharvest control of ochratoxin A production in coffee by A. carbonarius, but see the section on A. ochraceus, above.

7.3 By Penicillium verrucosum

Little published information exists about the time of invasion of cereal crops by P. verrucosum. It is commonly stated that this species is a storage fungus, invading after harvest, but that does not explain the paradox that P. verrucosum is a slowly growing, not notably xerophilic species which should compete poorly against many other species known to spoil grains. There have been few reports on the incidence of P. verrucosum in Canadian or northern European grain. Frisvad & Vuif (1986) found P. viridicatum Group II (= P. verrucosum) in each of 70 samples of Danish grain containing ochratoxin A. Holmberg et al. (1991) found that the incidence of storage fungi, and particularly P. verrucosum, was significantly higher in feed samples (barley and oats or cereal mash feed) of pig herds infected with ochratoxin A: Ochratoxin A-producing P. verrucosum was found in 60% of feed samples of infected herds and in only 5% of feed of uninfected herds. Moreover, as ochratoxin A is known to occur in grain in cold climates and no Aspergillus species is likely to occur in such grain, and as P. verrucosum is the only Penicillium species that produces ochratoxin A, it is likely that P. verrucosum is the source of ochratoxin A in Canadian and northern European grains.

The occurrence of ochratoxin A in such grains is attributed to insufficient drying or over-long storage before drying. Jonsson et al. (1997) studied the effect of moisture content, temperature, and time on the growth of moulds and the production of ochratoxin A in winter wheat. The maximum storage time without mould growth appeared to be halved when the moisture content at harvest was increased by 2–3% or if the storage temperature was increased by 5 °C. Ochratoxin A could generally be detected quite soon after microbial growth had begun.

The occurrence of ochratoxin A in dried grain used for human food can be controlled by analysis and segregation of defective lots. Little or no information exists about whether this procedure is practised anywhere in the world.

8. COMMENTS

Absorption, distribution, metabolism and excretion

Ochratoxin A is slowly absorbed from the gastrointestinal tract. It is distributed in a number of species via the blood, mainly to the kidneys, lower concentrations being found in liver, muscle, and fat. Transfer to milk has been demonstrated in rats, rabbits, and humans, but little is transferred to the milk of ruminants owing to metabolism of ochratoxin A by the rumenal microflora. The major metabolite of ochratoxin A in all species examined is ochratoxin alpha. This and minor metabolites that have been identified are all reported to be less toxic than ochratoxin A itself. Ochratoxin A is excreted in urine and faeces, and the relative contribution of each of these routes in different species is influenced by the extent of the enterohepatic recirculation of ochratoxin A and its binding to serum macromolecules. These factors are also important in the determination of the serum half-life of ochratoxin A, which varies widely among species. It has a long half-life in non-ruminant mammals, e.g. 24–39 h in mice, 55–120 h in rats, 72–120 h in pigs, 510 h in one macaque, and 840 h in a volunteer.

Toxicological studies

Ochratoxin A has been shown to be nephrotoxic in all mammalian species tested. Its main target is the renal proximal tubule, where it exerts cytotoxic and carcinogenic effects. Significant sex and species differences in sensitivity to nephrotoxicity were evident, in the order pig > rat > mouse. The doses at which carcinogenicity was observed in rodents were higher than those that caused nephrotoxicity. The Committee reconsidered the report of the study of carcinogenicity conducted by the National Toxicology Program (USA) in 1989 and noted the consistent presence and severity of karyomegaly in male and female rats and the aggressive nature of the renal tumours in this study. However, the biological and mechanistic significance of these observations was unclear.

Gene mutations were induced in bacteria and mammalian cells in a few studies of genotoxicity, but not in most. Ochratoxin A did, however, induce DNA damage, DNA repair, and chromosomal aberrations in mammalian cells in vitro and DNA damage and chromosomal aberrations in mice treated in vivo. Putative DNA adducts were found consistently with a 32P-postlabelling method in the kidneys of mice and rats dosed with ochratoxin A, but none of these adducts has been demonstrated to contain fragments of ochratoxin A. It was therefore uncertain whether ochratoxin A interacts directly with DNA or whether it acts by generating reactive oxygen species. There was no indication that a reactive metabolite of ochratoxin A is generated in vivo. Ochratoxin A is thus genotoxic both in vitro and in vivo, but the mechanism of genotoxicity is unclear and there was no evidence that it is mediated by direct interaction with DNA. The doses used in the studies of genetic toxicity were in the same range as those at which the incidence of renal tumours was increased in mice. In rats, however, the incidences of nephrotoxicity and renal tumours were increased at much lower doses; therefore the contribution of the genotoxicity of ochratoxin A to neoplasia in rats is unknown.

Ochratoxin A can cross the placenta and it is embryotoxic and teratogenic in rats and mice. It has been shown to have immunosuppressive effects in a number of species. Prenatal administration of ochratoxin A to rats caused immunosuppression, but perinatal administration stimulated certain aspects of the immune response in rats. Ochratoxin A inhibited the proliferation of B and T lymphocytes and affected the late stages of T-lymphocyte activation in vitro. However, both the immunological and teratogenic effects have been observed only at doses much higher than those that cause nephrotoxicity.

Observations in humans

Ochratoxin A has been found in human blood samples, most notably in a number of countries in the cool temperate climatic areas of the Northern Hemisphere; however, no cases of acute intoxication in humans have been reported. The Committee noted that ochratoxin A was found more frequently and at higher average concentrations in blood samples obtained from people living in regions where a fatal human kidney disease (known as Balkan endemic nephropathy) occurs and is associated with an increased incidence of tumours of the upper urinary tract. Nevertheless, similar average concentrations have been reported in several other European countries where this disease is not observed. The Committee concluded that the epidemiological and clinical data available do not provide a basis for calculating the likely carcinogenic potency in humans and that the etiology of Balkan endemic nephropathy may involve other nephrotoxic agents.

Analytical methods

Reliable, validated methods have been developed for the analysis of ochratoxin A in maize, barley, rye, wheat, wheat bran, wheat whole meal, roasted coffee, wine, and beer, which are based on liquid chromatography with fluorescence detection. The limit of quantification was 0.03 ΅g/kg for wine and beer and 0.3–0.6 ΅g/kg for other commodities. These methods have also been used successfully to analyse a number of other cereals, cereal products, and dried fruit. Two certified reference materials (blank and naturally contaminated wheat) are available, which improve quality assurance in laboratories. Screening methods based on TLC are available but have been used in only a few laboratories. Data obtained by these analytical methods, with a limit of quantification greater than 5 ΅g/kg, were not considered in this evaluation, as this was the lower concentration for which the Codex Committee on Food Additives and Contaminants requested a risk assessment. Furthermore, enzyme-linked immunosorbent assay (ELISA) techniques had not been used to produce the survey data considered by the Committee.

There are no formally validated methods for the analysis of ochratoxin A in human blood. The available methods are based on liquid chromatography with fluorescence detection and have different limits of quantification, ranging from about 0.1 to 2 ng/ml.

Sampling protocols

Adequate sampling procedures should be used in future surveys of cereals and cereal products for ochratoxin A. For example, an acceptable sampling procedure was used in 10 of 22 studies on cereals submitted to this Committee, whereas no description was reported in the remaining 12. No sampling plans for the determination of ochratoxin A in foods have been published, and details of sampling variability have not been reported.

Effects of processing

Milling has been reported to reduce substantially the concentration of ochratoxin A in white flour, but it had little effect on levels in wholemeal flour. Milling is a physical process: the ochratoxin A removed from the grain in the production of white flour remains in bran and other fractions, some of which may be used in foods. Ochratoxin A is relatively stable to heat: at 100 °C, a 50% reduction in the concentration was achieved after 2.3 h in wet wheat and 12 h in dry wheat. The process involved in the manufacture of breakfast cereals and biscuits resulted in substantial reductions in ochratoxin content, but little or no reduction was found in the manufacture of egg noodles and pasta. Decaffeination of coffee reduced the ochratoxin concentration by about 90%. The reduction obtained by roasting coffee varies but may also be as much as 90%.

Levels and patterns of contamination of food commodities

The data on ochratoxin A over the past 5 years that were reviewed by the Committee originated mainly from Europe (85%); 7% came from South America, 6% from North America, 1% from Africa, and 1% from Asia. The concentrations of ochratoxin A in the different commodities were highly variable; 1.4% and 0.6% of samples contained more than 5 ΅g/kg and 20 ΅g/kg, respectively. Within the cereals, 1.2% and 0.3% of samples contained more than 5 ΅g/kg and 20 ΅g/kg of ochratoxin A, respectively. Within cereal products, 0.3% and 0.05% of samples contained more than 5 ΅g/kg and 20 ΅g/kg of ochratoxin A, respectively. The weighted mean concentrations of ochratoxin A that were used for estimating intake were: 0.94 ΅g/kg for cereals, 0.19 ΅g/kg for cereal products, 0.32 ΅g/kg for wine, 0.86 ΅g/kg for coffee, 2.3 ΅g/kg for dried vine fruit, and 0.44 ΅g/kg for grape juice. The incidence of samples found to contain ochratoxin A depended on the commodity and was higher in the same commodity when analytical methods with lower limits of quantification were used.

Food consumption/intake assessment

Intake of ochratoxin A at the international level has been assessed on the basis of data on mean consumption combined with weighted mean levels of contamination. As ochratoxin A occurs mainly in the diet in European countries, data on food consumption in Europe obtained from the GEMS/Food database were considered the most relevant for risk assessment. The submitted data on levels of contamination were aggregated according to the recommendations of a FAO/WHO workshop to obtain a weighted mean. When this approach was used, the mean total intake of ochratoxin A was estimated to be 45 ng/kg bw per week, assuming a body weight of 60 kg.

Cereals and wine contributed about 25 and 10 ng/kg bw per week, respectively, to the mean intake, whereas grape juice and coffee each contributed 2–3 ng/kg bw per week. Other food products (dried fruits, beer, tea, milk, cocoa, poultry, and pulses) contributed less than 1 ng/kg bw per week. Most of the results submitted for pig meats and pig meat products were for samples of pig liver and kidney, whereas the figure for food consumption in the GEMS/Food database was based on pig meats. The resulting estimate of 1.5 ng/kg bw per week can therefore be considered a gross overestimate of intake.

A probabilistic approach was used to assess intake from cereals and cereal products, in which a simulated distribution of contamination and the distribution of cereal consumption in France were used. This example, which was considered to be realistic for European diets, showed that consumers of cereals at the 95th percentile would have an intake of ochratoxin A of 92 ng/kg bw per week. Use of a proposed maximum limit of 5 ΅g/kg as opposed to 20 ΅g/kg would have a statistically significant effect on intake of ochratoxin A only for consumers of quantities of cereals greater than the 95th percentile. However, the difference would be very small (84 vs 92 ng/kg bw per week at the 95th percentile) in view of the distribution of the level of contamination indicated by the available data.

Prevention and control

As formation of ochratoxin A depends on the fungal source, the type of crop, and its geographical location, control of ochratoxin A production by each fungal species was considered separately. Control of A. ochraceus, which occurs primarily in stored foods, consists of the standard methods for preventing growth of any fungus in dried foods. The major commodities in which A. ochraceus may produce ochratoxin A are stored grains. The traditional means of avoiding fungal growth in grains is to dry them rapidly and thoroughly and to keep them dry. Reduction of the moisture content of grains to provide a water activity below 0.8 is necessary to prevent formation of ochratoxin A by A. ochraceus. Further effective approaches to grain storage include fumigation, aeration and cooling, sealed storage, and controlled atmospheres, especially in tropical and subtropical regions where insect damage is a major problem. Controlled atmosphere storage is achieved by continuous application of atmospheres with a low oxygen or a high carbon dioxide concentration. Modified atmospheres to control insects may contribute to controlling fungi. Some fumigants used for insect control may also control fungi.

As ochratoxin A is apparently formed in green coffee beans after harvest, agricultural practice has little or no influence on the concentration of the toxin in dried beans. Control measures for ochratoxin A in coffee are therefore based on good manufacturing practice, i.e. rapid and effective drying, good storage practices, and, in some countries, colour sorting to reject defective beans.

The available evidence indicates that A. carbonarius and A. niger are not pathogens on fruit such as grapes and hence cannot gain entry to sound fruit. However, mechanical or chemical damage to fruit or damage caused by insects or microorganisms may permit fungal invasion of fruit tissue. Controlling the growth of these species in grapes before harvest therefore relies on controlling pathogenic fungi, mechanical damage, and splitting due to rain just before harvest. The occurrence of ochratoxin A from P. verrucosum in Canadian and European grains was attributed to insufficient drying or inadequate storage. Analysis and segregation of defective lots could be used to reduce the concentration of ochratoxin A in dried grain used for human food.

9. EVALUATION

The Committee concluded that the new data raised further questions about the mechanisms by which ochratoxin A causes nephrotoxicity and renal carcinogenicity and the interdependence of these effects. The mechanism by which ochratoxin A causes carcinogenicity is unknown, although both genotoxic and non-genotoxic modes of action have been proposed. The Committee noted that studies to resolve these issues are in progress and would wish to review the results when they become available. The Committee retained the previously established PTWI of 100 ng/kg bw per week, pending the results of on-going studies on the mechanisms of nephrotoxicity and carcinogenicity, and recommended a further review of ochratoxin A in 2004. In reaching this conclusion, the Committee noted the large safety factor applied to the NOEL for nephrotoxicity in deriving the PTWI, which corresponds to a factor of 1500 applied to the NOEL for carcinogenicity in male rats, the most sensitive species and sex for this end-point.

The adverse effect at the lowest effective dose in several mammalian species is nephrotoxicity, and this is likely also to be true in humans. Although an association between the intake of ochratoxin A and nephropathy in humans has been postulated, causality has not been established. The Committee noted that the intake of ochratoxin A by 95th percentile consumers of cereals may approach the PTWI from this source alone. Given the distribution of ochratoxin A contamination of cereals, application of a limit of 5 or of 20 ΅g/kg would make no significant difference to the average intake. The estimated intake at the 95th percentile of cereal consumers on a European diet would be about 84 and 92 ng/kg bw per week, respectively. Intakes below the PTWI would not present an appreciable risk. The Committee was unable, on the basis of the available data, to arrive at a quantitative estimate of the risk for nephrotoxicity if the PTWI were to be exceeded. Efforts are needed to ensure that intakes of ochratoxin A do not exceed the PTWI, and this could best be achieved by lowering overall contamination by appropriate agricultural, storage, and processing practices.

Recommendations

•

Studies should be conducted to clarify the mechanism by which ochratoxin A induces nephrotoxicity and carcinogenicity.

•

Appropriate sampling procedures should be developed for food commodities likely to be contaminated with ochratoxin A.

•

Better surveys are needed, particularly in regions of the world other than Europe, in order that intakes in those regions may be assessed.

•

Epidemiological investigations should be encouraged to explore the role of ochratoxin A in chronic renal disease.

•

Studies should be conducted to improve understanding of the occurrence and ecology of the fungi that produce ochratoxin A, especially in fresh produce.

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Appendix A

Results of surveys for ochratoxin A showing concentrations and distribution of
contamination in food commodities

Country/ Region

Commodity

Year/ Season

No. of samples

LOQ
(΅g/kg)

n < LOQ

Mean/Max (΅g/kg)

 

Wheat

 

 

 

 

 

Germany

Wheat grain

1995–98

35

0.01a

21

0.11/0.65

Wheat flour < T550

1995–98

98

0.01a

16

0.10/1

1995–98

83

0.01a

6

0.20/1.73

Wheat wholemeal flour

1995–98

18

0.01a

0

0.20/1.2

Wheat semolina

1995–98

25

0.01a

15

0.41/2.58

Wheat bran

1995–98

25

0.01a

22

0.22/1.59

Wheat germ

1995–98

19

0.01a

13

0.11/0.45

Netherlands

Wheat, domestic

1995

7

1.000

7

0/0

Wheat, imported

1995

24

1.000

23

0.36/8.7

White wheat flour

1999

31

0.250

30

0.048/1.5

Whole-wheat meal

1999

19

0.250

19

0/0

Norway

Wheat

1990

138

0.3a

122

0.17/1.5

1993

7

0.2a

6

0.77/4.7

1994

24

0.2a

20

0.3/3.4

1995

32

0.25a

20

0.15/0.57

1996

28

0.3a

28

0/0

1997

25

0.01a

22

0.22/3.5

1998

35

0.05a

30

0.7/20

Total wheat samples

1990–98

289

 

248

0.26/20

Wheat, imported

1990

28

0.3a

25

0.34/3.8

1993

11

0.2a

10

0.52/4.6

Norway

Wheat, imported

1994

9

0.2a

8

0.46/3.3

1995

13

0.25a

10

0.89/8.2

1996

14

0.3a

13

0.67/7.5

1997

10

0.01a

7

0.14/0.56

1998

24

0.05a

20

0.07/0.54

Total imported wheat

1990–98

108

 

93

0.4/8.2

Sweden

Wheat grain

1996–98

57

0.1

41

0.24/2.3

1999

75

0.05

36

0.37/5.2

Brazil

Wheat grain

1988–90

16

5a

15

2.5/40

1990

20

5a

20

0/0

Wheat products

1991

38

5a

38

0/0

USA

Wheat grain

1997

383

0.03a

327

NR/31.4

Finland

Wheat grain

1996

34

0.800

32

18.2/430

Denmark

Wheat grain

1986–92

402

0.05a

283

0.7/51

Wheat grain, organic

1986–92

73

0.05a

44

1.2/36

Wheat grain

1986–92

45

0.05a

28

0.9/13

Wheat bran

1986–92

120

0.05a

46

0.8/12

Wheat bran, organic

1986–92

22

0.05a

7

0.6/2.6

Dubai

Wheat flour

NR

11

0.500

10

0.023/0.25

United Kingdom

Wheat noodles

1995

4

0.1a

3

0.1/0.4

Wheat grain

1993

384

0.1a

NR

< 1/< 1

Wheat grain stored

1992–93

25

0.1a

NR

< 1/ 1

Wheat from millers

1993

129

0.1a

NR

NA/15

1994

250

0.1a

NR

NA/32

Wheat grain

1997-98

148

0.200

126

0.3/9.2

1996

76

0.200

74

0.042/2.4

Cereals and flours

1996

67

0.200

30

NA/6.4

Uruguay

Wheat grain

1993–95

123

50a

123

0/0

 

Buckwheat

 

 

 

 

 

Germany

Buckwheat

1995–98

23

0.01a

13

0.07/0.59

Buckwheat flour

1995–98

14

0.01a

3a

0.96/12.1

 

Barley

 

 

 

 

 

Germany

Barley

1995–98

22

0.01a

3

0.07/0.49

Pearl barley

1995–98

31

0.01a

10

0.094/0.95

Norway

Barley

1990

10

0.3a

10

0/0

Barley groats

1990

10

0.3a

10

0/0

USA

Barley

1997

103

0.03a

92

NA/17

United Kingdom

Barley

1997-98

131

0.1a

96

0.7/17.8

Barley

1996

37

0.2a

34

0.20/6.4

Barley

1993

73

0.1a

NR

NA/<1

stored in 1992

 

 

 

 

 

Barley

1994

150

0.1a

NR

NA/33

Barley stored in 1993

1994

50

0.1a

NR

NA/14

Uruguay

Barley and malt

1993-95

137

50a

137

0/0

Finland

Barley kernel

1996

45

0.800

42

0.55/12.3

Canada

Barley cereals

1998–99

20

0.500

NR

NA/0.57

Barley based cereals

1998–99

20

0.200

NR

NA/6.92

 

Maize

 

 

 

 

 

Germany

Maize and popcorn

1995–98

31

0.01a

12

0.17/3.35

United Kingdom

Raw maize, imported

1998–99

139

0.200

125

NA/1.5

Croatia

Raw maize

1996

105

0.2a

95

3.61/224

Raw maize

1997

104

0.2a

68

19.8/614

Brazil

Raw maize

1991

130

5a

130

0/0

Brazil

Raw maize

1993–94

292

5a

292

0/0

Uruguay

Maize and by-products

1993-95

147

50a

147

0/0

United Kingdom

Corn flour

1995

4

0.1a

3

0.15/0.6

 

Oats

 

 

 

 

 

Sweden

Oat grain

1996–98

23

0.1

16

0.32/3.6

Oat grain

1999

10

0.05

8

0.05/0.15

Norway

Oat groats

1990

20

0.3a

14

0.26/0.9

Oats

1990

20

0.3a

17

0.44/5.8

Oats

1993

3

0.2

2

0.17/0.26

Oats

1994

3

0.2a

2

3.47/10.2

Oats

1995

21

0.25a

20

0.32/4.2

Oats

1996

14

0.3a

14

0/0

Oats

1997

14

0.01a

?

0.053/0.23

Oats

1998

22

0.01a

19

0.065/0.47

Total oats samples

1990–98

97

 

 

0.46/10.2

Denmark

Oat kernels

1986–92

50

0.05a

29

0.5/5.6

Oat kernels, organic

1986–92

17

0.05a

11

0.3/4.2

Oat kernels, imported

1986–92

25

0.05a

12

0.5/4.6

Finland

Oat kernels

1996

34

0.80

32

1.7/56.6

United Kingdom

Oat kernels

1997–98

21

0.1a

15

0.1/2.2

1996

18

0.2a

17

0.33/5.9

Germany

Oats

1995–98

30

0.01a

6

0.06/0.14

 

Rice

 

 

 

 

 

Uruguay

Rice

1993-95

62

50a

62

0/0

United

Basmati rice

1995

4

0.1a

4

0/0

Kingdom

Chinese rice

1995

4

0.1a

4

0/0

Dubai

Rice

NR

15

0.50

14

0.017/0.25

Germany

Rice

1995–98

22

0.1a

18

0.11/0.28

Parboiled rice

1995–98

21

0.1a

21

 

Long-grain rice

1995–98

24

0.1a

24

 

Round-grain rice

1995–98

15

0.1a

15

 

 

Rye

 

 

 

 

 

Sweden

Rye grain

1996–98

28

0.1

8

0.56/2.3

1999

19

0.05

6

1.8/27

Norway

Rye, imported

1990

18

0.3a

13

0.22/0.8

1993

9

0.2a

5

0.31/1.0

1994

6

0.2a

4

0.28/1.0

1995

4

0.25a

3

0.75/2.5

1996

4

0.3a

4

0/0

Total

1990–98

41

 

29

0.31/2.5

Denmark

Rye kernels

1986–92

503

0.05a

326

1.2/121

Rye kernels, organic

1986–92

91

0.05a

20

5.4/120

Rye kernels, imported

1986–92

22

0.05a

14

0.1/0.7

United Kingdom

Rye kernels

1996

22

0.2a

21

0.05/1.1

Germany

Rye

1995–98

37

0.01a

23

0.11/0.8

Rye flour < T997

1995–98

26

0.01a

6

0.42/6.4

1995–98

71

0.01a

3

0.32/2.14

Rye wholemeal flour

1995–98

43

0.01a

11

0.11/1.46

 

Sorghum

 

 

 

 

 

Germany

Sorghum

1995–98

26

0.01a

3

0.11/0.83

 

Spelt

 

 

 

 

 

Germany

Spelt/spelt flour

1995–98

21

0.01a

3

0.66/9.43

 

Cereal products

 

 

 

 

 

United Kingdom

Pitta bread

1995

4

0.1a

NR

NA/0.8

Chapatti

1995

4

0.1a

NR

NA/0.9

Nan bread

1995

4

0.1a

4

0/0

Poppadoms

1995

4

0.1a

4

0/0

Tunisia

Cereal-derived food

1994

66

0.1a

0

1715/12 770

Dubai

Mixed cereals

NR

28

0.500

25

NA/4.3

Canada

Mixed grain cereals

1998–99

31

0.200

NR

0.25/0.54

Mixed cereals

1998–99

19

0.500

19

0/0

Europe, Tunisia

Bread

NR

141

0.008

0

NA/6.66

Germany

Bread, wheat mix

1995–98

125

0.01a

15

0.19/2.09

Germany

Bread, rye mix

1995–98

128

0.01a

7

0.24/2.24

White bread

1995–98

57

0.01a

9

0.11/1.9

Toast bread

1995–98

59

0.01a

7

0.081/0.58

Rye meal bread

1995–98

96

0.01a

7

0.22/5.49

Milk–water bread roll

1995–98

89

0.01a

10

0.09/0.52

Various cereals, bread

1995–98

49

0.01a

1

0.24/1.76

Various cereals, bread +oilseed

1995–98

101

0.01a

3

0.17/2.44

Crispbread

1995–98

87

0.01a

25

0.076/0.44

Wheat whole bread

1995–98

13

0.01a

0

0.134/0.40

Special bread

1995–98

64

0.01a

9

0.145/2.23

Wholemeal bread roll

1995–98

31

0.01a

0

0.17/0.77

Various cereals and muesli bread

1995–98

49

0.01a

1

0.36/5.54

Rye bread roll

1995–98

38

0.01a

1

0.16/0.44

Pasta without egg

1995–98

50

0.1a

21

0.28/1.75

Pasta with egg

1995–98

84

0.1a

57

0.199/0.95

Wholemeal pasta

1995–98

27

0.1a

17

2.0/29.77

Oat flakes

1995–98

66

0.01a

40

0.07/0.25

Oats bran

1995–98

26

0.01a

12

0.089/0.33

Polenta

1995–98

29

0.01a

23

0.20/1.53

Green corn

1995–98

17

0.01a

15

0.07/0.10

Infant food

1995–98

97

0.01a

31

0.12/2.13

Peas, lentil, beans

1995–98

103

0.01a

102

ND/0.84

Soya beans

1995–98

31

0.01a

5

0.06/0.10

FSIS185 pulses

1998

50

0.2a

50

 

FSIS185 pulses

1997

29

0.2a

27

1.1/15.4

 

Seeds

 

 

 

 

 

United Kingdom

Fennel

1995

3

0.1a

3

0/0

Sesame seeds

1995

3

0.1a

3

0/0

Coriander

1995

3

0.1a

2

1.33/4.0

 

Herbs and spices

 

 

 

 

 

Netherlands

Paprika powder

1996–98

12

0.250

3

1.7/9.8

Pepper

1996–97

14

0.250

7

3.73/14.5

United Kingdom

Chilli powder

1995

4

0.1a

NR

NA/50.4

Curry powder

1995

10

0.1a

NR

NA/21.3

Tandoori

1995

3

0.1a

NR

NA/23.9

Ginger

1995

4

0.1a

NR

NA/7.5

Garlic

1995

4

0.1a

4

0/0

Five spices powder

1995

4

0.1a

3

0.65/2.6

Tunisia

Dried vegetables

1994

6

0.1a

0

2934/7444

Dubai

Spices

NR

7

0.500

3

NA/3.56

 

Pickles and pastes

 

 

 

 

 

United Kingdom

Chilli pickle

1995

4

0.1a

NR

NA/1.2

Garlic pickle

1995

4

0.1a

NR

NA/2.5

United Kingdom

Curry paste

1995

4

0.1a

NR

NA/15.5

Chilli sauce

1995

4

0.1a

3

0.82/3.3

 

Canned foods

 

 

 

 

 

United Kingdom

Canned foods

1995

8

0.1a

NR

NA/0.3

 

Oils

 

 

 

 

 

United

Sesame oil

1995

3

0.1a

2

0.13/0.4

Kingdom

Chili, almond oils

1995

4

0.1a

4

0/0

Uruguay

Oilseed

1993-95

80

50a

80

0/0

Dubai

Oilseed

NR

5

0.500

5

0/0

 

Olive

 

 

 

 

 

Tunisia

Olives

1994

6

0.1a

0

7809/46 830

 

Beans

 

 

 

 

 

Uruguay

Soya beans

1993–95

19

50a

19

0/0

Sweden

Brown beans

1996–98

20

0.1

18

0.18/1.9

United Kingdom

Baked beans

1996

50

0.2a

49

0.006/0.3

Butter beans

1996

12

0.2a

11

1.14/13.7

 

Pulses

 

 

 

 

 

Denmark

Pulses

1993–94

22

0.1a

22

0/0

United Kingdom

Pulses

1998

50

0.200

50

0/0

 

Chickpeas

 

 

 

 

 

Sweden

Peas, dry

1996–98

30

0.1

28

0.12/1.2

United Kingdom

Dried chickpeas

1996

14

0.2a

14

0/0

 

Lentils

 

 

 

 

 

 

Dried lentils

1996

21

0.2a

21

0/0

 

Soya

 

 

 

 

 

Canada

Soya-based cereals

1998–99

16

0.200

NR

NA/0.92

Japan

Soya sauce

1996

5

0.003a

0

0.0068/0.026

 

Coffee

 

 

 

 

 

Netherlands

Roasted coffee

1999

22

0.1a

13

0.45/4.5

USA

Green coffee

1997

19

0.03a

10

NA/4.6

USA

Green coffee, imported

1995–99

180

4–10a

174

0.353/19.2

Soluble coffee, imported

1995–99

23

4–10a

23

0/0

Roasted coffee

1997

13

0.03a

4

NA/1.2

Denmark

Roasted coffee

1993-94

11

0.1a

0

0.51/3.2

United Kingdom

Green coffee, imported

NR

291

0.260

181

NA/27.3

Coffee products

1995

100

0.1a

19

NA/8

Europe

Coffee products

1999

633

0.2–1a

334

0.90/27.2

Eastern Europe

Adulterated soluble coffee

NR

15

0.2a

0

5.9/15.9

World

Soluble coffee

NR

101

0.2a

26

1.1/6.5

Canada

Instant coffee

1997–98

30

0.1a

NR

NA/3.1

Coffee, ground and beans

1997–98

71

0.1a

NR

NA/2.3

Dubai

Coffee beans

NR

8

0.500

5

NA/7.46

Sweden

Green coffee

1999

45

0.05

23

0.53/12.1

Roasted coffee

1999

37

0.05

29

0.40/3.86

Coffee granulate

1999

6

0.05

0

0.50/0.79

Spain

Coffee, roasted and soluble

1997

38

0.110

0

1.01/5.64

Decaffeinated coffee

1997

8

0.110

0

0.55/1.29

Japan

Canned coffee

1996

10

0.003a

1

0.028/0.133

Instant coffee

1996

12

0.06a

0

0.018/0.063

Regular coffee

1996

10

0.06a

10

0/0

European Union,

Roasted coffee

1995–96

86

NR

NR

0.8/NR

Roasted coffee

1995–97

504

NR

NR

0.8/NR

Switzerland

Roasted coffee

1996–98

232

NR

NR

0.6/NR

Roasted coffee

1999

107

NR

NR

0.4/NR

Brazil

Roasted coffee

2000

34

0.2a

11

0.93/6.5

Instant coffee

2000

16

0.2a

0

2.17/5.10

Germany

Roasted coffee

1997

34

0.3a

10

1.43/7.54

Germany

Roasted coffee

1995–98

113

0.3a

61

0.61/6.32

Roasted coffee

1995–98

60

0.3a

39

0.45/4.75

Roasted coffee

1995–98

67

0.3a

35

0.56/3.34

Soluble coffee

1995–98

52

0.3a

6

1.83/9.47

Soluble coffee

1995–98

32

0.3a

13

0.59/1.8

Malt coffee

1995–98

33

0.3a

28

< 0.3/0.96

Green coffee

1995–99

82

0.250

60

1.29/24.5

Roasted coffee

1995–99

419

0.250

228

0.99/12.1

Decaffeinated coffee

1995–99

71

0.250

45

0.49/2.7

Instant coffee

1995–99

41

0.250

12

1.0/4.8

 

Cocoa

 

 

 

 

 

Netherlands

Cocoa products

1996

19

0.250

19

0/0

Uruguay

Cocoa beans and by-products

1993-95

91

50a

91

0/0

United Kingdom

Cocoa powder

1998

20

0.200

0

1.67/2.4

 

Cocoa powder

1996

20

0.2a

3

0.67/1.1

Germany

Cocoa

1995–98

40

0.01a

0

NR/1.8

Cocoa powder

1995–98

56

0.01a

5

NR/0.63

Cocoa drinks

1995–98

34

0.01a

0

NR/0.05

FSIS185 powder

1998

20

0.01a

0

1.7/2.4

FSIS185 powder

1997

20

0.01a

5

0.68/1.1

 

Chocolate

 

 

 

 

 

United Kingdom

Chocolate

1998

40

0.020

10

0.16/0.6

Germany

Milk chocolate < 30%

1995–98

39

0.01a

3

NR/0.41

Germany

Plain chocolate > 60%

1995–98

78

0.01a

0

NR/0.66

Germany

Chocolate with nuts

1995–98

35

0.01a

4

NR/0.16

Germany

Filled chocolate

1995–98

58

0.01a

3

NR/0.324

Germany

FSIS185 milk chocolate

1998

28

0.01a

9

0.15/0.6

Germany

FSIS185 plain chocolate

1998

12

0.01a

1

0.27/0.6

 

Dried fruits

 

 

 

 

 

Uruguay

Dried fruits

1993–95

157

50a

157

0/0

USA

Raisins

NR

63

NR

14

1.56/11.5

USA

Raisins

1998–99

133

NR

43

1.27/29

Raisins

1998

114

NR

38

0.82/8.1

Raisins

1997

69

NR

19

0.42/3.1

United Kingdom

Sultanas

1998

100

0.200

8

3.42/25.1

Raisins

1998

101

0.200

3

2.87/29.8

Currants

1998

100

0.200

4

4.97/40.8

Currants

1996

20

0.2a

1

9.19/53.6

United Kingdom

Apricots

1996

20

0.2a

20

0/0

Dried, fresh coconut

1996

20

0.2a

20

0/0

Dried dates

1996

20

0.2a

19

0.01/0.2

Raisins

1996

20

0.2a

3

2.79/20

Sultanas

1996

20

0.2a

3

4.86/18.1

Figs

1998

20

0.200

18

0.05/0.8

Germany

FSIS 185 currants

1998

100

0.01a

4

NA/40.8

FSIS 185 sultanas

1998

100

0.01a

8

NA/53.6

Dried raisins

1995–98

117

0.01a

5

0.90/7.74

FSIS 185 raisins

1998

101

0.01a

3

NA/29.8

Dried plums

1995–98

31

0.01a

5

NR/0.07

Other dried fruit

1995–98

49

0.01a

23

NR/0.09

Dried figs

1995–98

34

0.01a

7

NR/3.95

 

Sweets

 

 

 

 

 

Germany

Marmelade

1995–98

42

0.01a

42

0/0

Nut-nugget cream

1995–98

33

0.01a

2

0.06/0.27

Other puddings and creams

1995–98

32

0.01a

25

< 0.01/0.09

Cocoa cream

1995–98

32

0.01a

25

0.03/0.08

 

Milk and milk products

 

 

 

 

 

Germany

Milk and milk products

1995–98

264

0.01a

242

<0.01/0.86

Norway

Milk

1995–98

87

0.01a

76

NA/0.058

Infant formula

1995–98

20

0.01a

20

0/0

Human milk

1995–96

80

0.01a

63

0.006/0.18

Sierra Leone

Human milk

NR

113

0.2a

73

7.9/337

 

Dried vegetables

 

 

 

 

 

Uruguay

Dried vegetables

1993–95

100

50a

100

0/0

 

Oil and oilseeds

 

 

 

 

 

Germany

Sunflower seed

1995–98

34

0.01a

14

NA/0.1

Sesame seed

1995–98

24

0.01a

15

NA/0.86

Germany

Linseed

1995–98

24

0.01a

12

NA/1.79

Poppy seed

1995–98

16

0.01a

16

0/0

Edible oils

1995–98 30

0.01a

30

0/0

NA

 

Meat

 

 

 

 

 

Uruguay

Meat products

1993–95

59

10a

59

0/0

Denmark

Pig kidney

1999

300

0.060

63

NA/14.72

Pig meat

1999

300

0.090

227

NA/2.88

Pork

1993–94

76

0.02a

12

0.11/1.3

Pork, organic

1993–94

7

0.02a

3

0.05/0.12

Duck

1993–94

19

0.03a

8

0.02/0.09

Duck liver

1993–94

7

0.03a

3

0.06/0.16

Goose

1993–94

12

0.03a

7

0.03/0.10

Goose liver

1993–94

12

0.03a

8

0.02/0.06

Turkey

1993–94

17

0.03a

7

0.02/0.11

Turkey liver

1993–94

17

0.03a

14

0.04/0.28

Chicken

1993–94

65

0.03a

29

0.03/0.18

United Kingdom

Pork liver

1996

10

0.2a

9

0.02/0.2

Pork salami

1996

9

0.2a

9

0/0

Germany

Raw sausage

1995–98

56

0.01a

28

0.04/0.27

Sausage

1995–98

40

0.01a

26

0.02/0.18

Germany

Sausage

1995–98

45

0.01a

24

0.04/0.38

Liver sausage

1995–98

53

0.01a

17

0.15/4.56

Blood sausage

1995–98

57

0.01a

13

0.16/3.16

Other meat products

1995–98

21

0.01a

18

0.01/0.04

Beef sausage

1995–98

31

0.01a

26

0.02/0.19

Poultry sausage

1995–98

40

0.01a

33

0.01/0.03

Beaf meat

1995–98

58

0.01a

57

0.01/0.03

Pig meat

1995–98

58

0.01a

48

0.02/0.14

Poultry meat

1995–98

41

0.01a

41

0/0

Pig kidney

1995–98

61

0.01a

34

0.43/9.33

Pig liver

1995–98

59

0.01a

49

0.07/2.72

France

Pig kidney

1997

300

1

297

0.01/1.4

Pig kidney

1998

710

1

656

NA/5.0

Nephropathic pig kidneys

1997

100

0

94

NA/0.48

 

Snacks

 

 

 

 

 

Germany

Bar

1995–98

32

0.01a

4

NR/0.11

Bar with nuts

1995–98

47

0.01a

7

NR/3.6

Muesli bar

1995–98

67

0.01a

28

NR/1.72

Muesli

1995–98

115

0.01a

44

NR/31.8

Breakfast cereals

1995–98

85

0.01a

20

NR/0.94

Corn flakes

1995–98

38

0.01a

26

NR/0.1

Biscuit

1995–98

102

0.01a

20

NR/3.81

Biscuit with chocolate

1995–98

67

0.01a

2

NR/0.39

Germany

Rusk

1995–98

37

0.01a

5

NR/2.26

Rye biscuit

1995–98

31

0.01a

8

NR/0.92

Chips, popcorn

1995–98

33

0.01a

23

NR/2.1

 

Nuts

 

 

 

 

 

Netherlands

Roasted peanuts

1996

12

0.250

12

0/0

Peanuts products

1996

4

0.250

4

0/0

Pistachio nuts

1996

3

0.250

3

0/0

Germany

Hazelnuts

1995–98

32

0.01a

13

NR/0.08

Groundnuts

1995–98

31

0.01a

28

NR/0.08

Other nuts

1995–98

125

0.01a

99

NR/0.27

 

Beer

 

 

 

 

 

Canada

Beer

NR

41

0.1a

15

0.04/0.65

Denmark

Beer

1993–94

21

0.001a

0

0.049/0.16

United Kingdom

Beer

1996

20

0.2a

20

0/0

Spain

Beer

1997

40

0.004a

1

0.024/0.075

Europe

Beer

1997

40

0.004a

0

0.025/0.121

Italy

Beer, imported

1999

61

0.01a

31

0.017/0.135

Japan

Beer

1998

22

0.001a

1

0.012/0.045

Beer, imported

1998

94

0.001a

8

0.01/0.066

Germany

Pils beer

1995–98

135

0.01a

34

0.026/0.137

Export beer

1995–98

31

0.01a

6

0.027/0.123

Wheat beer

1995–98

30

0.01a

7

0.031/0.293

Strong beer

1995–98

54

0.01a

9

0.031/0.126

Beer, alcohol-free

1995–98

24

0.01a

11

0.013/0.035

Light beer

1995–98

14

0.01a

8

0.012/0.047

Malt beer

1995–98

30

0.01a

16

0.016/0.081

 

Teas

 

 

 

 

 

Germany

Black tea

1995–98

32

0.3a

32

0/0

Green tea

1995–98

32

0.3a

31

< 0.3/1.33

Fruit tea

1995–98

32

0.3a

32

0/0

 

Wine

 

 

 

 

 

Netherlands

Red wine

1999

150

0.100

90

0.22/3.1

White wine

1999

20

0.100

18

0.12/2.1

Sweden

Wine

1998–99

32

0.005

3

0.21/2.5

United Kingdom

Red wine

1998

50

0.020

22

0.074/0.46

1996

10

0.2a

6

0.38/1.1

White wine

1996

10

0.2a

10

0/0

Switzerland

Wine

NR

18

0.005

5

NA/0.11

Japan

Wine

1996

46

0.003a

27

NA/0.245

Italy

Red wine

1992–94

8

0.001

1

0.54/1.29

Passito

1990–94

5

0.001

3

0.009/0.04

Red wine

1995

9

0.001

0

1.05/2.47

Passito

1995

2

0.001

1

1.92/3.86

Red wine

1996

23

0.001

7

0.54/1.78

Passito

1996

2

0.001

1

0.007/0.01

Passito

1997

5

0.001

1

1.42/3.48

Red wine

1997

13

0.001

0

0.76/2.15

Red wine

1998

18

0.001

4

0.66/3.17

Red wine

1999

115

0.01a

12

2.10/15.61

White wine

1999

21

0.01a

14

0.57/8.86

Rosé wine

1999

4

0.01a

2

0.13/0.28

Italy

Red wine

1997–98

38

0.01a

1

1.21/7.63

Rosé wine

1997–98

8

0.01a

1

0.63/1.15

White wine

1997–98

9

0.01a

0

0.16/0.97

North Italy

Red wine

1997–99

8

0.01a

4

0.102/0.54

South Italy

Red wine

1997–99

43

0.01a

15

0.193/2.55

Red wine

1997–99

20

0.01a

1

1.153/3.31

North France

Red wine

1997–99

68

0.01a

60

0.061/0.78

South France

Red wine

1997–99

40

0.01a

19

0.07/0.47

Germany

White wine

1995–98

58

0.01a

44

NA/1.4

Rosè wine

1995–98

51

0.01a

33

NA/2.4

Red wine

1995–98

172

0.01a

110

NA/7

FSIS 185 red wine

1998

50

0.01a

22

0.08/0.8

FSIS 185 red wine

1997

10

0.01a

6

0.44/1.1

North Germany

Red wine

1997–99

30

0.01a

23

0.022/0.23

White wine

1997–99

26

0.01a

22

0.012/0.04

South Germany

White wine

1997–99

18

0.01a

10

0.054/1.36

Germany

Red wine

 

40

 

20

0.17/1.90

Red wine

 

48

 

28

0.14/1.10

White wine

 

7

 

5

0.08/0.35

France

Wine

1998

29

0.01a

15

0.038/0.19

World

Red wine

1997–99

305

0.01a

140

0.20/3.31

Rosé wine

1997–99

55

0.01a

33

0.12/2.38

White wine

1997–99

60

0.01a

45

0.108/1.36

World

White wine

NR

41

0.00

27

NA/1.2

Rosé wine

NR

14

0.00

8

NA/2.4

Red wine

NR

89

0.00

49

NA/7.0

Europe

Wine

1998

40

NR

20

0.17/1.90

Europe

Red wine

1997

91

0.003a

7

0.054/0.603

Rosé wine

1997

32

0.003a

3

0.031/0.161

White wine

1997

69

0.003a

24

0.020/0.267

Aperitif wine

1997

47

0.003a

12

0.04/0.254

Sparkling wine

1997

12

0.003a

2

0.012//0.037

Dessert wine

1997

16

0.003a

1

1.05/2.54

Europe

Red wine

1994–95

79

0.005

NR

0.039/0.39

Rosé wine

1994–95

15

0.005

NR

0.025/0.12

White wine

1994–95

24

0.005

NR

0.011/0.18

Special wines

1994–95

15

0.005

NR

NA/0.45

 

Vinegar and mustard

 

 

 

 

 

Germany

Apple and fruit vinegar

1995–98

18

0.01a

17

NA/< 0.01

Wine vinegar

1995–98

38

0.01a

19

NA/1.9

Balsam vinegar

1995–98

29

0.01a

5

NA/4.35

Mustard

1995–98

4

0.01a

1

NA/0.34

 

Grape juice

 

 

 

 

 

United Kingdom

Grape juices

1998

20

0.020

1

0.48/2.05

Germany

White grape juice

1995–98

27

0.01a

6

NA/1.3

Red grape juice

1995–98

64

0.01a

8

NA/5.3

FSIS 185 white grape juice

1998

11

0.01a

1

0.27/0.6

FSIS 185 red grape juice

1998

9

0.01a

0

0.76/2.05

FSIS 185 grape juice

1998

20

0.01a

1

0.48/2.05

Europe

Grape juice

1994–95

8

0.005

3

0.137/0.31

Japan

Grape juice

1996

12

0.003a

10

NA/0.006

World

Grape juice

NR

20

000

6

NA/4.7

Other juices

 

 

 

 

 

Germany

Apple juice

1995–98

33

0.01a

33

0/0

Orange juice

1995–98

30

0.01a

30

0/0

Blackcurrant juice

1995–98

19

0.01a

16

NA/0.06

Tomato juice

1995–98

30

0.01a

27

NA/0.032

Carrot juice

1995–98

18

0.01a

17

NA/0.01

Other vegetable juice

1995–98

30

0.01a

30

0/0

 

Seasonings

 

 

 

 

 

Germany

Ketchup

1995–98

57

0.01a

41

NA/3.8

Herb sauce

1995–98

15

0.01a

13

NA/0.25

Pepper sauce

1995–98

50

0.01a

43

NA/0.72

 

Fermented beverages

 

 

 

 

 

Japan

Fermented beverages

1996

15

0.003a

15

0/0

 

Baby food

 

 

 

 

 

Canada

Baby food

1998–99

11

0.500

11

0/0

 

Diet

 

 

 

 

 

United Kingdom

Normal diet

NR

32

0.002

0

0.025/0.073

Vegetarian diet

NR

11

0.002

0

0.045/0.114

Traditional diet

NR

7

0.002

0

0.029/0.066

 

Dust

 

 

 

 

 

USA

Dust

2000

7

NR

0

278.8/1581.8

Country/ Region

Commodity

Year/ Season

90th %ile (΅g/kg)

n > 5 ­ 20
(΅g/kg

n > 20
΅g/kg)

References

Sampling procedure

 

Wheat

 

 

 

 

 

 

Germany

Wheat grain

1995–98

0.26

0

0

P,S,A, Wolff et al. (2000)

 

Wheat flour < T550

1995–98

0.26

0

0

1995–98

0.59

0

0

Wheat wholemeal flour

1995–98

0.66

0

0

Wheat semolina

1995–98

1.49

0

0

Wheat bran

1995–98

0.72

0

0

Wheat germ

1995–98

0.27

0

0

Netherlands

Wheat, domestic

1995

NR

0

0

P,S,A, Sizoo & van Egmond (1997) P,S,A, Inspectorate for Health Protection (personal communication, 1999)

 

Wheat, imported

1995

NR

1

0

White wheat flour

1999

NR

0

0

Whole-wheat meal

1999

NR

0

0

Norway

Wheat

1990

NR

0

0

P,S,A, Langseth (1999)*

 

1993

NR

0

0

1994

NR

0

0

1995

NR

0

0

1996

NR

0

0

1997

NR

0

0

1998

NR

1

0

Total wheat samples

1990–98

NR

5

0

Wheat, imported

1990

NR

0

0

1993

NR

0

0

Norway

Wheat, imported

1994

NR

0

0

1995

NR

1

0

1996

NR

1

0

1997

NR

0

0

1998

NR

0

0

Total imported wheat

1990–98

NR

2

0

Sweden

Wheat grain

1996–98

0.84

0

0

P,S, Thuvander et al. (2000); A, Larsson & Møller (1996)*

Mills; 1 kg sampled in national pesticide control programme/

1999

0.69

1

0

P, National Food Administration; S, Thuvander et al. (2000); A, Larsson (1996)b

 

Brazil

Wheat grain

1988–90

0.00

0

1

P,S, Furlong et al. (1995a); A, Soares et al. (1985)c

From experimental plots; all grain within 3 m x 6 rows/ 3.0–10

1990

0.00

0

0

P,S, Furlong et al. (1995); A, Soares et al. (1985)c

 

Wheat products

1991

0.00

0

0

P,S, Soares & Furlani (1996); A, Soares et al. (1985)c

 

USA

Wheat grain

1997

NA

3

1

P,S,A, Trucksess et al. (1999)

Sampled by GIPSA by unspecified USDA sampling plan

Finland

Wheat grain

1996

0.00

0

2

P,A, Solfrizzo et al. (1998)d

Random 1-kg samples from farms

Denmark

Wheat grain

1986–92

NR

6

3

P,S,A, Jorgensen et al. (1996)d

Random 1-kg samples from mills

Wheat grain, organic

1986–92

NR

3

1

Wheat grain

1986–92

NR

1

0

Wheat bran

1986–92

NR

2

0

Wheat bran, organic

1986–92

NR

0

0

Dubai

Wheat flour

NR

0.00

0

0

P,S,A, Rao (2000)

 

United Kingdom

Wheat noodles

1995

0.28

0

0

P,S,A, Patel et al. (1996)

Purchased in specialist food shops

Wheat grain

1993

NR

0

0

P, Scudamore (1999)

 

Wheat grain stored

1992–93

NR

0

0

A, Sharman et al. (1992)

 

Wheat from millers

1993

NR

2

0

S, NR

 

1994

NR

NR

3

 

 

Wheat grain

1997-98

NR

3

0

P,S,A, MAFF (1999a)

 

1996

0.00

0

0

P,S, MAFF (1997)
A, Sharman et al. (1992)

 

Cereals and flours

1996

NR

2

0

P,S, MAFF (1996b)
A, Sharman et al. (1992)

 

Uruguay

Wheat grain

1993–95

0.00

0

0

P,S, Pineiro et al. (1996)
A, Pineiro & Giribone (1994)c

 

 

Buckwheat

 

 

 

 

 

 

Germany

Buckwheat

1995–98

0.02

0

0

P,S,A, Wolff et al. (2000)

 

Buckwheat flour

1995–98

0.04

1

0

 

Barley

 

 

 

 

 

 

Germany

Barley

1995–98

0.10

0

0

P,S,A, Wolff et al. (2000)

 

Pearl barley

1995–98

0.10

0

0

Norway

Barley

1990

0.00

0

0

P,S,A, Langseth (1999)b

 

Barley groats

1990

0.00

0

0

USA

Barley

1997

NR

1

0

P,S,A, Trucksess et al. (1999)

Sampled by GIPSA with unspecified USDA sampling plan

United Kingdom

Barley

1997-98

NR

5

0

P,S,A, MAFF (1999a)

 

Barley

1996

0.00

1

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

Barley

1993

NR

0

0

P, Scudamore (1999); A, Sharman et al. (1992); S, NR

 

stored in 1992

 

 

 

 

Barley

1994

NR

0

1

Barley stored in 1993

1994

NR

1

1

Uruguay

Barley and malt

1993-95

0.00

0

0

P,S, Pineiro et al. (1996), A, Pineiro & Giribone (1994)c

 

Finland

Barley kernel

1996

0.00

2

0

P,A, Solfrizzo et al. (1998)d

Random 1-kg samples from farms

Canada

Barley cereals

1998–99

NR

0

0

P, Canada; S,A, NRb

 

Barley based cereals

1998–99

NR

NR

0

 

Maize

 

 

 

 

 

 

Germany

Maize and popcorn

1995–98

0.26

0

0

P,S,A, Wolff et al. (2000)

 

United Kingdom

Raw maize, imported

1998–99

NR

0

0

P,S, Scudamore (2000), A, Howell & Taylor (1981)

At ports; from conveyor between silos and mill or from ships' holds

Croatia

Raw maize

1996

0.00

0

2

P,S, Jurjevic et al. (1999); A, Solfrizzo et al. (1998)d

Random 1-kg samples from farms

Raw maize

1997

1.29

2

7

Brazil

Raw maize

1991

0.00

0

0

P,S, Pozzi et al. (1995) A, Soares et al. (1985)c

Samples from stored material collected from 60-kg sacks at monthly intervals

Brazil

Raw maize

1993–94

0.00

0

0

P,S, Gloria et al. (1997)
A, Soares et al. (1985)c

 

Uruguay

Maize and by-products

1993-95

0.00

0

0

P,S, Pineiro et al. (1996)
A, Pineiro & Giribone (1994)c

 

United Kingdom

Corn flour

1995

0.42

0

0

P,S,A, Patel et al. (1996)

 

 

Oats

 

 

 

 

 

 

Sweden

Oat grain

1996–98

0.81

0

0

P,S, Thuvander et al. (2000); A, Larsson & Møller (1996)b

 

 

Oat grain

1999

0.11

0

0

P, National Food Administration (2000); S, Thuvander et al (2000), A, Larsson & Møller (1996)b

 

Norway

Oat groats

1990

NR

0

0

P,S,A, Langseth (1999)b

 

Oats

1990

NR

1

0

Oats

1993

NR

0

0

Oats

1994

NR

1

0

Oats

1995

NR

0

0

Oats

1996

0

0

0

Oats

1997

NR

0

0

Oats

1998

NR

0

0

Total oats samples

1990–98

NR

2

0

Denmark

Oat kernels

1986–92

NR

1

0

P,S,A, Jorgensen et al. (1996)d

Random 1-kg samples from mills

Oat kernels, organic

1986–92

NR

0

0

Oat kernels, imported

1986–92

NR

0

0

Finland

Oat kernels

1996

0.00

0

1

P,A, Solfrizzo et al. (1998)d

Random 1-kg samples from farms

United Kingdom

Oat kernels

1997–98

NR

0

0

P,S,A, MAFF (1999a)

 

1996

0.00

1

0

P,S, MAFF (1997) A, Sharman et al. (1992)

 

Germany

Oats

1995–98

0.00

0

0

P,S,A, Wolff et al. (2000)

 

 

Rice

 

 

 

 

 

 

Uruguay

Rice

1993-95

0.00

0

0

P,S, Pineiro et al. (1996); A, Pineiro & Giribone (1994)c

Stratified random sampling to obtain 5-kg samples

United

Basmati rice

1995

0.00

0

0

P,S,A, Patel et al. (1996)b

 

Kingdom

Chinese rice

1995

0.00

0

0

Dubai

Rice

NR

0.00

0

0

P,S,A, Rao (2000)

 

Germany

Rice

1995–98

0.01

0

0

P,S,A, Wolff et al. (2000)

Parboiled rice

1995–98

 

0

0

Long-grain rice

1995–98

 

0

0

Round-grain rice

1995–98

 

0

0

 

Rye

 

 

 

 

 

 

Sweden

Rye grain

1996–98

1.2

0

0

P,S, Thuvander et al. (2000); A, Larsson & Møller (1996)b

 

1999

1.5

0

1

P, National Food Administration, S, Thuvander et al. (2000), A, Larsson & Møller (1996)b

 

Norway

Rye, imported

1990

NR

0

0

P,S,A, Langseth (1999)b

 

1993

NR

0

0

1994

NR

0

0

1995

NR

0

0

1996

0.00

0

0

Total

1990–98

NR

0

0

Denmark

Rye kernels

1986–92

NR

16

4

P,S,A, Jorgensen et al. (1996)d

Random 1-kg samples from mills

Rye kernels, organic

1986–92

NR

12

4

Rye kernels, imported

1986–92

NR

0

0

United Kingdom

Rye kernels

1996

0.00

0

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

Germany

Rye

1995–98

0.01

0

0

P,S,A, Wolff et al. (2000)

 

Rye flour < T997

1995–98

0.87

1

0

1995–98

0.06

0

0

Rye wholemeal flour

1995–98

0.03

0

0

 

Sorghum

 

 

 

 

Germany

Sorghum

1995–98

0.01

0

0

P,S,A, Wolff et al. (2000)

 

 

Spelt

 

 

 

 

 

 

Germany

Spelt/spelt flour

1995–98

0.06

NR

0

P,S,A, Wolff et al. (2000)

 

 

Cereal products

 

 

 

 

 

 

United Kingdom

Pitta bread

1995

NR

0

0

P,S,A, Patel et al. (1996)b

 

Chapatti

1995

NR

0

0

Nan bread

1995

000'

0

0

Poppadoms

1995

000'

0

0

Tunisia

Cereal-derived food

1994

4314.4

6

56

P,S,A, Maaroufi et al. (1995b)

From homes of nephropathy patients

Dubai

Mixed cereals

NR

NR

0

0

P,S,A, Rao (2000)

 

Canada

Mixed grain cereals

1998–99

NR

0

0

P, Canada (2000); S and A, NRb

 

Mixed cereals

1998–99

000'

0

0

Europe, Tunisia

Bread

NR

NR

NR

0

P, Burdaspal & Legarda (2000); A, Burdaspal & Legarda (2001); S, NRb

 

Germany

Bread, wheat mix

1995–98

000

0

0

P,S,A, Wolff et al. (2000)

 

Germany

Bread, rye mix

1995–98

0.04

0

0

P,S,A, Wolff et al. (2000)

 

White bread

1995–98

0.01

0

0

Toast bread

1995–98

0.20

0

0

Rye meal bread

1995–98

0.32

1

0

Milk–water bread roll

1995–98

0.21

0

0

Various cereals, bread

1995–98

0.73

0

0

Various cereals, bread +oilseed

1995–98

0.33

0

0

Crispbread

1995–98

0.01

0

0

Wheat whole bread

1995–98

0.02

0

0

Special bread

1995–98

0.02

0

0

Wholemeal bread roll

1995–98

0.02

0

0

Various cereals and muesli bread

1995–98

0.02

2

0

Rye bread roll

1995–98

0.03

0

0

Pasta without egg

1995–98

0.61

0

0

Pasta with egg

1995–98

0.04

0

0

Wholemeal pasta

1995–98

0.11

0

1

Oat flakes

1995–98

0.01

0

0

Oats bran

1995–98

0.01

0

0

Polenta

1995–98

0.03

0

0

Green corn

1995–98

0.01

0

0

Infant food

1995–98

0.01

0

0

Peas, lentil, beans

1995–98

 

0

0

Soya beans

1995–98

0.01

0

0

FSIS185 pulses

1998

 

0

0

FSIS185 pulses

1997

< 0.2

2

0

 

Seeds

 

 

 

 

 

 

United Kingdom

Fennel

1995

0.00

0

0

P,S,A, Patel et al. (1996)

 

Sesame seeds

1995

0.00

0

0

Coriander

1995

3.20

0

0

 

Herbs and spices

 

 

 

 

Netherlands

Paprika powder

1996–98

NR

1

0

P,S,A, Inspectorate for Health Protection (personal communication, 1999)

 

Pepper

1996–97

NR

4

0

United Kingdom

Chilli powder

1995

NR

NR

NR

P,S,A, Patel et al. (1996)

 

Curry powder

1995

NR

NR

NR

Tandoori

1995

NR

NR

NR

Ginger

1995

NR

NR

0

Garlic

1995

0.00

0

0

Five spices powder

1995

1.82

0

0

Tunisia

Dried vegetables

1994

3426.6

0

6

P,S,A, Maaroufi et al. (1995b)

From homes of nephropathy patients

Dubai

Spices

NR

NR

0

0

P,S,A, Rao (2000)

 

 

Pickles and pastes

 

 

 

 

 

 

United Kingdom

Chilli pickle

1995

NR

0

0

P,S,A, Patel et al. (1996)

 

Garlic pickle

1995

NR

0

0

United Kingdom

Curry paste

1995

NR

NR

0

P,S,A, Patel et al. (1996)

 

Chilli sauce

1995

2.31

0

0

 

Canned foods

 

 

 

 

 

 

United Kingdom

Canned foods

1995

NR

0

0

P,S,A, Patel et al. (1996)

 

 

Oils

 

 

 

 

 

 

United

Sesame oil

1995

0.32

0

0

P,S,A, Patel et al. (1996)

 

Kingdom

Chili, almond oils

1995

0.00

0

0

Uruguay

Oilseed

1993-95

0.00

0

0

P,S, Pineiro et al. (1996), A, Pineiro & Giribone (1994)

 

Dubai

Oilseed

NR

0.00

0

0

P,S,A, Rao (2000)

 

 

Olive

 

 

 

 

 

 

Tunisia

Olives

1994

32 782

2

1

P,S,A, Maaroufi et al. (1995b)

From homes of nephropathy patients

 

Beans

 

 

 

 

 

 

Uruguay

Soya beans

1993–95

0.00

0

0

P,S, Pineiro et al. (1996), A, Pineiro & Giribone (1994)

 

Sweden

Brown beans

1996–98

0.07

0

0

P,S, Thuvander et al. (2000), A, Larsson & Møller (1996)

 

United Kingdom

Baked beans

1996

0.00

0

0

P,S, MAFF (1997)

 

Butter beans

1996

0.00

1

0

A, Sharman et al. (1992)

 

 

Pulses

 

 

 

 

 

 

Denmark

Pulses

1993–94

NA

0

0

P,S,A, Jorgensen (1998)

'Random samples' from retail shops

United Kingdom

Pulses

1998

NA

0

0

P,S,A, MAFF (1999b)

 

 

Chickpeas

 

 

 

 

 

 

Sweden

Peas, dry

1996–98

0.08

0

0

P,S, Thuvander et al. (2000); A, Larsson & Møller (1996)

 

United Kingdom

Dried chickpeas

1996

NA

0

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

 

Lentils

 

 

 

 

 

 

 

Dried lentils

1996

NA

0

0

 

 

 

Soya

 

 

 

 

 

 

Canada

Soya-based cereals

1998–99

NR

0

0

P, Canada (2000); S,A, NR

 

Japan

Soya sauce

1996

NR

0

0

P,A, Ueno (1998); S, NR

 

 

Coffee

 

 

 

 

 

 

Netherlands

Roasted coffee

1999

NR

0

0

P,S,A, Inspectorate for Health Protection (personal communication, 1999)

 

USA

Green coffee

1997

NR

0

0

P,S,A, Trucksess et al. (1999)

Sampled by GIPSA with unspecified USDA sampling plan

USA

Green coffee, imported

1995–99

0.00

3

0

P, Ochratoxin A Monitoring Program, S,A, NRc

 

Soluble coffee, imported

1995–99

0.00

0

0

 

 

Roasted coffee

1997

NR

0

0

P,S,A, Trucksess et al. (1999)

'Random samples' from retail shops

Denmark

Roasted coffee

1993-94

NR

0

0

P,S,A, Jorgensen (1998)

'Random samples' from retail shops

United Kingdom

Green coffee, imported

NR

NR

11

2

P,S,A, MAFF (1996a)

 

Coffee products

1995

NR

NR

0

P,S,A, Patel et al. (1997)

 

Europe

Coffee products

1999

NR

3

1

P,S,A, Stegen et al. (1997)

 

Eastern Europe

Adulterated soluble coffee

NR

1.40

6

0

P,S,A, Pittet et al. (1996)

 

World

Soluble coffee

NR

NA

NR

0

P,S,A, Pittet et al. (1996)

 

Canada

Instant coffee

1997–98

NR

0

0

P, Canada (2000);S,A,

 

Coffee, ground and beans

1997–98

NR

0

0

NR

 

Dubai

Coffee beans

NR

NR

1

0

P,S,A, Rao (2000)

 

Sweden

Green coffee

1999

0.74

1

0

P,S,A, National Food Administration

 

Roasted coffee

1999

1.7

0

0

 

Coffee granulate

1999

0.68

0

0

 

 

Spain

Coffee, roasted and soluble

1997

NR

NR

0

P, Burdaspal & Legarda (1998b), A, Pittet et al. (1996); S, NR

 

Decaffeinated coffee

1997

NR

0

0

Japan

Canned coffee

1996

NR

0

0

P,A, Ueno (1998); S, NR

 

Instant coffee

1996

NR

0

0

Regular coffee

1996

000'

0

0

European Union,

Roasted coffee

1995–96

NR

NR

NR

Olsen (2000)

 

Roasted coffee

1995–97

NR

NR

NR

Switzerland

Roasted coffee

1996–98

NR

NR

NR

Roasted coffee

1999

NR

NR

NR

Brazil

Roasted coffee

2000

000

1

0

P,A, Leoni et al. (2000); S, NR

 

Instant coffee

2000

0'05

1

0

Germany

Roasted coffee

1997

NR

3

0

P,A, Koch et al. (1996); S, NR

 

Germany

Roasted coffee

1995–98

2

1

0

P,S,A, Wolff et al. (2000)

 

Roasted coffee

1995–98

1

0

0

Roasted coffee

1995–98

2

0

0

Soluble coffee

1995–98

4

5

0

Soluble coffee

1995–98

2

0

0

Malt coffee

1995–98

1

0

0

Green coffee

1995–99

NR

NR

NR

P,S, Ottender & Majerus (2001); A, Entwisle et al. (2000b)

 

Roasted coffee

1995–99

NR

NR

0

Decaffeinated coffee

1995–99

NR

0

0

Instant coffee

1995–99

NR

0

0

 

Cocoa

 

 

 

 

 

 

Netherlands

Cocoa products

1996

0.00

0

0

P,S,A, Inspectorate for Health Protection (personal communication, 1999)

 

Uruguay

Cocoa beans and by-products

1993-95

0.00

0

0

P,S, Pineiro et al. (1996), A, Pineiro & Giribone (1994)c

 

United Kingdom

Cocoa powder

1998

2.11

0

0

P,S,A, MAFF (1999b)

 

 

Cocoa powder

1996

1.00

0

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

Germany

Cocoa

1995–98

0.93

0

0

P,S,A, Wolff et al. (2000)

 

Cocoa powder

1995–98

0.03

0

0

Cocoa drinks

1995–98

0.00

0

0

FSIS185 powder

1998

NR

0

0

FSIS185 powder

1997

NR

0

0

 

Chocolate

 

 

 

 

 

 

United Kingdom

Chocolate

1998

0.31

0

0

P,S,A, MAFF (1999b)

 

Germany

Milk chocolate < 30%

1995–98

0.01

0

0

P,S,A, Wolff et al. (2000)

 

Germany

Plain chocolate > 60%

1995–98

0.02

0

0

Germany

Chocolate with nuts

1995–98

0.01

0

0

Germany

Filled chocolate

1995–98

0.01

0

0

Germany

FSIS185 milk chocolate

1998

NR

0

0

Germany

FSIS185 plain chocolate

1998

NR

0

0

 

Dried fruits

 

 

 

 

 

 

Uruguay

Dried fruits

1993–95

0.00

0

0

P,S, Pineiro et al. (1996), A, Pineiro & Giribone (1994)c

 

USA

Raisins

NR

3.48

5

0

P, Ochratoxin A Monitoring Program, S,A, NR

 

USA

Raisins

1998–99

3.96

3

1

P, Ochratoxin A Monitoring Program; S,A, NR

 

Raisins

1998

1.87

6

0

Raisins

1997

0.92

0

0

United Kingdom

Sultanas

1998

8.56

15

2

P,S,A, MAFF (1999b)

 

Raisins

1998

6.40

13

1

Currants

1998

11.47

20

5

Currants

1996

14.33

9

2

P,A, MacDonald et al. (1999); A, NR

From retail shops with MAFF sampling scheme

United Kingdom

Apricots

1996

NA

0

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

Dried, fresh coconut

1996

NA

0

0

Dried dates

1996

0.00

0

0

Raisins

1996

8.60

4

0

P,A, MacDonald et al. (1999); A, NR

 

Sultanas

1996

11.35

7

0

Figs

1998

0.15

0

0

P,S,A, MAFF (1999b)

 

Germany

FSIS 185 currants

1998

NA

29 (> 4)

5

P,S,A, Wolff et al. (2000)

 

FSIS 185 sultanas

1998

NA

18 (> 4)

2

Dried raisins

1995–98

NA

2

6

FSIS 185 raisins

1998

NA

19 (> 4)

1

Dried plums

1995–98

0.00

0

0

Other dried fruit

1995–98

0.00

0

0

Dried figs

1995–98

0.02

0

0

 

Sweets

 

 

 

 

 

 

Germany

Marmelade

1995–98

NA

0

0

P,S,A, Wolff et al. (2000)

 

Nut-nugget cream

1995–98

0.00

0

0

Other puddings and creams

1995–98

0.00

0

0

Cocoa cream

1995–98

0.00

0

0

 

Milk and milk products

 

 

 

 

 

 

Germany

Milk and milk products

1995–98

NA

0

0

P,S,A, Wolff et al. (2000)

 

Norway

Milk

1995–98

NA

0

0

P,S, Skaug (1999)

 

Infant formula

1995–98

NA

0

0

A, Breitholtz-Emanuelsson (1993b)

 

Human milk

1995–96

NA

0

0

Sierra Leone

Human milk

NR

NA

NR

4

P,S,A, Jonsyn et al. (1995)

 

 

Dried vegetables

 

 

 

 

 

 

Uruguay

Dried vegetables

1993–95

0.00

0

0

P,S, Pineiro et al. (1996), A, Pineiro & Giribone (1994)c

 

 

Oil and oilseeds

 

 

 

 

 

 

Germany

Sunflower seed

1995–98

0.00

0

0

P,S,A, Wolff et al. (2000)

 

Sesame seed

1995–98

1

0

0

Germany

Linseed

1995–98

1

0

0

P,S,A, Wolff et al. (2000)

 

Poppy seed

1995–98

NA

0

0

Edible oils

1995–98 30

0

0

 

 

Meat

 

 

 

 

 

 

Uruguay

Meat products

1993–95

0.00

0

0

P,S, Pineiro et al. (1996); A, Pineiro & Giribone (1994)c

 

Denmark

Pig kidney

1999

1.5b

5

0

P,S,A, Petersen (2000)

 

Pig meat

1999

0.3b

0

0

Pork

1993–94

NR

0

0

P,S, Jorgensen (1998); A, Jorgensen et al. (1996)

'Random samples' from slaughterhouses

Pork, organic

1993–94

NR

0

0

Duck

1993–94

NR

0

0

Duck liver

1993–94

NR

0

0

Goose

1993–94

NR

0

0

Goose liver

1993–94

NR

0

0

Turkey

1993–94

NR

0

0

Turkey liver

1993–94

NR

0

0

Chicken

1993–94

NR

0

0

United Kingdom

Pork liver

1996

0.02

0

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

Pork salami

1996

NA

0

0

Germany

Raw sausage

1995–98

0.00

0

0

P,S,A, Wolff et al. (2000)

 

Sausage

1995–98

0.00

0

0

Germany

Sausage

1995–98

0.00

0

0

P,S,A, Wolff et al. (2000)

 

Liver sausage

1995–98

0.00

0

0

Blood sausage

1995–98

0.00

0

0

Other meat products

1995–98

0.00

0

0

Beef sausage

1995–98

0.00

0

0

Poultry sausage

1995–98

0.00

0

0

Beaf meat

1995–98

0.00

0

0

Pig meat

1995–98

0.00

0

0

Poultry meat

1995–98

NA

0

0

Pig kidney

1995–98

0.00

3

0

Pig liver

1995–98

0.00

0

0

France

Pig kidney

1997

< 0.5

0

0

P,S,A, Dragacci et al. (1999)

 

Pig kidney

1998

NA

0

0

Nephropathic pig kidneys

1997

NA

0

0

 

Snacks

 

 

 

 

 

 

Germany

Bar

1995–98

0.097

0

0

P,S,A, Wolff et al. (2000)

 

Bar with nuts

1995–98

0.02

0

0

Muesli bar

1995–98

0.01

0

0

Muesli

1995–98

0.03

1

1

Breakfast cereals

1995–98

0.02

0

0

Corn flakes

1995–98

0.01

0

0

Biscuit

1995–98

0.02

0

0

Biscuit with chocolate

1995–98

0.02

0

0

Germany

Rusk

1995–98

0.03

0

0

P,S,A, Wolff et al. (2000)

 

Rye biscuit

1995–98

0.01

0

0

Chips, popcorn

1995–98

0.01

0

0

 

Nuts

 

 

 

 

 

 

Netherlands

Roasted peanuts

1996

0.00'

0

0

P,S,A, Inspectorate for Health Protection (personal communication, 1999)

 

Peanuts products

1996

0.00

0

0

Pistachio nuts

1996

0.00

0

0

Germany

Hazelnuts

1995–98

0.00

0

0

P,S,A, Wolff et al. (2000)

 

Groundnuts

1995–98

0.00

0

0

Other nuts

1995–98

0.024

0

0

 

Beer

 

 

 

 

 

 

Canada

Beer

NR

NA

0

0

P,S,A, Scott & Kanhere (1995)

 

Denmark

Beer

1993–94

NR

0

0

P,S,A, Jorgensen (1998)

'Random samples' from retail shops

United Kingdom

Beer

1996

NA

0

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

Spain

Beer

1997

NR

0

0

P,A, Burdaspal & Legarda (1998a); S, NR

 

Europe

Beer

1997

NR

0

0

Italy

Beer, imported

1999

0.05

0

0

P,S,A, Visconti et al. (2000b)

 

Japan

Beer

1998

NR

0

0

P and A, Nakajima et al. (1999); S, NR

 

Beer, imported

1998

NR

0

0

Germany

Pils beer

1995–98

0.058

0

0

P,S,A, Wolff et al. (2000)

 

Export beer

1995–98

0.059

0

0

Wheat beer

1995–98

0.041

0

0

Strong beer

1995–98

0.082

0

0

Beer, alcohol-free

1995–98

0.030

0

0

Light beer

1995–98

0.044

0

0

Malt beer

1995–98

0.033

0

0

 

Teas

 

 

 

 

 

 

Germany

Black tea

1995–98

NA

0

0

P,S,A, Wolff et al. (2000)

 

Green tea

1995–98

NA

0

0

Fruit tea

1995–98

NA

0

0

 

Wine

 

 

 

 

 

 

Netherlands

Red wine

1999

1

0

0

P,S,A, Inspectorate for Health Protection and van Egmond (personal communication, 1999)

 

White wine

1999

0.00

0

0

P,S,A, van Egmond (personal communication, 1999)

 

Sweden

Wine

1998–99

0.45

0

0

P,S, A, National Food Administration

 

United Kingdom

Red wine

1998

0.22

0

0

P,S,A, MAFF (1999b)

 

1996

1.10

0

0

P,S, MAFF (1997); A, Sharman et al. (1992)

 

White wine

1996

NA

0

0

Switzerland

Wine

NR

NR

0

0

P,A, Zimmerli & Dick (1995); S, NR

 

Japan

Wine

1996

NR

0

0

P,A, Ueno (1998), S, NR

 

Italy

Red wine

1992–94

0.91

0

0

P, Pietri (2000); A, Zimmerli & Dick (1995), S, NR

 

Passito

1990–94

0.028

0

0

Red wine

1995

2.34

0

0

Passito

1995

3.47

0

0

Red wine

1996

1.55

0

0

Passito

1996

0.01

0

0

Passito

1997

3.07

0

0

Red wine

1997

1.41

0

0

Red wine

1998

0.09

0

0

Red wine

1999

7.12

18

0

P, Pietri (2001); A, Visconti et al. (1999);S, NR

 

White wine

1999

2.03

1

0

Rosé wine

1999

0.27

0

0

Italy

Red wine

1997–98

2.28

1

0

P,S,A, Visconti et al. (1999)

 

Rosé wine

1997–98

1.09

0

0

White wine

1997–98

0.43

0

0

North Italy

Red wine

1997–99

NR

0

0

P,A, Ottender & Majerus (2000); S, NR

 

South Italy

Red wine

1997–99

NR

0

0

Red wine

1997–99

NR

0

0

North France

Red wine

1997–99

NR

0

0

South France

Red wine

1997–99

NR

0

0

 

 

Germany

White wine

1995–98

0.00

0

0

P,S,A, Wolff et al. (2000)

 

Rosè wine

1995–98

0.00

0

0

Red wine

1995–98

1

1

0

FSIS 185 red wine

1998

NA

0

0

FSIS 185 red wine

1997

NA

0

0

North Germany

Red wine

1997–99

NR

0

0

P,A, Ottender & Majerus (2000); S, NR

 

White wine

1997–99

NR

0

0

South Germany

White wine

1997–99

NR

0

0

Germany

Red wine

 

NR

0

0

P,A, Lehtonen (1999); S, NR

 

Red wine

 

NR

0

0

White wine

 

NR

0

0

France

Wine

1998

0.01

0

0

P,A, Ospital et al. (1998); S, NR

 

World

Red wine

1997–99

NR

0

0

P,A, Ottender & Majerus (2000); S, NR

 

Rosé wine

1997–99

NR

0

0

White wine

1997–99

NR

0

0

World

White wine

NR

NA

0

0

P,S,A, Majerus & Ottender (1996)

 

Rosé wine

NR

NA

0

0

Red wine

NR

NA

 

0

Europe

Wine

1998

0.02

0

0

P, www.elintarvikevirastofi/; A, S, NR

 

Europe

Red wine

1997

NR

0

0

P, Burdaspal & Legarda (1999);A, Zimmerli & Dick (1996);S, NR

 

Rosé wine

1997

NR

0

0

White wine

1997

NR

0

0

Aperitif wine

1997

NR

0

0

Sparkling wine

1997

NR

0

0

Dessert wine

1997

NR

0

0

Europe

Red wine

1994–95

NR

0

0

P,A, Zimmerli & Dick (1996);S, NR

 

Rosé wine

1994–95

NR

0

0

White wine

1994–95

NR

0

0

Special wines

1994–95

NR

0

0

 

Vinegar and mustard

 

 

 

 

 

 

Germany

Apple and fruit vinegar

1995–98

< 0.01

0

0

P,S,A, Wolff et al. (2000)

 

Wine vinegar

1995–98

0.00

0

0

Balsam vinegar

1995–98

3

0

0

Mustard

1995–98

0.00

0

0

 

Grape juice

 

 

 

 

 

 

United Kingdom

Grape juices

1998

0'02

0

0

P,S,A, MAFF (1999b)

 

Germany

White grape juice

1995–98

1

0

0

P,S,A, Wolff et al.

 

Red grape juice

1995–98

3

2

0

(2000)

 

FSIS 185 white grape juice

1998

NA

0

0

 

 

FSIS 185 red grape juice

1998

NA

0

0

 

 

FSIS 185 grape juice

1998

1.73

0

0

 

 

Europe

Grape juice

1994–95

0.30

0

0

P,A, Zimmerli & Dick (1996); S, NR

 

Japan

Grape juice

1996

NR

0

0

P,A, Ueno (1998); S, NR

 

World

Grape juice

NR

NA

0

0

P,S,A, Majerus & Ottender (1996)

 

Other juices

 

 

 

 

Germany

Apple juice

1995–98

NA

0

0

P,S,A, Wolff et al. (2000)

 

Orange juice

1995–98

NA

0

0

Blackcurrant juice

1995–98

0.048

0

0

Tomato juice

1995–98

000

0

0

Carrot juice

1995–98

< 0.01

0

0

Other vegetable juice

1995–98

NA

0

0

 

Seasonings

 

 

 

 

 

 

Germany

Ketchup

1995–98

1

0

0

P,S,A, Wolff et al. (2000)

 

Herb sauce

1995–98

0

0

0

Pepper sauce

1995–98

0

0

0

 

Fermented beverages

 

 

 

 

 

 

Japan

Fermented beverages

1996

0.00

0

0

P,A, Ueno (1998); S, NR

 

 

Baby food

 

 

 

 

 

 

Canada

Baby food

1998–99

0.00

0

0

P, Canada (2000); S,A, NR

 

 

Diet

 

 

 

 

 

 

United Kingdom

Normal diet

NR

0.03

0

0

P,S,A, MAFF (1999c)

 

Vegetarian diet

NR

0.09

0

0

Traditional diet

NR

0.05

0

0

 

Dust

 

 

 

 

 

 

USA

Dust

2000

816

0

3

P,A, Richard et al. (1999); S, NR

 

 

NA, not analysed; NR, not reported; MAFF, Ministry of Agriculture, Fisheries and Food (United Kingdom); USDA, Department of Agriculture of the USA; GIPSA, Grain Inspection, Packers and Stockyards Administration (United Kingdom)

a Limit of detection

b Sampling not described

c LOQ > 5 ΅g/kg; not used in calculation of weighted mean

dThe number of samples was divided by a factor of 3 for calculation of the weighted mean.



    See Also:
       Toxicological Abbreviations
       Ochratoxin A (WHO Food Additives Series 28)
       OCHRATOXIN A (JECFA Evaluation)
       Ochratoxin A (IARC Summary & Evaluation, Volume 56, 1993)