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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY


    ENVIRONMENTAL HEALTH CRITERIA 11





    MYCOTOXINS







    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the World Health Organization or the United Nations
    Environment Programme.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    and the World Health Organization


    World Health Organization
    Geneva, 1979

    ISBN 92 4 154071 0


    (c) World Health Organization 1979

         Publications of the World Health Organization enjoy copyright
    protection in accordance with the provisions of Protocol 2 of the
    Universal Copyright Convention. All rights reserved.

         The designations employed and the presentation of the material
    in this publication do not imply the impression of any opinion
    whatsoever on the part of the Secretariat of the World Health
    Organization concerning the legal status of any country, territory,
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         The mention of specific companies or of certain manufacturers'
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    World Health Organization in preference to others of a similar
    nature that are not mentioned. Errors and omissions excepted, the
    names of proprietary products are distinguished by initial capital
    letters.


    CONTENTS

    ENVIRONMENTAL HEALTH CRITERIA FOR MYCOTOXINS

    1.   SUMMARY AND RECOMMENDATIONS FOR FURTHER RESEARCH

         1.1  Summary
              1.1.1   Aflatoxins
                      1.1.1.1   Sources and occurrence
                      1.1.1.2   Effects and associated exposures
              1.1.2   Other mycotoxins
                      1.1.2.1   Ochratoxins
                      1.1.2.2   Zearalenone
                      1.1.2.3   Trichothecenes

         1.2  Recommendations for further studies
              1.2.1   General recommendations
              1.2.2   Recommendations for aflatoxins
              1.2.3   Recommendations for other mycotoxins

    2.   MYCOTOXINS AND HUMAN HEALTH

    3.   AFLATOXINS

         3.1  Properties and analytical methods
              3.1.1   Chemical properties
              3.1.2   Methods of analysis for aflatoxins in foodstuffs
                      3.1.2.1   Sampling
                      3.1.2.2   Methods of analysis
         3.2  Sources and occurrence
              3.2.1   Formation by fungi
                      3.2.1.1   Moisture content and temperature
                      3.2.1.2   Invasion of field crops by  A. flavus
              3.2.2   Occurrence in foodstuffs
                      3.2.2.1   Maize
                      3.2.2.2   Wheat, barley, oats, rye, rice, and
                                sorghum
                      3.2.2.3   Groundnuts (peanuts)
                      3.2.2.4   Soybeans and common beans
                      3.2.2.5   Tree nuts
                      3.2.2.6   Copra
                      3.2.2.7   Cottonseed
                      3.2.2.8   Spices and condiments
                      3.2.2.9   Animal feeds
                      3.2.2.10  Animal products
              3.2.3   Fate of aflatoxins during the handling and
                      processing of food
              3.2.4   Pathways and levels of exposure

         3.3  Metabolism
              3.3.1   Absorption
              3.3.2   Tissue distribution
                      3.3.2.1   Animal studies
                      3.3.2.2   Studies in man
              3.3.3   Metabolic transformation and activation
              3.3.4   Excretion
                      3.3.4.1   Animal studies
                      3.3.4.2   Studies in man

         3.4  Effects in animals
              3.4.1   Field observations
              3.4.2   Experimental studies
                      3.4.2.1   Acute and chronic effects:
                                hepatotoxicity
                      3.4.2.2   Hepatotoxicity connected with
                                extrahepatic effects
                      3.4.2.3   Carcinogenesis
                      3.4.2.4   Teratogenicity
                      3.4.2.5   Mutagenicity
                      3.4.2.6   Biochemical effects and mode of action
                      3.4.2.7   Factors modifying the effects
                                and dose-response relationships of
                                aflatoxins

         3.5  Effects in man -- epidemiological and clinical studies
              3.5.1   General population studies
                      3.5.1.1   Liver carcinogenesis
                      3.5.1.2   Other effects reported to be
                                associated with aflatoxins
              3.5.2   Occupational exposure

         3.6  Evaluation of the health risks of exposure to aflatoxins
              3.6.1   Human exposure conditions
                      3.6.1.1   Sources and levels of aflatoxins in
                                food
                      3.6.1.2   Dietary intake and levels in human
                                tissues
              3.6.2   Acute effects of exposure
                      3.6.2.1   Acute liver disease
                      3.6.2.2   Reye's syndrome
              3.6.3   Chronic effects of aflatoxin exposure
                      3.6.3.1   Cancer of the liver
                      3.6.3.2   Juvenile cirrhosis in India
              3.6.4   Guidelines for health protection


    4.   OTHER MYCOTOXINS

         4.1  Ochratoxins
              4.1.1   Properties and analytical methods
                      4.1.1.1   Chemical properties
                      4.1.1.2   Methods for the analysis of foodstuffs
              4.1.2   Sources and occurrence
                      4.1.2.1   Fungal formation
                      4.1.2.2   Occurrence in foodstuffs
              4.1.3   Metabolism
                      4.1.3.1   Absorption
                      4.1.3.2   Tissue distribution and metabolic
                                conversion
                      4.1.3.3   Excretion
              4.1.4   Effects in animals
                      4.1.4.1   Field observations
                      4.1.4.2   Experimental studies
              4.1.5   Effects in man
                      4.1.5.1   Ochratoxin A and Balkan nephropathy
              4.1.6   Conclusions and evaluation of the health risks
                      to man of ochratoxins
                      4.1.6.1   Experimental animal studies
                      4.1.6.2   Studies in man
                      4.1.6.3   Evaluation of health risks

         4.2  Zearalenone
              4.2.1   Properties, analytical methods, and sources
              4.2.2   Occurrence
              4.2.3   Effects in animals
                      4.2.3.1   Field observations
                      4.2.3.2   Experimental studies
              4.2.4   Conclusions and evaluation of health risks to
                      man of zearalenone
                      4.2.4.1   Animal studies
                      4.2.4.2   Evaluation of health risks

         4.3  Trichothecenes
              4.3.1   Properties and sources
              4.3.2   Occurrence
              4.3.3   Effects in animals
                      4.3.3.1   Field observations
                      4.3.3.2   Experimental studies
              4.3.4   Alimentary toxic aleukia
              4.3.5   Conclusions and evaluations of the health risks
                      to man of trichothecenes

    REFERENCES

    NOTE TO READERS OF THE CRITERIA DOCUMENTS

         While every effort has been made to present information in the
    criteria documents as accurately as possible without unduly delaying
    their publication, mistakes might have occurred and are likely to
    occur in the future. In the interest of all users of the
    environmental health criteria documents, readers are kindly
    requested to communicate any errors found to the Division of
    Environmental Health, World Health Organization, Geneva,
    Switzerland, in order that they may be included in corrigenda which
    will appear in subsequent volumes.

         In addition, experts in any particular field dealt with in the
    criteria documents are kindly requested to make available to the WHO
    Secretariat any important published information that may have
    inadvertently been omitted and which may change the evaluation of
    health risks from exposure to the environmental agent under
    examination, so that the information may be considered in the event
    of updating and re-evaluation of the conclusions contained in the
    criteria documents.

    WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR MYCOTOXINS

     Members

    Dr B. K. Armstrong, University Department of Medicine, Perth Medical
        Centre, Nedlands, Australiaa

    Dr A.D. Campbell, Food and Drug Administration, US Department of
        Health, Education and Welfare, Washington, DC, USAb

    Dr T. Denizel, Department of Agriculture Microbiology, Faculty of
        Agriculture, University of Ankara, Ankara, Turkeya

    Dr M. Jemmali, Service Mycotoxines de l'INRA, Station de Biochimie
        et Physico-Chimie des Crales, Institut National de la
        Recherche Agronomique, Paris, Francea

    Professor P. Krogh, The University Institute of Pathological
        Anatomy, Copenhagen, Denmark,a,b,c

    Professor V. Kusak, Institute of Experimental Medicine,
        Czechoslovak Academy of Sciences, Prague, Czechoslovakia
         (Vice-Chairman)a,b

    Dr V. Nagarajan, National Institute of Nutrition, Jamai-Osmania,
        Hyderabad, Indiaa

    Dr M. F. Nesterin, Institute of Nutrition, Academy of Medical
        Sciences of the USSR, Moscow, USSRb

    Professor P. Newberne, Department of Nutrition and Food Science,
        Massachusetts Institute of Technology, Cambridge, MA, USAa

    Dr D. S. P. Patterson, Central Veterinary Laboratory, Ministry of
         Agriculture, Fisheries and Food, Weybridge, England
         (Chairman)a,b

    Dr F. G. Peers, Tropical Products Institute, London, Englanda,b

    Professor A. C. Sarkisov, Laboratory of Antibiotics and Mycology,
        All-Union Institute of Experimental Veterinary Science, Moscow,
        USSRa

    Dr P. L. Schuller, Laboratory of Chemical Analysis of Foodstuffs,
        National Institute of Public Health, Bilthoven, Netherlands
         (Vice-Chairman)a

    Dr A. Rogers, Department of Nutrition and Food Science,
        Massachusetts Institute of Technology, Cambridge, MA, USA
         (Rapporteur)b

    Professor H. D. Tendon, All-India Institute of Medical Sciences,
        New Delhi, Indiaa

    Professor A. Wasunna, Department of Surgery, University of
        Nairobi, Kenyaa

     Representatives of other International Organizations

    Dr O. Alozie, United Nations Environment Programme,
        Nairobi, Kenyaa,b

    Dr D. Djordjevic, Occupational Safety and Health Branch,
        International Labour Office, Geneva, Switzerlanda

    Dr G. D. Kouthon, Food and Agriculture Organization of the
        United Nations, Rome, Italya

    Professor D. Reymond, Coordinating Committee on Food Chemistry,
        International Union of Pure and Applied Chemistry, La Tour
        de Peilz, Switzerlandb

    Mrs. M. Th. van der Venne, Commission of the European Communities,
        Health Protection Directorate, Luxembourga,b

     WHO Secretariat

    Dr C. Agthe, Environmental Health Criteria arid Standards, Division
        of Environmental Health, WHO, Geneva, Switzerland
         (Co-Secretary)a,b

    Dr L. Fishbein, US Public Health Service, National Centre for
        Toxicological Research, Chemistry Division, Jefferson, AR, USA
         (Temporary Adviser)a

    Dr J. Korneev, Environmental Health Criteria and Standards, Division
        of Environmental Health, WHO, Geneva, Switzerlanda,b

    Professor E. Lillehoj, US Department of Agriculture, Northern
        Research Laboratory, Peoria, IL, USA  (Temporary Adviser)a

    Dr C. A. Linsell, Interdisciplinary Programme and International
        Liaison, IARC, Lyons, Francea,b

    R. Lunt, Cancer Unit, Division of Noncommunicable Diseases, WHO,
        Geneva, Switzerlandb

    Dr Z. Matyas, Veterinary Public Health, Division of Communicable
        Diseases, WHO, Geneva, Switzerlanda,b

    Professor C. J. Mirocha, Department of Plant Pathology, University
        of Minnesota, St Paul, MA, USA  (Temporary adviser)a

    Dr J. Parizek, Environmental Health Criteria and Standards,
        Division of Environmental Health, WHO, Geneva, Switzerland
         (Co-Secretary)a,b

    Dr V. B. Vouk, Environmental Health Criteria and Standards, Division
        of Environmental Health, WHO, Geneva, Switzerlanda,b

                 
 
   a Attended the first meeting of the Task Group.
    b Attended the second meeting of the Task Group.
    c Present address: Department of Veterinary Microbiology,
      Pathology and Public Health, School of Veterinary Medicine,
      Purdue University, West Layayette, IN, USA.

    ENVIRONMENTAL HEALTH CRITERIA FOR MYCOTOXINS

        Members of the Task Group on Environmental Health Criteria for
    Mycotoxins met in Geneva from 1 to 7 March 1977 and from 19 to 23
    June 1978.

        The first meeting was opened on behalf of the Director-General
    by Dr B. H. Dieterich, Director, Division of Environmental Health,
    and the second by Dr C. Agthe, Division of Environmental Health.

        The first and second draft criteria documents were prepared by
    Professor C. J. Mirocha. The comments on which the second draft was
    based were received from the national focal points for the WHO
    Environmental Health Criteria Programme in Belgium, Czechoslovakia,
    Federal Republic of Germany, India, New Zealand, Poland, Sweden,
    Thailand, USSR, and USA, and from the International Agency for
    Research on Cancer (IARC), Lyons, the United Nations Industrial
    Development Organization (UNIDO), Vienna, and the Food and
    Agriculture Organization of the United Nations (FAO), Rome. Comments
    were also received from the Tropical Products Institute, London.

        Dr P. Krogh, Dr D. S. P. Patterson, and Dr P. L. Schuler
    assisted in the preparation of the third draft criteria document,
    which was submitted for review to all the members of the Task Group,
    and to Dr R. Plestina of the Institute for Medical Research and
    Occupational Health, Yugoslav Academy of Sciences and Arts, Zagreb,
    Yugoslavia before the second meeting of the Task Group. The final
    edited draft was kindly reviewed by Dr D. S. P. Patterson. The
    collaboration of these national institutions, international
    organizations, WHO collaborating centres, and individual experts is
    gratefully acknowledged.

        The document is based primarily on original publications listed
    in the reference section. However, several recent publications
    reviewing the occurrence, health effects, and other aspects of
    mycotoxins have also been used including monographs prepared by
    Purchase (1974), Pokrovskij et al. (1977) and Wyllie & Morehouse
    (1977), and the report on the joint FAO/ WHO/UNEP Conference on
    Mycotoxins in Nairobi 1977 (FAO, 1977). In addition, comprehensive
    data have been obtained from the proceedings of several symposia and
    meetings including the Conference on Mycotoxins in Human and Animal
    Health, held in Maryland, USA, in 1976 (Rodricks et al., 1977).

        Details of the WHO Environmental Health Criteria Programme
    including some of the terms frequently used in the documents, may be
    found in the general introduction to the Environmental Health
    Criteria Programme published together with the environmental health
    criteria document on mercury (Environmental Health Criteria 1,
    Mercury, Geneva, World Health Organization, 1976), and now available
    as a reprint.

    1.  SUMMARY AND RECOMMENDATIONS FOR FURTHER RESEARCH

    1.1  Summary

        The ingestion of food containing mycotoxins, the toxic products
    of microscopic fungi (moulds), may have serious adverse health
    effects in man. Occasionally, occupational exposure to airborne
    mycotoxins may also occur.

        The occurrence of mycotoxins in foodstuffs depends on their
    formation by specific strains of fungi and is influenced by
    environmental factors such as humidity and temperature. Thus,
    mycotoxin contamination of foodstuffs may vary with geographical
    conditions;, production and storage methods, and also with the type
    of food, since some food products are more suitable substrates for
    fungal growth than others.

        The present document contains an evaluation of health risks
    associated with four classes of mycotoxins. Aflatoxins are treated
    in most detail because more is known about them than about the other
    mycotoxins and because there is epidemiological evidence associating
    health effects in man with exposure to aflatoxins.

        For the other 3 classes (ochratoxins, zearalenone, and
    trichothecenes), toxic effects in animals have been established and
    there is well-documented evidence that human exposure may occur, at
    least for the first two classes.

    1.1.1  Aflatoxins

    1.1.1.1  Sources and occurrence

    Aflatoxins are produced by certain strains of  Aspergillus
     flavus and  Aspergillus parasiticus. These fungi are ubiquitous
    and the potential for contamination of foodstuffs and animal feeds
    is widespread. The occurrence and magnitude of aflatoxin
    contamination varies with geographical and seasonal factors, and
    also with the conditions under which a crop is grown, harvested, and
    stored. Crops in tropical and subtropical areas are more subject to
    contamination than those in temperate regions, since optimal
    conditions for toxin formation are prevalent in areas with high
    humidity and temperature. Toxin-producing fungi can infect growing
    crops as a consequence of insect or other damage, and may produce
    toxins prior to harvest, or during harvesting and storage.

        The chemical structures of aflatoxins have been elucidated, and
    analytical techniques are available for their identification and
    determination in foodstuffs and body tissues at the g/kg level and
    lower. Four aflatoxins (B1, G1, B2, G2, often occurring
    simultaneously, have been detected in foods of plant origin
    including maize, groundnuts (peanuts), and tree nuts as well as many
    other foodstuffs and feeds.

        In animals, ingested aflatoxins may be metabolically degraded.
    Aflatoxin B1 may be converted into aflatoxin M1 which may occur
    in the milk. The concentration of aflatoxin M1 in the milk of cows
    is about 300 times lower than the concentration of aflatoxin B1
    consumed in the feed. In certain experimental animals, only small
    amounts of administered aflatoxins have been found in tissues, 24 h
    after injection.

        In studies on pigs, aflatoxin residues were detected in the
    liver, kidney, and muscle tissues of animals given aflatoxins in the
    feed for several months. There do not appear to be any published
    works on aflatoxin residues in the tissues of slaughtered animals.

        The use of resistant varieties of seed and of pesticides, and
    careful drying and storing procedures can reduce fungal infestation
    and thus diminish food contamination by aflatoxins. The toxin is not
    eliminated from foodstuffs or animal feeds by ordinary cooking or
    processing practices and, since pre-and post-harvest procedures do
    not ensure total protection from aflatoxin contamination, techniques
    for decontamination have been developed. The toxin is generally
    concentrated in a small proportion of seeds that are often different
    in colour. Segregation of discoloured seeds by sorting can
    significantly reduce the aflatoxin levels in some crops, such as
    groundnuts. Visual inspection for mould growth before processing can
    serve as an initial screening technique but toxin-producing fungi
    can be present without detectable aflatoxins and vice versa. Because
    aflatoxin distribution in a contaminated, unprocessed commodity is
    uneven, adequate sampling is essential for effective monitoring. As
    aflatoxins can be chemically degraded in vitro by several oxidizing
    agents and alkalis, hydrogen peroxide and ammonia are currently used
    for the chemical decontamination of animal feeds.

    1.1.1.2  Effects and associated exposures

        Outbreaks of aflatoxicosis in farm animals have been reported
    from many areas of the world. The liver is mainly affected in such
    outbreaks and also in experimental studies on animals, including
    nonhuman primates. The acute liver lesions are characterized by
    necrosis of the hepatocytes and biliary proliferation, and chronic
    manifestations may include fibrosis. A feed level of aflatoxin as
    low as 300 g/kg can induce chronic aflatoxicosis in pigs within
    3-4 months.

        Aflatoxin B1 is a liver carcinogen in air least 8 species
    including nonhuman primates. Dose-response relationships have been
    established in studies on rats and rainbow trout, with a 10% tumour
    incidence estimated to occur at feed levels of aflatoxin B1 of
    1 g/kg, and 0.1 g/kg, respectively. In some studies, carcinomas of
    the colon and kidney have been observed in rats treated with
    aflatoxins. Aflatoxin B1 causes chromosomal aberrations and DNA
    breakage in plant and animal cells, and, after microsomal
    activation, gene mutations in several bacterial test systems. In
    high doses, it may be teratogenic.

        The acute toxicity and carcinogenicity of aflatoxins are greater
    in male than in female rats; hormonal involvement may be responsible
    for this sex-linked difference. Nutritional status in animals,
    particularly with respect to lipotropes, proteins, vitamin A, and
    lipids (including cyclopropenoid fatty acids), can modify the
    expression of acute toxicity or carcinogenicity or both.

        There is little information on the association of acute
    hepatoxicity in man with exposure to aflatoxins but cases of acute
    liver damage have been encountered that could possibly be attributed
    to acute aflatoxicosis. A recent outbreak of acute hepatitis in
    adjacent districts of two neighbouring states in north-west India,
    which affected several hundred people, was apparently associated
    with the ingestion of heavily contaminated maize, some samples of
    which contained aflatoxin levels in the mg/kg range, the highest
    reported level being 15 mg/kg.

        Liver cancer is more common in some regions of Africa and
    southeastern Asia than in other parts of the: world and, when local
    epidemiological information is considered together with experimental
    animal data, it appears that increased exposure to aflatoxins may
    increase the risk of primary liver cancer. Pooled data from Kenya,
    Mozambique, Swaziland, and Thailand, show a positive correlation
    between daily dietary aflatoxin intake (in the range of 3.5 to
    222.4 ng/kg body weight per day) and the crude incidence rate of
    primary liver cancer (ranging from 1.2 to 13.0 cases per 100 000
    people per year). There is also some evidence of a vital involvement
    in the etiology of the disease.

        In view of the evidence concerning the effects, particularly the
    carcinogenic effects, of aflatoxins in several animal species, and
    in view of the association between aflatoxin exposure levels and
    human liver cancer incidence observed in some parts of the world,
    exposure to aflatoxins should be kept as low as practically
    achievable. The tolerance levels for food products established in
    several countries should be understood as management tools intended
    to facilitate the implementation of aflatoxin control programmes,
    and not as exposure limits that necessarily ensure health
    protection.

    1.1.2  Other mycotoxins

    1.1.2.1  Ochratoxins

        Ochratoxins are produced by several species of the fungal genera
     Aspergillus and  Pencillium. These fungi are ubiquitous and the
    potential for contamination of foodstuffs and animal feed is
    widespread. Ochratoxin A, the major compound, has been found in more
    than 10 countries in Europe and the USA. Ochratoxin formation by
     Aspergillus species appears to be limited to conditions of high
    humidity and temperature, whereas at least some  Pencillium species
    may produce ochratoxin at temperatures as low as 5C.

        Analytical techniques have been developed for the identification
    and quantitative determination of ochratoxin levels in the g/kg
    range.

        Ochratoxin A has been found in maize, barley, wheat, and oats, as
    well as in many other food products, but the occurrence of
    ochratoxin B is rare. Residues of ochratoxin A have been identified
    in the tissues of pigs in slaughterhouses, and it has been shown,
    under experimental conditions, that residues can still be detected
    in pig tissues one month after the termination of exposure.

        Field cases of ochratoxicosis in farm animals (pigs, poultry)
    have been reported from several areas of the world, the primary
    manifestation being chronic nephropathy. The lesions include tubular
    atrophy, interstitial fibrosis, and, at later stages, hyalinized
    glomeruli. Ochratoxin A has been found to be nephrotoxic in all
    species of animals studied so far, even at the lowest level tested
    (200 g/kg feed in rats and pigs). It has also been reported to
    produce teratogenic effects in mice, rats, and hamsters.

        Human endemic nephropathy is a kidney disease of unknown
    etiology that has so far only been encountered in some areas of the
    Balkan Peninsula. The renal changes observed with this disease are
    comparable to those seen in ochratoxin A-associated nephropathy in
    pigs. High ochratoxin A exposure through diet has been found in some
    of the areas of the Balkan Peninsula, where endemic nephropathy is
    prevalent.

    1.1.2.2  Zearalenone

        Zearalenone, a metabolite produced by various species of
     Fusarium, has been observed as a natural contaminant of cereals,
    in particular maize, in many countries in Africa and Europe, and in
    the USA.

        It has been shown to produce estrogenic effects in animals, and
    field cases of a specific estrogenic syndrome in pigs and of
    infertility in cattle have been encountered in association with feed
    levels of zearalenone of 0.1-6.8 mg/kg and 14 mg/kg, respectively.
    The compound has also produced congenital malformations in the rat
    skeleton.

        In some countries, zearalenone has been found in samples of
    cornmeal and cornflakes destined for human consumption, at levels up
    to 70 g/kg, corresponding to doses 400-600 times lower than those
    causing effects in monkeys or mice under experimental conditions. In
    certain areas of Africa, substantially higher levels have
    occasionally been found in beer and sour porridge prepared from
    contaminated maize and sorghum.

        No adverse effects due to zearalenone intake have been reported
    in man, so far, but a possible health hazard connected with the
    dally intake of zearalenone at levels such as those reported for
    African fermented preparations needs further attention.

    1.1.2.3  Trichothecenes

        Trichothecene toxins belong to a group of closely related
    chemical compounds produced by several species of  Fusarium,
     Cephalosporium, Myrothecium, Trichoderma, and  Stachybotrys.
    Four trichothecenes (T-2 toxin, nivalenol, deoxynivalenol, and
    diacetoxyscirpenol) have been detected as natural contaminants in a
    small number of food samples.

        Alimentary toxic aleukia, a disease diagnosed in man about 40
    years ago, was apparently associated with the ingestion of grains
    invaded by  Fusarium species. No cases have been reported since the
    end of the Second World War and the disappearance of the disease is
    probably due to improved food production and storage conditions.
    There is no firm evidence connecting the recently identified
    trichothecenes with alimentary toxic aleukia occurring in the past,
    or with other human disease.

    1.2   Recommendations for Further Studies

    1.2.1  General recommendations

    (a)     There is a need for more information concerning the
            occurrence of mycotoxins in various parts of the world and
            the possible daily intake of mycotoxins by man.

    (b)     Further studies should be undertaken on factors affecting
            fungal growth and mycotoxin formation in foodstuffs, under
            preharvest, postharvest, and storage conditions.

    (c)     The effects of various cooking processes on the levels of
            mycotoxins in foods should be elucidated.

    (d)     Better methods should be developed for the rapid detection
            and measurement of mycotoxin levels in foodcrops.

    (e)     Sampling has proved to be the most difficult step in the
            surveillance of food commodities. The development of
            reliable, internationally accepted sampling procedures is
            strongly recommended.

    (f)     Better methods should be developed for the identification
            and measurement of mycotoxins in human tissues, body fluids,
            and excreta.

    (g)     A network of reference centres should be established to
            assist Member States in confirming the identity of
            individual mycotoxins found in human foods and tissues.
            These reference centres should also provide mycotoxin
            reference samples, upon request, to reinforce the
            inter-comparability of analytical results obtained in
            different parts of the world.

    (h)     Better understanding is needed of the role of mycotoxins in
            human diseases. Where association between exposure to
            mycotoxins and the incidence of certain diseases is
            suspected, detailed epidemiological studies should be
            carried out.

    (i)     Improved diagnostic methods for the effects on health of
            mycotoxins are needed, particularly methods for the
            detection of early changes that occur before the development
            of irreversible effects.

    (j)     Attempts should be made to monitor exposure levels and to
            search for effects in workers handling pure mycotoxins or
            contaminated materials. This could provide important
            information on the effects of chronic exposure to mycotoxins
            and also indicate the need for safety measures.

    1.2.2  Recommendations for aflatoxins

    (a)     The validity of the assumption of a causal relationship
            between aflatoxin ingestion and primary liver cancer should
            be examined further by introducing control measures to
            reduce aflatoxin exposure in areas of high liver cancer
            incidence and high aflatoxin exposure. This should be
            followed by the monitoring of liver cancer incidence in
            these areas and in comparable areas where the aflatoxin
            exposure has been low.

    (b)     The prevalence of hepatitis B antigen should be determined
            in areas with various levels of aflatoxin exposure and a
            high incidence of primary liver cancer.

    (c)     Suspected outbreaks of acute aflatoxicosis should be
            studied in detail. Such studies should include measurements
            of the exposure to aflatoxins through foods and other
            routes. The presence of aflatoxins and their derivatives in
            the tissues and excreta of individuals including both those
            affected and those apparently unaffected by the disease,
            should be investigated.

    (d)     A prolonged, continuous surveillance of the health status
            of exposed populations is considered essential in
            localities, where outbreaks of acute aflatoxicosis have
            occurred. Such follow-up studies are important to fill the
            gaps in knowledge on the late effects of short-term exposure
            to high levels of aflatoxin in man. Recently reported
            outbreaks of aflatoxin-associated hepatitis in southeastern
            Asia may provide an ideal opportunity for such studies.

    (e)     Reports from the various countries on the presence of
            aflatoxins in human tissues, body fluids, and excreta should
            be confirmed using specific assay methods with adequate
            limits of detection. The frequency of such events should be
            studied in appropriate samples of the general population of
            various countries and a search made for the sources of
            aflatoxin exposure.

    (f)     The implication of aflatoxin as a contributing factor in
            the development of Reye's syndrome should be further
            investigated using the case-control approach. Data should be
            obtained on the presence of aflatoxins in tissues, body
            fluids, and excretion products for each case and its
            control, and attempts should be made to identify dietary
            sources of aflatoxins.

    (g)     More information is needed on the gastrointestinal
            absorption of aflatoxins in animals and human subjects, as
            well as on the rate of disappearance of aflatoxins from
            farm-animal and human tissues. This is important both for
            the evaluation of aflatoxin residues in food of animal
            origin, and for the evaluation of aflatoxin levels in human
            tissues as a means of assessing exposure.

    (h)     The modifying effect of the dietary intake of lipotropes,
            protein, or vitamin A on aflatoxin-related carcinogenesis
            should be further studied in experimental animals. This
            aspect should also be included in epidemiological studies on
            the association between human liver cancer incidence and
            aflatoxin intake.

    1.2.3  Recommendations for other mycotoxins

    (a)     The levels of ochratoxin A and possibly citrinin should be
            measured in "food-on-the-plate" in areas of the Balkan
            Peninsula with different incidence rates of Balkan
            nephropathy.

    (b)     Further systematic investigations are needed on the levels
            of ochratoxin in foodstuffs and animal feeds in different
            parts of the world, and their association with nephropathy
            in farm animals. More work is needed in various parts of the
            world to confirm or exclude the strictly localized
            occurrence of endemic nephropathy affecting human subjects
            and considered so far to be confined to certain areas of the
            Balkan Peninsula.

    (c)     Further studies are required on the mechanisms of ochratoxin
            toxicity, and on possible interactions with other nephrotoxic
            agents.

    (d)     Studies should be made in different countries of the levels
            of zearalenone in human food and on total daily intake.

    (e)     More information is needed on the levels of zearalenone in
            foods prepared from fermented maize and sorghum, such as
            those found in certain parts of Africa, and on the possible
            adverse effects of the daily consumption of these products,
            particularly in view of the estrogen-like effects of
            zearalenone observed in animals.


    2.  MYCOTOXINS AND HUMAN HEALTH

        The toxicity of certain mushrooms has been known for a long
    time. However, the potential human hazard of the toxic products of
    other fungi was not recognized until the 1850s when an association
    between the ingestion of rye infected with  Claviceps purpurea and
    the clinical features of ergotism was discovered. This was followed
    by reports of other mycotoxicoses that affected man such as the
    identification of a syndrome associated with the ingestion of bread
    infected by  Fusarium graminearum, recognition of human
    stachybotryotoxicosis, and studies on the association between
    alimentary toxic aleukia (ATA) and the ingestion of over-wintered
    grains infested with  Fusarium poae and  Fusarium sporotrichioides
    (Sarkisov, 1954).

        Recognition of the association of ATA with the consumption of
    food contaminated by moulds and the corresponding preventive
    measures taken, resulted in the eradication of the disease (Leonov,
    1977) showing that, even before the isolation of the first
    mycotoxins, fungi-related foodborne diseases could be prevented.

        The discovery of the hepatotoxic and hepatocarcinogenic
    properties of Aspergillus flavus in the early 1960s quickly followed
    by the elucidation of the structure of the aflatoxins changed the
    control strategy in the whole field of mycotoxins. A more
    quantitative approach is now possible, based primarily upon the
    chemical determination of the toxins and on studies of their effects
    in relation to dose.

        In spite of increasing knowledge concerning human mycotoxicoses,
    the majority of data available on mycotoxins and mycotoxicoses have
    been obtained from veterinary medicine. Field studies, as well as
    studies on experimental animals indicate that the potential toxicity
    of mycotoxins is great. Future investigations may well establish a
    causal role of mycotoxins in other human diseases besides those
    considered so far.

        Almost all plant products can serve as substrates for fungal
    growth and subsequent mycotoxin formation, thus providing the
    potential for direct contamination of human food. When farm animals
    used for food production, ingest feed contaminated with mycotoxins,
    not only may a direct toxic effect on the animals occur but there
    may also be a carry-over of the toxins into milk and meat, thus
    creating a further avenue for human exposure to mycotoxins.
    Furthermore, occupational exposure may occur through other media
    such as air.

        In this document, the risks of health effects have been
    considered only for those mycotoxins for which there is evidence of
    human exposure and of well defined adverse effects, at least in
    animals. This category includes the aflatoxins, ochratoxins, and
    zearalenone. The trichothecenes have also been included, as these
    have been shown, more recently, to be produced by fungi, that were
    reported to be associated with outbreaks of human illness several
    decades ago (ATA).

        During recent years many other mycotoxins have been discovered
    such as: citreoviridin; citrinin; cyclochlorotine; luteoskyrin;
    maltoryzine; patulin; P R toxin; rubratoxin; rugulosin;
    sterigmatocystine; and tremorgens.

        Some of these toxins, which are not discussed in this document,
    have been suggested to be related to disease outbreaks in farm
    animals (Pier et al., 1977). Certain human diseases, suspected of
    being associated with mycotoxins (van Rensburg, 1977), have not been
    discussed in this document as the causative agents have not been
    identified.

        Of the four groups of mycotoxins considered only aflatoxins have
    been shown to be associated with well recognized human health
    effects. For this reason, they are treated separately from the other
    mycotoxins.

    3. AFLATOXINS

    3.1  Properties and Analytical Methods

    3.1.1  Chemical properties

        Although 17 compounds, all designated aflatoxins, have been
    isolated, the term aflatoxins usually refers to 4 compounds of the
    group of bis-furanocoumarin metabolites produced by  Aspergillus
     flavus and  A. parasiticus, named B1, B2, G1 and G2,
    which occur naturally in plant products. The 4 substances are
    distinguished on the basis of their fluorescent colour, B standing
    for blue and G for green with subscripts relating to the relative
    chromatographic mobility. Cows fed rations containing aflatoxin B1
    and B2 excrete metabolites in the milk called aflatoxin M1 and
    aflatoxin M2 (see section 3.3.4.1); M stands for milk, and again
    the subscripts relate to the relative chromatographic mobility.
    (Aflatoxin M1 is also a fungal metabolite.) Of the 4 major
    aflatoxins, B1 is usually found in the highest concentrations,
    followed by G1 while B2 and G2 occur in lower concentrations.

        The structures of a number of aflatoxins and of aflatoxin
    B1-related metabolites (see section 3.3.3) are illustrated in
    Fig. 1. The structure of aflatoxins B1 and G1 were determined
    by Asao et al. (1963, 1965) and that of B2 by Chang et al. (1963).
    Aflatoxins B2 and G2 are dihydroderivatives of the parent
    compounds (Hartley et al., 1963). Aflatoxins M1 and M2 are the
    hydroxylated metabolites of B1 and B2, respectively (Holzapfel
    et al., 1966; Masri et al., 1967; Buchi & Weinreb, 1969). Chemical
    properties of some naturally-occurring aflatoxins and metabolites
    are summarized in Table 1.

        The aflatoxins are intensely fluorescent, when exposed to
    long-wave ultraviolet (UV) light. This makes it possible to detect
    these compounds at extremely low levels (ca. 0.5 ng or less per spot
    on thin-layer chromatograms) and provides the basis for practically
    all the physicochemical methods for their detection and
    quantification. A concentration of aflatoxin M1 of 0.02 g/litre
    can be detected in liquid milk (Schuller et al., 1977).

        Aflatoxins are freely soluble in moderately polar solvents
    (e.g., chloroform and methanol) and especially in dimethylsulfoxide
    (the solvent usually used as a vehicle in the administration of
    aflatoxins to experimental animals); the solubility of aflatoxins in
    water ranges from 10-20 mg/litre.

        As pure substances, the aflatoxins are very stable at high
    temperatures, when heated in air. However, they are relatively
    unstable, when exposed to light, and particularly to UV radiation,
    and air on a TLC plate and especially when dissolved in highly polar
    solvents. Chloroform and benzene solutions are stable for years if
    kept in the: dark and cold.


    FIGURE 1

        Little or no destruction of aflatoxins occurs under ordinary
    cooking conditions, and heating for pasteurization. However,
    roasting groundnuts appreciably reduces the levels of aflatoxins
    (see section 3.2.3) and they can be totally destroyed by drastic
    treatment such as autoclaving in the presence of ammonia or by
    treatment with hypochlorite.


        Table 1.  Physical and chemical properties of some aflatoxins and their metabolites
                                                                                                                                          

    Aflatoxin    Molecular    Relative   Melting     Ultraviolet absorption (epsilon)c    Fluorescence    Reference
                 formula      molecular  point                                              emission
                              mass       C          265 nm            360-362 nm         nm
                                                                                                                                          

    B1a          C17H12O6     312        268-269     12 400            21 800             425             Asao et al. (1965)
    B2a          C17H14O6     314        286-289     12 100            24 000             425             Chang et al. (1963)
    G1a          C17H12O6     328        244-246     9 600             17 700             450             Asao et al. (1965)
    G2a          C17H14O7     330d       237-240d    8 200             17 100             450             Hartley et al. (1963)
    M1a          C17H12O7     328        299         14 150            21 250 (357 nm)    425             Holzapfel et al. (1966)
    M2a          C17H14O7     330        293         12 100 (264 nm)   22 900 (357 nm)      f             Holzapfel et al. (1966)
    P1b          C16H10O6     298        >320        11 200 (267 nm)   15 400 (362 nm)      f             Dalezios et al. (1971 ) and
                                                              14 900 (342 nm)                             Buchi et al. (1973)

    Q1           C17H12O7     328           e        11 450 (267 nm)   17 500 (366 nm)      f             Masri et al. (1974a,b)
    Aflatoxicol  C17H14O6     314        230--234d   10 800 (261 nm)   14 100 (375 nm)    425             Detroy & Hesseltine (1970)
                                                                                                                                          

    a   Molar absorption coefficient for aflatoxins B1, B2, G1, and G2 obtained from Rodricks et al. (1970) and those for M1 and M2
        from Stubblefield et al, (1972).
    b   P stands for phenolic products of O-demethylation of aflatoxin B1.
    c   Compounds dissolved in methanol except for aflatoxin P1 which in this case was dissolved in ethanol. Data on molar absorption
        coefficients for other peaks and on the ultraviolet absorption characteristics of aflatoxins in other solvents can be found
        in the original papers.
    d   Data from Butler (1974).
    e   Not available.
    f   Violet fluorescence of aflatoxin M2 and yellow-green fluorescence of aflatoxins P1 and Q1 reported in original papers.

    

        The presence of a lactone ring in the aflatoxin molecule makes
    them susceptible to alkaline hydrolysis (De Iongh et al., 1962).
    This characteristic is important in that any food processing
    involving alkali treatment can decrease the contamination of the
    products (section 3.2.3) although the presence of protein, the pH,
    and the duration of treatment may modify the results (Beckwith et
    al., 1975). However, if the alkaline treatment is mild,
    acidification will reverse the reaction to reform the original
    aflatoxin.

        The chemistry of the aflatoxins has recently been reviewed by
    Roberts (1974).

    3.1.2  Methods of analysis for aflatoxins in foodstuffs

    3.1.2.1  Sampling

        Sampling is an integral part of the analytical procedure and the
    sample drawn should be representative of the lot. The total error
    made in an analytical procedure consists of the sampling error, the
    subsampling error, and the error in analysis (Whitaker, 1977). The
    difficulty in sampling for aflatoxins arises because of the
    heterogeneity of aflatoxin distribution in contaminated unprocessed
    commodities. On the basis of a large number of analyses, Whitaker et
    al. (1974a) were able to calculate the contribution of each error to
    the total error. The total variance of the analytical procedure is
    primarily caused by sampling variability, whereas the subsampling
    variability and the analysis variability are more or less
    independent of aflatoxin concentration. The coefficient of variation
    associated with sampling is about 115% at a level of contamination
    of 20 g/kg and about 145% at a level of contamination of 10 g/kg.
    Whitaker et al. (1974b) and Whitaker (1977) have summarized a
    procedure for sampling and have developed a number of sampling plans
    used in the USA for the control of aflatoxin contamination in
    shelled groundnuts (peanuts). Other recent publications deal with
    aflatoxin-testing programmes for maize (Whitaker et al., 1978) and
    cottonseed (Whitaker & Whitten, 1977).

        The sampling of small grains, oilseed cakes, foodstuffs, and
    feeds is also difficult, although in most cases the aflatoxin
    distribution within a batch is not likely to be as uneven as in the
    case of groundnuts. Fluids and well-mixed processed products such as
    milk and milk products, beer, and cider do not present such a
    sampling problem.

        Peanut butter, flours, and cornmeal do not present the same
    problems as the original raw materials because a finely divided
    product is formed during processing from which it is much easier to
    obtain a representative analytical sample.

        Some practical aspects of sampling are dealt with in Chapter 26
    of "Official methods of analysis" of the Association of Official
    Analytical Chemists (Horwitz et al., 1975). For survey purposes,
    1-5 kg samples are usually taken and the size of the sample for
    analysis ranges from 20 to 100 g. A sample of 50 g ensures both a
    representative sample and solvent economy.

    3.1.2.2  Methods of analysis

        Biological and chemical procedure have been developed for the
    detection and determination of aflatoxins and other mycotoxins. The
    bioassay techniques that are currently available are not suitable
    for routine screening purposes and their detection levels are not
    low enough. The chemical assay techniques, although more accurate
    and faster, are not always specific. The presence of a certain toxin
    is usually confirmed by derivative formation and its toxicity
    verified by bioassay.

         Biological methods. In the original biological test (Carnaghan
    et al., 1963), one-day-old ducklings were used as test animals for
    determining the presence of aflatoxins in suspect food by measuring
    the degree of biliary proliferation as a semiquantitative index (see
    section 3.4.2.1). The lowest dose level of 0.4 g/day administered
    for 5 days represents the minimum intake required to induce a
    detectable biliary proliferation. The test is also effective for
    detecting aflatoxin M1 in both liquid and powdered milk (Purchase,
    1967). Little information is available on the sensitivity of this
    test (and other biological methods) to aflatoxins other than B1.

        A commonly used method in regulatory actions is the chicken
    embryo bioassay in which 0.1-0.2 g of aflatoxin B1 is applied to
    the egg membrane and the mortality rate recorded during the 23-day
    period of hatching (Horwitz, 1975).

        Several other biological procedures have been developed, using
    maize seedlings, zebra fish larvae, brine shrimps, bacteria etc.
    Detailed descriptions can he found in the reviews by Goldblatt
    (1969) and Ciegler et al. (1971).

         Chemical methods. Although procedures are continually
    changing, the basic steps remain; extraction, lipid removal,
    cleanup, separation, and quantification. Since there is considerable
    overlap in the various methods, most of the published reviews
    (Jones, 1972; Stoloff, 1972) examine the different procedures by
    these basic steps. Depending on the nature of the commodity, methods
    can sometimes be simplified by omitting unnecessary steps. The
    presence of specific interferences such as theobromine in cacao and
    gossypol in cottonseed, may require additional steps.

        Numerous methods of analysis have been reported for the
    determination of aflatoxins in human and animal foodstuffs. Many of
    them are minor modifications of the basic steps adapted to special
    commodities or problems.

        Collaborative studies designed to assess the performances of
    different laboratories give information on the accuracy, precision,
    and specificity of the method under consideration, as well as on the
    occurrence of false negative and false positive results. Only
    methods that have been subjected to such studies are reported in
    this document.

        Chemical methods have mainly been developed for such commodities
    as groundnuts. In the first method for the analysis of groundnuts,
    the aflatoxins were extracted from the contaminated sample using
    methanol; this was later replaced by chloroform. An improvement was
    made by Lee (1965) who showed that the addition of water to
    hydrophilic plant tissues during extraction with chloroform resulted
    in more effective removal of aflatoxin. The combination of
    liquid-liquid extraction techniques and partition chromatography led
    to a method, which is now one of the most widely used, known as the
    Contamination Branch (CB) method (Eppley, 1966). The sample is
    extracted with water and the water extracted with chloroform, the
    lipids and aflatoxins are transferred to a silica-gel column where
    the lipids are selectively eluted with hexane and the pigments and
    other interfering material eluted with absolute diethylether;
    finally the aflatoxins are eluted from the column with 3% methanol
    in chloroform.

        Because the CB method is time-consuming, attempts have been made
    to simplify it. Waltking et al. (1968) drew attention to the fact
    that a separation funnel was simpler and faster for liquid-liquid
    partition than the silica-gel column, and that centrifuging was a
    faster method of separating a solid than filtration. Thus the Best
    Foods (BF) method was developed, which is faster and more economical
    in terms of the amounts of solvents used but provides a poorer
    cleanup. The sample is extracted and defatted with a two-phase
    aqueous methanol-hexane system, the aflatoxins are then partitioned
    from the aqueous phase into chloroform, leaving lipids and pigments
    in the hexane and aqueous methanol.

        In both the CB and BF methods, the aflatoxins are concentrated
    by evaporation of the chloroform, and then separated by thin-layer
    chromatography (TLC). Aflatoxins are intensely fluorescent when
    exposed to long-wave ultraviolet radiation, which makes it possible
    to determine these compounds at extremely low levels. An analyst
    experienced in this field can detect 0.5 ng aflatoxin B1 on a TLC
    plate. In most methods, the intensity of fluorescence of the sample
    is compared with that of a standard. Under ideal conditions this
    technique; has a coefficient of variation of about 20% which can be
    reduced to 5%-9% by the use of a fluorodensitometer.

        It should be pointed out that quantification and confirmation of
    identity can only be obtained if pure authentic standards are
    available for reference. Current sources of aflatoxin standards and
    methods for the determination of mass concentration and purity cart
    be found in Chapter 26 of the AOAC "Official methods of analysis"
    (Horwitz et al., 1975).

        When the CB and BF methods were compared in a collaborative
    study (Waltking, 1970), the methods were found to be equivalent in
    accuracy and precision with a recovery of about 70% of added
    aflatoxin and an overall coefficient of variation of about 35% for
    total aflatoxin levels down to about 20 g/kg. This result has been
    confirmed by the latest International Aflatoxin Check Sample Study
    (Coon et al., 1972) in which 129 laboratories participated. However,
    the coefficient of variation was about twice as high as in the
    original collaborative studies. This illustrates the inadequacies of
    many laboratories.

        Another collaborative study was conducted (Stack, 1974) in which
    the CB and BF methods were compared at levels down to the 2-10 g/kg
    range, Again both methods proved to be equally accurate (about 80%
    recovery) for total aflatoxins at the 5-10 g/kg levels. The CB
    method, however, was as precise (coefficient of variation = 30%) at
    the lowest level of 2 g/kg as at the highest levels. The BF method
    lost precision at these low levels and the coefficient of variation
    was of the order of 100%.

        In spite of cleanup and separation procedures, there might still
    be problems with compounds that have fluorescent and chromatographic
    properties similar to those of the aflatoxins. Thus, the presence of
    a spot on a TLC plate is only presumptive evidence of identity and
    additional confirmatory tests are necessary. Probably the first step
    in confirming the presence of aflatoxins is to use additional
    solvent systems in the TLC. The developed TLC plate can be sprayed
    with 25% sulfuric acid (Schuller et al., 1967), which changes the
    fluorescence colour of the aflatoxin spots to yellow. This test, if
    negative, would rule out the presence of aflatoxins but does not
    provide confirmatory evidence. The formation of chemical derivatives
    was first described for aflatoxins B1 and G1 (Andrellos & Reid,
    1964). The reagents used were formic acid-thionyl chloride, acetic
    acid-thionyl chloride, and trifluoroacetic acid, the acid-catalysed
    addition products formed were a dimeric acetate and an addition
    product with water, respectively. The characteristic mobilities and
    fluorescent properties on thin-layer chromatograms can be compared
    with those of standard derivatives. Pohland et al. (1970) simplified
    the preparation of the derivatives by using a mixture of
    hydrochloric acid and acetic anhydride, and hydrochloric acid alone.
    Further improvement, by elimination of the preparative
    chromatography step in these procedures, has been achieved by
    Przybylski (1975). The water adduct is formed directly on a TLC
    plate from as little as 0.5 ng of aflatoxin B1 or G2.

        In addition to the procedures for groundnuts, methods of
    analysis have been developed for cottonseed, copra, maize, various
    tree nuts (pistachio, walnut, Brazil nuts, etc.) and for animal
    feeds. Many of these methods are modifications of the CB and BF
    methods. Milk and dairy products require a far greater sensitivity
    for the determination of M1 and M2 because these animal
    metabolites are usually only found at sub g/kg levels; additional
    cleanup to eliminate interferences and sometimes two-dimensional TLC
    techniques (Schuller et al., 1973) are necessary to attain
    satisfactory performance. These methods are described in Chapter 26
    of the AOAC "Official methods of analysis" (Horwitz et al., 1975).

        Several analytical methods for the detection of aflatoxin
    residues in animal tissues have been developed, and their detection
    limits evaluated (Jemmali & Murphy, 1976).

        Column detection methods are being used for control purposes in
    the field because of their simplicity. The method of Romer (1975) is
    of particular value because it combines column detection, TLC
    quantification, and TLC plate chemical derivative confirmation in a
    method that has a wide application for a number of foodstuffs and
    feeds including mixed feeds.

        It appears that methods using high-pressure liquid
    chromatography will become the methods of choice for mycotoxin
    analyses in the near future because of their sensitivity and
    improved accuracy, and because they can be applied to a number of
    mycotoxins including aflatoxins B1, B2, G1, and G2 (Panalaks
    & Scott, 1977).

    3.2  Sources and Occurrence

    3.2.1  Formation by fungi

        The ability to produce aflatoxins seems to be confined to
    strains of the two species  Aspergillus flavus Link and
     A. parasiticus Speare, both members of the  A. flavus group.

        Aflatoxin-producing strains of  A. flavus are common and
    widespread, and have been isolated from a host of different
    materials. As indicated in Table 2, a high proportion (from 20% to
    98%) of isolated strains of  A. flavus is able to produce
    aflatoxins.

    Table 2.  Aflatoxin-producing strains of A. flavus isolated from
              four field cropsa
                                                                     

    Source       Isolates    Isolates producing   Maximum yield
                 tested      aflatoxin            of aflatoxin B1
                 (No.)       (%)                  (g/flask)
                                                                     

    groundnut     100         98                  3300
    cottonseed     59         81                  3200
    rice          127         20                  1100
    sorghum        63         24                  3300
                                                                     

    a  Data from Schroeder & Boller quoted by Hesseltine (1976) in
       "Mycotoxins and other fungal related food problems".


    3.2.1.1  Moisture content and temperature

        The moisture content of the substrate and temperature are the
    main factors regulating fungal growth and mycotoxin formation.

        Koehler (1938) established that a moisture content of 18.3% on a
    wet weight basis, was the lower limit for the growth of  A. flavus
    in shelled corn. Extensive studies under precisely controlled
    conditions (Sanders et al., 1968; Diener & Davis, 1969; Davis &
    Diener, 1970) established a moisture content in equilibrium with a
    relative humidity of 85% (or water activity (aw) = 0.85) as the
    lower limit for growth of  A. flavus and for the production of
    aflatoxins. In starchy, cereal grain such as wheat, oats, barley,
    rice, sorghum, and maize, the lower limit is a moisture content of
    18.3%-18.5% on a wet weight basis and in groundnuts, Brazil nuts,
    other nuts, copra, and sunflower and safflower seeds, all of which
    have a high oil content, it is a moisture content of 9%-10%.

        The minimum, optimum, and maximum temperatures for aflatoxin
    production are 12 C, 27 C, and 40-42 C, respectively (Davis &
    Diener, 1970). Northolt et al. (1976) studied the effect of water
    activity and temperature on the growth and aflatoxin production of
     A. parasiticus and came to the conclusion that no detectable
    quantities of aflatoxin B1 were formed at an aw value below 0.83
    and at temperatures below 10C.

    3.2.1.2  Invasion of field crops by  A. flavus

        Groundnut seeds may be invaded by  A. flavus before harvest but
    are more likely to be invaded very rapidly after the plants have
    been pulled and piled for preliminary drying before the nuts are
    removed. This postharvest period is the "high hazard" time for

    aflatoxin production. On the other hand, studies of aflatoxin
    contamination in North Carolina (USA) (Dickens & Satterwhite, 1973)
    under conditions of drought, suggest that drought after groundnuts
    are formed but before they are dug is conducive to their infection
    with  A. flavus. Damage caused by the lesser cornstalk borer (LCB)
    might also aid in the infection process because insects may carry
    fungal spores, although many drought area fields infested with LCB
    did not produce groundnuts with a high aflatoxin content. Drought
    alone does not result in high levels of aflatoxin contamination. It
    must coincide with, or promote infestation by insects which in turn
    infect the groundnut. The LCB may act as a vector for  A. flavus.

        Pettit et al. (1971) reported that groundnuts grown under dry
    land conditions (drought stress) accumulated more aflatoxin before
    digging than those grown under irrigation. Dry land fresh-dug
    kernels contained a maximum aflatoxin level of 35 800 g/kg while a
    maximum of 50 g/kg was detected in kernels from irrigated plots.
    Apparently, the higher kernel moisture content occurring under
    irrigated conditions reduced the aflatoxin production potential,
    whereas a moisture content of about 31% under drought conditions was
    near optimum. Similar observations have been reported from West
    Africa.

        In some irrigated regions with moist weather at harvest time,
    cottonseed may be invaded by  A. flavus while still on the plant
    and after the bolls open and may contain large amounts of aflatoxins
    (Marsh et al., 1973). Stephenson & Russell (1974) related the high
    aflatoxin contamination in the field (in USA) to invasion by insects
    that provided a site of injury and served as vectors for  A. flavus.

        Insect injury in ears of maize in the field may also be
    accompanied or followed by infection with  A. flavus and by
    aflatoxin formation before harvest (Lillehoj et al., 1976). To what
    extent this constitutes a contamination problem in many regions of
    the world, where maize is an important crop, is not known.
    Aflatoxins have also been reported in heads of sorghum heavily
    infected with mould in India (Tripathi, 1973). Pistachio nuts can
    become contaminated with Aflatoxins prior to harvest but the cause
    of infection with aflatoxin-producing strains of fungi has not yet
    been found. The contamination of almonds and of walnuts has been
    traced to specific types of insect damage in the orchard (Stoloff,
    1977).

    3.2.2  Occurrence in foodstuffs

        This subject has been reviewed recently by Stoloff (1976). Of
    the four major aflatoxins (B1, B2, G1, G2) B1 is usually
    found in the greatest concentrations. Measurements of toxin
    concentration are based on the wet weight of the commodity in

    question. The four toxins may occur together, although they need
    not, and their concentrations in relation to each other and their
    occurrence may vary depending on the fungal strain and substrate.
    For example, Hesseltine et al. (1970) found that most fungal strains
    that produced aflatoxin G1 also produced aflatoxin B1, but that
    not all strains that produced aflatoxin B1 produced aflatoxin
    G1. One strain of A. flavus from black pepper produced only
    aflatoxin B2 on 2 natural substrates tested (Schroeder & Carlton,
    1973).

        Although aflatoxins have been found in a variety of foodstuffs,
    the most pronounced contamination has been encountered in groundnuts
    and other oilseeds including cottonseed and maize. The most
    frequently contaminated tree nuts are Brazil nuts and pistachios.

    3.2.2.1  Maize

        Surveys in the USA of more than 1500 samples of maize collected
    in crop years 1964-67, mainly from commercial channels, revealed
    that 2%-3% of the samples contained aflatoxins (total aflatoxin B1
    and G1) in the range of 3-37 g/kg (Shotwell et al., 1969a, 1970).
    In a subsequent survey of 293 samples, 8 samples (2.7%) contained
    aflatoxin B1 levels in the range of 6-25 g/kg, one of the samples
    containing aflatoxin G1 (25 g/kg) as well as aflatoxin B1
    (Shotwell et al., 1971). Aflatoxin B2 was also detected in some of
    the samples. In a further study of 60 samples from south-east USA
    (Shotwell et al., 1973), aflatoxin B1 was found in 21 samples
    (35%) at levels ranging from 6-308 g/kg in the 1969-70 period.
    Aflatoxin B2 at levels ranging from a trace to 40 g/kg was found
    in 15 of these samples and aflatoxin G1 was found in 5 samples at
    levels of a trace to 10 g/kg. In 2 samples, aflatoxin G2 (1 and
    <2 g/kg) was detected in addition to aflatoxin B1. In some of
    the southeastern states of the USA a high frequency of aflatoxin
    contamination was also found in maize in the field (Anderson et al.,
    1975; Lillehoj et al., 1976). In some of the field samples, the
    levels of aflatoxin B1 ranged up to several thousand g/kg or
    more. Most of the field contamination was associated with damage
    caused by insects such as the European corn borer, corn ear worms,
    or weevils, and it seems likely that wherever such damage is
    prevalent, field contamination with aflatoxins will occur. In
    Thailand, 35% of maize samples contained aflatoxin B1 (average
    level 400 g/kg) while 40% contamination by aflatoxin B1 (average
    level 133 g/kg) was found in Uganda (Stoloff, 1976), and 97% in the
    Philippine island of Sebu (average 213 g/kg) (Alpert et al., 1971;
    Campbell & Salamat, 1971; Shank et al., 1972a; Campbell & Stoloff,
    1974; Stoloff, 1976).

        Aflatoxin levels found in household maize samples in connexion
    with the outbreak of acute toxic hepatitis in north-west India
    (Krishnamachari et al., 1975a,b; Tandon et al., 1977) are discussed
    in section 3.5.1.2.

    3.2.2.2  Wheat, barley, oats, rye, rice, and sorghum

        Shotwell et al. (1968b) reported the presence of aflatoxins at
    levels of less than 19 g/kg in 9/1368 samples of wheat, sorghum,
    and oats in the USA. Shotwell et al. (1976b) did not detect any
    aflatoxin B1 (detection limit 1-3 g/kg) in 848 samples of wheat
    from various districts of the USA. The presence of aflatoxins B1,
    B2, G1, and G2 was reported by Tripathi (1973) in heads of
    sorghum heavily infected with mould, in field samples in India, but
    he did not apply any confirmatory tests. Aflatoxins were also found
    in sorghum in Uganda (Alpert et al., 1971) and, in a survey in the
    USA, aflatoxins were detected in 2/66 samples of sorghum grain (13
    and 50 g/kg) (Stoloff, 1976). Aflatoxins have been detected in less
    than 2% of more than 400 samples of rice from markets in Africa, the
    Philippines, and Thailand (Alpert et al., 1971; Campbell & Salamat,
    1971; Shank et al., 1972a). However, Lucas et al. (1970-71) reported
    that out of 139 samples of rice obtained from the Ho Chi Minh
    (Saigon) area in Viet Nam, 31% were found positive for aflatoxins,
    no confirmatory tests were included in this study. In surveys of
    wheat and other cereals in the USSR, Lvova et al. (1976) found
    aflatoxin B1 at a level of 100 g/kg in 1/169 samples (0.6%) in
    the 1972 crop. Aflatoxins (aflatoxin B1 at levels ranging from 20
    to 444 g/kg and aflatoxin G1 at levels of 10-333 g/kg) were
    found in 24/138 samples (17.4%) in the 1973 crop year. In this year,
    the samples to be analysed were specially selected from those that
    were mouldy or had undergone heating or both. In a survey of wheat
    in southern USSR (Kazakstan), Bucarbaeva & Nikov (1977) found
    aflatoxin B1 in 2/50 samples (4%) from one district and 3/50
    samples (6%) from another (levels ranging from 5 to 10 g/kg).

    3.2.2.3  Groundnuts (peanuts)

        In the 1973 survey in the USA of shelled consumer groundnuts,
    15% of 361 samples contained aflatoxins in the range of trace to
    50 g/kg (Stoloff, 1976). Krogh & Hald (1969) found aflatoxins in
    86.5% of 52 samples of groundnut products imported into Denmark for
    feed, one sample contained 3465 g/kg. Aflatoxins were found in 41%
    of 173 samples of groundnuts in the Sudan, 16% of the samples
    containing more than 250 g/kg, and 9%, more than 1000 g/kg (Habish
    et al., 1971). In the Philippines, all the samples of peanut butter,
    tested in 1967-69, contained aflatoxins with a median value of
    155 g/kg and a mean value of 500 g/kg. The highest level detected
    was 8600 g/kg (Campbell & Salamat, 1971). In Thailand, 49% of
    market samples contained an average level of aflatoxins of
    1530 g/kg (Shank et al., 1972a).

    3.2.2.4  Soybeans and common beans

        No significant degree of aflatoxin contamination has been found
    in soybeans or common beans in commerce in the USA (Stoloff, 1976),
    although aflatoxin contamination sufficient to be of public health
    concern has been found in various types of edible beans in Thailand
    (Shank et al., 1972a) and in Africa (Alpert et al., 1971).

    3.2.2.5  Tree nuts

        Aflatoxin has been found occasionally in Brazil nuts, almonds,
    walnuts, pistachio nuts, pecans, and filberts. In some of these,
    contamination occurs when the nuts are still on the tree and is
    usually associated with damage of one sort or another. However,
    apparently sound, undamaged pecans may contain aflatoxins (Stoloff,
    1976). Yndestad & Underdal (1975)in Norway found 66% of Brazil nuts
    contaminated with aflatoxin B1 and Nilsson et al. (1974) found
    that all of 23 batches of Brazil nuts intended for importation to
    Sweden were contaminated. Fourteen percent of 74 samples of
    California almonds were contaminated with aflatoxin B1 with levels
    of less than 20 g/kg in 90% of the contaminated samples (Schade et
    al., 1975).

    3.2.2.6  Copra

        Aflatoxins were found in 88% of 72 samples of copra and copra
    meal (Stoloff, 1976) in amounts ranging from a trace to 30 g/kg,
    and similar contamination was found by Krogh et al. (1970) in copra
    imported into Finland.

    3.2.2.7  Cottonseed

        In 3 successive crop years (1964-67), aflatoxin B1 was
    detected in 6.5%-8.8% of more than 3000 cottonseed samples and in
    12.8%-21.5% of more than 3000 samples of cottonseed meal (Stoloff,
    1976). In contrast, aflatoxin was not detected in cottonseed hulls
    (Whitten, 1969). Relatively high levels of aflatoxin contamination
    were found in an area in southern California. Aflatoxin levels
    increased from 1735 g/kg in some samples of seed harvested in
    November to 2578 g/kg in some samples going into storage in late
    January. The amount present in the stored seeds did not increase
    with time, even though fungi, including  A. flavus, could be seen
    growing on some of the seeds. Marsh et al. (1973) tested cottonseeds
    from 13 locations across the USA cotton belt in 1969 and from 11
    locations in 1970. Aflatoxins B1 and B2 were found in one or
    more samples from 3 regions in areas where boll rot caused by
     A. flavus had been repeatedly observed in previous years. Seeds
    from individual lots contained aflatoxin B1 levels ranging from
    200 000 to 300 000 g/kg indicating the high potential hazard that
    might occur from cottonseed.

    3.2.2.8  Spices and condiments

        Scott & Kennedy (1973) did not find any aflatoxins in 24 samples
    of ground black or white pepper. Low concentrations (up to 8 g/kg)
    were found in 10/33 samples of cayenne pepper and 6/6 samples of
    Indian chili powder, mainly as trace amounts.

    3.2.2.9  Animal feeds

        In studies by Strzelecki & Gasiorowska (1974), aflatoxins
    occurred in 12.7% of 306 samples of animal feed and feed components
    in Poland, 4.2% of the samples containing more than 100 g/kg and
    2.6% of the samples containing more than 1000 g/kg. Feed
    components, mainly groundnut meals, were contaminated by aflatoxins
    more frequently and with higher levels. On the other hand, aflatoxin
    was detected in only one sample (2.7%) of cattle and sheep feeds
    (300 g/kg) and in one sample (1.7%)of poultry feeds (30 g/kg).
    Swine feeds contained aflatoxins in 11.4% of samples, with 6 samples
    (5.7%) exceeding 250 g/kg. Two recent surveys of mixed feeds in the
    Federal Republic of Germany revealed that 1 in 60 samples contained
    aflatoxin B1 levels exceeding 20 g/kg (Seibold & Ruch, 1977); 45
    out of another 105 samples contained levels of between 7 and
    300 g/kg (Kiermeier et al., 1977). Similar results were obtained in
    the United Kingdom (Patterson, personal communication) where 95/172
    samples of dairy feed were contaminated with aflatoxin B1 levels
    of 1-350 g/kg, and 92.4% contained no more than 30 g/kg.

    3.2.2.10  Animal products

        Surveys in several countries have shown that aflatoxin M1 may
    be present in liquid or dried milk (Table 3) and in milk products
    (Kiermeier, 1977). In addition, highly exceptional aflatoxin levels
    in the range of 50-500 g/litre were reported by Suzanger et al.
    (1976) in half of the samples of cow's milk collected in villages
    around Isfahan, Iran (15/30 samples collected in 1973 and 21/37
    samples in 1974). In contrast, no aflatoxins were detected in 8
    samples of milk obtained from large-scale producers in the same area
    in 1974 and only 10% of such samples (2/20) contained aflatoxin M1
    (in the range 8-10 g/litre) in 1973. Aflatoxin M1 was identified
    in all the positive samples. Eight of the 36 village samples
    containing aflatoxin M1 also contained aflatoxin M2 and 2
    samples contained aflatoxin B1. Considerable differences in the
    handling and storage of animal feeds were thought by the authors to
    be responsible for the differences in the aflatoxin M1 contents of
    milk samples from villages and large-scale producers in this area.
    However, levels of aflatoxins in animal feeds were not reported in
    this paper, but they must have been exceptionally high.


        Table 3.  Selected surveys of aflatoxin M1 in cow's milk
                                                                                                                                          

    Milk       Country                        Total no.    No. containing    Range of concentrations    Reference
    samples                                   of samples   aflatoxin M1      in the positive samples
                                              analysed                       (g/litre or g/kg)
                                                                                                                                          

    Liquid     Belgium                            68            42           0.02-0.2                   Van Pee et al. (1977)
               German Democratic Republic         36             4a          1.7-6.5c                   Fritz et al. (1977)
               Germany, Federal Republic of       61            28           0.01-0.25                  Kiermeier (1973)
               Germany, Federal Republic of      419            79           trace-0.54                 Kiermeier et al. ( 1977)
               Germany, Federal Republic of      260           118a          0.05-0.33                  Polzhofer (1977)
               India                              21             3           up to 13.3                 Paul et al. (1976)
               Netherlands                        95            74           0.09-0.5                   Schuller et al. (1977)
               United Kingdom                    278            85a          0.03-0.52a                 Patterson et al. (in press)

    Dried      German Democratic Republic         18             0f          ---                        Fritz et al. (1977)
               Germany, Federal Republic of      166             8           0.67-2.0                   Neumann-Kleinpaul & Terplan (1972)
               Germany, Federal Republic of       52            35           trace-4.0                  Hanssen & Jung (1972)
               Germany, Federal Republic of      120             7a          0.05-0.13                  Jung & Hanssen (1974)
               South Africa                       56             0           ---                        Luck et al. (1977)
               USA                               320            24           0.1-0.4                    FDA (1977) 1973 survey
               USA                               302           192           trace-3.9e                 FDA (1977) 1977 survey
                                                                                                                                          

    a   Seasonal effect observed, i.e., concentration obviously, dependent upon level of concentrate feeding.
    b   Samples collected in retail outlets in 4 southeast States of the USA; it has been estimated that approximately two-thirds
        of the crops in these areas contained aflatoxin concentrations exceeding 20 g/kg because of unusual drought, insect
        damage, and high temperature conditions that occurred in 1977.
    c   Values for 4 positive samples collected in winter; aflatoxin was not detected in the other 32 winter milk samples as well as
        in 12 milk samples collected in summer (detection limit = 0.1 g/kg).
    d   92.5% samples contained aflatoxin M1 concentrations of less than 0.1 g/litre.
    e   Levels ranging from 0.1 to 0.4 g/kg reported in 158 samples; levels exceeding 0.5 g/kg reported in 19 samples.
    f   Aflatoxin B1 contamination detected in one sample.

    

        Aflatoxin residues have been found in animal tissues, eggs, and
    poultry following the experimental ingestion of aflatoxin-
    contaminated feed and this subject has been reviewed by Rodricks &
    Stoloff (1977). However, the toxins have not yet been found in these
    products on the market.

    3.2.3  Fate of aflatoxins during the handling and processing
           of food

        Aflatoxins are affected by some ordinary food processing
    procedures. In the roasting of groundnuts, approximately 50% of the
    aflatoxins are altered to such an extent that they can no longer be
    detected (Lee et al., 1969b; Waltking, 1971). The chemical nature of
    the alteration products has not been fully elucidated.

        The usual methods of processing groundnuts to make peanut butter
    and some nuts for confections may appreciably reduce aflatoxin
    contamination. The removal of undersized nuts (shrivels and pegs);
    the removal of nuts that resist splitting and blanching; and the
    removal of discoloured nuts by hand or electronic sorting are
    effective means of reducing contamination (Rodricks et al., 1977).

        In the removal of oil from oilseeds, most of the aflatoxins are
    found in the oilseed meal. Small amounts remaining in the crude
    vegetable oil are mainly taken out in the soap stock, the byproduct
    from the alkali refining step. The remaining traces of aflatoxins
    are removed in the bleaching refining steps to give aflatoxin-free
    refined oil (Parker & Melnick, 1966).

        The normal alkali processing of maize to produce tortilla-type
    foods, a common practice in some areas of the world including some
    Latin American countries, effectively reduces the levels of
    aflatoxins in contaminated maize (Ulloa-Sosa & Schroeder, 1969).
    Although the mechanism of this reduction has not been clarified,
    some of the aflatoxin is most likely washed out by the initial
    soaking of the maize in lye water and some is undoubtedly chemically
    changed by the alkali; although this process is reported to produce
    a substantial reduction in aflatoxin contamination, it is not enough
    to give a safe product, when highly contaminated maize is being
    processed.

        Jemmali & Lafont (1972) reported only partial destruction of
    aflatoxins during bread making, indicating the importance of the
    contamination of wheat with  A. flavus.

        The above treatments are normal steps in the processing of
    particular foods. In addition to such steps, procedures have been
    developed specifically for the destruction or removal of aflatoxins
    from grains, nuts, oilseeds, and oilseed meals (cake) (FAO, 1977).

    Treatments with ammonia or hydrogen peroxide (H2O2) have been
    the most effective procedures developed to date for the
    detoxification of foodstuffs and animal feeds (Goldblatt & Dollear,
    1977). The treatments with ammonia, developed for the industrial
    decontamination of aflatoxin-contaminated groundnut and cottonseed
    meal (cake) and maize, are limited to the production of animal
    feeds. These procedures have recently been discussed in detail at
    the joint FAO/WHO/ UNEP conference on mycotoxins in Nairobi (FAO,
    1977). The process of treating groundnut protein isolate with
    hydrogen peroxide to obtain a product suitable for use as a human
    food supplement was also discussed. This process has been developed
    in India and is reported to be operating on a small commercial
    scale.

        The feasibility of methods combining the physical separation of
    contaminated portions of produce (for detailed descriptions of
    segregation techniques see, for example, Rodricks et al., 1977) with
    chemical decontamination, was considered at the same conference
    (FAO, 1977).

    3.2.4  Pathways and levels of exposure

        From the previous discussion, it can be seen that a range of
    commodities may become contaminated with trace amounts of
    aflatoxins. In vegetable foods, this contamination results directly
    from fungal spoilage, maize and nuts being particularly susceptible.
    On the other hand, milk, and possibly meat and eggs can become
    indirectly contaminated through the absorption by farm animals of
    aflatoxins from contaminated feed resulting in residues of the
    parent toxin or its metabolites in body fluids or tissues.

        Thus, the level of man's exposure to dietary aflatoxins depends
    upon the food available and on eating habits and will vary from
    country to country according to the local conditions, including the
    traditions of different ethnic groups, and amongst individuals.
    Where contaminated groundnuts or maize make a significant
    contribution to the diet, the level of exposure will be relatively
    higher than where less commonly contaminated commodities take their
    place as the staple food or when milk is the sole
    aflatoxin-containing constituent of the diet.

        In this connexion, the Task Group felt it was important to
    identify the infant as being potentially at risk because:  (a) baby
    food products may be made from dried milk or even maize, commodities
    known to be prone to contamination by aflatoxins; and  (b) in terms
    of larger amounts of food consumed per kg body weight, any level of
    aflatoxin contamination is more significant for the child than for
    the adult.

        Attempts to quantify dietary exposure to aflatoxins are
    discussed in detail in section 3.6.1.1.

        Occupational exposure (section 3.5.2) to aflatoxins with its
    attendant high risks, concerns two groups of individuals: those who
    handle grain, animal feedstuffs, groundnuts, groundnut meal etc.
    (where exposure could occur largely through inhalation of
    contaminated dust), and those who work with toxins in experiments or
    with pure toxins as analytical standards.

        In one paper (van Nieuwenhuize et al., 1973) available to the
    Task Group, an attempt was made to quantify occupational exposure to
    airborne aflatoxins in an oil-mill crushing groundnuts and other
    oil-seeds. Based on airborne dust determinations (mean aflatoxin
    concentrations of 250 and 410 g/kg airborne dust), the estimated
    airborne aflatoxin levels ranged from 0.87 to 72 ng/m3 of air.

    3.3  Metabolism

    3.3.1  Absorption

        Although quantitative data on absorption are not available at
    present, there is no doubt that most of the field cases of
    aflatoxin-induced diseases in animals and man have been associated
    with ingestion of aflatoxin-contaminated foodstuffs and thus with
    the absorption of aflatoxins in the alimentary tract. In spite of
    reports of respiratory exposure (sections 3.2.4 and 3.5.2), there is
    no quantitative information available on aflatoxin resorption from
    the respiratory tract or on percutaneous absorption.

    3.3.2  Tissue distribution

    3.3.2.1  Animal studies

        Experiments with 14C-ring-labelled aflatoxin B1 have shown
    that rats retain about 20% of the 14C activity 24 h after a single
    intraperitoneal dose of 0.07 mg/kg body weight (Wogan et al., 1967).
    The highest concentration was found in the liver, which contained
    amounts of radioactivity equivalent to the entire remainder of the
    carcass (about 5%-8% of the total 14C recovered).

        When poultry were fed rations containing aflatoxins at
    concentrations ranging from 25 to 15 000 g/kg for 8 weeks, residues
    of aflatoxin B1 were found in the liver and in muscle tissue
    (Mintzlaff et al., 1974). The liver contained the highest
    concentration, with a mean value of 15 g/kg at the highest exposure
    level. Similarly the highest concentration of aflatoxin B1 was
    found in the livers of pigs (range: trace-137 g/kg) fed rations
    containing aflatoxins (both aflatoxin B1 and B2) at levels of
    300 and 500 g/kg for 4 months (Krogh et al., 1973a). Aflatoxin
    residues were also detected in kidneys, and muscle and adipose
    tissue.

    3.3.2.2  Studies in man

        Levels of aflatoxins in the tissues of children with Reye's
    syndrome are discussed in section 3.6.2.2. In a liver biopsy from a
    subject with carcinoma of the rectum and liver in the USA, Phillips
    et al. (1976) found 520 g/kg of aflatoxin B1. In France, Richir
    et al. (1976) found aflatoxin B1 in liver biopsies in 6 out of 100
    subjects suffering from various diseases. Concentrations observed
    ranged from 1.6-8 g/kg.

    3.3.3  Metabolic transformation and activation

        With one exception, all primary biotransformations of aflatoxin
    B1 involve its conversion to hydroxylated metabolites but only one
    such derivative, aflatoxin M1 has appreciable oral toxicity
    (Holzapfel et al., 1966). Even so, this metabolite may be detoxified
    by conjugation with taurocholic and glucuronic acids prior to
    excretion in the bile or urine (Bassir & Osiyemi, 1967). In this
    respect, two recently discovered metabolites, P1 (Dalezios et al.,
    1971; Buchi et al., 1973) and Q1 (Masri et al., 1974a,b) are
    similar in that they also undergo this type of detoxification
    (Dalezios et al., 1971; Dalezios & Wogan, 1972).

        The conversion in the liver (Fig. 2) of aflatoxin B1 to
    aflatoxicol (Patterson & Roberts, 1971) and to aflatoxicol H1 via
    aflatoxin Q1 (Salhab & Hsieh, 1975) is unusual in that, unlike
    other biotransformations that are catalysed by liver microsomal
    enzymes, a cytoplasmic NADH-dependent dehydrogenase is involved.
    Furthermore, the formation of aflatoxicol can be inhibited by
    17-ketosteroid sex hormones (Patterson & Roberts, 1972a) and this is
    the only metabolic transformation of aflatoxin  in vitro known to
    be sensitive to hormones.

        Liver homogenates of certain avian and rodent species are
    particularly active in converting aflatoxins B1 and G1 to their
    2-hydroxy, 2,3-dihydro derivatives or hemiacetals called also
    aflatoxins B2a and G2a (Patterson & Roberts, 1970). These
    metabolites bind strongly to protein and are probably sufficiently
    reactive, when formed  in vivo, to cause many of the acute effects
    of aflatoxin poisoning (Patterson & Roberts, 1972b; Patterson, 1973,
    1977).

        At present, there is only indirect evidence for the formation of
    the epoxides of aflatoxins B1 and G1 but this is probably the
    more important form of metabolic activation. When either of the
    parent toxins is incubated with microsomes prepared from the livers
    of many animal species including man, a metabolite is formed which
    appears to have only a transient existence, is highly reactive,

    binds covalently to DNA, and induces mutation in a bacterial
     in vitro test system (Garner et al., 1971, 1972; Ames et al.,
    1973). The metabolite of B1 has not been isolated but the
    2,3-dihydrodiol has been recovered following mild acid hydrolysis of
    an adduct formed when the microsomal metabolite was generated in the
    presence of added DNA or RNA (Swenson et al., 1,974) and, more
    recently, after  in vivo intraperitoneal injection of aflatoxin
    B1 (Swenson et al., 1977). This has been assumed to be indirect
    evidence of the formation of the 2,3-epoxide and, in view of the
    interaction with DNA, it is now generally accepted that the epoxide
    of aflatoxin B1 is the bacterial mutagen and the proximal
    carcinogen.

    FIGURE 2

        Certain of these biotransformations are better developed in some
    animal species than others (Patterson, 1977) and attempts have been
    made to correlate liver metabolism of aflatoxins with toxicity. In
    the first such attempt (Patterson, 1973), it was proposed that rapid
     in vitro formation of aflatoxin hemiacetal was correlated with
    susceptibility to acute aflatoxin poisoning. More recently (Hsieh et
    al., 1977), it has been suggested that the reversible formation of
    aflatoxicol, which is thought to provide a "metabolic reservoir" of
    aflatoxin (Patterson & Roberts, 1972b), is correlated with
    susceptibility to liver tumour induction. On the basis of this, it
    has been tentatively suggested (Hsieh, 1977; Salhab & Edwards, 1977)
    that the human liver might be relatively more resistant to aflatoxin
    carcinogenesis than that of some other species, particularly the
    rat.

    3.3.4  Excretion

    3.3.4.1  Animal studies

         Excretory pathways. Using aflatoxin B1, ring-labelled or
    methoxy-labelled with 14C, Wogan et al. (1967) have shown that
    rats excrete 7096-80% of a single intraperitoneal dose within 24 h.
    A major excretory route of the ring-labelled toxin was through
    biliary excretion into the faeces, accounting for about 60% of the
    administered dose; approximately 20% of administered radioactivity
    was excreted in the urine, and only negligible amounts in expired
    air in the form of 14CO2. In contrast, approximately 25% of
    radioactivity from methoxy-labelled material appeared in expired air
    as 14CO2 with a concomitant decrease in the faeces, indicating
    that  O-demethylation is a significant metabolic pathway for
    aflatoxin B1 in the rat.

         Excretion in the milk of farm animals. Several reviews deal
    with the excretion of aflatoxins in the milk of farm animals
    (Allcroft, 1969; Kiermeier, 1973, 1977; Patterson, 1977; Rodricks &
    Stoloff, 1977). When cattle (Allcroft et al., 1968), sheep (Nabney
    et al., 1967) or goats (Vesely, et al., 1978) are given feed
    contaminated with aflatoxin B1 their milk contains aflatoxin M1.
    In the cow, there is a linear relationship between the amount of
    aflatoxin B1 ingested daily and the level of aflatoxin M1 in the
    milk (Allcroft & Roberts, 1968; Purchase, 1972; Patterson, 1977; see
    Fig. 3), indicating that about 1.5% of aflatoxin B1 is excreted as
    the metabolite M1 (Kiermeier, 1973), and that the concentration of
    aflatoxin B1 in milk is approximately 1/300 of the concentration
    of aflatoxin B1 in the dairy ration (Rodricks & Stoloff, 1977).
    Smaller quantities of unmetabolized aflatoxin B1 have been found
    in cow's and sheep's milk (Nabney et al., 1967; Allcroft et al.,
    1968; Wogan, 1969).

    FIGURE 3

    3.3.4.2  Studies in man

        In the Philippines, aflatoxin M1 has been found (not
    measured)in the urine of human subjects known to have ingested
    aflatoxin-contaminated peanut butter (Campbell et al., 1970).

        Claims concerning an aflatoxin involvement in the etiology of
    juvenile cirrhosis in India (section 3.6.3.2) based on a
    blue-fluorescent B1 spot in the breast milk of mothers and the
    urine of children with the disease (Robinson, 1967) are largely
    discounted by the later studies of Yadgiri et al. (1970) who
    produced spectrophotometric evidence that, although such a spot
    could be identified in the urine of children with the overt disease,
    this was not aflatoxin B1 For other reports see section 3.5.1.

    3.4  Effects in Animals

    The effects of aflatoxins in animals have., been reviewed by
    Allcroft (1969), Newberne & Butler (1969) and Butler (1974).

    3.4.1  Field observations

        When foodstuffs are affected by microbial deterioration, man
    normally eats the less affected parts, whereas domestic animals may
    be exposed to more contaminated rations. This explains why the
    discovery of several mycotoxins has been based on field observations
    in domestic animals.

        The first observation of a disease in animals subsequently
    associated with aflatoxins was an acute outbreak of a lethal disease
    in turkey poults in England in 1960 causing an estimated loss of at
    least 100 000 birds. Extensive research eventually revealed that the
    disease was caused by aflatoxins contained in a batch of Brazilian
    groundnut meal. The concentration of aflatoxin B1 in the original
    groundnut meal was later estimated to be about 10 mg/kg. The disease
    was characterized by rapid deterioration in the condition of the
    birds, subcutaneous haemorrhages, and death. At postmortem, the
    livers of the birds were pale, fatty, and showed extensive necrosis
    and biliary proliferation (Butler, 1974). A similar case of acute
    disease was observed in day-old ducklings fed "toxic" groundnut meal
    (Asplin & Carnaghan, 1961), where the liver changes described were
    followed by cirrhosis. Outbreaks of liver disease in chickens have
    also been associated with aflatoxin-contaminated feed (Asplin &
    Carnaghan, 1961).

        Loosmore & Harding (1961) noted outbreaks in pigs fed groundnut
    meal in which the toxic factor was later identified as aflatoxins.
    The lesions in the pigs included haemorrhages, and liver damage
    characterized by dissecting fibrosis and biliary proliferation.
    Calves fed rations containing 15% toxic groundnut meal also
    developed liver lesions characterized by fibrosis and biliary
    proliferation (Loosmore & Markson, 1961). Outbreaks associated with
    "toxic" groundnut meal and characterized by similar liver lesions
    have been reported in older cattle even though they are more
    resistant (Clegg & Bryston, 1962); there was also a drop in milk
    production.

        A liver disease "hepatitis X" has been reported in dogs in
    southeastern USA (Seibold & Bailey, 1952; Newberne et al., 1955).
    Icterus and in some cases ascites were observed and the liver
    lesions included fatty changes with centrilobular parenchymal
    necrosis and biliary proliferation. Commercial dog food thought to
    be the cause of toxicity was later found to contain aflatoxin B1
    (up to 1.75 mg/kg) (Newberne et al., 1966a). Similar lesions have
    been reproduced in dogs by peroral administration of aflatoxins, and
    it has been suggested that the "hepatitis X" in dogs could be
    causally associated with aflatoxins in the diet (Newberne et al.,
    1966a). During an outbreak of toxic hepatitis affecting several

    hundred people in north-west India and considered to be possibly
    associated with the consumption of maize heavily contaminated by
    aflatoxins (section 3.5.2 and 3.6.2.1), dogs fed food remnants from
    households in affected villages manifested a disease characterized
    by jaundice, ascites, and frequently death (Krisnamachari et al.,
    1975a, b; Tandon et al., 1977). A nonportal type of micronodular
    cirrhosis, with less conspicuous parenchymal and cholangiolar
    changes was found on histological examination of the livers of two
    dogs (Tandon et al., 1977).

    3.4.2  Experimental studies

    3.4.2.1  Acute and chronic effects: hepatotoxicity

        Different species vary in their susceptibility to acute
    poisoning by aflatoxins, with LD50 values ranging from 0.3 to
    17.9 mg/kg body weight (Table 4). In all the animals studied, the
    liver was the principal target organ (see for example Butler, 1974).

    Table 4.  Acute toxicity of aflatoxin B1a
                                                                     

    Species                LD50                     Zone of
                           (mg/kg body weight)      liver lesion
                                                                     

    chick embryo           0.025d
    rabbit                 0.3                      midzonal
    duckling               0.335                    periportal
    cat                    0.55                     periportal
    pig                    0.62                     centrilobular
    dog                    0.5-1.0                  centrilobular
    sheep                  1.0                      centrilobular
    guineapig              1.4                      centrilobular
    baboonb                2.0                      centrilobular
    rat (male)             7.2                      periportal
    macaque femalec        7.8                      centrilobular
    mouse                  9.0
    hamster                10.2
    rat (female)           17.9                     periportal
                                                                     

    a   Adapted from: Newberne & Butler (1969) and Butler (1974).
    b   From: Peers & Linsell (1976).
    c   From: Shank et al. (1971 b).
    d   g/embryo.

        The lesions observed in field cases (section 3.4.1) in poultry,
    pigs, cattle, and dogs have all been reproduced in the same animal
    species by feeding experiments during periods of time ranging from a
    few weeks to a few months, using diets containing aflatoxins, or
    pure aflatoxins ranging from 0.3 to several mg/kg (Newberne &
    Butler, 1969).

        In the study by Carnaghan et al. (1966), chickens were fed a
    diet containing aflatoxin B1 at a level of 1.5 mg/kg. Groups of 3
    control and 3 test chicks were killed after 3 days, 7 days, and
    then at weekly intervals for 8 weeks. After 4 weeks, the liver
    lesions included fatty change, biliary proliferation, and fibrosis.

        In 20 pigs, aflatoxins (aflatoxins B1 and B2) at a feed
    level as low as 300 g/kg resulted in the development of
    centrilobular necrosis and fibrosis of the liver as well as growth
    depression, during a normal feeding period of 3-4 months (Krogh et
    al., 1973a). In cattle, the liver lesions (centrilobular
    degeneration, fibrosis, biliary proliferation) occurred in all 4
    animals after 4 months on a feed containing an aflatoxin level of
    2 mg/kg (Allcroft & Lewis, 1963). The hepatic lesions induced in the
    duckling by the aflatoxins formed the basis of a bioassay originally
    described by Sargeant et al. (1961). At sublethal doses of
    aflatoxins, the bioassay depends upon an assessment of the degree of
    biliary proliferation. Liver lesions similar to those observed in
    farm animals have been experimentally induced by the administration
    of aflatoxins in a number of laboratory animals, including the rat,
    cat, guinea-pig, and rabbit (Newberne & Butler, 1969).

        In a study of Madhavan et al. (1965b), 2 rhesus monkeys
     (Macaca mulatta) were given daily oral doses of aflatoxins at
    500 g/animal for 18 days (corresponding approximately to 250 g/kg
    body weight per day) and then 1 mg/animal per day (corresponding
    approximately to 500 g/kg body weight per day) until death occurred
    after 32 and 34 days. Three rhesus monkeys were given 1 mg each,
    daily, until death occurred after 19, 20, and 27 days respectively.
    The liver lesions included fatty infiltration, biliary
    proliferation, and portal fibrosis. Death or similar lesions were
    not observed in the 2 control monkeys.

        Deo et al. (1970) studied the effect on male rhesus monkeys of
    repeated administration by gastric tube of 3 different levels of
    aflatoxins (B1 + G1). At the highest dose level (1 mg/kg body
    weight daily for 3 weeks), 35/35 animals died within 22 days with
    extensive haemorrhagic necrosis of the liver. A dose level of
    0.25 mg/kg body weight, twice a week, for 5 months induced various
    degrees of liver changes in 24/24 animals characterized by biliary

    proliferation and focal appearance of the liver cells with multiple
    nuclei and giant-sized liver cells with enlarged hyperchromatic
    nuclei. At the lowest dose, 5 animals were given 62 g/kg body
    weight once a week for periods ranging from a few days to 2 years.
    Liver changes were similar to the changes seen in the second group
    but in a milder form.

        Cynomolgus monkeys  (Macaca fascicularis = M. irus) fed a
    dietary level of aflatoxin B1 of 5 mg/kg rapidly developed liver
    damage with biliary proliferation and all 6 animals died within 2
    months. When fed aflatoxin B1 at a dietary level of 1.8 mg/kg, 5
    animals died within 3 months showing liver damage characterized by
    centrilobular necrosis, biliary proliferation, and fibrosis. Two
    animals survived and were killed after 3 years; the liver of one
    animal had the appearance of nodular cirrhosis. Two groups of 4
    animals each were fed lower levels of aflatoxin B1 (0.07 and
    0.36 mg/kg, respectively) for 3 years without showing any signs of
    liver lesions (Cuthbertson et al., 1967).

        The relationship between the chemical structure of different
    aflatoxins and their biological activity (discussed also in section
    3.4.2.3) was investigated in a small number of experiments; more
    extensive studies were not possible because of the limited
    quantifies available of some of the pure aflatoxins. Carnaghan et
    al. (1963) compared 6-day mortality following single doses of
    different aflatoxins, administered by intubation to one-day-old
    Khaki Cambell ducklings, and concluded that both aflatoxins B2 and
    G2 were less toxic than aflatoxins B1 and G1, the ratio of
    LD50 values being 1:4.7 for B1:B2 and 1:4.4 for G1:G2.
    Aflatoxins G1 and G2 were less toxic than the corresponding
    aflatoxins B1 and B2, the ratio of the LD50 values being
    1:2.15 for B1:G1 and 1:2.03 for B2:G2. The corresponding
    LD50 values for aflatoxins B1, B2, G1, and G2 were 0.36,
    1.70, 0.78, and 3.45 mg/kg, respectively. Comparable results were
    obtained by Wogan et al. (1971) who recorded 14 day mortality after
    intubation of male Pekin ducklings and reported LD50 values of
    0.73, 1.76, 1.18 and 2.83 mg/kg for aflatoxins B1. B2, G1, and
    G2, respectively. In the same paper, a study was reported on the
    14-day mortality of male Fischer rats after a single
    (intra-peritoneal) dose of aflatoxin. The LD50 value for aflatoxin
    B1 was 1.16 mg/kg body weight (95% confidence interval 0.91 to
    1.48 mg/kg) whereas the LD50 for aflatoxin G1 was between 1.5
    and 2.0 mg/kg body weight. On the other hand, no deaths occurred in
    20 rats given 12-200 mg of aflatoxin B2 per kg body weight, and
    all 4 rats given 170-200 mg of aflatoxin G2 per kg body weight
    survived. A similar difference in the toxicity of aflatoxins was
    observed when male Fischer rats were given repeated doses of

    aflatoxins by stomach tube over a 4-week period. The 4-week
    mortality in rats given a total dose of 1 mg of aflatoxin B1 per
    rat was 8/10 whereas all 10 rats given the same dose of aflatoxin
    G1 survived and only 4/10 animals given double this dose of
    aflatoxin G1 died. In another trial, all 11 rats survived
    intragastric administration of 3.75 mg of aflatoxin B2 per rat
    repeated every second day for 4 weeks to give a total dose of
    52.5 mg per rat.

        Holzapfel et al. (1966) and Purchase (1967) reported 7-day
    mortality alter oral dosing of one-day-old Pekin ducklings with
    aflatoxins B1, M1, and M2. Five groups of 2-3 ducklings (body
    weight 40-50 g) were used for each of the aflatoxins tested, and the
    following LD50s were calculated (with 95% confidence limits given
    in brackets): aflatoxin B1, 12 (3.9-37.2) g per duckling,
    aflatoxin M1, 16 (5.4-51.5) g per duckling, and aflatoxin M2,
    61.4 (37-100) g per duckling. Ducklings receiving aflatoxin M2
    showed characteristic liver lesions indistinguishable from those
    observed after a similar dose of aflatoxin B1. Higher doses of
    aflatoxin M2 produced similar effects (Purchase, 1967). A study
    comparing the acute toxicity of synthetic (racetalc) aflatoxins B1
    and M1 and the natural optical isomer of aflatoxin B1 was
    reported by Pong & Wogan (1971), suggesting that only one isomer of
    each synthetized racemic mixture was biologically active.
    Fourteen-day mortality rates observed after a single intraperitoneal
    dose of 1.5 mg/kg body weight of synthetic aflatoxin B1 and
    synthetic aflatoxin M1 were 1/1 and 1/2, respectively. However, no
    deaths occurred in groups of rats (each consisting of 4 animals)
    given these synthetic aflatoxins at doses of 1, 0.8, 0.6, or
    0.4 mg/kg body weight. With the natural aflatoxin B1, the observed
    mortalities at these dose levels were 4/4, 2/4, 2/4, and 0/4
    respectively.

        For information on the toxicity of certain other aflatoxin
    metabolites or derivatives, see Wogan et al. (1971) and Patterson
    (1976).

    3.4.2.2  Hepatotoxicity connected with extrahepatic effects

        Many other organs besides the liver are more or less severely
    affected in acute experiments with high doses of aflatoxins (Butler,
    1964): in male and female rats, a single dose of aflatoxin B1
    proved lethal in half of the animals (7.2 mg/kg body weight in the
    male and 17.9 mg/kg body weight in the female, by garage). Frequent
    bilateral adrenal haemorrhages, petechial haemorrhages in many
    organs, particularly in the congested lungs, and occasionally patchy
    necroses in the myocardium and in other organs (kidney, spleen) were
    observed during the first few days following administration. These
    changes were not detected in male or female rats given aflatoxin B1

    at 3.5 mg/kg body weight. With higher doses, the haemorrhages seen
    in the lungs, kidneys, and adrenals were more extensive. Animals
    dying within the first few days often had altered blood in the whole
    of the small intestine and in the colon. Ascites and oedema of the
    omentum were observed in some of the animals a week or more (but not
    one month) after aflatoxin administration. After a month, with the
    exception of the liver damage, all the other organs appeared normal
    in surviving animals. Histologically, certain renal changes were
    detected in the loops of Henle at this stage, consisting of a few
    cells with large irregular hyperchromatic nuclei, very similar to
    those seen in the liver (Butler, 1964).

        Congested lungs with small petechial haemorrhages, haemorrhagic
    necroses in the adrenals (localized in the inner zone of the
    reticularis) and patchy necroses in the kidneys, pancreas, and
    spleen were observed in guineapigs 2-3 days after a single
    intraperitoneal injection of aflatoxin B1 at 1.4 mg/kg body weight
    (lethal in half of the males and females). Even at this dose, the
    small intestine was frequently filled with altered blood. At higher
    doses, the haemorrhagic disease was more marked, with pleural,
    pericardial, and peritoneal haemorrhages. The only change seen in
    the heart of the guineapig, 2-3 days after aflatoxin administration,
    was an occasional small area of fatty degeneration of the
    myocardium. Many animals showed marked ascites and oedema of the
    omentum and subcutaneous tissue during the first week after
    injection (Butler, 1966).

        Bourgeois et al. (1971) reported a special syndrome induced by
    oral administration of aflatoxin B1 in the macaque  (Macaca
     fascicularis). In 2 groups of 4 young females, each receiving a
    single oral dose of aflatoxin B1 at 13.5 or 40.5 mg/kg body
    weight, all animals died within 149 h. Death occurred in 1 out of 4
    other animals receiving a dose of 4.5 mg/kg body weight. Doses of
    toxin of 1.5 mg/kg or 0.5 mg/kg (4 animals in each group) did not
    result in death or unusual clinical signs. Cough, vomiting,
    diarrhoea, and coma were characteristic clinical findings in animals
    exposed to toxic doses. Analysis of blood serum revealed a
    dose-dependent decrease in serum levels of phospholipids within 24 h
    of administration of the aflatoxin. A dose-dependent decrease in
    serum levels of glucose and an increase in nonesterified fatty acids
    occurred within 72 h of aflatoxin administration. The liver lesions
    included centrilobular necrosis, some biliary proliferation, and
    massive fatty degeneration which was also observed in the heart and
    kidneys. Cerebral oedema with neuronal degeneration was seen. Some
    of these findings resemble those associated with Reye's syndrome in
    children (see section 3.5.1.2).

    3.4.2.3  Carcinogenesis

        The carcinogenesis of aflatoxins has been reviewed by Wogan
    (1973, 1977) and re-evaluated by IARC (1976).

         Hepatic and renal tumours. Orally administered aflatoxins,
    mainly B1, have been hepatocarcinogenic in all species of test
    animals studied so far (including nonhuman primates), with the
    exception of the mouse, in which carcinogenic effects have been
    demonstrated only following intraperitoneal administration of
    aflatoxin B1 to neonates (Tables 5 and 6). These studies were
    concerned with repeated or long-term exposure to aflatoxins. In a
    study by Carnaghan (1967), 2 groups consisting of 16 and 18 weanling
    female Wistar rats, respectively, were given single oral doses of
    crystalline aflatoxin B1 or a mixture of aflatoxins containing
    about 40% aflatoxin B1 and 60% aflatoxin G1, at the rate of
    0.5 mg/rat in 0.1 ml dimethylformamide. These doses corresponded to
    averages of 7.65 mg aflatoxin B1/kg body weight and 2.7 mg
    aflatoxin B1 plus 4.0 mg aflatoxin G1/kg body weight,
    respectively. Within 21-32 months, 7 rats out of each group
    developed hepatic tumours with metastases in half the cases. Hepatic
    rumours were not observed in 19 control rats given the solvent only.
    No hepatocellular carcinomas were found in 22 male Fischer rats
    killed successively 16 weeks (3 rats), 25 weeks (5 rats), 38 weeks
    (5 rats), 55 weeks (4 rats), and 69 weeks (5 rats) after a single
    dose of aflatoxin B1 at 5.0 mg/kg body weight, administered by
    garage (Wogan & Newberne, 1967).

        A linear dose-response relationship was observed by Wogan et al.
    (1974) for the development of liver-cell carcinomas in male Fischer
    rats fed dietary concentrations of aflatoxin B1 ranging from
    1-100 g/kg (Table 7). At 1 g/kg, a 10% tumour incidence was found,
    compared with no rumours in the control group and at 100 g/kg the
    tumour incidence was 100%. A linear log (dose)-response relationship
    has been demonstrated in trout fed dietary levels of aflatoxin B1
    ranging from 0.5 to 20.0 g/kg, for 20 months. Extrapolating this
    relationship to lower exposure levels, an incidence of approximately
    10% would be expected with a dietary concentration of 0.1 g/kg.


        Table 5.  Hepatocarcinogenicity of aflatoxin B1 in rodentsa
                                                                                                                                          

    Species                         Dosing regimen          Duration of      Period of      Liver         Reference
                                                            treatment        observation    tumour
                                                                                            incidence
                                                                                                                                          

    rat, Fischer                    1.0 mg/kg diet          33 weeks         52 weeks       3/6           Svoboda et al. (1966)
    rat, Fischer                    1.0 mg/kg diet          41-64 weeks      41-64 weeks    18/21         Wogan & Newberne (1967)
    rat, Porton                     1.0 mg/kg diet          20 weeks         90 weeks       19/30         Butler (1969)
    rat, Wistar                     1.0 mg/kg diet          21 weeks         87 weeks       12/14         Epstein et al. (1969)
    mouse, Swiss                    150 mg/kg dietb         80 weeks         80 weeks       0/60          Wogan (1973)
    mouse, C57Bl/6NB                1.0 mg/kg diet          80 weeks         80 weeks       0/30          Wogan (1973)
    mouse, C3HfB/HEN                1.0 mg/kg diet          80 weeks         80 weeks       0/30          Wogan (1973)
    mouse, hybrid F1, 4 days old    6.0 g/g body weight    3 doses (i.p.)   80 weeks       16/16         Vesselinovitch et al. (1972)
                                                                                                                                          

    a   From: Wogan (1977).
    b   A mixture of aflatoxins B1 and G1 was used in this experiment.


    Table 6.  Hepatocarcinogenicity of aflatoxin B1 in nonrodent speciesa
                                                                                                                           

    Species                  Dosing regimen         Duration of    Period of       Liver        Reference
                                                    treatment      observation     tumour
                                                                                   incidence
                                                                                                                           

    monkey, rhesus (M)       1.655 g totalb         5.5 years      8.0 years       1/1          Gopalan et al. (1972)
    monkey, rhesus (F)       1.855 g total          5.5 years      10.75 years     1/1          Tilak (1975)
    monkey, rhesus (F)       0.504 g total          6.0 years      8.0 years       1/1          Adamson et al. (1973)
    marmoset                 3.0 mg total           50-55 weeks    50-55 weeks     1/3          Lin et al. (1974)
                             5.04-5.84 mg totalc    87-94 weeks    87-94 weeks     2/3          Lin et al, (1974)
    tree shrew (M & F)       24-66 mg total         74-172 weeks   74-172 weeks    9/12         Reddy et al, (1976)
    ferret                   0.3-2.0 g/kg          28-37 months   28-37 months    7/9          Butler (1969)
    duck                     30 g/kg               14 months      14 months       8/11         Carnaghan (1965)
    rainbow trout            4 g/kg in diet        12 months      12 months       15%          Sinnhuber et al, (1968b)
                             8 g/kg in diet        12 months      12 months       40%          Sinnhuber et al. (1968b)
    rainbow trout embryos    0.5 mg/kg in water     1 h            296-321 days    38%          Sinnhuber & Wales (1974)
    salmon                   12g/kg in dietd       20 months      20 months       50%          Wales & Sinnhuber (1972)
    guppy                    6 mg/kg in diet        11 months      11 months       7/11         Sato et al. (1973)
                                                                                                                           

    a   Modified from: Wogan (1977).
    b   A mixture of aflatoxins B1 and G1 was used in this experiment.
    c   These animals were infected simultaneously with hepatitis virus.
    d   This diet also contained 50 mg/kg cyclopropenoid fatty acids.

    

    Table 7.  Dose-response characteristics of aflatoxin B1 carcinogenesis
              in male Fischer strain ratsa
                                                                                   

    Dietary       Duration        Liver             Time of appearance
    aflatoxin     of feeding      carcinoma         of earliest tumour
    level         (week)          incidenceb        (week)
    (g/kg)
                                                                                   

    0             74--109         0/18c             --
    1             78--105         2/22              104
    5             65--93          1/22              93
    15            69--96          4/21              96
    50            71--97          20/25d            82
    100           54--88          28/28e            54
                                                                                   

    a   From: Wogan et al. (1974).
    b   In animals at risk (surviving longer than 50 weeks).
    c   Animals surviving for maximum period.
    d   Two animals had pulmonary metastases.
    e   Four animals had pulmonary metastases.


        In a recent study (FDA, 1978), an attempt was made to calculate
    the lifetime liver cancer risk in rats, which could be connected
    with aflatoxin feed levels lower than those directly tested in
    animal experiments. Estimates of life-time liver cancer incidence
    rates corresponding to aflatoxin feed levels of 0.1 and 0.3 g/kg
    were derived and compared for several selected rat studies using the
    mathematical procedure developed by Mantel & Bryan (1961) and
    modified by Mantel et al. (1975). A more detailed description of
    this procedure can be found in other publications including WHO
    (1978) and Hoel et al. (1975). Thus, the estimated life-time liver
    cancer risk derived from the experimental results reported by Wogan
    et al. (1974) (see Table 7) corresponded to life-time liver cancer
    incidence rates of 70 (600) per 105 rats for an aflatoxin dietary
    level of 0.1 g/kg and 360 (2300) for a level of 0.3 g/kg. (Numbers
    in parentheses; are upper 99% confidence limits.) Estimates derived
    from different rat studies varied considerably. The lifetime
    incidence rates calculated for the combined studies were 240 (470)
    and 1100 (1900) per 105 rats for aflatoxin feed levels of 0.1 and
    0.3 g/kg, respectively (FDA, 1978).

        The studies in primates are included in Table 6. No attempts
    have been made to establish dose-response relationships in primates,
    but they are susceptible to aflatoxin hepatocarcinogenesis.

        The carcinogenic effects of different purified aflatoxins have
    been compared in a limited number of studies. The results of a study
    by Butler et al. (1969) in which 8-9 week old, male (M) and female
    (F) MRC rats were given aflatoxins B1, B2, or G1 in drinking
    water for 10 or 20 weeks, are shown in Table 8. The earliest renal
    neoplasm was seen 54 weeks after the discontinuation of aflatoxin
    treatment. Renal tumours were detected only in males. The 2 rats
    treated with aflatoxin B1 that developed renal tumours did not
    have hepatic carcinomas, but 5 of the 11 rats receiving aflatoxin
    G1 developed both renal and hepatic carcinomas.

        Wogan et al. (1971) studied the relationship between the
    chemical structures of aflatoxins and their hepatocarcinogenicity in
    male Fischer rats, and concluded that aflatoxin B1 was apparently
    more carcinogenic than aflatoxin G1 and that both were much more
    active than aflatoxin B2. A total intraperitoneal dose of
    aflatoxin B2 of 150 mg per rat given in 40 equal doses over 8
    weeks induced hepatocellular carcinomas in 3/9 rats. A similar
    regimen, containing a total dose of aflatoxin B1 of 1.3 mg,
    induced liver tumours in 9/9 animals. In other experiments to
    compare the carcinogenicity of aflatoxins G1 and B1 given by
    stomach tube to rats, aflatoxin G1 in a total dose of 1.4 mg per
    animal (divided into 14 equal doses over 2.5 weeks) induced
    hepatocellular carcinoma in 3/5 rats within 68 weeks. Hepatocellular
    carcinomas were observed in all 18 animals given a total dose of
    2 mg of aflatoxin G1 (divided in 40 equal doses over 8 weeks) and
    killed within 45-64 weeks. Six of these rats had pulmonary
    metastases.

        Hepatocellular carcinomas were also found in 7/7 rats given
    aflatoxin B1 in a total dose of 0.5 mg per animal (divided in 20
    equal doses over 4 weeks) and sacrificed within 74 weeks, in 18/18
    rats given 1 mg per animal (divided into 40 equal doses over 8
    weeks) and killed within 42-58 weeks, and in 17/17 given 1.5 mg per
    animal (divided into 40 equal doses over 8 weeks) and killed within
    42-46 weeks. With the 2 higher doses, 2 and 7 animals, respectively
    had pulmonary metastases. Renal adenocarcinomas were found in 4/26
    rats given aflatoxin G1.

        Synthetic racemic aflatoxin M1 induced hepatocarcinomas at 100
    weeks in 1/29 male Fischer rats given 1 mg of the compound
    intragastrically in divided doses over a period of 8 weeks. The
    incidence of hepatocarcinomas in 9 rats given the same dose of
    natural aflatoxin B1 was 100%, 1 year after treatment (Wogan &
    Paglialunga, 1974). Natural aflatoxin M1 was less effective than
    natural aflatoxin B1 in inducing tumours in trout, particularly in
    the males. Only 14% of male trout, fed natural aflatoxin M1 for a
    year at a dietary level of 4 g/kg, developed liver tumours compared
    with 68% of those receiving a similar feed level of aflatoxin B1


        Table 8.  Carcinogenesis in rats due to ingestion of aflatoxin B1, G1 or B2 in drinking watera
                                                                                                                                     
    Compound       Concentration   Daily        Duration      Total       No. and sex     No. of animals          No. of animals
                   (g/ml)         dose (g)    weeks         dose (mg)   of animals      with tumours            with other
                                                                           treated                                neoplasms
                                                                                          liver      kidney
                                                                                                                                     

    aflatoxin B1   1               20           20            2           15 M            8         2             4
                                                                          15 F            11        0             1
    aflatoxin B1   1               20           10            1           10 M            3         0             2
    aflatoxin G1   3               60           20            6           11 M            9         6             5
                                                                          15 F            12        0             3
    aflatoxin G1   1               20           20            2           15 M            2         5             2
                                                                          15 F            1         0             7
    aflatoxin G1   1               20           10            1           10 M            1         0             1
    aflatoxin B2   1               20           10            1           10 M            0         0             2
    controls       0               0            0             0           15 M            0         0             6
                                                                          15 F            0         0
                                                                                                                                     

    a From: Butler et al. (1969).

    

    (the experiment was terminated 8 months after the discontinuation of
    aflatoxin feeding). In females, the difference was less pronounced,
    liver tumours occurring in 48% of aflatoxin B1-treated trout and
    78% of aflatoxin B1-treated trout (see Table 9) (Sinnhuber et al.,
    1974). Haemorrhages within the cancerous liver resulting in death,
    which were observed in most females receiving dietary levels of
    aflatoxin M1 of 16-64 g/kg, were not observed in similarly
    treated males.

    Table 9.  Aflatoxin M1 liver carcinogenesis in rainbow trout
              (Salmo gairdneri)a
                                                                       

    Aflatoxin in the diet   Liver tumour incidence
                                                         

                            males              females
                                                                       

    M1  4 g/kg             4/28 (14%)         13/27 (48%)
    M1 16 g/kg             22/27 (81%)        11/14 (79%)
    M1 32 g/kg             24/25 (96%)        13/14 (93%)
    M1 64 g/kg             21/24 (88%)        9/10 (90%)
    B1  4 g/kg             15/22 (68%)        18/23 (78%)
                                                                       

    a  From: Sinnhuber et al. (1974).


        A probable effect of rat strain on aflatoxin carcinogenesis was
    seen in the high incidence of renal epithelial neoplasms reported in
    male Wistar strain rats fed diets containing aflatoxin B1, for 147
    days, and then maintained on a basal diet until death (Epstein et
    al., 1969). Renal tumours developed in 57% (8/14), 28% (5/18), and
    23% (3/13) of male rats exposed to diets containing aflatoxin B1
    levels of 1.0, 0.5, and 0.25 mg/kg feed, respectively. The
    incidences of malignant hepatomas in corresponding groups were 86%,
    72%, and 62% respectively. No renal tumours or malignant hepatomas
    were detected in a control group (24 animals). Approximately
    one-third of the aflatoxin-exposed rats with renal rumours did not
    have hepatomas. The first malignant hepatoma and the first renal
    tumour were detected 463 and 468 days after initiation of the
    experiment, respectively; both these tumours occurred in the group
    with the highest exposure. Approximately half of the renal tumours
    were bilateral.

         Early hepatic lesions possibly related to carcinogenesis.
    Aflatoxin B1 given to rats in repeated doses of 15-25 g/day, for
    3.5 weeks, making a total dose of 375 g, elicited increased DNA
    synthesis and mitosis in clusters of liver cells that could be
    distinguished from the surrounding cells by their histological
    appearance and biochemical activity (Newberne & Wogan, 1968a; Rogers
    & Newberne, 1969). Development of the abnormal foci was more
    prominent in rats fed a high-fat, lipotrope-deficient diet, which
    enhances aflatoxin carcinogenesis (section 3.4.2.7), than in rats
    fed a nutritionally adequate diet. The abnormal foci were already
    present in deficient rats at the end of carcinogen administration.
    Similar foci are found in the livers of rats exposed to other
    hepatic carcinogens and may be useful in studying pathogenesis or
    metabolic aspects of liver cancer, since they are thought to be the
    possible precursors of tumours. However, many of the foci disappear
    with time after treatment, and progression is not inevitable.

         Other tumours. Carcinoma of the colon have occasionally been
    reported following aflatoxin exposure (Newberne & Butler, 1969).
    Increased incidence of tumours in the distal half of the colon was
    observed in vitamin A-deficient rats fed aflatoxin B1 (section
    3.4.2.7) (Newberne & Rogers, 1973; Newberne & Suphakarn, 1977).
    Tumours of the colon were also found in more than 20% (12/53) of
    F344 rats (NIH) exposed, either from conception (7/34), or from 6-7
    weeks of age (5/19), to a diet containing an aflatoxin B1 level of
    2 mg/kg and an unspecified amount of vitamin A (Ward et al., 1975).
    The tumours developed in both males and females at 42-64 weeks of
    age and were primarily polyploid neoplasms in the ascending colon,
    although 2 rats had tumours in the descending colon. No colon
    tumours were found in 18 control rats.

        Carcinomas of the glandular stomach were observed in 1/6 young
    rats given a diet of groundnut meal containing an aflatoxin level of
    3-4 mg/kg for 3 weeks and in 1/16 animals given the same diet at the
    age of one year (Butler & Barnes, 1966). Two definite and one
    probable adenocarcinomas of the stomach had previously been observed
    in rats fed a diet prepared from the same batch of groundnut meal
    (Butler & Barnes, 1963).

        Other extrahepatic tumours have occasionally been reported after
    oral aflatoxin exposure, e.g., tumours of the lacrimal glands
    (Dickens et al., 1966; Goodall & Butler, 1969; Butler et al., 1969),
    squamous cell carcinoma of the tongue (Ward et al., 1975) and
    oesophagus (Butler et al., 1969). Tumours at various sites have been
    induced in several species of test animals by intratracheal,
    subcutaneous, or intraperitoneal administration of aflatoxins. The
    experimental results obtained by intratracheal administration are of
    particular interest in connexion with reported effects in man

    associated with airborne aflatoxins (section 3.5.2). Squamous-cell
    carcinoma of the trachea developed within 37-62 weeks in 3/6 rats
    given a mixture of aflatoxins (containing B1, B2, G1, and
    G2) at 300 g per animal intra-tracheally twice weekly, for 30
    weeks. Four of these rats also developed hepatomas within 49-62
    weeks (Dickens et al., 1966).

         Prenatal and early postnatal exposure. In a study of these
    effects, 6 groups of 10 female, Wistar rats were each fed a diet
    containing 25% or 50% groundnut meal contaminated with aflatoxin
    B1 at 10 mg/kg and aflatoxin B2 at 0.2 mg/kg. Dams received this
    diet from day 10 of pregnancy to parturition (exposure of offspring
     in utero); from 1 day post partum to 10 days post partum (exposure
    via milk); or from day 10 of pregnancy to 10 days post partum
    (exposure  in utero and via milk). Among 113 male and 95 female
    offspring observed for up to 36 months, 1 male exposed in utero, 1
    female exposed via milk, and 2 females exposed  in utero
    and via milk developed malignant liver tumours (Grice et al., 1973).
    No liver tumours were seen in control offspring (50 male and 50
    female rats).

    3.4.2.4  Teratogenicity

        The teratogenicity of aflatoxins has been reviewed by Ong
    (1975). The effect of aflatoxin B1 on embryos in hamsters was
    studied by Elis & Di Paolo (1967) who reported that a single
    intraperitoneal injection of aflatoxin B1 at 4 mg/kg body weight,
    given on day 8 of pregnancy, resulted in a high proportion of
    malformed and dead or reabsorbed fetuses. Approximately 50% of the
    fetuses in aflatoxin-treated mothers and over 85% in control mothers
    were normal. A dose of 2 mg/kg did not have any effects. In studies
    by Di Paolo et al. (1967), no teratogenic effects were observed when
    12 pregnant C3H mice were given repeated dally intraperitoneal
    injections of aflatoxin B1 at 4 mg/kg body weight. However, a high
    proportion of dead or reabsorbed fetuses was observed in
    aflatoxin-treated mice in comparison with controls.

    3.4.2.5  Mutagenicity

        Aflatoxin B1 causes chromosomal aberrations (chromosomal
    fragments with occasional bridges, chromatid bridges, chromatid
    breakage) and DNA breakage in plant and animal cells (Ong, 1975). It
    has also been shown to cause gene mutations in bacterial test
    systems (Ames test), when activated by microsomal preparations from
    rat and human liver (Wong & Hsieh, 1976). However, no mutagenic
    effects were observed in mice exposed intra-peritoneally to
    aflatoxin B1 at 5 mg/kg body weight (Leonard et al., 1975).

    3.4.2.6  Biochemical effects and mode of action

        Individual aflatoxins (B1, B2, G1, G2 etc.) with
    slightly different chemical structures affect experimental animals
    (sections 3.4.2.1 and 3.4.2.3) and interact with  in vitro test
    systems to different degrees (Wogan et al., 1971; Patterson, 1976).
    However, as aflatoxin B1 is the commonest and the most potent, it
    has been studied extensively and the majority of reported
    biochemical effects are specifically related to this toxin. As
    discussed in section 3.3.3, the aflatoxin molecule appears to be
    metabolically activated before exerting its acute and chronic
    effects.

        Interaction between these activated molecular species and the
    liver cell apparently occurs at several loci. In the nucleus,
    DNA-dependent RNA polymerase (EC 2.7.7.6)a is inhibited (Pong &
    Wogan, 1970), the toxin binds covalently to DNA  in vitro and
     in vivo (Clifford & Rees, 1967; Lijinsky et al., 1970; Gamer,
    1973, 1975; Swenson et al., 1974, 1977), DNA repair is stimulated
    (Seegers & Pitout, 1973; Stich & Laishes, 1975), and the aflatoxin
    is activated on the outer nuclear membrane to a form that inhibits
    RNA synthesis (Neal & Godoy, 1976).

        The permeability of mitochondria increases (Bababunmi & Bassir,
    1972; Doherty & Campbell, 1973) and electron transport is
    interrupted with a decline in respiration (Doherty & Campbell, 1972,
    1973). Lysosomal membranes are also rendered permeable and unbound
    acid hydrolases leak out (Tung et al., 1970; Pokrovsky et al., 1972;
    Adekunle & Elegbe, 1974). Activation of lysosomal enzymes and their
    effects on cellular structures may be a component of the toxic
    mechanism of aflatoxins (Pokrovsky et al., 1977).

        Aflatoxin is metabolized in the endoplasmic reticulum (section
    3.3.3) and the unmetabolized toxin competes with steroid sex
    hormones for polysome-binding sites (Williams & Rabin, 1971). The
    reticulum degranulates (Theron, 1965; Theron et al., 1965; Butler,
    1971, 1972) with the breakdown of polysome profiles (Villa-Trevino &
    Leaver, 1968; Pong & Wogan, 1969; Godoy et al., 1976) and the
    formation of helical polysome forms (Sarasin & Moule, 1976). RNA
    polymerase is inhibited (Pong & Wogan, 1970) and the toxin binds
    covalently to RNA (Swenson et al., 1977). Many metabolic functions
    are inhibited, including protein synthesis, enzyme induction (Wogan

                 
    
    a The numbers within parentheses following the names of enzymes are
      those assigned by the Enzyme Commission of the Joint IUPAC-IUB
      Commission on Biochemical Nomenclature.

    & Friedman, 1968; John & Miller, 1969; Kato et al., 1970) and the
    synthesis of blood clotting factors II and VII (Bassir & Bababunmi,
    1969). Glucose metabolism via the 6-phosphate pathway (Brown &
    Abrams, 1965; Feuer et al., 1965;; Shankaran et al., 1970), and the
    synthesis of fatty acids and phospholipids are also depressed
    (Clifford & Rees, 1969; Kato et al., 1969; Black et al., 1970;
    Donaldson et al., 1972; Lo & Black, 1972). Furthermore, feed-back
    control of cholesterol synthesis is lost (Horton et al., 1972), a
    change considered characteristic of the pre-cancerous state.

        It has been shown that aflatoxins have immunosuppressive
    properties, probably related to their inhibitory effect on protein
    synthesis. Thus, at levels in poultry feed of 0.25-0.5 mg/kg,
    aflatoxins have been found to reduce resistance to infection by
     Pasteurella multocida, Salmonella spp., Marek's disease virus,
    coccidia, and Candida albicans (Brown & Abrams, 1965; Smith et al.,
    1969; Pier & Heddleston, 1970; Hamilton & Harris, 1971; Edds et al.,
    1973).

        In the liver cytoplasm, there is a transient stimulation
    followed by a depression of glycogenolysis and the pentose shunt
    pathway of glucose metabolism (Shankaran et al., 1970). Aflatoxins
    also compete with further steroid binding sites in the cytoplasm,
    notably NADP-linked 17-hydroxy steroid dehydrogenase (EC 1.1.1.148)
    (Patterson & Roberts, 1971).

        Thus, acute hepatocellular necrosis appears to result from the
    interaction of aflatoxins at a number of intercellular sites whereas
    the mutagenic (section 3.4.2.5) and carcinogenic (section 3.4.2.3)
    properties of aflatoxins probably depend upon metabolic activation
    to a DNA alkylating agent presumably the 2,3-epoxide (section
    3.3.3).

    3.4.2.7  Factors modifying the effects and dose-response
             relationships of aflatoxins

        Numerous reports have dealt with factors of various types that
    modify the carcinogenic and other toxic effects of aflatoxins in
    experimental animals. These include host factors, particularly the
    sex-linked and endocrine characteristics, and interactions with
    other environmental factors. The effects of nutrients deserve
    particular attention in view of nutritional deficiencies occurring
    in certain parts of the world, where aflatoxins exposure can be
    considerable. The more recently discovered effect of exposure to
    (artificial) sunlight might also be of interest in this respect.

        The mechanisms by which hormones, nutrition, and other factors
    influence aflatoxin carcinogenesis are not known but are thought to
    include effects on DNA synthesis and cell division and
    differentiation, and/or effects on aflatoxin metabolism, and

    excretion. Animals with a severely restricted energy food intake do
    not grow and are less susceptible to the action of many carcinogens
    than normal animals. The retardation of growth induced by severe
    protein deficiency and also by hypophysectomy may explain reduced
    tumour incidence under these experimental conditions. Conversion of
    aflatoxin B1 to a bacterial mutagen is different in microsomal
    liver preparations from rats fed a marginal lipotrope diet compared
    with those from normal rats. Excretion of mutagens in the urine is
    also different in lipotrope-deficient rats (Suit et al., 1977).

        Changes in aflatoxin B1 metabolism and reduced levels of
    hepatic macro-molecule-bound aflatoxin B1 adducts were reported in
    rats pretreated with phenobarbital (Garner, 1975; Swenson et al.,
    1977), and also in hypophysectomized animals (Swenson et al., 1977).

         Sex-linked differences and endocrine status. Several studies
    indicate that in comparison with males, female rats are more
    resistant to both acute toxic and carcinogenic effects of
    aflatoxins. Thus, with a single administration of aflatoxin B1 by
    gavage, the LD50 was estimated to be 7.2 mg/kg body weight
    (fiducial limits 5.36-8.23) in male rats and 17.9 mg/kg body weight
    (fiducial limits 14.4-22.5) in female rats (Butler, 1964). The
    sex-dependent influence of vitamin A deficiency on the acute
    toxicity of aflatoxins is discussed later in this section.

        In another study (Newberne & Wogan, 1968a), Fischer rats of both
    sexes were kept on diets containing aflatoxin B1 levels of 0.015,
    0.3, or 1.0 mg/kg and killed successively for histological
    examination. The early hepatic lesions, considered as precancerous,
    appeared with almost the same rate of incidence and at approximately
    the same time in both sexes; however, there was a considerably
    longer period between the appearance of the pre-cancerous lesions
    and progression to liver carcinomas in females than in males. At a
    dietary level of aflatoxin B1 of 1 mg/kg, males developed
    carcinomas after 35 weeks of exposure while tumours in females were
    observed only after 64 weeks. A similar, if much less pronounced,
    sex-linked difference was observed at the 2 lower aflatoxin levels.
    Calculations of the approximate total intake of aflatoxin B1
    before the appearance of tumours (based on average intake of food
    containing a known quantity of aflatoxin) and the time over which
    these total amounts were consumed are given in Table 10. In a study
    by Ward et al. (1975), male rats (F344, NIH) kept on a diet
    containing aflatoxin B1 at 2 mg/kg, died with malignant
    haemorrhagic liver tumours significantly earlier than females.
    Kidney tumours observed in male but not female rats exposed to
    aflatoxins (Butler et al., 1969) have already been discussed in
    section 3.4.2.3.

        Newberne & Williams (1969) reported that fewer male rats
    (Charles River CD) fed for more than a year on a diet containing
    aflatoxin B1 at 0.2 mg/kg and diethylstilboestrol at 4 mg/kg
    developed liver tumours (8/40) than those fed the same aflatoxin
    diet without the estrogen (25/35).

        In a study on the influence of hypophysectomy on aflatoxin
    carcinogenesis, male albino (MRC) rats were fed a diet containing an
    aflatoxin B1 level of 4 mg/kg. All of 14 control rats developed
    liver tumours in 49 weeks whereas none of 14 hypophysectomized rats
    developed liver tumours in the same period. However, tumours of
    extrahepatic tissues (4/14 carcinomas of retro-orbital lacrimal
    glands-see also section 3.4.2.3) were observed in the
    hypophysectomized aflatoxin-treated rats (Goodall & Butler, 1969).


    Table 10.  Sex-linked difference in the carcinogenicity (liver cell
               carcinoma) of dietary aflatoxin B1a
                                                                                   

    Dietary level     Approximate total intake of    Average time (days over
    of aflatoxin B1   aflatoxin B1 before the        which the total amount was
                      appearance of tumours          consumed
                                                                        
                      males         females          males        females
                                                                                   

    1 mg/kg           2.9 mg/rat    5.9 mg/rat       245 days     448 days
    0.015 mg/kg       95 g/rat     11 5 g/rat      476 days     560 days
                                                                                   

    a  Data from Newberne & Wogan (1968a).


         Nutritional factors (food components). The influence of
    nutritional factors on the effects and dose-response relationships
    of aflatoxins has been reviewed by Wogan (1973, 1977), Newberne
    (1974, 1976), Newberne & Rogers (1976), and Newberne & Gross (1977).

         (a) Dietary protein and lipotropic agents. Rhesus monkeys were
    given daily doses of 100 g aflatoxin per animal by stomach tube.
    Two animals, fed a severely protein-deficient ration (1% casein) for
    8 weeks before and during aflatoxin administration, developed fatty
    liver and biliary proliferation and fibrosis and died with
    gastrointestinal haemorrhage within 30 days of aflatoxin treatment.
    Two animals fed a control ration (16% casein) survived in apparent
    good health up to the termination of the experiment (35 days of
    aflatoxin treatment) (Madhavan et al., 1965a).

        In a study on weanling male rats given 50 g aflatoxin per
    animal per day for 20 days, 2/6 animals, fed a diet containing 4%
    casein, died on days 18 and 19 of the experiment. Extensive liver
    damage was found in these rats and in others in the 4%a casein
    group within 20 days of aflatoxin treatment. All 12 rats fed a diet
    containing 20% casein survived similar aflatoxin dosing with only
    mild changes in the liver (Madhavan & Gopalan, 1965). In another
    study by Madhavan & Gopalan (1968) 2 groups of 12 male rats were fed
    the 2 diets (5%a or 20% casein) for 2 years from weaning and were
    given, from the beginning of the experiment, 232 daily doses of
    5 g aflatoxin per animal or 225 daily doses of 10 g aflatoxin per
    animal. All 12 animals fed the 20% casein diet survived the period
    of aflatoxin dosing and 50% developed hepatomas; lung metastases
    were observed in 2 of these rats. Five of the 12 animals fed on the
    5% casein diet died during the period of aflatoxin dosing. No
    hepatomas but one renal cell carcinoma were found in the remaining 7
    rats. Both diets used in these experiments were supplemented only
    with 0.01% choline.

        In experiments by Newberne & Wogan (1968b), rats fed a diet
    containing 9% protein developed a higher incidence of liver tumours
    (11/15) in a shorter period of time (8 months) than rats fed a diet
    containing 22% protein (incidence 7/14 after 10 months). Both groups
    of rats were given a total dose of 375 g of aflatoxin B1 per
    animal by gastric intubation, over 3 weeks, at the beginning of the
    experiment.

        In studies examining lipotropic effects on aflatoxin activity, a
    diet marginal in methionine and choline, deficient in folate, and
    high in fat protected male rats against the acute toxicity of a
    single dose of aflatoxin B1; however, susceptibility to the toxic
    effects of repeated doses of aflatoxin B1 increased, and the
    carcinogenicity of aflatoxin B1 was enhanced. The experimental
    diet (Rogers & Newberne, 1971; Rogers, 1975) contained peanut meal
    (12%, alcohol extracted), gelatine (6%), casein (3%, vitamin free)
    and fibrin (1%) as sources of protein, with a supplement of
    L-cystine (0.5%). This diet is marginally but not severely deficient
    in threonine, tryptophan, and arginine as well as in methionine. The
    diet contained choline chloride (0.2%), and was high in fat (beef
    fat 30%; corn oil 2%). The control, nutritionally complete diet
    contained casein (22%, vitamin free) as the protein source, 15% or
    16% oil (corn or mixed vegetable oil) and 0.3% choline chloride.
    Both diets were adequate in other essential nutrients.

                 

    a According to Madhavan & Gopalan (1968) the composition of the
      diets used in both studies was the same. However, their 1965 paper
      gives 4% whereas that of 1968 gives 5% as the level of casein in the
      diet.

        As shown in Table 11, the marginal lipotrope diet fed for 2
    weeks protected rats against acute aflatoxin B11 toxicity. The
    rats that died had haemorrhagic necrosis of the liver with various
    degrees of bile duct proliferation and, occasionally, haemorrhagic
    necrosis in the adrenals and kidneys. The surviving rats (killed 2
    weeks after aflatoxin administration) had focal necrosis of
    hepatocytes, bile duct proliferation, and an increase in the size of
    periportal hepatocytes. Approximately 20% of the rats fed the
    marginal lipotrope diet and given a single dose of aflatoxin B1
    had focal areas of abnormal hepatocytes which showed an increased
    uptake of 3H thymidine (section 3.4.2.3) (Rogers & Newberne,
    1971).


    Table 11.  Effect of a marginal lipotrope diet on the acute toxicity of
               aflatoxin B1 in rata
                                                                                   

         Aflatoxin B1  Route of           Diet                  No. of   Mortality
         (mg/kg)       administration                           rats     at 2 weeks
                                                                         (%)
                                                                                   

     Sprague-Dawley (males)
            7          Intragastric       control                 5         60
                                          marginal lipotrope      5          0
            9          Intragastric       control                 5         80
                                          marginal lipotrope     10          0
            7          Intraperitoneal    control                 5        100
                                          marginal lipotrope      5          0

     Fischer (males)
            7          intragastric       control                10        100
                                          marginal lipotrope     10          0
                                                                                   

    a From: Rogers & Newberne (1971).


        Although resistant to the toxicity of a single dose of aflatoxin
    B1, rats fed the marginal lipotrope diet for 2 weeks before and
    also during repeated aflatoxin exposure (136 males) were highly
    sensitive to repeated daily doses of aflatoxin B1 at 25 g per
    animal. One half of these rats died, most of them after having
    received 8 or 9 doses of aflatoxin B1 (200 or 225 g total).
    Various degrees of necrosis were observed in the livers with
    extensive proliferation of bile duct cells. The mortality in rats
    fed the control diet (66 males) was only 4% during the
    administration of the total dose of 350 lag of aflatoxin B1
    (Rogers & Newberne, 1971).

        Enhanced aflatoxin B1 carcinogenicity in rats fed marginal
    lipotrope diets, observed repeatedly in several experiments reviewed
    by Newberne & Gross (1977), is demonstrated in Fig. 4 (Rogers,
    1975). Cumulative probability of death from a tumour was calculated
    here by the method described by Saffiotti et al. (1972) i.e., from
    the number of animals at risk and the number of deaths from tumours
    each week. A total dose of aflatoxin B1 of 375 g, divided in 25
    intragastric doses of 15 g/animal per day over 7 weeks, was given
    to male Fischer rats. Hepatocarcinomas developed in 87% of 52
    animals fed the marginal lipotrope diet and in only 11% of 27 rats
    fed the nutritionally complete diet ( P < 0.001). Twenty-seven
    percent of tumours in rats fed the marginal diet metastasized to
    other abdominal organs or the lung; no metastases were detected in
    rats fed the control diet (Rogers, 1975). The groundnut meal fed in
    these experiments did not contain detectable aflatoxins.

    FIGURE 4

        Further studies were carried out to determine how far the high
    fat content of the marginal lipotrope diet contributed to the
    enhancement of carcinogenesis. Male Fischer rats were fed the
    marginal lipotrope diet, the nutritionally complete control diet, or
    the control diet with substitution of the fat from the marginal
    lipotrope diet (30% beef fat, 2% corn oil) for the fat in the
    control diet (15% mixed vegetable oils). Each rat was given a total
    of 375 g of aflatoxin B1 intragastrically, in divided doses over
    7 weeks and kept until moribund or dead, or until 90 weeks after
    treatment, and then necropsied. Hepatocarcinoma incidence was based
    on the number of rats that survived until the first death with
    hepatocarcinoma, i.e., 27-34 rats per group. Incidences were found
    of 39% in rats fed the marginal lipotrope diet, 15% in control rats,
    and zero in rats fed the control diet with beef fat (30%) and corn
    oil (2%) substituted for vegetable oil (15%) (Rogers et al.,
    unpublished data). Thus the high fat content of the deficient diet
    inhibited, rather than contributed to the enhancement of aflatoxin
    carcinogenesis.

        An enhancing effect on aflatoxin carcinogenicity was observed in
    the experiments in which male rats (Charles River CD Sprague-Dawley)
    were fed a low lipotrope diet containing 20% isolated soybean
    protein and supplemented with 0.1% DL-methionine and 0.1% choline
    chloride. Aflatoxin B1 was given intragastrically during the early
    weeks of the experiment in a total dose of 240 g/animal (divided in
    24 daily doses of 10 g, 5 days a week). Liver cell carcinomas were
    observed 5/17 animals fed this diet. No tumours were found in rats
    given the same dose of aflatoxin B1 and fed the same basal diet
    supplemented with 0.6% DL-methionine, 0.6% choline chloride, and
    vitamin B12 (50 g/kg diet) (Newberne et al., 1968).

        It should be noted that a more severe lipotrope deficiency may
    decrease rather than increase the incidence of liver carcinoma in
    aflatoxin-treated rats. A decrease in liver carcinoma was observed
    in aflatoxin-treated, male, Sprague-Dawley rats with severe
    lipotrope deficiency particularly if penicillin, at a level of 0.1%
    were added to the diet. The expected penicillin-induced inhibition
    of cirrhosis development was not observed and the interactions
    between penicillin and aflatoxins have not been elucidated. Both
    diets used in these experiments, the control, adequate, and the
    highly lipo-trope-deficient contained alcohol-extracted groundnut
    meal (25%) and casein (6%) as protein source. No cystine or
    methionine was added, and choline and vitamin B12 were added to
    the control diet at levels of 0.3% and 50 g/kg, respectively. When
    rats were killed 12 months after receiving a total dose of 375 tag
    aflatoxin B1 per animal (divided into 15 daily intra-gastric doses
    of 25 g per animal), hepatomas were found in 64% (9/14) control
    animals and in 41% (7/17) lipotrope-deficient animals. In rats fed
    diets containing penicillin, hepatomas were found in 70% (14/20) of
    the controls and in only 17% (3/18) of the lipotrope-deficient
    animals (Newberne & Rogers, 1971).

        The different effects of marginal and severe lipotrope
    deficiencies on aflatoxin carcinogenesis are of interest in
    connexion with apparent discrepancies between different studies on
    aflatoxin carcinogenesis in rats fed protein-deficient diets
    containing different levels of lipotropic agents discussed earlier.

        Vitamin B12 is a weakly lipotropic factor that has recently
    been found to increase tumour incidence in aflatoxin-treated rats.
    In a study by Temcharoen et at. (1978), male Fischer rats were fed
    diets containing 20% or 5% casein with or without vitamin B12
    (50 g/kg diet) for 33 weeks. The composition of the diets, with the
    exception of the contents of casein (and dextrin) and vitamin B12,
    was similar to that used in previous studies by Wogan & Newberne
    (1967) and Newberne & Wogan (1968a). Choline chloride was added at
    the level 0.036%. The casein content was similar to that in diets
    used in the study by Madhavan & Gopalan (1968) mentioned earlier. A
    mixture of crystalline aflatoxins (containing aflatoxins B1 and
    G1 approximately in the proportion of 1: 1 and about 5% of
    aflatoxins B2 and G2) was added to the diets at the level of
    1 mg/kg. As shown in Table 12, vitamin B12 supplementation
    increased liver rumour incidence in rats fed a diet containing
    aflatoxin and 20% casein. Severe protein deficiency affected the
    growth of the animals, the body weight of rats fed 5% casein being
    reduced to about one third of the controls at the termination of the
    experiment. Liver cirrhosis was observed only in the
    aflatoxin-treated, protein-deficient rats. As suggested by
    Temcharoen et al. (1978), a high incidence of hyperplastic nodules
    and cholangiofibrosis in the protein-depleted, aflatoxin-treated
    animals may indicate that the carcinogenic process was retarded but
    not entirely absent.

         (b) Vitamin A. In a study by Reddy et al. (1973), male and
    female albino rats fed a vitamin A-deficient diet for 9 weeks after
    weaning or fed the same diet with a daily oral supplement of 30 g
    (100 IU) of vitamin A/ rat were given a preparation of crystalline
    aflatoxins containing aflatoxins B1 (44%), G1 (44%), and B2
    and G2 (2%)in a single intraperitoneal dose of 3.5 mg/kg body
    weight. High mortality was observed in vitamin A-deficient males
    (Table 13). The vitamin A-deficient females and all the vitamin
    A-supplemented animals did not show any adverse reactions 40 h after
    aflatoxin injection. Histologically, severe liver damage was
    observed in vitamin A-deficient, aflatoxin-treated male rats, in
    contrast to minimal liver damage in female rats and male rats given
    vitamin A.


        Table 12. The effects of dietary protein and vitamin B12 on aflatoxin-induced liver changesa
                                                                                                                                

    Experimental diet            No. of animals       Average bodyb     No. of animals with
    (for details see text)       at the beginning/    weight (g) at                                                             
                                 termination of       the termination   cholangiofibrosis   cirrhosis   hyperplastic  hepatoma
                                 the experiment       of the                                            nodules
                                                      experiment
                                                                                                                                

    1. 5% casein                 12/6                 85                0                   0           0             0
    2. 5%casein + B12            12/12                91                0                   0           0             0
    3. 5% casein + aflatoxins    25/23                125               9                   21          17            3
    4. 5% casein + B12 +         25/24                129               8                   12          15            1
            aflatoxins
    5. 20% casein                12/9                 328               0                   0           0             0
    6. 20% casein + B12          12/10                369               0                   0           0             0
    7. 20% casein + aflatoxins   25/24                409               0                   0           0             1
    8. 20% casein + B12 +        25/25                375               0                   0           4             6
            aflatoxins
                                                                                                                                

    a  Modified from: Temcharoen et al. (1978).
    b  Calculated from data of the authors on average liver weight and liver weight/100 g body weight.

    

        In a study on rats fed diets containing aflatoxin B1 in the
    range of 15-100 g/kg and vitamin A in deficient, adequate, or
    excessive levels over a 2-year period, the vitamin A-deficient
    animals developed a similar incidence of liver tumours to the other
    2 groups but had an increased incidence of colon carcinomas
    (Newberne & Rogers, 1973). Thus, in a group of 50 male rats (Charles
    River CD Sprague-Dawley) exposed to a dietary level of aflatoxin
    B1 of 100 g/kg and 5 g vitamin A (retinyl palmitate) per animal
    per day, colon tumours and liver tumours were observed in 6 and 11
    rats, respectively, whereas with daily intakes of 50 or 500 g
    retinyl palmitate per rat, no colon cancers were observed and the
    incidence of liver tumours was 24/50 or 19/50, respectively. The
    results of a further study (Newberne & Suphakarn, 1977) are shown in
    Table 14. Charles River CD Sprague-Dawley male (M) and female (F)
    rats were fed a diet containing an aflatoxin B1 level of 1 mg/kg
    (AFB1) and 3 different dietary levels of vitamin A (retinyl
    acetate). Again, there was an increased incidence of colon
    carcinomas in vitamin A-deficient rats. Excessive vitamin A did not
    give protection against aflatoxin carcinogenesis in either the liver
    or the colon.


    Table 13.  The effect of vitamin A status on the acute toxicity of aflatoxins
               in male and female ratsa
                                                                                   

    Sex        Vitamin A          No. of    Mortality     Vitamin A
               daily supplement   animals   40 h after    liver content
               (IU/rat per day)             aflatoxin     (IU/whole liver)
                                            injection     means  SEMc
                                                                                   

    Male             0            6            100%        36.5  6.16b
                  100d            6              0%      2128.1  153.56
    Female           0            6              0%        18.6  3.42
                  100d            6              0%      2299.1  111.49
                                                                                   

    a From: Reddy et al. (1973).
    b Vitamin A values for 5 animals only.
    c SEM = standard error of the mean.
    d 30g.

    Table 14.  Vitamin A status, aflatoxin B1, and liver and colon
               tumors in rats
                                                                                   

    Dietary       AFB1   No.         Sex       Tumour incidence (%)
    retinyl              rats at                                    
    acetate              risk                  liver   colon   both
    (mg/kg)
                                                                                   

    control
       3.0         0       24        M          0.0     0.0     0.0
       3.0         0       26        F          0.0     0.0     0.0
       3.0         +       24        M         87.5     4.1     4.1
       3.0         +       24        F         79.1     8.3     8.3

    low
       0.3         0       10        M          0.0     0.0     0.0
       0.3         0       12        F          0.0     0.0     0.0
       0.3         +       66        M         89.4    28.8    25.7
       0.3         +       42        F         76.2    28.6    11.9

    high
       30.0        0       23        M          0.0     0.0     0.0
       30.0        0       20        F          0.0     0.0     0.0
       30.0        +       26        M         92.3     7.7     7.7
       30.0        +       31        F         83.9     9.7     6.4
                                                                                   

    From: Newberne & Suphakarn (1977).


         (c) Selenium. A single oral dose of aflatoxin B1 given to
    rats at 7 mg/kg bodyweight was less toxic in animals fed a high
    selenium (selenite) diet containing selenium at 1 mg/kg (2-week
    mortality 7/28) than in animals fed diets adequate or marginal in
    selenium, containing selenium (selenite) at 0.1 or 0.03 mg/kg feed,
    respectively (2-week mortalities 20/20 and 28/29). However, a
    further increase in selenium intake (5 mg/kg feed) reaching toxic
    levels predisposed the liver to aflatoxin injury and together with
    aflatoxin exposure resulted in kidney lesions (tubular necrosis at
    the cortico-medullary junction) (Newberne & Conner, 1974). The
    incidence of liver tumours in Sprague-Dawley rats given a total of
    500 g of aflatoxin B1 intragastrically, over a 4-week period, was
    not influenced by dietary selenium (as selenite) contents ranging
    from 0.03 to 5.0 mg/kg (Grant et al., 1977).

         (d) Cyclopropenoid fatty acids (CPFA). Cyclopropenoid fatty
    acids (CPFA) which occur for example in cottonseed oil, enhanced
    tumour induction in trout by both aflatoxin B1 and aflatoxin M1
    (Sinnhuber et al., 1968, 1974). Young trout were fed a purified diet
    which contained an aflatoxin B1 concentration of 4 g/kg with or
    without the addition of CPFA at 220 mg/kg diet. Hepatomas were found
    in 27/30 fish fed CPFA and necropsied after 6 months; at 9 months,
    20/20 bore hepatomas. Corresponding incidences of hepatomas in fish
    that did not receive CPFA were 0/30 and 4/20, respectively
    (Sinnhuber et al., 1968). In later experiments, fish were fed
    aflatoxin M1 at the rate of 4 g/kg diet, with or without the
    addition of CPFA at 100 mg/kg. The incidences of hepatomas in
    CPFA-fed fish were 6/40 at 4 months and 42/63 at 12 months.
    Corresponding incidences in fish not fed CPFA were 2/40 and 6/40
    respectively (Sinnhuber et al., 1974).

        On the other hand, the enhancing effect of CPFA on aflatoxin
    hepato-carcinogenesis was not clearly evident in several studies on
    rats (Friedman & Molar, 1968; Lee et al., 1969a; Nixon et al.,
    1974). No significant increase in liver tumours was observed in
    Wistar male (M) and female (F) rats when sources of CPFA such as;
    food grade cottonseed oil (CSO) or  Sterculia foetida oil (SFO)
    were added at levels of 10% and 0.04%, respectively, to diets
    containing aflatoxin B1 at concentrations of 20 or 100 g/kg.
    Feeding these diets for different periods (generally exceeding 500
    days) at the aflatoxin level of 20 g/kg resulted in hepatomas in
    4/36 rats M: 2/17; F: 2/19) with CSO exposure, in 1/37 rats
    (F: 1/19) with SFO exposure, and in 0/38 rats without CSO or SFO in
    the diet. With an aflatoxin B1 exposure level of 100 g/kg diet,
    the incidences of hepatomas in the CSO group, SFO group, and the
    group without CSO or SFO were 15/36 (M: 12/17; F: 3/19), 17/37
    (M: 10/17; F: 7/18), and 15/35 (M: 7/17; F: 8/18), respectively. In
    Fischer rats, CSO was tested only in combination with the lower
    concentration of aflatoxin; with an aflatoxin B1 level of
    20 g/kg diet, the hepatoma incidence was 6/31 (M: 4/15; F: 2/16)
    with CSO, and 6/28 (M: 5/13; F: 1/15) without CSO exposure (Nixon et
    al., 1974).

         Other chemicals. Sodium phenobarbital given to rats in the
    drinking water (1 g/litre) for 9 weeks, together with a diet
    contaminated with aflatoxin B1 (at the level of approximately
    5 mg/kg) resulted in a lower incidence (11/20) and delayed
    appearance of liver tumours within the following 2 years compared
    with the incidence in rats fed aflatoxin alone (17/20) (McLean &
    Marshall, 1971). The effects of phenobarbital were confirmed in
    experiments by Swenson et al. (1977) in which 2 groups of 18 male
    Fischer rats were each given a diet containing aflatoxin B1 at a
    level of 0.3 mg/kg and drinking water with or without sodium

    phenobarbital (1 g/litre) for a period of 15 months. Examination by
    laparotomy at the end of aflatoxin exposure revealed liver tumours
    in 11 aflatoxin-treated controls (61%) and in only 2 (15%) rats
    exposed to aflatoxin with phenobarbital. When the experiment was
    terminated 5 months later, hepatocellular carcinomas were detected
    in 18 (100%) aflatoxin-exposed rats and in 12 (67%) rats given
    aflatoxin and phenobarbital. On the other hand, no effect on
    aflatoxin hepatocarcinogenesis was observed in rats fed a similar
    aflatoxin diet with the addition of benz (a)anthracene (70 mg/kg)
    or ascorbic acid (25 g/kg) (Swenson et al., 1977).

         Viral infection. Liver tumours were observed in 2/7 marmosets
    fed aflatoxin B1 at a concentration of 2 mg/kg feed and injected
    with hepatitis-type candidate virus (G. Barker strain) during
    aflatoxin exposure. The animals survived 3-94 weeks of treatment.
    Tumours were found in 3/9 marmosets given the aflatoxin diet only
    (Lin et al., 1974). Exposure to both agents produced more severe
    effects on the liver (cirrhosis) than exposure to aflatoxin B1
    alone.

         Exposure to artificial sunlight. The effects of exposure to
    artificial sunlight on acute aflatoxin toxicity (Newberne et al.,
    1974) and carcinogenicity (Joseph-Bravo et al., 1976) were studied
    recently, using a long-arc xenon source and filter combination,
    which had a spectral distribution in the ultraviolet and visible
    ranges closely approximating the 6000 K colour temperature of
    natural light. The rats, located 1.5 metres from the source, were
    kept at an illumination level of about 29 600 1x corresponding to an
    irradiance level of approximately 160 W/m2 over a 2-h period. In
    groups consisting of 40 Sprague-Dawley male rats each, 18 control
    rats died within 2 weeks of a single intragastric dose of aflatoxin
    B1 at 7 mg/kg body weight whereas 23 of the rats exposed to
    artificial sunlight for 2 h after aflatoxin administration died.
    When an excessive dose of riboflavin was given intra-gastrically 30
    min before aflatoxin, the corresponding 2-week mortalities in
    animals unexposed and exposed to artificial sunlight were 20/40 and
    30/40, respectively (Newberne et al., 1974). In the second study, a
    total dose of 375 g of aflatoxin B1 was given to male
    Sprague-Dawley Charles River CD rats, in the form of 15 doses of
    25 g per rat, administered intra-gastrically over a 3-week period.
    Thirty min after each aflatoxin administration, half of the animals
    were exposed for 2 h to artificial sunlight as described earlier.
    When the animals were killed 53 weeks after the last aflatoxin dose,
    benign or malignant liver tumours were found in all 11
    non-irradiated animals and in only 5/12 of the irradiated group
    (Joseph-Bravo et al., 1976).

    3.5  Effects in Man---Epidemiological and Clinical Studies

    3.5.1  General population studies

    3.5.1.1  Liver carcinogenesis

        The data on aflatoxins and human cancer available before October
    1975 were reviewed by IARC (1976) and several other reviews have
    been published more recently (Linsell & Peers, 1977; Shank, 1977;
    Van Rensburg, 1977). A positive association between aflatoxin
    ingestion and liver cancer in man has been found in population
    studies in which estimates of aflatoxin intake and the incidence of
    primary liver cancer were made concurrently.

        In a study in different parts of Uganda, it was found that
    increased frequencies of detectable aflatoxin contamination of food
    samples (range: 10.8%-43%) were associated with increased incidence
    of primary liver cancer (range: 1.4-15.0 cases per 100 000 total
    population per year) (Alpert et al., 1971). Four hundred and eighty
    samples of foods were analysed from 8 areas in Uganda; the total
    intake was not calculated. Within Swaziland, Keen & Martin
    (1971 a, b) showed regional differences in liver cancer frequencies
    consistent with regional differences in the frequency of aflatoxin
    contamination of groundnuts. From the results of a questionnaire
    study, they also suggested that tribal differences in the
    preparation of groundnuts for food, and in eating habits, resulting
    in higher aflatoxin exposure, could explain the apparently higher
    liver cancer rate in the Shangaans living in Swaziland compared with
    the Swazis.a

        In the studies conducted by Shank et al. (1972a,b) in Thailand,
    Peers & Linsell (1973) in Kenya, Van Rensburg et al. (1974) in
    Mozambique, and Peers et al. (1976) in Swaziland, actual
    concentrations of aflatoxin in meals about to be eaten (food on the
    plate) were related to the incidence of primary liver cancer in the
    areas where the meal samples were collected. These studies are
    summarized in Table 15. A linear regression between the incidence of
    liver cancer and the logarithm to the base 10 of the estimated
    dietary intake of aflatoxin was found within the range of the
    aflatoxin exposure levels and the liver cancer incidence rates
    existing in the areas studied? Within Kenya and Swaziland, Peers &
    Linsell (1977) demonstrated a steeper rise in liver cancer incidence
    with increasing aflatoxin intake in men than in women (Fig. 5). A
    similar difference seems to exist also in the other areas studied
    (Shank, 1977).

                 

    a A positive association of fiver cancer incidence variations with
      the availability of aflatoxin contaminated staple foods was also
      reported recently from the Philippines (Bulatao-Jayme et al., 1976).

    Table 15.  Summary of available data on aflatoxin ingestion levels and primary
               liver cancer incidencea
                                                                                   

    Country      Area              Aflatoxin             Liver cancer
                                   Estimated average                               
                                   daily intakeb in      No. of         Incidence
                                   adults--ng/kg body    cases          per 105
                                   weight per day        registered     of total
                                                                        population
                                                                        per year
                                                                                   

    Kenya        High altitude          3.5                  4            1.2
    Thailand     Songkhla               5.0                  2            2.0
    Swaziland    High veld              5.1                 11            2.2
    Kenya        Middle altitude        5.9                 33            2.5
    Swaziland    Mid veld               8.9                 29            3.8
    Kenya        Low altitude          10.0                 49            4.0
    Swaziland    Lebombo               15.4                  4            4.3
    Thailand     Ratburi               45.0                  6            6.0
    Swaziland    Low veld              43.1                 42            9.2
    Mozambique   Inhambanec           222.1                --d           13.0
                                                                                   

    a From: Peers & Linsell (1977).
    b Excluding any aflatoxin present in native beers.
    c Revised incidence estimate taken from Van Rensburg (1977).
    d Number of cases not available, probably >100.


        The possibility that hepatitis B virus infection may confound
    the relationship between aflatoxin ingestion and liver cancer
    incidence has been considered (Linsell & Peers, 1977). Hepatitis B
    infection is common in countries with a high incidence of primary
    liver cancer and evidence of prior exposure to hepatitis B virus is
    more common in individuals with liver cancer in these countries than
    in normal subjects (Vogel et al., 1970; Reys & Sequeira, 1974;
    Prince et al., 1975; Chainuvati et al., 1975). Nevertheless, the
    present evidence favours aflatoxin as a possible major disease
    determinant in primary liver cancer but hepatitis B virus may well
    be a cofactor in the etiology (Peers & Linsell, 1977).

                 

    a When Stoloff & Friedman (1976) compared published reports on the
      incidence of cancer in rural and urban areas of southeastern USA,
      and in southeast states compared with other areas in the USA, they
      found lower incidences of liver cancer in the rural areas and in the
      southeast states, respectively, even though they expected that
      long-term exposure to aflatoxins would be higher in these areas.

    FIGURE 5



        Two studies discussed by the Task Group reported the presence of
    aflatoxins in the tissues of cancer patients.

        Pang et al. unpublished dataa reported the results of a 2-year
    study in Indonesia in which the aflatoxins contents were determined
    in liver tissue biopsy specimens from 71 patients with primary
    cancer of the liver (histologically verified hepatocellular
    carcinoma in 62 patients and cholangiohepatocellular cancer in 7
    patients). Dietary history indicated consumption of contaminated
    food, many patients having eaten groundnuts, almost daily, since
    childhood. Great variation was found in the aflatoxin contents of
    food samples (type of food and number of analyses not given) with
    aflatoxin B1 levels ranging from 17 to 1190 g/kg, and aflatoxin
    G1 levels ranging from 5 to 690 g/kg. In extracts of liver tissue
    biopsy samples obtained soon after the first visit to the hospital,
    spots corresponding to aflatoxins were chromatographically detected
    for 41 patients (57.7%) but not in extracts of liver tissues from 15
    patients without liver cancer serving as controls. The authors also
    reported the more frequent presence of aflatoxins in the urine of
    liver cancer patients compared with the controls. Aflatoxin B1 at
    an estimated level of 520 g/kg fresh weight was detected in the
    liver of a resident of the USA, suffering from carcinoma of the
    liver and the rectum (Phillips et al., 1976). No attempt was made to
    associate the aflatoxin with cancer in this case.

    3.5.1.2  Other effects reported to be associated with aflatoxins

         Reye's syndrome. The possibility that some cases of Reye's
    syndrome (encephalopathy with fatty degeneration of the viscera)
    (Reye et al., 1963), might be due to aflatoxin ingestion was first
    suggested by Becroft (1966), who subsequently reported the presence
    of aflatoxins B1 and G1 in the livers of 2 children who had died
    from Reye's syndrome in New Zealand (Becroft & Webster, 1972).
    Following this report, Dvorackova et al. (1974) in Czechoslovakia
    and Chaves-Caballo et al. (1976) in the USA detected aflatoxins in
    the livers of patients with Reye's syndrome. More recently, in the
    USA, Hogan et al. (1978) detected aflatoxin B1 in the blood serum
    of 2 patients with Reye's syndrome.

        A dietary source of aflatoxin was not identified in any of these
    case reports. Aflatoxins were not found in the livers of 5 further
    subjects with Reye's syndrome in the USA (Shank, 1976).

                 

    a PANG, R. T. L., HUSAINI, S. N., & KARYADI, D. (1974) Aflatoxin
      and primary hepatic cancer in Indonesia. Paper presented at the
       Vth World Congress of Gastroenterology, 1319 October 1974, Mexico.

        Clustering of Reye's syndrome cases, observed in north-east
    Thailand, occurred mainly in the villages but not within families
    and was geographically and seasonally related to high levels of
    aflatoxin contamination of market food samples (Olson et al., 1971;
    Bourgeois, 1975). In 2 cases, the presence of heavy aflatoxin
    contamination in food, eaten 2 or 3 days before death, was
    demonstrated (Bourgeois et al., 1971; Bourgeois, 1975).

        Shank et al. (1971a) reported trace amounts of aflatoxin B1 in
    tissues, body fluids, gastrointestinal contents or stools of 22/23
    Thai patients who had died from Reye's syndrome and 11/15 who had
    died from other causes. More than trace amounts of both aflatoxin
    B1 and B2 were found in at least 2 liver specimens (47 and 93
    lag aflatoxin B1/kg, respectively) from 2 of the 23 patients who
    had died from Reye's syndrome but not in any of the specimens from
    patients dying from other causes. These 2 series of patients are not
    entirely comparable, however, because of differences in the
    frequencies with which the various tissues and body contents were
    examined.

        Twenty-seven cases of Reye's syndrome collected over a 5-year
    period (age range: 3 days to 8 years) were investigated by
    Dvorackova et al. (1977). Aflatoxin B1 was found in the liver in
    all cases and aflatoxin M1 in 4 cases. No aflatoxin was found in
    the livers of 25 children, who had died from other causes.
    Contamination of milk powder with aflatoxin B1 in the home was
    suggested to be the source of exposure in 5 of the cases. No
    aflatoxin M1 was found in this milk.

        With the exception of the previously mentioned studies from
    Thailand (Shank et al., 1971 a; Shank, 1977), cases of Reye's
    syndrome have not been reported from other countries in which food
    is commonly contaminated with more than traces of aflatoxins. As
    Reye et al. (1963) initially suggested, encephalopathy with fatty
    infiltration of the viscera is probably a syndrome of varied
    etiology.

         Other liver diseases. Instances of human liver disease, other
    than Reye's syndrome and cancer, that had apparently followed
    presumed dietary exposure to aflatoxins are summarized in Table 16.
    This table also gives the levels of aflatoxins found in the
    suspected foods. In these reports, the data are insufficient to
    establish a definite association or causal relationship. In the
    studies of Ling et al. (1967), however, there was a geographical and
    temporal association between the availability of mouldy food for
    consumption and the development of disease.


        Table 16.  Reports of liver disease (other than Reye's syndrome and cancer) in individuals exposed to aflatoxins in food
                                                                                                                                               

    Country          No. of        Age          Suspected       Estimated        Nature and outcome of liver           Reference
                     cases         group        aflatoxin       aflatoxin        disease
                     of liver                   vehicle         concentration
                     disease                                    (mg/kg)
                                                                                                                                               

    Senegal          2             4-6 years    groundnut meal  0.5-1            Hepatitis leading to hepatatic        Payet et al. (1966)
                                                                                 fibrosis in one case.
    China (Province  26            all ages     rice            0.2              Acute liver disease, 3 deaths in      Ling et al. (1967)
    of Taiwan)                                                                   children.
    Uganda           1             15 years     cassava         1.7              Acute hepatitis leading to death.     Serck-Hansen (1970)
    India            20            1.5-5 years  groundnut meal  0.3              Hepatomegaly. Hepatic failure and     Amla et al. (1971 )
                                                                                 death in 3 cases. Subsequent
                                                                                 cirrhosis in some cases (juvenile
                                                                                 cirrhosis).
    India            several       infants not  maize           0.25-15          Acute toxic hepatitis; more than      Krishnamachari et al.
                     hundred       affected                                      100 deaths.                           (1975a,b);
                                                                                                                       Tandon et al. (1977)

                                                                                                                                               
    

        The recent outbreak of acute toxic hepatitis in India
    (Krishnamachari et al., 1975a,b; Tandon et al., 1977, 1978) is
    described in detail, because of the number of people affected and
    because of the evaluation of this outbreak by the Task Group (see
    section 3.6.2).

        During the last 2 months of 1974 an outbreak of epidemic
    jaundice with a high mortality rate affected more than 150 villages
    in adjacent districts of 2 neighbouring states, Gujarat and
    Rajasthan, in north-west India. Three reports from 2 independent
    studies of this outbreak were available for evaluation by the Task
    Group (section 3.6.2.1). The first, preliminary report
    (Krishnamachari et al., 1975a) mentioned 397 patients in both
    affected states with 106 deaths. In a later more detailed paper
    (Krishnamachari et al., 1975b), the same group reported 277 cases in
    the Panchamahals district of the state of Gujarat with 75 deaths,
    and 126 hospitalized patients with 38 deaths in the Banswada
    district of the state Rajasthan. In an even later study, a different
    group (Tandon et al., 1977) reinvestigating the outbreak in
    Rajasthan, reported 994 affected individuals with 97 deaths in the
    Banswada and Dungarpur districts of Rajasthan. As reported by
    Krishnamachari et al. (1975b), the outbreak started almost
    simultaneously in all affected villages, with only a few households
    affected in each village and several members of the same household
    becoming ill in some instances. Cases were confined to rural areas
    and to tribal populations whose staple food, particularly during the
    period October-February, was locally grown maize. The outbreak
    commenced with the consumption of recently harvested, badly stored
    maize, which had been affected by unusual rainfalls in October 1974.
    Although the maize was visibly spoiled, it was consumed, leaving
    relatively better cobs for seed purposes and for later use.
    Suspecting that the outbreak could have been caused by the massive
    consumption of maize heavily contaminated with fungi, Krishnamachari
    et al. (1975b) determined the mycoflora and the aflatoxin contents
    of 10 food samples.  A. flavus was detected in all 5 samples of
    maize that were obtained from households affected with the disease,
    and the aflatoxin B1 levels in these samples ranged from
    0.25 mg/kg to 15.6 mg/kg. In contrast, only traces of aflatoxin were
    found in maize supplied to a hostel by local shops in one affected
    village and no aflatoxin was detected in 4 samples of other
    foodstuffs from the same source.  A. flavus was not found in these
    5 food samples of commercial origin. Assuming a dally local
    consumption of maize of up to 400 g per adult per day, and with
    aflatoxin contamination up to 15 mg/kg, Krishnamachari et al.
    (1975b) concluded that the affected people could have been exposed
    to considerable quantities of aflatoxins (up to 6 mg/day), for
    several weeks.


        One liver sample obtained at necropsy and 7 blood serum and 7
    urine samples collected from affected persons were analysed for
    aflatoxin content. No information is given on the stage of the
    disease at which the samples were collected or on the time that had
    elapsed since the last exposure to food suspected to be contaminated
    by aflatoxins. Traces of aflatoxin B1 were reported in only 2
    blood serum samples, with negative results for all the other human
    tissue and fluid samples (Krishnamachari, 1975b).

        Tandon et al. (1977) reinvestigated the outbreak in Rajasthan,
    presenting results of a retrospective epidemiological survey in an
    area, where the largest number of patients with jaundice had been
    reported. Statements on dietary history obtained from members of 47
    affected families (304 household members, 70 of whom had manifested
    the disease) and 29 other families (185 members with no case
    reported) did not indicate any difference in the reported
    consumption of mouldy maize between affected and non-affected
    families or between affected or non-affected members of the same
    household. However,  A. flavus was detected in 85% of mouldy maize
    samples collected from 14 affected families compared with 12% in
    samples obtained from 17 families without manifestation of the
    disease and 3% in samples obtained from 2 grain dealers. Aflatoxins
    B1 and G1 were detected in 13 out of 14 samples from affected
    families and in 17 out of 19 samples from the other families
    investigated in this area. Aflatoxin B1 levels ranged from 0.1 to
    0.6 mg/kg in all positive maize samples, with the exception of 2
    from affected families where levels of 0.9 and 1.1 mg/kg were found.
    Information is not given in the paper concerning the time at which
    the samples were collected in relation to the occurrence of the
    disease, whether the samples were analysed for  Aspergillus and
    aflatoxin contamination, and how far the aflatoxin levels found
    reflected the levels that could have occurred in maize actually
    consumed before and during the outbreak.

        According to Krishnamachari et al. (1975b), all the cases
    occurred among subjects whose staple food was maize. Even if maize
    were also the staple food in the community studied by Tandon et al.
    (1977), statements obtained from members of families studied
    indicated that 31% of the members of non-affected families and 16%
    of members of affected families reportedly did not consume maize.
    From 70 cases of illness, 10 patients (14%) were reported not to
    have consumed maize at all but the paper does not indicate the
    staple food that they consumed instead.

        In another part of the study by Tandon et al. (1977), clinical
    data on 200 hospitalized patients were analysed, using hospital
    records (176 cases) or direct clinical observations (24 patients).
    The disease had a subacute onset starting with fever (in 86%
    patients) followed by rapidly developing jaundice (98% cases) and
    ascites (74%). In the patients where ascites was not massive it was
    possible to detect hepatosplenomegaly. Vomiting, at the onset or at
    the time of reporting to the hospital, was present in 46% of cases.

    Leukocytosis (mainly an increase in polymorphonuclear leukocytes)
    was detected in the initial stage in 87% of patients. Raised levels
    of predominantly direct reacting bilirubin and alkaline phosphatase
    (EC 3.1.3.1) were found in blood serum; transaminase elevation was
    only mild or moderate and even normal levels were observed in blood
    samples collected from 11 patients. From the 200 hospitalized
    patients studied, 10% died in hospital, usually within 6 weeks of
    the onset of illness.

        This description of the principal signs and symptoms is in good
    agreement with cases reported in the same outbreak by Krishnamachari
    et al. (1975b). Both Tandon et al. (1977) and Krishnamachari et al.
    (1975a,b) pointed out that two-thirds of affected people were males.
    The disease was not reported in infants at all, in the study of
    Krishnamachari (1975a,b). The youngest patient reported by Tandon et
    al. (1977) was 2 years old but very few cases were observed below
    the age of 5 years. Both studies pointed out the concurrent liver
    disease with jaundice and ascites observed in village dogs fed food
    remnants from households (see also section 3.4.1).

        Results of histopathological liver examinations are available
    from 10 patients in this outbreak. In one necropsy sample described
    by Krishnamachari et al. (1975a,b), microscopic examination revealed
    extensive bile duct proliferation with periductal fibrosis and
    cholestasis. Apparently normal liver cells were observed over wide
    areas, occasionally replaced in some areas by multinucleated giant
    cells or hepatocytes with foamy cytoplasm. Tandon et al. (1977,
    1978), who examined liver biopsy specimens obtained from 8 patients
    and one liver specimen from autopsy, pointed out that several
    histopathological liver changes were characteristic. These included:
     (a) oedema and collagenization of the central veins (thrombosis
    was not observed);  (b) cholangiolar proliferation;  (c) moderate
    to severe ballooning of the hepatocytes (giant cell transformation
    of the liver cells);  (d) perisinusoidal fibrosis;
     (e) cholestasis; and  (f) cirrhosis with reverse lobulation. On
    the basis of liver histopathology, Tandon et al. (1977, 1978)
    excluded any possibility of vital hepatitis and pointed out that the
    cholangiolar proliferation and syncytial giant cell transformation
    of hepatocytes (as well as the high prevalence of icterus in
    affected people) were also not pathognomonic of the veno-occlusive
    disease of the liver.

        In one study by Maleki et al. (1976), an attempt was made to
    assess the urinary excretion of aflatoxins in patients suffering
    from cirrhosis of the liver, who came from a rural area of Iran
    where this disease is considered to be frequent without any clear
    association with high consumption of alcohol or hepatotoxic spices.

    The authors reported that the urine of 6/25 patients with a clinical
    diagnosis of cirrhosis contained aflatoxin M, whereas no aflatoxin
    M, was detected in the urine of 30 non-cirrhotic patients. The urine
    for aflatoxin analysis was collected within 2 days of admission to
    hospital. The patients came from villages near Isfahan, Iran where
    exceptionally high levels of aflatoxin M, in cow's milk had been
    reported (section 3.2.2.10).

    3.5.2  Occupational exposure

        Three available papers deal with occupational exposure to
    aflatoxins. Eleven out of a group of 55 workers, exposed for 2-9
    years to dust containing aflatoxins in a mill crushing groundnuts
    and other oil seed, developed cancer of various organs within the
    observation period of up to 11 years. Primary liver cancer
    (cholangiocarcinoma) was reported in one of these patients. Two
    other workers were diagnosed to have died of another liver disease.
    On the basis of airborne dust determinations at various work places
    and dust analysis for aflatoxins (see section 3.2.4), the authors
    calculated that airborne aflatoxin levels could have ranged between
    0.87 ng/m3 and 72 ng/m3. Assuming respiratory exposure to
    airborne aflatoxins in the range of 39 ng to 3.2 g per worker per
    week, the authors concluded that, depending on the length of
    employment, the total amount of airborne aflatoxins, to which the
    patients had been exposed during the whole period of work in the
    mill, could have ranged in individual cases from 160 to 395 g. In
    an age-matched group of 55 workers from a different factory in the
    same area, 4 cancer patients were found and no cases of liver cancer
    or death from other liver diseases were recorded (Van Nieuwenhuize
    et al., 1973).

        Dvorackova (1976) reported that 2 men, who had previously been
    carrying out the same type of work on a method of sterilizing
    Brazilian groundnut meal contaminated by  A. flavus, died with a
    diagnosis of pulmonary adenomatosis. Analysis of lung samples
    obtained at autopsy from one of these patients suggested the
    presence of aflatoxin B1. Deger (1976) observed that carcinoma of
    the colon developed in 2 research workers, who, for several years,
    had been involved in the same type of work in the same laboratory,
    purifying substantial amounts of aflatoxins for research purposes.
    No other people were involved in this work in the institute.

    3.6  Evaluation of the Health Risks of Exposure to Aflatoxins

    3.6.1  Human exposure conditions

        The main source of human exposure to aflatoxins is contaminated
    food. Two pathways of dietary exposure have been identified:
     (a) direct ingestion of aflatoxins (mainly B1) in contaminated
    foods of plant origin such as maize and nuts and their products; and
     (b) ingestion of aflatoxins carried over from feed into milk and
    milk products including cheese and powdered milk, where they appear
    mainly as aflatoxin M1.

        Exposure by pathway  (a) is likely to be much greater than by
    pathway  (b) irrespective of some toxicological differences between
    aflatoxins B1 and M1. In tropical countries, where optimal
    conditions for fungal growth exist, many components of the diet may
    become contaminated. In the surveillance of the first of these
    pathways of exposure, sampling is very important, as errors from
    this source are much greater than those from the analytical methods
    used. Since surveillance programmes have been established in only
    very few countries (section 3.6.1.2), information on dietary
    exposure to aflatoxins is not yet available on a worldwide basis.

        Occasionally, workers may be exposed to the dust of agricultural
    commodities that contain aflatoxins. The only available quantitative
    data on such exposure (section 3.2.4) indicate that the
    concentration of aflatoxins in air under these conditions may be of
    the order of 0.1 g/m3.

    3.6.1.1  Sources and levels of aflatoxins in food

        Aflatoxins are fungal products of some moulds that belong to two
    species:  Aspergillus flavus and  A. parasiticus. These moulds are
    found all over the word, except in polar regions, but their growth
    and the formation of aflatoxins require humidity and temperature
    conditions (section 3.2.1.1) that are prevalent in tropical and
    subtropical areas, but may occasionally be found in colder regions
    such as Northern Europe. The formation of aflatoxins takes place
    mainly during harvesting and storage, but there is evidence that
    attacks by insects carrying fungal spores can result in the
    pre-harvest formation of aflatoxins (section 3.2.1.2).

        Although aflatoxin-producing moulds can grow on a large variety
    of foodstuffs, particularly on plant products, it appears that
    certain foodstuffs are more suitable substrates for aflatoxin
    formation than others and, thus, may be contaminated more frequently
    and with higher levels. These include oil seeds (groundnuts, some
    other nuts, cottonseed) and some cereals (maize) (section 3.2.2).

        Existing data on the contamination of foodstuffs by aflatoxins
    have been obtained by means of various analytical procedures
    (section 3.1.2). Collaboratively tested methods are now available,
    and as more countries develop adequate laboratory facilities and
    make use of these methods, more comparable survey data should become
    available.

        Several species of cereals and nuts can be contaminated with
    aflatoxins and, from published survey reports, it appears that
    contamination is highest in groundnuts, Brazil nuts, maize, and
    maize products (section 3.2.2). An extremely high value of about
    3500 g/kg was reported in a single sample of groundnuts imported
    into Europe for feed, and in a survey in Thailand, an average
    concentration of about 1500 g/kg was recorded. However, average
    values of 5 g/kg or less are more common in countries where control
    measures have been implemented. Peanut butter can also contain
    aflatoxins, depending on the quality of the groundnuts used in its
    manufacture. Roasting of groundnuts reduces but does not eliminate
    aflatoxins contamination (section 3.2.3); in general, cooking of
    food is not a safeguard against aflatoxin exposure.

    There is reliable evidence showing that the level of aflatoxin M1
    in milk is directly related to the daily intake of aflatoxin B1 in
    dairy feeds, (section 3.3.4.1) and it is generally recognized that
    groundnut and cottonseed meals, and maize are the major sources of
    this contaminant. However, the level of aflatoxin M1 in milk is
    approximately 300 times lower than the level of aflatoxin B1 in
    the feed consumed. Several surveys of liquid and dried milk powder
    have been carried out throughout the world (section 3.2.2.10). The
    highest reported level of aflatoxin M1 in cow's milk exceeds
    10 g/litre, but, in countries where the quality of dairy products
    is strictly controlled, levels of 0.1 g/litre or less are commoner.

        Animal experiments indicate that, in addition to the carry-over
    into milk, residues of aflatoxins may be present in the tissues of
    animals that consume contaminated feed (section 3.2.2.10). However,
    the Task Group was not aware of any data from surveys for aflatoxin
    residues in meat and meat products.

    3.6.1.2  Dietary intake and levels in human tissues

        Dietary exposure will depend on the levels of aflatoxins in food
    and on food consumption patterns. It is evident that the
    contamination of staple foods is of major concern, and that
    population segments exposed to monotonous diets based on such staple
    foods are at particular risk. Two methods are available for
    assessing dietary exposure to aflatoxins:  (a) determination of
    contamination levels in major food commodities, combined with
    nutritional surveys; and  (b) analysis of food eaten
    ("food-on-the-plate" analysis).

        In principle, the second method provides a better estimate of
    aflatoxin intake but involves many practical difficulties.

        Even though there are many reports from different parts of the
    world on the presence of aflatoxins in individual food items
    (section 3.2.2), the use of food consumption data for the assessment
    of aflatoxin intake seems to be limited, at present, to certain
    parts of the USA. "Food-on-the-plate" analysis data on aflatoxins
    are available only for certain restricted areas in the regions of
    the world where the incidence: of liver cancer is high (section
    3.5.1.1).

        A comprehensive nutritional and commodity survey conducted in
    the southeastern states of the USA gave an estimated average level
    of aflatoxin B1 in groundnuts and groundnut products of 2 g/kg,
    and an average level of 5-10 g/kg for maize products. Based on
    these data, the estimated average daily intake of aflatoxin B1 in
    these areas of the USA was reported (FDA, 1978) to amount to
    2.73 ng/kg body weight (maximum 9.03 ng/kg). In certain areas of
    Thailand and East Africa, the estimated average daily intake based
    on "food-on-the-plate" data ranged from 3.5 to 222.4 ng/kg body
    weight (see section 3.5.1.1). However, during the outbreaks of acute
    liver disease in south-east Asia, much higher estimates of daily
    intake were obtained (up to 120 g/kg body weight), and levels of
    aflatoxin B1 up to 15 mg/kg were found in the contaminated maize
    consumed (section 3.5.1.2).

        Aflatoxin B1 has been found in the liver and other tissues of
    human subjects at levels up to 500 g/kg or more (section 3.3.2.2).
    Some of these cases occurred in Europe and North America indicating
    that, at least in some individuals, significant intake of aflatoxins
    might occur in these areas.

    3.6.2  Acute effects of exposure

        Cases of acute human intoxication (section 3.5.1.2) have been
    reportedly associated with dietary aflatoxin levels substantially
    higher (in the mg/kg range) than the levels thought to be associated
    with liver cancer (g/kg total food range). The Task Group was not
    aware of any long-term follow-up study of human populations in which
    acute intoxication was reported to have occurred.

    3.6.2.1  Acute liver disease

        The association of an outbreak of liver disease in turkeys with
    aflatoxins (section 3.4.1) was of basic importance in the
    recognition of aflatoxins as an environmental hazard.

        Similar outbreaks of acute liver disease associated with the
    ingestion of aflatoxins were also observed in other species. The
    hepatotoxicity of aflatoxins has been confirmed by animal
    experiments and dose-response relationships have been obtained for
    different species (section 3.4.2).

        Acute aflatoxicosis in man has rarely been reported but such
    cases may not always have been recognized. Apart from the death of 3
    children in the Province of Taiwan, China and one child in Uganda,
    where acute liver necrosis was associated with the ingestion of rice
    and cassava contaminated with aflatoxins at levels of 200 g/kg and
    1700 g/kg, respectively, the most convincing case of association of
    aflatoxins with acute liver disease was an epidemic of toxic
    hepatitis in north-west India in 1974 (section 3.5.1.2). In this
    epidemic, several hundred villagers who consumed maize, presumably
    contaminated with aflatoxins at levels up to 15 mg/kg, exhibited
    signs and symptoms of poisoning and more than one hundred people
    died. Estimated daily ingestion of levels up to 6 mg per person were
    reported, corresponding approximately to dose rates up to 120 g/kg
    body weight per day. Such dose rates exceed those required to
    produce liver damage in non-human primates and this provides
    additional support for the assumption that this epidemic was indeed
    related to aflatoxin ingestion. Although the role of aflatoxin was
    not unequivocally demonstrated, the Task Group agreed that this
    incident represented the most acceptable evidence to date of acute
    human aflatoxicosis, supported by the information on liver
    histology, the space-time clustering of the cases, and the deaths
    among village dogs due to a similar form of acute toxic hepatitis.
    Examination of tissues and body fluids for aflatoxins was limited to
    15 samples (1 necropsy liver sample, 7 urine samples, and 7 blood
    samples); aflatoxin was detected only in 2 blood samples. However,
    available animal data suggest that the detectable residues of
    mycotoxins may remain in tissues and body fluids for only a
    relatively short time after ingestion and that, therefore, the
    absence of residues does not exclude the possibility of prior
    exposure.

        In the light of two earlier similar occurrences of aflatoxin
    intoxication associated specifically with children, it is somewhat
    surprising that, in the Indian epidemic, infants were completely
    spared and children under the age of 5 years were less commonly
    affected than adults.

        Similar epidemics could be expected in the future if the unusual
    harvesting circumstances, considered in this case to be responsible
    for the high contamination of the staple diet, recurred. The paucity
    of reports of epidemics of this type would suggest that massive
    contamination of human staple food is a rare occurrence.

    3.6.2.2  Reye's syndrome

    This syndrome (section 3.5.1.2) is found in many countries of the
    world and unlike liver cancer does not show a geographical
    association with areas of high aflatoxin intake. Out of four
    countries in which the relationship between aflatoxins and Reye's
    syndrome has been studied (Czechoslovakia, New Zealand, Thailand,
    and the USA) only Thailand belongs to an area with high aflatoxin
    levels in food. With the exception of the Thailand cases, the Task
    Group was not aware of any other similar reports of Reye's syndrome
    in association with aflatoxins in countries thought to be at
    increased risk from aflatoxin exposure.

        A disease showing many similarities to Reye's syndrome has been
    experimentally demonstrated in macaques (section 3.4.2.2). This
    resulted from single doses of aflatoxin B1 ranging from 4.5 to
    40.5 mg/kg body weight. Reports on numerous experimental studies in
    different animal species, including other nonhuman primates, did not
    mention brain lesions; it is, however, possible that brains were not
    examined for abnormalities.

        Among the reports on the presence of aflatoxins in the tissues
    of patients with Reye's syndrome, two studies deserve; attention
    because of the number of cases included, and because control
    subjects were available (section 3.5.1.2).

        In Thailand, appreciable amounts of aflatoxins were detected in
    autopsy specimens of 6 out of 23 cases of Reye's syndrome and trace
    amounts (1-4 g/kg) were found in a further 16 cases. Similar trace
    amounts of aflatoxins were detected in specimens from 11 out of 15
    children who had died from other causes.

        In a systematic study over several years in Czechoslovakia,
    aflatoxin B1 was unequivocally demonstrated in the liver tissue of
    26/27 cases, and M1 in 4 cases (in one case the liver tissue was
    not examined for aflatoxins). No aflatoxin was found in the liver
    tissue of 25 children who died from other causes.

        Cases where aflatoxins have been identified in the tissues or
    body fluids are sporadic, and the Task Group had no indication of
    the number of symptomatic cases in which it was not possible to
    demonstrate the association with aflatoxin exposure. As regards
    cases in which aflatoxin was detected in the tissues, it cannot be
    excluded that pathological changes connected with Reye's syndrome
    could have decreased the clearance of aflatoxins from tissues.

        In view of these considerations, aflatoxins cannot be excluded
    as a contributing factor to Reye's syndrome in some areas, although
    an exclusive causal relationship cannot be accepted. There is
    evidence that other factors, particularly influenza B virus, may be
    associated with this syndrome.

    3.6.3  Chronic effects of aflatoxin exposure

    3.6.3.1  Cancer of the liver

        Epidemiological data indicate an association between the level
    of daily aflatoxin ingestion and the incidence of primary liver cell
    cancer in certain areas of Kenya, Mozambique, Swaziland, and
    Thailand (section 3.5.1.1). This relationship is strongly supported
    by studies in experimental animals. In at least 8 species of
    experimental animals, aflatoxin has been shown to increase the
    incidence of liver cancer (section 3.4.2.3).

        If the data from the 4 epidemiological studies (section 3.5.1.1)
    are combined, the best fit to the data points is obtained by a
    linear regression of the crude liver cancer incidence rates on the
    logarithms of the dietary aflatoxin intake. The regression has been
    estimated for dietary intakes ranging from 3.5 to 222.4 ng/kg body
    weight per day and for crude liver cancer incidence rates from 1.2
    to 13 cases/100 000 population per year. At the lower ranges of
    aflatoxin intake, liver cancer rates are of the magnitude
    encountered in parts of the world in which liver cancer frequency is
    considered to be low.

        It may be recalled that the liver cancer incidence in rats can
    be increased by ingestion of diets containing aflatoxin B1 at a
    level of 1 g/kg (Table 7). The estimated levels of exposure to
    aflatoxins in the USA (see section 3.6.1.2) have been used in the
    assessment of corresponding life-time liver cancer risks in rats
    (section 3.4.2.3) (FDA, 1978). For combined rat studies, these risk
    estimates amounted to 240 and 1100 per 10 000 for aflatoxin exposure
    levels of 0.1 and 0.3 g/kg feed, respectively; the estimated
    lifetime risks of primary liver cancer in the human population of
    the USA from all causes is approximately 161 per 100 000 (FDA,
    1978). Comparison of epidemiological and experimental data would
    seem to indicate that man is not more but probably less susceptible
    to aflatoxins than the rat. This conclusion also seems to be
    supported by studies on the metabolic transformation of aflatoxins
    (see section 3.3.3).

        An association between aflatoxin intake and human liver cancer
    incidence was established in surveys that included areas with high
    estimated aflatoxin exposure and high liver cancer incidence. These
    studies compared the current average aflatoxin intake and the crude
    liver cancer incidence rate and did not allow for the period of
    latency between the beginning of exposure and cancer manifestation.
    The length of this latent period and factors that may modify its
    length are not well known. However, the studies were conducted in
    rural areas with stable populations and conditions of exposure
    thought not to have changed substantially. Data from intervention
    studies, in which populations are followed up after a reduction in
    exposure levels has been achieved, are not available.

        Published epidemiological studies have been limited in scope and
    only a single possible etiological factor, i.e., aflatoxin exposure
    has been examined. Other factors for which there is some evidence of
    an etiological or modifying role in liver cancer, such as
    nutritional status, cirrhosis, or viral hepatitis, or the
    possibility of interactions between these and still other factors
    have not been considered.

        In rats, dietary intake of lipotropes, protein, and vitamin A
    modified the carcinogenic potential of aflatoxins (section 3.4.2.7).
    Diet, marginally deficient in lipotropes (choline, methionine,
    folate), enhanced liver cancer induction by aflatoxin B1. A diet
    marginally deficient in protein (9% casein) also increased liver
    cancer incidence in aflatoxin-treated rats. Severe lipotrope
    deficiency or severe protein deficiency (4% casein, combined with
    lipotrope deficiency) decreased cancer incidence in
    aflatoxin-treated rats. Severe deficiencies which markedly inhibit
    the growth of experimental animals can reduce the cancer incidence
    after exposure to different carcinogens. This probably represents a
    general effect on growth rather than a specific effect on
    carcinogenesis. Vitamin A deficiency did not influence the incidence
    of liver cancer in aflatoxin-treated rats but altered the effect of
    aflatoxin so that the incidence of colon cancers increased. In view
    of the importance of these results in relation to human health and
    the shortcomings in some of the experiments reported, further animal
    studies are necessary to quantify these effects. Future
    epidemiological studies should consider such interactions.

        Other questions raised by the data from animal studies but not
    demonstrated in the epidemiological studies, and thought by the Task
    Group to require further study before they can be applied to the
    human risk assessment, are the possible induction of extrahepatic
    cancers by aflatoxin (section 3.4.2.3), and the possibility that
    cancer could be induced by short-time exposure to high
    concentrations of aflatoxin.

        In spite of the existing gaps in knowledge, it should be
    recognized that in animal experiments there is "strong evidence"
    of carcinogenicitya for aflatoxins with established dose-response
    relationships, and that epidemiological studies in some parts of the
    world, where liver cancer is more frequent, have indicated a highly
    significant positive correlation between the crude incidence rate of
    liver cancer and the estimated current ingestion of aflatoxin in
    these areas.b The Task Group, therefore, concluded that aflatoxin
    ingestion may increase the risk of liver cancer, that the risk depends
    on the amount of aflatoxin ingested, and that reduction in daily
    aflatoxin exposure could be expected to reduce the liver cancer risk.

    3.6.3.2  Juvenile cirrhosis in India

        The Task Group concluded that the postulated involvement of
    aflatoxins in juvenile cirrhosis in India has not been substantiated
    (section 3.3.4.2 and Table 16 in section 3.5.1.2) and that it is
    unlikely in view of the epidemiological and morphological evidence
    available. Preliminary data suggesting the involvement of aflatoxins
    in the etiology of this disease were not supported by later
    measurements of aflatoxin exposure and examination of urine and
    liver specimens.

    3.6.4  Guidelines for health protection

        The effect of aflatoxins which is of greatest concern is the
    possible induction of liver cancer in man. Even if it is not
    possible, at present, to quantify individual risk corresponding to a
    given exposure to aflatoxins, it is nevertheless prudent to attempt
    to reduce exposure as much as is practically achievable. Reduction
    of food contamination by aflatoxins, sufficient to significantly
    reduce liver cancer risk, would of course significantly reduce the
    risk of acute toxic effects.

        Although there are several aspects of the relationship between
    aflatoxin exposure and carcinogenic risks in man that require
    elucidation by further experimental and epidemiological studies,
    there is, at present, sufficient evidence to justify the
    implementation or strengthening of national aflatoxin control
    programmes. It is impractical to insist that staple foodstuffs be
    aflatoxin-free, but the level of aflatoxin contamination should be
    reduced gradually by programmes involving the following components:
    education of farmers to improve crop quality and storage;
    surveillance of foodstuffs and animal feeds for the presence of
    aflatoxins; and application of appropriate food-processing
    technology to separate contaminated, from noncontaminated food
    elements. These and other measures have recently been discussed
    elsewhere (FAO, 1977).

                 

    a "Strong evidence" of carcinogenicity is considered to exist when
      a chemical has been shown unequivocally to produce malignant
      neoplasms. Chemicals for which the evidence of carcinogenicity is
      based solely on the appearance of such neoplastic lesions as lung
      adenomas or hepatomas in mice belong to the class of chemicals for
      which there is "weak evidence" of carcinogenicity (IARC, 1977).

    b Of course, this association between liver cancer incidence and
      aflatoxin does not necessarily mean that these two variables are
      causally related. However, the existing animal data tend to support
      a causal relationship, although there may be other factors that
      contribute to the development of liver cancer in these areas.

        Several countries have established tolerance levels for
    aflatoxins in specific food items (for review see Stoloff, 1977;
    Krogh, 1978). It should be clearly understood that these tolerance
    limits are only management tools intended to facilitate the
    implementation of aflatoxin control programmes, and that adherance
    to these tolerance limits does not provide an absolute protection
    against the increased liver cancer risk associated with aflatoxin
    exposure.

    4. OTHER MYCOTOXINS

    4.1  Ochratoxins

    4.1.1  Properties and analytical methods

    4.1.1.1  Chemical properties

    FIGURE 6

        The ochratoxins are a group of structurally-related compounds
    (Fig. 6), classified according to biosynthetic origin as
    pentaketides within the group polyketides (Turner, 1971). The first
    compound discovered, ochratoxin A, was isolated from a strain of
     Aspergillus ochraceus (van der Merwe et al., 1965). It is a
    colourless, crystalline compound, exhibiting blue fluorescence under
    UV-light. The sodium salt of ochratoxin A is soluble in water; as an
    acid, it is moderately soluble in polar organic solvents (e.g.,
    chloroform and methanol). Some of the chemical and physical
    properties of three ochratoxins are summarized in Table 17. Only
    ochratoxin A, and very rarely ochratoxin B, have been encountered as
    natural contaminants of foodstuffs, the remaining ochratoxins listed
    in Fig. 6 have been isolated only from fungal cultures, under
    laboratory conditions. On acid hydrolysis, ochratoxin A yields
    phenylalanine and an optically active lactone acid, ochratoxin
    alpha, a metabolite which has been found in the urine of test
    animals ingesting ochratoxin A-contaminated feed. This subject has
    been reviewed by Chu (1974) and Harwig (1974), and the
    spectroanalytical parameters have been reviewed by Neely & West
    (1972).


        Table 17. Chemical and physical properties of some ochratoxins
                                                                                                                                          

    Ochratoxin      Molecular       Relative           Melting              Absorption maxima (nm)              Reference
                    formula         molecular mass     point C             absorption coefficient (Epsilon)
                                                                                                                                          

    A               C20H18CINO6     403                169 (xylene)         213(36 800); 322(6400)              Steyn & Holzapfel
                                                       89-95 (benzene)                                          (1967)
    B               C20H19NO6       369                221                  218(37 200); 318(6900)              van der Merwe et al.
                                                                                                                (1965)
    Alpha           C11H9CIO5       256                229                  212(30 000); 338(5600)              van der Merwe et al.
                                                                                                                (1965)
                                                                                                                                          

    

    4.1.1.2  Methods for the analysis of foodstuffs

        Methods of analysing foodstuffs for ochratoxins have been
    reviewed by Nesheim (1976). The distribution of ochratoxins in
    commodities has not been studied in detail and no specific sampling
    plans have been developed. However, the general principles of
    sampling, outlined in section 3.1.2.1 for aflatoxins, are also
    applicable to ochratoxin.

        Several chemical methods have been developed, with limits of
    detection as low as 2 g/kg (Nesheim, 1976). Ochratoxin A in
    acidified commodities is readily soluble in many organic solvents,
    and this characteristic has been used as the principle of extraction
    in several methods. The most widely used method, for cereals in
    particular, includes extraction with chloroformaqueous phosphoric
    acid followed by cleanup on an aqueous bicarbonate-diatomaceous
    earth column, and quantitative determination using thin-layer
    chromatography (Nesheim et al., 1973). This procedure has been
    recommended by the International Union of Pure and Applied Chemistry
    (IUPAC) as an international method (IUPAC, 1976), and has a limit of
    detection of a few g/kg, when improved by ammoniation.

        Minicolumn methods for screening purposes have been developed
    (Hald & Krogh, 1975; Holaday, 1976), as well as a spectrophotometric
    procedure based on cleavage of ochratoxin A to form ochratoxin a and
    phenylalanine (Hult & Gatenbeck, 1976).

        A number of bioassays involving zebra fish larvae, brine
    shrimps, and bacteria have been developed, but none of the assays
    has been used routinely, so far (Harwig, 1974).

    4.1.2  Sources and occurrence

    4.1.2.1  Fungal formation

        Ochratoxin A was first obtained from A. ochraceus, but
    subsequent investigations have revealed that a variety of moulds
    included in the fungal genera Aspergillus and Penicillium are able
    to produce ochratoxins (Table 18). The main producers appear to be
    A. ochraceus and P. viridicatum. This subject has been reviewed by
    Krogh (1976a).

         Moisture content and temperature. In studies of ochratoxin A
    production by A. ochraceus, optimal production occurred between 20
    and 30 C (Schindler & Nesheim, 1970; Bacon et al., 1973). Maximum
    production was observed at 30 C and a water activity (%) of 0.953
    (39% of water content, % dry weight). At lower temperatures, such as
    15 C, the moisture requirement was higher (aw = 0.997, or 52%
    moisture) (Table 19).

        The genus  Penicillium includes psychrophilic species, and
    investigations of the influence of low incubation temperatures have
    revealed that strains of  P. viridicatum are able to produce
    ochratoxin A at 5-10 C (Harwig & Chen, 1974) (Table 19). This
    indicates that the heavy ochtratoxin contamination observed in
    countries with cold climates such as Canada and the Scandinavian
    countries is mainly produced by the Penicillia.

    Table 18. Ochratoxin-producing fungia
                                                                                   

        Penicillium Link
        Monoverticillata:
           P. frequentans series:         P. purpurrescens Sopp
        Asymmetrica-Lanata:
           P. commune series:             P. commune Thom
        Asymmetrica-Fasciculata:
           P. viridicatum series:         P. viridicatum Westling
           P. palitans Westling
           P. cyclopium series:           P. cyclopium Westling
        Biverticillata-Symmetrica:
           P. purpurogenum series:        P. variabile Sopp

        Aspergillus Micheli
        Aspergillus ochraceus group:
                                          A. sulphureus (Fres.) Thom and Church
                                          A. sclerotiorum Huber
                                          A. alliaceus Thom and Church
                                          A. melleus Yukawa
                                          A. ochraceus Wilhelm
                                          A. ostianus Wehmer
                                          A. petrakii Voros
                                                                                   

    a From: Krogh (1978).

    Table 19.  Production of ochratoxin A by  A. ochraceus and
                P. viridicatum at various aw and temperatures
                                                                     

    (a)  A. ochraceus (after 2 weeks of incubation)a

         aw               15 C                22 C           30 C
                                        mg ochratoxin A/kg
                                                                     

         0.852               0                    0             60
         0.901               0                   46            111
         0.953              36                  201            302
         0.997              81                  156            218
                                                                     
                                                                     

    (b)  P. viridicatum (after 3 weeks of incubation)b

         aw               5 C                 12 C           25 C
                                        mg ochratoxin A/kg

         0.85-0.86                               4000         11 000
         0.90-0.93                            160 000        280 000
         0.95-0.97        17 000
                          (after
                          5 weeks)
                                                                     

    a Adapted from: Bacon et al. (1973).
    b Adapted from: Harwig & Chen (1974).


    4.1.2.2  Occurrence in foodstuffs

         Plant products. The occurrence of ochratoxins in foodstuffs
    has been reviewed by Chu (1974a), Krogh (1976a, 1977a), and Stoloff
    (1976). Naturally occurring ochratoxin A was first reported at a
    concentration of 110-150 g/kg in one sample of maize included in a
    survey of 283 samples from commercial markets in the USA (Shotwell
    et al., 1969c). Data from subsequent surveys of plant products in
    various areas of the world are summarized in Table 20.

        Residues in food of animal origin. In 1971, a farm was traced
    where pigs had been fed ochratoxin A-contaminated feed. When the
    bacon pigs, some of which suffered from nephropathy, were delivered
    to the slaughterhouse, samples of kidney, liver, and adipose tissue
    were collected for analysis. Residues of ochratoxin A were detected
    in 18/19 investigated kidneys, at levels up to 67 g/kg (Hald &
    Krogh, 1972). Residues were also detected in 7/8 livers, and in all
    8 samples of adipose tissue analysed.


        Table 20. Natural occurrence of ochratoxin A in plant products
                                                                                                                                               

    Commodity                               Country     No. of    Percentage    Range of         Reference
                                                        samples   contaminated  contamination
                                                        analysed                (g/kg)
                                                                                                                                               

    wheat, oats, barley, rye (feed)         Canada        32         56.3         30-27 000c     Scott et al. (1972)
    barley, oats                            Denmark       33         57.6         28-27 500b,c   Krogh et al. (1973b)
    malt barley                             Denmark       50          6.0          9-189         Krogh (1978)
    maize                                   France       463          2.6         15-200         Galtier (1975)
    barley, wheat, oats, rye, maize (feed)  Poland       150          5.3         50-200         Juszkiewicz & Piskorska-Pliszczynska (1976)
    mixed feed                              Poland       203          4.9         10-50          Juszkiewicz & Piskorska-Pliszczynska (1977)
    barley, oats (feed)                     Sweden        84          8.3         16-409         Krogh et al. (1974)
    maize, wheat, barley                    Yugoslavia    47         12.8          5-90          Krogh et al. (1977)
    barley                                  USA          127         14.2         10-40          Nesheim (1971)
    coffee beans                            USA          267          7.1         20-360         Levi et al. (1974)
    maize                                   USA          283          0.4        110-150         Shotwell et al. (1969c)
    maize                                   USA          293          1.0         83-166a        Shotwell et al. (1971 )
    wheat (red winter)                      USA          291          1.0          5-115         Shotwell et al. (1976a)
    wheat (red spring)                      USA          286          2.8          5-115         Shotwell et al. (1976a)
                                                                                                                                               

    a Two of these samples also contained ochratoxin B.
    b Ochratoxin B, as well as ochratoxin A detected in 2 additional samples of barley.
    c Most of the samples containing high levels had undergone "heating".

    

    Table 21.  Correlation between feed level and tissue levels
               (residues) of ochratoxin A in pigsa
                                                                     

    Tissue        Regression equation        r
                                                                     

    kidney        y = 2.15 + 0.0123x         0.86
    liver         y = 0.35 + 0.0095x         0.82
    adipose       y = 2.51 + 0.0099x         0.78
                                                                     

    a Modified from: Krogh et al. (1974).
    x = ochratoxin A in feed (g/kg)
    y = ochratoxin A residue (g/kg tissue)
    r = correlation coefficient
    The regression is calculated on feed levels of ochratoxin A in
    the range of 200-4000 g/kg.


        Surveillance studies based on data from meat inspection in
    Denmark have revealed prevalence rates of porcine nephropathy
    ranging from 10-80 cases per 100 000 slaughtered pigs (Krogh,
    1976b). A survey of kidneys from pigs with the disease collected at
    various slaughterhouses, showed that 35% of the affected kidneys
    contained residues of ochratoxin A, ranging from 2-68 g/kg (Krogh,
    1977b). A similar survey of porcine nephropathy in Sweden revealed
    that 25% of the affected kidneys contained ochratoxin A at levels
    ranging from 2 to 104 g/kg (Rutqvist et al., 1977). The carry-over
    of ochratoxin A from feed to animal tissues has been elucidated in
    studies in which groups of pigs were exposed for 3-4 months to
    dietary levels of ochratoxin A of 200, 1000, and 4000 g/kg (Krogh
    et al., 1974). At termination (slaughter), the highest levels of
    ochratoxin A residues were found in the kidneys (mean level 50 g/kg
    at the 4000 g/kg feed level) with slightly lower levels in the
    liver, and even lower levels in muscle and adipose tissue. Other
    tissues were not analysed. There was a high correlation between the
    feed level of ochratoxin A and the residue levels in the 4 tissues
    investigated (Table 21). In another study on pigs (Krogh et al.,
    1976a), a high correlation ( r = 0.74-0.94) was found between
    ochratoxin A levels in the kidney and in other organs and tissues
    including the liver, muscle, and adipose tissue (Table 22 and
    section 4.1.3.3).

    Table 22.  Correlation between ochratoxin A mass concentration
               residues in the kidney and certain other tissuesa
                                                                     

    Tissue     Regression equation     r
                                                                     

    liver      y = --0.650 + 0.706x    0.937
    muscle     y = --0.603 + 0.438x    0.888
    adipose    y = --0.775 + 0.309x    0.739
                                                                     

    a From: Krogh et al. (1976a).
    x = ochratoxin A mass concentration (g/kg) in the kidney
    y = ochratoxin A mass concentration (g/kg) in the other tissues
    r = correlation coefficient

        Ochratoxin A levels of up to 29 g/kg were found in the muscle
    of hens and chickens collected in one slaughterhouse (Elling et al.,
    1975). The birds had been condemned because of nephropathy. In
    another study, groups of hens were exposed for 1-2 years to dietary
    levels of ochratoxin A of 0.3 and 1 mg/kg (Krogh et al., 1976c). The
    kidneys contained the highest residues, with a mean value of
    19 g/kg tissue in the group fed ochratoxin A at 1 mg/kg: the liver
    and muscle contained lower levels of ochratoxin A residues.
    Ochratoxins were not detected in the eggs.

    4.1.3  Metabolism

    4.1.3.1  Absorption

        In a study on rats exposed by gavage to a single dose of
    ochratoxin A at 10 mg/kg body weight, Galtier (1974b) found the
    highest tissue level of unchanged ochratoxin A in the stomach wall
    during the first 4 h following administration. The small and large
    intestine and caecum contained small amounts of unchanged ochratoxin
    A, and it was concluded that ochratoxin A was absorbed mainly in the
    stomach. In the caecum and the large intestine, small amounts (1-3%
    of the total dose), were detected as the isocoumarin moiety
    (ochratoxin a) most likely as the result of the hydrolysing action
    of the intestinal microflora (Galtier & Alvinerie, 1976; Hult et
    al., 1976).

        In  in vitro studies, Pitout (1969) showed that ochratoxin
    alpha could also be formed from the hydrolysis of ochratoxin A by
    carboxypeptidase A (EC 3.4.12.2) and alpha-chymotrypsin. No
    quantitative information is available on the rate of absorption of
    ochratoxin A and ochratoxin a from the gastrointestinal tract.

    4.1.3.2  Tissue distribution and metabolic conversion

        In slaughterhouse cases of mycotoxic porcine nephropathy studied
    by Hald & Krogh (1972), residues of unchanged ochratoxin A were
    found in all tissues investigated (kidney, liver and muscle), the
    highest levels (up to 67 g/kg) occurring in the kidney. In
    experimental studies on pigs ingesting feed containing ochratoxin A,
    residues of this toxin were found in all 4 tissues in the decreasing
    order of kidney, liver, muscle, adipose tissue (Krogh et al., 1974).
    When rats were exposed perorally to an ochratoxin A dose of 10 mg/kg
    body weight, Galtier (1974b) recovered 0.3% of the administered dose
    in the whole kidneys, 0.9% in the whole liver, and 0.6% in the total
    muscle tissue, 96 h after exposure. Chang & Chu (1977), using a
    single intraperitoneal injection of I mg ochratoxin A per rat
    (labelled with 14C in phenylalanine), found that the kidney
    contained twice as much unchanged ochratoxin A as the liver after
    0.5 h, amounting to 4-5% of the total dose.

        It has been shown by in vitro studies that ochratoxin A binds to
    serum albumin (Chu, 1971, 1974b); this binding has also been
    observed in  in vivo studies of rats (Galtier, 1974a; Chang & Chu,
    1977). Ochratoxin it has been detected in the urine and faeces of
    rats intraperitoneally injected with ochratoxin A (Nel & Purchase,
    1968; Chang & Chu, 1977), indicating the cleavage of ochratoxin A
    into ochratoxin alpha and phenylalanine under these conditions.
    Studies with 14C-labelled ochratoxin A indicated that some other,
    not yet identified, metabolities are formed in the body. Less than
    half of the radioactivity excreted in the urine within 24 h of a
    single intra-peritoneal injection of 14C-phenylalanine-labelled
    ochratoxin A was identified as ochratoxin A (Chang & Chu, 1977).

    4.1.3.3  Excretion

        Using 14C-labelled ochratoxin A in studies on rats, it has
    been demonstrated that this toxin is excreted primarily in the urine
    (Chang & Chu, 1977) although faecal excretion also occurs to some
    extent (Galtier, 1974b; Chang & Chu, 1977). Ochratoxin A has been
    detected in the urine of bacon pigs suffering from nephropathy
    (Krogh, personal communication).

        In a study of the disappearance rates for various tissues,
    female bacon pigs were fed ochratoxin A at a level of 1 mg/kg feed
    for 1 month and then kept on a toxin-free diet for another month
    during which animals were sacrificed at regular intervals (Krogh et
    al., 1976a). Ochratoxin A disappeared exponentially (Table 23) from

    the 4 tissues investigated (kidney, liver, muscle, and adipose
    tissue), with residual life values (RL50)a in the range of
    3.3-4.5 days; the toxin could still be detected in kidneys one month
    after termination of exposure. When the level in the kidney is
    known, the ochratoxin A residues in the 3 other tissues can be
    calculated (Table 22). No data are available on ochratoxin levels in
    human tissues, urine, or faeces.

    Table 23.  The rate of disappearance of ochratoxin A residues from pig
               tissues after termination of one month exposure to
               ochratoxin A at 1 mg/kg feeda
                                                                                   

    Tissue            Ochratoxin A (g/kg tissue)
                      at time t (days) after
                      termination of exposure
                                                                                   

    kidney              28.22 exp (--0.1522t)
    liver               19.49 exp (--0.1598t)
    muscle              12.94 exp (--0.2096t)
    adipose              4.62 exp (--0.0565t)
                                                                                   

    a From: Krogh et al. (1976a),


    4.1.4  Effects in animals

    4.1.4.1  Field observations

         Pigs. The effects of ochratoxins in animals have been reviewed
    by Krogh (1976a, 1978). Cases of mycotoxic porcine nephropathy, have
    been regularly encountered in studies in Denmark since the disease
    was first discovered 50 years ago (Larsen, 1928). The disease is
    endemic in all areas of the country, although unevenly distributed.
    Prevalence rates in 1971 varied from 0.6 to 65.9 cases per 10 000
    pigs and epidemics were encountered in 1963 and 1971, associated
    with a high moisture content in the grain caused by unusual climatic
    conditions (Krogh, 1976b). Extensive etiological studies have

                 

    a RL50 = half-time of residues calculated from the exponential
      equations shown in Table 23.

    revealed that ochratoxin A is a major disease determinant of porcine
    nephropathy, although other factors such as citrinin are also
    involved as causal determinants (for review see Krogh, 1976a).
    Analyses of kidneys from cases of porcine nephropathy collected at
    slaughterhouses have revealed that 35% of these kidneys contained
    ochratoxin A in concentrations ranging from 2-68 g/kg (Krogh,
    1977b). As this compound has not been detected in healthy kidneys,
    it is indicated that it may play a causal role in the disease.

        The morphological changes in the kidneys in cases of mycotoxic
    porcine nephropathy are characterized by degeneration of the
    proximal tubules, followed by atrophy of the tubular epithelium,
    interstitial fibrosis in the renal cortex, and hyalinization of some
    glomeruli (Elling & Moller, 1973). Although mycotoxic porcine
    nephropathy has only been reported from one other European country
    besides Scandinavia (Buckley, 1971), there are indications that this
    disease also occurs in other countries in Europe and North America.

         Poultry. In a preliminary study in Denmark of chickens and
    hens condemned by meat inspectors because of renal lesions, 29% of
    14 birds were suffering from nephopathy associated with ingestion of
    ochratoxin A (Elling et al., 1975). The morphological renal lesions
    were characterized by degeneration of proximal and distal tubules of
    both reptilian and mammalian nephrons, and interstitial fibrosis.

    4.1.4.2  Experimental studies

         Acute and chronic effects. The acute and chronic effects of
    ochratoxins in experimental animals have been reviewed by Chu
    (1974a), Harwig (1974), and Krogh (1976a). Different species vary in
    their susceptibility to acute poisoning by ochratoxin A, with LD50
    values ranging from 3.4 to 30.3 mg/kg (Table 24). When administered
    orally to rats, the female is more sensitive to ochratoxin A than
    the male. The kidney is the target organ, but changes in the liver
    have also been noted during studies of acute effects.

    Table 24.  Acute toxicity of ochratoxin A
                                                                                   

    Animal              LD50 mg/kg          Route of           Reference
                        body weight         administration
                                                                                   

    mouse (female)       22               intraperitoneal   Sansing et al. (1976)
    rat, male            30.3             peroral           Galtier et al. (1974)
    rat, female          21.4             peroral           Galtier et al. (1974)
    rat, male            12.6             intraperitoneal   Galtier et al. (1974)
    rat, female          14.3             intraperitoneal   Galtier et al. (1974)
    guineapig, male       9.1             peroral           Thacker (1976)
    guineapig, female     8.1             peroral           Thacker (1976)
    white leghorn         3.4             peroral           Prior et al. (1976)
    turkey                5.9             peroral           Prior et al. (1976)
    Japanese quail       16.5             peroral           Prior et al. (1976)
    rainbow trout         4.7             intraperitoneal   Doster et al. (1972)
    beagle dog, male    < 9 (total dose)  perorala          Szczech et al. (1973a)
    pig, female         < 6 (total dose)  peroralb          Szczech et al. (1973b)

                                                                                   

    a All 3 dogs, dosed daily with 3 mg/kg, died within 3 days.
    b Both pigs receiving 2 mg/kg daily were moribund and killed within 3 days, and
      both pigs receiving 1 mg/kg daily were moribund and killed within 6 days.

        The lesions observed in field cases of mycotoxic porcine
    nephropathy (section 4.1.4.1) have been reproduced by feeding diets
    containing levels of ochratoxin A identical to those encountered in
    naturally contaminated products (section 4.1.2.2). Thus 39 pigs fed
    rations containing ochratoxin A at levels ranging from
    200-4000 g/kg developed nephropathy after 4 months at all levels of
    exposure (Krogh et al., 1974). Changes in renal function were
    characterized by impairment of tubular function, indicated
    particularly by a decrease in TmPAH/CIna and reduced ability
    to produce concentrated urine. These functional changes corresponded
    well with the changes in renal structure observed at all exposure
    levels including atrophy of the proximal tubules, and interstitial
    cortical fibrosis. Sclerotized glomeruli were also observed in the
    group receiving the highest dose of ochratoxin A of 4000 g/kg feed.
    No other organ or tissue exhibited any changes.

                 

    a TmPAH = transport maximum for para-aminohippuric acid, CIn =
      clearance of inulin.

        Kidney damage, identical to the naturally occurring porcine
    nephropathy, was produced in another study by feeding pigs (9
    animals) with crystalline ochratoxin A in amounts corresponding to a
    feed level of 1 mg/kg for 3 months. Similar damage was not observed
    in 9 controls. Significant renal tubular impairment was detected
    after only 5 weeks of ochratoxin exposure (Krogh et al., 1976b).

        In pigs and dogs given high peroral doses, corresponding to feed
    levels of more than 5-10 mg/kg (levels rarely found in nature)
    extrarenal effects, in addition to renal lesions, were observed,
    involving the liver, intestine, spleen, lymphoid tissue, and
    leukocytes (Szczech et al., 1973a,b,c). Three groups of rats, each
    consisting of 15 animals were exposed to feed levels of ochratoxin A
    ranging from 0.2 to 5 mg/kg for 3 months. Renal damage in the form
    of tubular degeneration was observed at all dose levels (Munro et
    al., 1974).

        Avian nephropathy similar to spontaneously occurring cases
    (section 4.1.4.1) developed in chickens and hens exposed to dietary
    levels of 0.3 and 1 mg/kg for 1 year (Krogh et al., 1976c). The
    renal changes included degeneration of the tubular epithelium,
    mainly confined to the proximal and distal tubules of both reptilian
    and mammalian nephrons; impairment of glomerular and tubular
    function was also observed. Acute necrosis and "visceral gout" was
    observed in chickens exposed to high levels of ochratoxin A (LD50
    values) (Peckham et al., 1971). The same authors reported that
    ochratoxin B, the other naturally occurring ochratoxin was not
    highly toxic to chickens (LD50: 54 mg/kg); no toxic effects have
    been reported in other animals.

        The toxic effects of ochratoxin A on the renal epithelial cells
    of the monkey were demonstrated in  in vitro studies, in the form
    of abnormal mitotic cells (Steyn et al., 1975).

         Teratogenic effects. Intraperitoneal injection of pregnant
    mice with ochratoxin A at 5 mg/kg body weight on one of gestation
    days 7-12 resulted in increased prenatal mortality, decreased fetal
    weight, and various fetal malformations including exencephaly and
    anomalies of the eyes, face, digits, and tail (Hayes et al., 1974).
    When rats were treated perorally with ochratoxin A at 0.75 and
    1.0 mg/kg body weight on gestation days 6-15, fetuses taken on day
    20 showed decreased weight and various anomalies (e.g., open eyes,
    wavy ribs, and agenesis of vertebrae) (Brown et al., 1976). In
    hamsters injected intraperitoneally with ochratoxin A at doses of
    5-20 mg/kg body weight on one of gestation days 7-9, increased
    prenatal mortality and malformations were observed, including
    hydrocephalus, micrognathia, and heart defects (Hood et al., 1976).

         Mutagenicity. No data were available on the mutagenicity of
    ochratoxins.

         Carcinogenesis. There have not been any recent data that would
    change the conclusion that an evaluation of the carcinogenic risk of
    ochratoxins cannot be made because of the inadequacy of available
    studies in terms of the numbers of animals used and survival rates
    (IARC, 1976).

         Biochemical effects. Ochratoxin A affects the carbohydrate
    metabolism in rats. Thus, a single oral dose of ochratoxin A at
    15 mg/kg body weight caused a decrease in the glycogen level in the
    liver and an increase in the heart glycogen level 4 h later (Suzuki
    & Satoh, 1973). In a more extensive study on rats, the decrease in
    liver glycogen level, 4 h after a single oral dose of ochratoxin A
    at 15 mg/kg body weight, was associated with an increase in serum
    glucose levels and a decrease in liver glucose-6-phosphate (Suzuki
    et al., 1975). At the same time, the liver glycogen synthetase (EC
    2.7.1.37) activity decreased and the liver phosphorylase (EC
    2.4.1.1) activity increased. Three daily oral doses of ochratoxin A
    at 5 mg/kg body weight caused a decrease in liver glycogen
    concentration, measured on the fourth day. The decrease was
    attributed to inhibition of the active transport of glucose into the
    liver, suppression of glycogen synthesis from glucose, and
    acceleration of glycogen decomposition.

        During  in vitro studies of rat liver mitochondria, it was
    observed that ochratoxin A inhibited the respiration of whole
    mitochondria by acting as a competitive inhibitor of transport
    carrier proteins located in the inner mitochondrial membrane
    (Meisner & Chan, 1974). Further experiments with mitochondrial
    preparations revealed that the mitochondrial uptake of ochratoxin A
    was an energy-using process that resulted in depletion of
    intramitochondrial adenosine triphosphate (ATP), and that ochratoxin
    A inhibited intramitochondrial phosphate transport, resulting in
    deterioration of the mitochondria (Meisner, 1976). This might
    explain the degeneration of liver mitochondria observed by Purchase
    & Theron (1968) in rats exposed perorally to a single dose of
    ochratoxin A at 10 mg/kg body weight. These authors observed
    accumulation of glycogen in the cytoplasm of the rat liver cells
    microscopically. This was in contrast to the previously discussed
    observations of Suzuki et al. (1975) who found a decrease in
    glycogen levels.

        In a study on mice, Sansing et al. (1976) found that ochratoxin
    A, administered intraperitoneally at 6 mg/kg body weight, inhibited
    orotic acid incorporation into both liver and kidney RNA, 6 h after
    toxin injection. Ochratoxin A acted synergistically, in this
    respect, with another nephro-toxic mycotoxin, citrinin.

    4.1.5  Effects in man

    4.1.5.1  Ochratoxin A and Balkan nephropathy

        Balkan endemic nephropathy is a kidney disease only observed so
    far in rural populations in Bulgaria, Romania, and Yugoslavia. In
    the past 2 decades, etiological investigations covering bacteria,
    viruses, toxic metals, genetic factors, etc. have been conducted but
    with unconvincing results (reviewed Puchlev, 1973, 1974). Balkan
    endemic nephropathy is a chronic disease that is commonest between
    30 and 50 years of age and progresses slowly up to death. The
    kidneys are remarkably reduced in size. Histologically, the renal
    disease is characterized by tubular degeneration, interstitial
    fibrosis, and hyalization of glomeruli in the more superficial part
    of the cortex (Heptinstall, 1966). Impairment of tubular function,
    indicated by a decrease in TmPAH, is a prominent and early sign
    (Dotchev, 1973).

        The disease occurs endemically and affects females more often
    than males (Hrabar et al., 1976, Chernozemsky et al., 1977). In
    Bulgaria and Yugoslavia, a high incidence of urinary tract tumours
    has been found to be closely correlated with the incidence and
    mortality rates of Balkan endemic nephropathy (Ceovic et al., 1976;
    Chernozemsky et al., 1977).

        Fungal growth in foodstuffs and subsequent mycotoxin formation
    is influenced by the water content of the foodstuffs, and can be
    changed by climatic conditions such as heavy rainfalls during
    harvest. Thus, the observation (Austwick, 1975) of a positive
    correlation ( r = 0.80) between excess rainfall and the number of
    people who died of nephropathy during the succeeding 2 years in the
    Balkan peninsula might be interpreted as suggesting a fungal
    involvement in the etiology of endemic nephropathy.

        Attention has been called to the striking similarities in the
    changes of renal structure and function found in Balkan endemic
    nephropathy and in ochratoxin A-induced porcine nephropathy,
    suggesting common causal relationships (Krogh, 1974). Furthermore,
    epidemiological similarities have been noted, in particular, the
    endemic occurrence (Krogh, 1976b). Preliminary results of a survey
    of foodstuff's indicate that exposure to food-borne ochratoxin A
    seems to be higher (12.8% contamination) in an area of Yugoslavia
    with a high prevalence of human endemic nephropathy than in
    nonendemic (control) areas (1.6% contamination) (Krogh et al.,
    1977).

    4.1.6  Conclusions and evaluation of the health risks to man
           of ochratoxins

    4.1.6.1  Experimental animal studies

        The toxic effects of ochratoxin A have been studied extensively
    in a variety of experimental animals. All the animals studied so far
    have been susceptible to orally administered ochratoxin A, but to
    various degrees, as indicated by the range of LD50 values
    (Table 24). At high levels of ochratoxin A, changes were found in
    the kidneys and also in other organs and tissues. However, only
    renal lesions were observed at exposure levels identical to those
    occurring environmentally. The renal lesions included degeneration
    of the tubules, interstitial fibrosis, and, at later stages,
    hyalinization of glomeruli, with impairment of tubular function as a
    prime manifestation. Feed levels as low as 200 g/kg produced renal
    changes in the course of 3 months in rats and pigs. Field cases of
    ochratoxin A-induced nephropathy are regularly encountered in pigs
    and poultry. Ochratoxin A is teratogenic in the mouse, rat, and
    hamster.

        Ochratoxin B, rarely found as a natural contaminant, is much
    less toxic; the other ochratoxins have never been encountered in
    natural products.

    4.1.6.2  Studies in man

        The ochratoxin A-induced nephropathy in farm animals is similar
    to Balkan endemic nephropathy in several aspects. In a preliminary
    study in an area where Balkan endemic nephropathy is prevalent, the
    ochratoxin A contamination of food appeared to be more frequent than
    in control areas. However, the hypothesis that ochratoxin A may be a
    causal determinant in this disease, needs further support.

    4.1.6.3  Evaluation of health risks

        The nephrotoxic potential of ochratoxin A is well documented
    from all experimental studies, with a feed level of 200 g/kg
    causing nephropathy in pigs and rats. Lower levels have not been
    tested. Field eases of ochratoxin A-induced nephropathy in farm
    animals have long been recognized. The toxin has been found in a
    variety of foodstuffs, with levels in commodities used as feed
    ranging up to 27 mg/kg, and with levels in foodstuffs used for human
    consumption in the range of trace to about 100 g/kg. In one area
    where endemic nephropathy was prevalent in the human population,
    home produced foodstuffs were more frequently contaminated with
    ochratoxin A than those from control areas. However, the total
    intake of ochratoxin A by man has not been assessed so far, and
    there is, at present, no proof that ochratoxin A is causally
    involved in human diseases.

    4.2  Zearalenone

    4.2.1  Properties, analytical methods, and sources

        The properties, analytical methods, sources, and occurrence of
    zearalenone have been reviewed by Mirocha & Christensen (1974),
    Pathre & Mirocha (1976), and Mirocha et al. (1977). Zearalenone is a
    phenolic resorcylic acid lactone (Fig. 5), classified, according to
    biosynthetic origin, as a nonaketide within the group polyketides
    (Turner, 1971). Zearalenone (C18H22O5) is a white crystalline
    compound with a relative molecular mass of 318, melting point
    164-165 C, and absorption maxima (and absorption coefficient) at
    236 nm (29 700), 274 nm (13 909) and 316 nm (6020).

        Zearalenone exhibits blue-green fluorescence when excited by
    long wavelength (360 nm) UV-light, and a more intense green
    fluorescence when excited with short wavelength (260 nm) UV-light.

        A number of derivatives of zearalenone have been isolated from
    fungal cultures (Fig. 7), but none of these derivatives has been
    encountered, so far, as a natural contaminant of foodstuffs.

        A multiple detection method for aflatoxin, ochratoxin, and
    zearalenone has been developed (Eppley, 1968) and tested
    collaboratively (Shotwell et al., 1976b). The procedure consists of
    water-chloroform extraction combined with sequential elution of the
    mycotoxins from a silica-gel column and the detection limit is in
    the range of 50-100 g/kg. A versatile method of analysis for
    zearalenone has been described by Mirocha et al. (1974) using
    thin-layer chromatography (TLC), gas-liquid chromatography (GLC),
    gas-liquid chromatography-mass spectrometry, or a combination of all
    these methods; the limit of detection is about 50 g/kg. The
    derivatives dimethoxyzearalenone and methyl oxime-di-TMS-zearalenone
    are used to confirm the identity of zearalenone. Two methods using
    high pressure liquid chromatography (HPLC) are now available (Scott
    et al., 1978; Ware & Thorpe, 1978). With HPLC, a concentration of
    zearalenone in cornflakes of 5 g/kg could be determined (Scott et
    al., 1978). Zearalenone is produced by strains of Fusarium
    graminearum, F. tricinctum, F. oxysporum, F. sporotrichioides, and
    F. moniliforme, and a period of low temperature ( 12-14C) during
    fungal formation seems essential for high yield.

    FIGURE 7



    4.2.2  Occurrence

        The occurrence of zearalenone in foodstuffs has been reviewed by
    Stoloff (1976). Zearalenone has been encountered as a natural
    contaminant, particularly in maize, but occasionally in other
    cereals and in feedstuffs. In a survey of maize in the USA during
    the period 1968-69, zearalenone was found in 6 out of 576 (1%)
    samples at levels ranging from 450 to 800 g/kg (Shotwell et al.,
    1971). In 1972, when conditions in the USA were conducive to
    Fusarium ear rot, zearalenone was found in 17% of 223 samples of
    maize at levels ranging from 0.1 to 5.0 mg/kg (Eppley et al., 1974).
    It has also been detected in maize in France and in barley and mixed
    feed in England, Finland, and Yugoslavia (Stoloff, 1976).

        Nine out of 11 commercial corn meal samples in the USA contained
    zearalenone at levels ranging from 12 to 69 g/kg (Ware & Thorpe,
    1977). The compound has been found in one sample of cornflakes
    (13 g/kg) (Scott et al., 1978), and in maize beer, in Zambia, in
    the range of 0.01-4.6 mg/litre (Lovelace & Nyathi, 1977).

        Zearalenone was detected in 11% of 55 samples of Swazi sour
    drinks, sour porridges, and beers (range: 8-53 mg/kg) and in 12% of
    140 beer samples from Lesotho (range: 0.3-2 mg/kg), but such high
    figures have not been reported elsewhere. No data on consumption
    were given (Martin & Keen, 1978).

    4.2.3  Effects in animals

    4.2.3.1  Field observations

        The effects of zearalenone in animals have been reviewed by
    Mirocha & Christensen (1974). Field cases of the estrogenic syndrome
    in pigs, associated with the use of mouldy feed, were first observed
    half a century ago. The disease was characterized by enlarged
    oedematous vulvae and mammary glands (McNutt et al., 1928).
    Subsequently, this syndrome has been encountered in a number of
    countries in North America and Europe and in Australia (Table 25),
    and in most cases zemralenone has been identified in associated
    feeds, indicating together with the result of experimental studies
    (4.2.2.2) a causal role of this compound. Zearalenone feed levels
    reported to be associated with the syndrome in swine (Mirocha et
    al., 1977) are listed in Table 26. Cases of reduced fertility in
    cattle indicated by an increase in the artificial insemination index
    have been reported to be associated with a feed (hay) content of
    zearalenone of 14 mg/kg (Mirocha et al., 1968b). Fertility
    disturbances and prolonged heat in a herd of cattle suggested to be
    associated with zearalenone-contaminated feed were reported by Roine
    et al. (1971).


    Table 25.  Occurrence of the estrogenic syndrome in pigs in
               various countriesa
                                                                                   
    Year          Country           Feedstuff
                                                                                   

    1928          USA               maize
    1937          Australia         maize
    1952          Ireland           barley
    1962          France            maize
    1962          Italy             maize
    1963          Yugoslavia        maize and barley
    1967          Romania           maize
    1968          Hungary           maize
    1968          Denmark           barley
    1971          Canada            maize
                                                                                   

    a From: Mirocha & Christenson (1974).


    Table 26.  Natural occurrence of zearalenone in feeds associated with
               hyperestrogenism in swinea
                                                                                   

    Feed sample                           Level of
                                          contamination
                                          (mg/kg)
                                                                                   

    maize kernels (Minnesota)b            0.1-0.15
    dry sow ration (Vancouver)            0.15
    farrowing ration (Vancouver)c         0.066
    dry sow ration (Vancouver)            0.15
    corn kernels (Vancouver)              0.20
    dry sow ration (Vancouver)c           0.25
    lactation ration (Vancouver)          1.00
    gestation ration (Vancouver)          0.6
    milo (Minnesota)                      2.5-5.6
    sesame meal (Univ. of Minn.)d         1.5
    corn kernels (Ohio)                   0.12
    mixed feed corn (Ohio)b               0.12
    corn kernels (Minnesota)              6.4
    commercial pelletted mixed feed       6.8
    (Minnesota)
                                                                                   

    a From Mirocha et al. (1977).
    b Rectal prolapse in gilts.
    c Diethylstilbestrol was also present in these samples.
    d Associated with hyperestrogenism in turkey poults.

    4.2.3.2  Experimental studies

        Under experimental conditions, pigs (6-week-old-gilts) exposed
    perorally to zearalenone at 5 mg per animal per day for 5 days
    developed enlarged vulvae and mammae, and prolapse of the vagina
    within a few days; effects were reversible on termination of
    exposure. In another experiment, oral administration of 8 mg of pure
    crystalline zearalenone to 6-week-old, pre-pubertal gilts (8 doses
    of 1 mg/day per animal) induced pronounced tumefaction of the vulva
    (Mirocha & Christensen, 1974). Histological changes in the genital
    tract of pigs exposed to zearalenone included metaplasia of the
    epithelium in the cervix and vagina, and oedema in the wall of the
    uterus (Kurtz et al., 1969).

        Observations on pigs (Miller et al., 197.3) suggested that
    ingestion of grain contaminated with zearalenone during late
    gestation might be related to still-birth and splayleg. Splayleg was
    observed in the offspring of one sow and one gilt given dally
    intramuscular injections of zearalenone at 5 mg per animal
    throughout the last month of pregnancy. However, in the experiment
    of Patterson et al. (1977), all 7 gilts fed zearalenone at a level
    of 2 mg/kg feed throughout pregnancy remained clinically normal and
    embryonic survival rates were not affected by the toxin. Splayleg
    was diagnosed in only 1/63 piglets and the condition appeared to
    have resolved within 24 h. Results in the 2 groups of 3 pigs fed the
    lower levels were inconclusive (unpublished data, Chang & Kurtz,
    quoted by Mirocha et al., 1977).

        No effects on egg production were observed when laying hens were
    fed rations containing zearalenone levels of 250 and 500 mg/kg
    (Speers et al., 1971).

        In a 2-generation study on rats exposed to dietary levels of
    zearalenone corresponding to daily intakes of 0.1, 1.0, and 10 mg/kg
    body weight, no teratogenic effects were observed at any level of
    intake but impaired fertility and resorptions and stillbirths
    occurred in animals receiving 10 mg/kg body weight daily with 56% of
    dams showing complete litter resorption (Bailey et al., 1976).

        Ruddick et al. (1976) found fetal skeleton anomalies in rats
    exposed orally to zearalenone at doses ranging from 1-10 mg/kg body
    weight during the gestation period, with defect incidences of 12.8%
    (11/86) at the 1 mg/kg level, 26.1% (18/69) at the 5 mg/kg level,
    and 36.8% (28/76) at the 10 mg/kg level. No effect was observed with
    exposure to zearalenone at a dose of 0.075-0.30 mg/kg body weight.

        The results of a study by Mirocha et al. (1968a) in which the
    dose-effect relationships for zearalenone and estrone were compared
    with regard to increase in uterus weight, are given in Table 27. The
    results of a study comparing the effects of estrogens and
    zearalenone in 24 adult female castrated rhesus monkeys with a
    previous history of regular menstrual cycles are recorded in Table
    28.

    Table 27.  Effect of peroral dosing of zearalenone and estrone in micea
                                                                                   

    Material        Total dose   No. of   Mean uterine
    administered    (g)         mice     ratio  S.E.
                                                                                   

    control           0            10     0.76  0.06
    estrone           0.5          10     1.21  0.12
                      1             9     1.19  0.10
                      2             9     2.12  0.23
                      4            10     2.93  0.21
    zearalenone      12.5          10     1.11  0.04
                     25            10     1.48  0,18
                     50            10     1.80  0.12
                    100             9     2.35  0.17
                                                                                   

    a From: Mirocha et al. (1968a).

    4.2.4  Conclusions and evaluation of the health risks to man of
           zearalenone

    4.2.4.1  Animal studies

        Field cases of the estrogenic syndrome in pigs have been
    encountered in many countries in association with zearalenone in
    feeds at levels ranging from 0.1 to 6.8 mg/kg (Table 26), in samples
    not known to be contaminated with other estrogens. The condition has
    been reproduced experimentally in pigs, with a dally dose of
    1 mg/animal for 8 days. Infertility, sporadically encountered in
    cattle, has been suggested to be causally associated with
    zearalenone in feed at a reported level of 14 mg/kg of hay.

        In one of two studies on teratogenic effects in rats, skeletal
    defects were detected in fetuses at a daily oral dose of 1 mg/kg
    body weight.

    Table 28.  The estimated minimum dose of estrogens that will depress
               serum gonadotropin (FSH or LH) in castrated rhesus monkeysa
                                                                                   

    Estrogen administered   Estimated minimum dose
                            (g/kg)
                                                                           

                            Subcutaneousb      Oralc
                                                                           

                            LHd      FSHd      LH        FSH

    estradiol-17            4        1         5        --
    diethylstilboestrol      0.5      2         2.5      --
    zearalenone             14       56       400        --
                                                                                   

    a From: Hobson et al. (1977).
    b Two injections given in oil.
    c Given on 4 consecutive days,
    d FSH = follicle-stimulating hormone; LH = luteinizing hormone.


    4.2.4.2  Evaluation of health risks

        There are no reports on the adverse effects of zearalenone in
    man. With the exception of 2 reports from Africa, levels of
    zearalenone ranging from 12 to 69 g/kg have been found in a limited
    number of maize products destined for human consumption. Even
    assuming that 1 kg of these products is consumed dally, it can be
    estimated that a 70 kg man would not receive more than 1 g
    zearalenone per kg body weight in food. This level of exposure is
    400 times lower than the lowest peroral dose causing effects in
    tests on monkeys (Table 28) and more than 600 times lower than the
    lowest dose (g/kg) used in assessing the estrogenic potency of
    perorally administered zearalenone in mice (Table 27).

        However, in the 2 reports from certain parts of Africa, high
    levels of zearalenone were found in beer and sour porridge prepared
    from maize and sorghum. Long-term exposure to such contaminated
    drinks could represent a health hazard.

    4.3  Trichothecenes

    4.3.1  Properties and sources

        The properties and sources of trichothecenes have been reviewed
    by Bamburg & Strong (1971), Smalley & Strong (1974), and Bamburg
    (1976). The trichothecenes possess the tetracyclic
    12,13-epoxytrichothec-9-ene skeleton. More than 30 trichothecene

    derivatives have been isolated from fungal cultures, but, so far,
    only 4 have been identified as natural contaminants of foodstuffs
    (Table 29). The compounds have been detected by various methods
    including thin-layer chromatography, gas chromatography, and
    bioassays, in particular the rabbit skin test.

        One or more of the trichothecenes have been isolated from
    strains of the following Fusarium species:  F. episphae,
     F. lateritium, F. nivale, F. oxysporum, F. rigidisculum,
     F. solani, F. roseum, and F. tricinctum (syn.
     F. sporotrichioides). In addition, species of  Cephalosporium,
     Myrothecium, Trichoderma, and  Stachybotrys have been found to
    produce trichothecenes.

    Table 29.  Natural occurrence of trichothecenes
                                                                                   

    Compound            Concentration    Commodity      Reference
                        (g/kg)
                                                                                   

    T-2 toxin           2                maize          Hsu et al. (1972)
    T-2 toxin           25               barley         Puls & Greenway (1976)
    T-2 toxin           0.076            mixed feed     Mirocha et al. (1976)
    nivalenol           n.q.a            barley         Morooka et al. (1972)
    deoxynivalenol      7.3              barley         Morooka et al, (1972)
    deoxynivalenol      n.m.b            maize          Vesonder et al. (1973)
    deoxynivalenol      0.1, 1.0, 1.8    maize          Mirocha et al. (1976)
                                         (3 samples)
    deoxynivalenol      1.0, 1.0, 0.06   mixed feed     Mirocha et al. (1976)
                                         (3 samples)
    diacetoxyscirpenol  0.38, 0.5        mixed feed     Mirocha et al. (1976)
                                         (2 samples)
                                                                                   

    a  n.q. = not quoted
    b  n.m. = not measured

    4.3.2  Occurrence

        So far, the trichothecenes have only been found very
    sporadically in natural products. Naturally occurring trichothecenes
    include the following, based on chemical identification: T-2 toxin,
    nivalenol, deoxynivalenol, (vomitoxin), and diacetoxyscirpenol. In
    available studies, these 4 compounds have only been found in a total
    of 14 samples (Table 29), sometimes (as in the case of
    deoxynivalenol) concomitantly with zearalenone. No information is
    available concerning the number of samples analysed in these studies
    and therefore on the frequency of positive results.

    4.3.3  Effects in animals

    4.3.3.1  Field observations

        A field case involving the death of 20% of a dairy herd was
    suggested to be associated with ingestion of mouldy corn in the feed
    containing a concentration of T-2 toxin of approximately 2 mg/kg dry
    weight (Hsu et al., 1972). The lesions in the cattle included
    extensive haemorrhages on the serosal surface of all internal
    viscera.

        An outbreak of a disease, observed in poultry (ducks, geese),
    horses, and pigs, was suggested to be associated with mouldy barley
    containing T-2 toxin at approximately 25 mg/kg (Greenway & Puls,
    1976). The lesions in the geese included necrosis of the mucosa of
    the oesophagus, proventriculus, and gizzards.

        Deoxynivalenol (vomitoxin) was isolated from a batch of maize
    that had caused vomiting in pigs (Vesonder et al., 1973).

    4.3.3.2  Experimental studies

        Acute effects of trichothecenes determined as LD50 values, are
    listed in Table 30. Sato et al. (1975) studied tile effects in cats
    of T-2 toxin, administered in several ways. Two cats weighing 1.5
    and 4.0 kg were given purified T-2 toxin in single subcutaneous
    doses of 0.5 and 1.0 mg/kg, respectively. Nausea and vomiting
    appeared after 1 h and the animals died 20 h after the injection.
    The autopsy revealed extensive necrosis of the mucosa in the small
    and large intestine, marked karyorrhexis in the germ centre of the
    lymph follicles of the spleen and lymph nodes, and diffuse vacuolar
    degeneration of the renal tubules. Damage to bone marrow cells was
    observed in the cervical, thoracic, and lumbar vertebrae. In two
    cats subcutaneously injected with T-2 toxin (repeated doses of 0.1
    and 0.05 mg/kg body weight, given over a 4-week period) a decrease
    in the white blood cell (WBC) count was observed and the WBC value
    remained low until death occurred during the fourth week of
    exposure. The clinical signs in the cats were nausea and vomiting a
    few hours after each injection and ataxia of the hind legs. At the
    postmortem examination, hypoplasia was observed in the thymus,
    spleen, lymph nodes, and bone marrow. Other changes included marked
    meningeal haemorrhage, extensive bleeding in the lung, and vacuolar
    degeneration of the renal tubular epithelium. A marked decrease in
    the WBC values was also observed in 3 cats given T-2 toxin
    subcutaneously at a daily dose of 0.05 mg/kg for 12 days (i.e.,
    total dose of 0.60 mg/kg); 2 of these cats died, 4 and 35 days,
    respectively, after the last injection and one cat survived with the
    WBC count returning to the normal level 17 days after the last
    injection. Leukopenia and death were also observed in 3 cats (body

    weight 2-3 kg) receiving a crude preparation containing 4% T-2 toxin
    and 1% neosolaniol. This crude preparation was first administered
    subcutaneously once a week for 5 weeks in repeated doses of 1 mg/kg
    body weight (corresponding to a repeated dose of T-2 toxin of
    0.04 mg/kg). This subcutaneous administration was then followed by
    daily oral dosing (15 mg of the crude preparation corresponding to
    0.6 mg of T-2 toxin per animal per day) for 17 days.


    Table 30. Acute toxicity of naturally occurring trichothecenesa
                                                                                   

    Compound             LD50                 Route of          Animal
                         (mg/kg body weight)  administration
                                                                                   

    T-2 toxin              3.04               intraperitoneal   mouse
    T-2 toxin              3.8                peroral           rat
    T-2 toxin              5.1                peroral           trout
    T-2 toxin              5.25               peroral           one-day old chickb
    nivalenol              4.0                intraperitoneal   mouse
    diacetoxyscirpenol    10.0                intravenous       mouse
    diacetoxyscirpenol     0.75               intraperitoneal   rat
    diacetoxyscirpenol     7.3                peroral           rat
                                                                                   

    a From: Bamburg & Strong (1971 ).
    b From: Chi at al. (1977).


        More recently Yagen et al. (1978) have mentioned an experiment,
    in which oral administration of gelatin capsules containing purified
    T-2 toxin to 10 cats resulted in vomiting, leukopenia, haemorrhagic
    diathasis, neurological disturbances, and death. The dose and
    frequency of administration are not given in the paper.

        The effects observed in these experiments, in particular
    leukopenia, resemble those produced when cats are fed cultures of
     F. sporotrichioides, which is thought to be causally associated
    with alimentary toxic aleukia (ATA) in man (section 4.3.4).

        Feeding laying hens T-2 toxin at a dietary level of 20 mg/kg for
    3 weeks, resulted in oral necrotic lesions, decreased leukocyte
    count, and reduced egg production (Wyatt et al., 1975). In mice,
    intraperitoneal injection with T-2 toxin at doses of 1.0 and
    1.5 mg/kg body weight on one of the days 7-11 of gestation resulted
    in a number of maternal deaths as well as in an increase in prenatal
    mortality. Malformations of the fetuses were observed including tail
    and limb malformations, exencephaly, and retarded jaw development
    (Stanford et al., 1975).

        Daily oral doses of crude or purified T-2 toxin (0.2 mg/kg body
    weight) continued for 79 days failed to produce ill effects in
    calves, although the total amount of toxin ingested by one calf was
    almost 1.8 g (Matthews et al., 1977).

    4.3.4  Alimentary toxic aleukia

        Studies on alimentary toxic aleukia (ATA), a disease encountered
    in man in the period 1931-43, have been reviewed by Sarkisov (1954)
    and more recently by Bilai (1977) and Leonov (1977). The dominant
    pathological changes were necrotic lesions of the oral cavity, the
    oesophagus, and stomach, and in particular a pronounced leukopenia.
    The disease was lethal in a high proportion of cases. An association
    was established with ingestion of grain invaded by some moulds, in
    particular Fusarium poae and  F. sporotrichioides. Effects similar
    to ATA have been reproduced in cats by feeding cultures of these
    species (Bilai, 1977). T-2 toxin and other trichothecenes have been
    identified in a submerged culture of  F. sporotrichioides by
    Mirocha & Pathre (1973).

    4.3.5  Conclusions and evaluation of the health risks to man of
           trichothecenes

        In recent years, a group of mycotoxins, the trichothecenes, has
    been isolated under experimental conditions from many fungi,
    including  Fusarium species. Isolated field cases of intoxication
    in farm animals have been suggested to be related to some of the
    trichothecenes.

        About 40 years ago, a disease in man known as alimentary toxic
    aleukia (ATA), occurred that was suggested to be related to the
    presence of toxic  Fusarium species in mouldy over-wintered grain.
    With improved harvesting, food production, and storage conditions,
    the disease disappeared and no new outbreaks have occurred. However,
    present knowledge is not sufficient to establish a causal
    relationship between any of the isolated trichothecenes and this
    outbreak. There are no reports on the exposure of man to
    trichothecenes.

    REFERENCES

    ADAMSON, R. H., CORREA, P., & DALGARD, D. W. (1973) Brief
    communication: Occurrence of a primary liver carcinoma in a rhesus
    monkey fed aflatoxin B1.  J. Natl Cancer Inst., 50:549-551.

    ADAMSON, R. H., CORREA, P., SIEBER, S. M., MCINTIRE, K. R., &
    DALGARD, D. W. (1976) Carcinogenicity of aflatoxin B1 in rhesus
    monkeys: Two additional cases of primary liver cancer.  J. Natl
     Cancer Inst., 57: 67-78.

    ADENKUNLE, A. A. & ELEGBE, R. A. (1974.) Lysosomal activity in
    aflatoxin-B1-treated avian embryos.  Lancet, 1: 991.

    ALLCROFT, R. (1969) Aflatoxicosis in farm animals. In: Goldblatt,
    L.A., ed.  Aflatoxin scientific background, control and
     implications, New York, Academic Press, pp. 237-264.

    ALLCROFT, R. & LEWIS, G. (1963) Groundnut toxicity in cattle:
    Experimental poisoning of calves and a report on clinical effects in
    older cattle.  Vet. Rec., 75: 487-493.

    ALLCROFT, R. & ROBERTS, B. A. (1968) Toxic groundnut meal: The
    relationship between aflatoxin B1 intake by cows and excretion of
    aflatoxin M1 in milk.  Vet. Rec., 82: 116-118.

    ALLCROFT, R., ROBERTS, B. A., & LLOYD, M. K. (1968) Excretion of
    aflatoxin in a lactating cow.  Food Cosmet. Toxicol., 6: 619-625.

    ALPERT, g. E., HUTT, g. S. R., WOGAN, G. N., & DAVIDSON, C. S.
    (1971) Association between aflatoxin content of food and hepatoma
    frequency in Uganda.  Cancer, 28: 253-260.

    AMES, B. N., DURSTON, W. E., YAMASAKI, E., & LEE, F. D. (1973)
    Carcinogens are mutagens: A simple test system combining liver
    homogenates for activation and bacteria for detection.  Proc. Natl
     Acad. Sci. USA, 70: 2281-2285.

    AMLA, I., KAMALA, C. S., GOPALAKRISHNA, G. S., JAYARAJ, A. P.,
    SREENIVASAMURTHY, V., & PARPIA, H. A. B. (1971) Cirrhosis in
    children from peanut meal contaminated by aflatoxin.  Am. J. clin.
     Nutr., 24: 609-614.

    ANDERSON, H. W., NEHRING, E. W., & WICHSER, W. R. (1975) Aflatoxin
    contamination of corn in the field.  J. agric. food Chem., 23:
    775-782.

    ANDRELLOS, P. J. & REID, G. R. (1964) Confirmatory tests for
    aflatoxin B1. J.  Assoc. Off. Anal. Chem., 47: 801-803.

    AMBRECHT, B. H., SHALKOP, W. T., ROLLINS, L. D., POHLAND, A. E., &
    STOLOFF, L. (1970) Acute toxicity of aflatoxin B1 in wethers.
     Nature (Lond.), 225: 1062.

    ASAO, T., BUCHI, G., ABDEL-KADER, M., CHANG, S. B., WICK, E. L., &
    WOGAN, G. N. (1963) Aflatoxins B and G.  J. Am. Chem. Soc., 85:
    1706-1707.

    ASAO, T., BUCHI, G., ABDEL-KADER, M. M., CHANG, S. B., WICK, E. L.,
    & WOGAN, G. N. (1965) The structures of aflatoxins B and G1.  J.
    Am. Chem. Soc., 87: 882-886.

    ASHWORTH, L. J., JR, MCMEANS, J. L., HOUSTON, B. R., WITTEN, M. E.,
    & BROWN, C. M. (1971) Mycoflora, aflatoxins and free fatty acids in
    California cottonseed during 1967-1968.  J. Am. Oil Chem. Soc., 48:
    129-133.

    ASPLIN, F. D. & CARNAGHAN, R. B. A. (1961) The toxicity of certain
    groundnut meals for poultry with special reference to their effect
    on ducklings and chickens.  Vet. Rec., 73: 1215-1218.

    AUSTWICK, P. K. C. (1975) Balkan nephropathy.  Proc. R. Soc. Med.,
    68: 219-221.

    BABABUNMI, E. A. & BASSIR, O. (1972) Effects of aflatoxin B1 on
    the swelling and adenosine triphosphatase activities of mitochondria
    isolated from different tissues of the rat.  Febs Lett., 26:
    102-104.

    BACON, C. W., SWEENEY, J. G., ROBBINS, J. D., & BURDICK, D. (1973)
    Production of penicillic acid and ochratoxin A on poultry feed by
     Aspergillus ochraceus: Temperature and moisture requirements.
     Appl. Microbiol., 26: 155-160.

    BAGSHAWE, A. F., GACENGI, D. M., CAMERON, C. H., DORMAN, J., & DANE,
    D. S. (1975) Hepatitis Bs antigen and liver cancer in Kenya.
     Br. J. Cancer, 31: 581-584.

    BAILEY, D. E., Cox, G. E., MORGAREIDGE, K., & TAYLOR, J. (1976)
    Acute and subacute toxicity of zearalenone in the rat.  TAP, 37:
    144.

    BAMBURG, J. R. (1976) Chemical and biochemical studies of the
    trichothecene mycotoxins. In: Rodricks, J.V., ed.  Mycotoxins and
     other fungal related food problems, Washington, DC, American
    Chemical Society, pp. 144-162 (Advances in Chemistry Series 149).

    BAMBURG, J. R. & STRONG, F. M. (1971) 12,13-epoxytrichothecenes. In:
    Kadis, S., Ciegler, A., & Ajl, S. J., ed. Microbial toxins, New
    York, Academic Press, Vol. VII, pp. 207-289.

    BASSIR, O. & BABABUNMI, E. A. (1969) Inhibition of blood clotting
    factors by aflatoxin.  West Aft. J. biol. appl. Chem., 12: 28-32.

    BASSIR, O. & OSIYEMI, F. (1967) Biliary excretion of aflatoxin in
    the rat after a single dose.  Nature (Lond.), 215: 882.

    BECKWITH, A. C., VESONDER, R. F., & CIEGLER, A. (1975) Action of
    weak bases upon aflatoxin B1 in contact with macromolecular
    reactants.  J. agric. food Chem., 23: 582-587.

    BECROFT, D. M. O. (1966) Syndrome of encephalopathy and fatty
    degeneration of viscera in New Zealand children.  Br. med. J., 2:
    135-140.

    BECROFT, D. M. O. & WEBSTER, D. R. (1972) Aflatoxins and Reye's
    disease.  Br. med. J., 14 Oct., p. 117.

    BILAI, V. I. (1977) [Fuzarii.] Kiev, Naukova Dumka, pp. 360-394 (in
    Russian).

    BLACK, H. S., SMITH, J. D., AUSTIN, B. J., & RAUSCHKOLB, E. W.
    (1970) Effect of aflatoxin B1 on the  in vitro incorporation of
    14C-acetate into human skin lipids.  Experientia (Basel), 26:
    1292-1293.

    BOURGEOIS, C. H. (1975) Encephalopathy and fatty viscera: A possible
    response to acute aflatoxin poisoning. In: Pollack, J. D., ed.
     Reye's Syndrome, Grune & Stratton, New York, pp. 131-134.

    BOURGEOIS, C. H., SHANK, R. C., GROSSMAN, R. A., JOHNSEN, D. O.,
    WOODING, W. L., & CHANDAVIMOL, P. (1971) Acute aflatoxin B1
    toxicity in the macaque and its similarities to Reye's Syndrome.
    Lab.  Invest., 24: 206-216.

    BROWN, J. M. M. & ABRAMS, L. (1965)Biochemical studies on
    aflatoxicosis. Onderstepoort  J. vet. Res., 32: 119-146.

    BROWN, M. H., SZCZECH G. M., & PURMALIS, B. P. (1976) Teratogenic
    and toxic effects of ochratoxin A in rats. Toxicol. appl.
    Pharmacol., 37: 331-338.

    BUCARBAEVA, A. S. & NIKOV, P.S. (1977) [Aflatoxin sources in
    foodstuffs from different regions of Kazachstan.] In: [ Mycotoxins
     (sources, chemistry, biosynthesis, analysis and effects on the
     organism), Scientific Papers of a Regional Symposium, 28-30
     September 1977.] Orenburg, Nutrition Institute of the USSR Academy
    of Medical Sciences, pp. 29-31 (in Russian).

    BUCHI, G. & WEINREB, S. M. (1969) The total synthesis of racemic
    aflatoxin-M1 (milk toxin).  J. Am. Chem. Soc., 91: 5408-5409.

    BUCHI, G., SPITZNER, D., PAGLIALUNGA, S., & WOGAN, G. N. (1973)
    Synthesis and toxicity evaluation of aflatoxin P1.  Life Sci.,
    13: 1143-1149.

    BUCKLEY, H. G. (1971) Fungal nephrotoxicity in swine.  Irish
     vet. J., 25: 194-196.

    BULATAO-JAYME, J., ALMERO, E. M., & SALAMAT, L,. (1976) Epidemiology
    of primary liver cancer in the Philippines with special
    consideration of a possible aflatoxin factor.  J. Philipp. Med.
     Assoc., 52: 129-150.

    BUTLER, W. H. (1964) Acute toxicity of aflatoxin B1 in rats.
     Br. J. Cancer, 18: 756-762.

    BUTLER, W. H. (1966) Acute toxicity of aflatoxin B1 in guinea
    pigs.  J. Pathol. Bacteriol., 91: 277-280.

    BUTLER, W. H. (1969) Aflatoxicosis in laboratory animals. In:
    Goldblatt, L.A., ed.  Aflatoxin: Scientific background, control
     and implications, New York, Academic Press, pp. 223-236.

    BUTLER, W. H. (1971-1972) Further ultrastructural observations on
    injury of rat hepatic parenchymal cells induced by aflatoxin B1.
     Chem. biol. Interactions, 4: 49-65.

    BUTLER, W. H. (1974) Aflatoxin. In: Purchase, I. F. H., ed.
     Mycotoxins, Amsterdam, Elsevier, pp. 1-28.

    BUTLER, W. H. & BARNES, J. M. (1963) Toxic effects of groundnut meal
    containing aflatoxin to rats and guinea pigs.  Br. J. Cancer, 17:
    699-710.

    BUTLER, W. H. & BARNES, J. M. (1966) Carcinoma of the glandular
    stomach in rats given diets containing aflatoxin.  Nature (Lond.),
    209: 90.

    BUTLER, W. H. & WIGGLESWORTH, J. S. (1966) The effects of aflatoxin
    B1 on the pregnant rat.  Br. J. exp. Pathol., 47: 242-247.

    BUTLER, W. H., GREENBLATT, M., & LIJINSKY W. (1969) Carcinogenesis
    in rats by aflatoxins B1, G1, and B2. Cancer Res., 29:
    2206-2211.

    CAMPBELL, T. C. & SALAMAT, L. (1971) Aflatoxin ingestion and
    excretion by humans. In: Purchase, I. F. H., ed. Mycotoxins in human
    health, London, Macmillan Press, pp. 271-280.

    CAMPBELL, T. C. & STOLOFF, L. (1974) Implication of mycotoxins for
    human health. J. agric. food Chem., 22: 1006-1015.

    CAMPBELL, T. C., CAEDO, J.P., JR, BULATAO-JAYME, J., SALAMAT, L., &
    ENGEL, R. W. (1970) Aflatoxin M1 in human urine.  Nature (Lond.),
    227: 403-404.

    CARNAGHAN, R. B. A. (1965) Hepatic tumours in ducks fed on low level
    of toxic ground-nut meal.  Nature (Lond.), 208, 308.

    CARNAGHAN, R. B. A. (1967) Hepatic tumours and other chronic liver
    changes in rats following a single oral administration of aflatoxin.
    Br. J. Cancer, 21: 811-814.

    CARNAGHAN, R. B. A., HARTLEY, R. D., & O'KELLY, J. (1963) Toxicity
    and fluorescence properties of the aflatoxins.  Nature (Lond.),
    200: 1101.

    CARNAGHAN, R. B. A., LEWIS, G., PATTERSON, D. S. P., & ALLCROFT, R.
    (1966) Biochemical and pathological aspects of groundnut poisoning
    in chickens. Pathol. Vet., 3: 601-615.

    CEOVIC, S., GRIMS, P., & MITAR, J. (1976) [The incidence of tumours
    of the urinary organs in a region of endemic nephropathy and in a
    control region.] Lijee vjesn., 98: 301-304 (in Croatian).

    CHAINUVATI, T., VIRANUVATT, V., & PONGPIPAT, D. (1975) Relationship
    of hepatitis B antigen in cirrhosis and hepatoma in Thailand. An
    etiological significance.  Gastroenterology, 68: 1261-1264.

    CHANG, F. C. & Car, F. S. (1977) The fate of ochratoxin A in rats.
    Food Cosmet. Toxicol., 15: 199-204.

    CHANG, S. B., ABDEL-KADER, M. M., WICK, E. L., & WOGAN, G. N. (1963)
    Aflatoxin B2: Chemical identity and biological activity.
     Science, 142: 1191-1192.

    CHAVES-CARBALLO, E., ELLEFSON, R. D., & GOMEZ, M. R. (1976) An
    aflatoxin in the liver of a patient with Reye-Johnson syndrome.
     Mayo Clin. Proc., 51: 48-50.

    CHERNOZEMSKY, I. N., STOYANOV, I. S., PETKOVA-BOCHAROVA, T. K.,
    NICOLOV, I. G., DRAGANOV, I. V., STOICHEV, I. I., TANCHEV, Y.,
    NAIDENOV, D., & KALCHEVA, W. D. (1977) Geographic correlation
    between the occurrence of endemic nephropathy and urinary tract
    tumours in Vratza district, Bulgaria.  Int. J. Cancer, 19: 1-11.

    CHI, M. S., MIROCHA, C. J., KURTZ, M. Y., WEAVER, G., BATES, F., SHIMODA,
    W., & BURMEISTER, H. R. (1977) Acute toxicity of T-2 toxin in
    broiler chicks and laying hens.  Poult. Sci., 56: 103-116.

    CHU, F. S. (1971) Interaction of ochratoxin A with bovine serum
    albumin.  Arch. Biochem. Biophys., 147: 359-366.

    CHU, F. S. (1974a) Studies on ochratoxins.  CRC crit. Rev.
     Toxicol., 2: 499-524.

    CHU, F. S. (1974b) A comparative study of the interaction of
    ochratoxins with bovine serum albumin.  Biochem. Pharmacol.,
    23: 1105-1113.

    CIEGLER, A., KADIS, S., & AJL, S. J., ed. (1971)  Microbial toxins,
     fungal toxins, New York, Academic Press, Vol. VI, p. 563.

    CLEGG, F. G. & BRYSON, H. (1962) An outbreak of poisoning in store
    cattle attributed to Brazilian groundnut meal.  Vet. Rec., 37:
    992-994.

    CLIFFORD, J. I. & REES, K. R. (1966) Aflatoxin: A site of action in
    the rat liver cell.  Nature (Lond.), 209: 312-313.

    CLIFFORD, J. I. & REES, K. R. (1967) The action of aflatoxin B, on
    the rat liver.  Biochem. J., 102: 65-75.

    COON, F. B., BAUR, F. J., & SYMMES, L. R. L. (1972) International
    aflatoxin check sample program: 1971 study.  J. Assoc. Off. Anal.
     Chem., 55: 315-327.

    CUTHBERTSON, W. F. J., LAURSEN, A. C., & PRATT, D. A. H. (1967)
    Effect of groundnut meal containing aflatoxin on Cynomolgus monkeys.
     Br. J. Nutr., 21: 893-908.

    DALEZIOS, J. I. & WOGAN, G. N. (1972) Metabolism of aflatoxin B1
    in rhesus monkeys.  Cancer Res., 32: 2297-2303.

    DALEZIOS, J. I., WOGAN, G. N., & WEINREB, S. M. (1971) Aflatoxin
    P1: A new aflatoxin metabolite in monkeys.  Science, 171:
    584-585.

    DAVIS, N. D. & DIENER, U. L. (1970) Environmental factors affecting
    the production of aflatoxin. In: Herzberg, M., ed.  Proceedings of
     the First US-Japan Conference on "Toxic Microorganisms",
    Washington, DC, US Govt Printing Office, pp. 43-47.

    DEGER, G. E. (1976) Aflatoxin-Human colon carcinogenesis?  Ann.
     int. Med., 85: 204.

    DE IONGH, H., BEERTHUIS, R. K., VLES, R. O., BARRETT, C. B., & ORD,
    W. O. (1962) Investigation of the factor in groundnut meal
    responsible for turkey X disease.  Biochim. Biophys. Acta, 65:
    548-551.

    DEO, M. G., DAYAL, Y., & RAMALINGASWAMI, V (1970) Aflatoxins and
    liver injury in the rhesus monkey.  J. Pathol., 101 47-56.

    DETROY, R. W. & HESSELTINE, C. W. (1970) Aflatoxicol: Structure of a
    new transformation product of aflatoxin B1  Can. J. Biochem.,
    48: 830-832.

    DICKENS, J. W. & SATTERWHITE, J. B. (1973) Aflatoxin-contaminated
    peanuts produced on North Carolina farms in 1968.  J. Am. Peanut
     Res. Educ. Assoc. Inc., 5: 11 pp.

    DICKENS, F., JONES, H. E. H., & WAYNFORTH, H B. (1966) Oral
    subcutaneous and intra-tracheal administration of carcinogenic
    lactones and related substances: The intratracheal administration of
    cigarette tar in the rat.  Br. J. Cancer, 20: 134-144.

    DIENER, U. L. & DAVIS, N. D. (1969) Production of aflatoxins on
    peanuts under controlled environments.  J. stored Prod. Res.,
    5: 251-258;.

    DI PAOLO, J. A., ELIS, J., & ERWIN, H. (1967) Teratogenic response
    by hamsters, rats and mice to aflatoxin B1.  Nature (Lond.), 215:
    638-639.

    DOHERTY, W. P. & CAMPBELL, T. C. (1972) Inhibition of rat-liver
    mitochondria electron-transport flow by aflatoxin B1.  Res.
     Commun. chem. Path. Pharmacol., 3: 601-612.

    DOHERTY, W. P. & CAMPBELL, T. C. (1973) Aflatoxin inhibition of rat
    liver mitochondria.  Chem. biol. Interactions, 7: 63-77.

    DONALDSON, W. E., TUNG, H-T., & HAMILTON, P. B. (1972) Depression of
    fatty acid synthesis in chick liver  (Gallus domesticus) aflatoxin.
    Comp. Biochem. Physiol., 41: 843-847.

    DOSTER, R. C., SINNHUBER, R. O., & WALES, J. H. (1972) Acute
    intraperitoneal toxicity of ochratoxins A and B in rainbow trout
     (Salmo gairdneri). Food Cosmet. Toxicol, 10: 85-92.

    DOTCHEV, V. D. (1973) [The endemic (Balkan) nephropathy in
    Bulgaria.]  Muench. med. Woenschr., 115: 537-541 (in German).

    DVORACKOVA I. (1976) Aflatoxin inhalation and alveolar cell
    carcinoma.  Br. med. J., 20: 691.

    DVORACKOVA I., BRODSKY, F., & GERMAN, J. (1974) Aflatoxin and
    encephalitic syndrome with fatty degeneration of viscera.  Nutr.
     Rep. Int., 10: 89-102.

    DVORACKOVA I., KUSAK, V., & NESNIDAL, P. (1977) Aflatoxin and
    encephalopathy with fatty degeneration of viscera (Reye).  Ann.
     Nutr. Ailment., 31: 977-989.

    EDDS, G. T., NAIR, K. P. C., & SIMPSON, C. F. (1973) Effects of
    aflatoxin B1 on resistance in poultry against cecal coccidiosis
    and Marek's disease.  Am. J. vet. Res., 34: 819-826.

    ELIS, J. & DI PAOLO, J. A. (1967) Aflatoxin B1. Arch. Pathol., 83:
    53-57.

    ELLING, F. & MOLLER, T. (1973) Mycotoxic nephropathy in pigs.  Bull.
     Worm Health Organ., 49: 411-418.

    ELLING, F., HALO, B., JACOBSEN, CHR., & KROGH, P. (1975) Spontaneous
    toxic nephropathy in poultry associated with ochratoxin A.  Acta
     path. microbiol. Scand. Sect. A, 85: 739-741.

    EPPLEY, R. M. (1966) A versatile procedure for assay and preparatory
    separation of aflatoxins from peanut products.  J. Assoc. Off.
     Anal. Chem, 49: 1218-1223.

    EPPLEY, R. M. (1968) Screening method for zearalenone, aflatoxin,
    and ochratoxin.  J. Assoc. Off. Anal. Chem, 51: 74-78.

    EPPLEY, R. M., STOLOFF, L., TRUCKSESS, M. W., & CHUNG, C. W. (1974)
    Survey of corn for  Fusarium toxins.  J. Assoc. Off. Anal. Chem,
    57: 632-635.

    EPSTEIN, S. M., BARTUS, B., & FARBER, E. (1969) Renal epithelial
    neoplasms induced in male Wistar rats by oral aflatoxin B1. Cancer
    Res., 29: 1045-1050.

    FAO (1977) Mycotoxins.  Report of the joint FAO/WHO/UNEP Conference
     on Mycotoxins, Nairobi, 19-27 September, 1977, Rome, Food and
    Agriculture Organization of the United Nations, 105 pp. (FAO Food
    and Nutrition Paper 2).

    FDA (1977)  Report on action level for aflatoxin M1 in milk.
    Washington DC, 21 November, Bureau of Foods, US Food and Drug
    Administration.

    FDA (1978)  Assessment of estimated risk resulting from aflatoxins
     in consumer peanut products and other food commodities.
    Washington, DC, 19 January, Bureau of Foods, US Food and Drug
    Administration.

    FEUER, G, GOLBERG, L., & LEPELLEY, J. R. (1965) Liver response
    tests. I. Exploratory studies on glucose-6-phosphatase and other
    liver enzymes.  Food Cosmet. Toxicol., 3: 235-249.

    FRIEDMAN, L. & MOHR, H. (1968) Absence of interaction between
    cyclopropenoid fatty acids and aflatoxin in rats.  Fed. Proc., 27:
    551 (Abstract no. 1882).

    FRITZ, W., DONATH, R., & ENGST, R. (1977) [Determination and
    occurrence of aflatoxin M1 and B1 in milk and dairy produce.]
     Nahrung, 21: 79-84 (in German).

    GALTIER, P. (1974a) Devenir de l'ochratoxine A dans l'organisme
    animal. I. Transport san Ruin de la toxine chez le rat.  Ann. Rech.
    vet., 5: 311-318.

    GALTIER, P. (1974b) Devenir de l'ochratoxine A dans l'organisme
    animal II. Distribution tissulaire et limination chez le rat.  Ann.
     Rech. vet., 5: 319-328.

    GALTIER, P. (1975) Metabolism et mode d'action d'ochratoxine A. In:
    Biquet, J. & Jemmali, M., ed.  Les Mycotoxines, Paris, Editions
    INSERM, pp. 69-78.

    GALTIER, P. & ALVINERIE, M. (1976)  In vitro transformation of
    ochratoxin A by animal microbial floras.  Ann. Rech. vet., 7:
    91-98.

    GALTIER, P., MOR., J., & BODIN, G. (1974) Toxine d' Aspergillus
     ochraceus Wilhelm. III. Toxicit aigue de l'ochratoxine A chez le
    rat et la souris adultes.  Ann. Rech. vet., 5: 233-247.

    GARNER, R. C. (1973) Chemical evidence for the formation of a
    reactive aflatoxin B1 metabolite, by hamster liver microsomes.
     Febs Lett., 36: 261-264.

    GARNER, R. C. (1975) Reduction in binding of [14C]aflatoxin B1
    to rat liver macromolecules by phenobarbitone pretreatment.
     Biochem. Pharmacol., 24: 1553-1556.

    GARNER, R. C., MILLER, E. C., MILLER, J. A., GARNER, J. V., &
    HANSON, R. S. (1971) Formation of a factor lethal for S. typhimurium
    TA 1530 and TA 1531 on incubation of aflatoxin B1 with rat liver
    microsomes.  Biochem. biophys. res. Comm., 45: 774-780.

    GARNER, R. C., MILLER, E. C., & MILLER, J. A. (1972) Liver
    microsomal metabolism of aflatoxin B1 to a reactive derivative
    toxic to  Salmonella typhimurium TA 1530.  Cancer Res., 32:
    2058-2066.

    GODOY, H. M., JUDAH, D. J., ARORA, H. L., NEAL, G. E., & JONES, G.
    (1976) The effects of prolonged feeding with aflatoxin B1 on adult
    rat liver. Cancer Res., 36: 2399-2407.

    GOLDBLATT, L. E., ed. (1969) Aflatoxin; scientific background,
    control, and implications, New York, Academic Press, pp. 472.

    GOLDBLATT, L. A. & DOLLEAR, F. G. (1977) Detoxification of
    contaminated crops. In: Rodricks, J. V., Hesseltine, C. W., &
    Mehlman, M. A., ed. Mycotoxins in human and animal health, Park
    Forest South, IL, USA, Pathotox Publishers Inc., pp. 139-150.

    GOODAL, C. M. & BUTLER, W. H. (1969) Aflatoxin carcinogenesis:
    Inhibition of liver cancer induction in hypophysectomized rats.
     Int. J. Cancer, 4: 422-429.

    GOPALAN; C., TULPULE, P. G., & KRISHNAMURTHI, D. (1972) Induction of
    hepatic carcinoma with aflatoxin in the rhesus monkey.  Food
     Cosmet. Toxicol., 10: 519-521.

    GRANT, K. E., COMER, M. W., & NEWBERNE, P.M. (1977) Effect of
    dietary sodium selenite upon lesions induced by repeated small doses
    of aflatoxin B1.  Toxicol. appl. Pharmacol, 41: 166 (Abstract no.
    84).

    GREENWAY, J. A. & PULS, R. (1976) Fusariotoxicosis from barley in
    British Columbia. I. Natural occurrence and diagnosis.  Can. J.
     comp. Med., 40: 12-15.

    GRICE, M. C., MOODIE, C. A., & SMITH, B. C. (1973) The carcinogenic
    potential of aflatoxin or its metabolites in rats from dams fed
    aflatoxin pre- and postpartum.  Cancer Res., 33: 262-268.

    HABISH, H. A., ABDULLA, M. H., & BROADBENT, J. H. (1971) The
    incidence of aflatoxin in Sudanese groundnuts.  Trop. Sci., 8:
    279-287.

    HALD, B. & KROGH, P. (1972) Ochratoxin residues in bacon pigs. In:
    Abstracts,  IUPAC-Sponsored Symposium, Control of Mycotoxins,
     Kungalr, Goteborg, Sweden, 21-22 Aug., 1972, Goteborg, Sweden,
    Swedish Institute for Food Preservation Research, p. 18.

    HALD, B. & KROGH, P. (1975) Detection of ochratoxin A in barley,
    using silica gel mini-columns.  J. Assoc. Off. Anal. Chem.,
    58:156-158.

    HALVER, J. E. (1969) Aflatoxicosis and trout hepatoma. In:
    Goldblatt, L. A., ed.  Aflatoxin: Scientific background, control
     and implications, New York, Academic Press, pp. 265-306.

    HAMILTON, P. B. & HARRIS, J. R. (1971)Interaction of aflatoxicosis
    with Candida albicans infections and other stresses in chickens.
     Poult. Sci., 50: 906-912.

    HANSSEN, E. & JUNG, M. (1972) [On the presence of aflatoxin in
    non-mouldy food and proposals for sampling.]
     Z. Lebensmitt.-Unters.-Forsch., 150: 141-145 (in German).

    HARTLEY, R. D., NESBITT, B. F., & O'KELLY, J. (1963) Toxic
    metabolites of  Aspergillus flavus. Nature (Load.), 198: 1056-1058.

    HARWIG, J. (1974) Ochratoxin A and related metabolites. In:
    Purchase, I. F. H., ed.  Mycotoxins, Amsterdam, Elsevier, pp.
    345-367.

    HARWIG, J. & CHEN, Y.-K. (1974) Some conditions favoring production
    of ochratoxin A and citrinin by  Penicillium viridicatum in wheat
    and barley.  Can. J. Plant. Sci., 54: 17-22.

    HAYES, A. W., HOOD, R. D., & LEE, H. L. (1974) Teratogenic effects
    of ochratoxin A in mice.  Teratology, 9: 93-98.

    HEPTINSTALL, R. H. (1966)  Pathology of the kidney, London,
    Churchill, pp. 457-464.

    HESSELTINE, C. W. (1976) Conditions leading to mycotoxin
    contamination of foods and feeds. In: Rodricks, J. V., ed.
     Mycotoxins and other fungal related food problems, Washington, DC,
    American Chemical Society, pp. 1-22 (Advances in Chemistry Series
    149).

    HESSELTINE, C. W., SORENSEN, W. G., & SMITH, M. (1970) Taxonomic
    studies of the aflatoxin producing strains in the  Aspergillus
     flavus group.  Mycologia, 62: 123-132.

    HOBSON. W., BAILEY, J., & FULLER, G. B. (1970 Hormone effects of
    zearalenone in non-human primates. J. Toxicol. environ. Health, 3:
    43-57.

    HOEL, D. G., GAYLOR, D. W., KIRSCHSTEIN, R. L., SAFFIOTTI, U., &
    SCHNEIDERMAN, M. S. (1975) Estimation of risks of irreversible
    delayed toxicity. J. Toxicol. environ. Health, 1: 133-151.

    HOGAN, G. R., RYAN, N.J., & HAYES, A. W. (1978) Aflatoxin B1 and
    Reye's syndrome.  Lancet, March 11, p. 561.

    HOLADAY, C. E. (1976) A rapid screening method For the aflatoxins
    and ochratoxin A.  J. Am. Oil Chem. Soc., 53: 603-605.

    HORWITZ, W, SENZEL, A., REYNOLDS, H., & PARK, D. L., ed. (1975)
    Natural poisons. In:  Chapter 26, Official Methods of analysis of
     the Association of Official Analytical Chemists, Washington, DC,
    AOAC, p. 24.

    HRABAR, A., SUJAGA, K., BORCIC, B., ALERAJ, B., CEOVIC, S., &
    CVORISCEC, D. (1976) [Morbidity and mortality from endemic
    nephropathy in the village of Kaniza.]  Arh. hig. rada, 27: 137-145
    (in Serbo-Croat).

    HSIEH, D. P. H., WONG, Z. A, WONt, J. J., MICHAS, C., & RUEBNER, B.
    H. (1977) Comparative metabolism of aflatoxin. In: Rodricks, J. V.,
    Hesseltine, C. W., & Mehlman, M. A., ed. Mycotoxins in human and
    animal health, Park Forest South, IL, USA, Pathotox Publishers Inc.,
    pp. 37-50.

    HSU I-CH., SMALLEY, E. B., STRONG, F. M., & RIBELIN, W. E. (1972)
    Identification of T-2 toxin in moldy corn associated with a lethal
    toxicosis in dairy cattle. Appl. MicrobioL., 24: 684-690.

    HULT, K. & GATENECK, S. (1976) A spectrophotometric procedure using
    carboxy-peptidase A, for the quantitative measurement of ochratoxin
    A.  J. Assoc. Off. Anal. Chem., 59: 128-129.

    HULT, K., TEILING, A., & GATENBECK, S. (1976) Degradation of
    ochratoxin A by a ruminant.  Appl. environ. Microbiol., 32:
    443-444.

    IARC (1976) Aflatoxins. In:  IARC Monographs on the evaluation of
     carcinogenic risk of chemicals to man: Some naturally occurring
     substances. Lyons, International Agency for Research on Cancer,
    Vol. 10, pp. 51-72.

    IARC (1977) IARC Monograph Programme on the evaluation of the
    carcinogenic risk of chemicals to humans. Preamble. Lyons,
    International Agency for Research on Cancer, 31 pp. (IARC Internal
    Technical Report No. 77/002).

    IUPAC (1976) Recommended methods of ochratoxins A and B in barley,
    Oxford, International Union of Pure & Applied Chemistry Secretariat,
    pp. 1-7 (Technical Reports-No. 14).

    JEMMALI, M. & LAFONT, P. (1972) Evolution de l'aflatoxine B1 au
    tours de la panification.  Cah. Nutr. Dit., 7: 319-322.

    JEMMALI, M. & MURTHY, T. R. K. (1976) A chemical assay method for
    the determination of aflatoxin residues in animal tissues.
     Z. Lebensm.-Unters.-Forsch., 161: 13-17.

    JOHN, D. W. & MILLER, L. L. (1969) Effect of aflatoxin B1 on net
    synthesis of albumin, fibrinogen and alpha1-acid glycoprotein by
    the isolated perfused rat liver.  Biochem. Pharmacol., 18:
    1135-1146.

    JONES, B. D. (1972)  Methods of aflatoxin analysis. London,
    Tropical Products Institute, 58 pp. (Report G 70).

    JOSEPH-BRAVO, P. I., FINDLEY, M., & NEWBERNE P.M. (1976) Some
    interactions of light, riboflavin, and aflatoxin B1  in vivo and
     in vitro. J. Toxicol. environ. Health, 1: 353-376.

    JUNG, M. & HANSSEN, E. (1974) [The occurrence of aflatoxin M1 in
    dried milk products.]  Food Cosmet. Toxicol., 12: 131-138 (in
    German).

    JUSZKIEWICZ, T. & PISKORSKA-PLISZCZNSKA, J. (1976) [Occurrence of
    aflatoxin B1, B2, G1, and G2, ochratoxin A and B,
    sterigmatocystin and zearalenone in cereals.] Med. Weter., 32:
    617-619 (in Polish).

    JUSZKIEWICZ, T. & PISKORSKA-PLISZCZYNSKA, J. (1977) [Occurrence of
    mycotoxins in mixed feed and concentrates.] Med. Weter., 53: 193-196
    (in Polish).

    KATO, R., ONODA, K., & OMORI, Y. (1969) The effect of aflatoxin B1
    on incorporation of 14C-acetate into cholesterol by rat liver.
    Experientia (Basel), 25: 1026.

    KATO, R., TAKANAKA, A., ONODA, K., & OMORI, Y. (1970) Different
    effect of aflatoxin on the induction of tryptophan oxygenase and of
    microsomal drug-hydroxylase system.  J. Biochem. (Tokyo), 68:
    589-592.

    KEEN, P. & MARTIN, P. (1971a) The toxicity and fungal infestation of
    foodstuffs in Swaziland in relation to harvesting and storage.
     Trop. geogr. Med., 23-35-43.

    KEEN, P. & MARTIN, P. (1971b) Is aflatoxin carcinogenic in man? The
    evidence in Swaziland.  Trop. geogr. Med., 23: 44-53.

    KIERMEIER, F. ( 1973) Aflatoxin residues in fluid milk.  Pure appl.
     Chem., 35: 271-274.

    KIERMEIER, R. (1977) The significance of aflatoxins in the dairy
    Industry.  Annu. Bull. Int. Dairy Fed., 98: 1-23, 25-43.

    KIERMEIER, F., WEISS, G., BEHNRINGER, G., MILLER, M., & RANFFT, K.
    (1977) [On the presence and the content of aflatoxin M1 in milk
    shipped to a dairy plant.] Z. Lebensmitt.- Unters.-Forsch., 163:
    171-174 (in German).

    KOEHLER, B. (1938) Fungus growth in shelled corn as affected by
    moisture  J. agric. Res., 56: 291-307.

    KRISHNAMACHARI, K. A. V. R., BHAT, R. V., NAGARAJAN, V., & TILAK, T.
    B. G. (1975a) Investigations into an outbreak of hepatitis in parts
    of Western India.  Indian J. med. Res., 63: 1036-1048.

    KRISHNAMACHARI, K. A. V. R., BHAT, R. V., NAGARAJAN, V., & TILAK, T.
    B. G. (1975b) Hepatitis due to aflatoxicosis.  Lancet, 1: 1061.

    KROGH, P. (1974) Mycotoxic porcine nephropathy: A possible model for
    Balkan endemic nephropathy. In: Puchlev, A., ed.  Endemic
     Nephropathy. Proceedings of the Second International Symposium on
     Endemic Nephropathy, Sofia, November 9-11, 1972. Sofia, Bulgarian
    Academy of Sciences, pp. 266-270.

    KROGH, P. (1976a) Mycotoxic nephropathy. In:  Advances in
     veterinary science and comparative medicine, New York, Academic
    Press, Vol. 20, pp. 147-170.

    KROGH, P. (1976b) Epidemiology of mycotoxic porcine nephropathy.
    Nord. Vet.-Med., 28: 452-458.

    KROGH, P. (1977a) Ochratoxins. In: Rodricks, J. V., Hesseltine, C.
    W., & Mehlman, M. A., ed. Mycotoxins in human and animal health,
    Park Forest South, IL, USA, Pathotox Publishers, Inc., pp. 489-498.

    KROGH, P. (1977b) Ochratoxin A residues in tissues of slaughter pigs
    with nephropathy. Nord. Vet.-Med., 29: 402-405.

    KROGH, P. (1977c) Mycotoxin tolerances in foodstuffs.  Pure appl.
     Chem., 49: 1719-1721.

    KROGH, P. (1978) Causal associations of mycotoxic nephropathy.
     Acta path. microbiol. Scand. Sect. A, Suppl. no. 269.

    KROGH, P. & ELLING, F. (1977) Mycotoxic nephropathy.  Vet. Sci.
     Commun., 1: 51-63.

    KROGH, P. & HALD, B. (1969) [Occurrence of aflatoxin in imported
    groundnut products.]  Nord. Vet.-Med., 21: 398-407 (in Danish).

    KROGH, P., HALD, B., & KORPINEN, E.-L. (1970) [Occurrence of
    aflatoxin in groundnut- and copra-products imported to Finland.]
     Nord. Vet.-Med., 22: 584-589 (in Norwegian).

    KROGH, P., HALD, B., HASSELAGER, E., MADSEN, A., MORTENSEN, H. P.,
    LARSEN, A. E., & CAMPBELL, A.D. (1973a) Aflatoxin residues in bacon
    pigs.  Pure appl. Chem., 35: 275-282.

    KROGH, P., HALD, B., & PEDERSEN, E. J. (1973b) Occurrence of
    ochratoxin A and citrinin in cereals associated with mycotoxic
    porcine nephropathy.  Acta path. microbiol. Scand. Sect. B, 81:
    689-695.

    KROGH, P., AXELSEN, N.H., ELLING, F., GYRD-HANSEN, N., HALD, B.,
    HYLDGAARD-JENSEN, J., LARSEN, A. E., MADSEN, A., MORTENSEN, H. P.,
    MOLLER, T., PETERSEN, O. K., RAVNSKOV, U., ROSTGAARD, M., & AALUND,
    O. (1974) Experimental porcine nephropathy: Changes of renal
    function and structure induced by ochratoxin A- contaminated feed.
     Acta path. microbiol. Scand. Sect. A, Suppl. No. 246, 9 pp.

    KROGH, P., ELLING, F., BALD, B., LARSEN, A. E., LILLEHOJ, E. B.,
    MADSEN, A., & MORTENSEN, H. P. (1976a) Time-dependent disappearance
    of ochratoxin A residues in tissues of bacon pigs. Toxicology, 6:
    235-242.

    KROGH, P., ELLING, F., GYRD-HANSEN, H., HALD, B., LARSEN, A. E.,
    LILLEHOJ, E. B., MADSEN, A., MORTENSEN, H. P., & RAVNSKOV, U.
    (1976b) Experimental porcine nephropathy: Changes of renal function
    and structure perorally induced by crystalline ochratoxin A. Acta.
    pathol. microbiol. Scand. Sect. A, 84: 429-434.

    KROGH, P., ELLING, F., HALD, B., JYLLING, B., PETERSEN, V. E.,
    SKADHAUGE, E., & SVENDSEN, C. K. (1976c) Experimental avian
    nephropathy: Changes of renal function and structure induced by
    ochratoxin A-contaminated feed. Acta path. microbiol. Scand. Sect.
    4, 84: 215-221.

    KROGH, P., HALD, B., PLESTINA, R., & CEOVIC, S. (1977) Balkan
    (endemic) nephropathy and foodborne ochratoxin A: Preliminary
    results of a survey of foodstuffs.  Acta path. microbiol. Scand.
     Sect. B, 85. 238-240.

    KURTZ, M. J., NAIRN, M. E., NELSON, G. H., CHRISTENSEN, C. M., &
    MIROCHA, C. J. (1969) Histologic changes in the genital tracts of
    swine fed estrogenic mycotoxin.  Am. J. vet. Res., 30: 551-556.

    LARSEN, S. (1928-1929) [On chronic degeneration of the kidneys
    caused by mouldy rye.]  Maanedsskr. Dyrl., 40: 259-284 & 289-300
    (in Danish).

    LE BRETON, E., FRAYSSINET, C., LAFARGE, C., & DE RECONDO, A.M.
    (1964) Aflatoxine-mechanisme de l'action.  Food Cosmet. Toxicol.,
    2: 675-676.

    LEE, D. J., WALES, J. H., & SINNHUBER, R. O. (1969a) Hepatoma and
    renal tubule adenoma in rats fed aflatoxin and cyclopropenoid fatty
    acids.  J. Natl Cancer Inst., 43: 1037-1044.

    LEE, L. S., CUCULLU, A. F., FRANZ, A. O., JR, & PONS, W. A., JR
    (1969b) Destruction of aflatoxins in peanuts during dry and oil
    roasting. J. agric. food Chem., 17: 451-453.

    LEE, W. V. (1965) Quantitative determination of aflatoxin in
    groundnut products.  Analyst, 90: 305-307.

    LEONARD, A., DEKNUDT, GH., & LINDEN, G. (1975) Mutagenicity tests
    with aflatoxins in the mouse. Mutat. Res., 28: 137-139.

    LEONOV, A. N. (1977) Current view of the chemical nature of factors
    responsible for alimentary toxic aleukia. In: Rodricks, J. V.,
    Hesseltine, C. W., & Mehlman, M. A., ed.  Mycotoxins in human and
     animal health, Park Forest South, IL, USA, Pathotox Publishers
    Inc., pp. 323-328.

    LEVI, C. P., TRENK, H. L., & MOHR, H. K. (1974) A study of the
    occurrence of ochratoxin A in green coffee beans.  J. Assoc. Off.
     Anal. Chem., 57: 866-870.

    LIJINSKY, W. & BUTLER, W. H. (1966) Purification and toxicity of
    aflatoxin G1.  Proc. Soc. Exp. Biol. Med., 123: 151-154.

    LIJINSKY, W., LEE, K. Y., & GALLAGHER, C. H. (1970) Interaction of
    aflatoxin B1 and G1 with tissues of the rat.  Cancer Res., 30:
    2280-2283.

    LILLEHOJ, E. B., FENNELL, D. I., & KWOLEK, W. F. (1976)
     Aspergillus flavus and aflatoxin in Iowa corn before harvest.
     Science, 193: 195-496.

    LIN, J. J., LIU, C., & SVOBODA, D. J. (1974) Long-term effects of
    aflatoxin B1 and viral hepatitis on marmoset liver. Lab. Invest.,
    30: 267-278.

    LING, K. H., WANG, J. J., WU, R., TUNG, T. C., LIN, C. K., LIN,
    S.S., & LIN, T. M. (1967) Intoxication possibly caused by aflatoxin
    B1 in mouldy rice in Shuang-Chih township. J.  Formosan Med.
     Assoc., 66: 517-525.

    LINSELL, C. A. & PEERS, F. G. (1977) Field studies on liver cell
    cancer. In: Hiatt, H. H., Watson, J. D., & Wingsten, J. A., ed.
     Origins of human cancer. Cold Spring Harbor, NY, Cold Spring
    Harbor Laboratory, pp. 549-556.

    LO, W-B. & BLACK, H. S. (1972) Effects of aflatoxin B1 on human
    skin lipids metabolism-a site of action on 14C-acetate
    incorporation.  Experientia (Basel), 28: 1278-1279.

    LOOSMORE, R. M. & HARDING, J. D. J. (1961) A toxic factor in
    Brazilian groundnut causing liver damage in pigs. Vet. Rec., 73:
    1362-11364.

    LOOSMORE, R. M. & MARKSON, L. M. (1961) Poisoning of cattle by
    Brazilian groundnut meal.  Vet. Rec., 73: 813-814.

    LOVELACE, C. E. A. & NYATHI, C. B. (1977) Estimation of the fungal
    toxins, zearalenone and aflatoxin, contaminating opaque maize beer
    in Zambia.  J. Sci. Food Agric., 28: 288-292.

    LUCAS, F. V., JR., MONROE, P., NGA, P. V., TOWNSEND, J. F., & LUCAS,
    F. V. (1970-1971) Mycotoxin contamination of South Vietnamese rice.
     J. trop. Med. Hyg., 74: 182-183.

    LUCK. H., STEYN, M., & WEHNER, F. C. (1917) A survey of milk powder
    for aflatoxin content.  S. Afr. J. dairy Technol., 8: 85-86.

    LVOVA, L. S., SOSEDOV, N. I., GARALL, W., SCHWARZMAN, M. I.,
    SHATILOVA, T. I., & SHULGINA, A. P. (1976) [Formation of aflatoxins
    in wheat grain induced by self-heating and change in its chemical
    composition by the development of storage moulds.]  Prikl. Biochem.
     Mikrobiol., 12: 741-749 (in Russian).

    MADHAVAN, T. V. & GOPALAN, C. (1975) Effect of dietary protein on
    aflatoxin injury in weanling rats.  Arch. Pathol.,
    80: 123-126.

    MADHAVAN, T. V. & GOPALAN, C. (1968) The effect of dietary protein
    on carcinogenesis of aflatoxin.  Arch. Pathol., 85: 133-137.

    MADHAVAN, T. V., SURYANARAYANA RAO, K., The effect of dietary
    protein level on susceptibility of monkeys to aflatoxin liver
    injury. Indian J. med. Res., 53: 984-989.

    MADHAVAN, T. V., TULPULE, P. G, & GOPALAN, C. (1965b)
    Aflatoxin-induced hepatic fibrosis in rhesus monkeys.  Arch.
     Pathol., 79: 466-469.

    MALEKI, M. F., SUZANGAR, M., JAMSHIDI, CH., TAHMASEBI, A., &
    BARNETT, R. (1976) Urinary aflatoxin detected in cirrhotic patients
    in Ispahan,  Iran. Clin. Res., 24: 630A.

    MANTEL, N. & BRYAN, W. R. (1961) Safety testing of carcinogenic
    agents. J. Natl Cancer Inst., 27: 455-470.

    MANTEL, N., BOHIDAR, N., BROWN, C., CIMINERA, J., & TUKEY, J. (1975)
    An improved "Mantel-Bryan" procedure for "safety" testing of
    carcinogens.  Cancer Res., 35: 865-872.

    MARSH, P. B., SIMPSON, M. E., CRAIG, G. O., DONOSO, J., & RAMEY, H.
    H., JR (1973) Occurrence of aflatoxins in cotton seeds at harvest in
    relation to location of growth and field temperatures.  J. environ.
     Qual., 2: 276-281.

    MARTIN, P.M. D. & KEEN, P. (1978) The occurrence of zearalenone in
    raw and fermented products from Swaziland and Lesotho.  Sabrouaudia,
    16: 15,22.

    MASRI, M. S., LUNDIN, R. E., PAGE, J. R., & GARCIA, V. V. (1967)
    Crystalline aflatoxin M1 from urine and milk.  Nature (Lond.),
    215: 753-755.

    MASRI, M. S., HADDON, W. F., LUNDIN, R. E., & HSIEH, D. P. H.
    (1974a) Aflatoxin Q1: A newly identified major metabolite of
    aflatoxin B1 in monkey liver.  J. agric. food Chem., 22: 512-514.

    MASRI, M. S., BOOTH, A. N., & HSIEH, D. P. H. (1974b) Comparative
    metabolic conversion of aflatoxin B1 to aflatoxin M1 and
    aflatoxin Q1 by monkey, rat and chicken liver.  Life Sci., 15:
    203-212.

    MATTHEWS, J. G., PATTERSON, D. S. P., ROBERTS, B. A., & SHREEVE, B.
    J. (1977) T-2 toxin and haemorrhagic syndromes in cattle.  Vet.
     Rec., 101 (19): 391.

    MCLEAN, A. E. M. & MARSHALL, A. (1971) Reduced carcinogenic effects
    of aflatoxin in rats given phenobarbitone.  Br. d. exp. Pathol.,
    52: 322-329.

    MCNUTT, S. H., PURWIN, P., & MURRAY, C. (1928) Vulvovaginitis in
    swine  J. Am. Vet. Med. Assoc., 73: 484;-492.

    MEISNER, H. (1976) Energy-dependent uptake of ochratoxin A by
    mitochondria.  Arch. Biochem. Biophys., 173: 132-140.

    MEISNER, H. & CHAN, S. (1974) Ochratoxin A, an inhibitor of
    mitochondrial transport systems.  Biochemistry, 13: 2795-2800.

    MERWE, K. J. van der, STEYNE, P.S., FOURIE, L., SCOTT, DE B., &
    THERON, J. J. (1965) Ochratoxin A, a toxic metabolite produced by
     Aspergillus ochraceus Wilh.  Nature (Lond.), 205: 1112-1113.

    MILLER, J. K., HACKING, A., & GROSS, V. J. (I[973) Stillbirths,
    neonatal mortality and small litters in pigs associated with the
    ingestion of Fusarium toxin by pregnant sows. Vet. Rec., 93:
    555-559.

    MINTZLAFF, H.-J., LOTZSCH, R., TAUCHMANN, F., MEYER, W., & LEISTNER,
    L. (1974) [Aflatoxin residues in the liver and muscle of broiler
    chicken given aflatoxin containing feed.]  Fleischwirtschaft, 54:
    774-778 (in German).

    MIROCHA, C. J. & CHRISTENSEN, C. M. (1974) Oestrogenic mycotoxins
    synthesized by Fusarium. In: Purchase, I. F. H, ed.  Mycotoxins,
    Amsterdam, Elsevier, pp. 129-148.

    MIROCHA, C. J. & PATHRE, S. (1973) Identification of the toxic
    principle in a sample of poaefusarin.  Appl. Microbiol., 26:
    719-724.

    MIROCHA, C. J., CHRISTENSEN, C. M., & NELSON, G. H. (1968a) Toxic
    metabolites produced by fungi implicated in mycotoxicoses.
     Biotech. Bioeng., 10: 469-482.

    MIROCHA, C. J., HARRISON, J., NICHOLS, A. A.., & MCCLINTOCK, M.
    (1968b) Detection of a fungal estrogen (F-2) in hay associated with
    infertility in dairy cattle.  Appl. Microbiol., 16: 797-798.

    MIROCHA, C. J., SCHAUERHAMER, B., & PATHRE, S. V. (1974) Isolation,
    detection and quantitation of zearalenone in maize and barley.
     J. Assoc. Off. Anal. Chem., 75: 1104-1110.

    MIROCHA, C. J., PATHRE, S. V., SCHAUERHAMER, B., & CHRISTENSEN, C.
    M. (1976) Natural occurrence of  Fusarium toxins in feedstuff. Appl
    environ. Microbiol., 32: 553-556.

    MIROCHA, C. J., PATHRE, S. W., & CHRISTENSEN, C. M. (1977)
    Zearalenone. In: Rodricks, J. V., Hesseltine, C. W., & Mehlman, M.
    A., ed.  Mycotoxins in human and animal health, Park Forest South,
    IL, USA, Pathotox Publishers Inc., pp. 345-364.

    MOROOKA, N., URATSUJI, N., YOSHIZAWA, T., & YAMAMOTO, H. (1972)
    [Studies on the toxic substances in barley infected with  Fusarium
    spp.]  Jpn. J. food Hyg, 13: 368-375 (in Japanese).

    MUNRO, I. C, MOODIE, C. A, KUIPER-GOODMAN, T., SCOTT, P.M., & GRICE,
    H. C. (1974) Toxicologic changes in rats fed graded dietary levels
    of ochratoxin A.  Toxicol. appl. Pharmacol, 28: 180-188.

    NABNEY, J., BURBAGE, M. B., ALLCROFT, R., & LEWIS, G. (1967)
    Metabolism of aflatoxin in sheep: Excretion pattern in the lactating
    ewe.  Food Cosmet. Toxicol., 5: 11-17.

    NEAL, G. E. & GODOY, H. M. (1976) The effect of pre-treatment with
    phenobarbitone on the activation of aflatoxin B1 by rat liver.
     Chem. biol. Interactions, 14: 279-289.

    NEELY, W. C. & WEST, A.D. (1972) Spectroanalytical parameters of
    fungal metabolites. III. Ochratoxin A.  J. Assoc. Off. Anal. Chem.,
    55: 1305-1309.

    NEL, W. & PURCHASE, I. F. M. (1968) Note. The fate of ochratoxin A
    in rats.  J.S. Afr. Chem. Inst., 21: 87-88.

    NESHEIM, S. (1971) Report on ochratoxins: Occurrence, production,
    analysis and toxicity. In:  Abstracts of the 85th Annual meeting of
     the Association of Official Analytical Chemists, Washington, DC,
     Oct. 11-14, 1971, p. 23.

    NESHEIM, S. (1976) The ochratoxins and other related compounds. In:
    Rodricks, J. V., ed.  Mycotoxins and other fungal related food
     problems, Washington, DC, American Chemical Society, pp. 276-295
    (Advance.,; in Chemistry Series 149).

    NESHEIM, S., HARDIN, N. F., FRANCIS, O. J., JR, & LANGHAM, W. S.
    (1973) Analysis of ochratoxins A and B and their esters in barley,
    using partition and thin layer chromatography. I. Development of the
    method.  J. Assoc. Off. Anal. Chem., 56: 817-821.

    NEUMANN-KLEINPAUL, A. & TERPLAN, G. (1972) [The presence of
    aflatoxin M1 in dried milk products.]  Arch. Lebensmittelhyg.,
    23: 128-132 (in German).

    NEWBERNE, P.M. (1974) Mycotoxins: Toxicity, carcinogenicity and the
    influence of various nutritional conditions.  Environ. Health
     Perspect., 9: 1-32.

    NEWBERNE, P.M. (1976) Environmental modifiers of susceptibility to
    carcinogenesis.  Cancer Detect. Prev., 1: 129-173.

    NEWBERNE, P.M. & BUTLER, W. H. (1969) Acute and chronic effects of
    aflatoxin on the liver of domestic and laboratory animals: A review.
     Cancer Res., 29: 236-250.

    NEWBERNE, P.M. & CONNER, M. W. (1974) Effect of selenium on acute
    response to aflatoxin B1. In: Hemphill, D. D., ed.  A Symposium.
     Trace Substances in Environmental Health-VIII, Columbia, MO,
    University of Missouri, pp. 323-328.

    NEWBERNE, P.M. & GROSS, R. L. (1977) The role of nutrition in
    aflatoxin injury. In: Rodricks, J. V., Hesseltine, C. W, & Mehlman,
    M. A., ed.  Mycotoxins in human and animal health, Park Forest
    South, IL, USA, Pathotox Publishers Inc., pp. 51-65.

    NEWBERNE, P.M. & ROGERS, A. E. (1971) Aflatoxin carcinogenesis in
    rats: Dietary effects. In: Purchase, I. F. H., ed.  Mycotoxins in
     human health, London, Macmillan, pp. 195-208.

    NEWBERNE, P.M. & ROGERS, A. E. (1973) Rat colon carcinomas
    associated with aflatoxin and marginal vitamin  A. J. Natl Cancer
     Inst., 50: 439-448.

    NEWBERNE, P.M. & ROGERS, A. E. (1976) Nutritional modulation of
    carcinogenesis. In: Magee, P. N. et al., ed.  Fundamentals in
     cancer prevention, Tokyo, Univ. of Tokyo Press, & Baltimore, Univ.
    Park Press, pp. 15-40.

    NEWBERNE, P.M. & SUPHAKARN, V. (1977) Preventive role of vitamin A
    in colon carcinogenesis in rats.  Cancer, 40: 2553-2556.

    NEWBERNE, P.M. & WILLIAMS, G. (1969) Inhibition of aflatoxin
    carcinogenesis by diethyl-stilbestrol in male rats.  Arch. environ.
     Health, 19: 489-498.

    NEWBERNE, P.M. & WOGAN, G. N. (1968a) Sequential morphologic changes
    in aflatoxin B1 carcinogenesis in the rat.  Cancer Res., 28:
    770-781.

    NEWBERNE, P.M. & WOGAN, G. N. (1968b) Potentiating effects of
    low-protein diets on effect of aflatoxin in rats.  Toxicol. appl.
     Pharmacol., 12: 309-310.

    NEWBERNE, J. W., BAILEY, W. S., & SEIBOLD, H. R. (1955) Notes on a
    recent outbreak and experimental reproduction of hepatitis X in
    dogs.  J. Am. Vet. Med. Assoc., 127: 59-62.

    NEWBERNE, P.M., RUSSO, R., & WOGAN, G. N. (1966a) Acute toxicity of
    aflatoxin B1 in the dog.  Pathol. vet., 3: 331-340.

    NEWBERNE, P.M., HARRINGTON, D. H., & WOGAN, G. N. (1966b) Effects of
    cirrhosis and other liver insults on induction of liver tumours by
    aflatoxin.  Lab. Invest., 15: 962-969.

    NEWBERNE, P.M., ROGERS, A. E., & WOGAN, G. N. (1968) Hepatorenal
    lesions in rats fed a low lipotrope diet and exposed to aflatoxin.
     J. Nutr., 94: 331-343.

    NEWBERNE, P.M., CHAN, W-CH. M., & ROGERS, A. E. (1974) Influence of
    light, riboflavin, and carotene on the response of rats to the acute
    toxicity of aflatoxin and mono-crotaline. Toxicol. appl. Pharmacol.,
    28: 200-208.

    NILSSON, G. (1974) [Aflatoxin in nuts.]  Vor Foda, 26: 56-62 (in
    Swedish).

    NIXON, J. E., SINNHUBER, R. O., LEE, D. J., LANDERS, M. K., & HARR,
    J. R. (1974) Effect of cyclopropenoid compounds on the carcinogenic
    activity of diethylnitrosamine and aflatoxin B1 in rats.  J. Natl
     Cancer Inst., 53: 453-458.

    NORTHOLT, M.D., VERHULSDONK, C. A. H., SOENTORO, P.S. S., & PAULSCH,
    W. E. (1976) Effect of water activity and temperature on aflatoxin
    production by  Aspergillus parasiticus. J. milk food Technol., 39:
    170-174.

    OLSON, L. C., BOURGEOIS, C. H., JR, COTTON, R. B., HARIKUL, S.,
    GROSSMAN, R. A., & SMITH, T. J. (1971) Encephalopathy and fatty
    degeneration of the viscera in northeastern Thailand. Clinical
    syndrome and epidemiology.  Pediatrics, 47: 707-716.

    ONG, T-M. (1975) Aflatoxin mutagenesis.  Mutat. Res., 32: 35-53.

    PAG FAG/WHO/UNICEF PROTEIN ADVISORY GROUP (1969) PAG Recommendation
    on aflatoxin. PAG Statement No. 2. New York, United Nations, 2 pp.

    PANALAKS, T. & SCOTT, P.M. (1977) Sensitive silica gel-packed
    flowcell for fluorometric detection of aflatoxins by high pressure
    liquid chromatography.  J. Assoc. Off. Anal. Chem., 60: 583-589.

    PARKER, W. A. & MELNICK, D. (1966) Absence of aflatoxin from refined
    vegetable oils.  J. Am. Oil Chem. Soc., 43: 635-638.

    PATHRE, S. V. & MIROCHA, C. J. (1976) Zearalenone and related
    compounds. In: Rodricks, J. V., ed.  Mycotoxins and other fungal
     related food problems, Washington, DC, American Chemical Society
    pp. 178-227 (Advances in Chemistry Series 149).

    PATTERSON, D. S. P. (1973) Metabolism as a factor in determining the
    toxic action of the aflatoxin in different animal species.  Food
     Cosmet. Toxicol., 11: 287-294.

    PATTERSON, D. S. P. (1976) Structure, metabolism and toxicity of the
    aflatoxins: A review.  Cah. Nutr. Dit., 11 (Suppl. 2): 71-76.

    PATTERSON, D. S. P. (1977) Aflatoxin and related compounds. In:
    Wyllie, T.D. & Morehouse, L.G., ed.  Mycotoxic fungi, mycotoxins,
     mycotoxicoses: An encyclopedic handbook, New York & Basel, Marcel
    Dekker, pp. 131-136 & 144-189.

    PATTERSON, D. S. P. & ROBERTS, B. A. (1970) The formation of
    aflatoxin B2a and G2a and their degradation products during the
     in vitro detoxification of aflatoxin by livers of certain avian
    and mammalian species. Food Cosmet. Toxicol., 8: 527-538.

    PATTERSON, D. S. P. & ROBERTS, B. A. (1971) The  in vitro reduction
    of aflatoxins B1 and B2 by soluble avian liver enzymes. Food
    Cosmet. Toxicol., 9: 829-837.

    PATTERSON, D. S. P. & ROBERTS, B. A. (1972a) Steroid sex hormones as
    inhibitors of aflatoxin metabolism in liver homogenates.
     Experientia (Basel), 28: 929-930.

    PATTERSON, D. S. P. & ROBERTS, B. A. (1972b) Aflatoxin metabolism in
    duck-liver homogenates: The relative importance of reversible
    cyclopentenone reduction and hemiacetal formation.  Food Cosmet.
     Toxicol., 10: 501-512.

    PATTERSON, D. S. P., ROBERTS, B. A., SHREEVE, B. J., WRATHALL, A.
    E., & GITTER, M. (1977) Aflatoxin, ochratoxin and zearalenone in
    animal feedstuffs: Some clinical and experimental observations.  Ann
     Nutr. Aliment., 31: 643-650.

    PATTERSON, D. S. P., SHREEVE, B. J., & ROBERTS, B. A. (in press)
    Mycotoxin residues in body fluids and tissues of food-producing
    animals. In:  Proceedings of the 12th International Congress
     Microbiology, Munchen, Federal Republic of Germany, 3-8 September
     1978.

    PAUL, R., KALRA, M. S., & SINGH, A. (1976) Incidence of aflatoxins
    in milk and milk products.  Indian J. dairy Sol., 29: 318-322.

    PAYET, M., CROS, J., QUENUM, C., SANKALE, M., & MOULANIER, M. (1966)
    Deux observations d'enfants avant consomm de faon prolonge des
    farines souilles par "Aspergillus flavus".  Presse med.,
    13: 649-651.

    PECKHAM, J. C., DOUPNIK, B., JR, & JONES, O. H., JR (1971) Acute
    toxicity of ochratoxins A and B in chicks.  Appl. Microbiol., 21:
    492-494.

    PEERS, F. G. & LINSELL, C. A. (1973) Dietary aflatoxins and liver
    cancer--a population based study in Kenya.  Br. J. Cancer, 27:
    73-484.

    PEERS, F. G. & LINSELL, C. A. (1976) Acute toxicity of aflatoxin
    B1 for baboons.  Food Cosmet. Toxicol., 14: 227-228.

    PEERS, F. G. & LINSELL, C. A. (1977) Dietary aflatoxins and human
    primary liver cancer. Gaucher, A., ed.  Ann. Nutr. Aliment., 31:
    1005-1018.

    PEERS, F. G., GILMAN, G. A., & LINSELL, C. A. (1976) Dietary
    aflatoxins and human liver cancer. A study in Swaziland.  Int. J.
     Cancer, 17: 167-176.

    PETTIT, R. E., TABER, R. A., SCHROEDER, H. W., & HARRISON, A. L.
    (1971) Influence of fungicides and irrigation practice on aflatoxin
    in peanuts before digging.  Appl. Microbiol., 22: 629-634.

    PHILLIPS, D. L., YOURTEE, D. M., & SEARLES, S. (1976) Presence of
    aflatoxin B1 in human liver in the United States.  Toxicol. appl.
     Pharmacol., 36: 403-406.

    PIER, A. C. & HEDDLESTON, K. L. (1970) The effect of aflatoxin on
    immunity in turkeys. I. Impairment of actively acquired resistance
    to bacterial challenge.  Avian Dis., 14: 797-809.

    PIER, A. C., CYSEWSKI, S. J., RICHARD, J. L., & THURSTON, J. R.
    (1977) Mycotoxins as a veterinary problem. In: Rodricks, J. V.,
    Hesseltine, C. W., & Mehlman, M. A., ed.  Mycotoxins in human and
     animal health, Park Forest South, IL, USA, Pathotox Publishers
    Inc., pp. 745-750.

    PITOUT, M. J. (1969) The hydrolysis of ochratoxin A by some
    proteolytic enzymes.  Biochem. Pharmacol., 18: 485-491.

    POHLAND, A. E., YIN, L., & DANTZMAN, J. G. (1970) Rapid chemical
    confirmatory method for aflatoxin B1. I. Development of the
    method.  J. Asoc. Off. Anal. Chem., 53: 101-102.

    POKROVSKY, A. A., KRAVCENKO, L. V., & TUTELJAN, V. A. (1972) Effect
    of aflatoxin on rat liver lysosomes.  Toxicon, 10: 25-30.

    POKROVSKY, A. A., KRAVCENKO, L. V., & TUTELIAN, V. A. (1977)
    [Aflatoxins.] Toksikologija, Moskva, Viniti, Vol. 8, 108 pp. (in
    Russian).

    POLZHOFER, K. (1977) [Determination of aflatoxins in milk and milk
    products.]  Z. Lebensmitt.-Unters.-Forsch., 163: 175-177 (in
    German). 

    PONG, R. S. & WOGAN, G. N. (1969) Time course of alterations of rat
    liver polysome profiles induced by aflatoxin B1.  Biochem.
     Pharmacol., 18: 2357-2361.

    PONG, R. S. & WOGAN, G. N. (1970) Time course and dose-response
    characteristics of aflatoxin B1 effects on rat liver RNA
    polymerase and ultrastructure.  Cancer Res., 30: 294-304.

    PONG, R. S. & WOGAN, G. N. (1971) Toxicity and biochemical and fine
    structural effects of synthetic aflatoxins M1 and B1 in rat
    liver.  J. Natl Cancer Inst., 47: 585-592.

    PRINCE, A.M., SZMUNESS, W., MICHON, J, DEMAILLE, J., DIEBOLT, G.,
    LINHARD, J., QUENUM, C., & SANKALE, M. (1975) A case/control study
    of the association between primary liver cancer and hepatitis B
    infection in Senegal.  Int. J. Cancer, 16: 376-383.

    PRIOR, M. G., SISODIA, C. S., & O'NEIL, J. B. (1976) Acute oral
    ochratoxicosis in day-old white leghorns, turkeys and Japanese
    quail.  Poult. Sci., 55: 786-790.

    PRZYBYLSKI, W. (1975) Formation of aflatoxin derivatives on thin
    layer chromatographic plates.  J. Assoc. Off. Anal. Chem., 58:
    163-164.

    PUCHLEV, A. (1973) La nphropathie endmique en Bulgarie.  Extrait
     du Symposium sur la nphropathie endmique de l'Acadmie serbe des
     sciences et des arts, Belgrade, pp. 15-27.

    PUCHLEV, A. (1974) Basic problems in endemic nephropathy. In:
    Puchlev, A., ed.  Endemic Nephropathy, Proceedings of the Second
     International Symposium on Endemic Nephropathy, Sofia, 9-11
     November, 1972, Sofia, Publishing House of the Bulgarian Academy
    of Sciences, pp. 15-18.

    PULS, R. & GREENWAY, J. A. (1976) Fusariotoxicosis from barley in
    British Columbia. II. Analysis and toxicity of suspected barley.
     Can. J. comp. Med., 40: 16-19.

    PURCHASE, I. F. H. (1967) Acute toxicity of aflatoxins M1 and M2
    in one-day-old ducklings.  Food Cosmet. Toxicol., 5: 339-342.

    PURCHASE, I. F. H. (1972) Aflatoxin residues in food of animal
    origin.  Food Cosmet. Toxicol., 10: 531-544.

    PURCHASE, I. F. H. (1974) Mycotoxins, Amsterdam, Elsevier.

    PURCHASE, I. F. H. & THERUN, J. J. (1968) The acute toxicity of
    ochratoxin A to rats. Food Cosmet. Toxicol., 6: 479-483.

    PURCHASE, I. F. H. & VORSTER, L. J. (1968) Aflatoxin in commercial
    milk samples.  S. Afr. med. J., 42: 219.

    REDDY, G. S., TILAK, T. B. G., & KRISHNAMURTHI, D. (1973)
    Susceptibility of vitamin A- deficient rats to aflatoxin.  Food
     Cosmet. Toxicol., 11: 467-470.

    REDDY, J. K. & SVOBOOA, D. J. (1975) Aflatoxin B1-induced liver
    tumors in  Tupaia glis (tree shrews) a nonhuman primate.  Fed.
     Proc., 34: 827 (Abstract no. 3432).

    REDDY, J. K., SVOBODA, D. J., & RAO, M. S. (1976) Induction of liver
    tumors by aflatoxin B1 in the tree shrew  (Tupaia glis), a
    nonhuman primate.  Cancer Res., 36: 151-160.

    REYE, R. D. K., MORGAN, G., & BARAL, J. (1963) Encephalopathy and
    fatty degeneration of the viscera: A disease entity in childhood.
     Lancet, 2: 749-752.

    REYS, L. L. & SEQUEIRA, O. A. (1974) Detection of Australia antigen
    (HBAg) in blood donors and hepatoma patients in Mozambique.
     S. Aft. med. J., 48: 267-269.

    RICHIR, CL., PACCALIN, J., LARCEBEAU, S., FAUGERES, J., MORARD,
    J.-L., & LAMANT, M. (1976) Prsence d'aflatoxine B1 dans le toie
    humain.  Cah. Nutr. Diet., 11: 223-226.

    ROBERTS, J. C. (1974) Aflatoxins and sterigmatocystins.  Fortschr.
     Chem. Org. Naturst., 31: 119-151.

    ROBINSON, P. (1967) Infantile cirrhosis of the liver in India. With
    special reference to probable aflatoxin etiology.  Clin. Pediatr.,
    6: 57-62.

    RODRICKS, J. V. & STOLOFF, L. (1970) Determination of concentration
    and purity of aflatoxin standards.  J. Assoc. Off. Anal. Chem., 53:
    92-95.

    RODRICKS, J. V. & STOLOFF, L. (1977) Food producing animals. In:
    Rodricks, J. V., Hesseltine, C. W., & Mehlman, M. A., ed. Mycotoxins
    in human and animal health, Park Forest South, IL, USA, Pathotox
    Publishers Inc., pp. 67-79.

    RODRICKS, J. V., STOLOFF, L., PONS, W. A., JR,. ROBERTSON, J. A., &
    GOLDBLATT, L. A. (1970) Molar absorptivity values for aflatoxins and
    justification for their use as criteria of purity of analytical
    standards.  J. Assoc. Off. Anal. Chem., 53: 96-101.

    RODRICKS, J. V., HESSELTINE, C. W., & MEHLMAN, M. A., ed. (1977)
    Mycotoxins in human and animal health, Park Forest South, IL, USA,
    Pathotox Publishers Inc., 807 pp.

    ROGERS, A. E. (1975) Variable effects of a lipotrope-deficient, high
    fat diet on chemical carcinogenesis in rats.  Cancer Res., 35:
    2469-2474.

    ROGERS, A. E. & NEWBERNE, P.M. (1969) Aflatoxin B1 carcinogenesis
    in lipotrope-deficient rats.  Cancer Res., 29: 1965-1972.

    ROGERS, A. E. & NEWBERNE, P.M. (1971) Diet and aflatoxin B1
    toxicity in rats.  Toxicol. appl. Pharmacol., 20: 113-121.

    ROINE, K., KORPINEN, E.-L., & KALLELA, K. (1971) Mycotoxicosis as a
    probable cause of infertility in dairy cows.  Nord. Vet.-Med., 23:
    628-633.

    ROMER, T. R. (1975) Screening method for the detection of aflatoxins
    in mixed feeds and other agricultural commodities with subsequent
    confirmation and quantitative measurement of aflatoxins in positive
    samples.  J. Assoc. Off. Anal. Chem., 58: 500-506.

    RUDDICK, J. A., SCOTT, P. M., & HARWIG, J. (1976) Teratological
    evaluation of zearalenone administered orally to the rat.  Bull.
     environ. Contain. Toxicol., 15: 678-681.

    RUTQVlST, L., BJORKLUND, N.-E., HULT, K., & GATENBECK, S. (1977)
    Spontaneous occurrence of ochratoxin residues in kidneys of
    fattening pigs.  Zentralbl. Vet.-Med. A, 24: 402-408.

    SAFFIOTTI, U., MONTESANO, R., SELLAKUMAR, A. R., CEFIS, F., &
    KAUFMAN, D. G. (1972) Respiratory tract carcinogenesis in hamsters
    induced by different numbers of administrations of benzo (a) pyrene
    and ferric oxide.  Cancer Res., 32: 1073-1081.

    SALHAB, A. S. & EDWARDS, G. S. (1977) Comparative  in vitro
    metabolism of aflatoxicol by liver preparations from animals and
    humans.  Cancer Res., 37: 1016-1021.

    SALHAB, A. S. & HSIEH, D. P. H. (1975) Aflatoxicol H1: A major
    metabolite of aflatoxin B1 produced by human and rhesus monkey
    livers  in vitro. Res. Commun. chem. Pathol. Pharmacol.,
    10: 419-431.

    SANDERS, T. H, DAVIS, N. D., & DIENER, U. L. (1968) Effect of carbon
    dioxide, temperature, and relative humidity on production of
    aflatoxin in peanuts.  J. Am. Oil Chem. Soc., 45: 683-685.

    SANSING, G. A., LILLEHOJ, E. B., DETROY, R. W., & MILLER, M. A.
    (1976) Synergistic toxic effect of citrinin ochratoxin A and
    penicillic acid in mice.  Toxicon, 14: 213-220.

    SARASIN, A. & MOULE, Y. (1976) Helical polysomes induced by
    aflatoxin B1  in vivo. A new hypothesis for helix formation by
    chemicals and carcinogens.  Exp. cell Res., 97: 346-358.

    SARGEANT, K., O'KELLY, J., CARNAGHAN, R. B. A., & ALLCROFT, R.
    (1961) The assay of a toxic principle in certain groundnut meals.
     Vet. Rec., 73: 1219-1223.

    SARKISOV, A. C. (1954)  [Mycoses.] Moscow, State Publishing House
    for Agricultural Literature, 216 pp. (in Russian).

    SATO, S., MATSUSHIMA, T., TANAKA, N., SUGIMURA, T., & TAKASHIMA, F.
    (1973) Hepatic tumors in the guppy  (Lebistes reticulatus) induced
    by aflatoxin B1, dimethylnitrosamine, and 2-acetylaminofluorene.
     J. Natl Cancer Inst., 50: 765-778.

    SATO, N., UENO, Y., & ENOMOTO, M. (1975) Toxicological approaches to
    the toxic metabolites of Fusaria. VIII. Acute and sub-acute
    toxicities of T-2 toxins in cats.  Jpn. J. Pharmacol., 25: 263-270.

    SCHADE, J. E., MCGREEVY, K., KING, A.D., MACKEY, B., & FULLER, G.
    (1975) Incidence of aflatoxin in California almonds.  Appl.
     Microbiol., 29: 48-53.

    SCHINDLER, A. F. & NESHEIM, S. (1970) Effect of moisture and
    incubation time on ochratoxin A production by an isolate of
     Aspergillus ochraceus. J. Assoc. Off. Anal. Chem., 53: 89-91.

    SCHROEDER, H. W. & CARLTON, W. W. (1973) Accumulation of only
    aflatoxin B2 by a strain of  Aspergillus flavus. Appl.
     Microbiol., 25: 146-148.

    SCHULLER, P. L., OCKHUIZEN, TH., WERRINGLOER, J., & MARQUARDT, P.
    (1967) [Aflatoxin B1 and histamine in wine.]
     Arzneimitteforschung, 17: 888-890 (in German).

    SCHULLER, P. L., VERHOLSHONK, C. A. H., & PAULSCH, W. E. (1973)
    Analysis of aflatoxin M1 in liquid and powdered milk.  Pure appl.
     Chem., 35: 291-296.

    SCHULLER, P. L., VERHULSDONK, C. A. H, & PAULSCH, W. E. (1967)
    Aflatoxin M1 in liquid and powdered milk (evaluation of methods
    and collaborative study).  Zesz. Probl. Postepow Nauk Roln.,
    189: 255-261.

    SCOTT, P.M. & KENNEDY, B. P. C. (1973) Analysis and survey of ground
    black, white, and capsicum peppers for aflatoxins.  J. Assoc. Off.
     Anal. Chem., 56: 1452-1457.

    SCOTT, P.M., VAN WALBEEK, W., KENNEDY, B, & ANYETI, D. (1972)
    Mycotoxins (ochratoxin A, citrinin, and sterigmatocystin) and
    toxigenic fungi in grains and other agricultural products.  Agric.
     food Chem., 20: 1103-1109.

    SCOTT, P.M., PANALAKS, T., KANHERE, S., & MILES, W. F. (1978)
    Determination of zearalenone in cornflakes and other corn-based
    foods by thin layer chromatography, high pressure liquid
    chromatography, and gas-liquid chromatography/high resolution mass
    spectrometry.  J. Assoc. Off. Anal. Chem., 61: 593-600.

    SEEGERS, J. C. & PITOUT, M. J. (1973) DNA repair synthesis in cell
    cultures exposed to low doses of aflatoxin B1 or sterigmatocystin
     S. Aft. lab. clin. Med., 19: 961 (abstr. 23).

    SEIBOLD, H. R. & BAILEY, W. S. (1952) An epizootic of hepatitis in
    the dog.  J. Am. Vet. Med. Assoc., 121: 201-206.

    SEIBOLD, R. & RUCH, W. (1977) [Aflatoxin content of mixed feeds of
    dairy cows.]  Kraftfutter, 60: 182-185 (in German).

    SERCK-HANSSEN, A. (1970) Aflatoxin-induced fatal hepatitis? A case
    report from Uganda. Arch. environ. Health, 20: 729-731.

    SHANK, R. C. (1976) The role of aflatoxin in human disease. In:
    Rodricks, J. V., ed.  Mycotoxins and other fungal related food
     problems. Washington, DC, American Chemical Society, pp. 51-57
    (Advances in Chemistry Series 149).

    SHANK, R. C. (1977) Epidemiology of aflatoxin carcinogenesis. In:
    Kraybill, H. F. & Mehlman, M. M., ed.  Environmental cancer.
     Advances in modern toxicology, Vol. 3, John Wiley and Sons, New
    York, pp. 291-318.

    SHANK, R. C., BOURGEOIS, C. H., KESCHAMRAS, N., & CHANDAVIMOL, P.
    (1971a) Aflatoxins in autopsy specimens from Thai children with an
    acute disease of unknown actiology.  Food Cosmet. Toxicol., 9:
    501-507.

    SHANK, R. C., JOHNSEN, D. O., TANTICHAROENYOS, P., WOODING, W. L., &
    BOURGEOIS, C. H. (1971b) Acute toxicity of aflatoxin B, in the
    macaque monkey.  Toxicol. appl. Pharmacol., 20: 227-231.

    SHANK, R. C., WOGAN, G. N., GIBSON, J. B., & NONDASUTA, A. (1972a)
    Dietary aflatoxins and human liver cancer. II. Aflatoxins in market
    foods and foodstuffs of Thailand and Hong Kong.  Food Cosmet.
     Toxicol., 10: 61-69.

    SHANK, R. C., GORDON, J. E., WOGAN, G. N., NONDASUTA, A., &
    SUBHAMANI, B. (1972b) Dietary aflatoxins and human liver cancer.
    III. Field survey of rural Thai families for ingested aflatoxins.
     Food Cosmet. Toxicol., 10: 71-84.

    SHANK, R. C., BHAMARAPRAVATI, N., GORDON, J. E., & WOGAN, O. N.
    (1972c) Dietary aflatoxins and human liver cancer. IV. Incidence of
    primary cancer in two municipal populations of Thailand.  Food
     Cosmet. Toxicol, 10: 171-179.

    SHANKARAN, R., RAJ, H. G., & VENKITASUBRAMANIAN, T. A. (1970) Effect
    of aflatoxin on carbohydrate metabolism in chick liver.
     Enzymology, 39: 371-378.

    SHOTWELL, O. L., HESSELTINE, C. W., BURMEISTER, H. R., KWOLEK, W.
    F., SHANNON, G. M., & HALL, H. H. (1969a) Survey of cereal grains
    and soybeans for the presence of aflatoxin: II. Corn and soybeans.
     Cereal Chem., 46. 454-463.

    SHOTWELL, O. L., HESSELTINE, C. W., BURMEISTER, H. R., KWOLEK, W.
    F., SHANNON, G. M., & HALL, H. H. (1969b) Survey of cereal grains
    and soybeans for the presence of aflatoxin: I. Wheat, grain sorghum,
    and oats.  Cereal Chem., 46: 446-454.

    SHOTWELL, O. L., HESSELTINE, C. W., & GOULDEN, M. L. (1969)
    Ochratoxin A: Occurrence as natural contaminant of a corn sample.
     Appl. Microbiol., 17: 765-766.

    SHOTWELL, O. L., HESSELTINE, C. W., GOULDEN, M. L., & VANDEGRAFT, E.
    E. (1970) Survey of corn for aflatoxin, zearalenone and ochratoxin.
     Cereal Chem., 47: 700-707.

    SHOTWELL, O. L., HESSELTINE, C. W., VANDEGRAFT, E. E., & GOULDEN, M.
    L. (1971) Survey of corn from different regions for aflatoxin,
    ochratoxin, and zearalenone.  Cereal Sci. Today, 16: 266-273.

    SHOTWELL, O. L., HESSELTINE, C. W., & GOULDEN, M. L. (1973)
    Incidence of aflatoxin in southern corn, 1969-1970.  Cereal Sci.
     Today, 18, 192-195.

    SHOTWELL, O. L., GOULDEN, M. L., & HESSELTINE, C. W. (1976a) Survey
    of US wheat for ochratoxin and aflatoxin.  J. Assoc. Off. Anal.
     Chem., 59: 122-124.

    SHOTWELL, O. L., GOULDEN, M. L., & BENNETT, G. A. (1976b)
    Determination of zearalenone in corn: Collaborative study.
     J. Assoc. Off. Anal. Chem., 59: 666-670.

    SINNHUBER, R. O. & WALES, J. H. (1974) Aflatoxin B1
    hepatocarcinogenicity in rainbow trout embryos.  Fed. Proc., 33:
    247 (Abstract no. 253).

    SINNHUBER, R. O., LEE, D. J., WALES, J. H., & AYRES, J. L. (1968a)
    Dietary factors and hepatoma in rainbow trout  (Salmo gairdneri).
    II. Cocarcinogenesis by cyclopropenoid fatty acids and the effect of
    gossypol and altered lipids on aflatoxin-induced liver cancer.
     J. Natl Cancer Inst., 41: 1293-1301.

    SINNHUBER, R. O., WALES, J. H., AYRES, J. L., ENGEBRECHT, R. H., &
    AMEND, D. L. (1968b) Dietary factors and hepatoma in rainbow trout
     (Salmo gairdneri) I. Aflatoxins in vegetable protein feedstuffs.
     J. Natl Cancer Inst., 41: 711-718.

    SINNHUBER, R. O., LEE, D. J., WALES, J. H., LANDERS, M. K., KEYL, A.
    C. (1974) Hepatic carcinogenesis of aflatoxin M1 in rainbow trout
     (Salmo gairdneri) and its enchancement by cyclopropene fatty
    acids.  J. Natl Cancer Inst., 53: 1285-1288.

    SMALLEY, E. B. & STRONG, F. M. (1974) Toxic trichothecenes. In: 
    Purchase, I. F. H., ed.  Mycotoxins, Amsterdam, Elsevier, pp.
    199-228.

    SMITH, J. W., PRINCE, W. R., & HAMILTON, P. B. (1969) Relationship
    of aflatoxicosis to  Salmonella gallinarum infections of chickens.
     Appl. Microbiol., 18: 946-947.

    SPEERS, G. M., MERONUCK, R. A., BARNES, D. M., & MIROCHA, C. J.
    (1971) Effect of feeding  Fusarium roseum f. sp. graminearum
    contaminated corn and the mycotoxin F-2 on the growing chick and
    laying hen.  Poult. Sci., 50: 627-633.

    STACK, M. E. (1974) Collaborative study of AOAC methods I and III
    for the determination of aflatoxins in peanut butter.  J. Assoc.
     Off. Anal. Chem., 57: 871-874.

    STANFORD, G. K., HOOD, R. D., & HAVES, A. W. (1975) Effect of
    prenatal administration of T-2 toxin to mice.  Res. Commun. chem.
     Path. Pharmacol., 10: 743-746.

    STEPHENSON, L. W. & RUSSELL, T. E. (1974) The association of
     Aspergillus flavus with hemipterous and other insects infesting
    cotton bracts and foliage.  Phytopathology, 64: 1502-1506.

    STERN, P.S., & HOLZAPFEL, C. W. (1967) The synthesis of ochratoxins
    A and B, metabolites of  Aspergillus ochraceus Wilh.  Tetrahedron
     Lett., 23: 4449-4461.

    STEYN, P.S., VLEGGAAR, R., Du PREEZ, N. P., BLYTH, A. A., & SEEGERS,
    J. C. (1975) The  in vitro toxicity of analogs of ochratoxin A in
    monkey kidney epithelial cells.  Toxicol. appl. Pharmacol., 32:
    198-203.

    STICH, H. F. & LAISHES, B. A. (1975) The response of  Xeroderma
     pigmentosum cells and controls to the activated mycotoxins,
    aflatoxins and sterigmatocystin.  Int. J. Cancer, 16: 266-274.

    STOLOFF, L. (1972) Analytical methods for mycotoxins.  Clin.
     Toxicol., 5: 465-494.

    STOLOFF, L. (1976) Occurrence of mycotoxins in foods and feeds. In:
    Rodricks, J. V., ed.  Mycotoxins and other fungal related food
     problems, Washington, DC, American Chemical Society, pp. 23-50
    (Advances in Chemistry Series 149).

    STOLOFF, L. (1977) Aflatoxins-an overview. In: Rodricks, J. V.,
    Hesseltine, C. W., & Mehlman, M. A., ed.  Mycotoxins in human and
     animal health, Park Forest South, IL, USA, Pathotox Publishers
    Inc., pp. 7-28.

    STOLOFF, L. & FRIEDMAN, L. (1976) Information bearing on the
    evaluation of the hazard to man from aflatoxin ingestion.
     PAG Bulletin, 6: 21-32.

    STRZELECKI, E. L. & GASIOROWSKA, U. W. (1974) Aflatoxin B1 in
    feedstuffs. Zentralbl.  Vet.-Med. B, 21: 395-400.

    STUBBLEFIELD, R.D., SHOTWELL, O. L., & SHANNON, O. H. (1972)
    Aflatoxins M1 and M2 and parasiticol: Thin layer chromatography
    and physical and chemical properties.  J. Assoc. Off. Anal. Chem.,
    55: 762-767.

    SUIT, J. L., ROGERS, A. E., JETTEN, M. E. R., & LURIA, S. E. (1977)
    Effects of diet on conversion of aflatoxin B1 to bacterial
    mutagen(s) by rats  in vivo and by rat hepatic microsomes
     in vitro. Mutat. Res., 46: 313-323.

    SUZANGAR, M., EMAMI, A., & BARNETT, R. (1976) Aflatoxin
    contamination of village milk in Isfahan, Iran.  Trop. Sci., 18:
    155-159.

    SUZUKI, S. & SATOH, T. (1973) Effects of ochratoxin A on tissue
    glycogen levels in rats.  Jpn. J. Pharmacol., 23: 415-419.

    SUZUKI, S., SATOH, T., & YAMAZAKI, M. (1975) Effect of ochratoxin A
    on carbohydrate metabolism in rat liver.  Toxicol. appl.
     Pharmacol., 32: 116-122.

    SVOBODA, D., GRADY, H. J, & HIGGINSON, J. (1966) Aflatoxin B1
    injury in rat and monkey liver.  Am. J. Pathol., 49: 1023-1051.

    SWENSON, D. H., MILLER, E. C., & MILLER, J. A. (1974) Aflatoxin
    B1-2, 3-oxide: Evidence for its formation in rat liver  in vivo
    and by human liver microsomes  in vitro. Biochem. Biophys. res.
     Comm., 60: 1036-1043.

    SWENSON, D. H., LIN, J.-K, MILLER, E. C., & MILLER, J. A. (1977)
    Aflatoxin B1-2, 3-oxide as a probable intermediate in the covalent
    binding of aflatoxins B1 and B2 to rat liver DNA and ribosomal
    RNA  in vivo. Cancer Res., 37: 172-181.

    SZCZECH, G. M., CARLTON, W. W., & TUITE, J. (1973a) Ochratoxicosis
    in beagle dogs. I. Clinical and clinicopathological features.
     Vet. Pathol., 10: 135-154.

    SZCZECH, G. M., CARLTON, W. W., TUITE, J., & CALDWELL, R. (1973b)
    Ochratoxin A toxicosis in swine.  Vet. Pathol., 10: 347-364.

    SZCZECH, G. M., CARLTON, W. W, & TUITE, J. (1973c) Ochratoxicosis in
    beagle dogs. II. Pathology.  Vet. Pathol., 10: 219-231.

    TANDON, B. N., KRISHNAMURTHY, L., KOSHY, A., TANDON, H. D.,
    RAMALINGASWAMI, V., BHANDARI, J. R., MATHUR, M. M., & MATHUR, P. D.
    (1977) Study of an epidemic of jaundice, presumably due to toxic
    hepatits, in northwest India.  Gastroenterology, 72: 488-494.

    TANDON, H. D., TANDON, B. N., & RAMALINGASWAMI, V. (1978) Epidemic
    of toxic hepatitis in India of possible mycotoxic origin.  Arch.
     Pathol. lab. Med., 102. 372-376.

    TEMCHAROEN, P., ANUKARAHANONTA, T., & BHAMARAPRAVATI, N. (1978) The
    influence of dietary protein and vitamin B12 on the toxicity and
    carcinogenicity of aflatoxins in rat liver.  Cancer Res.,
    38: 2185-2190.

    THACKER, H. L. (1976)  Citrinin and ochratoxin A mycotoxicosis in
     guinea pigs, Lafayette, Indiana, Purdue University (A thesis).

    THERON, J. J. (1965) Acute liver injury in ducklings as a result of
    aflatoxin poisoning.  Lab. Invest., 14: 1586-1603.

    THERON, J. J., LIEBENBERG, N., & JOUBERT, H. J. B. (1965)
    Histochemistry and electron microscopy of acute liver lesions
    induced by aflatoxin B1 in ducklings.  Nature (Lond.), 206:
    908-909.

    TILAK, T. B. (1975) Induction of cholangiocarcinoma following
    treatment of a rhesus monkey with aflatoxin.  Food Cosmet.
     Toxicol., 13: 247-249.

    TRIPATHI, R. K. (1973) Aflatoxins in sorghum grains infested with
    head moulds.  Indian J. exp. Biol., 11: 361-362.

    TUNG, H.-T., DONALDSON, W. E., & HAMILTON, P. B. (1970) Effects of
    aflatoxin on some marker enzymes of lysosomes.  Biochem. Biophys.
     Acta, 222. 665-667.

    TURNER, W. B. (1971)  Fungal metabolites, New York, Academic Press,
    446 pp.

    ULLOA-SOSA, M. & SCHROEDER, H. W. (1969) Note on aflatoxin
    decomposition in the process of making tortillas from corn.
     Cereal Chem., 46: 397-400.

    VAN NIEUWENHUIZE, J.P., HERBER, F. M., DE BRUIN, A., MEYER, P. B., &
    DUBA, W. C. (1973) [Cancers in men following a long-term low-level
    exposure to aflatoxins in indoor air.] T. Soc. Geneeskd., 51:
    754-760 (in Dutch).

    VAN PE, W., VAN BRABANT, J., & JOOSTENS, J. (1977) La dtection et
    le dosage de l'aflatoxine M1 dans le lait et le lait en poudre.
     Rev. Agrie. (Bruxelles), 30: 403-415.

    VAN RENSBURG, S. J. (1977) Role of epidemiology in the elucidation
    of mycotoxin health risks. In: Rodricks, J. V., Hesseltine, C. W., &
    Mehlman, M. A., ed.  Mycotoxins in human and animal health, Park
    Forest South, IL, USA, Pathotox Publ., pp. 699-711.

    VAN RENSBURG, S. J., VAN DER WATT, J. J., PURCHASE, I. F. H.,
    PEREIRA COUTINHO, L., & MARKHAM, R. (1974) Primary liver cancer rate
    and aflatoxin intake in a high cancer area.  S. Afr. Med. J., 48:
    2508a-2508d.

    VANYI, A. & SZAILER, E. (1974) Investigation of the cytotoxic effect
    of F-2 toxin (zearalenone) in various monolayer cell cultures.
     Acta Vet. Acad. Sci. Hung., 24: 407-412.

    VERRETT, M. J., MARLIAC, J.-P., & MCLAUGHLIN, J., JR (1964) Use of
    the chicken embryo in the assay of aflatoxin toxicity.
     J. Assoc. Off. Anal. Chem., 47: 1003-1006.

    VESELY, D., VESELA, D., KUSAK, V., & NESNIDAL, P. (1978)
    [Distribution of perorally administered aflatoxin B1 in tissues
    and organs of the goat.] Veterinarni medicina, 23 (LI): 555-558 (in
    Czech).

    VESONDER, R. F., CIEGLER, A., & JENSEN, A. H. (1973) Isolation of
    the emetic principle from  Fusarium-infected corn.
     Appl. Microbiol., 26: 1008-1010.

    VESSELINOVITCH, S. D., MIHAILOVICH, N., WOGAN, G. N., LOMBARD, L.
    S., & RAO, K. V. N. (1972) Aflatoxin B1, a hepatocarcinogen in the
    infant mouse.  Cancer Res., 32: 2289-2291.

    VILLA-TREVINO, S. & LEAVER, D. D. (1968) Effects of the hepatotoxic
    agents retrorsine and aflatoxin B1 on hepatic protein synthesis in
    the rat.  Biochem. J., 109: 87-91.

    VOGEL, C. L., ANTHONY, P. P., MODY, N., & BARKER, L. F. (1970)
    Hepatitis-associated antigen in Ugandan patients with hepatocellular
    carcinoma.  Lancet, 2: 621-624.

    WALES, J. H. & SINNHUBER, R. O. (1972) Hepatomas induced by
    aflatoxin in the sockeye salmon  (Oncorhynchus nerka). J. Natl
     Cancer Inst., 48: 1529-1530.

    WALTKING, A. E. (1970) Collaborative study of three methods for
    determination of aflatoxin in peanuts and peanut products.
     J. Assoc. Off. Anal. Chem., 53: 104-113.

    WALTKING, A. E. (1971) Fate of aflatoxin during roasting and storage
    of contaminated peanut products.  J. Assoc. Off. Anal. Chem., 54:
    533-539.

    WALTKING, A. E., BLEFFERT, G., & KIERNAN, M. (1968) An improved
    rapid physio-chemical assay method for aflatoxin in peanuts and
    peanut products.  J. Am. Oil Chem. Soc., 45: 880-884.

    WARD, J. M., SONTAG, J. M., WEISBURGER, E. K., & BROWN, C. A. (1975)
    Effect of lifetime exposure to aflatoxin B1 in rats.
     J. Natl Cancer Inst., 55: 107-113.

    WARE, G. M. & THORPE, C. W. (1977) Analysis for zearalenone in corn
    by high pressure liquid chromatography with a fluorescence detector.
     Abstracts 91st Annual Meeting, Association of Official Analytical
     Chemists, Washington, DC, p. 47 (Abstract no. 148).

    WARE, G. M. & THORPE, C. W. (1978) Determination of zearalenone in
    corn by high-pressure liquid chromatography and fluorescence
    detection.  J. Assoc. Off. Anal, Chem., 61 (5): 1058-1062.

    WHITAKER, T. B. (1977)Sampling granular foodstuffs for aflatoxin.
     Pure appl. Chem., 49: 1709-1717.

    WHITAKER, T. B. & WHITTEN, M. E. (1977) Evaluation of cottonseed
    aflatoxin testing programs.  J. Am. Oil Chem. Soc., 54: 436-441.

    WHITAKER, T. B., DICKENS, J. W., & MONROE, R. J. (1974a) Variability
    of aflatoxin test results.  J. Am. Oil Chem. Soc., 51: 214-218.

    WHITAKER, T. B., DICKENS, J. W., WISER, E. H., & MONROE, R. J.
    (1974b)  Development of a method to evaluate sampling plans used to
     estimate aflatoxin concentrations in lots of shelled peanuts.
    I.U.P.A.C. Information Bulletin, Oxford, International Union of Pure
    & Applied Chemistry Secretariat, pp. 1-14 (Technical Report-No. 10).

    WHITAKER, T. B., DICKENS, J. W., & MONROE, R. J. (in press)
    Variability associated with testing corn for aflatoxin.  J. Am. Oil
     Chem. Soc.

    WHITTEN, M. E. (1969) Aflatoxins in cottonseed hulls.  J. Am. Oil
     Chem. Soc., 47: 5-6.

    WHO (1978) Environmental Health Criteria 6-- Principles and methods
     for evaluating the toxicity of chemicals. Part I, Geneva, World
    Health Organization, 272 pp.

    WILLIAMS, D. J. & RABIN, B. R. (1971) Disruption by carcinogens of
    the hormone dependent association of membranes with polysomes.
     Nature (Lond.), 232: 102-105.

    WOGAN, G. N. (1966) Chemical nature and biological effects of the
    aflatoxins.  Bacteriol. Rev., 30: 460-470.

    WOGAN, G. N. (1969) Metabolism and biochemical effects of
    aflatoxins. In: Goldblatt, L. A., ed.  Aflatoxin: Scientific
     background, control and implications, New York, Academic Press,
    pp. 151-186.

    WOGAN, G. N. (1973)Aflatoxin carcinogenesis. In: Bush, M., ed.
     Methods in cancer research, New York, Academic Press, pp. 309-344.

    WOGAN, G. N. (1977) Mycotoxins and other naturally occurring
    carcinogens. In: Kraybill, H. F. & Mehlman, M. A., ed.
     Environmental cancer, Advances in modern toxicology, Washington,
    DC, Hemisphere Publishing Corporation, Vol. 3, pp. 263-290.

    WOGAN, G. N. & FRIEDMAN, M. A. (1968) Inhibition by aflatoxin B1
    of hydrocortisone induction of rat liver tryptophan pyrrolase and
    tyrosine transaminase.  Arch. Biochem. Biophys., 128: 509-516.

    WOGAN, G. N. & NEWBERNE, P.M. (1967) Dose-response characteristics
    of aflatoxin B1 carcinogenesis in the rat.  Cancer Res., 27:
    2370-2376.

    WOGAN, G. N. & PAGLIALUNGA, S. (1974) Carcinogenicity of synthetic
    aflatoxin M1 in rats.  Food Cosmet. Toxicol., 12: 381-384.

    WOGAN, G. N., EDWARDS, G. S., & SHANK, R. C. (1967) Excretion and
    tissue distribution of radioactivity from aflatoxin B1-14C in
    rats.  Cancer Res., 27: 1729-1736.

    WOGAN, G. N., EDWARDS, G. S., & NEWBERNE, P.M. (1971)
    Structure-activity relationships in toxicity and carcinogenicity of
    aflatoxins and analogs.  Cancer Res., 31: 1936-1942.

    WOGAN, G. N., PAGLIALUNGA, S., & NEWBERNE, P.M. (1974) Carcinogenic
    effects of low dietary levels of aflatoxin B1 in rats.  Food
     Cosmet. Toxicol., 12: 681-685.

    WONG, J. J. & HSIEH, D. P. H. (1976) Mutagenicity of aflatoxins
    related to their metabolism and carcinogenic potential.  Proc. Natl
     Acad. Sci. (USA), 73: 2241-2244.

    WYATT, R. D., DOERR, J. A., HAMILTON, P. B., & BURMEISTER, H. R.
    (1975) Egg production, shell thickness and other physiological
    parameters of laying hens affected by T-2 toxin.  Appl. Microbiol.,
    29: 641-645.

    WYLLIE, W. D. & MOREHOUSE, L. D., ed. (1977)  Mycotoxic fungi,
     mycotoxins, mycotoxicosis: An encyclopaedic handbook. New York and
    Basel, Marcel Dekker.

    YADGIRI, B., REDDY, V., TULPULE, P. G., SRIKANTIA, S. G., & GOPALAN,
    C. (1970) Aflatoxin and Indian childhood cirrhosis;  Am. J. clin.
     Nutr., 23: 94-98.

    YAGEN, B., JOFFE, A. Z., HORN, P., MOR, N., & LUTSKY, I. I. (1977)
    Toxins from a strain involved in ATA. In: Rodricks, J. V.,
    Hesseltine, C. W., & Mehlman, M. A., ed.  Mycotoxins in human and
     animal health, Park Forest South, IL, USA, Pathotox Publishers
    Inc., pp. 329-336.

    YNDESTAD, M. & UNDERDAL, B. (1975) [Aflatoxin in foods on the
    Norwegian market.]  Nord. Vet.-Med., 27: 42-48 (in Norwegian).


    See Also:
       Toxicological Abbreviations