First draft prepared by
    Drs T. Kuiper-Goodman and D.L. Grant,
    Toxicological Evaluation Division,
    Health and Welfare Canada.


         Ochratoxin A (OA) is a mycotoxin produced by  aspergillus
     ochraceus, after which it was named, as well as by other molds,
    notably  Penicillium viridicatum.  OA consists of a
    dihydroisocoumarin moiety linked through its 7-carboxyl group by an
    amide bond to one molecule of L-ß-phenylalanine (Fig. 1).  OA has
    not been evaluated previously by the Joint FAO/WHO Expert Committee.

         OA has both antibiotic and toxic properties, the most important
    of which are its nephrotoxic, teratogenic, carcinogenic, and
    immunotoxic properties.  It has been the cause of a nephropathy
    affecting many pigs in Scandinavian countries.  In Denmark carcasses
    are condemned if residue levels of OA in the kidney exceed 25 ng/g
    (previous 10 ng/g).  Its presence in Yugoslavian and Bulgarian
    foodstuffs has been speculatively associated with a human
    nephropathy endemic in certain parts of those countries.  OA
    residues are known to occur in food and feed grade cereal crops and
    in pig tissues and pig blood at levels that may be of health

         A detailed risk assessment on OA, which discusses the
    chemistry, mycology, and natural occurrence of OA as well as its
    toxicity, metabolic disposition, and its role in porcine, avian and
    human nephropathy has recently been published (Kuiper-Goodman &
    Scott, 1989) and the present review has made considerable use of
    that paper.  OA was also recently reviewed by WHO/IPCS (draft
    document was not available to the reviewers) and was a subject for 2
    working groups of the International Agency for Research on Cancer
    (IARC, 1976, 1983).


    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

    FIGURE 1

              R1                    R2    R3    R3    R5

    Phenylalanyl                Cl    H     H     H    Ochratoxin A
    Phenylalanyl                H     H     H     H    Ochratoxin B
    Phenylalanyl ethyl ester    Cl    H     H     H    Ochratoxin C
    Phenylalanyl methyl ester   Cl    H     H     H    Ochratoxin A methyl ester
    Phenylalanyl methyl ester   H     H     H     H    Ochratoxin B methyl ester
    Phenylalanyl ethyl ester    H     H     H     H    Ochratoxin B ethyl ester
    OH                          Cl    H     H     H    Ochratoxin alpha
    OH                          H     H     H     H    Ochratoxin ß
    Phenylalanyl                Cl    H     OH    H    4R-Hydroxyochratoxin A
    Phenylalanyl                Cl    OH    H     H    4S-Hydroxyochratoxin A
    Phenylalanyl                Cl    H     H     OH   10-Hydroxyochratoxin A

    Fig. 1   Chemical structures of ochratoxin A and related metabolites  Absorption

         It has been suggested that in most species OA is absorbed from
    the stomach, aided by the acidic properties of the mycotoxin
    (pKa=7.1) (Galtier, 1978; Roth  et al., 1988).

         However, in studies with ligated gastro-intestinal loops, 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 is
    dependent on the pH at the mucosal surface of the jejunum.  OA so
    transferred is in lipid soluble, non-ionized form (Kumagai & Aibara,
    1982; Kumagai, 1988).

         Recent studies, with a low dose of 3H-OA given by intubation
    to mice, were interpreted by the authors as indicating rapid
    absorption of OA from the stomach (Roth  et al., 1988), but the
    reviewers felt that the data could also be interpreted as supporting
    intestinal absorption of OA as the major route, based on rapid
    transit of OA from the stomach to the intestine.  These authors also
    found that secondary distribution peaks of OA in the intestinal
    content and serum may be a consequence of enterohepatic circulation
    since the biliary excretion of OA is very efficient (Roth  et al.,
    1988, Fuchs  et al., 1988).

         The overall percentage of OA absorbed is 66%, 56%, 56%, and
    40%, respectively, for the pig, rat, rabbit and chicken (Galtier  et
     al., 1981; Suzuki  et al., 1977).

         Phenylalanine, given to mice by gavage together with OA in a
    10:1 molar ratio, appeared to increase absorption of OA from the
    stomach and intestine, and increase gastrointestinal transit.  This
    resulted in 8-fold and 4-fold higher levels of OA in serum and
    liver, respectively, during the first 12 hours (Roth  et al.,

         OA that has been transferred across the intestinal mucosa
    reaches the liver via the portal vein (Kumagai & Aibra, 1982; Storen
     et al., 1982a).

         Its relative bioavailability, estimated from a comparison of
    the maximal serum concentration after oral and intravenous exposure,
    was estimated to be very low in fish, but 44 and 97% for several
    mammalian species investigated (Hagelberg  et al., 1989).

         Once it reaches the blood, OA readily binds to serum albumin
    (Galtier  et al., 1980), and other serum macromolecules (Hult &
    Fuchs, 1986).  Red blood cells contained only traces of OA (Galtier,

         The association constants for OA binding to serum albumins were
    7.1 x 104 M-1, 5.1 x 104 M-1 and 4.0 x 104 M-1 for porcine,
    chicken and rat albumins respectively (Galtier  et al., 1981).

         The fraction of OA bound to serum albumin and other serum
    macromolecules constitutes a mobile reserve of mycotoxin which can
    be available for release to the tissues for a long time (Galtier,
    1978;; Hult  et al., 1982).

         Studies with albumin deficient rats have shown that the primary
    effect of OA binding or serum albumin is to retard its elimination
    by limiting transfer of OA from the blood stream to hepatic and
    renal cells (Kumagai, 1985).

         In studies on the stability of OA bound to porcine albumin, the
    acidic drug phenylbutazone displaced OA from serum albumin so that
    more free toxin was available.   In vivo studies in male rats
    showed greater toxicity of OA in the presence of phenylbutazone,
    with a significant decrease in the LD50 value from 33.4 to 21.1
    mg/kg bw (Galtier  et al., 1980).

         OA was found to have higher affinity  for an as yet
    unidentified serum macromolecule (MW=20,000) with association
    constants of 2.3 x 1010 M-1 and 0.59 x 1010 M-1 in human and
    porcine sera respectively.  Saturation of this specific binding
    macromolecule occurs at low levels of OA, 10 to 20 ng per ml serum. 
    Significant serum albumin binding takes place at higher
    concentrations of OA, with saturation taking place above several
    hundred µg of OA per ml serum (Stojkovic  et al., 1984; Hult &
    Fuchs, 1986).

         Binding constants of OA to two identified plasma proteins and
    the fraction of unbound toxin in the sera of different species were
    also determined.  The latter values were 0.02% (man, rat), 0.08%
    (monkey), 0.1% (mouse, pig), and 22% (fish) (Hagelberg  et al.,
    1989).  Tissue residues and half lives of OA in various species

         Once OA has been absorbed, tissue and plasma residues of OA and
    its metabolites depend on a number of factors such as: the length of
    time of feeding, the dose, the use of naturally contaminated grain
    versus crystalline OA, the route, the degree of serum binding, the
    half life of OA, and the length of time on an OA-free diet prior to
    sacrifice.  These factors are of importance in assessing data on the
    natural occurrence of residues in animal tissues (Kuiper-Goodman &
    Scott, 1989).

         With a single oral exposure, maximum serum levels of OA were
    found within 10 to 48 hours in the pig and rat (Mortensen  et al.,
    1983b; Suzuki  et al., 1977; Galtier, 1978; Galtier  et al., 
    1981), at 2 to 4 hours in the ruminant calf (Sreemannarayana  et
     al., 1988), and more rapidly in rabbits and chickens, 1 and 0.33
    hrs, respectively (Galtier  et al., 1981).  Maximum tissue residues
    were also found within 48 hours in the rat.

         Wide species differences in the serum half life of OA have been
    reported.  After oral administration in the monkey  (Macaca mulata),
    510 hr (Hagelberg  et al., 1989), in the pig, 72-120 hours (Galtier
     et al., 1981; Mortensen  et al., 1983a), in the pre-ruminant calf
    77 hours (Sreemannarayana  et al., 1988), in rats 55-120 hours
    (Galtier  et al., 1979; Ballinger  et al., 1986; Hagelberg  et
     al., 1989), in mice 24-39 hours (Fukui  et al., 1981), in quail
    6.7 hours (Hagelberg  et al., 1989) and in chickens 4.1 hour
    (Galtier  et al., 1981).

         In the above species which were so tested, the serum half life
    was longer after intravenous administration of OA (Hagelberg  et
     al., 1989).  Differences in serum half life could be related in
    part to differences in absorption (Galtier  et al., 1981);
    differences in peak plasma values (see above); and species
    differences in degree of binding to serum macromolecules, including

         The disappearance rate of OA from blood was slower than from
    kidney, liver and other tissues in the pig (Hult  et al., 1979).

         Whole body autoradiography using a single i.v. dose of 14C-
    labeled OA in mice (approximately 200 µg/kg bw), showed that OA
    persisted for a long time (> 4 days) in the blood.  This was
    attributed to OA being present mainly in bound form at this low dose
    level (Fuchs  et al., 1988).

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

         Tissue distribution in pigs, rats, chickens and goats generally
    follows the order kidney > liver > muscle > fat (Harwig  et al.,
    1983), or in some recent studies kidney > muscle > liver > fat
    (Mortensen  et al., 1983b; Madsen  et al., 1982).

         Very few data are available on the metabolic disposition of OA
    in humans.  It has been suggested that OA in humans has a long serum
    half life, based on the strong binding of OA to human serum
    macromolecules (Bauer & Gareis, 1987; Hagelberg  et al., 1989).  Excretion

         Both biliary excretion and glomerular filtration play an
    important role in the plasma clearance in OA in rats.  This can be
    related to its molecular weight of 403.8, since for this species
    both pathways are used for substances with molecular weights between
    350 and 450.  Thus in the rat 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).

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

         In rats, the major excretory products were Oalpha (Fig. 1)
    (both in urine and foeces), OA and the 4R-OH-OA epimer, and in the
    urine these represented 25-27%, 6%, and 1-1.5% of the administered
    dose respectively (Storen  et al., 1982b).

         Up to 33% of radioactivity of an orally administered dose of OA
    was excreted into the bile of rats up to 6 hours after dosing; only
    trace amounts of Oalpha were detected in the bile (Suzuki  et al.,

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

         When OA was administered to rats by i.p. injection, only traces
    of OA and Oalpha were identified in faeces, whereas after oral
    administration 12% and 9% of OA and Oalpha were found in faeces
    (Storen  et al., 1982b).

         In pre-ruminant and ruminant calves 85-90% of orally
    administered OA was excreted as Oalpha, most of it in the urine
    (Sreemannarayana  et al., 1988).  Metabolic Disposition during pregnancy  Mouse

         Whole body autoradiography studies by i.v. route, using high
    doses of 14C-labeled OA, showed that OA could cross the placenta
    more readily at days 8 and 9 than at day 10 of gestation, with
    radioactive label appearing within 20 minutes in the uterine wall,
    placental and fetal tissues.  OA given to mice later during
    gestation (day 17) resulted in very low fetal radioactive label
    (Appelgren & Arora, 1983a, 1983b).

         Differences in fetal uptake of OA during different times of
    gestation were suggested to be due to differences in the placenta,
    which was considered to be completely developed by day 9 of
    gestation.  After i.p. injection of OA at days 11 or 13 of
    gestation, fetal residues appeared more slowly, and reached maximum
    values at 30 to 48 hours after dosing.  Residues in the placenta
    were high around 2 to 6 hours after injection and then decreased
    more slowly than from other tissues.  Serum half lives of OA were 29
    and 24 hours at days 11 and 13 of gestation respectively.  The
    authors considered the embryo as a "deep compartment" (Fukui  et
     al., 1987).  Rat

         3H-labeled OA given s.c. to rats at day 12 of gestation also
    showed a delayed fetal uptake of OA, with maximum residues appearing
    at 48 to 72 hours after dosing, and representing approximately 0.1%
    of the administered dose (Ballinger  et al., 1986).  Pig

         OA given at 0.38 mg/kg bw to pregnant sows from day 21 to 28 of
    pregnancy did not cross the placenta (Patterson  et al., 1976).

         Similarly, no residues were found in piglets when low levels of
    OA, 7-16 µg/kg bw, were fed during the whole period of gestation
    (Mortensen  et al., 1983a).

         However, in more recent studies,  in utero transmission of OA
    to 6 piglets was observed in a sow which had been fed naturally
    contaminated feed; blood levels in newborn piglets were 0.075 - 0.12
    ng OA/ml compared to 0.20 ng/ml in the blood of the sow (Barnikol &
    Thalmann, 1988).

    2.1.2  Biotransformation

         OA is hydrolyzed to the non-toxic Oalpha (Fig. 1) at various
    sites.  In rodents detoxification by hydrolysis to Oalpha is a
    function of the bacterial microflora in the rat caecum (Galtier,
    1978).  The enzymes responsible for hydrolysis to Oalpha are
    carboxypeptidase A and chymotrypsin, both in the cow and rodent
    (Pitout, 1969a, 1969b; Pitout & Nel, 1969), with other mycotoxins
    such as penicilloic acid inhibiting this reaction (Parker  et al.,

         Studies with rat tissue homogenates have shown 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), or non-existent in rat hepatocytes (Hansen
     et al., 1982) and rabbit and rat liver (Stormer  et al., 1983;
    Kanisawa  et al., 1979).

         Distribution studies in rats with 14C-labeled OA showed that
    most radioactivity was due to OA, indicating that major efficient
    metabolism of OA is lacking in most tissues other than the intestine
    (Galtier  et al., 1979).

          In vitro incubation studies with the contents from the four
    stomachs of the cow indicated effective hydrolysis of OA to Oalpha
    by the cow's ruminant protozoa; assuming a similar reaction velocity
     in vivo, it was estimated that up to 12 mg OA per kg feed can be
    degraded (Hult  et al., 1976; Pettersson  et al., 1982), so that
    this species is assumed to be relatively resistant to the effects of
    OA in the feed.  Similarly sheep have a good capacity to detoxify OA
    before it reaches the blood (Kiesling  et al., 1984).

         It has been suggested, from studies conducted in mice, that OA
    circulates from the liver into the bile and into the intestine,
    where it is hydrolyzed to Oalpha (Moroi  et al., 1985).

         About 25-27% of OA, given either i.p. or orally to rats, was
    present as Oalpha in the urine.  Its presence in rat urine can be
    explained by reabsorption from the intestine following its formation
    in the intestine (Storen  et al., 1982b) .

         A similar mechanism of intestinal reabsorption of Oalpha has
    recently been suggested for ruminant calves (Sreemannarayana  et
     al., 1988).

         Other minor urinary metabolites of OA are 4-OH-(4R-and
    4S)epimers (Fig. 1) produced in rat and rabbit liver (Stormer  et
     al., 1981) and rat kidney (Stein  et al., 1985) under the
    influence of cytochromes P-450 (Stormer  et al., 1981; 1983).  The
    4R-OH-OA epimer, which is considered less toxic than OA, is the
    major of these two metabolites formed from OA in human and rat liver
    microsomal systems (Stormer  et al., 1981), whereas the 4S-OH-OA
    epimer is more prevalent with pig liver microsomes.  No data are
    available on its toxicity (Moroi  et al., 1985).

         The 10-OH derivative (Fig. 1) was formed from OA with a rabbit
    liver microsomal system (Stormer  et al., 1983).  OC (Fig. 1), a
    metabolite of OA produced in rumen fluid, is equally as toxic as OA
    (cited by Galtier  et al., (1981)).  OB (Fig. 1), a dechloro
    derivative of OA, may co-occur with OA in cereal products.  In the
    rat it is less toxic than OA and is metabolized to 4-OH-OB and Oß
    (Stormer  et al., 1985).

         OB was not found to act as an antagonist to OA, with respect to
    the effects of OA on the formation of phenylalanyl-tRNA and protein
    synthesis (Roth  et al., 1989).

         Many researchers have considered that the toxicity of OA was
    due to one of its metabolites.  From the research findings cited
    above, however, it appears that in the rat OA itself, rather than
    one of the metabolites mentioned above, may be the active toxic
    agent, since the known metabolites are less toxic than or equally
    toxic to OA itself.  This agrees with findings in mice where the
    LD50 of OA increased 1.5- to 2-fold after feeding phenobarbital at
    500 mg/kg diet for one week prior to oral or i.p. administration
    (Moroi  et al., 1985).

         Similarly, pretreatment with sodium phenobarbital (80 mg/kg bw
    by gavage) for 5 days, or 3-methylcholanthrene (20 mg/kg bw by
    gavage) for 2 days resulted in increased LD50 values for OA given
    by gavage.  For phenobarbital the difference was, however, less
    large at 144 hours post dosing with OA, compared to the 48-hr LD50. 
    The administration of piperonyl butoxide, an inhibitor of microsomal
    mono-oxygenases, decreased the 144-hr LD50 of OA from 40 to 18.9
    mg/kg bw (Chakor  et al., 1988).

         On the other hand, preliminary studies with mice showed that
    simultaneous feeding of phenobarbital slightly increased the
    incidence of liver tumours seen after OA alone, and that mice
    developed large and multiple hepatomas (Suzuki  et al., 1986).

    2.1.3  Effects on enzymes and other biochemical parameters

         The biochemistry and molecular aspects of the action of OA in
    both prokaryotes and eukaryotes were recently reviewed
    (Röschenthaler  et al., 1984).  It was noted that not all findings
    are consistent, due to limitations in experimental models and
    procedures as well as interfering factors, especially in more
    complex organisms.  Based on work in prokaryotes (Konrad &
    Röschenthaler, 1977), eukaryotic microorganisms (Creppy  et al.,
    1979b) mammalian cell cultures (Creppy  et al., 1980a; 1983b) and
    on  in vivo animal studies (Creppy  et al., 1980b; 1984), it is
    established that the primary effect of OA is inhibition of protein
    synthesis.  Secondary to this, RNA and DNA synthesis may be

         The inhibition of protein synthesis is specific and occurs at
    the post-transcription level, with OA having a direct effect on the
    translation step in protein synthesis.  This involves a 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 5 times more than transfer to tRNA, the second part.  In
    this reaction OA 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 an acute dose of 0.8 mg OA
    injected i.p. was completely prevented by the simultaneous injection
    of 1 mg phenylalanine (Creppy  et al., 1980b).

         In yeast the rR-OH-OA epimer, a metabolite of OA, had a
    similar effect to that of OA on protein synthesis, but Oalpha,
    lacking the phenylalanine moiety, had no effect (Creppy  et al.,

         Analogues of OA in which phenylalanine has been replaced by
    other amino acids, i.e., tyrosine, have similar inhibitory effect on
    the respective amino acid specific tRNA synthetases (Creppy  et al.,

         The binding affinity of phenylalanine-tRNAPhe synthetase for
    OA is lower than for phenylalanine and ranges from 1/300 in yeast
    (KM = 1.3 mM for OA and 3.3 µM for phenylalanine) (Creppy  et al.,
    1983b), to 1/20 in rat liver (Km = 0.28 mM for OA and 6 µM for
    phenylalanine) (Röschenthaler  et al., 1984).  Despite these
    differences in binding affinity, the inhibition of phenylalanine-
    tRNAPhe by OA is very effective, since OA is more readily
    concentrated by cells than phenylalanine.  In HTC cells the
    concentration of OA inside the cells was 200- to 300-fold that in
    the medium (Creppy  et al., 1983b).

         There was a dose related inhibition of protein synthesis in
    mice given OA i.p. at a dose of 1 mg/kg bw or more.  The degree of
    inhibition of protein synthesis, 5 hours after administration of 1
    mg OA/kg bw, was found to vary within different organs, and for
    liver, kidney, and spleen was 26%, 68% and 75% as compared to
    controls (Creppy  et al., 1984).

         It is possible that OA also acts on other enzymes which use
    phenylalanine as a substrate, but no direct effect of OA on the
    activity of other isolated enzyme systems has been demonstrated
    (Röschenthaler  et al., 1984).

         However, in kidney slices from rats, two days after feeding 2
    mg/kg bw OA, the activity of renal phosphoenolpyruvate
    carboxykinase, a key enzyme in the gluconeogenic pathway, was
    lowered by 50% (Meisner and Krogh, 1986).  It was found that the
    inhibition was indirectly due to a specific degradation of mRNA
    coding for the above enzyme.  This effect was not seen in rat liver
    (Meisner  et al., 1983).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         A comparison of LD50 values in different species and using
    different routes of exposure is shown in Table 1.  These results
    indicate that in acute toxicity studies with OA, the dog and pig
    were the most sensitive species, and rats and mice were the least
    sensitive.  Simultaneous oral administration of 100 mg/kg bw
    phenylalanine 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, the neonate rat was considerably more susceptible than
    the adult rat.

    Table 1:  LD50 values for ochratoxin A in various speciesa
                         LD50 values (mg/kg body weight) 


    Species              Oral            i.p.          i.v.
    Mouse               46-58.3          22-40.1       25.7-33.8

    Rat                 20-30.3          12.6          12.7

    Rat neonate           3.9

    Dog                   0.2

    Pig                    1

    Chicken               3.3

    a    Based on literature compilations in 
         Harwig  et al., (1983) and NIOSH (1986).

         Histopathological and electron microscopic studies were
    conducted in groups of 10 male Long Evans and Sprague-Dawley rats
    administered by gavage a single dose of 0, 17, or 22 mg/kg bw
    benzene free OA in 0.1 M sodium bicarbonate and examined for up to
    48 hours 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.  This was postulated to be
    due to the 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.  Myocardial changes were thought to be related to
    shock and subsequent ischemic injuries (Albassam  et al., 1987).

    2.2.2  Short term studies

         OA has been demonstrated to have a nephrotoxic effect in all
    monogastric mammalian species which have been tested so far (Kuiper-
    Goodman & Scott, 1989).  For their risk assessment of OA, these
    authors summarized the results of about 12 short studies in rats,
    dogs and pigs (see Table 2, adapted from their report).  The most
    relevant of these studies are presented here.  Rat

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

         Similar results were seen when OA was administered to groups of
    4 to 6 adult Sprague-Dawley and Wistar rats by intraperitoneal
    injection for 5 to 7 days at dose levels of 0, 0.75, and 2 mg/kg
    bw/day.  Decreased body weight, increased urine flow, increased
    urinary protein, increased urinary glucose, and impaired urinary
    transport of organic substances were seen at all dose levels. 
    Sprague-Dawley rats were found to be 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).

         Groups of 5 weanling  male and female Fischer F344/N rats were
    administered OA in corn oil by gavage at dose levels of 0, 1, 4 or
    16 mg/kg bw 5 days per week for a total of 12 doses over a 16 day
    period.  All rats that received 16 mg/kg bw OA 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 doses above 1 mg/kg bw.  Bone marrow hypoplasia
    and nephropathy were seen at all dose levels, and involved renal
    tubular degenerative and regenerative changes (NTP, 1989).  Groups
    of 15 weanling rats were administered OA in 0.1 M sodium bicarbonate
    by gavage at dose levels of 0 or 100 µg/rat (equivalent to 1.25

    mg/kg bw/day) for 8 weeks.  Fasting blood samples of OA treated rats
    contained about twice the level of glucose as control rats.  After a
    glucose tolerance test, insulin levels did not reach the level seen
    in control rats.  Total carbohydrates and glycogen in liver tissue
    of treated rats were reduced, in agreement with earlier observations
    (Kuiper-Goodman, personal observations; Suzuki  et al., 1975). 
    Activities of glycolytic enzymes were reduced, whereas gluconeogenic
    enzymes were increased.  The diabetogenic effect of OA was thought
    to be due to inhibited synthesis and/or release of insulin from
    pancreatic cells, thereby suppressing glycolysis, glycogenesis, and
    enhancing gluconeogenesis and glycogenolysis (Subramanian  et al.,

         Semi-purified diets containing 0, 0.2, 1, or 5 mg OA/kg,
    equivalent to 0, 0.015, 0.075, or 0.37 mg OA/kg bw/day, were fed to
    groups of 15 weanling Wistar rats of both sexes for 90 days. At this
    time, 8 animals from each group were sacrificed, and the remaining
    rats were subsequently fed control diet for an additional 90 days. 
    No changes in BUN, urinalysis, or haematological parameters were
    seen at any of the dosage levels.  After 90 days at the two highest
    dietary levels, relative kidney weights were reduced in both sexes,
    but returned to control values after the 90-day recovery period,
    except for males that had received the highest dose level.  Dose
    related changes in morphological parameters were seen after 90 days
    of treatment at doses as low as 0.2 mg/kg in the diet and involved
    karyomegaly and increased eosinophilia in cells of the proximal
    convoluted tubules.  The authors considered the latter change a
    phenomenon of ageing which had been accelerated by OA
    administration.  Desquamation of proximal tubular cells, autolysis,
    changes in rough endoplasmic reticulum (RER), smooth endoplasmic
    reticulum (SER), and tubular basement membrane thickening up to 4 µm
    were noted at the highest dose level after 90 days of treatment.  In
    animals from the highest dose group that were subsequently given
    control diet for 90 days, karyomegaly and tubular basement membrane
    thickening persisted, but otherwise the kidneys appeared normal
    (Munro  et al., 1974).

         Groups of 10 male and female weanling Fischer F344/N rats were
    administered OA in corn oil by gavage at doses of 0, 0.0625, 0.125,
    0.25, 0.50 and 1 mg/kg bw for 5 days per week for 91 days.  Growth
    retardation and a reduced relative kidney weight were seen in males
    at the two highest dose levels.  The NOEL for kidney tubular
    necrosis was 0.0625 mg/kg bw, but karyomegaly, with dose related
    severity, was observed in proximal tubules at all dose levels.  Less
    severe renal changes consisting of tubular atrophy were seen at
    lower doses (NTP, 1989).

        Table 2:  Subacute and Subchronic Toxicity of Ochratoxin A
    Species,            N       Route    mg/kg b.w./day     Time       NOEL                Effects                        Reference
    Strain, sex                          [mg/kg in diet]    (days)     (mg/kg b.w.)

    Rat, Wistar,        10      Diet     0.24-2.4           14         -0.48               Growth retardation             Munro et al., 
    M, [Weanling]                        [2.4-24]                      -0.48               increased serum BUN            1974
                                                                       -0.96               increased kidney wt. 
                                                                       <0.24               Decreased urine vol. 
                                                                       <0.24               Kidney pathology

    Rat, Wistar,        15      Diet     0.015-0.37         90         approx. 0.075       Reduced weight gain.           Munro et al., 
    M.F [Weanling]                       [0.2-5]                       -0.016              Reduced kidney wt. No          1974
                                                                       >0.37               change in BUN, 
                                                                                           Desquamation, increase 
                                                                                           in SER, changes in RER, 
                                                                                           basement membrane 
                                                                                           thickening of PCT 
                                                                                           cells. Increased 
                                                                                           eosinophilia and 
                                                                                           karyomegaly in PCT cells 

    Rat, Wistar, M       5      Gavage    5.15               3         <5                  Reduced p-amine hippuric       Suzuki et al., 
    [Adult]                                                                                acid clearance, basement       1975
                                                                                           membrane thicking

    Rat, Wistar, M      10      Gavage    0.5-2             10          1                  Increased BUN.                 Hatey & Galtier,
    [Adult]                                                            <0.5                Increased urine volume         1977


    Table 2 (contd)
    Species,            N       Route    mg/kg b.w./day     Time       NOEL                Effects                        Reference
    Strain, sex                          [mg/kg in diet]    (days)     (mg/kg b.w.)

    Rat, Sprague-       4-6     i.p.     0.475-2             5-7       <0.75               Decreased body weight,         Berndt & Hayes,
    Dawley and                                                                             Increased urine flow.          1979
    Wistar, M,F                                                                            Decreased urine 
    [Adult]                                                                                osmolality. Increased 
                                                                                           urinary protein. 
                                                                                           Increased urinary 
                                                                                           glucose. Impaired 
                                                                                           urinary transport of 
                                                                                           organic substances, 
                                                                                           (Sprague-Dawley more 
                                                                                           sensitive than Wistar, 
                                                                                           females less sensitive)  

    Rat, Wistar, M      14      Gavage    4                             4-10               Decreased factors              Galtier et al.,
    [Adult]                                                            <4                  II, VI, X. Decreased           1979a
                                                                                           plasma fibrinogen. 
                                                                                           Decreased thrombocyte, 
                                                                                           megakaryocyte counts

    Rat, Wistar, M       9      Gavage    4                 10         <4                  Hypothermia cachexia,          Galtier et al.,
    [Adult]                                                                                tremors, diarrhoea             1980

    Rat, Wistar, M       3      Gavage    0.145             56-84      <0.145              Decrease in kidney             Kane et al., 
    [Adult]                              approx. 2                                         enzymes. Increase in           1986a
                                                                                           urinary enzymes


    Table 2 (contd)
    Species,            N       Route    mg/kg b.w./day     Time       NOEL                Effects                        Reference
    Strain, sex                          [mg/kg in diet]    (days)     (mg/kg b.w.)

    Rat, F344/N, M,F     5      Gavage    1-6               16          1                  Increased relative             NTP (1989)
    [Weanling]                                              (12                            kidney, heart, and brain 
                                                            doses)                         weight.
                                                                        1                  Thymus atrophy.
                                                                        1                  Forestomach necrosis. 
                                                                        1                  Adrenal gland 
                                                                       <1                  Bone marrow hyplasia. 
                                                                       <1                  Kidney nephropathy

    Rat, F344/N M,F     10      Gavage    0.06-1            91          0.125,M            Growth retardation             NTP (1989)
    [Weanling]                                                          0.125,M            Reduced relative kidney 
                                                                        0.062              Kidney tubular necrosis
                                                                       <0.062              Karyomegaly

    Dog, Beagle, M.     3-6     Cap.      0.1-0.2           14         >0.2                No change in kidney            Kitchen et al.,
    [Young]                                                                                function                       1977b

                                                                       <0.1                Kidney tubular                 Kitchen et al..
                                                                                           necrosis                       1977b

                                                                       <0.1                Proximal tubes, EM             Kitchen et al.,
                                                                                           changes                        1977a

                                                                       <0.1                Thymus, lymphoid necrosis      Kitchen et al.,

                                                                                           functions                      1988


    Table 2 (contd)
    Species,            N       Route    mg/kg b.w./day     Time       NOEL                Effects                        Reference
    Strain, sex                          [mg/kg in diet]    (days)     (mg/kg b.w.)

    Pig. F              3-6     Diet     approx. 0.008-     5-90       <0.008              Renal enzyme changes;          Elling, 1979b;
    [8-12 weeks]                         [0.2 0.2, 1.5]                                    changes in renal               Krogh et al., 


    Note:  number of animals per group.

         Groups of 3 to 6 young Beagle dogs were administered OA by
    capsule at dose levels of 0, 0.1 and 0.2 mg/kg bw/day for 14 days. 
    At these dose levels no changes were observed in kidney function,
    but kidney tubular necrosis and ultrastructural changes in proximal
    tubules were observed at all dose levels.  Necrosis of lymphoid
    tissues of the thymus and tonsils was also seen at all dose levels
    (Kitchen  et al., 1977a,b,c).  Pig

         In a series of experiments, groups of 3 to 6 female pigs were
    administered OA at levels of 0, 0.2, 1, and 5 mg/kg feed, equivalent
    to approximately 0, 0.008, 0.04 and 0.2 mg/kg bw/day, for periods of
    5 days, 8 and 12 weeks, and up to 2 years.  A decrease in kidney
    function (see 2.2.6), nephropathy and reduction in kidney enzymes
    were reported.  Progressive nephropathy but no renal failure was
    seen in female pigs given feed containing 1 mg OA/kg feed for 2
    years.  No 2-year toxicity studies in male pigs have been reported. 
    (Krogh & Elling, 1977; Elling, 1979a, 1979b, 1983; Elling  et al.,
    1985; Krogh  et al., 1988).

    2.2.3  Long-term/carcinogenicity studies  Mouse

         Diets containing 0 or 40 mg/kg OA, equivalent to an intake of
    approximately 5.6 mg/kg bw/day, were fed to groups of 10 ddY male
    adult mice for 44 weeks, followed by 5 weeks of basal diet.  Of the
    9 surviving OA-fed mice, 5 had hepatic cell tumours, 9 had renal
    cystic adenomas, and 2 had solid renal cell tumours (terminology as
    used by the authors).  No liver or renal tumours were observed in
    control mice, and no data on the incidence of these tumours in
    historical controls of this strain of mice were presented.  It was
    not clearly indicated whether liver tumours were benign or malignant
    (Kanisawa & Suzuki, 1978).

         A second study from the same laboratory confirmed the results
    of the above study.  Diets containing 0 or 25 mg/kg, equivalent to
    an intake of approximately 3.5 mg/kg bw/day were fed to groups of 20
    6-week old male DDD mice for 70 weeks.  All of the 20 surviving OA-
    treated mice had renal cystic adenomas, 6 had solid renal tumours,
    and 8 had hepatic cell tumours.  One of the 17 control mice had a
    hepatic cell tumour (Kanisawa, 1984).

         A third study from the same laboratory was not a lifetime
    exposure study.  Diets containing 0 or 50 mg/kg OA, equivalent to an
    intake of approximately 7 mg/kg bw/day, were fed to groups of 16
    adult male ddY mice for periods of 5 to 30 weeks, followed by a

    control diet for the remainder of the study (total length of study
    was 70 weeks).  The length of time on control diet ranged from 65
    down to 40 weeks.  No renal or liver tumours were observed in
    control mice or in mice fed OA for 10 weeks or less.  The incidences
    of renal cell tumours were 3/15, 1/14, 2/15 and 4/17 after 15, 20,
    25, and 30 weeks on an OA diet, respectively.  The incidence of
    renal cystic adenomas was not indicated.  A significant increase in
    liver tumours was observed after mice had been fed OA for 25 weeks
    (5/15) and 30 weeks (6/17).  These results indicated that the renal
    and liver tumours persisted through long term subsequent feeding of
    control diet (Kanisawa, 1984).

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

         Diets containing 0, 1, or 40 mg/kg OA were fed to groups of 50
    weanling B6C3F1 mice of each sex for 24 months.  The test compound
    contained about 84% OA, 7% OB, and 9% benzene.  Inspections of dead
    or moribund mice were made daily.  The mice were examined, weighed,
    and food consumption recorded weekly for the first 4 weeks, then
    monthly.  At the 40 mg/kg dietary level, body weights were decreased
    by 25 and 33% in female and male mice respectively, indicating that
    the Maximum Tolerated Dose (MTD) 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 40
    mg/kg OA, and was more severe in males than in females.  There was
    no nephropathy in males or females given a control diet, or diets
    containing 1 mg/kg OA.  Benign and malignant renal tumours were seen
    only in male mice fed diets containing 40 mg/kg OA, and their
    incidence was 53% and 28.6% respectively (combined incidence 63%). 
    No metastases attributable to renal tumours were found.

         When compared to concurrent controls, the combined incidence of
    hepatocellular adenomas and carcinomas was statistically significant
    in both male and female mice administered 40 mg OA per kg diet;
    however, for males the 20% incidence was within the historical
    control range of 0-21.6% for this strain of mice (Ward  et al.,
    1979); for females the 14% incidence was greater than the incidence
    of 0-3.9% for historical controls (Ward  et al., 1979).  The
    authors noted that the OA 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
    survival.  In fact, survival of males in the control and 1 mg/kg

    dietary groups at 18 months was only 75% and 65%, respectively,
    compared to 98% in the 40 mg/kg dietary group, due to a high
    incidence of fatal obstructive urinary tract disease (uropathy) in
    the 0 and 1 mg/kg dietary dose groups, with an onset as early as 4
    months (Bendele  et al., 1985a).

         The protective effect of the 40 mg/kg dietary level of OA may
    have been due to a growth inhibitive effect on Gram positive
    bacteria, and to the OA induced polyuria, as a result of renal
    proximal tubular damage (Bendel & Carlton, 1980).  Group caging and
    fighting-related lesions of the prepuce/penis may have contributed
    to the chronic uropathy (Rao, 1987).  Rat

         Groups of 80 male and female Fischer F344/N rats were
    administered OA by gavage in corn oil at 0, 21, 70, or 210 µg/kg
    bw/day, 5 days per 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 sacrificed after 9 and 15 months.  At the
    highest dose level, body weight was decreased from 4-7% between 18-
    77 weeks for male rats, and between 6-89 weeks for female rats.  No
    compound related clinical signs were noted, and the results of
    haematological and serum chemical analysis showed no effects of
    biological significance.  Urinalysis indicated a mild to moderate
    change in the ability to concentrate urine, with no other changes in
    kidney function (see 2.2.6).

         The incidences of renal adenomas and renal carcinomas in males
    administered 0, 21, 70, and 210 µg OA were 1/50, 1/51, 6/51 and
    10/50 and 0/50, 0/51, 16/51 and 30/50 respectively.  The combined
    incidences of renal tubular cell adenomas and carcinomas were 36/50
    and 20/51 at 210 µg and 70 µg respectively.  At the highest dose
    level many renal adenomas and carcinomas were multiple or bilateral. 
    There was a dose related increase in the number of males that were
    dead or moribund (7, 19, 23, and 26, respectively, in the 0, 21, 70,
    and 210 µg/kg bw dose groups) before the time of terminal sacrifice. 
    In the two highest dosage groups, decrease in survival was
    attributed by the authors to the presence of kidney tumours since 15
    out of 23, and 18 out of 26 rats which died had kidney tumours.  As
    well, a larger proportion of animals that died prior to the terminal
    sacrifice had carcinomas that had become metastatic (3/8 and 11/15
    at the mid- and high dose respectively) compared to animals killed
    at terminal sacrifice (0/7 and 3/15 at the mid- and high dose
    respectively).  However in male rats given the low dose of OA, only
    one kidney tumour was present, although the decrease in survival was
    similar to that of the two higher doses.  Lower survival in 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 for the 0, 21, 70, and 210
    µg OA groups, respectively.

         The significance of the OA-induced rat renal carcinoma is
    increased by the presence of a high frequency of metastases,
    attributed to renal cell carcinomas, mainly in the lungs and lymph

         In high dose female rats there was also an increased incidence
    in multiplicity of fibroadenomas in the mammary gland (14/50
    compared to 4-5/50 in controls and lower doses). 

         Non-neoplastic lesions involved mainly the kidney.  Chronic
    diffuse nephropathy, common to old rats, was seen with about the
    same incidence in all groups of males and females, but the extent
    and grade were not reported.

         At the two highest dose levels karyomegaly or karyocytomegaly
    (large kidney epithelial cells with giant polyploid nuclei and
    prominent nucleoli) was seen in all males and females, and it was
    the most consistent finding at the two highest dose levels in the
    interim 9- and 15-month sacrifices, as well as in the 13-week NTP
    preliminary study (NTP, 1989).

    2.2.4  Reproduction studies

         No adequate reproduction studies with OA have been reported to

    2.2.5  Special studies on embryotoxicity/teratogenicity  Mouse

         Groups of 4 to 26 pregnant CBA mice were administered a single
    dose of OA in corn oil by gavage at dose levels of 0, 1, 2, or 4
    mg/kg bw on days 8 or 9 of gestation (vaginal plug day = post
    conception day 1), or of 4 mg/kg bw on days -2 (2 days prior to
    mating), 2, 4, 6, 7, 10, and 14 of gestation and observed until day
    19.  At this time the number 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
    whether maternal toxicity was present.  Prenatal survival was
    decreased for groups that had received 4 mg/kg bw on days 7 (24%
    deaths), 8 (17.3% deaths), and 9 (22.2% deaths) of gestation.  Overt
    craniofacial anomalies were produced only by exposure on days 8 or
    9, and their incidence, multiplicity, and severity increased with
    increasing dosage, the peak effect being on day 9.  The incidences
    of malformed pups among surviving pups were 0%, 0%, 8.1%, and 16.4%
    for mice administered 0, 1, 2, or 4 mg/kg bw on day 8 of gestation,

    and 0%, 29.3%, 41.8%, and 91.1% for mice administered these same
    dosages on day 9 of gestation.  The mean number of malformations per
    fetus was approximately 0.3 and 2.3 on days 8 and 9 of gestation in
    the 4 mg/kg dose group, and 1.7 and 3.9 respectively when
    administered 8 mg/kg bw (separate study).  The central nervous
    system, the eye and the axial skeleton were mainly 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, median cleft face.  On day 9 of
    gestation at the 4 mg/kg dose level, the incidences for the various
    major anomalies were exencephaly (89.3%), anophthalmia (44.6%),
    microphthalmia (26.8%), open eye lids (16.1%), agenesis of external
    nares (21.4%), cleft lip (7.1%), median cleft face (8.9%), and
    malformed jaws/short maxilla with protruding tongue (41.1%).  The
    craniofacial anomalies were thought to arise from a 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 OA were studied in groups of 10 to 13 CD-1 mice, maintained on
    diets providing 26% (control), 16%, 8%, and 4% purified protein
    (casein), following mating and throughout gestation.  A single dose
    of OA in 0.1 N sodium bicarbonate was administered by gavage at dose
    levels of 0, 2, or 3 mg/kg bw on day 8 of gestation (vaginal
    plug=day 1), and the mice were sacrificed at day 18 of gestation for
    teratologic examination.  Dams were monitored twice daily and food
    consumption was monitored.  Protein diets and water were available
     ad libitum.

         OA treatment did not affect maternal food consumption, but in
    some of the 3 mg OA groups (26% and 4% protein) maternal deaths were
    significantly more frequent (5 and 4 respectively versus 0 in the
    two OA free groups).  There were also 9 maternal deaths in the 4%
    protein group given 2 mg OA/kg.  The percentage of litters with
    grossly malformed fetuses and the percentage of malformed fetuses
    (in brackets) for each of the 4 protein diets (26, 16, 8, and 4%,
    respectively were 58 (25), 50 (17), 75 (45), and 100 (81.3) at 3 mg
    OA/kg bw, 25 (5), 50 (21), 30 (12.6), and 100 (77.7) at 2 mg OA/kg
    bw, and 0 (0), 0 (0), 18 (3), and 31 (9.8) at 0 mg OA/kg bw.  Fetal
    weights were reduced as a result of OA and protein deprivation. 
    Cranofacial malformations were the most common, but at lower protein
    levels gross malformations affecting limbs and tail were also seen
    (Singh & Hood, 1985).  Rat

         Five groups of 12 to 20 pregnant Wistar rats were administered
    a total of 5 mg/kg bw OA in 0.16 M sodium bicarbonate by gavage as

    follows: at each of days 8 and 9 of gestation (vaginal plug=day 1)
    single doses of 2.5 mg/kg bw, on each of days 8 to 11 of gestation
    doses of 1.25 mg/kg bw, on each of days 8 to 13 of gestation doses
    of 0.83 mg/kg bw, and on each of days 8 to 15 of gestation doses of
    0.63 mg/kg bw, or vehicle control.  In a similar way, three groups
    of 20 rats were administered single doses of 2.5 mg OA/kg bw on each
    of days 8 and 9 of gestation, or on each of days 8 to 10 of
    gestation doses of 1.67 mg OA/kg bw, or vehicle control.  Rats were
    sacrificed on day 20 of gestation.  There were no significant
    differences in the number of implantations per female for the
    various groups.  Females that had received the same total amount of
    OA, divided into fewer single doses, and early in gestation were
    most affected.  There was a single-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 single-dose related incidence of fetal haemorrhages (seen at
    the 2 times 2.5 and 4 times 1.25 mg/kg dose levels) and celosome
    with or without oedema were considered teratogenic responses (Moré &
    Galtier, 1974).

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

         Levels as low as 0.5 mg OA/kg b.w. given by gavage to rats on
    days 11 to 14 of gestation caused learning deficits in pups which
    were tested over a 26-week period (Kihara  et al., 1984).

         Other studies on the teratogenicity in mice and rats given OA
    by i.p. or s.c. route were reviewed by Kuiper-Goodman & Scott

    2.2.6  Special studies on nephrotoxicity

         As seen in the short-term studies, kidney function and
    morphology are greatly affected at higher dose levels of OA as
    indicated by increases in kidney weight, urine volume, blood urea
    nitrogen (BUN) (Hatey & Galtier, 1977), urinary glucose and
    proteinuria (Berndt and Hayes, 1979). The latter two findings
    indicate that the site of reabsorption, i.e. the proximal convoluted

    tubules, is damaged.  NOELs for changes in renal function depend on
    the species and on the parameter tested.

         At lower dose levels of OA, no increases in BUN, creatinine or
    glucose were found in the urine of male and female rats given 210
    µg/kg b.w./day by gavage for 6-12 months, but a mild to moderate
    decreased ability to concentrate urine was seen.  The NOEL for this
    effect was 70 µg/kg b.w. for male rats and 21 µg/kg b.w. for female
    rats (NTP, 1989).

         Different groups of investigators have shown that this specific
    toxic effect is due to an OA induced defect on the organic anion
    transport mechanism located on the brush border of the proximal
    convoluted tubular cells and basolateral membranes (Endou  et al.,
    1986; Sokol  et al., 1988).

         The organic ion transport system is also the mechanism by which
    OA 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 OA (0.05 mM), 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 OA on the
    release of enzymes from the kidney into the urine.  Changes in
    enzyme and protein pattern can be used to distinguish different
    types of renal injury (Stonard  et al., 1987).

         Subcutaneous doses of OA, at a dose level of 10 mg/kg bw for 5
    days, decreased first the level of muramidase, followed by decreases
    in the levels of lactate dehydrogenase, alkaline phosphatase,
    glutamate dehydrogenase, and acid phosphatase in the kidney (Ngaha,

         The levels 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 mM OA
    (Endou  et al., 1986).

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

         More recently, it was shown that in rats given OA by gavage at
    a dose level of 0.145 mg/kg bw every 48 h (equivalent to about 2
    mg/kg diet) for 8 to 12 weeks, the level of lactate dehydrogenase,
    alkaline phosphatase, leucine amino peptidase, and gamma-glutamyl
    transferase decreased significantly.  The latter three enzymes are
    located in the brush border of the proximal convoluted tubules,
    indicating damage at that site.  Concomitant with the decrease of
    enzyme activity in the kidney was the appearance of these enzymes in
    the urine.  A late event was the urinary increase in N-acetyl ß-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 the exfoliation
    of necrotic proximal convoluted tubular cells releasing lysosomal
    enzymes (Stonard  et al., 1987).

         In the above 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 OA on renal enzyme
    activity.  In kidneys of pigs fed 0.2 to 1 mg/kg OA in the diet
    (equivalent to about 0.008 to 0.041 mg/kg bw/day), a dose related
    decrease in the activity of PEPCK and gamma-glutamyl transpeptidase
    was accompanied by a dose related decrease of renal function, as
    indicated by a reduction of maximal tubular excretion of para-
    aminohippurate per clearance of inulin and an increase in glucose
    excretion.  Only cytosolic PEPCK activity was inhibited, with
    mitochondrial PEPCK activity not affected by OA (Meisner & Krogh,
    1986; Krogh  et al., 1988).

    2.2.7  Special studies on genotoxicity

         The genotoxicity of OA was recently reviewed (Bendele  et al.,
    1985b; Kuiper-Goodman & Scott 1989).  The following is taken from
    the latter review.  OA has been shown to be non-mutagenic in various 
    microbial and mammalian gene mutation assays, both with and without
    exogenous metabolic activation.  A single positive result in a
    bacterial assay was attributed to the presence of 15% OB in the OA
    (Kuczuk  et al., 1978) (Table 3).

         While evidence for DNA damage/repair in microbial systems has
    been negative, a weakly positive response was found for induction of
    unscheduled DNA synthesis (UDS) in ACI strain rat and C3H strain
    mouse primary hepatocytes, each treated at 2 dose levels for 20
    hours with OA (purity not stated) (Mori  et al., 1984) (Table 3). 
    The positive results were reported at approximately 0.4 and 4.0
    µg/ml, respectively; OA was cytotoxic at 4.0 and 40.0 µg/ml,

         On the other hand, Bendele  et al., (1985b) tested 2 lots of
    highly purified OA over a 7 1/2 log concentration range, and used 15
    dose levels (treatment duration not stated) for induction of UDS in
    Fischer 344 primary rat hepatocytes.  They found that OA was
    cytotoxic at ±0.05 µg/ml concentration, and that OA did not induce
    UDS at dose levels up to cytotoxic doses.

         OA has caused a small but significant dose-related increase in
    sister chromatid exchange (SCE) in CHO cells in the presence, but
    not in the absence, of rat liver S9 activation (NTP, 1989).

         Negative effects on SCE frequency were found in HPBL cells
    (Cooray, 1984) and in an  in vivo assay (Bendele  et al., 1985b).

         OA did not induce chromosome aberrations in CHO cells (NTP,
    1989) (Table 3).

         OA has caused DNA damage (single strand breaks)  in vitro in
    CHO cells, rat fibroblasts (Stetina & Votava, 1986) and in mouse
    spleen cells (Creppy  et al., 1985).

         DNA strand breaks were induced by OA treatment  in vivo in
    mouse spleen, kidney and liver cells, and rat kidney and liver cells
    after a single i.p. injection, at fairly high dose levels (Creppy
     et al., 1985), or after gavage treatment for 12 weeks at levels
    equivalent to low (4 mg/kg) dietary concentrations (Kane  et al.,
    1986b) (Table 3).

    2.2.8  Special studies on immune response

         Several studies have shown that OA affects structural
    components of the immune system in several species.  In chickens fed
    2-4 mg OA/kg in the diet for 20 days, OA was found to decrease the
    lymphoid cell population of immune organs (Dwivedi & Burns, 1984a).

         The size of the mouse thymus was reduced to 33% of controls
    after four i.p. injections of 20 mg OA/kg bw on alternate days, a
    dose which caused minimal nephrotoxicity.  There was also bone
    marrow depression, as shown by a dose-related and significantly
    (p<0.01) decreased marrow cellularity, including a reduction of
    bone marrow macrophage-granulocyte progenitors, a decrease in the
    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, 3 weeks post exposure, was demonstrated by an
    increased sensitivity to irradiation, even though bone marrow
    cellularity and the peripheral blood count had returned to normal
    (Hong  et al., 1988; NTP, 1989).

         Bone marrow hypocellularity and a reduction in thymic size were
    also seen in Fischer rats given 1 or 4 mg OA/kg bw/day by gavage for
    16 days (NTP, 1989).

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

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

         Several studies have shown that OA affects both humoral and
    cell-mediated immunity.  In chickens fed OA at 5 mg/kg diet for 56
    days, the content of alpha1 alpha2, beta and gamma globulin in
    blood plasma was reduced (Rupic  et al., 1978).

         In chickens, fed 2-4 mg OA per kg diet for 20 days, there was a
    depression of IgG, IgA and IgM in lymphoid tissues and serum
    (Dwivedi & Burns, 1984b), and complement activity was slightly
    affected when fed at 2 mg OA per kg diet for 5-6 weeks (Campbell  et
     al., 1983).

         OA also reduced IgG and increased IgM in the bursa of Fabricius
    in chick embryos that had been injected with 2.5 µg OA/embryo on day
    13.  This did not however affect immunocompetence, 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., 1987b).

         OA administered to 8-10 week old Swiss mice at 5 mg/kg bw/day
    by i.p. injection for 50 days, reduced the antibody response to
     Brucella abortus, a cell mediated immune response, and this was
    postulated to be due to a suppression of IgM synthesis (Prior &
    Sisodia, 1982).

         The same treatment also reduced mitogen (con A)-induced blast
    formation in mouse spleen derived lymphocytes (Prior & Sisodia,

        Table 3: Results of Genotoxicity Tests with Ochratoxin A
    Endpoint       Organism/         Details      Activation   Value           Units         Result   Comment          Reference
                   cell type


    Gene           S. typhimurium    TA98,        +/-          0.4-400         µg/plate      -/-      Highly           Wehner et al., 1978
    mutation                         100,                                                             variable         Kuzuk et al., 1978
                                     1535,                                                            TA 100           
                                     1537,                                                            controls,        
                                     1538                                                             not tested to 

    Gene           S. typhimurium    TA100,       +/-          approx. 198     µg/plate      -/-      Mouse            Bartsch et al., 1980
    mutation                         1538                                                             liver and        
                                                                                                      rat liver        

    Gene           S. typhimurium    TA98,        +/-          50-600          µg/plate      -/-      Tested to        Bendele et al., 
    mutation                         100,                                                             cytotoxicity/    1985b)
                                     1535,                                                            solubility       

    Gene           S. typhimurium    TA98,        +/-          0.1-100         µg/ml         -/-      Non              Bendele et al., 1985b
    mutation                         100,                      (in log-                               quantitative     
                                     1535,                     arithmic                               assay            
                                     1537,                     gradients)

    Table 3 (contd)
    Endpoint       Organism/         Details      Activation   Value           Units         Result   Comment          Reference
                   cell type

    Gene           S. typhimurium    TA1538,      +            0.1-500         µg/plate      +        Positive >       Kuczuk et al., 1978
    mutation                                                   (OA:OB =                               100 µg/plate     

    Gene           S. typhimurium    TA97,        +/-          1-100           µg/plate      -/-      Hamster or       NTP, 1989
    mutation                         98,                                                              rat liver        
                                     100,                                                             activation       

    DNA damage/repair

    DNA repair     E. coli           SOS          +/-          1-2             mg/100 µl     -/-      Qualitative      Reiss (1986);
                                                                                                      colorimetric;    Auffray & Bouti-
                                                                                                      spot test        bonnes (1986)

    DNA repair     E. coli           WP2          +/-          Gradient        Not           -/-      Qualitative      Bendele et al., 
                                                               plate           stated                 assay            (1985b)

    DNA damage     B. subtilis       rec                       20-100          µg/disk       -        Inhibition       Ueno & Kubota 
                                                                                                      zone             (1976)

    Eukaryotes                                                                               -
    Gene mutation  S. cerevisiae     D3           -            200             µg/plate                                Kuczuk et al., 1978
                                                  +             75             µg/plate

    Mammalian  In vitro

    Gene mutation  Mouse lymphoma    TK           +/-          0.1-12.5        µg/ml         -/-      >12.5 µg/ml,     Bendele et al., 1985b

    Table 3 (contd)
    Endpoint       Organism/         Details      Activation   Value           Units         Result   Comment          Reference
                   cell type

    Gene mutation  C3H mouse         8-AG                      5-10            µg/ml         -/-      10 µg/ml,        Umeda et al., 1977b
                   mammary                                                                            cytotoxic        

    DNA damage/repair

    UDS, repair    Rat primary       Fisher 344   -            0.000025-       µg/ml         -        >0.05 µg/ml      Bendele et al., 1985b
                   hepatocytes       strain                    500; 2 lots                            cytotoxic        
                                                               tested, 15 

    UDS, repair    Rat primary       ACI strain   -            0.4, 4.0        µg/ml         +        At approx.       Mori et al., 1984
                   hepatocytes                                                               (weak)   0.4 µg/ml;       
                                                                                                      cytotoxic; at    
                                                                                                      approx. 4        

    UDS, repair    Mouse primary     C3H strain   -            4.0, 40.0       µg/ml         +        At 4.0 µg/ml;    Mori et al., 1984
                   hepatocytes                                                               (weak)   cytotoxic at     
                                                                                                      40 µg/ml         

    SCE            Human, HPBL                    +/-          5-10            µg/ml         -        Mitotic          Cooray (1984)
                                                                                                      inhibition at    
                                                                                                      10 µg/ml         

    SCE            CHO cells         26 h with    -            0.5-5           µg/ml         -                         NTP (1989)

    Table 3 (contd)
    Endpoint       Organism/         Details      Activation   Value           Units         Result   Comment          Reference
                   cell type

    SCE            CHO cells         2 h with OA  +            5-160           µg/ml         +        SCE frequency    NTP (1989)
                                                                                             (weak,   up to 37%        
                                                                                             dose     above control    

    Chromosome     CHO cells         8-10 h with  -            30-160          µg/ml         -                         NTP (1989)
    aberration                       OA

                                     2 h with OA  +            100-300         µg/ml         -                         NTP (1989)

    DNA strand     CHO cells; rat    Alkaline                  200             µg/ml         +        1.2 breaks/109   Stetina & Votava
    break          fibroblasts       elution                                                          Da               (1986)

    DNA damage     Mouse spleen      48 h                      10              µg/ml         +        replicated 6     Creppy et al., 1985
                   PHA stimulated    treatment                                               (dose    times in pairs
                                     at 1-10                                                 related)

     In vivo

    SCE            Chinese                                     Gavage,         mg/kg         -        >100 mg/kg       Bendele et al., 1985b
                   hamster, bone                               25-400          body wt.               body wt.         
                   marrow                                                                             cytotoxic        

    DNA damage     Balb/c mouse

    single strand  Spleen            4, 16,                    i.p. 2500       µg/kg         +        Max. response    Creppy et al., 1985
    breaks                           24 h after                                body wt                at 24 h

    Table 3 (contd)
    Endpoint       Organism/         Details      Activation   Value           Units         Result   Comment          Reference
                   cell type

                   Kidney            24, 48 h                  i.p. 2500       µg/kg         +        Max. response    Creppy et al., 1985
                                     after                                     body wt                at 24 h          

                   Liver             24, 48,                   i.p. 2500       µg/kg         +        Max. response    Creppy et al., 1985
                                     72 h after                                body wt                at 48 h;         
                                                                                                      Recovery at      
                                                                                                      72 h             

    DNA damage-    Wistar rat        6-12 weeks                Gavage 144      µg/kg         both+    No recovery      Kane et al., 1986b
    single strand  kidney, liver                               (289 µg/kg      body wt                seen between     
    breaks                                                     body wt                                treatments       
                                                               every 48 h 
                                                               equiv. to 
                                                               4 ppm in 

    Chromosome     human             48hr         +/-          4.5             µg/ml         +/+      4.5-5 fold       Manolova et al., 1990
    aberration     lymphocytes                                                                        increase in

    a    Aberrations on x chromosomes of similar types to those previously detected in lymphocytes from patients suffering from 
         endemic nephropathy.

         In the above-mentioned mouse studies, immune response to sheep
    red blood cells (SRBC), measured as the number of antibody forming
    cells in the spleen using the indirect plaque assay, was not
    affected.  When OA was administered at  4 mg/kg diet (equivalent to
    about 0.5 mg/kg bw), a dose which is about 10-fold lower and close
    to that which can be found to occur naturally, none of these
    responses were affected (Prior & Sisodia, 1982).

         In contrast to these studies, very low levels of OA (1 µg
    kg/bw) given once by i.p. route to BALB mice, 8-12 weeks of age, had
    an immuno-suppressive effect on both IgM and IgG response to a
    single injection of SRBC in the standard plaque counting assay for
    the estimation of antibody producing spleen lymphocytes (Creppy  et
     al., 1982).

         No explanation, other than differences in route of exposure, is
    available for the differences in response to SRBC between these
    studies and those of Prior and Sisodia.

         A reduction in blast cell formation was also seen in human
    peripheral lymphocytes treated with 5-20 µg OA/ml (Cooray, 1984).

         At even lower concentrations of OA, similar and dose related
    inhibition of con-A-induced blastogenesis of porcine blood
    lymphocytes was observed, with concentrations of >1 µg, 0.5 µg, and
    0.06 µg OA/ml causing almost complete, 50% and 10% inhibition,
    respectively (Holmberg  et al., 1988).

         PHA-induced proliferation of highly purified human t-
    lymphocytes was inhibited by 12.5 to 50 µM concentrations of OA
    (equivalent to 5-20 µg OA/ml).  This was attributed to a low
    interleukin-2 receptor expression and/or production.  OA also
    impaired the ability of purified human B-lymphocytes to proliferate
    in response to anti-µ antibodies in the presence of BCGF-1 (Lea  et
     al., 1989).  The immunosupressive effects of OA could be prevented
    by i.p. administration of phenylalanine at 10 µg/kg b.w. (Haubeck
     et al., 1981; Creppy  et al., 1982).  Thus the immunosuppressive
    action of OA could be due to its action on protein synthesis,
    although the dose employed was very low.  Immunocompetent cells
    require activation, differentiation and proliferation, and all these
    steps could be affected if protein synthesis in lymphocytes is

         The OA metabolite, 4R-OH-OA, was found to be almost as
    effective as OA, and Oalpha was found to be ineffective (Creppy  et
     al., 1983c).

         Protein synthesis inhibition occurred in lymphocytes in culture
    at 0.5 mg OA/ml after 2 hrs, and in hepatoma cells at 10-15 mg/ml,
    compared to an  in vivo immunosuppressive dose in the above studies
    of 1 µg/kg bw (Creppy  et al., 1982).

         Female B6C3F1 mice, 6-8 weeks of age, administered OA at 3.4,
    6.7, and 13.4 mg/kg bw by gavage or i.p. injection (6 doses over 12
    days) had decreased natural killer (NK) cell activity.  OA also
    caused an increase in the growth of transplantable tumor cells
    without altering T-cell- or macrophage-mediated antitumor activity. 
    Suppression of NK activity appears to be due to a decreased
    production of basal interferon; OB was much less toxic in this
    system (Luster  et al., 1987).

    2.3  Observations in humans

         Chronic human nephropathy, endemic in the Balkan area, has been
    associated with OA exposure, as indicated by the presence of OA
    residues in local foodstuffs as well as in the blood of patients
    with nephropathy.  In the last 10 years, direct evidence of human
    exposure to OA has been obtained in six countries (Table 4). 
    Published surveys have shown up to 40 ng/ml OA in human blood serum. 
    In a preliminary study on lymphocytes from healthy women, OA
    treatment at 6 ng/ml resulted in an increased frequency of numerical
    chromosome aberrations, mainly affecting the X chromosome (Manolova
     et al., 1990)

         In Bulgaria, a significant proportion of serum samples from
    patients with endemic nephropathy and/or urinary system tumours
    contained more than 2 ng OA/ml serum compared to samples from people
    in a non-endemic area (Petkova-Bocharova  et al., 1988).

         A survey conducted in 1980 in Yugoslavia showed a higher upper
    range of OA in serum in a hyper-endemic village than in a non-
    endemic village, although the incidence of positive samples (> 1
    ng/ml serum) was about the same in both villages, 6% and 7.8%
    respectively.  In 1979, the incidence of positive OA samples in the
    same hyper-endemic village was 16.6%, whereas the non-endemic
    village had an incidence of 7.8% positive samples (Hult  et al.,

         Since that time, one exceptionally high level of 1800 ng/ml
    serum has also been found.  Thus it is apparent that there are
    fluctuations in human serum OA levels, that probably reflect local,
    seasonal or yearly fluctuations in the level of OA in food.  Further
    long term studies are underway to investigate serum levels of OA in
    Yugoslavia (Hult & Fuchs, 1986).

         The mean level of OA in Polish human sera was estimated as 0.27
    ng/ml, and the average daily human exposure from food was estimated
    to be 0.448 ng/kg (Golinski & Grabarkiewicz-Szcaesna, 1989).

        Table 4  Occurrence of Ochratoxin in Humans
    Sample                   Location            Incidence    Ochratoxin A     Reference
                                                              ng/g or ng/ml    

    Blood serum              Bulgaria            26%          1-35             Petkova-Bocharova 
    (from patients with                                                        et al., 1988
    urinary system 
    tumours and/or 
    endemic nephropathy)

    Blood serum              Bulgaria            7.7%         1-2              Petkova-Bocharova 
    (from non-endemic                                                          et al., 1988

    Blood serum              Yugoslavia          25/420       1-40             Hult et al., 
    (from village with                                                         1982a,b
    endemic nephropathy)

    Blood serum              Yugoslavia          17/219       1-10             Hult et al., 
    (from non-endemic                                                          1982a,b

    Blood serum              Poland              9/216        1.3-4.8          Goliśski & 
                                                                               Grabarkiewicz -
                                                                               Szczesna, 1985 

    Blood serum              Germany (FRG)       173/306      0.1-14.4         Bauer & Gareis, 
    (1977, 1985)                                                               1987

    Kidneys                  Germany (FRG)       3/46         0.1-0.3          Bauer & Gareis, 
    (1982, 1983)                                                               1987


    Table 4 (contd)
    Sample                   Location            Incidence    Ochratoxin A     Reference
                                                              ng/g or ng/ml    

    Milk (1986)              Germany (FRG)       4/36         0.017-0.03       Bauer & Gareis, 
                                                                               1987; Gareis et 
                                                                               al., 1988

    Blood serum              Denmark             ?            <0.1-9.7         Hald, 1989
    (obtained from                                            mean 1.5-2.3     
    blood bank 
    (1986, 1987)

    Blood serum              Czechoslovakia      35/143       <0.1-1.26        Fukal & Reisnerova,


    ? - incidence not given

         A high incidence of OA in human blood serum, as well as kidneys
    and milk, reported in the Federal Republic of Germany reflects the
    use of a very sensitive analytical method (sensitivity = 0.1 ng/ml
    serum) (Bauer & Gareis, 1987; Gareis  et al., 1988).  It also
    reflects continuous and widespread exposure of humans to OA.

         Mean serum levels in 96 randomly collected Danish human blood
    bank samples collected during 1986 and 1987 were 1.5 to 2.3 ng/ml,
    and ranged from < 0.1 (detection limit) to 9.7 ng/ml (incidence not
    reported) (Hald, 1989).

         The maximum level of OA detected in human sera obtained from
    two hospitals in Czechoslovakia was 1.26 ng/ml (Fukal & Reisnerova,

         About one third of the patients dying from Balkan endemic
    nephropathy (BEN) have been reported to have papillomas and/or
    carcinomas of the renal pelvis, ureter or bladder.  In one endemic
    area in Bulgaria the relative risk of patients with BEN developing
    urinary tract tumours is 90-fold higher than in the population from
    non-endemic areas (Castegnaro & Chernozemsky, 1987).

         Besides a possible association with OA, genetic factors may
    also be involved in this disease.  More analytical epidemiology
    studies, and studies on oncogene activation in urothelial neoplasms
    are required (Castegnaro & Chernozemsky, 1987; Radovanovic, 1989).


         The Committee reviwed studies on the metabolic disposition and
    toxicology of OA, as well as limited information on the association
    of OA exposure and chronic human nephropathy, endemic in Yugoslavia,
    Bulgaria and Rumania.

         Metabolic studies indicated that OA is absorbed mainly from the
    proximal jejunum and stomach.  Absorption varied from 40-60% and
    serum half-life ranged from 4 to > 500 hours, depending on the
    species.  In blood, OA was predominantly bound to serum albumin and
    other yet unidentified macromolecules, Tissue distribution of OA
    residues followed the order kidney > liver > muscle > fat.  OA
    was excreted via the urine and faeces.  Cows and sheep had a high
    capacity to hydrolyse OA to the relatively non-toxic ochratoxin

         The underlying mechanism of the toxic action of OA is believed
    to be specific competitive inhibition of phenylalanine-tRNA ligase
    (phenylalanyl-tRNA synthetase).

         Acute toxicity studies indicated that the pig and dog were the
    most sensitive species and that the cause of death was attributed to
    widespread multifocal haemorrhages, intravascular coagulation and
    necrosis of the liver, kidney and lymphoid organs.  Short-term
    studies in rats, dogs and pigs showed that the dominant pathological
    effects were found in the kidneys.  Progressive nephropathy was
    observed in each species, characterized by a deterioration in 
    kidney function and, histologically, by karyomegaly and necrosis of
    tubular cells, and thickening of tubular basement membranes.  The
    severity of the effects depended on the dose and sensitivity of the
    animal species used.  Long-term studies with mice and rats
    demonstrated that, in addition to nephropathy, there was a dose
    related incidence of benign and malignant tumours.  Rats appeared to
    be more sensitive than mice.  The majority of the genotoxicity
    assays on OA were negative.

         OA also exhibited teratogenic activity in rats and mice with
    the CNS being the predominant target tissue.


         In experimental animals treated with ochratoxin A, both humoral
    and cell-mediated immunity as well as structural components of the
    immune system were adversely affected.

         The effects of ochratoxin A that were considered to be most
    significant by the Committee are summarized in Table 5.  The kidney
    appeared to be the primary target organ and the most sensitive
    species was the pig.  As no-observed-effect levels were frequently
    not demonstrated and since the effects were observed in a small
    proportion of the pig's lifetime, the Committee concluded that, in
    assessing the tolerable intake of ochratoxin A, a 500-fold margin of
    safety should be applied to the lowest-observed-effect levels of
    0.008 mg per kg of body weight per day.  On this basis, a
    provisional tolerable weekly intake of 112 ng per kg of body weight
    was established.

         Chronic human nephropathy, endemic in some areas of the
    Balkans, has been lined with exposure to ochratoxin A, as indicated
    by the presence of ochratoxin A residues in local foodstuffs as well
    as in the blood of inhabitants.  On the other hand, some individuals
    and village populations have had detectable ochratoxin A residues in
    the blood, but have shown no evidence of nephropathy.  This suggests
    that either the effects of ochratoxin A are delayed or the disease
    is caused by more than one factor.  About one-third of those dying
    with Balkan endemic nephropathy have had papillomas and/or
    carcinomas of the renal pelvis, ureter or bladder.  No quantitative
    estimates of ochratoxin A dietary intake were available.

         Data on the occurrence of ochratoxin A have demonstrated
    significant levels in a variety of foods although the overall
    incidence of positive samples is low.  As a result, it is extremely
    difficult to estimate total dietary exposure to ochratoxin A for the
    general population, although worst-case intakes of the order of 1 to
    5 ng per kg of body weight per day have been estimated in
    populations when there is no evidence of nephropathy.

         The Committee was informed that the occurrence of elevated
    ochratoxin A levels in foodstuffs in areas with endemic nephropathy
    was associated with poor conditions for grain storage; this factor
    has been recognized as being important in the production of
    ochratoxin A.

         The Committee therefore recommended that efforts be made to
    highlight the need for instituting proper storage conditions for
    grain and grain products.  Furthermore, monitoring of appropriate
    ochratoxin A residues should be undertaken to obtain better
    estimates of dietary exposure and to identify populations at greater
    risk with a view to implementing preventive measure.  The Committee
    also encouraged further studies to elucidate the role of ochratoxin

    A and other mycotoxins in nephropathy in pigs and humans, the
    mechanism of induction of tumours, and the role of phenylalanine in
    antagonizing the adverse effects of ochratoxin A.      

        Table 5

    Summary of effects observed in laboratory animal studies, following 
    oral administration of ochratoxin A

    Effect               Species       Duration of     Lowest-observed-   No-observed-
                                       treatment       effect level       effect level
                                                       (mg/kg bw/day)     (mg/kg bw/day)

    Deterioration        Pig           90 days              0.008               -a
      in renal

    Karyomegaly of       Rat           90 days              0.015               -a
      the proximal
      tubular cells

    Progressive          Pig           2 years              0.04                0.008

    Overt fetal          Mouse         -b                   1                   -a

    Kidney tumours       Mouse         2 years              4.4                 0.13
                         Rat           2 years              0.07                0.02

    Necrosis of          Dog           14 days              0.1                 -a
      tissues of 
      thymus and

    Table 5 (contd)
    Effect               Species       Duration of          Lowest-observed-    No-observed-
                                       treatment            effect level        effect level
                                                            (mg/kg bw/day)      (mg/kg bw/day)

    Decreased            Mouse         50 days              -c                  0.5


    a  No-observed-effect levels were not demonstrated in these studies.
    b  Results refer to a teratogenicity study in which ochratoxin A was
       administered on day 9 of gestation.
    c  Only one dose level was used.


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    See Also:
       Toxicological Abbreviations
       Ochratoxin A (JECFA Food Additives Series 47)
       OCHRATOXIN A (JECFA Evaluation)
       Ochratoxin A (IARC Summary & Evaluation, Volume 56, 1993)