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 µg/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.

2.1.1 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 (pK_a = 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 × 10⁴ per mol for pigs, 5.1 × 10⁴ per mol for chickens, and 4.0 × 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 × 10¹⁰ per mol in human serum and 0.59 × 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 µg/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 µg/kg in those given 5 mg/kg bw and and 34 µg/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 µg/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 µg/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 µg/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 µg/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 (K_M = 1.3 mmol/L for ochratoxin A; 3.3 µmol/L for phenylalanine) (Creppy et al., 1983a) to 1/20 in rat liver (K_m = 0.28 mmol/L for ochratoxin A; 6 µmol/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 µg/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).

2.2.1 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.

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.

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 µm 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 µg/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).

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).

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).

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

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).

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 µg/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 µg/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 µg/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 µg/kg bw per day and in 30/50 at 210 µg/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.

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.

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.

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 µg/ml. In the studies with negative results, concentrations of 10 µg/ml (C3H mouse mammary cells) and > 12 µg/ml (mouse lymphoma L5178Y cells) were cytotoxic. The positive result therefore requires confirmation. No studies of mutation in vivo have been reported.

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.

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).

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).

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 µg 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.

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 µg/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

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 µg/kg for 28 or 90 days, equivalent to 1, 40, and 400 µg/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 µg/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).

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 µg/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 µg/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 µg/kg bw. In contrast, exposure to 10–50 µg/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 µg/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 µg/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.

Groups of six weanling hybrid pigs received either pure or crude ochratoxin A at doses of 7–50 µg/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.

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 µg 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).

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 µmol/L. In contrast, incubation of ochratoxin A with phytohaemagglutinin-stimulated lymphocytes resulted in inhibition of DNA synthesis at concentrations > 6.4 µmol/L. Protein synthesis in resting lymphocytes was markedly inhibited by 12 µmol/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 µg/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

Three male Wistar rats received 1 nmol (about 400 ng) of ochratoxin A by intracerebral administration and four received a diet containing 290 µg/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 µg/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 µg/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).

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 µmol/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

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 µg/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 µg/kg bw per day for male rats and 21 µg/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 µmol/kg bw or six intraperitoneal injections of 1.2 µmol/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 µmol/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).

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 µmol/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 µmol/L but not at 0.1 µmol/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 µmol/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 µmol/L, and their viability was decreased by 10 µmol/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 µmol/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 µmol/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 µmol/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 µmol/L increased the IC₅₀ for inhibition of protein synthesis from 14 µmol/L to 22 µmol/L. An increase to 34 µmol/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 µg/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 µg 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 µg/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).

2.3.1 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 µg/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).

2.4.1 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).

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.

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).

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 µg/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 µg/kg in maize and barley, has been accepted as final-action AOAC International Official Method 991.44.

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 µg/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.

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 µg/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 µg/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.

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.

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.

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).

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.

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.

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.

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).

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).