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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

        WORLD HEALTH ORGANIZATION



        SAFETY EVALUATION OF CERTAIN
        FOOD ADDITIVES AND CONTAMINANTS



        WHO FOOD ADDITIVES SERIES 40





        Prepared by:
          The forty-ninth meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998



    AFLATOXINS

    First draft prepared by
    S. Henry1, F.X. Bosch2, J.C. Bowers1, C.J. Portier3, 
    B.J. Petersen4 and L. Barraj4
    1 US Food and Drug Administration, Washington, DC
    2 Institut d'Oncologia, Unitat d'Epidemiologia, Hospitalet del
    Llobregat, Barcelona, Spain
    3 National Institute of Environmental Health Sciences, Research
    Triangle Park, NC, USA
    4Novigen Sciences Inc., Washington, DC, USA (authors of section 4)

    1.  Explanation
    2.  Biological data
        2.1 Biochemical aspects
            2.1.1   Metabolism of aflatoxins
        2.2 Toxicological studies
            2.2.1   Acute toxicity
            2.2.2   Special studies on reproductive toxicity
            2.2.3   Special studies on genotoxicity
            2.2.4   Special studies on immunosuppression
            2.2.5   Factors modifying carcinogenicity of aflatoxins
            2.2.6   Special studies on covalent binding of aflatoxin
                    residues with nucleic acids and proteins
            2.2.7   Special studies on glucose tolerance
            2.2.8   Special studies on effect of ammoniation of AFBI in
                    contaminated cottonseed
            2.2.9   Special studies on aflatoxin and hepatitis B virus
                    infection in woodchucks, ducks, ground squirrels and
                    tree shrews
            2.2.10  Observations in humans
                2.2.10.1    Biomarkers of aflatoxin exposure
                2.2.10.2    Mutations in p53 tumour-suppressor gene in
                            human hepatocellular carcinoma
                2.2.10.3    Epidemiology of primary liver cancer
            2.2.11  Summary of information on other aflatoxins
                2.2.11.1    Aflatoxin B2
                2.2.11.2    Aflatoxin G1 
                2.2.11.3    Aflatoxin G2
                2.2.11.4    Aflatoxin M1
    3.  Estimating carcinogenic risks from the intake of aflatoxins
        3.1 Information from various scientific disciplines and its
            contribution to aflatoxin carcinogenic risk
            3.1.1   Laboratory animal, mutagenicity and metabolic studies
            3.1.2   Studies on the p53 gene 
            3.1.3   Epidemiological studies
            3.1.4   Aflatoxin biomarker studies
        3.2 General modelling issues
            3.2.1   Choice of data
            3.2.2   Measure of exposure
            3.2.3   Measure of response
            3.2.4   Choice of mathematical model

        3.3 Potency estimates
            3.3.1   Potency estimates based upon epidemiological data
            3.3.2   Potency estimates not accounting for HBV infection
            3.3.3   Potency estimates accounting for HBV infection
            3.3.4   Potency estimates based on biomarker studies
            3.3.5   Potency estimates from test species
    4.  Aflatoxin dietary intake estimates
        4.1 Introduction
        4.2 Background
        4.3 Methods
            4.3.1   Period of intake of relevance
            4.3.2   Estimated levels of aflatoxins in foodstuff
            4.3.3   Estimated intakes
        4.4 Results
            4.4.1   Aflatoxin levels in foods: general
            4.4.2   Aflatoxin levels in foodstuffs: Occurrence data by
                    commodity
                4.4.2.1 Amount of commodity imported
                4.4.2.2 Accounting for the change in aflatoxin levels
                        during processing
            4.4.3   National estimates of aflatoxin intake
                4.4.3.1 Australia
                4.4.3.2 China
                4.4.3.3 European Union
                4.4.3.4 USA
                4.4.3.5 Zimbabwe
            4.4.4   Relative impact of establishing maximum limits on
                    estimate of intake
                4.4.4.1 Average aflatoxin concentrations using four
                        possible scenarios
                4.4.4.2 Intake of total aflatoxins using four scenarios
                4.4.4.3 Intake of aflatoxin b1 within four scenarios
            4.4.5   Summary
    5.  Comments and evaluation
        5.1 Aflatoxin potencies
        5.2 Population risks
        5.3.    Conclusions
    6.  References

     List of abbreviations

    AAT        alpha-1-antitrypsin
    ADA       aflatoxin-DNA adduct
    AF        aflatoxin (general)
    AF-alb    aflatoxin-albumin (adduct)
    AFB1      aflatoxin B1
    AFB2      aflatoxin B2
    AFG1      aflatoxin G1
    AFG2      aflatoxin G2
    AFL       aflatoxicol
    AFM1      aflatoxin M1
    AFP        alpha-fetoprotein
    AL         ad libitum
    ALT       alanine aminotransferase
    AM        alveolar macrophage
    APAT      ambient temperature ammoniation procedure
    BNF        beta-naphthoflavone
    CMI       cell-mediated immunity
    CR        calorically restricted
    CYP       cytochrome P450
    DHBV      duck hepatitis B virus
    DTH       delayed type hypersensitivity
    eAAIR     estimated age adjusted incidence rate
    EPHX      epoxide hydrolase
    GGT        gamma-glutamyltranspeptidase
    GHIS      Gambia hepatitis intervention trial
    GSHV      ground squirrel hepatitis virus
    GST       glutathione S-transferase
    GSTM1     glutathione S-transferase M1
    HBV       hepatitis B virus
    HC        high carbohydrate (diet)
    HCC       hepatocellular carcinoma
    HCV       hepatitis C virus
    HF        hypercaloric fat-containing (diet)
    HPHT      high temperature ammoniation procedure
    IC        isocaloric fat-containing (diet)
    I3C       indole-3-carbinol
    LC        liver cancer
    LDH       lactate dehydrogenase
    MDA       malonaldehyde
    OECD      Organisation for Economic Co-operation and Development
    Orm       matched odds ratio
    PCR       polymerase chain reaction
    PHC       primary hepatocellular carcinoma
    PLC       primary liver cancer
    ROS       reactive oxygen species
    SeY       selenium-enriched yeast extract
    WHV       woodchuck hepatitis virus

    1.  EXPLANATION

         Aflatoxins B1, B2, G1, and G2 are mycotoxins that may be
    produced by three moulds of the  Aspergillus species:  A. flavus, 
     A. parasiticus and  A. nomius, which contaminate plants and plant
    products. Aflatoxins M1 and M2, the hydroxylated metabolites of
    aflatoxin B1 and B2, may be found in milk or milk products obtained
    from livestock that has ingested contaminated feed. Of these
    six aflatoxins, aflatoxin B1 is the most frequent one present in
    contaminated samples and aflatoxins B2, G1 and G2 are generally not
    reported in the absence of aflatoxin B1. Most of the toxicological
    data relate to aflatoxin B1. Dietary intake of aflatoxins arises
    mainly from contamination of maize and groundnuts and their products.

         Aflatoxins were evaluated at the thirty-first meeting of the
    Committee (Annex 1, reference 77), at which time the Committee
    considered aflatoxin to be a potential human carcinogen. Sufficient
    information was not available to establish a figure for a tolerable
    level of intake. The Committee urged that the intake of dietary
    aflatoxin be reduced to the lowest practicable levels so as to reduce,
    as far as possible, the potential risk. A working group convened by
    the International Agency for Research on Cancer also concluded that
    naturally occurring aflatoxins are carcinogenic to humans1.

         At the forty-sixth meeting (Annex 1, reference 122), potency
    evaluations and population risk estimates were considered, and the
    Committee recommended that these analyses be completed and presented
    in an updated toxicological review.

         At its present meeting, the Committee reviewed a wide range of
    studies in both animals and humans that provided qualitative and
    quantitative information on the hepatocarcinogenicity of the
    aflatoxins. This monograph reviews the experimental evidence
    concerning the carcinogenicity of the aflatoxins, evaluates the
    potencies of these contaminants, links these potencies to intake
    estimates, and discusses the impact of hypothetical standards on
    sample populations and their overall risks.

         The scientific literature on aflatoxins in the past thirty years
    includes more than 3000 research articles. In 1971 aflatoxins were
    reviewed in Volume 1 of the International Agency for Research on
    Cancer (IARC) Monographs on the Evaluation of Carcinogenic Risk and
    again in Volume 56 of the IARC monographs in 1993. Aflatoxins were
    last reviewed by the JECFA in 1987. A key recent publication in the
    aflatoxin field was the review by Eaton & Groopman (1994). Eaton &

                   

    1 Some naturally occurring substances: food items and constituents,
    heterocyclic aromatic amines and mycotoxins. Lyon, International
    Agency for Research on Cancer, 1993 (IARC Monographs on the Evaluation
    of Carcinogenic Risks to Humans, Vol. 56): 245-395.

    Gallagher (1994) wrote a review of the mechanisms of aflatoxin
    carcinogenesis. This review for JECFA will focus on key reports that
    have appeared in the literature since the publication of the Eaton &
    Groopman review and the 1993 IARC review.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Metabolism of aflatoxins

    An excellent review on the cellular interactions and metabolism of
    aflatoxin has been produced by McLean & Dutton (1995). Gorelick (1990)
    compared metabolism of aflatoxin by different species. Guengerich 
     et al. (1996) discussed the involvement of cytochrome P450,
    glutathione-S-transferase and epoxide hydrolase in the metabolism of
    aflatoxin B1 (AFB1) and the relevance to risk of human liver cancer. A
    wide variety of vertebrates, invertebrates, plants, bacteria and fungi
    are sensitive to aflatoxins, but the range of sensitivity is wide for
    reasons not yet fully understood (Cullen & Newberne, 1994). Two
    important factors in species and strain variation of sensitivity are
    1) the proportion of AFB1 that is metabolized to the 8,9-epoxide,
    relative to other metabolites that are considerably less toxic, and 2)
    the relative activity of phase II metabolism, which forms non-toxic
    conjugates and inhibits cytotoxicity1. The 8,9-epoxide of AFB1 is
    short-lived but highly reactive, and is believed to be the principal
    mediator of cellular injury (McLean & Dutton, 1995).

         Formation of DNA adducts of AFB1-epoxide is well characterized
    (Cullen & Newberne, 1994). The primary site of adduct formation is the
    N7 position of the guanine nucleotide.

         It has been hypothesized that viral infection and associated
    liver injury alter expression of carcinogen-metabolizing enzymes.
    Kirby  et al. (1994) tested this hypothesis in a hepatitis B virus
    (HBV)-transgenic mouse model in which a synergistic interaction occurs
    between AFB1 and HBV in the induction of hepatocellular carcinoma
    (HCC). In this transgenic mouse lineage, overproduction of the HBV
    large envelope protein results in progressive liver cell injury,
    inflammation, and regenerative hyperplasia. Initially, two cytochrome
    P450s important in AFB1 metabolism in the mice were identified -
    CYP2a-5 and CYP3a, using specific antibodies and chemical inhibitors.
    The expression of these P450 isoenzymes and an alpha-class glutathione

                   

    1 Phase I enzymes of major importance to carcinogen metabolism are
    certain members of the superfamily (primarily within families 1-3) of
    CYPs. In general, P450 enzymes catalyse the formation of more polar,
    non-toxic products; however, bioactivation is sometimes a sequela. The
    phase II enzymes of primary importance are the GST, which catalyse
    conjugation of potentially toxic electrophiles to the tripeptide GSH,
    generally rendering them non-toxic.

    S-transferase (GST) isoenzyme, YaYa, was examined. Increased
    expression and altered distribution of CYP2a-5 were demonstrated, by
    immunohistochemical analysis, to be associated with the development of
    liver injury in mice and to increase with age between 1 and 12 months.
    CYP3a expression was also increased in HBV-transgenic mice, but the
    increase was not as clearly related to age. GST YaYa levels were the
    same in HBV-transgenic mice and their non-transgenic littermates of
    all ages.

         These results show that expression of specific cytochrome P450s
    is altered in association with over-expression of HBV large envelope
    protein and liver injury in this model. These findings may have
    general relevance to human HCC, which is associated with a diverse
    range of liver-damaging agents.

         Judah  et al. (1993) studied an aldehyde reductase in the rat,
    which, in contrast with fractions from control animals, catalysed the
    reduction of AFB1-dihydrodiol, in the dialdehyde form at physiological
    pH values, to AFB1-dialcohol. This aldehyde reductase was expressed in
    cytosolic fractions prepared from rat livers bearing pre-neoplastic
    lesions, or following treatment with the anti-oxidant ethoxyquin. This
    expression paralleled the development of resistance to the toxin. This
    enzymatic mechanism might also have relevance in terms of the
    development of resistance to other cytotoxic agents, the mechanism of
    which involves metabolism to a reactive aldehyde. The authors
    suggested that other systems, in particular human, be examined to
    determine if this enzyme activity is expressed, and if so in what
    circumstances, before its potential significance in the carcinogenic
    process can be evaluated. For example, do the livers of humans
    consuming diets contaminated with aflatoxins express such enzymes?

         The monkey CYP1A1 has been expressed in BALB 3T3 A31-1-1 cells
    and the expressed proteins were assayed for their capacity to activate
    AFB1 and benzo[a]pyrene (B[a]P (Itoh  et al., 1993). The transformed
    cells were approximately 5.4- to 4.7-fold more sensitive to AFB1 and
    B[a]P than the parental cells, respectively. The authors concluded
    that monkey CYP1A1-cDNA encoded a functional protein and that the
    expressed CYP1A1 enzyme is active in the activation of B[a]P as well
    as AFB1 to produce toxic metabolites.

         The combined presence of CYP1A2 and 3A4, both of which oxidize
    AFB1 to the reactive AFB1-8,9-epoxide and to hydroxylated inactivation
    products aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), substantially
    complicates the kinetic analysis of AFB1 oxidation in human liver
    microsomes. Gallagher  et al. (1996) examined the reaction kinetics
    of AFB1 oxidation in human liver microsomes (N = 3) and in human
    CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the
    purpose of identifying the CYP isoform(s) responsible for AFB1
    oxidation at low substrate concentrations approaching those
    potentially encountered in the diet. CYP3A4 with AFB1 was found to
    have sigmoidal kinetics such that the rate of product formation fell
    off quickly as the substrate concentration was reduced. CYP1A2 obeyed
    Michaelis-Menten kinetics. Thus, at the low substrate concentrations

    that probably occur  in vivo, the formation of AFB oxide, as well as
    AFB1 clearance, were predicted to be dominated by CYP1A2. Even at a
    relatively higher substrate concentration, CYP1A2 formed approximately
    three times as much AFB-exo-epoxide and generated three times as much
    DNA binding as an equivalent amount of cDNA-expressed CYP3A4.

         The authors pointed out that because AFB1 is highly lipophilic,
    it is difficult to know how nominal concentrations of AFB in  in 
     vitro microsomal preparations relate to concentrations  in vivo. 
    The authors also discussed the work of Ueng  et al. (1995), who
    reported that CYP1A2 formed less AFB oxide than CYP3A4, using human
    CYP1A2 and 3A4 proteins that were expressed in a bacterial expression
    system. The discrepancy between the two studies, according to
    Gallagher  et al. (1996), may have been due to different sources of
    P450s used in the two experiments.

         Gallagher  et al. (1996) concluded that the dominant route for
     in vivo AFB1 activation at dietary concentrations obtained in human
    liver is primarily thorough CYP1A2. Evidence that both CYP1A2 and 3A4
    are involved in AFB1 metabolism  in vivo is substantiated by
    biomarker studies indicating the presence of AFM1 and AFQ1 in the
    urine of individuals exposed to dietary AFB1 (Ross  et al., 1992;
    Qian  et al., 1994). The ratio of activation:inactivation products
    catalysed by CYP1A2 (roughly 2.5:1, AFBO:AFM1) and CYP3A4 (1:10;
    AFBO:AFQ1) is likely to be a key determinant of the pathway and
    biological consequences of  in vivo AFB1 exposure. Unfortunately, the
    actual urinary and faecal levels of these two metabolites, (in
    particular, AFQ1 and possible secondary metabolites) following
    exposure to AFB1 are not known. Thus, the relative ratio of these two
    metabolites in individuals exposed to dietary AFB1, a key ratio, is
    also unknown.

         Sawada  et al. (1993) show that human placental microsomes
    activated AFB1, AFB1 showed relatively high mutagenic activity in the
    Ames test when incubated with human placental microsomes. Addition of
     alpha-naphthoflavone or aminoglutethimide, known inhibitors of
    cytochrome P450 1A and P450 19, respectively, into the test system
    partially inhibited the mutagen-producing activity.

         Induction of glutathione-S-transferase placental form (GST-P)
    positive hepatic foci has been examined by immunohistochemical
    analysis in young male Fischer rats 3 weeks after a single i.p.
    injection of AFB1 (Gopalan  et al., 1993). Pretreatment of rats with
    L-buthionine sulfoximine (BSO), a GSH depleter, at a dose of 4 mmol/kg
    bw 4 and 2 hours before 1.0 mg AFB1 treatment enhanced both the number
    of AFB1-induced hepatic foci and the area occupied by these foci by
    approximately 400 and 575% above their respective controls without
    affecting the mean diameter of these foci. Pretreatment of rats with
    0.1% phenobarbital (PB) in their drinking water for 1 week before AFB1
    (1 mg) treatment, inhibited AFB1-induced foci almost completely.
    However, the number of AFB1-induced foci in PB-treated rats was not
    significantly increased by BSO pretreatment.

         Fetal rat liver has been shown to possess substantial levels of
    glutathione-S-transferase (GST) activity toward AFB1-8,9-epoxide. The
    enzyme responsible for this activity was an alpha-class GST
    heterodimer comprising Yc1 and Yc2 subunits (Hayes  et al., 1994).
    The cDNAs encoding these polypeptides have been cloned and shown to
    share approximately 91% identity over 920 base pairs, extending from
    nucleotide -23 to the AATAAA polyadenylation signal. GST Yc2Yc2
    expressed in  Escherichia coli was found to exhibit 150-fold greater
    activity toward AFB1-8,9-epoxide than GST Yc1Yc1. Comparison between
    the structures of alpha-class GST suggested that tyrosine at residue
    108 and/or aspartate at residue 208 is responsible for the high AFB1
    detoxification capacity of Yc2. Immunoblotting and enzyme assays have
    shown that liver from adult female rats contains about 10-fold greater
    levels of Yc2 than is found in liver from adult male rats. This
    sex-specific expression of Yc2 in adult rat liver may contribute to
    the relative insensitivity of female rats to AFB1. Dietary
    administration of oltipraz, a synthetic antioxidant which protects
    against aflatoxin-hepatocarcinogenesis served as an inducer of GST
    Yc2.

         Gallagher & Eaton (1995) have investigated the biotransformation
    of AFB1 in hepatic microsomal and cytosolic fractions from channel
    catfish, an aquatic species shown to be refractory to AFB1 toxicity
    and reported to be resistant to AFB1 hepatocarcinogenesis, and in
    rainbow trout, a species sensitive to AFB1 toxicity and
    hepatocarcinogenesis. AFB1 was poorly oxidized by channel catfish
    microsomes, suggesting that the lack of microsomal AFB1 activation
    together with the rapid conversion of AFB1 to aflatoxicol (AFL)
    contributes to the apparent resistance of channel catfish to AFB1
    toxicity and hepatocarcinogenesis.

         Oltipraz is currently under evaluation as a possible
    chemopreventive agent in humans. Primiano  et al. (1995) investigated
    the chemopreventive efficacy achieved by administration of
    intermittent doses of oltipraz in rats. Fischer 344 rats were treated
    with oltipraz (0.5 mmol/kg, p.o.) once weekly, twice weekly, or daily
    over a 5-week period. After the first week, all rats were gavaged with
    20 g/kg AFB1 for 28 consecutive days. Livers were analysed 2 months
    after the last AFB1 dose, and the volume of liver occupied by
    glutathione-S-transferase (GST)-P positive foci, a presumptive marker
    of neoplasia, was observed to be decreased by >95%, >97% or >99% in
    livers of rats receiving once-, twice-weekly or daily oltipraz
    treatments, respectively. The chemopreventive actions of oltipraz have
    been associated with increases in the levels of phase 2 detoxifying
    isozymes. Accordingly, GST conjugation activity measured with
    1-chloro-2,4-dinitrobenzene as a substrate increased 1.5, 1.8 or
    2.4-fold for the once-weekly, twice-weekly or daily treatments,
    respectively, throughout a 7-day period. The authors suggested that
    the protracted pharmacodynamic actions of oltipraz on enzyme induction
    may account from the marked reduction in the hepatic burden of
    AFB1-induced putative preneoplastic tumours after intermittent dosing.
    Consequently, scheduling of intermittent dosing protocols may sustain

    efficacy while improving drug tolerance and patient compliance over
    long-term treatments. These properties of oltipraz increase its
    attractiveness for clinical chemopreventive interventions, the authors
    emphasized.

         Langouet  et al. (1995) investigated metabolism of AFB1 in
    primary human hepatocytes with or without pretreatment by oltipraz.
    AFM1, glutathione conjugates of AFB1 oxides and unchanged AFB1 were
    quantified in cultures derived from eight human liver donors.
    Parenchymal cell obtained from the three GST M1-positive livers
    metabolized AFB1 to AFM1 and to AFB1 oxides derived from the isomeric
    exo and endo-8,9-oxides, whereas no AFB1 oxides were formed in the GST
    M1-null cells. Pretreatment of the cells with 3-methylcholanthrene or
    rifampicin, inducers of CYP1A2 and CYP3A4 respectively, caused a
    significant increase in AFB1 metabolism. Although oltipraz induced GST
    A2, and to a lesser extent GST A1 and GST M1, it decreased formation
    of AFM1 and AFB1 oxides, which involves CYP1A2 and CYP1A2. Inhibition
    by oltipraz of AFB1 metabolism through a reduction in CYP1A2 and
    CYP3A4 was also shown by decreased activity of their monooxygenase
    activities toward ethooxyresorufin and nifedipine, respectively. The
    significant inhibition by oltipraz of human recombinant yeast CYP1A2
    and CYP3A4 was also shown. These results demonstrated that AFB1 oxides
    can be formed by GST M1-positive human hepatocytes only, and suggested
    that chemoprotection with oltipraz is due to an inhibition of
    activation of AFB1 in addition to a GST-dependent inactivation of the
    carcinogenic  exo-epoxide.

         AFB1-induced carcinogenesis has been shown to be both inhibited
    and promoted by indole-3-carbinol (I3C), found in cruciferous
    vegetables. Stresser  et al. (1994a) examined the influence of
    dietary treatment with I3C and the well-known Ah receptor agonist
     beta-naphthoflavone (BNF) on the relative levels of different
    cytochrome P-450 (CYP) isoforms known to metabolize AFB1 in male
    Fischer 344 rats. After 7 days of feeding 0.3% I3C or 0.04% BNF alone
    or in combination, the relative levels of hepatic CYP1A1, 1A2, 2B1/2
    2C11 and 3A were assessed by laser densitometry of Western blots. Both
    diets containing I3C markedly increased band densities of CYP1A1, 1A2,
    and 3A1/2 with less effects on 2B1/2 and no effect on CYP2C11. BNF
    also strongly increased band densities of CYP2C11, but had no effect
    on CYP2C11 or 3A1/2, and repressed CYP2C11. In addition the  in 
     vitro hepatic microsomal metabolism of AFB1 was examined at 16, 124,
    and 512 TM substrate levels. The authors' results suggested that BNF
    inhibits AFB1 carcinogenesis, in part by enhancing net production of
    less toxic hydroxylated metabolites of AFB1, as a result of elevated
    levels of P450, and that I3C may share this mechanism. However, other
    mechanisms, such as direct inhibition of P450 bioactivation by I3C
    oligomers, or induction of phase II enzymes, also appeared to
    contribute.

         Stresser  et al. (1994a) also examined the influence of I3C and
    BNF on the AFB1 glutathione detoxication pathway and AFB1-DNA
    induction in rat liver. After 7 days of feeding approximately equally
    inhibitory doses of I3C (0.2%) or BNF (0.04%) alone or in combination,

    male Fischer 344 rats were administered [3H]AFB1 (0.5 mg/kg, 480
    TCi/kg) i.p. and killed 2 hours later. All three diets inhibited 
     in vivo AFB1-DNA adduction. Using an improved HPLC method for
    separation of the two isomeric forms of AFB1 8,9-epoxide-glutathione,
    both I3C diets were shown to induce GST activities strongly toward
    AFB1  exo-epoxide, whereas BNF alone induced activity weakly. Data
    suggest that enhanced detoxication of AFB1 via increased glutathione
    conjugation efficiency, as a result of elevated levels of the Yc2 GST
    subunit, is one mechanism that contributes to a protective effect of
    I3C against AFB1-induced preneoplastic lesions in the rat, and that
    this mechanism also participates to a lesser degree in protection by
    BNF.

         The role of reactive oxygen species (ROS) in AFB1-induced cell
    injury was investigated using cultured rat (male Fischer 344)
    hepatocytes (Shen  et al., 1995). Malonaldehyde (MDA) generation and
    lactate dehydrogenase (LDH) release were determined as indices of
    lipid peroxidation and cell injury, respectively. Exposure to AFB1 for
    up to 72 hours resulted in significantly elevated levels of LDH being
    released into the medium as well as the MDA generation in cultured
    hepatocytes. These effects were dose-dependent, indicating that AFB1
    was capable of inducing oxidative damages in the cell. Further, MDA
    generation and LDH release were effectively inhibited by the addition
    of the following: 1) superoxide dismutase (500 units/ml); 2) catalase
    (1500 units/ml); 3) 10 mM desferrioxamine (a specific iron chelator),
    or 4) 260 mM dimethyl sulfoxide (a hydroxyl radical scavenger). This
    evidence therefore suggests that ROS, such as superoxide radicals,
    hydroxyl radicals and hydrogen peroxides, are involved in AFB1-induced
    cell injury in cultured rat hepatocytes, the authors concluded.

         Kirby  et al. (1993) examined liver tissues from 20 liver cancer
    patients from Thailand, an area where the incidence of this tumour is
    high and where exposure to aflatoxin occurs. The expression of hepatic
    cytochrome P450s and GST was examined and this expression was compared
    to the  in vitro metabolism of AFB1. There was a >10-fold
    inter-individual variation in expression of the various P450s
    including CYP3A4 (57 fold), CYP2B6 (56-fold), and CYP2A6 (120 fold).
    Microsomal metabolism of AFB1 to AFB1 8,9-epoxide and AFQ1, the major
    metabolite produced, was statistically significantly correlated with
    CYP3A3/4 expression and, to a lesser extent, with CYP2B6 expression.
    There was a significantly reduced expression of major P450 proteins in
    microsomes from liver tumours compared to microsomes from the paired
    normal liver when analysed by Western immunoblot analysis.

         The immunoreactive expression of the major human classes of
    cytosolic GSTs  (alpha, mu and  pi) was also analysed in normal and
    tumorous liver tissue. The expression of GSTA  (alpha) and GSTM 
     (mu) class proteins was markedly decreased and GSTP  (pi) increased
    in the majority of tumour cytosols compared to normal liver. Cytosolic
    GST activity was significantly lower in liver tumours compared to
    normal liver. There was no detectable conjugation of AFB1 8,9-epoxide
    to glutathione by cytosol either from tumorous or normal liver. Thus,

    capacity of human cytosols to conjugate reactive AFB1 metabolites to
    GSH resembled AFB1-sensitive species such as rat, trout and duck
    rather than resistant species such as mouse and hamster. These data
    indicate a strong capacity of multiple forms of human hepatic P450s to
    metabolize AFB1 to both the reactive intermediate AFB1-8,9-epoxide and
    the detoxification product AFQ1. The authors suggested, that, in view
    of the lack of significant GST-mediated protection against AFB1 in
    human liver, variations in expression of hepatic P450, due either to
    genetic polymorphisms or to modulation by environmental factors, may
    be important determinants in the risk of liver cancer development in
    AFB1-exposed populations.

         Liu  et al. (1991) evaluated the functional significance of the
    glutathione transferase (GST)  mu polymorphism by measuring its
    effect on AFB1-DNA adduct formation  in vitro. Human liver cytosols
    prepared from persons having low or high glutathione transferase
    toward  trans-stilbene oxide were incubated with human liver
    microsomes, calf thymus DNA, and AFB1. AFB1-DNA binding was inhibited
    to a greater extent in high conjugators than low conjugators; the
    correlation between AFB1-DNA adduct concentrations and GST  mu 
    activity was highly statistically significant. The authors suggested
    that GST  mu plays an important role in detoxifying DNA reactive
    metabolites of AFB1, and this enzyme may be a susceptibility marker
    for AFB1-related liver cancer.

         Heinonen  et al. (1996) studied the profile of AFB1 metabolism
    and the extent of AFB1 binding to cell macromolecules in human liver
    slices under experimental conditions that would allow direct
    comparison to similar end-points in the rat, a species sensitive to
    the carcinogenic actions of AFB1. Liver slices were prepared from
    three individual human liver samples with a Krumdieck tissue slicer
    and incubated with 0.5 M [3H]AFB1 for 2 hours. Significant
    inter-individual variations were observed in the rates of oxidative
    metabolite formation and in specific binding to cell macromolecules.
    The rates of oxidative metabolism of AFB1 to AFQ1, AFP1 and AFM1 in
    the human liver samples were similar to those previously observed in
    rat liver slices. AFB1-GSH conjugate formation was not detected in any
    of the human liver samples, and yet specific binding of AFB1 to cell
    macromolecules was considerably lower in the human liver slices
    relative to that in rat liver slices. The authors postulated that
    these studies suggest that an as yet unidentified protective pathway
    may exit in human liver. These studies support the hypothesis that
    humans do not form as much aflatoxin B1-8,9-epoxide as the rat, but
    humans do not possess GST isozymes with high specific activity toward
    the epoxide. Significant interindividual differences in AFB1
    metabolism and binding between humans suggest the presence of genetic
    and/or environmental factors that may make some humans more or less
    susceptible to AFB1.

         Gallagher  et al. (1994) studied the metabolism of AFB1 in
    microsomes derived from human lymphoblastoid cell lines expressing
    transfected CYP1A2 or CYP3A4 (cytochrome P450) and in microsomes

    prepared from human liver donors (n=4). The authors summarized their
    findings as follows. Both CYP3A4 and CYP1A2 were involved in the
    activation of AFB1 to the AFB1-8,9-epoxide; 1A2 appeared to have a
    higher affinity for AFB1 and produced a higher ratio of activation
    (AFB1-8,9-epoxide) to detoxification (AFM1) products, relative to 3A4.
    3A4 may be expressed in human liver at a much higher level than 1A2,
    such that in some individuals, 3A4 may be the predominant source of
    AFB1-8,9-epoxide at low substrate concentrations, even though 3A4
    produces AFQ1 predominantly. Such differences in the apparent kinetics
    of these two P450s toward AFB1 indicate that the most important
    determinant of individual susceptibility to AFB may well be the level
    of expression of 1A2. Individuals with relatively high 1A2 expression
    may be at particular risk for AFB1-induced DNA damage, since human
    GSTs are relatively ineffective in detoxifying AFB1-8,9-epoxide.
    Inhibition of 1A2 may prove to be an effective means of
    chemointervention in AFB1-exposed populations. Of course,  in vivo 
    human toxicity is ultimately determined by a complex set of processes.

         In experiments by Ueng  et al. (1995), human cytochromes P450
    1A2 and 3A4 were expressed in  Escherichia coli, purified, and used
    in reconstituted oxidation systems. Relatively high catalytic
    activities were obtained with such a system for AFB1
    3 alpha-hydroxylation and 8,9-epoxidation. P450 3A4 was more active
    than P450 1A2 in forming genotoxic AFB1 oxidation products; P450 3A4
    formed AFQ1 and the  exo-8,9-epoxide; P450 1A2 formed AFB1, some
    AFQ1, and both the  exo- and  endo-8,9-epoxides. Plots of AFB1
    3 alpha-hydroxylation and 8,9-epoxidation  vs. AFB1 concentration
    were sigmoidal in both human liver microsomes and the reconstituted
    P450 3A4 system. The results were consistent, the authors
    hypothesized, with the view that P450 3A4 is a major human liver P450
    enzyme involved in AFB1 activation, although the  in vivo situation
    may be more complex due to the presence of the enzyme in the
    gastrointestinal tract.

    Guengerich  et al. (1996) have reviewed a series of studies to show
    the complexities encountered with metabolism of AFB1; the complexity
    demonstrates the difficulties in doing molecular epidemiology studies,
    even when a single chemical carcinogen has been identified. Figure 1
    shows these metabolism complexities. With all the enzymes,
    stereochemistry of the epoxide must be considered. In addition, the
    P450s both activate and detoxify AFB1, and the effect of inducing
    individual P450s is not easy to predict. P450 3A4 is expressed in the
    small intestine, the site of absorption of orally ingested AFB1, where
    the extent of detoxification is unknown. Even activation of AFB1 and
    DNA alkylation in the small intestine may be considered to be a
    detoxification process since the cells are sloughed rapidly, and
    cancers of the small intestine are very rare.

    FIGURE 1

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         No additional acute toxicity studies have been reported in the
    literature since the review by Eaton & Groopman (1994).

    2.2.2  Special studies on reproductive toxicity

         Ankrah  et al. (1993) exposed ddy mice to AFB1 and AFG1 via
    their feed (4.8 ng AFG1, 0.8 ng AFB1 (or both) per kg bw per day while
     in utero. Levels of aflatoxin used were realistic relative to the
    level of human exposure currently seen in Ghanaian foods. Offspring of
    these animals (control and aflatoxin-fed) were continued on the
    respective diets received by the parent stock until sacrifice at six
    months of age. Blood obtained by cardiac puncture was used to
    determine haema-tological indices and the sera were used to determine
    glucose, triglyceride, total protein and albumin. AFG1 caused
    significant accumulation of only neutral fat in the liver, a slight
    rise in serum triglyceride and intensified hepatorenal inflammation,
    necrosis and bile duct proliferation. AFB1 caused the accumulation of
    both neutral fat and fatty acids in the liver, and was cytotoxic to
    the liver and kidney. Iron storage in the liver, haematological
    indices, serum total protein and albumin levels were not affected by
    the aflatoxins. At the level used, AFG1 was six times in excess of
    AFB1, but the latter was more severe in the observed hepatorenal
    effects.

         The authors pointed out that the mouse liver has been shown to
    metabolize aflatoxin in a manner similar in some ways to the human
    liver, although not all investigators would agree on this point. Hence
    they postulated that the action of aflatoxin on mouse organs may shed
    light on aflatoxin cytotoxicity in humans; results of this study are
    of particular relevance to population groups that ingest foods known
    to contain mainly AFG1 and to some extent AFB1.

    2.2.3  Special studies on genotoxicity

         AFB1 covalently binds to DNA and efficiently induces G to T
    trans-versions; codon 249, one site in p53, is a striking hot spot for
    AFB1 mutagenesis (Sengstag & Wurgler, 1994). Often, such mutations are
    followed by the loss of the second functional alleles of tumour
    suppressor genes, a phenomenon called loss of heterozygosity. To test
    whether mitotic recombination leading to loss of heterozygosity is
    induced by certain carcinogens, the authors genetically engineered a
     Saccharomyces cerevisiae tester strain so that it metabolized two
    important classes of carcinogens including AFB1. Human cDNAs coding
    for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination
    with NADPH-CYP oxidoreductase in a strain heterozygous for two
    mutations in the trp5 gene were inserted. AFB1, when activated
    intracellularly in microsomes isolated from the yeast strains
    containing either human CYP enzyme, significantly induced mitotic
    recombination. The authors concluded that activated AFB1 is a potent

    inducer of DNA recombination in  S. cerevisiae strains harbouring
    various heterologous xenobiotic-metabolizing systems.

         Young weanling Swiss albino mice were orally administered crude
    AFB1 in a dose mimicking human exposure, i.e., at 0.05 g/kg bw per
    day for 14 weeks (Sinha & Dharmshila, 1994). Vitamin A (retinol) was
    orally administered along with the toxin at double (132 IU/kg bw per
    day) the human equivalent therapeutic dose. The authors concluded that
    vitamin A minimized the frequency of toxin-induced clastogeny in both
    mitotic and meiotic chromosomes. The decreases in sperm count, as well
    as increases in abnormality in the gross morphology of the sperm head,
    as observed upon toxin treatment, was ameliorated by the vitamin A.

         Marquez-Marquez  et al. (1993) evaluated the effects of an AFB1
    inactivating system with ammonia on the genotoxicity of AFB1 measuring
    micronucleus (MN) and sister chromatid exchange (SCE) analyses. Four
    groups of CD1 male mice were fed for 8 weeks with a special diet
    mainly composed of maize: 1) uncontaminated; 2) uncontaminated/
    inactivated; 3) contaminated/ inactivated; and 4) contaminated. The
    inactivating treatment was performed with ammonium hydroxide by
    homogeneously impregnating the grain and leaving it for 20 days in
    hermetically closed plastic bags and then heating in an oven for 24
    hours to eliminate the residual ammonia. AFB1 was quantified before
    and after inactivation. MN was evaluated at weekly intervals in
    peripheral blood; SCE was quantified in bone marrow cells at weeks 4
    and 8. The results showed that mice fed with AFB1 contaminated/
    inactivated maize had a 45% lower level of induced cytogenetic damage
    than those animals fed with AFB1 contaminated (but not inactivated)
    maize. A residual amount of AFB1 remaining after the inactivating
    treatment and the reconversion back to AFB1 in the organism may
    account for the remaining increased levels of SCE and MN.

         Marquez-Marquez  et al. (1995) evaluated the efficiency of the
    AFB1 inactivating system with ammonia, as described above, and using
    mice (male CD-1) and micronucleus (MN) and sister chromatid exchange
    (SCE) analysis. Apparently this study was the same as that published
    earlier.

         Occupational exposures to respirable dusts contaminated with the
    mycotoxin AFB1 have been associated with an increased incidence of
    upper airway tumours. To investigate this possible etiology Ball 
     et al. (1995) compared the abilities of tracheal and lung S9 from
    rabbit (male, New Zealand white), hamster (male Syrian Golden) and rat
    (male Sprague-Dawley) to activate AFB1 to mutagens in  Salmonella 
     typhimurium TA98. These species differ in airway morphology with
    respect to numbers of metabolically active non-ciliated tracheal
    epithelial cells. Tracheas from hamster and rabbit and lung from
    rabbit were active in converting AFB1 to bacterial mutagens. Tracheas
    from hamster were more efficient in activating AFB1 to mutagens than
    lung, while rabbit lung was over 4 times more active in converting
    AFB1 to mutagens than that from trachea. In all cases, AFB1 was more
    mutagenic than B[a]P. The relative capabilities of trachea to activate
    AFB1 were in agreement with the ability of cultured tracheas from

    these species to form AFB1-DNA adducts. These results demonstrate that
    AFB1 is activated more efficiently than B[a]P in the lung, and that
    the metabolic capabilities of airway epithelium to activate AFB1 are
    not predictable by airway morphology.

         A study by Shi  et al. (1995a) examined the effect of two
    selenium compounds, namely, sodium selenite and selenium-enriched
    yeast extract (SeY) on the cytotoxicity, DNA binding, and mutagenicity
    of AFB1 in cultured Chinese hamster ovary (CHO) cells. CHO cells,
    after treatment with 2 g/ml selenite or 80 g/ml SeY, exhibited
    increased resistance to AFB1-induced cell killing. At a concentration
    of 50 g/ml AFB1, cell survival, measured by the clonogenicity assay,
    was increased by 21- and 10-fold in selenite- and SeY-treated cells,
    respectively. However, selenium treatment did not appear to affect
    AFB1-DNA binding. Similarly, no effect was observed on AFB1
    mutagenicity, as determined by the hypoxanthine-guanine phosphoribosyl
    transferase (HPRT) gene mutation assay. The results showed that
    selenium could effectively protect cells from AFB1 cytotoxicity in
    cultured cells, but had no effect on AFB1-DNA adduct formation or
    mutagenesis. The authors suggested that there are multiple pathways of
    AFB1 toxicity and that selenium can modulate AFB1-induced cell killing
    independent of its genotoxicity.

         Rats and mice differ markedly in sensitivity to AFB1
    hepatocarcinogenicity, the former being sensitive and the latter
    resistant. The purpose of this study was to determine whether the
    formation of AFB1-albumin (AF-alb) adducts was related to the
    induction of cytogenetic changes  in vivo as a step to understanding
    whether such markers of exposure may be informative with respect to
    genetic alterations important in the carcinogenic process (Anwar 
     et al., 1994). The comparison was made at two levels: between
    species and between individuals within a species. Animals (male Helwan
    Wistar albino rats and Swiss albino mice) were treated with single
    doses of different concentrations of AFB1 between 0.01 and 1.0 g
    AFB1/g bw. The frequency of chromosomal aberrations and micronuclei in
    the bone marrow was measured and compared to the level of AFB1 bound
    covalently to albumin in the peripheral blood. Both chromosomal
    aberrations and micronuclei were significantly increased in treated
    rats compared to the control group at doses above 0.1 g/g. In
    contrast, in mice, a slight increase in chromosome aberrations was
    seen in the highest dose group (1.0 g/g), but no increase in
    micronuclei was observed at any of the doses. The level of chromosomal
    aberrations was about 10 times higher in rats than in mice at the
    highest dose of AFB1. AFB1-albumin increased linearly with dose of
    AFB1, and there were strong statistically significant correlations at
    the individual rat level with both chromosomal aberrations.

         The level of AF-alb adducts was higher for a given dose in rats
    than mice, as has been seen for the level of liver DNA adducts in the
    two species. The metabolic basis of these differences has been
    investigated and has been shown to be associated with the expression
    of a specific glutathione-S-transferase isoenzyme in mice, which
    efficiently conjugates the AFB1-epoxide to glutathione.

         In rats, the level of AF-alb adducts was strongly correlated with
    the frequency of both micronuclei and chromosomal aberrations in the
    bone marrow. An increase in adduct levels was seen with exposures as
    low as a few ng AFB1/g bw, whereas the genetic alterations were only
    increased above control levels at doses around 0.1 g/g.

         The authors suggested two considerations for interpretation of
    the present studies. First, the cells in which the micronuclei and
    chromosomal aberrations were examined are not the target cells for
    AFB1 hepatocarcinogenesis, and second, that this type of genetic
    marker is relatively non-specific. Thus, the genetic alterations being
    measured are not directly relevant to the carcinogenic process; this
    limitation may be overcome as sensitive molecular techniques are
    developed to measure mutation induced by aflatoxin in specific gene
    sequences in somatic cells (See Aguilar  et al., 1993). Recent
    studies by these authors (Wild  et al., in preparation) suggest that
    AF-alb adducts reflect the differing species sensitivity to AFB1
    carcinogenesis. This peripheral blood marker could be an indicator of
    risk of liver cancer development in addition to being a marker of
    exposure, the authors suggest, as has been further supported by the
    study of Ross  et al. (1992), in which the level of AFB1-N7 guanine
    adduct in the urine was related to the subsequent risk of developing
    hepatocellular carcinoma in a Chinese cohort. This study will be
    discussed in section 2.2.10.

    2.2.4  Special studies on immunosuppression

         The immunosuppressive potential of AFB1 was evaluated in growing
    rats (Raisuddin  et al., 1993). The weanling rats (species
    unspecified) were sub-chronically exposed to 60, 300 or 600 g AFB1/kg
    bw for four weeks on alternate days by oral feeding. Various
    parameters of cell-mediated immunity (CMI) and humoral immunity were
    assessed in control and treated animals. CMI was evaluated by
    measuring delayed type of hypersensitivity (DTH) response and humoral
    immunity was measured by plaque forming (PFC) assay. The
    lympho-proliferative response assay for T- and B-cells was also
    performed. It was observed that AFB1 selectively suppressed
    cell-mediated immunity in growing rats. AFB1 suppressed CMI at the 300
    and 600 g dose levels only as measured by DTH response assay. The
    authors concluded that continuous low level exposure of aflatoxin to
    the growing host may enhance its susceptibility to infection and
    tumorigenesis.

         Jakab  et al. (1994) conducted experiments to demonstrate the
    immunosuppressive effects of AFB1 ingestion, in this case respiratory
    tract exposure to AFB1. Rats (male Fischer 344) and mice (female
    Swiss) were exposed either by aerosol inhalation or intratrachael
    instillation to AFB1. Nose-only inhalation exposure of rats to AFB1
    aerosols suppressed alveolar macrophage (AM) phagocytosis at an
    estimated dose of 16.8 g/kg with the effect of persisting for
    approximately 2 weeks. To determine whether another mode of
    respiratory tract exposure, intratrachael instillation, reflected
    inhalation exposure, animals were treated with increasing

    concentrations of AFB1, which also suppressed AM phagocytosis in a
    dose-related manner, albeit at doses at least an order of magnitude
    more than that obtained by aerosol inhalation. Intratrachael
    administration of AFB1 also suppressed the release of tumour necrosis
    factor-alpha from AMs and impaired systemic innate and acquired immune
    defences as shown, respectively, by suppression of peritoneal
    macrophage phagocytosis and the primary splenic antibody response. The
    authors concluded that experimental respiratory tract exposure to AFB1
    suppressed pulmonary and systemic host defences; they indicated that
    inhalation exposure to AFB1 is an occupational hazard where exposure
    to AFB1-laden dust is common, such as in grain dust.

    2.2.5  Factors modifying carcinogenicity of aflatoxins

         Young adult male Fischer rats maintained on a reduced calory diet
    (60% of  ad libitum food consumption) for 6 weeks showed a decrease
    in the binding of AFB1 to hepatic or renal nuclear DNA and a reduction
    of AFB-induced hepatocellular damage (Chou  et al., 1993). Repeated
    dosing of rats with AFB1 resulted in the inhibition of hepatic and
    renal DNA synthesis as measured by [3H]thymidine incorporation.
    However, the rate of DNA synthesis was greater in  ad libitum (AL)
    rats than in calorically restricted (CR) animals. Three days after
    AFB1 dosing, the rate of DNA synthesis had recovered to the control
    level. Cell cycle analyses measured by a flow cytometric method on
    kidney cells of both AL and CR rats showed that there were no
    significant changes in cell populations in the S phase between these
    two groups of rats. AFB1 inhibited the cell proliferation by 33% (on
    average). The restoration of the cell proliferation in kidney cells
    was found on the third day after AFB1 dosing. The rate of regenerative
    cell proliferation was found to be slightly greater in AL rats than in
    CR animals. The AFB1-induced regenerative DNA synthesis in both liver
    and kidney was retarded by CR.

         Youngman & Campbell (1992) demonstrated that with young Fischer
    344 rats the post-initiation development of AFB1-induced 
     gamma-glutamyltranspeptidase-positive (GGT+) hepatic foci was
    markedly inhibited by low protein feeding, even though the energy
    intake was greater. These investigators also studied this dietary
    effect upon the development of hepatic tumours and the correlation of
    foci development with tumour development. Following AFB1 dosing (15
    daily doses of 0.3 mg/kg each), animals were fed diets containing 6,
    14 or 22% casein (5.2, 12.2 or 19.1% protein) for 6, 12, 40, 58 or 100
    weeks. Foci at 12 weeks and tumours at 40, 58 and 100 weeks developed
    dose-dependently to protein intake. Foci development, tumour
    incidence, tumour size and the number of tumours per animal were
    markedly reduced, while the time to tumour emergence was increased
    with low-protein feeding. Non-hepatic tumour incidence also was lower
    in the animals fed the lowest protein diet. Foci development indices
    (foci number, per cent liver volume occupied) were highly correlated
    with tumour incidence at 58 and 100 weeks (r = 0.90-1.00). Tumour and
    foci inhibition occurred in spite of the greater energy intake.

         Previous results from a large ecological study in 65 rural
    counties in China suggested that primary liver cancer in humans
    primarily is associated with chronic HBV infection, coupled with
    nutritional factors (e.g., animal protein) that elevate plasma
    cholesterol level and encourage cancer growth (Campbell  et al., 
    1990). To test this hypothesis, the authors investigated the effect of
    dietary animal protein on tumour development in HBV transgenic mice.
    Male F2 offspring of 50-4 HBV transgenic mice were randomly assigned
    to 6, 14 and 22% dietary casein. Serum was collected from the retro-
    orbital vein and was analysed for the level of hepatitis B virus
    antigen (HBsAg), the products of the S-transgene. The increases from
    baseline in S-gene product observed for the normal protein animal
    (22%) at 3 months was inhibited in the mid- and low-protein animals by
    42% and 72%, respectively, with a highly significant dose-response
    relationship (P<0.001). Serum glutamic-pyruvic transaminase activity
    was not affected by diet treatment. The authors concluded that their
    results strongly suggest that dietary casein controls, in a
    dose-response manner, S-transgene expression in these experimental
    animals.

         Hasler  et al. (1994) fed Fischer 344 rats a low-fat high
    carbohydrate (HC) diet, an isocaloric fat-containing (IC) diet, a
    hypercaloric fat-containing (HF) diet or a commercial rodent chow. The
    effects of these diets were studied on the binding of AFB1 to
    exogenous DNA and on the activities of hepatic glutathione
    transferases (GSTs), cytochromes 2B1 and 1A1. Microsome-mediated
    binding of [3H]AFB1 to exogenous DNA was significantly lower in the
    HC rats than in the chow- and IC-fed rats. No significant differences
    were noted between HF and either HC or IC rats. There was no
    significant difference in hepatic GST activity of rats fed the
    different diets. The authors suggested that high carbohydrate/low fat
    diets may reduce microsome-mediated epoxidation of AFB1 to a larger
    extent than high-fat diets. In general, high-fat diets increased
    cytochrome 1A1 and 2B1 activities relative to chow and
    high-carbohydrate diet. This suggested greater detoxification of AFB1,
    thus reducing the amount of AFB1 available for hepatic macromolecular
    binding, the authors concluded.

         An excellent review by Massey  et al. (1995) covers the
    biochemical and molecular aspects of mammalian susceptibility to AFB1
    carcinogenicity. Important considerations include: 1) different
    mechanisms for bioactivation of AFB1 to its ultimate carcinogenic
    epoxide metabolite; 2) the balance between bioactivation to and
    detoxification of the epoxide; 3) the interaction of AFB1 epoxide with
    DNA and the mutational events leading to neoplastic transformation; 4)
    the role of cyto-toxicity in AFB1 carcinogenesis; 5) the significance
    of non-epoxide metabolites in toxicity; and 6) the contribution of
    mycotoxin-unrelated disease processes.


    2.2.6  Special studies on covalent binding of aflatoxin residues with
    nucleic acids and proteins

         Shi  et al. (1994) studied the effect of selenium on AFB1-DNA
    binding and adduct formation. Male Fischer 344 rats, fed with up to 8
    mg/litre sodium selenite in drinking-water for 8 weeks, were given a
    single i.p. dose of AFB1. The rats were killed 24 hours later and the
    amount of AFB1 bound to hepatic DNA and the amount of DNA adducts
    formed were determined. Selenium pretreatment resulted in a
    dose-dependent inhibition of AFB1-DNA binding as well as adduct
    formation. This was accompanied by an increase of reduced glutathione
    (GSH) in the liver of selenium-treated animals. These results
    suggested that selenium could effectively inhibit AFB1-induced DNA
    damage, which may be partially responsible for its anticarcinogenic
    effect against AFB1.

         Choy (1993) has reviewed the dose-response induction of DNA
    adducts by AFB1 and its implication to quantitative cancer risk
    assessment. Dose-response curves of DNA adduct formation after
    ingestion or injection treatments in the rat were reviewed; a linear
    dose-response relationship was observed in both injection and
    ingestion studies at low doses. The author concluded that this
    observation is consistent with the assumption of the linear
    dose-response risk assessment model for genotoxic agents and justifies
    the use of this model for quantitative cancer risk assessment for
    aflatoxins. The author also concluded that although AFB1-DNA adducts
    generated in rats, mice and humans reflect the "molecular dose" and
    DNA damage in the target organ, bypassing the need for interspecies
    pharmacokinetic dose adjustments, it is not possible to extrapolate
    from rodents to humans at this time because human DNA adduct data are
    incomplete.

    2.2.7  Special studies on glucose tolerance

         Glyoxalase-1 activity plays an important role in glucose
    metabolism and has been reported to be depressed in mice fed low
    levels of AFB1 (Ankrah, 1995). In the present study, glyoxalase-1
    activity, glucose tolerance and pancreatic beta cell sensitivity were
    examined in mice (male and female ddy) fed 0.045 ng AFB1 plus 0.450 ng
    ABG1/g feed prenatally and for 6 months after birth. After glucose
    challenge, the ratios between 0- and 2-hour serum glucose levels were
    significantly higher than controls, indicating an increase in
    tolerance of glucose in the aflatoxin-fed mice with lower glyoxalase-1
    activity. Pancreatic beta cell sensitivity to stimulation by
    tolbutamide was similar in both groups. However, liver malonic
    dialdehyde was significantly higher in the aflatoxin-fed mice,
    suggesting that the altered tolerance for glucose in the aflatoxin-fed
    mice might be a consequence of aflatoxin-mediated peroxidative action
    in the liver, the authors suggested.

    2.2.8  Special studies on effect of ammoniation of AFB1 in
    contaminated cottonseed

         The effectiveness of ammonia in inactivating aflatoxin in
    contaminated cottonseed was investigated (Bailey  et al., 1994). Two
    aflatoxin-contaminated cottonseed lots were treated separately using
    atmospheric pressure, ambient temperature ammoniation procedure (APAT)
    or a high pressure, high temperature ammoniation procedure (HPHT), and
    incorporated into dairy cow rations. Isocalorific diets containing 25%
    defatted, dried milk from cows fed aflatoxin-contaminated cottonseed
    without or with APAT or HPHT treatment, or an aflatoxin-free human
    grade commercial milk powder, were then fed for 12 months to rainbow
    trout  (Oncorhynchus mykiss). AFM1 concentrations in milk powders
    without and with seed treatment were: APAT, 85 and <0.05 g/kg; HPHT,
    32 and <0.05 g/kg. In the APAT experiment, trout consuming the diet
    containing milk from cows fed the aflatoxin-contaminated cottonseed
    had a 42% incidence of hepatic tumours; APAT cottonseed treatment
    reduced this to 2.5%. Positive controls were included to demonstrate
    trout responsiveness. AFB1 fed continuously for 12 months at 4 g/kg
    resulted in a 34% tumour incidence, whereas positive controls fed 20
    g AFB1/kg, 80 g AFM1/kg, or 800 g AFM1/kg for 2 weeks and killed 9
    months later had a 37, 5.7 and 50% incidence of tumours, respectively.

         The authors concluded that APAT ammonia treatment of
    aflatoxin-contaminated dairy cattle cottonseed feedstock abolished the
    detectable transfer of AFM1 or AFB1 into milk powder, and greatly
    reduced the carcinogenic risk posed by any carry-over of aflatoxins or
    their derivatives into milk.

         In addition, the results confirm AFM1 to be a lower level
    hepatocarcinogen in comparison with AFB1 in the trout carcinogenicity
    assay. In the separate HPHT experiment, no tumours were observed in
    the livers of trout fed diets containing milk from either the
    ammonia-treated or untreated source, or the control diet containing 8
    g AFM1/kg. Positive controls fed 64 g AFB1/kg for 2 weeks exhibited
    a 29% tumour incidence 12 months later. Thus in this experiment,
    neither AFM1 at 8 g/kg nor any HPHT-derived aflatoxin derivatives
    that might have been carried over into milk represented a detectably
    carcinogenic hazard to trout, the authors conclude.

    2.2.9  Special studies on aflatoxin and hepatitis B virus infection in
    woodchucks, ducks, ground squirrels and tree shrews

         Interactive hepadnavirus and chemical hepatocarcinogenesis has
    been studied in woodchucks inoculated as newborns with woodchuck
    hepatitis virus (WHV), which is closely related to the human hepatitis
    B virus (Bannasch  et al., 1995). When the woodchucks reached 12
    months of age, AFB1 was administered in the diet at dose levels of 40
    g/kg bw per day for 4 months and subsequently 20 g/kg bw per day
    (5 days/week) for a lifetime. WHV DNA was demonstrated by Southern
    blot hybridization in the serum and by PCR in the serum and/or liver
    tissue. The histomorphology and cytomorphology of the liver were

    investigated by light and electron microscopy. WHV carriers with and
    without AFB1 treatment developed a high incidence of preneoplastic
    foci or altered hepatocytes, hepatocellular adenoma and hepatocellular
    carcinomas that appeared 6-26 months after the beginning of the
    combination experiment. Administration of AFB1 to WHV carriers
    resulted in a significantly earlier appearance of hepatocellular
    neoplasms and a higher incidence of hepatocellular carcinomas compared
    to WHV carriers not treated with AFB1. Neither hepatocellular adenomas
    nor carcinomas (but preneoplastic foci of altered hepatocytes) were
    detected in woodchucks receiving AFB1 alone, and no preneoplastic or
    neoplastic lesions were found in untreated controls.

         The authors pointed out that these results provide conclusive
    evidence of a synergistic hepatocarcinogenic effect of hepadnaviral
    infection and dietary AFB1. The striking similarities in altered
    cellular phenotypes of preneoplastic hepatic foci similarities in
    altered cellular phenotypes of preneoplastic hepatic foci emerging
    after both hepadnaviral infection and exposure to AFB1 suggested
    closely related underlying molecular mechanisms that may be mainly
    responsible for the synergistic hepatocarcinogenic effect of these
    oncogenic agents.

         In addition, the authors observed that the decisive role of the
    chronic WHV infection for hepatocarcinogenesis became particularly
    evident in those animals that seroconverted after 1 year and showed
    neither a chronic active hepatitis nor hepatocellular neoplasms, no
    matter when AFB1 was given. From this observation, the authors
    concluded that chronic hepatitis is not an absolutely necessary
    condition for the development of HCC in WHV carriers.

         To determine whether p53 mutations are common to HCCs of hosts
    infected with related hepadnaviruses with and without treatment with
    aflatoxin, Rivkina  et al. (1994) studied the occurrence of mutations
    in the p53 gene in HCCs of ground squirrels and woodchucks with a
    history of infection with ground squirrel hepatitis virus (GSHV) and
    woodchuck hepatitis virus (WHV). Sequencing of wild type p53 genes
    from ground squirrels and woodchucks revealed remarkable homology
    between the two species; using direct polymerase chain reaction
    sequencing, the investigators analysed the state of the p53 gene in 20
    HCCs from ground squirrels (2 uninfected, 7 with past and 11 with
    ongoing infection with GSHV) and in 11 HCCs from woodchucks
    persistently infected with WHV. Five GSHV carrier and two uninfected
    ground squirrels received i.p. administration of AFB1. Only one
    mutation - located in codon 176 of exon 5 - in the p53 gene of the
    tested animals was detected and that in a GSHV-positive ground
    squirrel treated with AFB1. The investigators suggested that in view
    of the considerably lower apparent rate of mutations in comparison to
    human HCCs, other etiological factors may be of greater significance
    in the development of HCC in ground squirrels and woodchucks.

         The unique mutation from G to T at the third base in codon 249
    observed in human hepatocellular carcinoma has been suggested to be

    linked to aflatoxin exposure. Imazeki  et al. (1995) studied six
    ducks with HCC, three of which were infected with duck hepatitis B
    virus and five of which were fed a diet containing AFB1 for 1-2 years.
    Liver tissues were analysed for the presence of point mutations at
    this codon of the p53 gene by polymerase chain reaction and direct
    nucleotide sequencing. None of the six ducks with HCC showed the
    change at this codon regardless of duck hepatitis B virus infection.
    Integration of duck hepatitis B virus DNA into the host genome was not
    observed in two ducks that were chronically infected with the virus
    and treated with AFB1. A third duck from Qitong Province in China,
    where HBV and AFB1 are risk factors for HCC in humans, did show viral
    integration. This suggested, in the opinion of the authors, that AFB1
    itself might not be involved in the unique mutation at codon 249 in
    hepatocarcinogenesis, or that other factors coincident with aflatoxin
    may be responsible for this unique mutation.

         Cova  et al. (1996) used a Pekin duck model to examine the
    effect of congenital duck hepatitis B virus (DHBV) infection and AFB1
    exposure in the induction and development of liver cancer. The study
    of the two major risk factors in the development of HCC, i.e.,
    persistent hepatitis virus infection and exposure to dietary
    aflatoxins, has been hampered by lack of an animal model, and these
    experiments were undertaken to this end. AFB1 was administered to
    groups of 13 DHBV infected or non-infected ducks at two doses (0.08
    and 0.02 mg/kg) by i.p. injection once a week from the third month
    posthatch until they were sacrificed 2.3 years later. Two control
    groups of ducks not treated with AFB1 (one of which was infected with
    DHBV) were observed for the same period. Higher mortality was observed
    in ducks infected with DHBV and treated with AFB1 compared to
    non-infected ducks treated with AFB1 and other control ducks. In the
    groups of non-infected ducks treated with high and low doses of AFB1,
    liver tumours developed in 3 out of 10 and 2 of 10 ducks,
    respectively. In infected ducks treated with the high dose, 3 of 6
    showed liver tumours; there were none with the low dose of AFB1. No
    liver tumours were observed in the two control groups. Ducks infected
    with DHBV and treated with AFB1 showed more pronounced periportal
    inflammatory change, fibrosis and focal necrosis compared to other
    groups. All DHBV carrier ducks showed persistent viraemia throughout
    the observation period. An increase of viral DNA titres in livers and
    sera of AFB1-treated animals compared to infected controls was
    frequently observed.

         No DHBV DNA integration into the host genome was observed,
    although in one hepatocellular carcinoma from an AFB1-treated duck, an
    accumulation of viral multimer DNA forms was detected. Unlike the
    situation observed for woodchuck and ground squirrel, HCC has rarely
    been associated with DHBV infection or integration of viral DNA in the
    duck. HCC has to date been reported only in Chinese ducks from
    Chi-tung County, not always associated with detectable virus, and with
    only a single reported case of integrated DHBV. Colonies of
    DHBV-infected ducks from other parts of the world do not develop HCC.
    Prevalence of liver tumours observed in Chi-tung County ducks

    reportedly correlated with the AFB1 food contamination and with the
    incidence of primary liver cancer in these areas.

         The authors observed a lower level of AFB1 binding to liver DNA
    and plasma protein in the DHBV-infected ducks compared to non-infected
    ducks after a single dose of AFB1; this finding appeared inconsistent
    with the hypothesis that DHBV infection could increase the metabolic
    activation of AFB1, as has been observed in woodchucks and in some
    human data. The investigators noted that their observations were made
    at a single dose at a single exposure and using one specific age of
    ducks; all of these factors could have influenced the AFB1-DNA adduct
    level.

         Yan  et al. (1996) reported the successful establishment of an
    animal model in tree shrews  (Tupaia belangeri chinensis) captured
    from the wild and experimentally infected with human hepatitis B
    virus. In animals exposed to AFB1 and infected with HBV, the incidence
    of HCC was significantly higher than in the animals solely infected
    with HBV or exposed to AFB1. AFB1-exposed animals received a total
    dosage of 15-16 mg/animal. No HCC or precancerous lesions were found
    in the controls that were neither HBV-infected nor AFB1-exposed. HBV
    DNA and the protein it encodes were detected in the cancer cells
    and/or the surrounding hepatocytes. Integration of HBV DNA into the
    host liver genome was found during hepatocarcinogenesis among the
    animals infected by HBV.

         The investigators pointed out that the cumulative dose of AFB
    used in their experiment was much lower than those (24-66 mg/animal)
    used in previous experiments on tree shrews where HCC was seen. This
    suggested that HBV infection might increase the hepatocarcinogenic
    effect of AFB1. The occurrence of precancerous GT foci in the tree
    shrews exposed only to AFB1 was much more frequent than in those
    infected by HBV alone. Among the animals exposed to the same dose of
    AFB1, the  gamma-glutamyltranspeptidase (GGT) foci were more numerous
    and larger in HBV-infected than in uninfected animals during the late
    state (after the 83rd week), but not at the early state. This suggests
    that, although both AFB1 and HBV may induce GGT foci and have a
    synergistic effect, the effect of HBV is weaker and slower than that
    of AFB1.

    2.2.10  Observations in humans

    2.2.10.1  Biomarkers of aflatoxin exposure

         A key issue in the use of aflatoxin biomarkers is whether the
    ratio of AFB1-albumin adduct to DNA adduct suggested in rodent
    experiments is the same in humans (Wild  et al., 1996). Direct
    evidence for this is not available due to the limitation of measuring
    DNA adducts in human liver. The human populations where AFB1 intake
    vs. AFB1-albumin adduct relationship has been examined are populations
    in which aflatoxin intake is relatively high. Examination of the
    relationship in low-exposure populations would be important to test
    whether the linear dose-response relationships seen in rats at

    exposures as low as 1 ng AFB1/kg bw are also observed in man. There
    are some data discussed in Wild  et al. (1996) indicating that the
    amounts of AFB1 intake bound to albumin are similar for rats and
    humans; assuming that the majority of AFB1-DNA adducts are formed in
    liver, then the initial ratio between the serum albumin and liver DNA
    adducts would be expected also to be similar in humans and Fischer
    rats. However, the capacity of human intestine to metabolize AFB1 must
    be further explored to clarify this point.

         It would appear that the AFB1-albumin adduct in peripheral blood
    is a reliable marker of AFB1-DNA adducts in the liver in rodents (Wild
     et al., 1996). Both of these parameters are at least qualitatively
    associated with species susceptibility to AFB1 hepatocarcinogenesis.
    Cross-species extrapolation to man suggests that the amount of
    AFB1-albumin formed for a given exposure more closely approximates
    that in the sensitive species rather than the resistant, and indicates
    that the Fischer rat may be a more appropriate model than the mouse
    for molecular dosimetry studies of AFB1 when, for example, validating
    approaches for chemoprevention studies.

         However, carcinogenesis is a multistep process; as pointed out in
    Wild  et al. (1996) AFB1-albumin adduct is acting as a surrogate
    marker only for one critical step, the formation of AFB1-DNA adducts
    in the target cell. The relationship between this marker and the
    genetic consequences of exposure as well as the quantitative
    association with HCC risk in man remain to be determined. In addition,
    HBV and possibly HCC infection, are major risk factors. The
    availability of more reliable markers of biologically effective dose
    of AFB1 should contribute to improving attempts to understand the
    mechanism of interaction between these two and other risk factors.

    2.2.10.2  Mutations in p53 tumour-suppressor gene in human
    hepatocellular carcinoma

         Molecular epidemiological studies have found that a G to T
    mis-sense mutation at the third base of codon 249 of the p53 gene,
    effecting an arginine to serine substitution, occurs in high frequency
    (up to 67%) in human liver tumours in regions with high risk of
    aflatoxin exposure, but not in regions of low aflatoxin exposure
    (Ozturk, 1991). Hsieh & Atkinson (1995) performed experiments to
    confirm this, using liver tissue from liver cancer patients in Taiwan
    and Japan. This was analysed for the presence of aflatoxin-DNA adducts
    (ADA) as a marker for aflatoxin exposure and an AGG to AGT
    transversion at codon 249 of the p53 gene. Ten per cent of samples
    containing ADA, indicating definite exposure of the subjects to
    aflatoxin, were found to harbour the codon 249 mutation, whereas 18%
    of the samples with no detectable adducts also contained the mutation.
    Since the presence of ADA in the liver tissue samples is an indication
    of definite recent exposure of the liver cancer patients to aflatoxin,
    these data indicated that the codon 249 mutation is not a high
    frequency event associated with recent aflatoxin exposure. If recent
    exposure to aflatoxin is indeed involved in the late stage
    hepatocarcinogenesis, these data suggested that it is through some

    mechanism other than codon 249 mutation. If either mutation at codon
    249 of the p53 gene or exposure to aflatoxin is involved in earlier
    stages of hepatocarcinogenesis, whether codon 249 of the p53 gene is a
    "hot spot" for aflatoxin attack could be shown by the present
    experiment, the authors concluded.

         The tumour suppressor p53 exerts important protective functions
    towards DNA-damaging agents (Gerbes & Caselmann, 1993). Its
    inactivation by allelic deletions or point mutations within the p53
    gene as well as complex formation of wildtype p53 with cellular or
    viral proteins is a common and crucial event in carcinogenesis.
    Mutations increase the half-life of the p53 protein allowing the
    immunohistochemical detection and anti-p53 antibody formation.
    Distinct  G to  T mutations in codon 249 leading to a substitution
    of the basic amino acid arginine by the neutral amino acid serine are
    responsible for the altered functionality of the mutation gene product
    and were originally identified in 8 of 16 Chinese and 5 of 10 African
    HCC patients, both groups living in regions with traditionally high
    exposure to mycotoxins. None of these mutations was detectable in 20
    patients with HCCs recently studied in the United Kingdom; only two of
    13 HCC DNAs from Germany displayed a C to T and a T to A transversion,
    respectively, in codons 257 or 273, but not in codon 249. An average
    p53 gene nutation rate of 25% is currently assumed for high-AFB1
    exposure regions; this is double the rate observed in low-AFB1
    exposure countries. The authors concluded that although many HCC
    patients displaying P53 mutations also suffer from HBV infection,
    which itself can lead to rearrangement of P53 coding regions or induce
    the synthesis of viral proteins possibly interacting with p53, the
    specific G to T transversion within codon 249 of the P53 gene seems to
    directly reflect the extent of AFB1 exposure and is not pathognomonic
    for all HCCs.

         Yap  et al. (1993) analysed 24 HCC liver biopsy samples from
    patients in Durban, South Africa, for p53 mutations and HBV infection.
    One patient was negative for HBV (Hbsag, anti-HBcAb, anti-HBsAb) and
    possessed the p53 249 mutation (which results in an arginine to serine
    substitution). The authors suggested that HBV infection or integration
    increases the likelihood of, but is not essential for, this p53
    "hot-spot" mutation in HCC. AFB1 or other as yet unidentified
    environmental carcinogens and cofactors are implicated; the mechanisms
    by which cells exposed to these agents acquire such a specific
    mutation and then expand clonally remains to be elucidated.

         The subject of the mutation at codon 249 of the p53 tumour
    suppressor gene has continued to be the subject of much research.
    Fifty-eight per cent of HCCs from Quidong, China, contain this
    mutation which is rarely seen in HCCs from Western countries (Aguilar
     et al., 1994). The population of Qidong is exposed to high levels of
    AFB1 and this toxin has been shown to induce the same mutation in
    cultured human HCC cells. To investigate the role of AFB1 and of these
    p53 mutations in hepatocarcinogenesis, normal liver samples from the
    USA (5), Thailand (3), and Qidong (14) (where AFB1 exposures are
    negligible, low, and high, respectively), were examined for p53

    mutations. The frequency of the AGG to AGT mutation at codon 249
    paralleled the level of AFB1 exposure, which supports the hypothesis
    that this toxin has a causative - and probably early - role in
    hepatocarcinogenesis. However, a role for other carcinogens cannot be
    ruled out, the authors point out; bulky heterocyclic amines in cooked
    foods and oxidants released by inflammatory leukocytes possess the
    same specificity for G to T transversion and HBV infection is
    associated with inflammation. All of the liver samples from Qidong and
    Thailand were from HBV-infected individuals.

         The presence of elevated frequencies of codon 249 AGT mutations
    in the non-malignant tissue of HCC patents from Qidong suggested that
    the mutagenic event occurred early in hepatocarcinogenesis. In
    contrast, p53 mutations in HCCs from geographic areas with low
    exposure to AFB1 could be late events. For example, p53 mutations have
    been observed more frequently in large tumours and in advanced grades
    of malignancy in HCCs from Japan. In other organs, such as the colon
    and the bladder, p53 mutations are thought to occur late in
    tumorigenesis. However, the methods used in previous work may not have
    been sensitive enough to detect mutations at early stages of
    tumorigenesis.

         Fujimoto  et al. (1994) tested the hypothesis that exposure to
    AFB1 alone or coincident with other environmental carcinogens might be
    associated with allelic losses occurring during development of human
    hepatocarcinogenesis (HCC) in China. The HCCs were obtained from two
    different areas in China: Qidong, where exposure to HBV and AFB1 is
    high; and Beijing, where exposure to HBV is high, but that to AFB1 is
    low. Tumours were analysed for mutations in the p53 gene and loss of
    heterozygosity for the p53, Rb and APC genes and at marker loci on
    chromosomes 4, 13 and 16. The data indicated that mutation and/or loss
    of heterozygosity in the p53 gene, independent of the 249 mutation,
    played a critical role in the development of HBV-associated HCCs in
    China. The authors postulated that different mechanisms appeared to be
    responsible for the development of HCC in Beijing and may have
    resulted from exposure to unknown environmental carcinogens or a
    different subtype of HBV. Also, the results demonstrated that multiple
    alterations in DNA located on different chromosomes may be involved in
    the development of HCC.

         Additional support for the etiological role of AFB1 in
    hepatocarcinogenesis in regions of the world with AFB1-contaminated
    food has come from the studies of Aguilar  et al. (1993). These
    investigators studied the mutagenesis of codons 247-250 of p53 by rat
    liver microsome-activated AFB1 in human HCC cells HepG2 by restriction
    fragment length polymorphism/polymerase chain reaction genotypic
    analysis. AFB1 preferentially induced the transversion of G to T in
    the third position of codon 249, and also induced G to T and C to A
    transversions into adjacent codons, albeit at lower frequencies. Since
    the latter mutations are not observed in HCC, the investigators
    concluded that both mutability on the DNA level and altered function
    of the mutant serine 249 p53 protein are responsible for the observed
    mutational hot spot in p53 HCC from AFB1-contaminated areas. The fact

    that this mutation is only rarely found in HCC from low AFB1 regions
    indicates that it is not a prerequisite for hepatocarcinogenesis;
    perhaps HBV and the mutant serine 240 p53 protein play a synergistic
    role.

         In a later study, Aguilar  et al. (1995) examined normal liver
    samples from the USA, Thailand and Qidong, where AFB1 exposures are
    negligible, low and high, respectively, for p53 mutations. The
    frequency of the AGG to AGT mutation at codon 249 paralleled the level
    of AFB1 exposure, which, according to the authors, provides additional
    support for the hypothesis that this toxin has a causative and
    probably early role in hepatocarcinogenesis.

         Hulla  et al. (1993) analysed the p53 gene at the site
    corresponding to codon 249 of the human gene in AFB-induced
    preneoplastic hepatic nodules from rats. No mutations were detected in
    the tissues examined. Thus, at least in the rat, the authors suggested
    that AFB exposure alone may not be sufficient for the specificity of
    p53 mutations observed in HCC. The selective mutations have been
    identified only in populations at risk for hepatitis B; it is possible
    that both AFB1 and chronic hepatitis are essential for mutation at
    codon 249 in the human p53 gene.

         In another study in rats, Liu  et al. (1996) looked at the
    effects of AFB1 on the p53 locus at the preneoplastic stage of rat
    liver oncogenesis. Male Wistar rats received a single dose of 1.5 mg
    AFB1/kg bw by a gastric tube. Liver biopsies over a period of one year
    were examined for aberrations of the p53 gene together with the
    expression of placental GST, a marker for preneoplasia.
    Immunohistochemistry, Western blot, polymerase chain
    reaction-single-strand conformation polymorphism and DNA sequencing
    techniques were used. AFB1 induction resulted in GST overexpression,
    forming GST-positive multi-foci and nodules of hepatocytes but no
    aberrations in the p53 expression and the microstructure of exons 5-8
    of the p53 gene. Thus, the authors concluded that p53 mutations might
    not occur at this early stage of AFB1-induced hepatocarcinogenesis.

         Shi  et al. (1995b) characterized p53 mutations in 44
    hepatocellular carcinomas from Chinese patients residing in a
    high-incidence area. In contrast to HCCs from other high HCC incidence
    areas with endemic aflatoxin exposures, in which codon 249 is a
    mutational hotspot, no mutations were observed at codon 249. The
    authors concluded that risk factors other that dietary exposure to
    aflatoxin may contribute to the high HCC incidence in Singapore.

         Liang (1995) recently reviewed the relationship of p53 proteins
    and AFB1. He pointed out firstly that the murine mutant p53 gene
    p53Ser249 appears to have a hepatocyte-specific phenotype, which
    suggests that this gene may interact with cellular factors(s) in a
    liver-specific manner to alter the growth property of hepatocytes. It
    is not known if the human form of p53Ser249 exhibits the same
    properties. Secondly, cooperative interaction of this p53 mutation and
    viral-induced cellular changes are probably involved in the

    transformation of hepatocytes in situations where aflatoxin exposure
    and hepatitis viral infection are evident. Recent studies of
    non-aflatoxin-associated HCC showed that p53 mutations are not as
    common as other human malignancies. This difference could be explained
    by the relatively low proliferation rate of hepatocytes as compared
    with other epithelial cells, such as colonic mucosa and mammary gland.
    Because p53 plays a critical role in "damage control" of proliferating
    cells and in regulation of abnormal proliferation, it is reasonable to
    speculate that p53 mutations may play a lesser role in
    hepatocarcinogenesis. However, dysregulated p53 function may still be
    an important step in this process, in view of the recent observation
    that HBX protein encoded by HBV appears to interact with p53 and
    inhibit its function. 

         Thirdly, it has not been possible to induce the same p53 mutation
    with aflatoxin exposure in a murine model, which casts a shadow of
    doubt on the applicability of studies in the murine model to human
    hepatocarcinogenesis. Liang (1995) recommended using human p53 genes
    to perform parallel experiments.

         Harris (1995) has also reviewed this subject. He reiterated that
    in high-incidence liver cancer areas such as China and Mozambique, the
    high frequency of G:C to T:A transversions in human hepatocellular
    carcinomas in this region could be due to the high mutability of the
    third base of codon 249 by AFB1 or a selective growth advantage of
    hepatocyte clones carrying this specific p53 mutant in liver
    chronically infected with HBV. The third base of codon 240 in a human
    liver cell line exposed to AFB1 has been shown to be preferentially
    mutated, and transfected 240Serp53 mutant enhances the growth rate of
    the p53 null hepatocellular carcinoma cell line Hep3B.

         The hypothesis that some of the mutations observed in the p53
    tumour-suppressor gene may be specific markers of exposure to
    aflatoxin may represent a real breakthrough in the field of liver
    cancer epidemiology. In particular, the confirmation of the
    specificity of the p53/aflatoxin association could be useful in
    assessing and quantifying the responsibility of aflatoxin as an
    independent cause of liver cancer and in evaluating the likely
    interactions with the hepatitis viruses in humans. A word of caution
    should be raised regarding the interpretation of the early studies
    because of: 1) the small sample size and limited methodology as to the
    criteria of specimen inclusion; 2) inadequate adjustment of the
    correlations for exposures to other viral and non-viral risk factors
    at the individual level; 3) limited information on the sensitivity and
    specificity of the proposed genetic markers; in particular, some
    animal data and cell system data are inconsistent in showing a
    specific association between p53 codon 249 mutations and previous
    exposure to aflatoxin; and 4) insufficient knowledge of the additional
    genetic changes in p53 and other genes (i.e., N-ras, C-myc, c-fos,
    alpha-TGF) associated with liver cancer development.

    2.2.10.3  Epidemiology of primary liver cancer

     (a) Descriptive epidemiology.

         Liver cancer is a disease prevalent in some of the developing
    parts of the world. It is frequent in China, South East Asia and
    subsaharan Africa. In some of these regions, like the Qidong area in
    Southern China, liver cancer is the major cause of death to cancer
    among men. It is relatively common in Japan and in the countries in
    the Mediterranean basin and it is rare in the Americas and Northern
    Europe. Pockets of high risk populations have been described in the
    Amazonian basin, among Eskimos and in special populations like the
    renal transplant patients. The incidence of liver cancer is
    consistently higher in men than in women with a sex ratio ranging from
    2 to 3 in most countries. Within countries, further variation in
    incidence rates is observed across cancer registries, men showing
    greater variation than women. Worldwide, the incidence of liver cancer
    in men and women shows a strong correlation.

         Migration from high risk areas to lower risk areas tends to
    reduce the risk to the levels of the host country, and this is
    observable within first and second generations.

     (b) Etiology

         The etiology of primary liver cancer is nowadays largely
    understood. Table 1 summarizes the range and the point estimates of
    the attributable fractions in two different settings, the low-risk
    areas in Europe and the USA and the high-risk areas in Africa and
    Asia.

         In both scenarios, viral infections to hepatitis B or C virus are
    associated with liver cancer in a range from 65% to 100% of cases. In
    low-risk countries HBV predominates and the other relevant factors are
    alcohol, tobacco and oral contraceptives. In high-risk areas HBV
    predominates and aflatoxins play a role, although quantification has
    been difficult.

         The evidence points to a synergistic interaction between HBV and
    AF in the etiology of liver cancer and some debate exists as to the
    independency of AF as an etiologic agent in humans.

         It is noteworthy that the large majority of the available
    epidemiological studies including data on aflatoxin exposure are based
    on high-risk countries where both HBV and AF are highly prevalent.
    Since the nature of the interaction at low levels of exposure is
    unknown, extrapolation of results from available studies to other
    settings is questionable.

    In addition to these established factors, studies have identified
    other factors that may modulate the incidence of the disease. Risk
    factors identified are the use of contaminated drinking-water, liver
    flukes and severe malnourishment.  Protection from liver cancer has



        Table 1.  Causal factors of liver cancer and estimates of the attributable fractions

                                                                                          
    Factor                        Low-risk countries               High-risk countries
                              Japan Europe and the USA               Africa and Asia
                         Estimate   Range    Estimate   Range      Estimate      Range
                                                                                          

    Hepatitis B            <15%     4-50%       20%     18-44%       60%         40-90%
    Hepatitis C3            60%     12-64%      50%     40-80%      <10%         NE
    Aflatoxin             limited exposure     limited exposure     important exposure1
    Alcohol                <15%4               <20%     11-30%5      NE
    Tobacco                <12%4                40%     38-51%5      NE
    Oral contraceptive    10-50%2               NE                   NE
    Other                  <5%                                      <5%
                                                                                          

    1  Attributable risk not quantified. One study suggested attributable fraction close 
    to 50%.
    2  Restricted to liver cancer in women. Likely to increase in future generations. 
    Uncertain if hepatitis infections (notably HCV) are necessary co-factors.
    3  Not including double infections with HBV and HCV. Very few studies available using 
    second-generation assays.
    4  Estimates for the USA
    5  Estimates from three studies of LC in men

    NE non evaluated.
    Note: attributable fractions do not necessarily add to 100% due to multiple exposures and 
    possible interactions between risk factors.

    Adapted from CDC, 1989; Bosch & Muoz, 1991; Thomas, 1991; Tanaka et al., 1993; IARC, 
    1994; Bosch, 1995
    


    been reported in the case of diets rich in retinol and protein.
    Associations have been reported between liver cancer and blood
    testosterone levels, HLA types, and predisposition due to
    polimorphisms in some of the SGT and CYT metabolic regulatory genes.

     (c) Vaccination against HBV as a preventive measure against liver
    cancer

        In 1983, the World Health Organization proposed as a medium-term
    objective trials of immunization against hepatitis B to prevent liver
    cancer. Since then more than 70 countries have introduced HBV
    vaccination into their routine vaccination schemes. A recently
    published study in Taiwan (Chang  et al., 1997) has described the
    rigorous application of universal immunization against hepatitis B and
    the prevention of the carrier state in children; these data provide
    further evidence of a direct causal relationship between HBV and liver
    cancer.

        The immunization programme against hepatitis B in Taiwan, an area
    of hyperendemic infection and moderate to high aflatoxin exposure,
    reduced the rate of HBV carriage in six-year-old children from about
    10% in the period from 1981 to 1986 to between 0.9 and 0.8% in the
    period from 1990 to 1994. The drop in the rate of carriage occurred as
    the proportion of infants immunized against hepatitis B increased from
    15% of children born to mothers at high risk during the earlier period
    to 84-94% of all newborn infants during the later period. This
    significant reduction in the prevalence of hepatitis B surface antigen
    was accompanied by a decline in the average annual incidence of
    hepatocellular carcinoma in children 6 to 14 years of age, from 0.7
    per 100 000 between 1981 and 1986 to 0.57 between 1986 and 1989 and
    0.36 between 1990 and 1994. The incidence of hepatocellular carcinoma
    in children 6 to 19 years old fell even more dramatically, from 0.52
    among those born between 1974 and 1984 to 0.13 among those born
    between 1984 and 1986. As the investigators pointed out, since the
    incidence of hepatocellular carcinoma in Taiwan peaks in the sixth
    decade of life, it may take 40 years or longer to see an overall
    decrease in the rate of hepatocellular carcinoma as a result of the
    vaccination programme.

        The Committee noted that studies like this one need to be observed
    carefully in coming years for the light they may shed on the
    relationship between aflatoxin, HBV and liver cancer.

     (d)    Effects of exposure to aflatoxins

        Ahmed  et al.(1995) undertook two prospective studies to
    determine a possible relationship between perinatal aflatoxin exposure
    and neonatal jaundice. First, cord blood samples from 37 neonates who
    subsequently developed jaundice and from 40 non-jaundiced (control)
    babies were analysed for six major aflatoxins and aflatoxicol.
    Peripheral blood samples of both groups were also analysed postnatally
    for aflatoxins. In a second study, serum aflatoxin levels of 64
    jaundiced neonates admitted from outside the hospital were compared

    with levels in 60 non-jaundiced control babies. Aflatoxins were
    detected in 14 (38%) cord blood samples of jaundiced neonates and in
    nine (23%) of the controls. The mean cord aflatoxin concentration was
    highest in jaundiced neonates with septicaemia, but the difference was
    not statistically significant. The frequency of detection of
    aflatoxins in peripheral blood was not significantly different in
    jaundiced and non-jaundiced babies. Aflatoxins were detected in the
    blood of over 50% of neonates with jaundice of unknown etiology. There
    was no correlation between severity of hyperbilirubinaemia and serum
    aflatoxin levels. Further studies are needed to determine the extent
    of pre-and postnatal exposure to aflatoxin in Nigerian infants and the
    effects of such exposure on fetal and neonatal health, the authors
    concluded.

        In October 1988, 13 Chinese children died of acute hepatic
    encephalopathy in the northwestern state of Perak in peninsular
    Malaysia (Lye  et al., 1995). Symptoms included vomiting,
    haematemesis, seizures, diarrhoea, fever and abdominal pain. All had
    liver dysfunction with increased aspartate aminotransferase and
    alanine aminotransferase levels greater than 100 IU/litre. The
    acuteness of the illness differed from previously reported outbreaks
    described in Kenya, India and Thailand; median incubation period for
    this outbreak was 8 hours, whereas the exposure was over a period of
    days to weeks of consumption of highly contaminated food such as maize
    in outbreaks in Kenya and India. Epidemiological investigations
    determined that the children had eaten a Chinese noodle,  loh see 
     fun, hours before they died. The attack rates among those who had
    eaten the noodles were significantly higher than those who had not 
    (P < 0.0001). The cases were geographically scattered in six towns in
    two districts along the route of distribution of the noodle supplied
    by one factory in Kampar town. Aflatoxins were confirmed in the
    postmortem samples from patients, but the noodles or their ingredients
    were not analysed for aflatoxins. The authors questioned the etiology
    of the outbreak.

        Ibeh  et al. (1994) examined the relationship between aflatoxin
    levels in serum of infertile men in comparison with random controls
    from the community. The subjects were 100 adult males, yielding 50
    semen samples, from men attending infertility clinics at a university
    teaching hospital and 50 normal men in the same community. The staple
    foods of the men were assayed for aflatoxin content. Aflatoxin was
    found in 20 semen samples from the infertile group (40 %) with a mean
    concentration of 1.7 g/ml and four samples from the fertile group
    (8%) with a mean concentration of 1.0 g/ml. Infertile men showed a
    higher percentage of spermatozoa abnormality (50%) than the fertile
    men (10-15 %).

        In a parallel experiment, adult male rats were given an aflatoxin-
    contaminated diet (8.5 g purified AFB1/g of feed) for 14 days while
    10 control age-matched rats were fed a normal aflatoxin-free diet
    during the same period. Seven days after the withdrawal of aflatoxin
    from the diet of test rats, two rats were randomly selected from the
    test and control groups, killed and their semen harvested from the

    epididymis and vasa deferentia and analysed. The process was repeated
    at weekly intervals until four rats were left in the test and control
    groups. Thereafter, four fertile adult female rats were introduced to
    mate with test and control rats, and rats were observed for 90 days
    with an adequate, non-aflatoxin-contaminated diet. Results
    showed that rats exposed to dietary aflatoxin experienced changes in
    spermatozoal profile which differed in a statistically significant
    manner from the control rats; test rats showed depression in the
    motility, viability and number of sperm cells which resembled features
    seen in semen of infertile men exposed to aflatoxin. The four test
    rats who were mated in the conclusion of the study were unable to
    effect conception of fertile female rats, while the four control rats
    were able to do so.

        The authors hypothesized that aflatoxin may affect the
    reproductive system by its toxic effect on the liver, leading to the
    desquamation of the membranes of hepatocytes, the mitochondria, the
    cytosol and the endoplasmic reticulum. This cellular damage could
    include inhibition of enzyme synthesis and/or enzyme activities or
    inhibition of lipid metabolism or fatty acid synthesis, which may
    derail the capacity of the hepatocytes to handle the conversion of
    intermediate biomolecules, such as precursor molecules for hormones,
    e.g., testosterone and progesterone. Depression or absence of normal
    hormone levels could cause a wide range of degenerative changes in
    sexual organs. Aflatoxin may also affect the male reproductive system
    by causing lysis of sperm cells as a result of constant reversible
    reaction with the mycotoxin, binding of the toxin to free and/or bound
    amino acids in the seminal fluid, depressing the motility of
    spermatozoa and the formation of aflatoxin adducts with nucleic acids,
    giving a risk of mutations of the spermatogonia.

        El-Nazami  et al. (1995) examined the exposure of infants to
    aflatoxin M1 (AFM1) and of lactating mothers to AFB1, using AFM1 in
    breast milk as a biomarker for exposure to AFB1. Prevalence of AFM1 in
    breast milk samples from 73 women from Victoria, Australia
    (low-exposure area) and 11 women from Thailand (high-exposure area)
    was also compared. Assays were done by both HPLC and by ELISA. AFM1
    was detected in 11 samples from Victoria and five samples from
    Thailand at median concentrations of 0.071 mg/ml (range 0.028 - 1.031)
    and 0.664 ng/ml (range 0.039 - 1.736). Levels of AFM1 were
    significantly higher in milk samples from Thailand than in milk
    samples from Victoria.

        Ankrah  et al. (1994) attempted to ascertain if the presumed
    intake of dietary aflatoxins (AFB1 and AFG1) has adverse effect on the
    liver; aflatoxins were measured in serum, urine and faecal specimens
    obtained from a group of 40 apparently healthy adults (11 females and
    29 males) from the Greater Accra region of Ghana. Liver status of the
    subjects was monitored with serum  alpha-fetoprotein (AFP),
    alpha-1-antitrypsin (AAT) and direct: total bilirubin ratio. Aliquots
    of serum were tested for HBsAg. AFG1, AFB1, AFQ1, and AFM1 were
    detected in one or more of the body specimens in 35% of the subjects
    (AFB1+ group). Sixty-five per cent of the subjects had only AFG1 in

    their body specimens (AFB-group). Serum levels of AFP (greater than 20
    ng/ml), AAT (greater than 170 ng/dl) and direct: total bilirubin ratio
    (greater than 0.5), which indicated absence of predisposition to liver
    cancer in all the subjects but were suggestive of liver inflammation,
    were noted in both the AFB+ and AFB1-subjects. None of the subjects
    had malaria or hepatitis B virus infection. The authors suggested that
    the pattern of distribution of the aflatoxins in the subjects
    indicates that the suspected liver inflammation may involve other
    factors and may not be only due to present intake levels of
    aflatoxins.

     (e) Epidemiological studies on dietary aflatoxins and liver cancer

        A number of important epidemiological studies have been published
    since the Committee's last review of aflatoxins at its thirty-first
    meeting (Annex 1, reference 77).

        Yeh  et al. (1989) examined the roles of the hepatitis B virus
    and AFB1 in the development of primary hepatocellular carcinoma (PHC)
    in a cohort of 7917 men aged 25 to 64 years old in southern Guangxi,
    China, where the incidence of PHC is among the highest in the world.
    After accumulating 30 188 person-years of observation, 149 deaths were
    observed, 76 (51%) of which were due to PHC. Ninety-one per cent (69
    of 76) were HBsAg+ at enrollment into the study in contrast to 23% of
    all members of the cohort. Three of the four patients who died of
    liver cirrhosis were also HBsAg+ at enrollment. There was no
    association between HBsAg positivity and other causes of death. Within
    the cohort, there was a 3.5-fold difference in PHC mortality by place
    of residence.

        To estimate AFB1 exposure, between 1978 and 1984, the Fusui Liver
    Cancer Institute regularly sampled and tested staple foods consumed in
    the counties of southern Guangxi for contamination by AFB1. Twice a
    year, samples of raw foods were collected from all over the region and
    analysed for AFB1 content by TLC. An estimated mean level was computed
    for each commune as follows. The yearly amount consumed of a given raw
    foodstuff was multiplied by the average AFB1 content as determined
    from tested samples of raw foodstuffs. These cross-product terms were
    then summed over all staple foods, and the resultant figure was
    divided by the total population to obtain an estimated intake per
    person per year. These population-based levels of AFB1 were correlated
    with mortality rates of PHC among members of the cohort by the
    communes from which the subjects were derived.

        When estimated AFB1 levels in the subpopulations were plotted
    against the corresponding mortality rates of PHC, a positive and
    almost perfectly linear relationship was observed. On the other hand
    the prevalence of HBsAg was very high and homogeneous across the study
    areas (range 21.6%-24.7%) and therefore, no significant association
    was observed when the prevalence of HBsAg positivity in the
    subpopulations was compared with their corresponding rates of PHC
    mortality. The authors conclude that despite the "crudeness" of their
    exposure estimate, (i.e., population-based instead of personal

    exposure assessments), it is reasonable to conclude that AFB1 seems to
    play a role in the unusually high rates of PHC in southern Guangxi. 

        The population prevalence of HBsAg is extraordinarily high in this
    study population, almost one in every four adult men being a
    positive carrier of HBV. Primary infection occurs very early in this
    high-risk population, possibly through vertical transmission from
    carrier mothers to infants during the perinatal period, based on a
    survey of serum HBsAg in children ages 1 to 9 years in a county
    adjacent to Fusui. Even though most cases of liver cancer in this
    study did not have histopathological confirmation, the authors
    indicate that probably all were PHCs.

        The Yeh  et al. (1989) report is an early important study showing
    that, in a region where HBV is highly prevalent and PLC is common, the
    HBV carriers are at very high risk. It further indicates that in an
    area of high AFB1 exposure, the PLC mortality rates are higher than in
    areas of lower AFB1 exposure. This study provides the basic
    information for most potency estimates. Most of the early correlation
    studies (with or without HBV consideration) are consistent with the
    basic conclusion of the study of Yeh  et al. (1989), but other
    studies are not (Campbell  et al., 1990; Hsing  et al., 1991).
    However, the study has the general limitations of correlation studies
    in which: i) exposure to AFB1 is estimated from raw foodstuffs
    available to populations and attributed to individuals; ii) the
    correlation between PLC and AFB1 was not adjusted for any of the
    possible confounders such as HCV, alcohol, tobacco or nutritional
    status as shown in Taiwan by Yu  et al. (1995); iii) HBV exposure may
    have been underestimated due to lack of use of PCR methodology; iv)
    HBsAg prevalence was measured in a 25% sample of the cohort and
    attributed to the region.

        Campbell  et al. (1990) conducted a comprehensive cross-sectional
    survey in the People's Republic of China of possible risk factors for
    primary liver cancer (PLC) to include 48 survey sites, an
    approximately 600-fold aflatoxin exposure range, a 39-fold range of
    HCC mortality rates, a 28-fold range of hepatitis B virus surface
    antigen (HBsAg+) carrier prevalence, and estimation of exposures for a
    large number of other nutritional, dietary and life-style features
    (Campbell  et al., 1990). PLC mortality was unrelated to aflatoxin
    intake, but was positively correlated with HBsAg+ prevalence, plasma
    cholesterol, frequency of liquor consumption, and mean daily intake of
    cadmium from foods of plant origin. Multiple regression analysis for
    various combinations of risk factors showed that aflatoxin exposure
    consistently remained unassociated with PLC mortality regardless of
    variable adjustment. In contrast, associations of PLC mortality with
    HBsAg+, plasma cholesterol, and cadmium intake remained, regardless of
    model specifications, while the association with liquor consumption
    was markedly attenuated (nonsignificant) with adjustment for plasma
    cholesterol.

        The authors commented on the lack of an association between
    aflatoxin exposure and PLC mortality in this study, in view of the
    findings of most previous investigations. The absence of an
    aflatoxin-PLC association was consistent with a similar lack of
    association of PLC mortality with the consumption of the two foods
    most commonly contaminated with aflatoxin i.e. maize and mouldy
    groundnuts. In contrast to the lack of an association with aflatoxin,
    PLC mortality was highly correlated with HBsAg+ prevalence and not
    with past HBV infection, as assessed by the prevalence of antibody to
    the HBV core protein. In this study, the association of plasma
    cholesterol with PLC mortality was even more consistent than the
    association of PLC mortality with HBsAg+ prevalence. Mortalities from
    colon cancer, rectal cancer, lung cancer, leukemia, brain cancer and
    total aggregate cancer are also known to be associated with plasma
    cholesterol. This association was even more surprising in China where
    plasma cholesterol in this cohort ranged up to about 190 mg/dl, which
    is near the low end of the range for comparable Western subjects (Chen
     et al., 1990).

        The authors offered several explanations for the lack of an
    association between aflatoxin intake and PLC mortality, which
    contrasts with the finding of other previous studies. First, Chinese
    people might respond differently to aflatoxin, perhaps because of
    unique genetic or environmental characteristics. This is unlikely
    given the previously shown positive association between aflatoxin
    intake and PLC mortality in Chinese subjects (Yeh  et al., 1985; Yeh
     et al., 1989) and because major ethnic differences in risk for other
    cancers are greatly reduced or eliminated after migration to new
    environments.

        A second argument could be that the lack of an effect in this
    study may have been because measurement of aflatoxin exposure during
    the survey period was not representative of past intakes when the
    cancers were forming. However, a similar limitation existed for all
    other Chinese studies; this study is more reliable, in the opinion of
    the authors, because it is based on urinary aflatoxin metabolite
    excretion which directly represents and integrates over a day or so
    actual consumption. In addition, aflatoxin contamination rates in a
    county in the Guangxi Autonomous Region were relatively stable during
    the years 1972-1983.

        A third line of reasoning suggests that aflatoxin may not be a
    significant human carcinogen, in the opinion of the authors. The
    present study has greater statistical power and more comprehensive
    range, diversity and inclusiveness of risk factors than other previous
    studies. Humans may also be resistant to aflatoxin carcinogenesis, a
    finding which is supported by  in vitro aflatoxin studies on species
    of varying resistance (Booth  et al., 1981). Humans may also be more
    refractory when consuming lower protein diets; whereas acute toxicity
    of aflatoxin is increased in protein-malnourished children (Hendrickse
     et al., 1982).

        The authors pointed out that data from animal studies have shown
    that when animals were fed either lower levels of animal protein (5-
    or the same level (20%) of plant protein after completion of aflatoxin
    dosing, development or preneoplastic lesions and tumours was markedly
    inhibited (Appleton & Campbell, 1983; Schulsinger  et al., 1989).
    Protein in the Chinese diet is primarily of plant or fish origin, as
    compared to protein in the USA diet which is primarily of animal
    origin (Food and Nutrition Board, 1989; Chen  et al., 1990). 

        The authors continued with a critique of previous aflatoxin
    epidemiology studies and offered the following model to explain the
    etiology of PLC. The vast majority of individuals who are susceptible
    to PLC are those who are persistently infected with HBV. Within this
    HBsAg+ population, additional risk is contributed chiefly by
    nutritional and dietary practices that enhance liver cell
    proliferation, such as diets containing significant amounts of animal
    protein. Aflatoxin may act as a carcinogenic initiator, but
    contributes only a very small proportion of the initiating activity
    routinely exposing the liver. Therefore, HBsAg+ is a necessary but
    insufficient cause of PLC, aflatoxin is an unnecessary and
    insufficient cause, and sustained nourishment causing liver cell
    proliferation (and elevated plasma cholesterol) is a necessary and
    insufficient cause for HBsAg negative carriers, but a necessary and
    sufficient cause for HBsAg positive carriers. Why is PLC so much more
    common in undernourished and impoverished societies? The authors
    concluded that PLC is more common because HBsAg+ carriers are more
    common.

        In evaluating the significance of this study by Campbell  et 
     al. (1990), a number of issues, both statistical and
    non-statistical, should be considered. For example, PLC rates were
    determined for the years 1973-1975 and the biochemical analyses
    (covariate ascertainment) was conducted in 1983. With regard to the
    statistical analysis presented in the paper, there is some indication
    that the sample data do not adequately satisfy the normality
    assumptions upon which the univariate correlation and multiple
    regression analyses are based. Finally, the urinary aflatoxin
    measurements were of total aflatoxin metabolites, which have been
    shown not to correlate well with levels of AFB1 consumed (Wild 
     et al., 1992; Groopman  et al., 1993).

     (f) What can we learn from epidemiological studies that considered
     HBV, HCV and AFB in relation to liver cancer?

        Viral hepatitis is a major worldwide public health problem. It is
    estimated that over 300 million individuals are chronically infected
    with HBV and perhaps 100 million with HCV. Chronic infection with
    either virus has been linked to cirrhosis and liver cancer. HBV is
    prevalent in the developing parts of the world, and HCV is emerging as
    a major cause of hepatocellular cancer in Japan and western societies
    (Table 1).

        Tests to detect HBV markers have increased in sensitivity, largely
    due to the use of the polymerase chain reaction (PCR) to amplify HBV
    DNA in serum and liver tissues. HBV infection has been shown to
    persist in the serum (49.7%) or in the liver cancer tissue (24.9%) in
    a number of patients with liver cancer that are at the same time HBSAg
    negative (Bosch & Muoz, 1991). This pattern has been documented in
    cases from areas at low and high risk for HBV infection.

        Although the significance of detecting low levels of HBV DNA in a
    patient with liver cancer is not fully understood, from an
    epidemiological viewpoint, these subjects could easily be classified
    as persistently exposed to HBV and grouped with the HBsAg carriers in
    computing risk estimates. Although data are sparse on the prevalence
    of equivalent markers in the general population, it is likely that
    among controls the prevalence of PCR-detected HBV DNA in the absence
    of any other HBV marker is extremely low. If this is the case, the
    case control studies that use PCR would increase the Risk Ratio
    estimates for HBV as well as the estimates of the Attributable
    Fraction. There are no case control studies that have used PCR methods
    (in serum or liver tissue) to detect HBV exposure.

        Ramesh & Panda (1993) have questioned the hypothesis that HBV
    causes chronic liver disease and liver cell carcinoma in
    HBsAg-positive individuals only. The presence of HBV in patients with
    HCC who are seropositive for the envelope antigen (HBsAg) is well
    established. Epidemiological studies have shown a small percentage of
    patients with HCC with past HBV infection, positive for anti-HBsAb or
    HBcAb. However, the role of HBV in HCC cases who seroconverted from
    HBsAg to HBsAb is unclear. The authors described a study of 36 HCC
    cases where four cases negative for HBsAg and with underlying
    cirrhosis were found. Biopsy tissue was investigated by polymerase
    chain reaction; all four samples tested were positive for a portion of
    the surface region (nucleotide position: 636-735), but were negative
    for the "X" and the "C" regions of HBV genome. Since hepatitis C virus
    (HCV) has been associated with HCC, the authors tested serum samples
    of the four cases for anti-HCV; one out of four was positive for
    anti-HCV. The authors concluded that these observations indicate that
    parts of the HBV genome can persist in liver cells of individuals who
    have recovered from clinical illness and seroconverted to HBsAg
    positive. However, the significance of these sub-genomic fragments in
    the development of HCC is not clear.

        The identification of HCV in the last decade has been a major step
    forward in the understanding of the origins of liver cancer and in the
    quantification of the proportion of cases related directly to viral
    infections (IARC, 1994). Epidemiological studies are largely
    consistent in showing a strong association between carriers of
    anti-HCV and liver cancer. The specific potential to induce PLC by
    each of the HVC types and variants of types as well as the impact of
    other factors from the host and the environment still requires further
    research. Likewise, few studies are available exploring the role of
    aflatoxin in the presence of HBV and HCV. High estimates of the
    Relative Risk for carriers of anti-HCV have also been reported in

    areas of HBV endemicity. The risk linked to HCV is independent of HBV
    and persons who are carriers of both HBsAg and anti-HCV are at a very
    high risk of developing liver cancer. HCV is likely to be the major
    cause of liver cancer in countries at low/intermediate risk like the
    USA and Europe.

     (g) Epidemiological studies including aflatoxins in countries where 
     the risk of liver cancer is low

        In countries where liver cancer is rare and aflatoxin exposure is
    low, most etiological studies on liver cancer have not considered
    aflatoxins as a risk factor. The populations with higher exposures are
    the workers occupationally exposed to grain dust in the animal feed
    processing plants. In studies conducted in the Nordic countries in
    Europe, Sweden, Denmark, the Netherlands and in the USA, aflatoxins
    have been isolated from dust samples and an excess of mortality of
    several such cohorts has been documented for liver cancer (risk, 2.4
    times the expected rates) liver and biliary tract cancer (risk, 2.5
    times the expected rates), lung cancer and lymphomas (risk, 1.5-3
    times the expected rates (Hayes  et al., 1984; Alavanja  et al., 
    1987; Olsen  et al., 1988). It should be noted that some of these
    studies did not evaluate other relevant exposures such as hepatitis
    infections and alcohol.

        It is of interest that few studies are available on liver cancer
    in Latin America. In this extensive region of the world, agricultural
    products are prone to mould growth, and consumption of maize is part
    of the staple food in many countries. Yet liver cancer is rare in
    these populations as is HBV infection. If that is the case, Latin
    America would be potentially a very informative field to investigate
    the occurrence of liver cancer in populations exposed to AF as the
    central risk factor.

     (h) Epidemiological studies that used biomarkers of exposure to 
     aflatoxins including studies on genetic susceptibility to aflatoxins

        Biomarkers have been developed and are being introduced in
    epidemiological studies with the purpose of increasing the accuracy of
    the assessment of exposure to aflatoxins. Various biomarkers have been
    developed, including urinary total aflatoxins, aflatoxin adducts in
    urine, aflatoxin albumin adduct in serum, aflatoxin adducts in liver
    cancer tissue and more recently p53 specific mutations in liver cancer
    specimens. Other studies are investigating genetic polymorphisms in
    some key genes involved in the metabolism of aflatoxin that may
    introduce some variability in the response to aflatoxin. (Groopman 
     et al., 1994; Wild  et al., 1996; IARC, 1997).

        The importance of the major aflatoxin-nucleic acid adduct,
    AFB-N7-guanine, in urine as a biomarker was enhanced by the finding
    that this metabolite is excreted exclusively in urine of exposed rats,
    thus simplifying pharmacokinetic considerations. The aflatoxin-albumin
    adduct in serum has also been examined as a biomarker of exposure;
    because of the longer half-life  in vivo of albumin compared to the

    urinary AFB-N7-guanine, the serum albumin adduct can integrate
    exposures over longer time periods. Data from human exposure studies
    have shown that the excretion of the urinary aflatoxin nucleic acid
    adduct and formation of the serum albumin adduct are highly
    correlated. In the rat, validation studies for the dose-dependent
    excretion of urinary aflatoxin biomarkers were conducted in rats
    following a single exposure to AFB1; excellent linear correspondence
    between oral AFB1 dose and excretion of AFB-N7-guanine in urine was
    shown (Scholl  et al., 1995).

        Aflatoxin metabolites in urine or adducts in serum can be a useful
    tool to evaluate exposure but with the currently available methods
    remain relatively short-term exposure markers; biomarkers are of much
    less use in predicting long-term or lifetime human exposure. As such,
    they reflect poorly the natural pattern of exposure to aflatoxin
    (i.e., seasonality, manual sorting of foodstuffs, age at exposure,
    etc.); therefore, it is not surprising that studies conducted using
    aflatoxin biomarkers as markers of exposure show conflicting results.

        At present, it is not fully understood how the functional status
    of the liver or the coexistence of other risk factors for liver cancer
    may affect the different biomarkers that are being proposed for
    epidemiological studies. Few studies have described the natural
    history of these markers in patients with chronic liver disease
    including chronic hepatitis and liver cirrhosis, conditions that
    usually precede liver cancer by months or years (Wild  et al., 1993;
    Wang  et al., 1996a). In this circumstance, the interpretation of the
    findings is complicated since the aflatoxin biomarker may be
    confounded by the presence of some risk factors, (e.g., HBV, HCV,
    alcohol), the presence of some protective factors (e.g., retinol in
    the diet) or by the presence of liver disease (i.e. chronic active
    hepatitis B infection).

        The mutations in the p53 gene claimed to be specific markers of
    exposure to aflatoxin are being actively investigated to confirm the
    strength and the specificity of the association in human populations
    (Harris, 1995). Recent data have been summarized earlier in this
    paper.

        The best evidence of an interaction between HBV and aflatoxin in
    the causation of human liver cancer is the cohort study in Shanghai
    (Ross  et al., 1992, Qian  et al., 1994; Yuan  et al., 1995). This
    is an ongoing prospective study of 18 244 middle-aged men in Shanghai,
    China. Assays for urinary AFB1, its metabolites AFP1 and AFM1 and DNA
    adducts have been performed to assess the relationship between
    aflatoxin exposure and liver cancer. After 35 299 person-years of
    follow-up, 22 cases of liver cancer had been identified. For each
    case, 5 or 10 controls were randomly selected from cohort members
    without liver cancer on the date the disorder was diagnosed in the
    case and matched to within 1 year of age, within 1 month for sample
    collection, and for neighbourhood of residence. Each subject provided
    a blood sample and a urine sample. A positive result was defined as
    the presence of at least 1 ng of an individual aflatoxin compound in

    the sample. Hepatitis B surface antigen was measured by a standard
    radioimmunoassay method.

        Subjects with liver cancer were significantly more likely than
    were controls to have detectable concentrations of any of the
    aflatoxin compounds; the strongest association was for AFP1.
    Positivity for HBsAg was strongly associated with risk of liver
    cancer. The authors concluded that their results are based on too few
    cases to give a reliable estimate of attributable risk, but they
    estimated that up to 50% of cases of liver cancer in Shanghai may be
    due to aflatoxin exposure.

        In further follow-up of the Shanghai study, Qian  et al. (1994)
    reported on 70 000 person-years of follow-up and 55 cases of HCC.
    Levels of urinary AFB1 and the oxidative metabolites, including the
    major aflatoxin nucleic acid adduct, aflatoxin-N7-guanine, were
    determined for 50 of the 55 identified cases of HCC; 267 controls were
    matched against the 50 cases as above. A nested case-control analysis
    showed highly significant association between the presence of at least
    one of the four urinary aflatoxin metabolites, serum HBsAg positivity
    and HCC risk. Risk was especially elevated in individuals who were
    positive for both of these biomarkers. However, the number of liver
    cancer cases in which the interaction was explored is small (i.e., 13
    cases of AFB1 positive and HBsAg negative), and there is room for
    misclassification of cases in relation to their viral exposure. Thus
    risk estimates become unstable and the barely significant increase in
    the ORs for the AFB1 exposed may be easily lost if only 2/3 of cases
    now considered HBV negative turn out to be HBV (or HCV) positive. On
    the other hand, a cohort analysis using all 55 cases of HCC revealed
    no statistically significant association between HCC risk and dietary
    aflatoxin consumption, as determined from the in-person food frequency
    interview combined with the survey of market foods in the study
    region, adding additional uncertainty of the value of the biomarkers
    used. HCV prevalence was low in this cohort (Yuan  et al., 1995).

        An exchange of correspondence on this Shanghai study has occurred.
    Campbell (1994) has pointed out that the HCC risk putatively
    attributed to aflatoxin in the study of Ross  et al. (1992) appears
    to be accounted for mostly by the urinary AFB-N7-guanine adduct.
    Instead Campbell postulates that this risk could also be caused by
    factors that enhance enzymatic activation of AFB1 by the hepatic P450
    enzyme system to produce more AFB-N7-guanine. These enzyme-inducing
    factors could readily be nutritional, especially those that also are
    associated with elevated plasma cholesterol. This interpretation would
    be in accord with the ecological study by Youngman  et al. (1992) in
    48 survey counties in China showing that the most significant and
    robust determinants of HCC risk were elevated cholesterol levels and
    HBsAg positivity, not aflatoxin. This finding is given further
    plausibility by animal experiments (Preston  et al., 1976; Hu 
     et al., 1994). These experiments have shown that  in vivo 
    activation of AFB1 to form hepatic DNA adducts could be markedly
    enhanced by a modest elevation in the intake of animal protein. This
    same modest animal protein intake that markedly elevates AFB1

    activation also markedly increased the over-expression of a hepatitis
    B virus transcript in mice.

        Ross  et al. have responded to the comments of Campbell (1994).
    There is an overwhelming amount of experimental data across species
    and in experimental models demonstrating the potency of AFB1 as a
    carcinogen and mutagen (IARC, 1993). There is also evidence that
    humans have the metabolic capacity to activate AFB1 to the same
    DNA-damaging products that occur in animal models. A well-established
    major risk factor for liver cancer is hepatitis B virus; however,
    there is at least a 5-8 fold variation in liver cancer incidence
    across regions of the world where the prevalence of hepatitis B viral
    markers is comparable. Ross  et al. emphasize that their Shanghai
    study using aflatoxin-specific biomarkers and HBV markers has provided
    the first direct evidence in human studies that aflatoxins are major
    risk factors for HCC and that a synergistic interaction between HBV
    and aflatoxin exposure occurs. Ross  et al. criticize the Chinese
    study of Campbell (1994) as an ecological study, "well known to be
    highly limited in their ability to address cause and effect
    relationships". In addition, a questionnaire administered to the
    Shanghai subjects showed no difference in daily intake of animal
    protein between liver cancer cases and their matched controls, or
    between Hbsag positive cases and controls.

        Groopman  et al. (1993) stated that, based on urinary measures of
    AFB-N7-guanine and dose-response characteristics of people living in
    China and The Gambia, that 1) levels of daily urinary excretion of
    total aflatoxin metabolites are unrelated to risk of aflatoxin-induced
    disease; 2) the AFB-N7-guanine adduct in urine is a good,
    non-invasive, short-term biomarker for determining both aflatoxin
    exposure and risk of genetic damage in target organs.

        However, the Shanghai study is clearly limited for purposes of
    quantitative risk assessment of the risk to humans of aflatoxin
    exposure. By the authors' own admission (Qian  et al., 1994), no
    dose-dependent association between the dietary aflatoxin index and
    either liver cancer risk or biomarker status was found. This is due at
    least in part to the facts that urinary levels of aflatoxin accurately
    reflect intake levels of the past 24 hours and dietary assessment is
    inadequate to reflect lifetime aflatoxin exposure. An unexplained
    observation was the rather marked decline in the prevalence of
    unmetabolized AFB1 with longer follow-up. Urinary adducts were
    measured with inadequate precision; a patient was scored "positive" or
    "negative" if an adduct was detected. Levels of adducts were not
    considered, nor was the fact that one measurement represents one
    "snapshot" out of a lifetime. One might question whether or not the
    increased excretion of aflatoxin-DNA adducts represents the activity
    of a diseased liver rather than a causal relationship. Exposure to
    AFB1 may be expected to fluctuate greatly on a day-to-day basis (as a
    result of varying behaviour and AFB1 concentrations); exposure for
    each individual was evaluated at a single time point. The authors are
    correct in their call for pharmacokinetic investigations of ingested
    aflatoxins in humans. Until this work is done, AFB adducts cannot be

    considered to be a true indicator of aflatoxin exposure, especially
    over the probable lengthy timeframe required for human cancer
    induction.

        This study strongly suggests that aflatoxin exposure in the
    presence of a persistent HBV infection increases the risk of liver
    cancer. It is less convincing support for the conclusion that AFB1 is
    capable of independently inducing liver cancer and provides limited
    quantitative data on aflatoxin's relationship to liver cancer.
    Follow-up of this cohort is awaited with great interest.

        In a study by Wild  et al. (1993), blood samples were collected
    over a one-month period from 117 children aged 3 to 4 years residing
    in Kuntair or Kerr Cherno in the Upper Niumi District of The Gambia.
    Samples were analysed for aflatoxin-albumin (AF-alb) adducts, markers
    of HBV infection, liver enzymes (serum alanine aminotransferase (ALT))
    as markers of liver damage, and glutathione-S-transferase (GST) M1
    genotype. All but two children showed detectable serum AF-alb with
    levels ranging from 2.2 to 250 pg AFB1-lysine equivalent/mg albumin.
    There was a statistically significant positive correlation between
    AF-alb and ALT. HBV carriers showed moderately higher levels of AF-alb
    than non-carriers, but the difference was not statistically
    significant and the association between AF-alb and ALT was unchanged
    when the HBV carriers were excluded from the analysis, suggesting that
    factors other than HBV infection contributed to the association. The
    null GSTM1 genotype was infrequent in this population and was not
    associated with any difference in AF-alb adduct levels compared to
    GSTM1-positive subjects. However, the percentage of individuals with
    the null genotype varied significantly between ethnic groups. The
    association between AF-alb and ALT could be a result of the
    hepatotoxicity of aflatoxin, but the data are also consistent with the
    hypothesis that liver damage resulting from HBV and/other factors can
    alter aflatoxin metabolism resulting in an increased binding to
    cellular macromolecules including DNA. The authors recommended more
    study of this hypothesis.

        Srivatanakul  et al. (1991) conducted a case control study on
    hepatocellular carcinoma in Thailand using the aflatoxin-albumin
    adduct as a marker of recent exposure to aflatoxin. HBV exposure and
    anti-HCV were assessed using standard methods. HBV was the predominant
    risk factor; neither aflatoxin-albumin nor HCV were associated with
    liver cancer. The Committee concluded that the study lacked sufficient
    statistical power to detect aflatoxin or HCV as independent risk
    factors. In addition, aflatoxin-albumin samples were taken from liver
    cancer cases; levels or kinds of aflatoxin metabolites might have been
    affected by illness.

        Hatch  et al. (1993) conducted a survey in eight areas in Taiwan
    with a gradient in the estimates of exposure to aflatoxin and in the
    incidence of PLC. Exposure to aflatoxin was assessed using urinary
    tests, and a regression model was used to predict aflatoxin urinary
    metabolites using mortality due to PLC as a predictor (as well as five
 
    other variables). The conclusion of the study was that aflatoxin
    played an independent role in PLC in Taiwan.

        Monoclonal antibodies recognizing the stable imidazole ring-opened
    form of the major N7-guanine aflatoxin B1-DNA adduct have been used in
    competitive enzyme-liked immunosorbent assays (ELISA) and indirect
    immunofluorescence assays to quantify adduct levels in liver tissue.
    Santella  et al. (1993) developed methods in AFB1-treated animals,
    then applied these to paired tumour and non-tumour liver tissues of
    hepatocellular carcinoma patients from Taiwan. An avidin-biotin
    complex staining method was also used for the detection of HBsAg and
    HBxAg antigens in liver sections. In all, 8 (30%) HCC samples and 7
    (26%) adjacent non-tumour liver tissue samples from Taiwan were
    positive for AFB1-DNA adducts. For HBsAg, 10 (37%) HCC samples and 22
    (81%) adjacent non-tumorous liver samples were positive, and 9 (33%)
    HCC samples and 11 (41%) adjacent non-tumour liver samples were HBsAg
    positive. No association with AFB1-DNA adducts was observed for HBsAg
    and HBxAg. The authors concluded that these results were compatible
    with the conclusion that HBV and AFB1 do not act synergistically in
    the genesis of HCC, but called for further investigation to define the
    relationship between HBV and AFB1.

        Wang  et al. (1996a) conducted studies in Qidong, China, where
    liver cancer accounts for 10% of all adult deaths and both HBV and
    AFB1 exposures are common. Serum samples were collected during a
    longitudinal study designed to measure aflatoxin molecular biomarkers
    in residents of Daxin Township, Qidong City, China. In this study, the
    temporal modulation of aflatoxin adduct formation with albumin over
    multiple lifetimes of serum albumin was examined in both HBV-positive
    and HBV-negative people in two periods: September-December 1993 (wave
    1) and June-September 1994 (wave 2). During the 12-week monitoring
    period of wave 1, 120 persons (balanced by gender and HBV status)
    provided a total of 792 blood samples. AFB1-albumin adducts were
    detected in all but one of the serum samples. During wave 2, 103
    individuals from wave 1 provided 396 blood samples collected monthly
    over wave 2. Using linear regression models, the mean
    aflatoxin-albumin adduct levels increased during the 12 weeks of wave
    1 and decreased over the 4 months of wave 2.

        Neither HBV status nor gender modified either the baseline mean or
    the temporal trend. High-performance liquid chromatography
    confirmation was done on a subset of serum samples, and the results
    showed an excellent association between the immunoassay data and
    high-performance liquid chromatography. The investigators concluded
    that AFB1-albumin is a sensitive and specific biomarker for assessing
    exposure to AFB1 in the Qidong population.

        The authors noted that aflatoxin-albumin binding is a longer term
    biomarker of aflatoxin exposure than any of the urinary markers; the
    rate of turnover of these adducts is similar to that of the blood
    protein. The half-life of albumin in normal people is about 14-20
    days, but there is some information to indicate that people with
    serious liver disease have a much more variable turnover time. In this

    study, an 8-fold range of adduct formation existed among individuals
    within a cycle. Such factors as liver disease may account for the lack
    of tracking shown in this study between HBV status and adducts. The
    authors stressed the need to follow-up on this investigation with
    studies that have more frequent and longer sampling intervals for
    albumin adducts.

        The authors discussed the data supporting the hypothesis that HBV
    enhances aflatoxin metabolism to genotoxic derivatives. This study did
    not seem to support this hypothesis nor did previous results in adult
    populations in West Africa and Taiwan. HBV may affect the metabolism
    of aflatoxin in children at a time when hepatocytes are maximally
    dividing; more data are needed in this regard.

        Wang  et al. (1996b) investigated the carcinogenic effect of
    aflatoxin exposure in Taiwan. Fifty-six cases of HCC diagnosed between
    1991 and 1995 were identified and individually matched by age, sex,
    residence and date of recruitment to 220 healthy controls from the
    same large cohort in Taiwan. Blood samples were analysed for hepatitis
    B and C virus markers and for aflatoxin-albumin adducts. Urine was
    tested for aflatoxin metabolites. Information was obtained about
    sociodemographic characteristics, habitual alcohol drinking, cigarette
    smoking and diet in a structured interview.

        HBsAg carriers had a significantly increased risk for HCC. After
    adjustment for HBsAg serostatus, the matched odds ratio (ORm) was
    significantly elevated for subjects with high levels of urinary
    aflatoxin metabolites. When stratified into tertiles, a dose-response
    relationship with HCC was observed. The ORm for detectable
    aflatoxin-albumin adducts was not significant after adjustment for
    HBsAg serostatus. HBsAg-seropositive subjects with high aflatoxin
    exposure had a higher risk than subjects with high aflatoxin exposure
    only or HBsAg seropositivity only. The OR for developing HCC was found
    to increase in the presence of anti-HCV alone, HBsAg alone and both
    anti-HCV and HBsAg. There was a poor correlation between
    aflatoxin-albumin adducts and urinary metabolites in the same
    controls, although both were related to HCC risk. The investigators
    suggested that environmental aflatoxin exposure may enhance the
    hepatic carcinogenic potential of hepatitis B virus, expressed concern
    about the small sample size in their study, and urged the mounting of
    a large-scale study to evaluate the effect of aflatoxin exposure on
    HBsAg non-carriers.

        Olubuyide  et al. (1993) screened for the presence of HBsAg and
    aflatoxin in the sera of 100 non-hospitalized individuals from the
    rural population of Igobo-Ora and 89 non-hospitalized individuals from
    the urban population of Ibadan, Nigeria. Controls were 31 healthy
    British Caucasians who had not travelled to the tropics or subtropics
    in the six months before the venipuncture. Forty-nine per cent of
    rural subjects and 47% of urban subjects were consistently and
    reproducibly seropositive for HBsAg (as determined by the ELISA test).
    Two of the former subjects and five of the latter were positive for
    both HBV DNA (as measured by spot hybridization) and HBsAg. Total

    aflatoxin levels were less than 17 pg/ml in the British controls;
    serum levels of aflatoxins greater than this were detected in 8% of
    rural subjects and in 9% of urban subjects. The types and amounts of
    aflatoxins and the amounts of aflatoxins found were so widely
    dispersed that it was not possible to draw any conclusions about
    differences in types and amounts of aflatoxins between rural and urban
    populations. The authors intend to follow their subjects to determine
    their propensity to develop HCC.

        Groopman  et al. (1994) have reviewed the subject of molecular
    biomarkers for aflatoxins and their application to human cancer
    prevention. Cancer prevention trials that use biological markers as
    intermediate end-points provide the ability to assess the efficacy of
    promising chemoprotective agents in an efficient manner by reducing
    sample size requirements, as well as reducing the time required to
    conduct the studies, compared to trials that have cancer incidence or
    mortality as end-points. The key issue in trials that use biomarkers
    as the outcome of interest is to use a marker that is directly
    associated with the evolution or development of neoplasia. The authors
    briefly discussed the possible impact of a short-term intervention
    with oltipraz (a substituted dithioethione, which is a potent
    inhibitor of AFB1-induced tumorigenesis and carcinogenesis in rats) on
    levels of two aflatoxin biomarkers in individuals exposed to
    aflatoxin-contaminated foods.

     (i) Oltipraz chemoprevention trial

        In 1995, 234 adults from Qidong, Jiangsu Province, China, where
    hepato-cellular carcinoma is the leading cause of cancer death and
    exposure to dietary aflatoxins is widespread, were enrolled and
    followed in a Phase II chemo-prevention trial (Jacobson  et al., 
    1997). The goals of the study were to define a dose and schedule of
    oltipraz for reducing levels of validated aflatoxin biomarkers and to
    characterize dose-limiting toxicities. Healthy eligible individuals,
    including those infected with hepatitis B virus, were randomized to
    receive either 125 mg of oltipraz daily, 500 mg of oltipraz weekly, or
    placebo. Blood and urine specimens were collected to monitor
    toxicities and evaluate biomarkers over the 8-week intervention period
    and subsequent 8-week follow-up period. The authors reported excellent
    compliance (>70%); 21% of subjects reported clinical adverse events.
    The oltipraz arms did not differ in symptom type or severity, most
    commonly an extremity syndrome. There were no indications of
    exacerbated drug intolerance among the few participants infected with
    hepatitis B virus. The authors concluded that chemoprevention trials
    with biomarker end-points are feasible in such populations.

     (j) Genetic susceptibility

        AFB1 is metabolized via the phase I and II detoxification pathway;
    hence, genetic variation at those loci may predict susceptibility to
    the effects of AFB1. To test this hypothesis, McGlynn  et al. (1995)
    contrasted genetic variation in two AFB1 detoxification genes, epoxide
    hydrolase (EPHX) and GSTM1 with the presence of serum AFB1-albumin

    adducts, the presence of hepatocellular carcinoma (HCC), and with p53
    codon 249 mutations. Subjects were 40 unrelated Ghanaian males
    (healthy gold miners employed by the Ashanti Goldfields Corp. in
    Obuasi, Ghana) and 52 patients with HCC and 116 healthy controls from
    the Zhong Shan Hospital in Shanghai, China.

        Mutant alleles at both loci were significantly over represented in
    individuals with serum AFB1-albumin adducts in a cross-sectional
    study. Mutant alleles of EPHX were significantly over-represented in
    subjects with HCC and also in a case-control study. The relationship
    of EPHX to HCC varied by hepatitis B surface antigen status and
    indicated that a synergistic effect may exist. p53 codon 249 mutations
    were observed only among HCC patients with one or both high risk
    genotypes. These results indicate that individuals with mutant
    genotypes at EPHX and GSTM1 may be at greater risk of developing
    AFB1-adducts, p53 mutations and HCC when exposed to AFB1. Hepatitis B
    carriers with the high-risk genotypes may be an even greater risk than
    carriers with low-risk genotypes. The authors concluded that these
    findings support the existence of genetic susceptibility in humans to
    the environmental carcinogen AFB1 and indicate that there is a
    synergistic increase in risk of HCC with the combination of hepatitis
    B virus infection and susceptible genotype. However, it has been
    pointed out that the control and experimental samples came from
    different populations, which may weaken the case for genetic
    susceptibility (J. Groopman, personal communication).

        The Committee concluded that the currently available studies
    utilizing aflatoxin biomarkers do not provide a reliable quantitative
    measure of aflatoxin exposure in humans, especially over the
    long-term. Biomarker levels in relation to cancer risk in the Wang 
     et al. and Qian  et al. studies have been used for modelling data;
    however, the interpretation is limited.

        At present, it seems reasonable to subscribe to the 1993
    conclusion of the IARC in qualitative terms that AFB1 is carcinogenic
    (group 1), and as such to recommend reducing exposure of human
    populations as much as possible. There is still some uncertainty
    concerning the independent status of aflatoxin as a human carcinogen
    and concerning the relationship between aflatoxin dose and liver
    cancer incidence.

     (k) Conclusions from epidemiology studies

        The potential carcinogenicity in humans of the aflatoxins (either
    total or AFB1) has been examined in a large number of epidemiology
    studies, generally carried out in Africa and Asia, where substantial
    quantities of aflatoxin occur in basic foodstuffs. Exposure to
    aflatoxins appears to present an additional risk which is enhanced by
    simultaneous exposure to hepatitis B virus, and possibly hepatitis C
    virus. This relationship, which may affect not only carcinogenic
    potency but also the metabolism, biochemistry and pharmacology of the
    aflatoxins, and other multiple etiological agents for primary liver
    cancer makes it difficult to interpret the epidemiological studies in


    the context of the risk of primary liver cancer from aflatoxins.
    Perhaps, further development of biochemical and pharmacological
    markers will help to clarify exposure, although these can cause other
    problems.

        Further clarification of the relative roles of hepatitis and
    aflatoxin in liver cancer awaits studies that comply with the
    following requirements: 1) the studies should be cohort studies with
    long-term follow-up; 2) the studies should be conducted in countries
    with variability in the exposure to aflatoxins; 3) the studies should
    provide for storage and analyses of biological specimens from repeated
    sampling, preferably with concurrent sampling of aflatoxin in the
    diet; 4) the studies should provide evidence that the aflatoxin
    biomarker used is not affected by the presence of chronic liver
    disease; this will be difficult to achieve; the different measures of
    aflatoxin exposure, i.e., biomarker vs. dietary analysis, should
    correlate with liver cancer; 5) a large number of liver cancer cases
    should be included, preferably confirmed by biopsy; 6) liver cancer
    cases, if shown to contain the p53 specific aflatoxin mutation, would
    strengthen the case.

        The Committee identified some on-going studies that comply with
    some of these requirements and may produce relevant results in the
    near future.

    i)  A study in Qidong, China, screened 45 000 males (ages 30-59) of
    which 20% were HBsAg positive. Questionnaires, urine and serum were
    collected at different intervals; 260 cases of liver cancer have been
    identified although the number of biopsies is small. Laboratory tests
    and statistical analyses are required; funds are needed.

    ii) A cohort study in Thailand collected questionnaires, blood and
    urine specimens at different intervals in a cohort of HBsAg carriers
    who were regularly screened for AFP, ALT and ultrasound. Field work is
    completed at this point and lab results are pending.

    iii) The Shanghai study described in the text may be of value if
    sampling and follow-up continue.

    iv) Finally, the on-going HBV vaccination trials and campaigns in
    China, Taiwan and The Gambia may provide evidence in the future for
    the occurrence of AFB1-induced liver cancer cases in individuals
    vaccinated against HBV.

    2.2.11  Summary of information on other aflatoxins

    2.2.11.1  Aflatoxin B2

        Aflatoxin B2 (AFB2) has not been studied extensively, and most
    data are derived from single reports. AFB2 becomes bound to DNA of
    rats treated  in vivo, after its metabolic conversion to AFB1. In
    rodent cells, AFB2 induced DNA damage, sister chromatid exchange and
    cell transformation, but not gene mutation. AFB2 produces gene

    mutation in bacteria. IARC concluded in 1993 that there is limited
    evidence for carcinogenicity of AFB2 in experimental animals.

        No additional toxicological information on AFB2 has appeared in
    the literature since IARC (1993).

    2.2.11.2  Aflatoxin G1

        Aflatoxin G1 (AFG1) binds to DNA and produces chromosomal
    aberrations in rodents treated  in vivo. In cultured human and animal
    cells, it induces DNA damage, and also induces chromosomal anomalies
    in single studies. AFG1 induces gene mutation in bacteria. IARC
    concluded in 1993 that there was sufficient evidence in experimental
    animals for the carcinogenicity of AFG1.

        No additional toxicological information on AFG1 has appeared in
    the literature since IARC (1993).

    2.2.11.3  Aflatoxin G2

        Aflatoxin G2 (AFG2) has been the subject of very little research.
    IARC concluded in 1993 that there was inadequate evidence for the
    carcinogenicity of AFG2.

        No additional toxicological information on AFG1 has appeared in
    the literature since IARC (1993).

    2.2.11.4  Aflatoxin M1

        Aflatoxin M1 (AFM1) is a metabolic hydroxylation product of AFB1,
    and can occur in the absence of the other aflatoxins. Human exposure
    occurs primarily via milk and milk products from animals that have
    consumed contaminated feed. IARC concluded in 1993 that there was
    sufficient evidence in experimental animals for the carcinogenicity of
    AFM1 and inadequate evidence for the carcinogenicity of AFM1 in
    humans. Although AFM1 has been tested less extensively, it appears to
    be toxicologically similar to AFB1. AFM1 is considered to be a
    genotoxic agent, based on its activity  in vitro and its structural
    similarity with AFB1. It is a less potent liver carcinogen, with a
    probable carcinogenic potency in laboratory animals within a factor of
    10 of AFB1 (Cullen  et al., 1987).

        No additional toxicological information on AFM1 has appeared in
    the literature since IARC (1993).

    3.  ESTIMATING CARCINOGENIC RISKS FROM THE INTAKE OF AFLATOXINS

        Risks from specific exposures to aflatoxins are difficult to
    estimate and predict, despite extensive information available from
    epidemiological studies, mutagenicity tests, animal bioassays, 
     in vitro and  in vivo metabolic studies, and p53 mutation studies.
    Many questions remain regarding the independence of aflatoxin as a


    human carcinogen, the extent to which hepatitis B, hepatitis C and
    other factors modify the effect of aflatoxin, how findings from
    countries with high liver cancer rates and high prevalence of
    hepatitis B may be compared to those from countries with low rates,
    how to deal with the wide range of susceptibility to aflatoxin
    carcinogenesis among experimental animals, and how to describe the
    dose-response curve over the wide range of aflatoxin exposure found
    worldwide.

    3.1  Information from various scientific disciplines and its
    contribution to aflatoxin carcinogenic risk

    3.1.1  Laboratory animal, mutagenicity and metabolic studies

        The liver is the primary target organ in most species, but tumours
    of other organs have also been observed in aflatoxin-treated animals.
    The effective dose of AFB1 for induction of liver tumours varied over
    a wide range in different animals species when the carcinogen was
    administered by continuous feeding, generally for the lifetime of the
    animal. Effective doses were 10-30 g/kg in the diet in fish and
    birds. Rats responded according to strain at levels of 15-1000 g/kg,
    while some strains of mice showed no response at doses up to 150 000
    g/kg. Tree shrews responded to 2000 g/kg. In subhuman primate
    species, AFB1 potency in induction of liver tumours differed widely,
    squirrel monkeys developing liver tumours when fed AFB1 at 2000 g/kg
    for 13 months, and rhesus, African green and cynomolgus monkeys
    developing a low (7-20%) incidence of liver tumours when fed average
    doses of 99-1225 mg/animal over 28-179 months (Wogan, 1992).

        The aflatoxins are among the most potent mutagenic and
    carcinogenic substances known. Much of the information available
    regarding mutagenesis has been performed in bacterial systems, but
    also to a lesser extent in eukaryotes. Aflatoxin falls in the category
    of bulky mutagens, including the polycyclic aromatic hydrocarbons and
    the aromatic amines. A large body of literature suggests that a
    chemical causes a cell to become tumorigenic by reacting readily with
    DNA to give DNA adducts and these adducts or their breakdown products
    must then cause mutations efficiently (Loechler, 1994).

        Much of the recent aflatoxin metabolic data has been discussed in
    more detail in section 2.1.3. In brief, it has been demonstrated that
    many isoforms of P450 are able to biotransform AFB1 to
    DNA-binding/mutagenic species. Differences in P450 isoform activities,
    due either to genetic polymorphisms or to environmental alteration in
    expression, may be important contributors to human susceptibility to
    AFB1 (Massey  et al., 1995). For example, there is some evidence that
    AFB1 is strongly metabolized to DNA-binding species in areas of
    damaged liver or in individual cells where CYP2A5 activity is high
    (Camus-Randon  et al., 1996). It is well known that a host of other
    risk factors affecting metabolism exist, including infection with
    hepatitis B and C, parasites such as liver flukes, alcohol
    consumption, cigarette smoking, long-term use of oral contraceptives,
    and nutritional status.

        There is increasing evidence that AFB1 can be activated by lipid
    hydroperoxide-dependent mechanisms, involving microsomal prostaglandin
    H synthase and lipoxygenases. Although the maximum activity of this
    co-oxidation is low relative to P450, these processes may contribute
    significantly to bioactivation of AFB1  in vivo in humans, who are
    generally exposed to low levels of AFB1. Co-oxidation may be
    particularly important for AFB1 carcinogenicity in extrahepatic
    tissues, in view of relatively low cytochrome P450 activity in these
    organs. In the search for target cell types in the human lung, a
    thorough analysis of the cellular distribution of potential AFB1
    metabolizing systems will be necessary (Massey  et al., 1995).

        Detoxification (mediated by cytochrome P450 as well as conjugation
    of the epoxide with glutathione) must also be considered. Animal
    studies suggest that GST-catalysed detoxification is the crucial
    factor in susceptibility to AFB1, and humans appear to lack
    significant GST-mediated protection against AFB1 (Massey  et al., 
    1995). There is suggestive evidence that human GSTs in the  alpha, 
     mu and  theta families may all have roles in the detoxification of
    the epoxide. It is not yet known with certainty whether there is a
    role for epoxide hydrolase.

        A possibly important factor in the assessment of aflatoxin risk is
    the variation of human susceptibility due to individual differences in
    human metabolism, such as polymorphisms in cytochrome P450s, GSTs, and
    epoxide hydrolase. Data are beginning to become available; it is
    unclear as yet what impact gene polymorphisms may have on human
    activating as well as detoxifying enzymes, and therefore on aflatoxin
    risk (Cardis  et al., in press). With all the enzymes, it is
    necessary to consider the stereochemistry of the aflatoxin epoxide,
    which is critical in genotoxicity (Guengerich  et al., 1996).

        Most studies comparing AFB1 metabolism in different species have
    been conducted  in vitro, using subcellular fractions such as
    microsomes or cytosol, or purified components of either fraction. The
    exclusion of enzymes and cofactors for competing metabolic pathways
    restricts quantitative comparisons of metabolism between different
    species; therefore, conclusions based on data from  in vitro 
    experiments are limited to qualitative comparisons of individual
    pathways of AFB1 metabolism. Although specific metabolism pathways may
    be associated with increasing sensitivity to AFB1 carcinogenicity,
    quantification of sensitivity requires a supply of metabolic factors
    for competing reactions found by either reconstitution of total
    cellular fractions, use of primary cell cultures with representative
    metabolic capacities, or whole animal studies (Gorelick, 1990).

        The P450s both activate and detoxify AFB1 and the effect of
    inducing individual P450s is not easily predicted. Also, the small
    intestine, site of absorption of orally ingested AFB1, expresses P450
    3A4. Activation of AFB1 and DNA alkylation in the small intestine may
    be considered also to be a detoxification process since the cells are
    sloughed rapidly and cancers of the small intestine are very rare
    (Guengerich  et al., 1996).

        After reviewing the available data from the metabolic,
    mutagenicity and laboratory animal studies, the Committee concluded
    that there is at the present time insufficient quantitative
    information available about competing aspects of metabolic activation
    and detoxification of AFB1  in vivo in various species to describe
    quantitatively a species-dependent effect of metabolism on AFB1
    carcinogenicity (Gorelick, 1990; Massey  et al., 1995; Guengerich 
     et al., 1996; Wild  et al., 1996). It is, however, probable, that
    differential sensitivity to AFB1-induced tumours between species can
    be partially attributed to differences in metabolism. 

    3.1.2  Studies on the p53 gene

        Studies on p53 mutations have been extensively discussed earlier
    in this paper. The Committee concluded that there is currently
    insufficient straightforward information available on the specificity
    of the aflatoxin/p53 association to assess and quantify the
    independence of aflatoxin as a cause of human liver cancer.

    3.1.3  Epidemiological studies

        The relevant epidemiological studies have been discussed earlier;
    only the conclusions are presented here. Most of the epidemiological
    studies show a correlation between exposure to aflatoxins and liver
    cancer; some studies suggest that aflatoxin exposure poses no
    detectable independent risk and other studies suggest that it poses a
    risk only in the presence of other risk factors such as HBV infection.
    Several ongoing studies are likely to improve further the estimates of
    human risks from aflatoxin exposures; most notable among these are
    cohort studies in Shanghai, Thailand and Qidong, China, and the HBV
    vaccination trials in The Gambia, Taiwan and Qidong. When these
    studies are complete, JECFA may want to re-evaluate the risks of
    aflatoxins in humans.

        A number of factors influence the risk of primary liver cancer,
    most notably carriage of HBV; the potency of aflatoxins appears to be
    significantly enhanced in individuals with simultaneous HBV infection.
    Most of the epidemiological data are from geographical areas where
    both the prevalence of HBsAg+ individuals and aflatoxins are high; the
    relationship between these risk factors in areas of low aflatoxin
    contamination and low HBV prevalence is unknown. This interaction
    makes it difficult to interpret the epidemiological studies in the
    context of aflatoxin as an independent risk. The Committee therefore
    has made decisions contingent upon the dynamics of HBV infection in a
    human population for which aflatoxin potency is to be determined.

        The identification of HCV is a major breakthrough in understanding
    the etiology of liver cancer. Two studies have investigated
    interactions between HCV infection, aflatoxins and liver cancer; the
    evidence so far is inconclusive. As shown in Table 1, it has been
    estimated that 50 to 100% of liver cancer cases are associated with
    persistent infection with HBV and/or HCV.

        In Latin America both liver cancer and HBV infection are rare, yet
    aflatoxin exposure is relatively high. Unfortunately, few studies are
    available on the occurrence of liver cancer in Latin America; much
    could be learned about aflatoxin as a risk factor in liver cancer by
    conducting appropriately designed epidemiology studies in Latin
    America.

    3.1.4  Aflatoxin biomarker studies

        The Committee concluded that the currently available studies
    utilizing aflatoxin biomarkers do not provide a quantitative measure
    of aflatoxin exposure in humans especially over the long term.
    Biomarker levels in relation to cancer risk in the studies of Wang 
     et al. (1996a) and Qian  et al. (1994) studies have been used for
    modelling data; however, the interpretation is limited.

    3.2  General modelling issues

        Quantitative risk assessment for food contaminants involves four
    basic issues: 1) choice of data; 2) measure of exposure; 3) measure of
    response; and 4) choice of a mathematical relationship between dose
    and response for a given data set. General comments can be made for
    each of these areas, as well as specific comments concerning what has
    been done regarding estimating risk from exposure to aflatoxin.

    3.2.1  Choice of data

        In general, the best data set to use for dose-response analysis
    would be a human study in which dose is accurately measured, response
    is determined without error and there are no confounding factors which
    are unexplained. It is rare to find an epidemiological study without
    one of these factors causing difficulty in the interpretation and
    utility of the data. In contrast to the human data, test species data
    are generally devoid of confounders. There is a clear and accurate
    measure of response, and dose is an integral part of the design of the
    study. Numerous dose-response assessments have been conducted by
    modelling animal data and extrapolating the results to humans.
    However, the extrapolation may be problematical, given outstanding
    questions concerning the overall relevance of the animal data. For
    aflatoxin, several epidemiological studies are capable of providing a
    dose-response assessment. However, the study of Yeh  et al. (1989)
    has some limitations as described in section 2.2.10.3(e). The cohort
    study in Shanghai (Ross  et al., 1992; Qian  et al., 1994)
    considered both biomarker information and dietary questionnaires as
    sources of aflatoxin exposure information. The study conducted by Wang
     et al. (1996b) in Taiwan considered HCV, but results were
    inconclusive.

    3.2.2  Measure of exposure

        In all of the risk assessments performed for aflatoxin, dose has
    been expressed as lifetime average exposure in ng/kg per day. Note
    that if peak exposure or early lifetime exposure has an impact on the

    risk other than through the increase in the lifetime average ng/kg per
    day, this choice of exposure measure could bias the risk estimates.

    3.2.3  Measure of response

        The major toxicological impact of aflatoxin on humans and animals
    is an increase in primary liver cancer; that is the focus of this risk
    assessment and all others performed to date.

    3.2.4  Choice of mathematical model

        Two basic risk models are routinely used in cancer epidemiology to
    describe the relationship between dose of a contaminant and the risk
    of disease or death. These models are of the form:

        rM(t,E) = rO(t)  fM(E)             multiplicative model

    and 

        rA(t,E)  =  rO(t)  +  fA(E)         additive model

    where rM(t,E) and rA(t,E) are functions that describe disease
    incidence as a function of age (t) and exposure (E). Exposure is
    used here generically to include factors other than age that could
    affect on the incidence rate. For unexposed individuals the incidence
    rate is rO(t), and fM(E) and fA(E) are functions describing
    the effect of exposure on the background. Typically, the forms for
    fM(E) and fA(E) are assumed to be either linear or log-linear
    (exponential). For example, 
    fM(E) = 1 + a1E1 + a2E2 + a3E3E2 or log[FM(E)] = a1E1
    + a2E2 + a3E1E2, where a1, a2 and a3 are parameters to be
    estimated. The multiplicative model with log-linear effect of exposure
    is commonly known as the Cox model and is related to logistic
    regression. Tests of significance for any one effect (say E1) are
    performed by testing whether its associated parameter (a1 for E1)
    differs significantly from 0. The term a3E1E2 describes an
    interaction between the two factors E1 and E2 and allows one to test
    for such an interaction amongst the exposures. Choice of an additive
    or multiplicative model can have a substantial impact upon resulting
    risk estimates, particularly when extrapolating to a different
    population. In the case of the additive model, differences in
    background incidence have no impact on predictions of additional risk
    (i.e., r(t,E) - r(t,0) = fA(E) and does not include rO(t)). On
    the other hand, in the multiplicative model, predictions of additional
    risk depend on the background incidence rate (i.e., r(t,E) - r(t,0)
    = r0(t)(fM(t,E)-1), which is proportional to r0(t)).

        Other plausible models not of the additive or the multiplicative
    form include "mechanistically-based" models such as the two-stage
    model for cancer. Although such models are not additive or
    multiplicative  per se, dose effects on the parameters that drive the
    background cancer rate are usually modelled as linear or exponential,

    as described earlier, and the choice of the relationship can affect
    the risk estimates.

    3.3  Potency estimates

    3.3.1  Potency estimates based upon epidemiological data

        In analysing any epidemiological study, there are many plausible
    alternatives as to the form of the mathematical relationship between
    exposure and response. For aflatoxin, the range of potencies derived
    by using different models provides an indication of the uncertainty in
    risk when one extrapolates from human data based upon studying areas
    with relatively high background incidence of liver cancers and with
    relatively high prevalence of HBV. In the following sections, selected
    risk analyses will be reviewed briefly and the resulting potency
    estimates presented and compared. In all of the analyses cited, it
    should be noted that the potential effect of misspecification of the
    dose that went into the derivation of the potency has not been
    quantitatively addressed. As for all retrospective constructions of
    exposure, use of recent levels of aflatoxin exposure to describe
    current incidence rates assumes that current exposures are comparable
    to past exposures. Owing to the long latency period predicted for most
    cancers, uncertainty in the lifetime dose is an additional source of
    variability that could lower (if the historical exposures were
    actually higher than reported) or raise (if the historical exposures
    were lower than actually reported) the resulting potency.

    3.3.2  Potency estimates not accounting for HBV infection

        Table 2 summarizes potency estimates based on analyses of
    epidemio-logical studies in which regional cancer rates were compared
    to estimates of aflatoxin intake without regard to differences in HBV
    infection rates. As a reality check, the values in Table 2 can be
    applied to the average aflatoxin exposure in the USA to obtain a
    prediction of added incidence for the population. Using the largest
    potency value in Table 2 (0.375) and assuming an average aflatoxin
    intake of 0.26 ng/kg per day (Henry  et al., 1997) for the USA
    population, the added incidence is calculated to be approximately
    0.375  0.26 or 0.0975 per 100 000 per year. Since this calculation is
    based on the highest predicted potency, none of the potency estimates
    in Table 2 are overtly inconsistent with the current USA rate of
    approximately 3.4 per 100 000 and estimates of current levels of
    aflatoxin intake (assumed to reflect past exposure levels). However,
    as already indicated, these potency estimates are largely based on
    studies in Africa and Southeast Asia where HBV infection rates are
    much higher than in the USA or other Western countries.

        Table 2.  Potency estimates of the risk of liver cancer in humans based upon epidemiological
    data with no correction for HBV status assuming an exposure of 1 ng/kg per day

                                                                                         
    Author                                                  Incidence/year per 100 0001
                                                                                         

    Peers & Linsell (1977)                                  0.11
    Stoloff & Friedman (1976)                               0
    Carlborg (1979)                                         0.21
    Bruce (1990)
       based upon Stoloff (1983)                            0
       based upon van Rensburg et al. (1985),
       Shank et al. (1972a,b), Peers et al. (1976, 1987)    0.10
    Croy & Crouch (1991)
       based on Peers et al. (1976)                         0.15  (0.09, 0.23)
       based on Yeh et al. (1989)                           0.14  (0.08, 0.21)
    Calif. Dept. Health Serv. (1990)                        
       based on Peers et al. (1976)                         0.38  (0.15, 0.60)
       based on van Rensburg et al. (1985)                  0.14  (0.10, 0.17)
       based on Peers et al. (1987)                         0.17  (NA, 0.3)
       based on Yeh et al. (1989)                           0.18  (NA)
                                                                                         

    1  Numbers in parentheses represent (lower, upper) 95% confidence limits on the predicted
    risk when available from the authors.
    
    3.3.3  Potency estimates accounting for HBV infection

        The epidemiology study by Yeh  et al. (1989) has been the focus
    of several recent quantitative risk assessments and is described in
    section 2.2.10.3(e). This study took place in Guangxi Province in
    southern China and was a prospective cohort study of 7917 men.

        In the analysis of their study, Yeh  et al. (1989) adjusted
    mortality rates for each region based on the age distribution of the
    composite study cohort as an internal standard. Wu-Williams  et al. 
    (1992) calculated that the age-adjusted PLC rate for the total cohort
    was 121.5 per 100 000 when standardized to the age distribution of the
    world population versus 226.3 per 100 000 when standardized to the age
    distribution of the study cohort. The ratio of these rates (0.54) was
    then used to adjust the regional PLC mortality rates reported by Yeh
     et al. (1989) to obtain expected incidence rates for a
    (hypothetical) cohort with age-distribution similar to the world
    population. Adjusted person-years of observation (APY) were calculated
    in each region as the number of PLC deaths observed in that region
    divided by the adjusted mortality rate. Adjusted person-years of
    observation were assumed to be distributed among HBsAg+ and
    HBsAg-carriers according to the regional prevalence of hepatitis B.
    The data are summarized in Table 3.

    Table 3.  Epidemiological data from Yeh et al. (1989)

                                                             

    Dose aflatoxin        PLC cases               APY1
    (ng/kg per day)    HBsAg-    HBsAg+      HBsAg-    HBsAg+
                                                             

    12                   0        12          9932     2727
    90                   1         7          6114     2017
    705                  4        12          7733     2537
    2028                 2        23          5803     1743
     -2                  7        54         29582     9034
                                                             

    1 Adjusted person-years (see text)
    2 No data are available for this group

        Croy & Crouch (1991) separately analysed the HBV negative and HBV
    positive cancer mortality rates in the Yeh  et al. (1989) study using
    additive linear models. They estimated potencies of 0.036 cancers per
    100 000 per year for every ng/kg per day exposure for the HBV
    negatives and 0.50 cancers per 100 000 per year for every ng/kg per
    day exposure for the HBV positives.

        Their analysis did not look at the combined data under a single
    model and has been criticized for the use of only the small numbers of
    cancers in the HBV negatives. Hoseyni (1992) analysed the Yeh  et 
     al. (1989) data using regression techniques applied to several

    different models including multiplicative and additive background
    combined with linear and linear-exponential (multiplicative only)
    models. He compared these various models based upon goodness-of-fit as
    well as rejection by a likelihood ratio test and concluded that the
    multiplicative model with a linear-exponential effect on mortality
    rates by aflatoxin and HBV status (an added constant in the model if
    HBsAg was positive) best fit these data. He did not explicitly include
    an interaction term in this model (although the multiplicative model
    implies a specific type of interaction) nor did he include an
    interaction term in the additive linear model (there is no implicit
    interaction in this model). The potency of the preferred
    multiplicative model changes as a function of the background (in the
    absence of aflatoxin and HBV) so that potencies can only be given with
    respect to an explicit population liver cancer rate. Focusing on risk
    prediction for the USA population, Hoseyni (1992) chose a background
    cancer rate of 3.4 per 100 000 in deriving potency estimates. The
    resulting estimates were 0.0018 cancers per 100 000 per year for every
    ng/kg per day exposure to aflatoxin in HBV negative individuals and
    0.046 cancers per 100 000 per year for every ng/kg per day exposure in
    HBV positives.

        Wu-Williams  et al. (1992) examined the fit of a variety of
    multiplicative and additive models that incorporated interaction
    terms. These models were fit to the adjusted person-years data as
    discussed above. Two models were found to fit the data adequately and
    equally; an additive-linear model that includes an interaction term
    and a multiplicative-linear model (very similar to that of Hoseyni)
    with no interaction term. Under the additive-linear model, the potency
    estimates were 0.031 and 0.43 for HBV-negative and HBV-positive
    populations, respectively. Under the multiplicative-linear model, the
    same risks for a USA population with a background cancer risk of 2.8
    per 100 000 were 0.0037 and 0.094, respectively.

        Finally, Bowers  et al. (1993) applied an approximation to the
    two-stage model of carcinogenesis (discussed in Kopp-Schneider &
    Portier, 1989) to the adjusted person-years data. This model is
    similar to a mixed additive model suggested by Bowers (1993), but the
    parameters are tied to the biological concepts of induction of
    mutations and growth of mutated cells (Thorslund  et al., 1987). In
    their analysis, it was assumed that aflatoxin had a linear effect on
    the formation of mutations while HBV had no effect on the mutation
    rate. For the growth of mutated cells, Bowers  et al. (1993) assumed
    a linear effect of HBV (presence or absence) and an interaction effect
    of HBV and AFB1. The resulting potencies for the HBV-negative and
    HBV-positive populations were 0.013 and 0.328 cancers per 100 000 per
    year for every ng/kg per day exposure, respectively.

        Potencies for all these studies are summarized in Table 4. Also
    summarized in Table 4 are new analyses performed for the Committee
    that analysed aflatoxin biomarker data from Qian  et al. (1994) and
    Wang  et al. (1996b).

    3.3.4  Potency estimates based on biomarker studies

        Recent studies in Shanghai (Qian  et al., 1994) and Taiwan (Wang
     et al., 1996b) have measured biomarkers of aflatoxin exposure on the
    individual level. The Committee has calculated potency estimates based
    on these studies for comparison to estimates determined on the basis
    of the data of Yeh  et al. (1989). Concerning these studies, an
    additional difficulty is that the internal markers of exposure were
    frequently below the level of analytical quantification and
    consequently individual determinations were necessarily classified on
    an ordinal scale. Estimating potency requires estimating quantitative
    mean levels of internal biomarkers and daily aflatoxin intake
    corresponding to these classifications.


    Table 4.  Potency estimates of the risk of liver cancer in humans
    based upon epidemiological data with correction for HBV status
    assuming an exposure of 1 ng/kg per day

                                                                       
    Study                       HbsAg status    Incidence per 100 0001
                                                                       

    Croy & Crouch (1991)             -          0.036      (0.079)
                                     +          0.50       (0.77)

    Wu-Williams et al.(1992)
    multiplicative-linear            -          0.0037     (0.006)
                                     +          0.094      (0.19)
    additive-linear                  -          0.031      (0.06)
                                     +          0.43       (0.64)

    Hosenyi (1992)
    (background=3.4/100 000)         -          0.0018     (0.0032)
                                     +          0.046      (0.08)

    Bowers et al. (1993)             -          0.013
                                     +          0.328

    Qian et al. (1994)
    (background=3.4/100 000)         -          0.011
                                     +          0.11

    Wang et al. (1996b)
    (background=3.4/100 000)         -          0.0082
                                     +          0.37
                                                                       

    1 Numbers in parentheses represent upper 95% confidence limits on
    the predicted risk when available from the authors.

        In the study of Qian  et al. (1994), 18 out of 50 (36%) cases and
    31 out of 267 (12%) controls had quantified levels of urinary AFB1-
    N7-Gua above the detection limit of 0.07 ng/ml. The overall range of
    quantified levels was 0.3-1.81 ng/ml but the ranges for cases and
    controls were not reported separately and the individual
    determinations are no longer readily available (J.D. Groopman,
    personal communication). The HBV-adjusted relative risk associated
    with detectable levels of AFB1-N7-Gua was 9.1 (95% CI = 2.1, 29.2).
    Though not reported, the HBV-adjusted relative risk associated with
    detectable versus non-detectable levels of AFB1-N7-Gua was 4.6 (95%
    CI = 1.8, 11.3) with a corresponding relative risk of 10.2 (95% CI =
    4.9, 21.2) for HBV positivity.

        Assuming an exponential distribution, estimates of mean levels of
    AFB1-N7-Gua were obtained by fitting the cumulative probability
    below the limit of detection to the proportion of non-detectables. The
    estimated conditional mean values corresponding to non-detectable and
    detectable classifications are 0.031 and 0.18 ng/ml for cases and 0.02
    and 0.095 ng/ml for controls. For the logistic regression
    (multiplicative linear-exponential) model, potency is the product of a
    regression coefficient for AFB effect (on a ratio scale) and the
    background incidence rate. Assuming that the distribution of
    AFB1-N7-Gua in the general population is similar to controls and
    adjusting the regression coefficient by dividing by the difference in
    estimated mean levels corresponding to detectable versus
    non-detectable levels gives:

    J = log(4.6)/(0.095-0.02) = 20 (ng/ml)-1.

        Adjusting for the relative molecular mass of AFB1-N7-Gua versus
    AFB1, and assuming an average body weight of 70 kg, a daily urine
    volume of 1400 ml (ICRP, 1975) and that 0.2% of daily AFB1 intake is
    excreted as AFB1-N7-Gua (Groopman  et al., 1992), the regression
    coefficient is equivalently expressed as 0.0031 (ng AFB1/kg per
    day)-1. For an annual background cancer rate of 3.4 per 100 000, the
    corresponding potency estimate is 0.011 cancers per 100 000 per year
    for every ng/kg per day exposure in HBV negative individuals and
    10-fold higher for HBV-positive individuals.

        In the study of Wang  et al. (1996b), urinary metabolites were
    fully quantified for all individual samples analysed and AFB1-albumin
    adduct levels were quantified for only 93 of 232 (40%) blood samples
    tested with a detection limit of 0.01 fm adduct/g albumin. Estimated
    HBV-adjusted relative risks were 1.6 (95% CI = 0.4, 5.5) for
    detectable versus non-detectable AFB1-albumin adducts and 3.8 (95% CI
    = 1.1, 12.8) for high versus low levels of urinary metabolites.
    Although the urinary biomarker was fully quantified, levels of
    AFB1-albumin adducts better reflect average AFB1 intake. Mean levels
    corresponding to detectable and non-detectable classifications of
    AFB1-albumin were calculated by fitting a log-normal distribution to
    the quantified levels (Santella, personal communication) by log-probit
    analysis (Travis & Land, 1990). For controls, the estimated mean

    values corresponding to non-detectable and detectable classifications
    were 0.0048 and 0.035 fm adduct/g albumin.

        Assuming that the distribution of AFB1-albumin adduct levels in
    the general population is similar to controls, the regression
    coefficient adjusted with respect to quantified levels of
    AFB1-albumin is:

    J = log(1.6)/(0.035-0.0048) = 16 (fm/g albumin)-1

    Based on data from China (Gan  et al., 1988), 1.05 ng AFB1-albumin
    adduct per g albumin corresponds to 1 g AFB1 intake per day.
    Correcting for the relative molecular mass of the AFB1 adduct, the
    conversion factor is 0.00015 fm adduct/g albumin per 1 ng/kg per day
    AFB1 intake and the regression coefficient is equivalently expressed
    as 0.0024 (ng/kg per day)-1. For a population with an annual
    background cancer rate of 3.4 per 100 000, the corresponding potency
    estimate is 0.0082 cancers per 100 000 per year for every ng/kg per
    day exposure in HBV-negative individuals. The estimated relative risk
    of 45.5 for HBV reported in the study of Wang  et al. (1996b)
    suggests a potency of about 0.37 cancers per 100 000 per year for
    every ng/kg per day exposure for HBV-positive individuals.

        The similarity of the estimates suggests that the magnitude of the
    associations detected in Shanghai and Taiwan are relatively consistent
    with that observed in Guangxi. However, the calculations presented
    here are subject to a number of reservations. First, the estimates of
    mean levels corresponding to detectable and non-detectable
    classifications of AFB1-N7-Gua or AFB1-albumin are based on very
    limited data. Furthermore, the conversion factors relating internal
    exposure (AFB1-N7-Gua or AFB1-albumin) to dietary AFB1 intake are
    based on studies in human populations that may have different genetic
    characteristics than the study populations to which the conversion
    factor is applied. For the Taiwan study, there is the additional
    consideration of how the case series was obtained. About half of the
    identified cases were prevalent cases diagnosed at the onset of the
    study. Consequently, the AFB1 exposure determinations for these cases
    may reflect alterations in metabolism directly related to the presence
    of PLC  per se.

    3.3.5  Potency estimates from test species

        Several investigators have studied the carcinogenic potential of
    aflatoxins  in vivo using laboratory animals (Wieder  et al., 1968;
    Butler  et al., 1969; Epstein  et al., 1969; Merkow  et al., 1973;
    Newberne & Rogers, 1973; Wogan  et al., 1974; Vesselinovitch  et 
     al., 1972; Ward  et al., 1975; Reddy & Svoboda, 1976; Sieber  et 
     al., 1979; Stoner  et al., 1986; Angsubhakorn  et al., 1981a,b;
    Butler & Hempsall, 1981; Nixon  et al., 1981; Moore  et al., 1982;
    Cullen  et al., 1987). In most of these studies, hepatocarcinogenesis
    was the main focus although other cancers have been noted such as
    colon, kidney, lung and lymphoreticular system. The majority of these

    studies focused on aflatoxin B1 with one study of aflatoxin M1
    (Cullen  et al., 1987), one study comparing aflatoxins B1, G1 and
    B2 (Butler  et al., 1969) and another study considering the
    aflatoxin metabolite aflatoxicol (Nixon  et al., 1981). All of these
    laboratory results are amenable to quantitative estimation of risks;
    however, some only contain one experimental dose group, have little
    indication of dose-response due to 100% response in all dosed animals
    or include the use of other agents (e.g., vitamin A) in their
    protocols. Cardis  et al. (1997) summarized the calculated potencies
    from aflatoxin exposure in these test species. With regard to
    quantitative estimations and prediction of risks for aflatoxin B1,
    the study by Epstein  et al. (1969) contains the most experimental
    dose-groups and the most complete data for fitting a model. Using a
    simple multistage model of carcinogenesis (Cardis  et al., 1997),
    these data predict an added incidence of 0.97 cancers per 100 000 per
    year for an exposure of 1 ng/kg per day of aflatoxin B1 (scaled from
    the animal data to human risk estimates using body weight raised to
    the 0.75 power). Other potency estimates (extrapolated to humans)
    ranged from as low as 0.05 per 100 000 per year for the Syrian golden
    hamster (Moore  et al., 1982) to as high as 37 per 100 000 per year
    for the Fischer 344 rat (Cullen  et al., 1987), with median estimate
    across all experiments of 1.4 per 100 000 per year.

        The human potency estimates for aflatoxin B1 alone (Table 4 shows
    that these are in the range of 0.002-0.036 per 100 000 per year for
    exposure to 1 ng/kg per day of aflatoxin) fall well below this range,
    suggesting that humans are considerably less sensitive than other
    species. There are several possible explanations for this. First, it
    is possible that humans are in fact less sensitive than species tested
    in laboratory experiments. Considering the proposed mechanism by which
    aflatoxin induces liver tumours at low levels of exposure (DNA damage)
    and the efficiency by which humans repair DNA damage, it is plausible
    that humans are less sensitive. For this to be a reasonable
    explanation, the rate per day of DNA repair in the human system for
    the critical aflatoxin B1 lesions would have to be approximately 5
    times more efficient than that of other species on a surface area
    basis. A second possibility is that estimated exposures in the Yeh  et
    al. (1989) study were larger than the true exposures. While exposure
    estimates in epidemiological studies are generally a point of concern
    for any dose-response assessment, it is unlikely that the estimates
    obtained in the Yeh  et al. (1989) study are biased to this degree. A
    third possibility is misclassification of the HBV cases, with a large
    proportion of the HBV negatives actually being classified as positives
    (the misclassification would need to be about 50% for the human
    potency estimates to move into the range of the test species
    estimates). This level of misclassification is highly unlikely; in
    fact it is more likely that HBV positives have been incorrectly
    classified as negatives. A fourth possibility is that many of the HBV-
    positive individuals with liver tumours were actually infected late in
    life and that, consequently, there was not sufficient time for HBV to
    contribute to development of liver cancer in these individuals. If
    true, this would suggest that the risk of liver cancer due to exposure

    to aflatoxin in the absence of HBV could be as high as the risks seen
    in Table 2 (approximately 0.15 cancers per 100 000 per year for an
    exposure of 1 ng/kg per day). However, once again, this would seem to
    be an unlikely explanation, and still falls well below the animal-
    based estimates.

        Finally, it is possible that the usual conversion factor for
    converting potency estimates in experimental species to potency
    estimates in humans is inappropriate for these data. For the case of
    malignant hepatomas in Wistar rats (Epstein  et al., 1969), as
    mentioned above, conversion based upon surface area scaling using body
    weight raised to the 0.75 power yielded a potency of 0.97 per 100 000
    per year for an exposure of 1 ng/kg per day. If no conversion had been
    made, the potency estimate in the Wistar rat would have been 0.23 per
    100 000 per year. Considering the different sizes of the test species
    involved, the potencies range from 0.014 per 100 000 per year (Syrian
    golden hamster) to 1 per 100 000 per year (Fischer rat), with most of
    the larger mammals being on the low range of the potency scale (0.029
    per 100 000 per year for the tree shrew; 0.057 per 100 000 per year
    for the rhesus and cynomolgus monkeys). In this case, although the
    human estimates are still at the lowest end of the potency scale, they
    would be comparable to the values estimated for other primates.

        Figure 2 presents potencies estimated from animal studies and
    epidemi-ological studies. Epidemiological data for which HBV infection
    status was unknown and for which potencies were estimated gave
    potencies in between the range predicted by the HBV
    infected/non-infected numbers. Potencies given in Figure 2 do not
    generally apply to aflatoxin M1, since exposure estimates given in
    many of the epidemiological studies ignored the contributions to total
    aflatoxins exposure from milk and milk products. From one comparative
    toxicology study in rats, it has been possible to estimate that
    aflatoxin M1 has a potency approximately one order of magnitude less
    than that of aflatoxin B1 in that species (Cullen  et al., 1987).

    FIGURE 2

    4.  AFLATOXIN DIETARY INTAKE ESTIMATES

    4.1  Introduction

        This report summarizes the results of monitoring and available
    national estimates of intake of aflatoxins in order to provide a
    framework for the task of estimating increments in intake of
    aflatoxins. Estimates are based on the results of available monitoring
    data. Total aflatoxin intake based on the GEMS/FOODS regional diets
    are used to evaluate the impact of four different scenarios: no limit,
    and limits set at 20, 15 and 10 g/kg. This evaluation was conducted
    for ground-nuts and for maize for total aflatoxins and for aflatoxin
    B1 alone. Generally they are not considered by the submitters to be
    representative because sampling has focused on those lots that are
    more likely to contain the highest levels of aflatoxin. However, this
    analysis provides useful qualitative comparisons between regulator
    options.

    4.2  Background

        Aflatoxins are found as contaminants in human and animal food as a
    result of fungal contamination both pre- and post-harvest, with the
    rate and degree of contamination being dependent on temperature,
    humidity, soil and storage conditions. Though a wide range of foods
    may be contaminated with aflatoxins, they have been most commonly
    associated with groundnuts (groundnuts and groundnut products), dried
    fruit, tree nuts, spices, figs, crude vegetable oils, cocoa beans,
    maize (maize), rice, cottonseed and copra. There are practices that
    reduce but do not completely eliminate aflatoxins in grains,
    groundnuts, figs and other crops. However, even in the most tropical
    of climates, many lots of these crops do not contain detectable levels
    of aflatoxins.

    4.3  Methods

    4.3.1  Period of intake of relevance

        Chronic (lifetime) intake is assumed to be the period of
    relevance. Therefore, the average concentrations in the diet are of
    primary interest.

    4.3.2  Estimated levels of aflatoxins in foodstuff

        Data were available for this analysis from at least one country on
    every continent. Typically the data were uniformly judged by the
    submitters not to be representative. In most instances, the data were
    thought to be biased towards the upper end of intake. Nonetheless,
    caution must be exercised in using the data to generate intake
    estimates and in interpreting the results of such analyses. However,
    the data did provide a framework to explore the relative impact of
    regulatory activities. The data are summarized in Table 5.

        For some of the analyses used in this paper individual data points
    were required in order to generate distributions and to evaluate the
    impact of imposing upper limits on aflatoxins in foodstuffs.
    Specifically, a series of analyses was conducted to determine the
    impact of truncating the distribution at 10, 15 and 20 g/kg,
    respectively, in order to simulate the potential impact of proposed
    limits. For these analyses, data reported by the USA, China and Europe
    were evaluated because the raw data were available. These analyses
    were conducted for maize and groundnuts for total aflatoxins and for
    aflatoxin B1.

    4.3.3  Estimated intakes

        Four pieces of information are required to estimate the potential
    intakes due to aflatoxins in crops that are imported: (1) the levels
    of aflatoxin in imported crops; (2) the amount of each imported crops
    consumed; (3) the impact of any subsequent processing on aflatoxins
    levels; and (4) methods for combining the first 3 to estimate intake.



        Table 5.  Summary of aflatoxin monitoring data submitted for consideration by the Committee

                                                                                                                                     
    Commodity             Country/Region    Number of samples      Results                      Comments            Reference
                                                                                                                                     

    NUTS

    Groundnuts
    (groundnuts and
    groundnut products)   Australia         9 samples           Mean = 2 g/kg;                                     Australia Market
                                                                Max = 10 g/kg                                      Basket (1992)

                                            913 lots            83.1% <5 g/kg               presorted              Read (1997)
                                            (b1)                14.2% 5-10 g/kg
                                                                2.7% 10-15 g/kg

                          Brazil            199 samples         Mean = 14 g/kg                                     Sabino (1997)
                                            (b1)                Max = 181 g/kg
                                                                51%> LOD

                          China             174 groundnuts      3 >30 g/kg               selected to be worst      Chen (1997)
                                            40 groundnut meal   1 >251 g/kg              case for research
                                            (b1)                (see Table 3)             purposes

                          Cuba              1114 samples        49% >LOD                                            Regueiro
                                            (b1)                                                                    (no date)

                          European Union    data from 12        Mean B1 35-64 g/kg       data submitters           SCOOP (1996)
                                            countries           (country means)           emphatically state that
                                                                Max = 789 g/kg           DATA ARE NOT
                                                                (see Tables 3 and 4)      REPRESENTATIVE

                          Japan             22 789 samples      >97% >LOD                 imported from >25         Japanese Ministry
                                            (1972-1989)         238 >10 g/kg (B1)        countries                 of Health (1995)
                                                                Max = 8070 g/kg

                          Mexico            107 samples         104 <20 g/kg                                       Mexico (1996)
                                            (1992-1995)         3 >20 g/kg
                                                                                                                                     

    Table 5.  Continued...

                                                                                                                                     
    Commodity             Country/Region    Number of samples   Results                         Comments            Reference
                                                                                                                                     

                          Nicaragua         9 samples (1996)    1 >15 g/kg (B1)                                    Nicaragua (1996)

                          USA               >600 000 lots       >90% <15 g/kg                                      Wood (1995)
                                            (1975-1992)         (total); see Table 6

                          Zimbabwe          286 samples         39% <5 g (1995)          analysed B1 and total     Zimbabwe Government
                                            (1995-1996)         54% <10 g (1995)                                   Analyst Laboratory
                                                                46% >10 g (1995)                                   (1995-1996)
                                                                85% <5 g (Jan-Jun 96)
                                                                92% <10 g/kg
                                                                8% >D12 10 g/kg+D24

    Brazil nuts           European Union    not available       Max = 15 g/kg (B1)       see comment under         SCOOP (1996)
                                                                Max = 35 g/g (total)     groundnuts

                          Japan             74 samples          70 <LOD                                             Japanese Ministry
                                                                2 >10 g/kg                                         of Health (1995)
                                                                2 >10 g/kg
                                                                Max = 123 g/kg

    Pistachio nuts        European Union    11 countries        Mean = 2-23 g/kg
                                            provided data       (B1; country means)
                                                                Mean = 44-27 g/kg
                                                                (total; country means)
                                                                Max = 450 g/kg (B1)
                                                                Max = 813 g/kg (total)

                          Japan             2422 samples        2339 <LOD (B1)                                      Japanese Ministry
                                            (1972-1989)         48 >10 g/kg                                        of Health (1995)
                                                                35 <10 g/kg
                                                                Max = 8030 g/kg

                          Mexico            244 samples         5 samples >20 g/kg                                 Mexico (1996)
                                            (1993-1996)
                                                                                                                                     

    Table 5.  Continued...

                                                                                                                                     
    Commodity             Country/Region    Number of samples   Results                         Comments            Reference
                                                                                                                                     

    Sunflower seeds       Argentina         20 samples          no detects                                          Argentina (1996)

    Almonds               Australia         9 samples           no detects                                          Australian Market
                                                                                                                    Basket Survey (1992)

                          Japan             93 749 samples      >98% <LOD                 reported all              Japanese Ministry
                                            Max = 128 g/kg                               miscellaneous nuts        of Health (1995)
                                                                                          in one statistics;
                                                                                          covered years 1972-
                                                                                          1989; all were imported

    Cashews               Japan             1227 samples        >98% <LOD                                           Japanese Ministry
                                                                                                                    of Health (1995)

    Walnuts               Japan             321 samples         >98% <LOD                                           Japanese Ministry
                                                                                                                    of Health (1995)

    Macadamia nuts        Japan             149 samples         >98% <LOD                                           Japanese Ministry
                                                                                                                    of Health (1995)

    Hazel nuts            Japan             103 samples         >98% <LOD                                           Japanese Ministry
                                                                                                                    of Health (1995)

    FIG PRODUCTS
                          European Union                        Mean = 0.5-26 g/kg       see comment for           SCOOP (1996)
                                                                (country mean B1)         groundnuts

    CEREAL & CEREAL PRODUCTS

    rice, wheat, maize
                          Bolivia           number unknown      2 rice 25-168 g/kg (B1)
                                            (1992-1995)         1 wheat 10 g/kg (B1)
                                                                1 wheat 18 g/kg (B1 + g1)
                                                                                                                                     

    Table 5.  Continued...

                                                                                                                                     
    Commodity             Country/Region    Number of samples   Results                         Comments            Reference
                                                                                                                                     

                                            2460 samples
                                            (1986-1997)         1273 >LOD
                                                                Mean = 34 g/kg
                                                                (range = 7-144 g/kg)

    Maize                 Brazil            321 samples         179 <LOD                                            Sabino (1997)
                                                                27 <30 g/kg
                                                                116 >30 g/kg
                                                                (B1 or total not reported)
                                                                Max = 2440 g/kg

    Maize                 Brazil            2546 samples        Mean = 35 g/kg
                                                                (51% >LOD)
    Rice                  Brazil            401 samples         Mean = 2 g/kg
                                                                (10% >LOD)
    Wheat                 Brazil            237 samples         Mean = 2 g/kg
                                                                (19% >LOD)
    Malt                  Brazil            30 samples          Mean = 30 g/kg
    Popcorn               Brazil                                32% >LOD
    Sorghum               Brazil            59 samples          Mean = 3 g/kg
                                                                (33% >LOD)

    Maize                 China             486 samples         Mean 5-251 g/kg
                                                                (117 >LOD)                                          Chen (1997)
    Wheat                 China             597 samples         Max = < 31 g/kg
                                                                (9 >LOD)                        see also Table 7    Chen (1997)
    Sorghum               China             58 samples          1 >LOD                                              Chen (1997)
    Rice                  China             747 samples         7 >LOD                                              Chen (1997)

    Sorghum               Columbia          45 samples          11 >LOD (B1)                                        Diaz (1996)
                                            (1995-1996)         (1.4-43 g/kg)
                                                                Mean = 11 g/kg
                                                                                                                                     

    Table 5.  Continued...

                                                                                                                                     
    Commodity             Country/Region    Number of samples   Results                         Comments            Reference
                                                                                                                                     

    Maize                 Columbia          33 samples          4 >LOD (B1)                                         Diaz (1996)
                                                                (4-66 g/kg)
                                                                Mean = 21 g/kg

    Soybeans              Columbia          25 samples          0 >LOD (B1)                                         Diaz (1996)

    Rice                  Columbia          22 samples          8 >LOD (B1)                                         Diaz (1996)
                                                                (1-53 g/kg)
                                                                Mean = 21 g/kg

    Cotton                Columbia          17 samples
                                            (1995-1996)         15 >LOD (B1)                                        Diaz (1996)
                                                                (2-11 g/kg)
                                                                Mean = 5 g/kg

    Maize                 Costa Rica        49 samples          1 >15 g/kg (B1)                                    Pacin (no date)
                                                                48 <10 g/kg

    Maize                 Cuba              4620 samples        20% >LOD (B1)                                       Regueiro (no date)
    Rice                  Cuba              340 samples         16% >LOD                                            Regueiro (no date)
    Sorghum               Cuba                                  12% >LOD                                            Regueiro (no date)
    Wheat                 Cuba                                  1% >LOD                                             Regueiro (no date)

    CEREAL PRODUCTS
                          European Union                        0.1-7.6 g/kg                see comment for        SCOOP (1996)
                                                                (country means B1)           groundnuts
                                                                0.25-5.9 g/kg
                                                                (country means total)
                                                                (see Tables 6 and 7)

    Maize                 Japan             371 samples         16 >LOD                                             Japanese Ministry
                                                                Max B1 = 1.5 g/kg                                  of Health (1995)
                                                                                                                                     

    Table 5.  Continued...

                                                                                                                                     
    Commodity             Country/Region    Number of samples   Results                         Comments            Reference
                                                                                                                                     

    Maize                 Mexico            1710 samples        265 >20 g/kg                                       Mexico (1996)
                                            (1992-1996)         35 tortilla samples
                                                                >20 g/kg

    Maize                 Thailand          18 samples          <LOD - 606 g/kg           Aflatoxin was visible    Yoshizawa et al., 
                                                                                                                    1996

    PULSES

    Soybeans              Brazil            143 samples         17% >LOD
                                                                Mean = 1 g/kg

    Soybeans              China             388 samples         11 >LOD                    Jiangu Province          Chen (1977)
                                                                (51-100 g/kg)

    Soy sauce             China             308 samples         4.6% >LOD                  Jiangu Province          Chen (1977)

    Bean paste            China             1 sample            <100 g/kg                 Jiangu Province          Chen (1977)
                                                                3.3% > LOD
                                                                Max = >251 g/kg

    Soy oil               China             379 samples         10 samples >LOD            Jiangu Province          Chen (1977)
                                                                Max = <251 g/kg

    Soybean meal          China                                 6.7% >LOD                  Jiangu Province          Chen (1977)
                                                                Max = <50 g/kg

    Frijoles (beans)      Cuba              413 samples         13% >LOD (B1)                                       Regueiro (no date)

    Pulses                Japan             >2000 samples       2 samples >LOD (B1)                                 Japanese Ministry
                                                                Max = <10 g/kg                                     of Health (1995)
                                                                                                                                     

    Table 5.  Continued...

                                                                                                                                     
    Commodity             Country/Region    Number of samples   Results                         Comments            Reference
                                                                                                                                     

    SPICES
                          European Union                        Max B1 = 323 g/kg              reported in at      SCOOP (1996)
                                                                (0.5-17 g country              least one sample 
                                                                mean levels)                    of every type of 
                                                                Max total = 72 g/kg            spice analysed- see 
                                                                                                comment re: data 
                                                                                                under groundnuts


                          Japan             1804                >LOD in chilli, nutmeg,         none found in 25    Japanese Ministry
                                            (1979-1988)         white pepper, paprika,          other spices        of Health (1995)
                                                                turmeric, cardamon,
                                                                pimento, ginger, celery
                                                                seed spices

    CONFECTIONARY PRODUCTS
                                            few samples         0.05 = - 1.1 g/kg                                  SCOOP (1996)
                                                                (country means B1)

    FATS, OILS, OILSEEDS
                          France            56 samples          Mean 4.5 g/kg (France)
                                                                Max = <25 g/kg

    MILK AND MILK PRODUCTS                                                                                          
                          see text                                                                                  
                                                                                                                                     
    

     a) National intakes

        Available national intake studies were reviewed to provide
    estimates of intake levels. This information was obtained from reports
    submitted by members of the EU (SCOOP, 1996), China (Chen, 1997), USA
    (1992), Brazil, Australia, Costa Rica, Argentina, Mexico, Nicaragua,
    Colombia and Thailand. Thirteen European countries provided data for
    the EU SCOOP project.

     b) International intakes

        The available data provide a general idea of the range of
    aflatoxin levels in foodstuffs and the frequency of detection.
    Information on the proportion of the crop imported was not available
    for most countries, nor was it possible to determine the distribution
    of aflatoxin levels in imported crops versus domestic crops.
    Therefore, the data from individual countries were reviewed to obtain
    a general overview of the likely levels in foods and to determine the
    impact on average intakes if extreme levels of aflatoxins could be
    removed from foodstuffs.

         Impact on dietary intake if upper concentrations of aflatoxin 
     in foodstuffs are successfully limited to 10, 15 or 20 g/kg 
     foodstuff versus no limit

        It was assumed that methods are available to ensure that aflatoxin
    levels above the specified limit are excluded from the food supply and
    that the same proportion of the commodity would be imported (versus
    domestic) regardless of the limit.

        For these analyses all of the food consumed in the country was
    assumed to contain the average residue concentration under that
    scenario. Each analysis was repeated twice: (1) using European
    monitoring data (b1 and total aflatoxin; and (2) using either Chinese
    monitoring data (aflatoxin b1) or USA monitoring data (total
    aflatoxin). Using these assumptions, the GEMS/FOODS regional diets
    were used to evaluate the difference in dietary intake to aflatoxin
    from groundnuts and maize under different limits.

     c) Amounts consumed

        The GEMS/FOODS regional diets were used as estimates of the
    consumption of each of the commodities (Tables 4 and 5).

    4.4  Results

    4.4.1  Aflatoxin levels in foods: general

        The 1995 FAO compendium, Worldwide regulations for mycotoxins
    (FAO, 1995), summarized reports from 48 countries. The data submitted
    by 33 countries for aflatoxin B1 and total aflatoxins (B1,B2,G1,G2)
    were used to estimate median levels of 4 and 8 g/kg, respectively, in
    foodstuffs. The range of levels reported for B1 was from 0 to 30 g/kg

    and for total from 0 to 50 g/kg. Seventeen countries provided
    information on aflatoxin M1 in milk with a median of 0.05 g/kg and a
    range of 0-1 g/kg. The FAO report did not provide additional details
    about sampling, treatment of non-detects, imported versus domestic
    foodstuffs, etc.

        Reports from individual countries that were submitted to FAO and
    to JECFA have been used to provide additional detail. The data are
    summarized in Table 5 by commodity and by country.

        The participants in the European Union Scientific Co-operation
    Assessment of aflatoxin (SCOOP) reviewed data submitted by member
    countries and by Norway. The participants concluded that the results
    were unlikely to be representative and should not be used to estimate
    total aflatoxin intake for individual countries or for Europe.
    However, the studies did provide some insight into issues surrounding
    aflatoxin intake assessments. Based on the data and subsequent
    discussions, SCOOP concluded: (1) aflatoxins are found in a broader
    range of foods than had been previously assumed; (2) most of the
    samples did not contain detectable aflatoxin; (3) sampling methods are
    important in accurately estimating aflatoxin levels; and (4) different
    methods of collecting food consumption data may make a difference in
    estimating aflatoxin intakes.

    4.4.2  Aflatoxin levels in foodstuffs: Occurrence data by commodity

        Most countries provided data for aflatoxin B1 in selected crops.
    Some countries also provided an estimate of the level of total
    aflatoxins. Total aflatoxin estimates typically included aflatoxin B1,
    B2 and G. M1 was the common aflatoxin reported for milk and milk
    products. The data are summarized in Table 1 for crops.

        The data for aflatoxin M1 are summarized below:

        Australia: No aflatoxins were detected in 34 whole milk samples.

        European Union: Ten countries reported results of sampling for M1
    in milk. The maximum level of 0.37 g/kg M1 was reported by France.
    The United Kingdom reported the highest level (0.22 g/kg) in cheese.

        Brazil: 204 samples of milk, cheese and yoghurt were analysed. Of
    these only four samples of pasteurized milk contained detectable
    levels of aflatoxin M1 (de Sylos, 1996). The levels ranged from 73-370
    ng/litre.

        Spain: Aflatoxin M1 was estimated in 19 total diet studies in 1990
    and 1991. All but one sample was below the limit of detection. The one
    sample contained 0.025 g/kg.

    4.4.2.1  Amount of commodity imported

        The proportion of any commodity that is imported varies from 0 to
    100%. For example, in the case of groundnuts all groundnuts, are

    imported into the Nordic countries while virtually no groundnuts are
    imported into China and the USA. If import/export data were available,
    aflatoxin intakes could be more accurately estimated. However, this
    information was not available for most countries.

    4.4.2.2  Accounting for the change in aflatoxin levels during
    processing

     a) Maize

        Processing of maize causes reduction in aflatoxin levels. Wet
    milling reduces the concentration of aflatoxin in maize starch to 1%
    of the levels found in the raw grain (Yahl, 1971). Similarly dry
    milling reduces aflatoxin in food products (grits, low-fat meal and
    low-fat flour) to 6-10% of the original concentrations (Brekke, 1975).

     b) Groundnuts

        The roasting of groundnuts reduces aflatoxin levels by 50-80%
    (Waltking, 1971; Read, 1989; Billy, 1996).

     c) Milk

        Aflatoxin M1 is a metabolite of aflatoxin B1 and is found
    associated with the casein in milk. Pasteurization does not affect the
    level of aflatoxin M1 in milk or yogurt (Wiseman, 1983). Aflatoxin M1
    has been reported to concentrate 3-6 fold during cheese making (Van
    Egmond, 1983). MAFF (1994-5, No. 22) reported that aflatoxin was not
    destroyed under domestic cooking conditions (microwave or heading in
    gas oven).

        The effects of processing were considered in many of the national
    estimates. Processing factors have not been included in any of the
    distributions generated for the estimates of aflatoxin intake.
    However, it would be appropriate to adjust further the international
    estimates of intake to reflect the impact of processing where the
    commodity is always processed/cooked prior to consumption.

    4.4.3  National estimates of aflatoxin intake

    4.4.3.1  Australia

        Australia conducts market basket surveys and estimates intake for
    average and extreme consumers. The average diet was estimated to
    contain 0.15 ng aflatoxin/kg bw per day and the upper 95th percentile
    diet to contain approximately twice that level. Children's diets were
    estimated to be somewhat higher - up to approximately 0.45 ng/kg bw
    per day for the 95th percentile 2-year-old (Australia Market Basket
    Survey, 1992).

    4.4.3.2  China

        A series of intake and market basket studies have been conducted
    since 1980 to estimate the aflatoxin B1 intake. The reported intakes
    have ranged from 0 to 91 g aflatoxin B1/kg bw per day (Chen, 1997).

    4.4.3.3  European Union

        Estimates of aflatoxin intake were provided to the EU SCOOP
    project by 9 countries. However, it should be noted that in every
    instance, it was clearly stated that these estimates were not
    representative. They were regarded as being no more than indicators of
    intake of aflatoxin and as clearly not useful for predictive purposes.
    These indicators of intake ranged from 2 to 77 ng/person per day for
    aflatoxin B1 and from 0.4 to 6 ng/person per day for aflatoxin M1.
    Although these may be useful to guide understanding of potential
    intake, they are not to be used as an estimate of intake either for a
    particular country or for Europe.

        Of the countries that computed intakes of M1, the computed intakes
    ranged from 0.04 g/kg bw per day M1 (France, Germany) to 0.19 ng/kg
    bw per day (Netherlands). The value reported by the Netherlands was
    from baby food. In addition, the Netherlands also estimate 0.04 ng/kg
    bw per day M1 from milk.

        France provided additional information on intakes from cereals,
    nuts, spices and milk. Two estimates of aflatoxin intake were
    computed. The first was the product of the maximum level of aflatoxin
    reported and the maximum consumption for each category of food. The
    second estimate was the product of the average aflatoxin concentration
    reported and the maximum consumption. The resulting estimates are
    given in Table 6.

    Table 6.  Estimated aflatoxin intake in France (g per day)

                                     
                Maximum        Mean
                                     
    Cereal      13.33          2.42
    Nuts        7.13           0.04
    Spices      0.68           0.01
    Milk        0.12           0.06
                                     


    4.4.3.4  USA

        The US FDA estimated intakes using data from the National
    Compliance program for maize, groundnut and milk products using Monte
    Carlo simulation procedures. The data were from the 1980s. The
    eaters-only mean lifetime intake of total aflatoxin was 18 ng/person
    per day and intake for the 90th percentile individuals was 40

    ng/person per day. Mean aflatoxin M1 intake was 44 ng/person per day
    and for the 90th percentile individuals 87 ng/person per day. The
    authors noted that many assumptions were made that bias these
    estimates upwards. The same analysis was repeated in 1992 with only
    slightly different results (DiNovi, 1992).

    4.4.3.5  Zimbabwe

        The theoretical maximum intake of aflatoxin B1 was estimated for a
    child's diet containing 150 grams maize with 5 g/kg aflatoxin B1 and
    30 grams ground-nuts with 10 g/kg aflatoxin B1. The total aflatoxin
    intake per day would be 1.05 g per day if all maize consumed
    contained 5 g/kg and all groundnuts contained 10 g/kg. If all maize
    were to contain 15 g per day the intake would be 2.55 g per day.

    4.4.4  Relative impact of establishing maximum limits on estimate of
    intake

    4.4.4.1.  Average aflatoxin concentrations using four possible
    scenarios

        Data from the EU, China and the USA were used to assess the
    potential impact of successfully eliminating aflatoxin levels above 20
    g/kg versus 15 g/kg versus 10 g/kg versus no limit for maize and
    groundnuts. For each commodity, two sets of analyses were conducted,
    (1) total aflatoxins and (2) aflatoxin B1, in order to determine
    whether different conclusions would be reached. For each analysis the
    data were evaluated as reported. In all cases in which samples
    contained no detectable aflatoxins it was assumed that aflatoxins were
    present at the LOD - obviously an overestimation. No impact of
    processing was included, which again is an overestimation since
    processing is known to result in 50-90% reduction in concentrations.

        Table 6 summarizes the impact of successfully limiting aflatoxin
    concentrations to less than 10, 15 or 20 g/kg foodstuff on the mean
    total aflatoxin concentrations in maize and groundnuts.



        Table 6.  Anticipated mean residues of total aflatoxins in maize and groundnuts under four assumptions for
    acceptable residue levels in samples:
         Scenario 1:    no samples excluded
         Scenario 2:    samples > 10 g/kg excluded
         Scenario 3:    samples > 15 g/kg excluded
         Scenario 4:    samples > 20 g/kg excluded

                                                                                                            
    Scenario              Samples      USA maize   European cereals   European groundnuts   USA groundnuts
                          excluded                                                       
                                                     g total aflatoxin/kg commodity
                                                                                                            

    1 No limit            Mean           4.7             0.2                13.3                  14.3
                          SD             30.7            0.4                97.2                  58.8

    2 Limit = 10 g/kg    Mean           0.6             NA                 0.9                   0.4
                          SD             1.4                                1.4                   0.9

    3 Limit = 15 g/kg    Mean           0.7             NA                 0.9                   0.5
                          SD             2.0                                1.6                   1.4

    4 Limit = 20 g/kg    Mean           0.9             NA                 1.0                   0.6
                          SD             2.6                                2.0                   1.7
                                                                                                            

    Note:  Samples were taken in either USA or Europe but the crop may have come from other geographic locations
    


        No aflatoxins levels above 5 g/kg have been reported on cereals
    in Europe, so there would be no impact of imposing a regulatory
    programme that would reduce aflatoxin levels1. In contrast, for
    groundnuts and for groundnuts and maize in the USA, the largest
    difference in mean aflatoxins levels is between no limit and a maximum
    aflatoxin level of 20 g/kg. In the USA, the greatest impact is
    achieved by establishing a maximum level of 20 g/kg for both maize
    and ground-nuts. For example, using data for USA maize, the average
    total aflatoxin levels would drop from 4.7 g/kg to 0.9 g/kg if all
    maize with aflatoxin levels above 20 g/kg were rejected (Table 6).
    Small additional declines in average aflatoxin levels would be found
    if the acceptable limit were 15 or 10 g/kg. The average aflatoxin
    levels would be 0.7 and 0.6 g/kg if 15 and 10 g/kg limits were
    established (Table 7) for aflatoxin B1 in maize.

        The same analysis was conducted for groundnuts. The reported total
    afla-toxin levels in groundnut and groundnut products sampled in
    Europe and USA are presented separately Table 6. If all groundnuts are
    included, the average aflatoxin concentration would be 14 g/kg. The
    average aflatoxin concentration would be 0.6 g/kg if all samples with
    levels above 20 g/kg were excluded and 0.5 and 0.4 g/kg if all
    samples with levels above 15 and 10 g/kg, respectively, were
    excluded.

        The distribution of total aflatoxins in crops sampled in the USA
    and Europe is provided in Table 8 and the distribution of aflatoxin B1
    in crops in Europe and China is provided in Table 9.

                   

    1 The major part of cereals for human consumption in Europe are
    domestically grown. If the source of cereals were to change this might
    no longer be the situation.

        Table 7.  Anticipated mean residues of aflatoxin B1 in maize and groundnuts under four
    assumptions for acceptable residue levels in samples:
         Scenario 1:    no samples excluded
         Scenario 2:    samples > 10 g/kg excluded
         Scenario 3:    samples > 15 g/kg excluded
         Scenario 4:    samples > 20 g/kg excluded

                                                                                        
    Scenario              Samples      European    Chinese    European      Chinese
                          excluded     cereals     maize      groundnuts    groundnuts
                                            g total aflatoxin B1/kg commodity
                                                                                        

    1 No limit            Mean         1.5         11.8       6.9           8.3
                          SD           2.3         52.3       72.2          33.1

    2 Limit = 10 g/kg    Mean         1.5         2.8        0.6           2.7
                          SD           2.3         1.9        0.9           1.6

    3 Limit = 15 g/kg    Mean         1.5         3.1        0.6           2.7
                          SD           2.3         2.5        1.2           1.8

    4 Limit = 20 g/kg    Mean         1.5         3.5        0.7           2.9
                          SD           2.3         3.3        1.7           2.3
                                                                                        

    Note:  Samples were taken in either China or Europe but the crop may have come from
    other geographic locations
    
    Table 8.  Distribution of total aflatoxins in maize and groundnuts
    sampled in the USA and Europe1

                                                                   
    Percentile   USA maize   European     European         USA
                             cereals      groundnuts    groundnuts
                         g total aflatoxin/kg commodity
                                                                   
    10.0%           0.1        0.0           0.1            0.1
    20.0%           0.1        0.0           0.2            0.1
    30.0%           0.2        0.0           0.3            0.2
    40.0%           0.2        0.0           0.5            0.2
    50.0%           0.3        0.1           0.6            0.3
    60.0%           0.3        0.1           0.7            0.3
    70.0%           0.4        0.1           0.8            0.4
    80.0%           0.5        0.1           1.0            0.4
    90.0%           4.0        0.1           3.3            1.9
    95.0%           15.1       1.1           11.5           106
    97.5%           38.1       1.5           55.3           267
    99.0%           93.8       2.1           379            304
    99.5%           149        2.6           767            314
    99.8%           247        3.2           1110           323
    99.9%           482        3.4           1320           327
                                                                   

    1 European cereals include other crops in addition to maize.

    Table 9.  Distribution of aflatoxin B1 in maize and groundnuts sampled
    in Europe1 and China

                                                                   
    Percentile      European   Chinese    European      Chinese
                    cereals    maize      groundnuts    groundnuts
                             g aflatoxin B1/kg commodity
                                                                   

    10.0%             0.1        0.6         0.0           0.6
    20.0%             0.1        1.2         0.1           1.2
    30.0%             0.3        1.8         0.1           1.7
    40.0%             0.5        2.5         0.2           2.3
    50.0%             0.7        3.0         0.4           3.0
    60.0%             0.9        3.6         0.6           3.5
    70.0%             1.2        4.3         0.7           4.0
    80.0%             1.9        4.9         0.8           4.6
    90.0%             5.4        16.1        1.2           7.8
    95.0%             7.6        43.5        4.2           43.8
    97.5%             8.8        75.9        16.9          69.9
    99.0%             9.4        169         61.4          90.8
    99.5%             9.7        453         367           97.3
    99.8%             9.9        630         809           150
    99.9%             10.0       720         1220          488
                                                                   

    1 European cereals include other crops in addition to maize
    From: Chen (1997) and SCOOP (1996)

        The proportion of samples that would be excluded under each
    scenario is identified in Table 10 for total aflatoxins. For example,
    if all maize that contains total aflatoxin levels > 20 g/kg is to be
    eliminated, 4% of USA maize would be rejected. If the limit is 10
    g/kg, 6% of USA maize would be rejected. No European maize would be
    rejected under either limit. Six per cent of USA and 4% of European
    groundnuts would be rejected with a limit of 20 g/kg and 7% and 5%,
    respectively, with a limit of 10 g/kg. The same information is
    provided in Table 11 for aflatoxin B1. If all maize that contains
    aflatoxin B1 levels > 20 g/kg is eliminated, 8% of Chinese maize
    would be rejected. If the limit is 10 g/kg, 13% of Chinese maize
    would be rejected. No European maize would be rejected under either
    limit. A limit of 20 g/kg would result in rejection of 2% of European
    groundnuts and 8% of Chinese groundnuts. A limit of 10 g/kg, would
    rejected 9% of Chinese groundnuts and 3% of European groundnuts.

    4.4.4.2  Intake of total aflatoxins using four scenarios

        The four scenarios are:

        Scenario 1: no change in current aflatoxin levels;
        Scenario 2: exclude groundnuts and maize with > 10 g/kg total
                    aflatoxin;
        Scenario 3: exclude groundnuts and maize with > 15 g/kg total
                    aflatoxin;
        Scenario 4: exclude groundnuts and maize with > 20 g/kg total
                    aflatoxin).

        This analysis was conducted using the average aflatoxin levels
    that would result under each scenario and was repeated using only the
    data sampled in the USA and only the European data (for total
    aflatoxins and only the data in Europe and China for aflatoxin B1).

        The range of estimated intakes of total aflatoxin from maize is
    shown in Table 12. The estimated range of intakes is 2-501 ng total
    aflatoxin/person perday. These data should not be used as true
    estimates of likely intake but rather as a measure of the relative
    impact of establishing limits. Thus with an upper limit of 20 g/kg
    aflatoxin, the estimated intake using the European WHO regional diet
    and European monitoring data would be 2 ng/person per day. There would
    be no impact in establishing a limit since there are no European maize
    data with levels > 5 g/kg. Using the USA monitoring data and the
    European WHO diet (which includes North America) the intake would be
    47 ng/person per day with no limit, 9 ng/person with a 20 g/kg limit
    and 7 ng/person per day with a 15 g/kg limit. A limit of 10 ng/person
    per day would lower the estimated intake to 6 ng/person per day (using
    the USA monitoring data). This exercise is repeated using the other
    four WHO regional diets and either the European or USA maize/cereal
    monitoring data.

    Table 10. Distribution of estimated concentrations of total 
    aflatoxin in maize and groundnuts sampled in the USA and Europe1

                                                               
    Aflatoxin  USA maize   European    European        USA
    (g/kg)                cereals     groundnuts   groundnuts
                                       percentiles
                                                               

    0.5          87.4%      91.5%         44.2%        89.1%
    1            88.0%      94.7%         81.8%        89.1%
    2.5          88.9%      99.2%         88.8%        90.3%
    5            90.6%      NA            91.7%        92.0%
    7.5          92.1%      NA            93.8%        92.6%
    10           93.8%      NA            94.9%        93.0%
    12.5         94.2%      NA            95.1%        93.4%
    15           95.0%      NA            95.3%        93.7%
    17.5         95.7%      NA            95.5%        94.0%
    20           96.1%      NA            95.8%        94.1%
    30           96.8%      NA            96.4%        94.6%
    40           97.6%      NA            96.8%        94.7%
    50           98.0%      NA            97.4%        94.8%
                                                               

    1 European cereals include other crops in addition to maize
    From: Chen (1997) and Wood (1995)


    Table 11.    Distribution of estimated concentrations of aflatoxins B1
    in maize and groundnuts sampled in Europe1 and China

                                                                       
    Aflatoxin B1       European     Chinese     European     Chinese
    levels (g/kg)     cereals      maize       groundnuts   groundnuts
                                                                       

    0.5                 41.6%         7.5%        56.9%          8.3%
    1                   66.2%        16.3%        89.8%         17.3%
    2.5                 83.4%        40.4%        93.1%         43.0%
    5                   89.3%        80.9%        96.4%         88.0%
    7.5                 94.8%        83.5%        96.7%         89.8%
    10                   NA          86.5%        97.1%         90.6%
    12.5                 NA          88.2%        97.2%         90.9%
    15                   NA          89.5%        97.4%         91.3%
    17.5                 NA          90.7%        97.6%         91.8%
    20                   NA          91.7%        97.9%         92.1%
    30                   NA          92.7%        98.3%         93.4%
    40                   NA          94.4%        98.5%         94.7%
    50                   NA          96.0%        98.9%         95.8%
                                                                       

    1 European cereals include other crops in addition to maize.

        The range of estimated intakes of total aflatoxin from groundnuts
    was 2-162 ng/person per day (Table 12). However, these data should not
    be used as true estimates of likely intake. Rather they should be used
    as a measure of the relative impact of establishing limits. Thus, for
    example, establishing a programme that successfully limits aflatoxin
    levels to 20 g/kg would reduce the estimated intake of aflatoxin from
    groundnuts to 5 ng per day from 66 ng/person per day (European diet).
    Likewise, reducing aflatoxin levels to no more than 15 g/kg limit
    would maintain the estimated intake at 5 ng/person per day (Table 12).
    Similarly, the estimated intake using the European monitoring data and
    a 10 g/kg limit would be 4.4 ng/person per day. Similar comparisons
    are shown in Table 12 for each of the regional diets combined with
    either the European or USA aflatoxin monitoring results.

    4.4.4.3  Intake of aflatoxin B1 within four scenarios

        These analyses were repeated for aflatoxin B1 using the results of
    the European and Chinese monitoring results. The results are presented
    in Table 13.



        Table 12.  Estimated intake of total aflatoxin under 4 different scenarios
                          Scenario 1:       no samples excluded
                          Scenario 2:       samples >10 g/kg excluded
                          Scenario 3:       samples >15 g/kg excluded
                          Scenario 4:       samples >20 g/kg excluded

                                                                                                                          
    I.  FOOD CONSUMPTION ESTIMATES (GEMS/Foods, World Health Organization) + 431

                                                                                                                          
                    Middle Eastern          Far Eastern           African           Latin American          European
                  (g/person per day)    (g/person per day)   (g/person per day)   (g/person per day)   (g/person per day)
                                                                                                                          

    Maize                 50                    31                  106                   42                   10
    Groundnuts             0.3                   6                   11.3                  2                    5
                                                                                                                          

    II.  MEAN TOTAL AFLATOXIN RESIDUE CONCENTRATIONS UNDER EACH SCENARIO (from Table 6)

                                                                                                  
    A.  MAIZE/CEREALS                                               Mean residues
                                                 European monitoring data    USA monitoring data
                                                 (g/kg)                     (g/kg)
                                                                                                  

    Scenario 1: no samples excluded                        0.2                       4.7
    Scenario 2: samples >10 g/kg excluded                 0.2                       0.6
    Scenario 3: samples >15 g/kg excluded                 0.2                       0.7
    Scenario 4: samples >20 g/kg excluded                 0.2                       0.9
                                                                                                  

    B.  GROUNDNUTS                                                  Mean residues
                                                 European monitoring data    USA monitoring data
                                                 (g/kg)                     (g/kg)
                                                                                                  
    Scenario 1: no samples excluded                       13                        14
    Scenario 2: samples >10 g/kg excluded                 0.9                       0.4
    Scenario 3: samples >15 g/kg excluded                 0.9                       0.5
    Scenario 4: samples >20 g/kg excluded                 1.0                       0.6
                                                                                                  

    Table 12.  Continued...

    III.  ESTIMATED DIETARY INTAKE OF AFLATOXIN (TOTAL) UNDER THE FOUR SCENARIOS AND DIFFERENT RESIDUE DATA SETS

                                                                                                                   
    A.  TOTAL AFLATOXIN INTAKE FROM MAIZE IF RESIDUES ARE THOSE REPORTED IN EUROPE

                                                                                                                   
                                                Middle Eastern   Far Eastern   African   Latin American   European
                                                                  (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                  10               6          21            8.4            2
    Scenario 2: samples >10 g/kg excluded           10               6          21            8.4            2
    Scenario 3: samples >15 g/kg excluded           10               6          21            8.4            2
    Scenario 4: samples >20 g/kg excluded           10               6          21            8.4            2
                                                                                                                   

                                                                                                                   
    B.  TOTAL AFLATOXIN INTAKE FROM MAIZE IF RESIDUES ARE THOSE REPORTED IN THE USA
                                                                                                                   
                                                Middle Eastern   Far Eastern   African   Latin American   European
                                                                  (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                 240             150         500          200             47
    Scenario 2: samples >10 g/kg excluded           30              19          64           25              6
    Scenario 3: samples >15 g/kg excluded           35              22          74           29              7
    Scenario 4: samples >20 g/kg excluded           45              28          95           38              9
                                                                                                                   
    GROUNDNUTS
                                                                                                                   
    A.  TOTAL AFLATOXIN INTAKE FROM GROUNDNUTS IF RESIDUES ARE THOSE REPORTED IN EUROPE
                                                Middle Eastern   Far Eastern   African   Latin American   European
                                                                  (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                  39              78         150           26             65
    Scenario 2: samples >10 g/kg excluded            0.3             3.6        10            1.8            4.5
    Scenario 3: samples >15 g/kg excluded            0.3             3.6        10            1.8            4.5
    Scenario 4: samples >20 g/kg excluded            0.3             6.0        11            2.0            5.0
                                                                                                                                     (ng total

    Table 12.  Continued...

                                                                                                                   
    B.  TOTAL AFLATOXIN INTAKE FROM GROUNDNUTS IF RESIDUES ARE THOSE REPORTED IN THE USA
                                                Middle Eastern   Far Eastern   African   Latin American   European
                                                                  (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                   4.2            84         160           28             70
    Scenario 2: samples >10 g/kg excluded            0.12            2.4         4.5          0.8            2.0
    Scenario 3: samples >15 g/kg excluded            0.15            3.0         5.7          1.0            2.5
    Scenario 4: samples >20 g/kg excluded            0.18            3.6         6.8          1.2            3.0
                                                                                                                   

    Table 13.  Estimated intake of aflatoxin B1 under 4 different scenarios with 2 residue datasets
    Scenario 1:  no samples excluded
    Scenario 2:  samples >10 g/kg excluded
    Scenario 3:  samples >15 g/kg excluded
    Scenario 4:  samples >20 g/kg excluded

                                                                                                                            
    I.  FOOD CONSUMPTION ESTIMATES (GEMS/Foods, World Health Organization)
                   Middle Eastern        Far Eastern            African           Latin American          European
                 (g/person per day)   (g/person per day)   (g/person per day)   (g/person per day)   (g/person per day)
                                                                                                                            
    Maize                50                   31                 106                    42                   10
    Groundnuts            0.3                  6                  11.3                   2                    5
                                                                                                                            

    II.  MEAN AFLATOXIN B1 RESIDUE CONCENTRATIONS UNDER EACH SCENARIO (from Table 7)

                                                                                                      
    A.  MAIZE/CEREALS                                              Mean residues
                                                European monitoring data    Chinese monitoring data
                                                          (g/kg)                    (g/kg)
                                                                                                      
    Scenario 1:  no samples excluded                        1.6                        12
    Scenario 2:  samples >10 g/kg excluded                 1.6                         2.8
    Scenario 3:  samples >15 g/kg excluded                 1.6                         3.1
    Scenario 4:  samples >20 g/kg excluded                 1.6                         3.5
                                                                                                      

    B.  GROUNDNUTS                              Mean residues
                                                European monitoring data    Chinese monitoring data
                                                          (g/kg)                    (g/kg)
                                                                                                      
    Scenario 1:  no samples excluded                        6.9                         8.3
    Scenario 2:  samples >10 g/kg excluded                 0.6                         2.7
    Scenario 3:  samples >15 g/kg excluded                 0.6                         2.7
    Scenario 4:  samples >20 g/kg excluded                 0.7                         2.9
                                                                                                      

    Table 13.  Continued...

    III.  ESTIMATED DIETARY INTAKE OF AFLATOXIN (B1) UNDER THE FOUR SCENARIOS AND DIFFERENT RESIDUE DATASETS

                                                                                                                   
    A.  AFLATOXIN B1 INTAKE FROM MAIZE IF RESIDUES ARE THOSE REPORTED IN EUROPE
                                                                                                                   
                                                Middle Eastern   Far Eastern   African   Latin American   European
                                                                  (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                   75              46         160           63            15
    Scenario 2: samples >10 g/kg excluded            75              46         160           63            15
    Scenario 3: samples >15 g/kg excluded            75              46         160           63            15
    Scenario 4: samples >20 g/kg excluded            75              46         160           63            15
                                                                                                                   

    B.  AFLATOXIN B1 INTAKE FROM MAIZE IF RESIDUES ARE THOSE REPORTED IN CHINA
                                                                                                                   
                                                Middle Eastern   Far Eastern   African   Latin American   European
                                                                  (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                  600             370        1270          500           120
    Scenario 2: samples >10 g/kg excluded           140              87         300          120            28
    Scenario 3: samples >15 g/kg excluded           160              96         330          130            31
    Scenario 4: samples >20 g/kg excluded           180             108         370          150            35
                                                                                                                   

    GROUNDNUTS
                                                                                                                   
    A.  AFLATOXIN B1 INTAKE FROM GROUNDNUTS IF RESIDUES ARE THOSE REPORTED IN EUROPE
                                                                                                                   
                                                Middle Eastern   Far Eastern   African   Latin American   European
                                                (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                    2.1            41          78           14            34
    Scenario 2: samples >10 g/kg excluded             0.2             3.6         6.8          1.2           3.0
    Scenario 3: samples >15 g/kg excluded             0.2             3.6         6.8          1.2           3.0
    Scenario 4: samples >20 g/kg excluded             0.2             4.2         7.9          1.4           3.5
                                                                                                                   

    Table 13.  Continued...

                                                                                                                   
    B.  AFLATOXIN B1 INTAKE FROM GROUNDNUTS IF RESIDUES ARE THOSE REPORTED IN CHINA
                                                                                                                   
                                                Middle Eastern   Far Eastern   African   Latin American    European
                                                                  (ng total aflatoxin/person per day)
                                                                                                                   
    Scenario 1: no samples excluded                    2.5            50          94           17            42
    Scenario 2: samples >10 g/kg excluded             0.8            16          30            5.4          14
    Scenario 3: samples >15 g/kg excluded             0.8            16          30            5.4          14
    Scenario 4: samples >20 g/kg excluded             0.9            17          33            5.8          15
                                                                                                                   
    

    4.4.5  Summary

        The values for aflatoxin levels presented above are not considered
    to be representative of the food supply in any country nor of the
    commodities moving in international trade. Quantitative estimates of
    intake of aflatoxin at the international level are severely limited by
    the lack of representative data. Although intake estimates are
    available at the national level for many countries, the submitters of
    all of these studies are emphatic in stating that the results are not
    truly "representative." In general, the results appear to be biased
    upwards because monitoring studies focus on lots of commodity that are
    thought to be contaminated.

        However, the data do provide sufficient information to evaluate
    the likely impact of limiting aflatoxin levels in foodstuffs. Of the
    scenarios considered, the greatest relative impact on estimated
    average aflatoxin levels is achieved by establishing a programme that
    would limit aflatoxin contamination to less than 20 g/kg. This
    assumes that the controls would successfully exclude all samples
    containing aflatoxin above that limit. Depending upon assumptions
    regarding the distribution of residues, some small incremental
    reductions can be achieved by limiting aflatoxin levels to no more
    than 15 or 10 g/kg, respectively.

        Additional data that would better simulate the actual aflatoxin
    levels in foods moving in international trade would provide much more
    accurate estimates of intake. Most likely these estimates would result
    in lower estimates of the average intake since the available data
    appear to be biased upwards. The incorporation of data on the effects
    of food processing on aflatoxin levels would also improve the accuracy
    of estimates of intake since aflatoxin is removed during many
    procedures.

    5.  COMMENTS AND EVALUATION

        The aflatoxins are among the most potent mutagenic and
    carcinogenic substances known. Extensive experimental evidence in test
    species shows that aflatoxins are capable of inducing liver cancer in
    most species studied. In addition, most epidemiological studies show a
    correlation between exposure to aflatoxin B1 and increased incidence
    of liver cancer. Aflatoxins are metabolized in humans and test species
    to an epoxide, which usually is considered to be the ultimate reactive
    intermediate. There is some evidence suggesting that humans are at
    substantially lower risk to aflatoxins than test species. The
    Committee was aware of epidemiological studies suggesting that intake
    of aflatoxin poses no detectable independent risk and studies that
    suggest it poses risks only in the presence of other risk factors such
    as hepatitis B infection. Several ongoing studies are likely to
    improve further the estimates of human risks from the intake of
    aflatoxin, most notably cohort studies in Shanghai, Thailand and
    Qidong and hepatitis B vaccination trials in The Gambia, Taiwan and
    Qidong. When these studies are complete, the Committee may want to
    reevaluate the risks of aflatoxins in humans.

        A number of factors influence the risk of primary liver cancer,
    most notably carriage of hepatitis B virus as determined by the
    presence in serum of the hepatitis B surface antigen (presence denoted
    HBsAg+ and absence denoted HbsAg-). The potency of aflatoxins
    appears to be significantly enhanced in individuals with simultaneous
    hepatitis B infection. This interaction makes it difficult to
    interpret the epidemiological studies in the context of aflatoxin as
    an independent risk factor. The conclusions of the Committee regarding
    aflatoxin potency therefore are contingent upon the dynamics of
    hepatitis B infection in a human population.

        The identification of hepatitis C virus is an important recent
    advance in understanding the etiology of liver cancer. Two studies
    have investigated interactions between hepatitis C infection,
    aflatoxins and liver cancer; the evidence so far is inconclusive. It
    is estimated that 50 to 100% of liver cancer cases are associated with
    persistent infection with hepatitis B and/or hepatitis C.

        The Committee considered that the weight of scientific evidence,
    which includes epidemiological data, laboratory animal studies and 
     in vivo and  in vitro metabolism studies, supports a conclusion
    that aflatoxins should be treated as carcinogenic food contaminants,
    the intake of which should be reduced to levels as low as reasonably
    achievable.

    5.1  Aflatoxin potencies

        The Committee reviewed dose-response analyses that have been
    performed on aflatoxins. All of these analyses suffer limitations,
    three of which predominate. First, all of the epidemiological data
    from which a dose-response relationship can be developed are
    confounded by concurrent hepatitis B infection. The epidemiological
    data are from geographical areas where both the prevalence of HBsAg+
    individuals and aflatoxins are high; the relationship between these
    risk factors in areas of low aflatoxin contamination and low hepatitis
    B prevalence is unknown. Second, the reliability and precision of the
    estimates of aflatoxin exposure in the relevant study populations are
    unknown. For example, aflatoxin biomarkers in humans do not reflect
    long-term aflatoxin intake; analysis of crops for aflatoxins do not
    reflect levels of aflatoxins consumed in foods after selection and
    processing. Finally, the shape of the dose-response relationship is
    unknown, which introduces an additional element of uncertainty when
    choosing mathematical models for interpolation.

        Observations concerning the interaction of hepatitis B and
    aflatoxins suggest two separate aflatoxin potencies in populations in
    which chronic hepatitis infections are common versus populations in
    which chronic hepatitis infections are rare. In analyses based on
    toxicological and epidemiological data, potency estimates for
    aflatoxin were divided into two basic groups, potencies applicable to
    individuals without hepatitis B infection and those applicable to
    individuals with chronic hepatitis B infection. The Committee found

    these estimates useful even though, through the use of differing
    mathematical models, they covered a broad range of possible values
    (Figure 2). Epidemiological data for which hepatitis B infection
    status was unknown and for which potencies were calculated were also
    reviewed and found to be in the range of potencies for hepatitis B
    infected/non-infected individuals. The review also considered the
    extrapolation of animal data to estimate potency in humans; these also
    generally fell within the range of the potency estimates derived from
    the epidemiological data.

        Some discussion is warranted on the potential biases in the
    potencies depicted in Figure 2: (i) only studies showing a positive
    association between aflatoxins and liver cancer were used, as opposed
    to considering all studies (positive as well as negative), leading to
    overestimation of the aflatoxin potency; (ii) by relating current
    levels of intake (i.e. using biomarkers or dietary surveys) to current
    levels of liver cancer (presumably with a long induction period),
    historical levels of intake are ignored; they are likely to have been
    higher, in which case aflatoxin potency will be overestimated; (iii)
    the earliest studies systematically underestimated hepatitis B
    prevalence in cases of liver cancer by a factor as high as 20-30%,
    owing to limitations in the methodology used to detect hepatitis B,
    which also leads to an overestimate of the relative potency of any
    other factor, including aflatoxins; (iv) histological confirmation of
    the liver cancer cases is limited in most epidemiological studies,
    allowing the possibility that non-primary liver cancer cases have been
    included, which could lead to an underestimation or overestimation of
    the aflatoxin potency. Considering these biases, the values in Figure
    2 should be viewed as overestimates of the potency of the aflatoxins,
    leading to the hypothesis that it is possible that humans are in fact
    less sensitive to aflatoxins than the species tested in laboratory
    experiments.

        The Committee reviewed the extensive data available on the
    metabolism of aflatoxins in various species. It was agreed that
    differential potency to aflatoxins between species can be partially
    attributed to differences in metabolism. However, there is at the
    present time insufficient quantitative information available about
    competing aspects of metabolic activation and detoxification of
    aflatoxin B1 in various species to identify an adequate animal model
    for humans and to explain the apparent species differences in potency.

        Intake assessments used in many of the epidemiological studies
    ignored the contributions to total aflatoxin intake through milk and
    milk products. Thus, the potencies shown in Figure 2 do not generally
    apply to aflatoxin M1. From one comparative toxicity study in rats,
    it is possible to estimate that aflatoxin M1 has a potency
    approximately one order of magnitude less than that of aflatoxin B1
    in this species.

        The Committee reviewed the potencies estimated from the positive
    epidemiological studies and chose separate central tendency estimated

    potencies and ranges for HBsAg+ and for HBsAg- individuals. Potency
    values of 0.3 cancers/year per 100 000 population per ng aflatoxin/kg
    bw per day with an uncertainty range of 0.05 to 0.5 in HBsAg+
    individuals and of 0.01 cancers/year per 100 000 population per ng
    aflatoxin/kg bw per day with an uncertainty range of 0.002 to 0.03 in
    HBsAg- individuals were chosen.

    5.2  Population risks

        The fraction of the incidence of liver cancer in a population
    attributable to intake of aflatoxins is derived by combining aflatoxin
    potency estimates (risk per unit dose) and estimates of aflatoxin
    intake (dose per person). The Committee reviewed the frequency and
    amount of aflatoxin contamination in a variety of products (e.g.,
    groundnuts, cereals and maize) in numerous countries (e.g., China,
    Denmark, Italy and the USA). Many of the data on contamination levels
    were derived from non-random samples, which appeared to be biased
    upwards because monitoring studies focus on lots of commodities that
    are thought to be contaminated. Some of the data on contaminant levels
    are unlikely to be based on current Codex sampling recommendations for
    aflatoxins. These contamination levels can only be used with caution
    to infer patterns of importance in setting standards and not to
    provide exact contamination estimates.

        Through the use of hypothetical standards, it was noted that the
    magnitude of the difference between two hypothetical standards is
    substantially larger than the magnitude of the difference in the mean
    contamination levels resulting from the separate standards. This point
    is illustrated in Figure 3 in which the derived distribution of
    aflatoxin contamination in maize in the USA is shown. Application of a
    hypothetical 20 g/kg standard would result in rejection of 4% of the
    maize crop and a mean aflatoxin level in maize of 0.9 g/kg. Imposing
    the stricter hypothetical standard of 10 g/kg would result in
    rejection of 6.2% of the samples to achieve a drop in the mean
    aflatoxin contamination level by 0.3 g/kg to 0.6 g/kg. Similar
    results were obtained when examining aflatoxin B1 levels in maize and
    also for total aflatoxins or B1 alone in groundnuts.

        Using the Global Environment Monitoring System - Food
    Contamination Monitoring and Assessment Programme (GEMS/Food) regional
    diets combined with contamination levels, the Committee was able to
    provide relative estimates of mean dietary intake of aflatoxins for
    various regions under differing standard dietary choices. Linking
    these intakes to the potencies shown in Figure 2 allows for the
    calculation of overall population risks based upon the prevalence of
    hepatitis B infection in various regions.

    FIGURE 3

        From its analysis the Committee noted that the application of a
    hypothetical standard removes from human consumption the samples most
    highly contaminated, thus greatly reducing average estimated intakes.
    Use of standards by all countries should be encouraged. Assuming a
    standard is in place, the Committee considered the effect of modifying
    that standard through the use of several hypothetical calculations.
    Two illustrations are given below.

        The first example pertains to areas with low contamination of food
    by aflatoxins and with a population having a small prevalence of
    carriers of hepatitis B. Aflatoxin levels based on European monitoring
    of aflatoxin B1 in groundnuts, maize and products derived from
    groundnuts and maize1 were used. In this example a population with
    1% carriers of hepatitis B was assumed. From the potencies given
    earlier, this yields an estimated population potency of 0.01  99% +
    0.3  1% = 0.013 cancers/year per 100 000 population per ng
    aflatoxin/kg bw per day with a range of 0.002 to 0.035. Based on
    European monitoring, if all samples with contamination above 20 g/kg
    are removed and it is assumed that these foods are ingested according
    to the "European diet", the mean estimated intake of aflatoxin is 19
    ng/person per day. Assuming an adult human weight of 60 kg, the
    estimated population risk (potency  intake) is 0.0041 cancers/year
    per 100 000 people with a range of 0.0006 to 0.01. In contrast, using
    the same assumptions but applying a 10 g/kg hypothetical standard,
    the average aflatoxin intake is 18 ng/person per day, resulting in an
    estimated population risk of 0.0039 cancers/year per 100 000 people
    with a range of 0.0006 to 0.01. Thus, reducing the hypothetical
    standard from 20 g/kg to 10 g/kg yields a drop in the estimated
    population risk of approximately 2 additional cancers/year per 109
    people.

        The second example pertains to areas with higher contamination
    (for these purposes, Chinese monitoring data of aflatoxin B1 in
    groundnuts, maize and their products were used) and areas with a
    larger population fraction as carriers of hepatitis B (in this case,
    25% hepatitis B carriers was assumed). The estimated potency for this
    population is 0.01  75% + 0.3  25% = 0.083, with a range of 0.014 to
    0.15. Using a 20 g/kg hypothetical standard and the "Far Eastern
    diet", the average estimated intake is 125 ng/person per day yielding
    an average population risk of 0.17 cancers/year per 100 000 people
    with a range of 0.03 to 0.3. Using a 10 g/kg hypothetical standard,
    the average estimated intake drops to 103 ng per person, yielding an
    estimated population risk of 0.14 cancers/year per 100 000 people with
    a range of 0.02 to 0.3. Thus, reducing the hypothetical standard for
    this population from 20 g/kg to 10 g/kg yields a drop in the
    estimated population risk of 300 cancers/year per 109 people.

                    

    1 The Committee noted that aflatoxin data for Europe was for "all
    cereals". However, in these calculations, it was assumed that the
    aflatoxin level for "all cereals" applied to maize consumption only.

    5.3  Conclusions

    1. Aflatoxins are considered to be human liver carcinogens. Aflatoxin
    B1 is the most potent carcinogen of the aflatoxins; most of the
    toxicological data available are related to aflatoxin B1. Aflatoxin
    M1, the hydroxylated metabolite of B1, has a potency approximately
    one order of magnitude less than that of B1.

    2. The potency of aflatoxins in HBsAg+ individuals is substantially
    higher than the potency in HBsAg- individuals. Thus, reduction of the
    intake of aflatoxins in populations with a high prevalence of HBsAg+
    individuals will have greater impact on reducing liver cancer rates
    than reductions in populations with a low prevalence of HBsAg+
    individuals.

    3. Vaccination against hepatitis B will reduce the prevalence of
    carriers. The present analysis suggests that this would reduce the
    potency of the aflatoxins in vaccinated populations and consequently
    reduce liver cancer risks.

    4. Analyses of the application of hypothetical standards (10 mg/kg or
    20 g/kg aflatoxin in food) to model populations indicate that: (i)
    populations with a low prevalence of HBsAg+ individuals and/or with
    a low mean intake (less than 1 ng/kg bw per day) are unlikely to
    exhibit detectable1 differences in population risks for standards in
    the range of the hypothetical cases; and (ii) populations with a high
    prevalence of HBsAg+ individuals and high mean intake of aflatoxins
    would benefit from reductions in aflatoxin intake.

    5. The Committee has previously noted that reductions can be achieved
    through avoidance measures such as improved farming and proper storage
    practices and/or through enforcing standards for food or feed within
    countries and across borders (Annex 1, reference 77).

    6. In considering two competing standards, if the fraction of the
    samples excluded under the two standards is similar, the higher
    standard will yield almost the same liver cancer risks as the lower
    standard. When a substantial fraction of the current food supply is
    heavily contaminated, reducing the aflatoxin contamination levels may
    detectably lower liver cancer rates. Conversely, when only a small
    fraction of the current food supply is heavily contaminated, reducing
    the standard by an apparently substantial amount may have little
    appreciable effect on public health.

                   

    1 In the context of this statement "detectable" refers to an
    aflatoxin-induced change in liver cancer rates that exceeds the year-
    to-year variability around the current incidence and mortality rates.
    Hence "detectable" refers to our ability to observe a significant
    effect in the occurrence of liver cancer following intervention and
    will depend upon the quality of the data available on historical
    trends in incidence and mortality.

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    See Also:
       Toxicological Abbreviations
       Aflatoxins (IARC Summary & Evaluation, Volume 56, 1993)
       Aflatoxins (IARC Summary & Evaluation, Volume 56, 1993)
       Aflatoxins (IARC Summary & Evaluation, Volume 82, 2002)