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    Published under the joint sponsorship of
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    World Health Organization
    Geneva, 1990

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    WHO Library Cataloguing in Publication Data

    Selected mycotoxins : ochratoxins, trichothecenes, ergot.

        (Environmental health criteria ; 105)

        1.Ochratoxins 2.Trichothecenes 3.Ergot alkaloids 

        ISBN 92 4 157105 5        (NLM Classification: QW 630)
        ISSN 0250-863X

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1. Ochratoxin A
    1.1. Natural occurrence
    1.2. Analytical methods
    1.3. Metabolism
    1.4. Effects on animals
    1.5. Effects on man
2. Trichothecenes
    2.1. Natural occurrence
    2.2. Analytical methods
    2.3. Metabolism
    2.4. Effects on animals
    2.5. Effects on man
3. Ergot
    3.1. Natural occurrence
    3.2. Analytical methods
    3.3. Effects on animals
    3.4. Effects on man

4. Recommendations for further research  
    4.1. General recommendations
    4.2. Ochratoxin A
    4.3. Trichothecenes
    4.4. Ergot

    I.1  Properties and analytical methods  
         I.1.1  Chemical properties
         I.1.2  Methods for the analysis of foodstuffs and 
                biological samples 
    I.2  Sources and occurrence
         I.2.1  Fungal formation
         I.2.2  Occurrence in foodstuffs
                I.2.2.1  Plant products
                I.2.2.2  Residues in food of animal origin
    I.3  Metabolism
         I.3.1  Absorption
         I.3.2  Tissue distribution
                I.3.2.1  Animal studies
                I.3.2.2  Studies on man
         I.3.3  Metabolic transformation
         I.3.4  Excretion
    I.4  Effects on animals
         I.4.1  Field observations
                I.4.1.1  Pigs
                I.4.1.2  Poultry

         I.4.2  Experimental animal studies
                I.4.2.1    Acute and chronic effects
                I.4.2.2    Teratogenicity
                I.4.2.3    Mutagenicity  
                I.4.2.4    Carcinogenicity
                I.4.2.5    Biochemical effects and mode of action
    I.5  Effects on man
         I.5.1  Ochratoxin A, Balkan endemic nephropathy, and 
                tumours of the urinary system
    I.6  Evaluation of the human health risks


    II.1  Properties and analytical methods
          II.1.1  Physical and chemical properties
                  II.1.1.1  Physical properties  
                  II.1.1.2  Chemical properties  
          II.1.2  Analytical methods for trichothecenes
                  II.1.2.1  Chemical methods
                  II.1.2.2  Immunological methods
                  II.1.2.3  Biological methods  
    II.2  Sources and occurrence
          II.2.1  Taxonomic considerations
          II.2.2  Ecology of trichothecene-producing fungi
          II.2.3  Natural occurrence
                  II.2.3.1  Agricultural products
                  II.2.3.2  Trichothecenes in human foodstuffs
    II.3  Metabolism
          II.3.1  Absorption and tissue distribution
                  II.3.1.1  Animal studies
          II.3.2  Metabolic transformation
          II.3.3  Excretion
                  II.3.3.1  Animal studies
                  II.3.3.2  Excretion in eggs and milk
    II.4  Effects on animals
          II.4.1  Field observations
          II.4.2  Effects on experimental animals
                  II.4.2.1  General toxic effects
                  II.4.2.2  Haematological and haemostatic changes
                  II.4.2.3  Disturbances of the central nervous 
                  II.4.2.4  Dermal toxicity
                  II.4.2.5  Impairment of immune response
                  II.4.2.6  Carcinogenicity  
                  II.4.2.7  Mutagenicity
                  II.4.2.8  Teratogenicity and reproductive effects
          II.4.3  Biochemical effects and mode of action
                  II.4.3.1  Cytotoxicity
                  II.4.3.2  Inhibition of protein synthesis
                  II.4.3.3  Inhibition of nucleic acid synthesis
                  II.4.3.4  Alterations of cellular membranes
                  II.4.3.5  Other biochemical effects
          II.4.4  Structure-activity relationships
          II.4.5  Prevention and therapy of trichothecene toxicosis

    II.5  Effects on man
          II.5.1  Contemporary episodes of human disease
          II.5.2  Historical  Fusarium-related diseases
          II.5.3  Skin irritation
          II.5.4  Studies of haemostasis
          II.5.5  Airborne trichothecene-related diseases
          II.5.6  Toxicological information on man, obtained from 
                  therapeutic uses
    II.6  Evaluation of the human health risks


      III.1  Properties and analytical methods
             III.1.1  Chemical properties
             III.1.2  Analytical methods for ergot and ergot 
                      III.1.2.1  Ergot
                      III.1.2.2  Ergot alkaloids  
     III.2  Sources and occurrence
            III.2.1  Fungal producers
            III.2.2  Biosynthesis
            III.2.3  Occurrence in foodstuffs
            III.2.4  Fate of ergolines during food processing
     III.3  Metabolism
     III.4  Effects on animals
            III.4.1  Field studies
            III.4.2  Experimental animal studies
                     III.4.2.1  Cattle
                     III.4.2.2  Sheep
                     III.4.2.3  Poultry
                     III.4.2.4  Swine
                     III.4.2.5  Primates
     III.5  Effects on man
            III.5.1  Ergometrine-related outbreaks
            III.5.2  Clavine-related outbreaks 
     III.6  Evaluation of the human health risks





Professor W.W. Carlton, Department of Veterinary Pathobiology, 
School of Veterinary Medicine, Purdue University, West Lafayette, 
Indiana, USA 

Mr T. Demeke, Health Service Department, Ministry of Health, Addis  
Ababa, Ethiopia 

Dr J. Gilbert, Ministry of Agriculture, Fisheries and Food, 
Norwich, United Kingdom 

Professor P. Krogh, Department of Microbiology, Royal Dental 
College, Copenhagen, Denmark  (Co-Rapporteur) 

Dr M. Nakadate, Section of Information and Investigation, Division 
of Information on Chemical Safety, National Institute of Hygienic 
Sciences, Tokyo, Japan 

Dr J. Parizek, Institute of Nuclear Biology and Radiochemistry, 
Czechoslovak Academy of Sciences, Prague, Czechoslovakia 

Dr A.E. Pohland, Division of Chemical Contaminants, Center for Food 
Safety and Applied Nutrition, Food and Drug Administration, US 
Department of Health and Human Services, Washington DC, USA 

Professor H.D.Tandon, ex-President, National Academy of Medical 
Sciences, New Delhi, India  (Chairman) 

Professor Y. Ueno, Department of Toxicology and Microbial 
Chemistry, Faculty of Pharmaceutical Sciences, Science University 
of Tokyo, Tokyo, Japan  (Co-Rapporteur) 


Dr K. Ohtsubo, Department of Clinical Pathology, Tokyo Metropolitan 
Institute of Gerontology, Tokyo, Japan 

Dr T. Yoshizawa, Department of Bioresource Sciences, Faculty of 
Agriculture, Kagawa University, Kagawa, Japan 


Dr M. Gilbert, International Programme on Chemical Safety, World 
Health Organization, Geneva, Switzerland 

Dr A. Prost, Division of Environmental Health, World Health 
Organization, Geneva, Switzerland 


    Every effort has been made to present information in the 
criteria documents as accurately as possible without unduly 
delaying their publication.  In the interest of all users of the 
environmental health criteria documents, readers are kindly 
requested to communicate any errors that may have occurred to the 
Manager of the International Programme on Chemical Safety, World 
Health Organization, Geneva, Switzerland, in order that they may be 
included in corrigenda, which will appear in subsequent volumes. 


    A WHO Task Group on Environmental Health Criteria for Selected 
Mycotoxins met in London on 14-18 November, 1988.  Dr Malcolm 
HUTTON opened the meeting on behalf of the Director of the 
Monitoring and Assessment Research Centre (MARC), King's College, 
London, which hosted the meeting on behalf of the three cooperating 
organizations of the International Programme on Chemical Safety 
(WHO/ILO/UNEP). The Task Group reviewed and revised the draft 
criteria document and made an evaluation of the health risks of 
exposure to selected mycotoxins. 

    The draft documents for Ochratoxins and Ergot were prepared by 
Professor P. KROGH.  Those for Trichothecenes were prepared by Dr 
M. NAKADATE AND HIS COLLEAGUES in the National Institute of 
Hygienic Sciences and by Professor Y. UENO AND HIS COLLEAGUES in 
Tokyo. During the task group meeting, several members of the Group 
agreed to undertake a substantial revision of the draft.  Dr A. 
PROST was responsible for the overall scientific content of the 
document and Mrs M. O. HEAD of Oxford, England, for the editing. 

    The Secretariat wishes to acknowledge the contributions of:  Dr 
J. GILBERT (Chemistry and analytical methods for trichothecenes); 
Dr A.E. POHLAND (Sources and natural occurrence of trichothecenes); 
and Professor W.W. CARLTON (Animal studies and metabolism of 

    The Secretariat also wishes to thank Professor Y. UENO, Co-
rapporteur of the Task Group, for his significant contributions and 
revisions of the draft document during the meeting. Professor H. 
TANDON, Chairman of the Task Group, and Professor P. KROGH and 
Professor Y. UENO, Co-rapporteurs, met with members of the 
Secretariat in Tokyo, 25-30 July, 1989, to review the final 
document before its release. 

    The efforts of all who helped in the preparation and 
finalization of the document, especially those of Dr H. KURATA and 
Dr M. ICHINOE (National Institute of Hygienic Sciences, Tokyo) and 
Dr K. OHTSUBO (Tokyo Metropolitan Institute of Gerontology), are 
gratefully acknowledged. 

                               * * *

    Partial financial support for the publication of this criteria 
document was kindly provided by the United States Department of 
Health and Human Services, through a contract from the National 
Institute of Environmental Health Sciences, Research Triangle Park, 
North Carolina, USA -- a WHO Collaborating Centre for Environmental 
Health Effects. 


    A decade has passed since the publication of Environmental 
Health Criteria 11: Mycotoxins (WHO, 1979), but this field of 
research has expanded rapidly, and recent data indicate that the 
health effects of several of the mycotoxins dealt with in the above 
publication should be updated. More than 200 mycotoxins are now 
known to exist. They are present in the environment and, in some 
cases, human exposure has been documented, mainly through food 
contamination or occasionally through inhalation. However, 
information on adverse human health effects is often lacking and, 
in many cases, the association between exposure to selected 
mycotoxins and the occurrence of health disorders remains 

    Ochratoxin A has been found as a contaminant in foods with a 
frequency in the range of 2-30% in all countries where attempts to 
perform food analysis have been made.  Field cases of ochratoxin A-
associated nephropathy in farm animals have been encountered in 
many countries, underlining the nephrotoxic potential of this 
compound. More recently, ochratoxin A has been detected in the 
blood of 6-18% of the human population in some areas where Balkan 
endemic nephropathy is prevalent. Ochratoxin A has also been found 
in human blood samples outside the Balkan peninsula. In some 
studies, more than 50% of the samples analysed have been 
contaminated.  A high incidence of tumours of the urinary system is 
strongly correlated with the prevalence of Balkan endemic 
nephropathy. For these reasons, the human health effects of 
ochratoxin A have been re-evaluated. 

    A considerable increase in trichothecenes research has been 
seen over the last decade.  The potential for the production of a 
number of trichothecenes among  Fusarium species is well documented, 
and the toxicology of a few trichothecenes and the natural 
occurrence of some of these compounds in food is fairly well 
established.  Thus, food-borne exposure of human beings to some 
trichothecenes, in particular deoxynivalenol (vomitoxin) and 
nivalenol, is likely to occur. Several reports have recently 
associated outbreaks of human disease with the presence of 
trichothecenes in food. For this reason, the health effects of 
trichothecenes have been re-evaluated. 

    Ergotism is by far the oldest known mycotoxicosis in man and 
animals. Recent episodes of  Claviceps purpurea-associated 
intoxication in Ethiopia, as well as episodes of  Claviceps 
 fusiformis-associated intoxications in areas of India indicate that 
ergotism is still a disease of public health importance, 
particularly in developing countries. The evaluation of ergot as a 
food contaminant has therefore been included in the present updated 
environmental health criteria dealing with selected mycotoxins. 
However, the review of available documentation has concentrated on 
studies dealing with the naturally occurring ergot alkaloids. 
Derivatives produced by the pharmaceutical industry have been 
deliberately excluded. No attempt has been made to review the 
literature on the pharmacology of derivatives of lysergic acid. The 

section on ergot in this publication aims at alerting the 
scientific community about the present status of ergotism as a 
disease of our time, and about the differences in clinical symptoms 
that are observed between Asia and Africa in relation to the 
chemical differences in responsible toxins. 

    Studies on the etiology of hepatocellular carcinoma, in 
particular those indicating that there is a consistent and specific 
causal association between hepatitis B virus and hepatocellular 
carcinoma, as well as the existence of other etiological factors 
that may cause hepatocellular carcinoma independently, have 
attracted much attention since the publication of the previous EHC 
on mycotoxins (WHO, 1979). A report by WHO (1983) recognized the 
value of available methods for the direct assessment of individual 
exposure to aflatoxins, and the methods and their use in field 
studies were considered in a later report by IARC (1984). Since the 
evaluation of the carcinogenic risks of aflatoxins for human beings 
has recently been reviewed by IARC (1987a), the present update on 
mycotoxins will not assess the issue and readers should refer to 
the IARC evaluation. 


1.  Ochratoxin A

1.1  Natural occurrence

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

    The highest incidences of ochratoxin A contamination have been 
found in cereals, and to a lesser extent in some beans (coffee, 
soya, cocoa).  Ochratoxin B occurs extremely rarely. 

1.2  Analytical methods

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

1.3  Metabolism

    Residues of unchanged ochratoxin A have been found in the 
blood, kidney, liver, and muscle of pigs in slaughter houses and in 
the muscle of hens and chickens.  However, residues of ochratoxin 
A  have not generally been found in ruminants.  The  in vitro 
binding of ochratoxin A to serum albumin is particularly strong in 
cattle, pigs, and man.  Experimental studies on pigs and hens have 
shown that higher levels of ochratoxin A occur in the kidneys.  
Microsomal hydroxylation might represent a detoxification reaction 
in pigs, rats, and man.  In experimental studies, residues could 
still be identified in pig kidneys, one month after the termination 
of exposure. 

1.4  Effects on animals

    Field cases of ochratoxicosis in farm animals (pigs, poultry) 
have been reported from several European countries, the primary 
manifestation being chronic nephropathy.  The lesions include 
tubular atrophy, interstitial fibrosis and, at later stages, 
hyalinized glomeruli.  Ochratoxin A has also been found in pig 
blood collected at Canadian slaughterhouses.  It has produced 
nephrotoxic effects in all species of single-stomach animals 
studied so far, even at the lowest level tested (200 µg/kg feed in 
rats and pigs). 

    Teratogenic effects were observed in mice exposed orally to 3 
mg/kg body weight.  Fetal resorption was observed in rats given 
doses from 0.75 mg/kg body weight orally.  Teratogenic effects, 

which in the rat were enhanced by a diet low in protein, have also 
been observed in hamsters. 

    There is no evidence of ochratoxin A activity in short-term 
tests for mutagenicity (bacteria and yeasts).  Rats exposed orally 
showed single-strand breaks in DNA in renal and hepatic tissues.  
Ochratoxin A induced renal cell neoplasms in male mice and in both 
sexes of rats dosed orally.  Hepatic cell neoplasms were reported 
in only one mouse strain and not in the rat. 

    Ochratoxin A is an inhibitor of protein synthesis and tRNA 
synthetase in microorganisms, hepatoma cells, and in renal mRNA in 
the rat. 

    Ochratoxin A can inhibit macrophage migration.  In mice, a dose 
of 0.005 µg/kg body weight suppressed the immune response to sheep 
erythrocytes; however, contradictory results have also been 

    Ochratoxin A has been shown to be carcinogenic to the renal 
tubular epithelium in male mice and in both sexes in rats. 

1.5  Effects on man

    Human exposure, as demonstrated by the occurrence of ochratoxin 
A in food, blood, and in human milk, has been observed in various 
countries in Europe.  Available epidemiological information 
indicates that Balkan nephropathy may be associated with the 
consumption of foodstuffs contaminated by this toxin. 

    A highly significant relationship has been observed between 
Balkan nephropathy and tumours of the urinary tract, particularly 
with tumours of the renal pelvis and ureters.  However, no data 
have been published that establish a direct causal role of 
ochratoxin A in the etiology of such tumours. 

2.  Trichothecenes

2.1  Natural occurrence

    To date, 148 trichothecenes, characterized chemically as having 
the same basic tetracyclic scirpenol ring system, are known.  These 
compounds are produced primarily by moulds belonging to the genus 
 Fusarium, though other genera, including  Trichoderma, 
 Trichothecium, Myrothecium, and  Stachybotrys, are also known to 
produce metabolites now characterized as trichothecenes.  Only a 
few of the known trichothecenes have been found to contaminate food 
or animal feed including: deoxynivalenol (DON), nivalenol (NIV), 
diacetoxyscirpenol (DAS), and T-2 toxin and, less frequently, 
certain derivatives (3-Ac-DON, 15-Ac-DON, fusarenon-X and HT-2 
toxin).  Of these, by far the most commonly encountered in food and 
animal feed is DON, with lesser amounts of NIV usually found as co-
contaminants.  Some macrocyclic trichothecenes, such as satratoxins 
G and H, and the verrucarins, occur occasionally in animal feed 
(straw, hay) but there are no reports of their presence in foods. 

    Surveys for the presence of trichothecenes have revealed the 
world-wide occurrence of DON, primarily in cereals, such as wheat 
and corn, at levels occasionally as high as 92 mg/kg, though 
average levels are considerably lower and vary with commodity.  
There are isolated reports of the occurrence of DON in barley, 
mixed feeds, potatoes, etc.  NIV, though not normally reported in 
cereals in Canada or the USA, is commonly found in conjunction with 
DON in Asian and European grains; the highest concentration 
recorded to date for NIV is 37.9 mg/kg.  T-2 toxin and DAS have 
been reported infrequently and at much lower concentrations. 

    Processing and milling studies have shown little reduction in 
DON levels from the cereal to the finished product.  Similarly, 
baking is not effective in destroying DON.  In general, 
commercially available human foodstuffs rarely contain detectable 
levels of DON and NIV. 

2.2  Analytical methods

    Analytical methods based on TLC, GC, HPLC, and immunological 
techniques are available for the determination of the four most 
frequently encountered toxins (DON, T-2 toxin, DAS, NIV) with 
detection limits below 1 µg/g.  Several of these methods have been 
tested collaboratively.  In addition, research methods, such as 
GC/MS and LC/MS, are available for confirmation of identity. 

2.3  Metabolism

    Metabolic studies have been carried out on animals, principally 
with T-2 toxin, but a few with DON.  These trichothecenes are 
rapidly absorbed from the alimentary tract, but quantitative data 
are not available.  The toxins are distributed fairly evenly 
without marked accumulation in any specific organ or tissue.  
Trichothecenes are metabolically transformed to less toxic 
metabolites by such reactions as hydrolysis, hydroxylation, 
de-epoxidation, and glucuronidation.  Trichothecenes, such as T-2 
toxin and DON, are rapidly eliminated in the faeces and urine.  For 
example, almost 100% of an  oral dose of T-2 toxin in cattle was 
eliminated within hours of dosing; in chickens, about 80% had been 
eliminated 48 h after dosing. In the rat,  25% of DON was 
eliminated in the urine and 65% in the faeces, 96 h after dosing.  
The results of transmission of T-2 toxin in the laying hen and 
lactating cow showed that less than 1% of the administered dose of 
this toxin and its metabolites was present in eggs and milk. Tissue 
residues of oral T-2 toxin and metabolites in chicken meat were 
below 2% of the dose, 24 h after dosing. 

2.4  Effects on animals

    Ingestion of animal feed of plant origin is the main route of 
exposure to trichothecenes.  T-2 toxin and DAS, which are the most 
potent for laboratory animals of the trichothecenes commonly 
reported as feed contaminants (T-2 toxin, DAS, NIV, and DON), 
induce a similar toxic response.  NIV is less potent in some 
systems than the previous two compounds and DON is the least toxic 

of the four (examples of potency include the oral LD50s in the 
mouse: T-2 toxin, 10.5 mg/kg body weight and DON, 46.0 mg/kg). 

    The more potent trichothecenes, such as T-2 toxin and DAS, 
produce acute systemic effects when administered experimentally to 
rodents, pigs, and cattle, via the oral, parenteral, or inhalation 
(pig, mouse) route. Epithelionecrosis is a lesion produced by 
contact exposure with potent trichothecenes, such as T-2 toxin and 
DAS (dose of 0.2 µg per spot for T-2 toxin).  Larger doses of other 
trichothecenes (NIV, 10 µg per spot) are required to produce an 
irritant effect.  The cytotoxic trichothecenes, such as T-2 toxin, 
produce necrosis of the intestinal crypt epithelium and of lymphoid 
and haematopoietic tissues after oral, parenteral, or inhalation 
exposure.  Haematological and coagulopathic abnormalities follow 
exposure to cytotoxic trichothecenes, such as T-2 toxin and DAS. 
Severe toxicosis can result in pancytopenia.  Suppression of cell-
mediated and humoral immunity has been demonstrated in studies with 
T-2 toxin, DON, and DAS, and observations include effects such as 
reduced concentrations of immunoglobulins and depressed phagocytic 
activity of both macrophages and neutrophils. The results  of  
experimental animal studies have indicated that the immunodepressive 
effect of such trichothecenes as T-2, DAS, and DON, results in 
decreased resistance to secondary infection by  bacteria 
(Mycobacteria,  Listeria monocytogenes), yeasts  (Cryptococcus 
 neoformans), and viruses (Herpes simplex virus). 

    T-2 toxin has been reported to be teratogenic in the mouse, 
when given by intraperitoneal injection (unusual route of 
administration for teratogenic studies).  DON was reported to be 
teratogenic in mice after gastric intubation, but was not 
teratogenic in rats when the toxin was provided in the feed.  NIV 
was not teratogenic in mice.  T-2 toxin, DAS, and DON were not 
mutagenic in an Ames-type assay.  T-2 toxin had weak clastogenic 
activity in some assays.  There is no evidence from the  published 
long-term toxicity studies in animals to indicate that T-2 toxin, 
fusarenon-X, and NIV are tumorigenic in animals.  No long-term 
studies of DON toxicity have been published. 

    Trichothecenes are toxic for actively dividing cells, such as 
the intestinal crypt epithelium and the haematopoietic cells.  The 
cytotoxicity has been associated with either impairment of protein 
synthesis by the binding of the compounds to the ribosomes of 
eukaryotic cells, or the dysfunction of cellular membranes.  
Inhibition of protein synthesis has been associated with the 
induction of labile and regulatory  proteins, such as IL-2 in 
immunocytes.  Transport of small molecules is impaired in cell 
membranes by extremely low concentrations of trichothecenes. 

2.5  Effects on man

    Ingestion of contaminated foods of plant origin is the main 
route of exposure to trichothecenes, but other routes have been 
reported occasionally, such as accidental skin contact amongst 
laboratory research workers, and airborne trichothecenes in dust. 

    Reported cases of illness associated with exposure to 
trichothecenes are scarce and none has been established as being 
due to trichothecenes.  However, a causative role is suggested by 
the two outbreaks referred to below. 

    One disease outbreak was reported from China and was associated 
with the consumption of scabby wheat containing 1.0-40.0 mg DON/kg.  
The disease was characterized by gastrointestinal symptoms.  No 
deaths occurred in human beings.  Swine and chicks fed the leftover 
cereals were also affected. 

    An analogous outbreak was reported from India and was 
associated with consumption of baked bread made from contaminated 
wheat.  The disease was characterized by gastrointestinal symptoms 
and throat irritation, which developed within 15 minutes to one 
hour following ingestion of the bread.  The following mycotoxins 
were detected in samples of refined wheat flour used in the 
preparation of the bread:  DON (0.35-8.3 mg/kg), acetyldeoxynivalenol 
(0.64-2.49 mg/kg), NIV (0.03-0.1 mg/kg) and T-2 toxin (0.5-0.8 
mg/kg).  However, there was no confirmation of the identity of the 
detected trichothecenes.  The concomitant occurrence of DON and NIV 
with T-2 toxin is unusual. 

    Two diseases of historical interest, alimentary toxic aleukia 
(ATA) in the USSR and scabby wheat toxicosis in Japan and Korea, 
have been associated with the consumption of grain invaded by 
 Fusarium moulds. Some trichothecenes have since been identified 
under laboratory conditions in fungal cultures of  Fusarium moulds 
isolated from grains involved in the incidents.  Studies linking 
ATA and scabby grain toxicosis to trichothecenes exposure could not 
be made at the time that the disease occurred, because the toxins 
were not known. 

3.  Ergot

3.1  Natural occurrence

    Ergot is the name given to sclerotia of fungal species within 
the genus  Claviceps.  Biologically active alkaloids contained in 
the sclerotia cause the development of toxicoses when the sclerotia 
are consumed by man and animals through contaminated food or animal 

    Ergot alkaloids produce two different patterns of diseases, 
depending on the fungal organism involved  (C. Purpurea, C. 
 fusiformis) and hence the alkaloids produced.  Ergotism, induced by 
ergotamine-ergocristine alkaloids produced by  C. purpurea, is 
characterized predominantly by gangrene of the extremities as well 
as gastrointestinal symptoms.  Intoxication induced by millet 
contaminated with  C. fusiformis is mainly characterized by 
gastrointestinal symptoms, and is related to clavine alkaloids.  
There are no signs or symptoms suggesting vaso-occlusion. 

3.2  Analytical methods

    Ergot alkaloids (ergolines) are derivatives of lysergic acid.  
The individual alkaloids vary in the magnitude of their biological 
activity.  Determination of  C. purpurea ergot alkaloids has been 
carried out by HPLC with fluorescence detection.  Concentrations of 
0.2 µg ergoline per litre of human plasma can be measured.  
Ergotamine and ergocristine can be determined very specifically by 
radioimmunoassays at levels of 3.5 picomoles and 0.8 picomoles, 

3.3  Effects on animals

    Ergolines, mainly ergotamine and ergotaminine, have been 
associated with outbreaks of bovine abortion.  Sheep, administered 
ergotamine orally, rapidly became ill and intestinal inflammation 
was observed.  Orally exposed poultry, pigs, and primates 
experienced slight effects.  No data on the mutagenicity, 
teratogenicity, and carcinogenicity of ergolines were available to 
the task group. 

3.4  Effects on man

    Claviceps-infected grain is a source of human exposure to 
ergolines.  In most toxicological studies, identification of 
specific alkaloids has not been undertaken.  The published 
information from only one survey of cereals and cereal products 
indicates a total daily human intake of ergolines in Switzerland of 
approximately 5.1 µg per person, the contents of certain 
commodities being up to 140 µg/kg. Baking reduces the ergolines 
present in contaminated flour by 25-100%. 

    An outbreak of ergotism in Ethiopia in 1978 resulted from 
exposure to ergolines from  C. purpurea sclerotia.  The grain 
contained up to 0.75% ergot; ergometrine was detected specifically.  
Symptoms included dry gangrene with loss of one or more limb (29% 
of cases), feeble or absent peripheral pulses (36%), and 
desquamation of the skin.  Gastrointestinal symptoms occurred in 
only a few cases. Lower extremities were involved in 88% of 

    In India, several outbreaks have occurred since 1958 as a 
result of ingesting pearl millet containing clavine-type ergot from 
 C. fusiformis.  Symptoms included nausea, vomiting, and giddiness.  
Pearl millet containing 15-26 mg ergoline/kg caused the toxic 

    Since autopsies were not performed in either of the episodes, 
no information is on record of the pathological effects on human 

4.  Recommendations for further research

4.1  General recommendations

    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 intercomparability of analytical results obtained in 
different parts of the world. 

4.2  Ochratoxin A

(a)  Extended retrospective, as well as focal prospective, 
     epidemiological studies on the association of ochratoxin A 
     with Balkan type endemic nephropathy and with urinary-tract 
     tumours should be conducted in the Balkan peninsula and the 
     Mediterranean region. 

(b)  Blood analysis for ochratoxin A should be performed on 
     patients with urinary-tract tumours, outside the Balkan 

(c)  The source of ochratoxin A exposure, as indicated by human 
     blood analysis, should be elucidated in countries outside the 
     Balkan peninsula. 

(d)  The mechanism of the sex differences in renal, neoplastic, and 
     non-neoplastic disease, caused by ochratoxin A in experimental 
     animals, should be elucidated. 

(e)  Extended surveys on the ochratoxin A contents of foods in 
     different parts of the world are required.  Such surveys are 
     particularly important in regions of the world where high 
     incidence rates of urinary-tract tumours, renal tumours, or 
     nephropathy occur. 

4.3  Trichothecenes

(a)  Follow-up studies should be performed in the areas of India 
     and the People's Republic of China in which episodes of 
     trichothecenes intoxication in human beings have been 
     encountered recently. The unusual pattern of trichothecene 
     occurrence in these episodes should be further elucidated. 

(b)  The effects of long-term exposure of experimental animals to 
     DON, including the carcinogenic effects, should be studied.  
     Because the response to DON by different species varies 
     greatly, the test species must be chosen carefully. 

(c)  Secondary microbial infection in experimental animals 
     following trichothecenes exposure should be further 

(d)  The influence of environmental conditions, including the 
     presence of insecticides and other man-made chemicals, on the 
     fungal production of trichothecenes should be studied. 

(e)  The effects of food processing on trichothecenes should be 

(f)  Agricultural plants, resistant to infection by trichothecene-
     producing fungi, should be developed, using biotechnological 

(g)  The possible synergistic effects in experimental animals of 
     combined exposure to trichothecenes, aflatoxins, ochratoxin A, 
     and other mycotoxins should be studied. 

(h)  Studies on the intake of trichothecenes by human beings should 
     be performed. 

(i)  Rapid and sensitive screening procedures for trichothecenes 
     should be developed, and surveys for trichothecenes in grain 
     and processed foods in temperate zones of the world should be 

4.4  Ergot

(a)  Methods of analysis for agroclavines should be developed. 

(b)  Information should be made available to developing countries 
     on the use of pathological seed screening and milling 
     procedures for minimizing the problems of ergot. 

(c)  Epidemiological studies should be performed on the possible 
     effects of low levels of ergolines on the human population. 

(d)  Pharmacological and toxicological studies should be performed 
     using individual and combined ergolines on experimental 

(e)  The possible transmission of ergolines through the mother's 
     milk to the infant should be elucidated. 



I.1.1  Chemical properties

    The ochratoxins constitute a group of closely-related 
derivatives of isocoumarin linked to L-phenylalanine (Fig. 1), and 
classified according to biosynthetic origin as pentaketides within 
the group of polyketides (Turner, 1971).  The topic has been 
reviewed by Scott (1977) and Steyn (1977, 1984). 


    The first compound discovered, ochratoxin A, was isolated from 
a culture of  Aspergillus ochraceus, hence the name (Van der Merwe 
et al., 1965).  The acids, including 4-hydroxy-ochratoxin A, the 
methyl and ethyl esters, and the isocoumarin part of ochratoxin B 
(ochratoxin) have all been isolated from fungal cultures under 
experimental conditions.  On acid hydrolysis, ochratoxin A yields 
phenylalanine, and the isocoumarin part, ochratoxin alpha, a 
cleavage product also found in the intestines, faeces, urine, and 
liver of rodents experimentally fed an ochratoxin A-containing diet 
(Galtier & Alvinerie, 1976).  Ochratoxin A and, very rarely, 
ochratoxin B are the only compounds found as natural contaminants 
in plant material, and most of the information available concerns 
ochratoxin A.  It can be stored in ethanol in the refrigerator for 
more than a year without loss (Chu & Butz, 1970); however, such 
solutions should be protected from light, since decomposition 
occurs on exposure to fluorescent light for several days (Neely & 
West, 1972). 

    Ochratoxin A is a colourless, crystalline compound, obtained by 
crystallization from benzene, with a melting point of about 90 °C, 
and containing approximately one mole of benzene.  After drying for 
1 h at 60 °C, it has a melting point in the range of 168-173 °C.  
It is soluble in polar organic solvents, slightly soluble in water, 
and soluble in dilute aqueous bicarbonate.  Physical data on 
ochratoxin A, based on a collaborative study, have been published 
by IUPAC (Pohland et al., 1982).  Ochratoxin A is optically active:  
[alpha]21D:  -46.8 °C (c = 2650 µmol/litre in chloroform).  The 

publication cited above includes information on the following 
spectra of ochratoxin A:  ultraviolet absorption spectrum; infrared 
absorption spectrum; electron impact mass spectrum; nuclear 
magnetic resonance spectrum. 

I.1.2  Methods for the analysis of foodstuffs and biological 

    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. 

    A number of methods using thin-layer chromatography have been 
published, one of the most commonly used being the official AOAC 
method developed for barley (Nesheim et al., 1973).  In this 
method, ochratoxins A and B are extracted from ground samples with 
chloroform, after acidification.  The toxins are trapped in a 
column containing diatomaceous earth impregnated with a basic 
aqueous solution.  After column clean-up, the toxins are eluted and 
thin-layer chromatography is performed using long-wave UV 
irradiation for visualization of the fluorescent ochratoxin spots 
(limit of detection:  12 µg/kg).  The method has been 
collaboratively studied, revealing coefficients of variation 
(between laboratories) in the range of 31-54% (Nesheim, 1973).  
This method has been published as an IUPAC recommended procedure 
(IUPAC, 1976).  The sensitivity can be improved by exposing the 
developed plate to ammonia fumes, resulting in a limit of detection 
of a few µg/kg.  A slightly modified method, developed for green 
coffee (Levi, 1975), has a coefficient of variation in the range of 
33-49%, based on a collaborative study. 

    The two methods have been combined into one procedure, 
published as an IARC procedure (Nesheim, 1982).  In an 
international check sample survey on ochratoxin A in animal feed, 
the AOAC procedure was used by 61% of the participants, and the 
performance was slightly better than that of the other methods, 
with a coefficient of variation of 69% compared to 79% for the 
other methods combined (Freisen & Garren, 1983). Paulsch et al. 
(1982) developed a procedure for the determination of ochratoxin A 
in the kidneys of swine, using a liquid-liquid partitioning step 
instead of column clean-up, followed by two-dimensional thin-layer 
chromatography using an acidic and an alkaline developing solvent.  
In addition, the procedure included a confirmatory test, based on 
the formation of ochratoxin A methyl ester on the plate. 

    High-performance liquid chromatographic procedures have been 
developed for the determination of ochratoxin A in food of plant 
origin (Hunt et al., 1978; Josefsson & Moller, 1979), with limits 
of detection in the range of 1-12 µg/kg, and with recoveries in the 
55-92% range.  A procedure for the determination of ochratoxin A 
residues in renal tissue has been published, in which enzymic 
digestion of the sample tissue is followed by dialysis and high 
performance liquid chromatography (Hunt et al., 1979).  The limit  
of detection is about 1 µg/kg, and recoveries of 77-78% have been 
observed.  Rapid screening methods for ochratoxin A are  available,  

based on minicolumn  chromatography, with limits of detection in 
the 8-12 µg/kg range (Hald & Krogh, 1975; Holaday, 1976).  By using 
antisera to ochratoxin A, an enzyme-linked immunosorbent assay 
(ELISA) has been constructed in which the toxin in barley can be 
determined with only 0.5% cross-reaction for ochratoxin B, and with 
a lower level of detection of 10 pg ochratoxin A/well of the ml 
plate (Morgan et al., 1982).   Immunological methodology has been 
further improved by the use of monoclonal antibody (IgG)  in a 
radioimmunoassay with high specificity for ochratoxin A, which can 
detect levels of this toxin as low as 0.2 µg/kg in swine kidney 
tissue (Candlish et al., 1986; Rousseau et al., 1987).  A method 
has been described in which ochratoxin A is cleaved to ochratoxin 
alpha and phenylalanine using the enzyme carboxypeptidase.  The 
quantification of ochratoxin A is based on the loss of fluorescence 
intensity at 380 nm, the limit of detection being 4 µg/kg  of 
barley (Hult & Gatenbeck, 1976).  The procedure has also been 
applied to swine blood with a limit of detection of 2 µg/litre 
(Hult et al., 1979). 

    The same procedure has been used in screening human blood for 
the presence of ochratoxin A, with a limit of detection of 1-2 
µg/litre serum for a 2-g sample.  High-performance liquid 
chromatography was used as confirmation (Hult et al., 1982).  A 
screening method involving flow injection has been developed by 
which ochratoxin A concentrations of more than 10 µg/litre can be 
determined, based on 50-µlitre samples of serum (Hult et al., 
1984).  As the specificity of the method is limited, positive 
results have to be confirmed by conventional methods requiring much 
larger blood samples. 

    A method has been developed for the detection of ochratoxin 
A (and aflatoxin B and citrinin) in human urine using hydrolysis 
of urine, solid-phase extraction, and reversed-phase liquid 
chromatography with fluorescence detection (Orti et al., 1986).  
The detection limit for ochratoxin A was approximately 10 ng/ml in 
samples of 10 ml urine. 


I.2.1  Fungal formation

    The ochratoxins were isolated in 1965 from a culture of 
 Aspergillus ochraceus, hence the name (Van der Merwe et al., 1965), 
but subsequent investigations have revealed that a variety of 
fungal organisms included in the genera  Aspergillus and  Penicillium 
are able to produce ochratoxins (Table 1). 

    The effects of water activity (aw) and temperature, the main 
factors controlling mycotoxin formation, have been elucidated in 
relation to growth and ochratoxin production for 3 fungal 
organisms:   A. ochraceus, P. cyclopium, and  P. viridicatum 
(Northolt et al., 1979).  The minimum aw values for ochratoxin 
production ranged between 0.83 and 0.87, 0.87 and 0.90, and 0.83 
and 0.86, respectively.  At 24 °C, optimum aw values for  A. 
 ochraceus and for both  P. cyclopium and  P. viridicatum were 0.99 
and 0.95-0.99, respectively (Fig. 2).  

Table 1.  Ochratoxin-producing fungia
 Penicillium link

   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. varibile Sopp

 Aspergillus Micheli

 Aspergillus ochraceus group:     A. sulphureus (Fres.)
                                Thom & Church
   A. sclerotiorum Huber
   A. alliaceus Thom & Church
   A. melleus Yukawa
   A. ochraceus Wilhelm
   A. ostianus Wehmer
   A. petrakii Vörös
a  From: Krogh (1978).


    At optimum aw, the temperature range for ochratoxin production 
by A. ochraceus was 12-37 °C, whereas that of  P. cyclopium and  P. 
 viridicatum was 4-31 °C.  These laboratory data correspond with 
those from field observations on ochratoxin contamination in crops.  
Thus, the more frigophilic  Penicillia, particularly  P. viridicatum, 
are the major ochratoxin producers in crops in colder climatic 
zones, such as Scandinavia (Krogh, 1978; Rutqvist et al., 1978; 
Häggblom, 1982), and in Canada (Scott et al., 1972). 

    In contrast, ochratoxin-producing fungal potential has been 
found in 28-50% of  A. ochraceus strains isolated from crops in 
warmer climatic zones, such as Australia (Connole et al., 1981), 
and Yugoslavia (Pepeljnjak & Cvetnic, 1981), and strains isolated 
from coffee beans (Stack et al., 1982). 

I.2.2  Occurrence in foodstuffs

I.2.2.1  Plant products

    Ochratoxin A was first encountered as a natural contaminant in 
maize (Shotwell et al., 1969), and subsequent surveys have 
established that ochratoxin A is a contaminant of cereals and some 
beans (coffee beans, soya beans, cocoa beans) in many areas of the 
world (Table 2).  Although the mean level of ochratoxin A in all 
reported surveys up to 1979 was 1035 µg/kg (Fig. 3), 83% of the 
samples contained less than 200 µg/kg (Krogh, 1980). 


Table 2.  Natural occurrence of ochratoxin A in foodstuffs and animal feed of plant origin
Commodity            Country           Number of   Percentage     Range of       Reference
                                       samples     contaminated   ochratoxin A
                                       analysed                   levels (µg/kg)
maize                USA               293         1.0            83-166         Shottwell et al. (1971)
maize (1973)         France            463         2.6            15-200         Galtier et al. (1977b)
maize (1974)         France            461         1.3            20-200         Galtier et al. (1977b)
wheat (red winter)   USA               291         1.0            5-115          Shottwell et al. (1976)
wheat (red spring)   USA               286         2.8            5-115          Shottwell et al. (1976)
barley (malt)        Denmark           50          6.0            9-189          Krogh (1978)
barley               USA               127         14.2           10-40          Nesheim (1971)
coffee beans         USA               267         7.1            20-360         Levi et al. (1974)
maize                Yugoslaviaa       542         8.3b           6-140          Pavlovic et al. (1979)
wheat                Yugoslaviaa       130         8.5b           14-135         Pavlovic et al. (1979)
wheat bread          Yugoslaviaa       32          18.8b                         Pavlovic et al. (1979)
barley               Yugoslaviaa       64          12.5b          14-27          Pavlovic et al. (1979)
barley               Czechoslovakia    48          2.1            3800           Vesela et al. (1978)
bread                United Kingdomc   50          2.0            210            Osborne (1980)
flour                United Kingdomc   7           28.5           490-2900       Osborne (1980)
beans                Sweden            71          8.5            10-442         Akerstrand & 
peas                 Sweden            72          2.8            10             Josefsson (1979)
maize                United Kingdom    29          37.9           50-500         Ministry of 
cornflour            United Kingdom    13          30.8           50-200         Agriculture (1980)
soya bean            United Kingdom    25          36.0           50-500         Ministry of
soya flour           United Kingdom    21          19.0           50-500         Agriculture (1980)
cocoa beans (raw)    United Kingdom    56          17.9           100-500        Ministry of
  (roasted)          United Kingdom    19          15.8           100            Agriculture (1980)
grain                German Democratic 49          4.1            18-22          Fritz et al.
                     Republic                                                    (1979)
grain (barley,       Poland            296         6.8            20-470         Szebiotko et al. (1981)
wheat, rye)
grain (wheat, rye)   Denmark           151         1.3            15-50          Pedersen & 
bran (wheat)         Denmark           57          10.5           5-20           Hansen (1981)
maize                Bulgariaa         22          27.3           25-35          Petkova-Bocharova &
maize                Bulgaria          22          9.0            10-25          Castegnaro (1985)
beans                Bulgariaa         24          16.7           25-27          Petkova-Bocharova &
beans                Bulgariaa         28          7.1            25-50          Castegnaro (1985)

Table 2 (contd.)
Commodity            Country           Number of   Percentage     Range of       Reference
                                       samples     contaminated   ochratoxin A
                                       analysed                   levels (µg/kg)
FOOD (continued)
barley, wheat,       Poland            150         5.3            50-200         Juszkiewicz &
oats, rye, maize                                                                 Piskorska-
                                                                                 Pliszczynska (1976)
mixed feed           Poland            203         4.9            10-50          Juszkiewicz &
                                                                                 Pliszczynska (1977)
maize                Yugoslavia        191         25.7           45-5125        Balzer et al. (1977)
barley, oats         Sweden            84          8.3            16-409         Krogh et al. (1974b)
wheat, hay           Canada            95          7.4            30-6000        Prior (1976)
wheat, oats,         Canadac           32          56.3           30-27 000      Scott et al. (1972)
barley, rye
barley, oats         Denmarkc          33          57.6           28-27 500      Krogh et al. (1973)
mixed feed           Canada            474         1.1            30-100         Prior (1981)
mixed feed           Canadac           51          7.8            48-5900        Abramson et al. (1983)
mixed feed           Australia         25          4.0            70 000         Connole et al. (1981)
mouldy bread         Italyd            1                          80 000         Visconti &
                                                                                 Bottalico (1983)
a  From an area with endemic nephropathy.
b  Average values for a period of 2-5 years.
c  All samples suspected of containing mycotoxins.
d  This sample contained in addition 9600 µg ochratoxin B/kg.
    Ochratoxin B has only been found in 3 samples and, thus, occurs 
extremely rarely; the other ochratoxins have never been found in 
plant products.  Levels of ochratoxin A and the frequency of 
contamination are generally higher in animal feed than in foodstuffs 
(Table 2).  Although the mean level of ochratoxin A in all reported 
surveys up to 1979 was 1035 µg/kg (Fig. 3), 83% of the samples 
contained less than 200 µg/kg (Krogh, 1980). 

I.2.2.2  Residues in food of animal origin

    Residues of ochratoxin A are not generally found in ruminants, 
because ochratoxin A is cleaved in the forestomachs by protozoan and 
bacterial enzymes (Galtier & Alvinerie, 1976; Hult et al., 1976; 
Patterson et al., 1981).  The non-toxic cleavage product, ochratoxin 
alpha, has been found in the kidneys at levels below 10 µg/kg and in 
the blood of calves fed a diet containing 300-500 µg ochratoxin A/kg 
(Patterson et al., 1981).  In half of the calves, the kidneys 
contained low levels of ochratoxin A (up to 5 µg/kg), a feature that 
may reflect that the calves were not yet functioning as ruminants.  
When 2 milking cows were fed a ration containing 317-1125 µg 
ochratoxin A/kg for 11 weeks, a residue of 5 µg ochratoxin A/kg was 
found in the kidneys of one of the animals but not in any other 
tissue or the milk.  Ochratoxin  alpha was not found in any tissue 
(Shreeve et al., 1979).  Residues of ochratoxin A have been detected 
in a number of tissues in single-stomach food animals, such as pigs.  
The carry-over of ochratoxin A from feed to animal tissues was 
elucidated in a study in which groups of pigs were exposed for 3-4 
months to dietary levels of ochratoxin A of 200, 1000, or 4000 µg/kg 
(Krogh et al., 1974a).  At termination (slaughter), the highest 
levels of ochratoxin A residues were found in the kidneys (mean 
levels 50 µg/kg at the 4000 µg/kg  feed  level), with lower levels 
in the liver, muscle, and adipose tissue; other tissues, including 
blood, 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 3). 

Table 3.  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. (1974a).
X = ochratoxin A in feed (µg/kg).
Y = ochratoxin A residue (µg/kg tissue).
 r = correlation coefficient.
The regression is calculated on feed levels of ochratoxin A 
in the range of 200-4000 µg/kg.

    Ochratoxin A is present in the blood bound to serum-albumin 
(Chu, 1971; Galtier, 1974a) and as free ochratoxin A; saturation (in 
the rat) occurs at 70 mg ochratoxin A/litre plasma.  The binding of 
ochratoxin A to serum-albumin is particularly strong in cattle, 
pigs, and man, based on  in vitro studies (Table 4). 

Table 4.   In vitro binding of ochratoxin A to 
serum-albumin in several speciesa
Animal          Number of       Intrinsic            
species         binding         Association   
                sites           constant (M-1)
Cattle          0.58            94 600
Pig             0.56            71 100
Man             0.58            63 500
Horse           0.57            57 400
Chicken         0.51            52 700
Rat             0.68            40 100
Sheep           0.88            22 600
a  Adapted from: Galtier (1979).

    The presence of ochratoxin A in the blood of pigs has been 
elucidated in experimental animal studies as well as by surveys of 
blood samples from pigs at farms (Hult et al., 1979, 1980).  A 
total of 1200 pig blood samples, collected from slaughterhouses 
over various periods during 1986 in western Canada, was screened 
for the presence of ochratoxin A using an HPLC procedure (Marqhardt 
et al., 1988). It was shown that 3.6-4.2% of the blood samples 
contained the toxin at concentrations higher than 20 ng/ml.  It 
appears that the concentration of ochratoxin A in the blood is 
higher than that in any other tissue.  In feeding studies, bacon  
pigs were exposed for various periods to rations containing  
ochratoxin A concentrations in the range of 58-1878 µg/kg;  blood, 
kidney, liver, muscle, and adipose tissues were analysed, at 
slaughter, for residues of ochratoxin A (Mortensen et al., 1983).  
The statistical association between residue levels in various 
tissues is indicated by the regression analyses (Table 5). 

Table 5.  Regression between residues of ochratoxin A
in the serum and certain other tissuesa
Tissue          Regression       r       sb
kidney          Y = 0.0651X     0.89    0.002
muscle          Y = 0.0346X     0.88    0.001
liver           Y = 0.0259X     0.89    0.001
adipose         Y = 0.0191X     0.84    0.001
a  Adapted from: Mortensen et al. (1983).
X  = µg ochratoxin A/litre serum.
Y  = µg ochratoxin A/kg in the other tissues.
 r  = correlation coefficient
sb = Standard error of slope

    Epidemiological studies on the basis of data from meat 
inspection in Danish slaughterhouses revealed prevalence rates of 
porcine nephropathy ranging from 10 to 80 cases per 100 000 
slaughtered pigs (Krogh, 1976a).  Surveys in a number of European 

countries for residues of ochratoxin A in kidneys from cases of 
porcine nephropathy revealed that 25-39% of the cases contained 
ochratoxin A levels in the range 2-100 µg/kg (Table 6). 

Table 6.  Surveys for ochratoxin A residues in kidneys from
cases of porcine nephropathy, based on meat inspection data
Country      No. of porcine  Percentage    Range of    Reference
             nephropathy     containing    ochratoxin 
             kidneys         residues of   A residues
             investigated    ochratoxin    (µg/kg wet
                             A             weight)
Denmark      60              35            2-68        Krogh (1977)

Germany,     104             21            0.1-1.8     Bauer et al.
Federal                                                (1984)
Republic of

Hungary      122             39            2-100       Sandor et 
                                                       al. (1982)
Poland       113             24            1-23        Golinski et
                                                       al. (1984)
Sweden       129             25            2-104       Rutqvist et
                                                       al. (1977)
Sweden       90              27            2-88        Josefsson 

    Ochratoxin A levels of up to 29 µg/kg were found in the muscle 
of hens and chickens collected at 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 or 1 mg/kg (Krogh et al., 
1976b).  The kidneys contained the highest residues with a mean 
value of 19 µg/kg tissue in the group fed 1 mg ochratoxin A/kg; the 
liver and muscle contained lower levels of residues and no 
ochratoxin A was found in the eggs.  These results are in 
accordance with those of subsequent experimental animal studies.  
For example, in a study in which 4 groups of hens were fed diets 
containing 0, 0.5, 1, or 4 mg ochratoxin A/kg, the kidneys 
contained the highest level of ochratoxin A residues (31 µg/kg) in 
the highest dose group;  eggs were not analysed (Prior & Sisodia, 

    In another study, hens were fed 1 mg ochratoxin A/kg feed for 8 
weeks; the kidneys contained 3-10 µg ochratoxin A/kg and the liver, 
1.5-2.5 µg/kg.  The eggs were not analysed (Reichmann et al., 
1982). When groups of hens were fed diets containing 2.5 or 10 
mg/kg, the kidneys contained 1-6 µg ochratoxin A/kg, lower levels 
being found in the plasma, muscle, and liver (Juszkiewicz et al.,  
1982).  Eggs from the high-dose group contained 0.7-1.3 µg 
ochratoxin A/kg, but none was detected in eggs from the low-dose 

    White Leghorn hens (54 birds), divided into four groups, were 
fed diets for one month containing the following concentrations of 
ochratoxin A (mg/kg): 0, 1.3, 2.6, and 5.2 (Bauer et al., 1988).  
At termination, three tissues were analysed for residues.  The 
following ranges of dose-dependent residues were found:  serum, 
4.7-11.7 ng/ml, liver, 9.1-18.0 ng/g, and yolk, 1.6-4.0 ng/g; very 
little ochratoxin A was found in the egg white, and none was 
detected in the tissues of the control group. 


I.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.  Small amounts (1-3% of the total dose) were 
detected in the caecum and the large intestine, as the isocoumarin  
moiety (ochratoxin alpha), most likely as the result of the 
hydrolysing action of the intestinal microflora (Galtier & 
Alvinerie, 1976; Hult et al., 1976). 

    In a study on intestinal absorption using the same animal 
species, Kumagai & Aibara (1982) came to the conclusion that the 
site of maximal absorption of ochratoxin A was the proximal 
jejunum, and that the portal vein was the primary route of 
transport from the intestinal tract, though part of the 
transportation took place through the lymphatics. 

    Using a highly specific antibody against ochratoxin A, which is 
used in a peroxidase-antiperoxidase detection method, Lee et al. 
(1984) studied absorption and tissue distribution in Swiss mice 
over a 48-h period, after the administration of a single dose of 25 
mg ochratoxin A/kg body weight.  Ochratoxin A was found in large 
amounts, indicated by staining, in epithelial cells as well as in 
macrophages of the lamina propria in the duodenum.  Smaller amounts 
were found in jejunal epithelial cells and lamina propria, and much 
smaller amounts in the epithelial cells in the esophagus and 
stomach; no toxin was found in the ileum.  These findings suggest 
that absorption mainly takes place in the duodenum and the jejunum. 

I.3.2  Tissue distribution

I.3.2.1  Animal studies

    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 level (up to 67 µg/kg) occurring in the kidney.  In 
experimental studies on pigs ingesting feed containing ochratoxin 
A, residues of the toxin were found in all 4 tissues in the 

decreasing order of kidney, liver, muscle, adipose tissue (Krogh et 
al., 1974a).  A subsequent study revealed that the concentration of 
ochratoxin A residues in the blood of the pig was higher than those 
in the other tissues mentioned above (Mortensen et al., 1983).  
When rats were exposed orally 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 1 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 
30 minutes, amounting to 4-5% of the total dose. 

    In the study by Lee et al. (1984), the largest amounts of 
ochratoxin A found in the kidney (as indicated by staining 
intensity) were in the epithelium of the proximal convoluted 
tubules, and to a lesser extent in the distal convoluted tubules, 
the descending loop of Henle, and glomeruli and Bowman's capsule.  
Small amounts were found in hepatocytes as well as in the lumina of 
bile ducts, but not in biliary epithelium, indicating biliary 

    Using pig renal cortical slices, it was found that ochratoxin A 
enters the proximal tubule cells by the  common organic anion 
transport system (Friis et al., 1988).  Ochratoxin A inhibited  p-
aminohippurate (PAH) and phenolsulfophthalein uptake in a dose-
dependent manner. 

I.3.2.2  Studies on man

    In a study in Yugoslavia near Slavonski Brod, where endemic 
nephropathy is prevalent, 639 samples of serum from the inhabitants 
of 2 villages were screened for the presence of ochratoxin A; 42 
(6.6%) were positive for the toxin "with concentrations in the 
range of 1-57 ng ochratoxin A/g" (Hult et al., 1982).  Detection 
was carried out using the enzymic spectrofluorometric procedure, 
and positives were confirmed by the esterification of ochratoxin A 
in serum and of ochratoxin alpha obtained from the enzymetic 
hydrolisates; the esters were measured by high performance liquid 

    In a screening of serum samples in Poland using the same method 
of analysis, 77 out of 1065 samples (7.2%) contained ochratoxin A,  
with a mean concentration of 0.27 ng/ml and a maximum value of 40 
ng/ml (Colinski, 1987).  In the Federal Republic of Germany, 173 
out of 306 serum samples (56.6%) contained ochratoxin A, as 
measured by an HPLC procedure, with a mean of 0.6 ng/g and a range 
of 0.1-14.4 ng/g (Bauer & Gareis, 1987).  Three out of 46 kidneys 
(6.6%) contained ochratoxin A, with a mean of 0.2 ng/g and a range 
of 0.1-0.3 ng/g.  In a study of blood plasma in Denmark, 46 out of 
96 samples (47.9%) contained ochratoxin A, as measured by an HPLC 
procedure, with a mean of 1.7 ng/g and a range of 0.1-9.2 ng/g 
(Hald, 1989).  In Bulgaria, where endemic nephropathy also occurs 
in some areas, 45 out of 312 blood samples contained ochratoxin A 
(14.4%), with a mean of approximately 14 ng/g (Petkova-Bocharova et 
al., 1988). 

I.3.3  Metabolic transformation

    It has been shown from  in vitro studies that ochratoxin A binds 
to serum-albumin (Chu, 1971, 1974b); this binding has also been 
observed in  in vivo studies on rats (Galtier, 1974a; Chang & Chu, 
1977).  Ochratoxin alpha has been detected in the urine and faeces 
of rats injected intraperitoneally with ochratoxin A (Nel & 
Purchase, 1968; Chang & Chu, 1977), indicating the cleavage of 
ochratoxin A to ochratoxin alpha and phenylalanine, under these 

    Studies with 14C-labelled ochratoxin A indicated that some 
other, not yet identified, metabolites are formed in the body.  
Less than half of the radioactivity excreted in rat urine within 24 
h of a single intraperitoneal injection of 14C-phenylalanine-
labelled ochratoxin A was identified as ochratoxin A (Chang & Chu, 

    In both albino rats and brown rats given ochratoxin A orally or 
intraperitoneally, 1-1.5% of the dose was excreted as (4R)-4-
hydroxyochratoxin A and 25-27% as ochratoxin alpha in the urine 
(Storen et al., 1982).  In  in vitro studies using liver microsomes 
from the pig, rat, and man, both (4R)-and (4S)-4-hydroxy-ochratoxin 
A were produced in a hydroxylation process involving cytochrome 
P-450 (Stormer & Pedersen, 1980; Stormer et al., 1981).  
4-Hydroxyochratoxin A is non-toxic for rats in amounts up to 40 mg/kg 
body weight (Hutchison et al., 1971); thus, it has been concluded 
that the microsomal hydroxylation most likely represents a 
detoxification reaction. 

I.3.4  Excretion

    The results of studies in which 14C-labelled ochratoxin A was 
injected intraperitoneally in rats demonstrated that the toxin was 
excreted primarily in the urine (Chang & Chu, 1977), though faecal 
elimination also occurred to some extent (Galtier, 1974b; Chang & 
Chu, 1977).  In a study in which 14C-labelled ochratoxin A was 
given orally to rats as a single dose (15 mg/kg body weight), the 
cumulative elimination after 120 h was 11% ochratoxin A and 23% 
ochratoxin alpha in the faeces; 11% ochratoxin A and 12% ochratoxin 
alpha in the urine; and 33% ochratoxin A in the bile (Suzuki et 
al., 1977).  Absorption was influenced by enteritis, which was 
caused by the high ochratoxin A doses given (75% of the LD50 
value).  Ochratoxin A injected intravenously as a single dose (4.1 
mg/kg) in albumin-deficient and normal rats was excreted in the 
bile and urine 20-70 times faster in the albumin-deficient rats 
than in normal rats, indicating that the binding of ochratoxin A to 
blood albumin delays the excretion of the compound through the 
liver and kidney (Kumagai, 1985). 

    In rats given unlabelled ochratoxin A orally, Storen et al. 
(1982) found 6% ochratoxin A, 1.5% (4R)-4-hydroxyochratoxin A, and 
25-27% ochratoxin alpha in the urine; 12% ochratoxin A and 9% 
ochratoxin alpha were found in the faeces. 

    The excretion of ochratoxin A in the milk was studied in 
rabbits intravenously injected with 1-4 mg/kg body weight, as 
single dose (Galtier et al., 1977a).  At the highest dose injected, 
the milk contained 1 mg ochratoxin A/litre; ochratoxin  alpha and 
4-hydroxy-ochratoxin A were not detected.  Goats were given a 
single dose of tritium-labelled ochratoxin A (0.5 mg/kg) and the 
cumulative excretion (in terms of radioactivity) after 7 days 
amounted to 53% in the faeces, 38% in the urine, 6% in the milk, 
and 2% in the serum (Nip & Chu, 1979).  Only a small fraction of 
the radioactivity in milk was ochratoxin A, amounting to 0.026% of 
the total ochratoxin A given. 

    In the Federal Republic of Germany, a study of human milk 
obtained from women in two hospitals (patient category not stated) 
revealed that 4 out of 36 samples (11.1%) contained ochratoxin A, 
with a mean value of 0.024 ng/ml and a range of 0.017-0.030 ng/ml 
(Bauer & Gareis, 1987; Gareis et al., 1988). 

    In mice given 14C-labelled ochratoxin A intravenously at 
various stages of pregnancy, the toxin was shown to cross the 
placental barrier on day 9 of pregnancy, at which time it is most 
effective in producing fetal malformations (Appelgren & Arora, 
1983).  The highest toxin concentration was found in the bile, 
which contained 5 times as much as the blood. 

    Ochratoxin A has been detected in the urine of bacon pigs 
suffering from nephropathy (Krogh, unpublished information 
communicated to the Task Group). 

    In a study on the disappearance rates for various tissues, 
female bacon pigs were fed ochratoxin A at a level of 1 mg/kg feed 
for one 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 7) from 
the 4 tissues investigated (kidney, liver, muscle, and adipose 
tissue) with residual life values (RL50)a in the range of 3.5-4.5 
days; the toxin could still be detected in the kidneys one month 
after termination of exposure. 

Table 7.  The rate of disappearance of ochratoxin A residues
from pig tissues after termination of a one-month exposure to
ochratoxin A at 1 mg/kg feeda
Tissue       Ochratoxin A (µg/kg tissue) at time  t (days) 
             after termination of exposure
kidney       28.22 exp (-0.1522 t)
liver        19.49 exp (-0.1598 t)
muscle       12.94 exp (-0.2096 t)
adipose      4.62 exp (-0.0565 t)
a  From: Krogh et al. (1976a).

a  RL50 = half residual life, calculated from the exponential 
equations shown in Table 7.

    No data are available on ochratoxin levels in human urine or 

    When the level in the serum is known, the ochratoxin A residues 
in the four other tissues can be calculated (Table 5). 


I.4.1  Field observations

I.4.1.1  Pigs

    The effects of ochratoxins on 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, though unevenly distributed.  
Prevalence rates in 1971 varied from 0.6 to 65.9 cases per 10 000 
pigs, and epidemics encountered in 1963 and 1971 were associated 
with a high moisture content in the grain caused by unusual 
climatic conditions (Krogh, 1976b).  On the basis of these studies, 
Krogh (1978) concluded that ochratoxin A is the substance most 
frequently associated with porcine nephropathy, though other 
factors, such as citrinin, may also be involved. 

    A survey on porcine nephropathy was conducted at six 
slaughterhouses in Sweden during the spring months of 1978 
(Josefsson, 1979).  A prevalence rate of 4.4 cases per 10 000 pigs 
was encountered corresponding to the endemic level of prevalence 
rates in Denmark; 26.7% of nephropathic kidneys contained residues 
of ochratoxin A.  In Hungary, an epidemiological study on porcine 
nephropathy and the association with ochratoxin A was conducted in 
1980-81 covering 4 areas in the country (Sandor et al., 1982).  A 
prevalence rate of 2.0 cases per 10 000 pigs was measured, 
comparable to endemic prevalence rates in Scandinavia; 39% of 
nephropathic kidneys contained residues of ochratoxin A.  The 
morphological changes in the kidneys in cases of mycotoxic porcine 
nephropathy were 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). 

    In Poland, surveys for porcine nephropathy in 1983 and 1984 
revealed prevalence rates of 4.7-5.7 cases per 10 000 pigs, with 5-
55% of the nephropathic kidneys containing detectable residues of 
ochratoxin A, apparently depending on the season of the year 
(Golinski et al., 1984, 1985).  Porcine nephropathy has been 
encountered in the Federal Republic of Germany (Bauer et al., 1984) 
and in Belgium (Rousseau & van Peteghem, 1989), with respectively, 
21% and 18% of the affected kidneys containing residues of 
ochratoxin A, in the range of 0.1-12 ng/g.  In Canada, 1200 samples 
of pig blood, collected at a slaughterhouse, were screened for 
ochratoxin A using HPLC (Marquardt et al., 1988). Levels exceeding 
10 ng/ml (11.3%) were found in 136 samples; detection was confirmed 
by derivative formation and spectrometry.  No kidney examination 

was conducted, but the ochratoxin A concentrations detected in the 
blood suggest that nephropathy might have been present in some of 
the pigs. 

I.4.1.2  Poultry

    In a preliminary study in Denmark on chickens condemned by meat 
inspectors because of renal lesions, 4 out of 14 birds (29%) were 
found to have nephropathy associated with the ingestion of 
ochratoxin A, as revealed by the presence of residues of ochratoxin 
A in tissues (Elling et al., 1975).  The renal lesions were 
characterized by degeneration of proximal and distal tubules of 
both reptilian and mammalian nephrons and interstitial fibrosis. 

I.4.2  Experimental animal studies

I.4.2.1  Acute and chronic effects

    The acute and chronic effects of ochratoxins on 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 8).  When ochratoxin A was administered orally to rats 
and guinea-pigs, the female was more sensitive than the male.  In 
rats, the kidney is the target organ, but necrosis of periportal 
cells in the liver has also been noted during studies on acute 
effects (Purchase & Theron, 1968).

Table 8.  Acute toxicity of ochratoxin A
Animal       LD50 (mg/kg        Route of                Reference
             body weight)       administration
mouse        22                 intraperitoneal         Sansing et
(female)                                                al. (1976)

rat (male)   30.3               oral                    Galtier et 
                                                        al. (1974)

rat          21.4               oral                    Galtier et
(female)                                                al. (1974)

rat (male)   28                 oral                    Kanizawa et
                                                        al. (1977)

rat (male)   12.6               intraperitoneal         Galtier et
                                                        al. (1974)

rat          14.3               intraperitoneal         Galtier et
(female)                                                al. (1974)

guinea-pig   9.1                oral                    Thacker &
(male)                                                  Carlton 

Table 8 (contd.)
Animal       LD50 (mg/kg        Route of                Reference
             body weight)       administration
guinea-pig   8.1                oral                    Thacker &
(female)                                                Carlton

white        3.4                oral                    Prior et 
leghorn                                                 al. (1976)

turkey       5.9                oral                    Prior et
                                                        al. (1976)

Japanese     16.5               oral                    Prior et
quail                                                   al. (1976)

rainbow      4.7                intraperitoneal         Doster et
trout                                                   al. (1972)

beagle dog   9                  orala                   Szczech et
(male)       (total dose)                               al. (1973a)

pig          6                  oralb                   Szczech et
(female)     (total dose)                               al. (1973b)
a  All 3 dogs, dosed daily with 3 mg/kg body weight, 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 nephropathy 
have been reproduced by feeding diets containing levels of 
ochratoxin A identical to those encountered in the naturally 
contaminated products.  In a study by Krogh et al. (1974a), 39 pigs 
fed rations containing ochratoxin A, at levels ranging from 200 to 
4000 µg/kg, developed nephropathy after 4 months at all levels of 
exposure.  Changes in renal function were characterized by 
impairment of tubular function, indicated particularly by a 
decrease in TmPAH/Cin,a 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.  
Sclerosed glomeruli were also observed in the group receiving the 
highest dose of ochratoxin A of 4000 µg/kg feed.  Changes were not 
seen in any other organs or tissues. 

a  TmPAH = transport maximum for para-aminohippuric acid. 
   Cin = clearance of insulin.

    Kidney damage, identical to 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.  Significant renal tubular 
functional impairment as measured by a decrease in TmPAH Cin was 
detected after only 5 weeks of ochratoxin exposure (Krogh et al., 
1976b).  The study was continued for a 2-year period during which 
the renal impairment aggravated slightly without reaching a state 
of terminal renal failure (Krogh et al., 1979). 

    In 2 pigs and 9 rats dosed orally with ochratoxin A (400 and 
250 µg/kg body weight, respectively) for 5 days, ochratoxin A was 
detected in the epithelial cells of the proximal convoluted tubules 
of the nephron of all animals.   The method of detection was 
immunofluorescence microscopy using an antibody against ochratoxin 
A that had been formed in rabbits after injection of an albumin-
ochratoxin A conjugate (Elling, 1977). 

    Groups of 80 F 344/N rats of each sex were administered 0, 21, 
70, or 210 µg ochratoxin A/kg in corn oil by gavage, 5 days per 
week for 103 weeks (Boorman, 1988).  The administration of 
ochratoxin A to male and female rats caused a spectrum of 
degenerative and proliferative changes in the kidney.  The 
predominant non-neoplastic lesion in treated rats was degeneration 
of the renal tubular epithelium in the inner cortex and the outer 
stripe of the outer medulla (nephropathy). 

    In four pigs given 0.8 mg ochratoxin A/kg body weight orally 
for 5 consecutive days, the activity of catalase and CN-insensitive 
palmitoyl-CoA-dependent NAD (nicotinamide adenine di-nucleotide) in 
renal homogenates decreased, but that in hepatic homogenates did 
not, suggesting peroxisomal changes.  This was confirmed by 
ultrastructural observations of peroxisomes in the proximal  
tubules in kidneys of  ochratoxin A-treated  animals (Elling et 
al., 1985). 

    When 11 SPF pigs and 23 beagle dogs were given high oral doses 
of ochratoxin A corresponding to feed levels of more than 5-10 
mg/kg (levels rarely found in nature), pathological effects, mainly 
necrosis, were observed in the liver, intestine, spleen, lymphoid 
tissue, leukocytes, and kidney (Szczech et al., 1973a,b,c).  Three 
groups of Wistar 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).  A decrease in 
urinary osmolality, glycosuria, and proteinuria were  observed in 
an unstated number of Sprague-Dawley and Wistar rats administered 
daily doses intraperitoneally of 0.75-2 mg ochratoxin A/kg body 
weight (Berndt & Hayes, 1979). 

    In a study of coagulation factors, rats were given daily oral 
doses of 4 mg/kg body weight over 4-10 days, resulting in decreases 
in plasma-fibrinogen levels, at levels of factors II, VII, and X, 
and in the platelet and megakaryocyte counts (Galtier et al., 

    In 4 calves fed rations containing 0.1-2 mg ochratoxin A/kg 
body weight for 30 days, the only signs observed were polyuria, 
increased levels of glutamic oxalacetic transaminase (GOT) in 
serum, and mild enteritis (Pier et al., 1976); there was mild 
tubular degeneration in the kidneys.  Cows given ochratoxin A 
orally for 4 days at doses ranging from 0.2 to 1.66 mg/kg body 
weight remained clinically normal (Ribelin et al., 1978).  A cow 
given a single dose of 13.3 mg/kg body weight (corresponding to 865 
mg/kg feed) developed diarrhoea, anorexia, and cessation of milk 
production, one day after. 

    Avian nephropathy, similar to that in spontaneously occurring 
cases, developed in Leghorn hens (27 birds per group) exposed to 
dietary levels of 0.3 or 1 mg ochratoxin A/kg for one 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 
nephrosis" and "visceral gout" were 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 for chickens (LD50, 54 
mg/kg) or other animals. 

    Groups of chicks (40 birds per group) were fed diets containing 
ochratoxin A levels in the range of 0-8 mg/kg for 3 weeks (Huff et 
al., 1974).  The group receiving the highest dose (8 mg/kg feed) 
showed decreased packed blood cell volume, haemoglobin 
concentration, and serum-iron and serum-transferrin saturation 
percentages.  In a similar study, Chang et al.(1979) observed that 
lymphocytopenia developed at all dose levels.  In the same study, 
decreased bone strength, as measured physically by resistance to 
fracturation, was observed at feed levels of 2-4 mg ochratoxin A/kg 
(Huff et al., 1980).  When groups of chicks were fed ochratoxin A 
at levels of 0, 2, or 4 mg/kg for 20 days, concentrations of serum-
immunoglobulins (IgA, IgG, IgM) were reduced to 57-66% of normal 
values in the toxin-exposed groups (Dwivedi & Burns, 1984). 

    In B6C3F1 female mice, groups of 6-7 animals were administered 
a total of 0, 20, 40, or 80 mg ochratoxin A/kg body weight ip on 
alternate days over an 8-day period.  A dose-related decrease in 
thymic mass was observed as well as myelotoxicity, indicated by 
bone marrow hypocellularity, due to decreased marrow pluripotent 
stem cells, and granulocyte-macrophage progenitors (Boorman et al., 

    When determining the LD50 in mice following intraperitoneal 
injection, synergistic effects were observed when ochratoxin A was 
combined with citrinin as well as with penicillic acid (Sansing et 
al., 1976).  An additive effect was observed between ochratoxin A 
and citrinin in terms of embryotoxicity in chicken embryos (Vesela 
et al., 1983).  In beagle dogs, when combined oral doses of 
ochratoxin A (0.1-0.2 mg/kg body weight) and citrinin (5-10 mg/kg)  
were injected intraperitoneally, synergism was observed with regard 

to severity of clinical disease and mortality (Kitchen et al., 
1977a,b).  Increased toxicity in terms of LD50 values and 
pathological changes was observed in rats, when the ochratoxin A 
was given orally combined with either of the drugs biscoumacetate 
or phenylbutazone, apparently because of displacement of the toxin 
from binding sites on plasma-proteins (Galtier et al., 1980).  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). 

I.4.2.2  Teratogenicity

    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 a 
combination of ochratoxin A (2 or 4 mg/kg body weight) and T-2 
toxin (0.5 mg/kg body weight) was injected intraperitoneally in 
mice on gestation day 8 or 10, ochratoxin A exacerbated the 
incidence of T-2-induced gross malformations (tail and limb 
anomalies); increased fetocidal effects were also noted (Hood et 
al., 1978).  Mice were given 3-5 mg ochratoxin A/kg body weight 
intraperitoneally or orally on gestation days 8, 9, and 10 or 15, 
16, and 17 (Szczech & Hood, 1981).  Cerebral necrosis was found in 
most fetuses from dams treated on days 15-17, but no cerebral 
necrosis developed after treatment on days 8-10, when ochratoxin A 
is overtly teratogenic.  Pups of mice given 1.25 and 2.25 mg 
ochratoxin A/kg orally on gestation days 15, 16, and 17 were tested 
for surface righting, swimming, and pivoting (Poppe et al., 1983).  
The results of all 3 tests indicated that a delay in development 
had occurred; no dose-related pathological alterations were found. 

    Pregnant mice were administered 1-2 mg ochratoxin A/kg body 
weight, and/or 5-20 mg zearalenone/kg body weight or 0.125 mg 
diethylstilboestrol/kg body weight, orally, on day 9 of pregnancy, 
either individually or in combination, and the offspring were 
examined on day 19 (Arora et al., 1983).  Teratogenic effects 
produced by ochratoxin A, such as exencephaly, open eyelids, and 
microphthalmia, were reduced or absent when the toxin was given in 
combination with one of the 2 non-steroidal estrogenic substances, 
zearalenone or diethylstilboestrol. 

    Rats were treated orally with ochratoxin A at 0.25, 0.50, 0.75, 
1, 2, 4, or 8 mg/kg body weight on gestation days 6-15 (Brown et 
al., 1976).  Maternal toxicity was not observed below 4 mg/kg body 
weight, but an increased incidence of fetal resorptions was 
observed from 0.75 mg/kg body weight.  All fetuses from dams given 
0.25-0.75 mg/kg body weight weighed significantly less than control 
fetuses, and fetuses from dams given 0.75 or 1 mg/kg body weight 
were stunted.  Reduced litters and decreased fetal weight were 
observed in rats administered 5 mg ochratoxin A/kg body weight, 
orally, on gestation day 8 (Moré et al., 1978). 

    Subcutaneous administration of ochratoxin A to rats (1.75 mg/kg 
body weight) on gestation days 5-7 resulted in the highest number 
of malformations, including hydrocephaly, omphalocele, and 
anophthalmia as well as a shift in position of the oesophagus 
(Mayura et al., 1982); lower doses (0.5 and 1 mg ochratoxin A/kg 
body weight) did not have any teratogenic effects, and higher doses 
(5 mg/kg) caused all fetuses to be resorbed.  In a subsequent study 
by the same authors (Mayura et al., 1983), using the same 
ochratoxin A exposure conditions, it was shown that a diet low in 
protein (10% of protein concentration in normal rat feed) enhanced 
the teratogenic action of ochratoxin A in the rat.  The combined 
action of ochratoxin A exposure and a low protein diet also 
resulted in decreases in mating and fertilization rates (22% and 
39%, respectively) compared with the control group. 

    When rats were exposed to ochratoxin A and citrinin (another 
nephrotoxic mycotoxin produced by species of the  Aspergillus and 
 Penicillium genera) either singly or combined, enhanced teratogenic 
effects were observed in terms of gross malformations, visceral 
anomalies, and skeletal defects following combined oral 
administration of 1 mg ochratoxin A/kg body weight and 30 mg 
citrinin/kg body weight (Mayura et al., 1984).  Maternal deaths 
(22-40%) occurred after the administration of the combined dose on 
days 5, 6, 7, and 14 of gestation, whereas administration of 
individual toxins did not cause any maternal deaths and only 
minimal malformations no matter which gestation day they were 

    Increased prenatal mortality and malformations, including 
hydrocephaly, micrognathia, and heart defects, were observed in 
hamsters injected intraperitoneally with ochratoxin A at doses of 
5-20 mg/kg body weight on one of gestation days 7-9 (Hood et al., 

I.4.2.3  Mutagenicity

    Ochratoxin A did not have any effects in a  Bacillus subtilis 
Rec- assay, measuring DNA damage when tested at 20 and 100 µg/plate 
(Ueno & Kubota, 1976).  Ochratoxin A was not mutagenic to 
 Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, or TA 100 
at doses of up to 500 µg/plate, with or without exogenous metabolic 
activation (Kuczuk et al., 1978; Wehner et al., 1978b).  No 
increase was observed in genetic changes at the ade 2 locus of 
 Saccharomyces cerevisiae after treatment with 50 or 100 µg/plate 
ochratoxin A, with or without exogenous metabolic activation 
(Kuczuk et al., 1978).  Ochratoxin A did not induce  mutations  to 
8-azaguanine resistance in C3H mouse mammary carcinoma cells (FM3A) 
treated with doses of 5 or 10 µg/litre (Umeda et al., 1977). 

    When rats were orally exposed to ochratoxin A every 48 h for 12 
weeks (corresponding to a feed level of 4 mg/kg), single-strand 
breaks of DNA in renal and hepatic tissue (the only tissues 
investigated) were more pronounced than those in control animals 
(Kane et al., 1986a,b). 

    Contradictory results have been obtained by testing ochratoxin 
A in the  Salmonella assay (SOS chromo test without metabolic 
activation) (Ueno et al., in press). 

I.4.2.4  Carcinogenicity

    Kanizawa & Suzuki (1978) have indicated that ochratoxin A is a 
hepatic and renal carcinogen in male mice.  A group of 10 male ddY 
mice were fed a diet containing 40 mg ochratoxin A/kg for 44 weeks.  
A group of 10 untreated controls were fed the basal diet.  All 
survivors were killed after 49 weeks.  Hepatic cell tumours were 
found in 5 out of 9 treated mice; no tumours were found in the 10 
controls.  Solid renal cell tumours were found in 2 out of 9 
treated mice and none in the 10 controls.  Cystic renal adenomas 
were found in 9 out of 10 treated mice compared with none in the 10 

    Two groups of 50 male and 2 groups of 50 female B6C3F1 mice 
were fed diets containing 1 or 40 mg ochratoxin A/kg, respectively; 
one group (control) was fed the basal diet.  All survivors were 
killed after 24 months.  Eleven out of 49 male mice in the 40 mg/kg 
group had renal carcinomas; 24 out of 49 male mice in the 40 mg/kg 
groups showed renal adenomas.  All male mice in the 40 mg/kg group 
had microscopic evidence of nephropathy.  A few females in the 40 
mg/kg group exhibited nephropathic changes but no carcinomas or 
adenomas.  Compound-related lesions were absent in the controls  
and the 1 mg/kg groups (Bendele et al., 1985).  In a test for the 
development of hyperplastic liver nodules in rats, ochratoxin A was 
characterized as having both an initiating and a promoting activity 
(Imaida et al., 1982). 

    Seven groups of 16 male ddY mice each were fed a diet 
containing 50 mg ochratoxin A/kg for various periods ranging from 0 
to 30 weeks followed by feeding of the basal diet until the end of 
70 weeks (Kanizawa, 1984).  After 15 weeks of ochratoxin A 
exposure, 3 out of 15 animals had renal cell tumours; after 20 
weeks, 1 out of 14 mice had renal cell tumours and 2 had hepatomas; 
after 25 weeks of exposure, 2 out of 15 mice had renal cell tumours 
and 5 had hepatomas; and after 30 weeks of exposure, 4 out of 17 
mice had renal cell tumours and 6 had hepatomas; these tumours were 
not observed in the controls.  The nature of the renal cell tumours 
was not further defined, but such tumours are mostly malignant.  In 
addition, pulmonary tumours were found in all groups, including the 
controls, the incidence ranging from 20 to 73%.  In a second study, 
the effects of a combination of ochratoxin A and citrinin were 
elucidated.  Groups of 20 male ddY mice were fed diets containing 
25 mg ochratoxin A/kg in  combination  with  100  or  200  mg  
citrinin/kg  for  70 weeks.  Control groups were fed diets that did 
not contain any toxins or the individual toxins at the 
concentrations indicated above.  In addition, one group was fed 25 
mg ochratoxin A/kg feed for the first 25 weeks followed by 200 mg 
citrinin/kg feed for the remaining period of time;  another group 
was exposed to the 2 toxins in the reverse order.  Exposure to 
citrinin alone did not produce any tumours.  Exposure to ochratoxin 
A alone resulted in renal cell tumours in 6 out of 20 mice, and in 

hepatomas in 8 mice.  Exposure to ochratoxin A and 100 mg 
citrinin/kg feed did not result in any renal cell tumours, but 10 
out of 19 mice had hepatomas.  Exposure to ochratoxin A and 200 mg 
citrinin/kg feed resulted in renal cell tumours in 10 out of 18 
mice, and in hepatomas in 7 mice.  Exposure to one toxin followed 
by the other toxin did not produce any renal cell tumours, but 
hepatomas were observed in less than 20% of the mice. 

    On the basis of these studies, IARC concluded that there was 
limited evidence of carcinogenicity for animals, and inadequate 
evidence of carcinogenicity for human beings (IARC, 1987a). 

    Groups of 80 F 344/N rats of each sex were administered 0, 21, 
70 or 210 µg ochratoxin A/kg in corn oil by gavage, 5 days per 
week, for 103 weeks (Boorman, 1988).  In the male rats, renal 
carcinomas were found in 16 out of 51 animals dosed with 70 µg/kg 
and in 30 out of 50 animals dosed with 210 µg/kg; no carcinomas 
were found in lower dose groups.  In the female rats, renal 
carcinomas were less common, as 1 out of 50 animals dosed with 70 
µg/kg and 3 out of 50 animals dosed with 210 µg/kg had carcinomas; 
no carcinomas were found in the lower dose groups.  Renal adenomas 
were found in all groups of male rats, with increasing frequencies 
associated with increased doses.  In the female groups, renal 
adenomas were only found in the two highest dose groups.  In the 
female rats, fibroadenomas in the mammary gland were found in 45-
56% in the treated groups, a significantly higher percentage than 
in the control group. 

I.4.2.5  Biochemical effects and mode of action

    Ochratoxin A is an inhibitor of tRNA synthetase and protein 
synthesis in several microorganisms ( Bacillus subtilis, B. 
 stearothermophilus, Streptococcus faecalis, yeasts) as well as in 
rat hepatoma cells (Konrad & Roschenthaler, 1977; Bunge et al., 
1978; Heller & Roschenthaler, 1978; Creppy et al., 1979a,b).  The 
competitive inhibitor effect of ochratoxin A on tRNA synthetase and 
protein synthesis in rat hepatoma cells can be prevented by the 
addition of phenylalanine in the cell culture medium at a molar 
ratio of phenylalanine: ochratoxin A of 1.7:1 (Creppy et al., 
1979b).  This observation suggested the possibility of preventive 
measures for ochratoxin A-induced disease.  Thus, the acute 
intraperitoneal effect of ochratoxin A (LD100) in mice was 
prevented by concomitant injection of phenylalanine (Creppy et al., 
1980; Moroi et al., 1985). 

    However, in the study on the reproduction of porcine 
nephropathy previously mentioned (Krogh et al., 1974a), the molar 
ratio of phenylalanine: ochratoxin A in the feed exceeded 4600:1, 
implying that, in the field situation, phenylalanine does not 
prevent ochratoxin A from inducing the development of nephropathy. 

    Ochratoxin A in the concentration range studied (20-1667 µmol/ 
litre) caused a 47-50% inhibition of macrophage migration (Klinkert 
et al., 1981); this effect could be prevented by the simultaneous 

addition of phenylalanine.  In BALB/c mice, a dose of ochratoxin A 
as low as 0.005 µg/kg body weight was able to suppress the immune 
response to sheep erythrocytes (Haubeck et al., 1981); the effect 
could be prevented by the simultaneous addition of phenylalanine.  
Studying the same effect in Swiss mice, Prior & Sisodia (1982) were 
unable to show any suppression of the immune response to sheep 
erythrocytes, even after daily injection of 5 mg ochratoxin A/kg 
for 50 days.  (4R)-4-Hydroxyochratoxin A at a dose of 1 µg/kg body 
weight caused an 80% reduction in the number of cells producing IgM 
and a 93% reduction in cells producing IgG in BALB/c mice compared 
with 90% and 92%, respectively, for ochratoxin A (Creppy et al., 

    Female B6C3F1 mice (6 per group) were administered ochratoxin A 
in amounts of 0.34, 6.7, or 13.4 mg/kg body weight or ochratoxin B 
(13.4 mg/kg body weight) 6 times during 12 days.  Ochratoxin A 
inhibited the natural killer cell activity at all dose levels, and  
increased the growth of transplantable tumour cells without 
affecting T-cell or macrophage-mediated anti-tumour activity 
(Luster et al., 1987).  Ochratoxin B did not influence immune 
function.  The inhibition by ochratoxin A of natural killer cell 
activity appeared to be caused by reduced production of basal 

    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, 

    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 activity decreased and the liver phosphorylase (EC activity increased.  Three daily oral doses of ochratoxin 
A at 5 mg/kg body weight caused a decrease in the liver glycogen 
concentration, which was 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 on 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 studies 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 orally 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.  In this respect, ochratoxin A acted synergistically 
with another nephrotoxic mycotoxin, citrinin. 

    When neonatal rats were exposed orally to a single dose of 1 mg 
ochratoxin A/kg or of 25 mg citrinin/kg or both doses within 24 h 
of birth, a synergistic effect of the 2 mycotoxins was observed on 
cytochrome P-450, NADPH-dependent dehydrogenase, and NADPH-
cytochrome  c reductase (Siraj et al., 1981). 

    In rats fed 2 mg ochratoxin A/kg body weight per day for 2 
days, renal gluconeogenesis from pyruvate was decreased by 26%, and 
renal phosphoenol-pyruvate carboxy kinase (PEPCK) (EC 
activity was reduced by 55%, whereas hepatic PEPCK was unchanged 
(Meisner & Selanik, 1979).  In a subsequent study on rats, it was 
found that even lower dose levels (0.3-0.5 mg ochratoxin A/kg body 
weight) caused a 50% reduction in PEPCK activity (Meisner & 
Meisner, 1981).  A number of other enzymes located in the proximal 
tubule of the nephron were unaffected.  When longer exposure 
periods (8-12 weeks) were used, and 145 ng ochratoxin A/kg body 
weight were administered orally to groups of 3 rats each, increased 
urinary excretion and corresponding renal tubular depletion of the 
following enzymes were observed:  gamma-glutamyl transferase, 
alkaline phosphatase, leucine aminopeptidase, lactate 
dehydrogenase, and  N-acetyl-beta- D-glucosaminidase (Kane et al., 
1986a).  Renal PEPCK in pigs was also sensitive to ochratoxin A 
with a feed level of 100 µg/kg causing a significant decrease in 
PEPCK activity (Meisner & Krogh, 1982). 

    In a study on pigs fed ochratoxin A at 0, 0.2, or 1 mg/kg for 5 
weeks, enzyme activities were measured in renal biopsies, collected 
1, 3, and 5 weeks after initiation (Krogh et al., 1988).  After one 
week, the activities of renal PEPCK and gamma-glutamyltranspeptidase 
were decreased by 40%.  The dose-related decrease in the activity 
of PEPCK and gamma-glutamyltranspeptidase was accompanied by a 
dose-related increase in renal impairment, as measured by the 
reduction of TmPAH/Cin, suggesting that these enzymes are sensitive 
indicators of ochratoxin-induced porcine nephropathy.  Thus, the 
renal biopsy-based measurements of enzyme activities might prove 
diagnostically useful in ochratoxin-induced disease in human 

    Ochratoxin A reduced the total renal mRNA concentration in male 
Sprague-Dawley rats and certain mRNA species, notably PEPCK, were 
reduced to a greater extent than the bulk of the RNA pool (Meisner 
et al., 1983). 


I.5.1  Ochratoxin A, Balkan endemic nephropathy, and tumours of the 
urinary system

    This topic has been reviewed by Krogh (1979, 1983) who called 
attention to the striking similarities in the changes in renal 
structure and function induced experimentally in animals by the 
administration of ochratoxin A, and the clinical and pathological 
features of a localized endemic disease known as Balkan endemic 
nephropathy.   So far, the disease has been observed only in rural 
populations of Bulgaria, Romania, and Yugoslavia, but information 
on the present magnitude of the problem was not available to the 
Task Group. 

    The Balkan endemic nephropathy is a chronic disease that 
predominantly affects women and progresses slowly up to death 
(Hrabar et al., 1976, Chernozemsky et al., 1977).  Age-specific 
incidence rates are highest above the age of 40.  Younger cases 
occur in the 10-19-year-old age group, and the mean age of new 
patients is in the early 50s (Stoyanov et al., 1978). 

    The disease is characterized by an extreme geographical 
clustering with a tendency for familial aggregation of cases 
(Nicolov et al., 1978).  However, sporadic cases occur outside 
endemic areas.  Age-adjusted incidence rates of 555 per 100 000 
population in females and 322 in males over a ten-year period have 
been recorded from a population sample of 147 000 in an endemic 
area of Bulgaria (Stoyanov et al., 1978).  In one of several 
endemic regions in Yugoslavia, the prevalence varied from 3% to 8% 
(Hrabar et al., 1976). 

    Autopsy has shown that kidneys are notably reduced in size.  
The histological lesions are interstitial fibrosis, tubular 
degeneration, and hyalization of glomeruli in the more superficial 
part of the cortex (Heptinstall, 1974). Impairment of tubular 
function, indicated by a decrease in TmPAH, is a prominent and 
early sign (Dotchev, 1973). 

    A high incidence of tumours of the urinary system is strongly 
correlated with the prevalence of Balkan endemic nephropathy 
(Ceovic et al., 1976; Chernozemsky et al., 1977; Nicolov et al., 
1978).  In one instance in Bulgaria, 46.6% of patients with tumours 
of the urinary system were also affected by endemic nephropathy.  
Among the tumours of the urinary system, cancers of the renal 
pelvis and ureters are more frequently associated with endemic 
nephropathy than urinary bladder tumours. 

    The relative risk for developing cancer of the renal pelvis and 
ureters is 88:1 in patients with nephropathy compared with controls 
in non-endemic areas.  The relative risk for the development of any 
tumour of the urinary system is only 28:1 in the same sample 
(Stoyanov et al., 1978). 

    Over the past 2 decades, investigations have been carried out 
to verify a variety of etiological assumptions with unconvincing 
results (review by Puchlev, 1973, 1974).  However, the assumption  
of the possible role of ochratoxin A, on the basis of similarities 
with the animal disease, has received increasing epidemiological 

    In Yugoslavia, surveys indicated that the contamination of 
foodstuffs (grains, maize, pork meat) with ochratoxin A occurred in 
12.8% of samples in an area where the prevalence of endemic 
nephropathy was 7.3%, compared with only 1.6% of contaminated 
samples in areas free of the disease (Krogh et al., 1977).  The 
concentration of ochratoxin A in maize was 5-90 µg/kg and that in 
pork meat, 5 µg/kg (Krogh et al., 1977) with levels of up to 27 
µg/kg in pig kidneys (Pepeljnjak et al., 1982).  Similarly, studies 
in Bulgaria have revealed that 16.7% of beans and 27.3% of maize 
from an endemic area were contaminated with ochratoxin A compared 
with 7.1% and 9%, respectively, from a non-endemic area (Petkova-
Bocharova & Castegnaro, 1985). 

    In a subsequent survey of home-produced foodstuffs (cereal and 
bread) from the same endemic area in Yugoslavia over a 5-year 
period, a mean contamination of 8.7% was found with pronounced 
annual variations, which probably reflect climatic conditions 
during the crop harvesting periods (Pavlovic et al., 1979). 

    Surveys on the presence of ochratoxin A in blood samples are 
difficult to compare, as different analytical methods have been 
used with different levels of sensitivity.  Prevalences of 16.6% 
and 5.9% with a one-year interval were reported in the same endemic 
village in Yugoslavia (Hult et al., 1982).  It is not possible to 
determine whether this difference reflects annual variations in the 
content of the blood, or the result of a less sensitive analytical 
method in the second instance.  However, higher prevalences of 
ochratoxin A and higher concentrations in blood are generally 
present in people from endemic areas, especially in persons 
suffering from Balkan nephropathy. 

    A survey in Yugoslavia reported a prevalence in the blood of 
16.6% in an endemic village and 6% in a non-affected one (Hult et 
al., 1982).  In Bulgaria, reported rates were 17.7% in an endemic 
area and 7.7% in a non-endemic one (Petkova-Bocharova et al., 
1988).  Table 9 illustrates the trend towards higher concentrations 
in endemic situations and in patients.  The similarity of results 
in healthy families from affected villages and healthy persons from 
unaffected villages (groups III and IV) suggests than an 
environmental or a behavioural determinant plays a role at the 
household level. 

    Thus, available epidemiological information seems to indicate 
that Balkan endemic nephropathy is associated with consumption 
patterns involving foodstuffs contaminated with ochratoxin A and 
with a higher frequency of positive blood samples of ochratoxin A.  
However, the association does not permit the establishment of a 

causal relationship. Cross sectional surveys, such as those 
reported in the literature so far, are probably not the appropriate 
means to determine this relationship. 

    The results of experimental animal studies suggest that Balkan 
nephropathy, a chronic condition, may require a long latency period 
between exposure and the onset of symptoms, or more likely a 
prolonged exposure or repeated exposure over a long period of time. 

    Investigations based on individual exposure time sequences 
together with follow-up cohort surveys could provide a clue to the 
causal role of ochratoxin A in Balkan endemic nephropathy.  In view 
of the lack of this information, such a causal relationship cannot 
be established or rejected. 

Table 9.  Ochratoxin A in blood samples from people in endemic and 
non-endemic areas in Bulgariaa
Groupb             No. of       No. of          Mean concentration
                   persons      ochratoxin A    ± S.D. (µg/kg)
                   assayed      positive cases
I. Persons with    61           16 (26.3)       20.3 ± 9.7
UST and/or EN

II. Healthy        63           10 (15.8)       14.5 ± 7.6
persons from
families with UST
and/or EN cases

III. Healthy       63           7 (11.1)        12.5 ± 3.5
persons from
families in
endemic villages

IV. Healthy        60           7 (11.6)        15.0 ± 4.2
persons from
unaffected villages
in endemic areas

V. Healthy         65           5 (7.7)         10.0
persons from
villages in
non-endemic areas
a  From: Petkova-Bocharova et al. (1988).
b  UST: urinary system tumours; EN: endemic nephropathy.
   The differences between group I and groups III and IV are 
   statistically significant ( P <0.002).  Difference between 
   groups I and V is statistically significant ( P <0.001).


    Human exposure, as demonstrated by the occurrence of ochratoxin 
A in food and in the blood, has been observed in various countries 
in Europe.  The Task Group was not aware of attempts to detect 
ochratoxin A in human blood in other parts of the world. 

    The causal role of ochratoxin A in porcine nephropathy has been 
established, based on studies of field cases as well as 
reproduction of the disease with ochratoxin A.  Using the porcine 
model, it has been postulated that Balkan endemic nephropathy may 
result from exposure to ochratoxin A.  Available epidemiological 
information indicates that Balkan nephropathy may be associated 
with the consumption of foodstuffs contaminated by this toxin.  
Since the publication of Environmental Health Criteria 11, in 1979, 
epidemiological studies on the concentration of ochratoxin A in 
human blood in affected and non-affected areas, have provided 
additional support for the relationship between Balkan nephropathy 
and exposure to ochratoxin A. 

    It has been shown that both the prevalence of ochratoxin A in 
the blood and the blood concentrations are higher in residents in 
endemic areas.  However, a direct causal relationship cannot be 
established on the basis of indirect evidence provided by the above 
retrospective studies alone.  Neither can it be excluded in view of 
the long latency period between the exposure and the onset of 

    Ochratoxin A has been demonstrated to be carcinogenic to the 
renal tubular epithelium in male mice and both sexes of rats.  A 
highly significant relationship has been observed between Balkan 
nephropathy and tumours of the urinary tract, particularly with 
tumours of the renal pelvis and ureters.  However, there are no 
published data to establish a direct causal role of ochratoxin A in 
the etiology of such tumours. 



II.1.1  Physical and chemical properties

    The  sesquiterpenoid trichothecenes possess the tetracyclic 
12,13-epoxytrichothecene skeleton.  A total of 148 trichothecenes, 
83 non-macrocyclic and 65 macrocyclic, have been isolated from 
fungal cultures and plants (Drove, 1988).  They can be conveniently 
divided into 4 categories according to similarity of functional 
groups (Ueno, 1977).  The first class is characterized by a  
functional group other than a ketone at C-8 (type A).  This is the 
largest category containing members such as T-2 toxin and 
diacetoxyscirpenol (DAS).  The second category of trichothecenes 
has a carbonyl function at C-8 (type B) typified by 4-deoxynivalenol 
(DON) and nivalenol (NIV).  The third category is characterized by 
a second epoxide group at C-7,8 or C-9,10 (type C), and the fourth 
contains a macrocyclic ring system between C-4 and C-15 with two 
ester linkages (type D).  The structures of representative 
trichothecenes of each category are illustrated below. 





II.1.1.1  Physical properties

    The trichothecenes are colourless, mostly crystalline solids 
that have been well characterized by physical and spectroscopic 
techniques (Cole & Cox, 1981).  The type A trichothecenes are 

soluble in moderately polar solvents, such as chloroform, diethyl 
ether, ethyl acetate, and acetone, whereas the more polar type B 
trichothecenes require higher polarity solvents, such as aqueous 
methanol or aqueous acetonitrile.  Some physical properties of the 
main trichothecenes are summarized in Table 10. 
Table 10.  Some physical properties of main trichothecenes
Trichothecenes     Molecular   Relative   Melting   (alpha)20D   References
                   formula     molecular  point
                               mass       (°C)
T-2 toxin          C24H34O9    466        151-152   +15          Bamburg et
                                                                 al. (1968b)
HT-2 toxin         C22H26O8    424        -         -            Bamburg &
                                                                 Strong (1969)
Diacetoxyscirpenol C19H26O7    366        162-164   -27          Sigg et al.
Neosolaniol        C19H26O8    382        171-172   -            Ishii et al.
Deoxynivalenol     C15H20O6    296        151-153   +6.35        Yoshizawa &
                                                                 Morooka (1973)
Nivalenol          C15H20O7    312        222-223   +21.54       Tatsuno et
                                                                 al. (1968)
Trichothecin       C19H24O5    332        118       +44          Freeman (1955)

Fusarenon-X        C17H22O8    354        91-92     +58          Ueno et al.
Roridin A          C29H40O9    532        198-204   +130         Harri et al.
Satratoxin H       C29H36O9    528        162-166   -            Eppley &
                                                                 Bailey (1973)
Verrucarin A       C27H34O9    502        360       +20.6        Gutzwiller &
                                                                 Tamm (1965)
    Most of the trichothecenes lack conjugated unsaturation in their 
structures with a consequent absence of absorption in the ultraviolet 
(UV) spectrum, except for end absorption due to unsaturation at C-9.  
This lack of absorbance is a source of difficulty in achieving 
sensitive and specific detection in HPLC analysis.  In contrast, the 
type D trichothecenes give characteristic ultraviolet spectra. 

II.1.1.2  Chemical properties

    When trichothecenes containing an ester group are treated with a 
base, they are hydrolysed to their corresponding parent alcohol (Wei 
et al., 1971).  Free hydroxyl groups are readily acylated.  The 
12,13-epoxy group is itself extremely stable to nucleophilic attack.  
However, prolonged boiling under highly acidic conditions causes an 
intramolecular rearrangement of the trichothecene skeleton to the 
apotrichothecene ring.  Detailed discussion of the chemistry of 
trichothecenes can be found in reviews by Bamburg & Strong (1971), 
Bamburg (1976), and Tamm (1977). 

    The trichothecenes are generally stable; for example, DON can be 
stored in organic solvents, such as ethyl acetate, for a long time 
without any significant deterioration (Shepherd & Gilbert, 1988).  
They remain unaffected when refluxed with various organic solvents 
and also under mildly acidic conditions. 

II.1.2  Analytical methods for trichothecenes

    Analytical methods have been reviewed by Scott (1982) and Pohland 
et al. (1986).  Selected examples of recently published analytical 
methods for type A trichothecenes and type B trichothecenes are 
summarized in Tables 11 and 12 respectively.  Although some of the 
multi-trichothecene methods included in Table 11 may also be 
applicable to certain of the type B compounds, the procedures in 
Table 12 have been developed exclusively for the type B toxins. 

II.1.2.1  Chemical methods

 (a)  Extraction

    The most commonly used extraction solvents for trichothecenes are 
chloroform, ethyl acetate, methanol, acetonitrile, aqueous methanol, 
and aqueous acetonitrile.  Chloroform, ethyl acetate, and 
acetonitrile have been successfully used for the extraction of T-2 
toxin, DAS, and some of their partially hydrolysed derivatives in 
naturally contaminated cereals.  Aqueous methanol and aqueous 
acetonitrile are the solvents of choice for the extraction of several 
trichothecenes of widely differing polarity as well as for the 
extraction of type B toxins alone.  Methods differ according to the 
type of solvent used, whether samples are homogenised in a blender 
with the solvent or agitated with a wrist action shaker, and in the 
length of time of the extraction process.  Spiking of samples with 
standards is not an adequate way of demonstrating the efficiency of 
extraction, and only methods validated with naturally contaminated 
material can be regarded as having been rigorously tested.  
Extraction procedures have been assessed for DON (Trenholm et al., 
1985) and it has been demonstrated that longer extraction times are 
required for naturally contaminated samples than for those that have 
been spiked (at least 120 min shaking).  It has also been shown that 
aqueous acetonitrile gives a cleaner extract than aqueous methanol. 

 (b)  Clean-up procedures

    The extent of sample clean-up required for a particular assay 
depends on the specificity of the detection procedure and the nature 
of the sample matrix.  The less specific detection methods, such as 
TLC, require extensive sample clean-up while more sophisticated  
approaches,  such  as  mass  spectrometry  and immunoassay, may only 
require minimal sample preparation. 

Table 11.  Selected methods for the determination of T-2 toxin and diacetoxyscirpenol in 
biological materials 
Matrix       Extractiona  Clean-upb     Assayc         Detection  Toxins assayed     References
                                                       limit      -----------------
                                                       (µg/g)     T-2   DAS  Others
Cereals      MeOH/H2O     XAD-4; flor.  TLC            0.5        +     +    7       Kamimura et 
                                                                                     al. (1981)
Cereals      EtOAC        prep. TLC     HPTLC          0.2        +     -    2       Ilus et 
                                                                                     al. (1981)
Foods        MeCN/H2O     char/alum     TLC            1.0        +     +    5       Romer 
Cereals      MeCN/KCl     Sep P         HPLC(RI)       1.0        +     -    1       Schmidt & 
                                                                                     Dose (1984)
Wheat/rye    MeCN         Bond-Elut     LC/MS (therm)  0.04       +     +    2       Rajakyla et 
                                                                                     al. (1987)
Plasma       EtOAC        Sep P/sil.g   LC/MS (therm)  0.002      +     +    2       Voyksner et 
                                                                                     al. (1985)

Cereals      EtOAC        sil.g         GC (FID)-TMS   0.1        +     +    3       Bata et al.
Cereals      MeOH/H2O     sil.g/cy      GC (ECD)-HFB   0.05       +     +    1       Cohen & 
Plasma       benzene      flor.         GC (ECD)-HFB   0.02       +     -    -       Swanson et 
                                                                                     al. (1983)

Milk         EtOAC        prep. TLC     GC/MS-TMS      0.003      +     -    -       Collins &
Corn         MeOH         Sep P/sil.g   GC/MS-TMS      0.02       +     +    1       Rosen & 
Foods        MeOH         flor.         GC/MS-HFB      0.01       +     +    11      Black et 
                                                                                     al. (1987)
Urine        EtOAC/MeOH   Sep P         GC/MS-HFB      0.005      +     +    5       Black et 
                                                                                     al. (1986)
Blood        acetone      Sep P         GC/MS-PFP      0.005      +     +    9       Begley et 
                                                                                     al. (1986)

Table 11 (contd.)
Matrix       Extractiona  Clean-upb     Assayc         Detection  Toxins assayed     References
                                                       limit      -----------------
                                                       (µg/g)     T-2   DAS  Others
Milk/urine   EtOAC        Sep P         RIA            0.002      +     -    -       Lee & Chu
Corn/wheat   MeOH         Sep P         RIA            0.001      +     -    -       Lee & Chu
Urine        none         Sep P         ELISA          0.0005     +     -    -       Fan et al.
a  Extraction: MeOH (methanol); H2O (water); EtOAC (ethyl acetate); MeCN (acetonitrile);
   KCl (potassium chloride aqueous soln).
b  Clean-up: XAD-4 (amberlite XAD-4 resin); flor. (florisil); char/alum (charcoal/alumina 
   columns); Sep P (C18-Sep Pak); sil.g (silica gel); cy (cyano extraction column).
c  Assay: TLC (thin layer chromatography); HPTLC (high performance TLC); HPLC (high performance
   liquid chromatography); RI (refractive index); LC/MS (combined HPLC/mass spectrometry); 
   GC (gas chromatography); FID (flame ionisation detector); ECD (electron capture detector);
   TMS (trimethylsilyl derivative); HFB (hepafluorobutyrl ester derivative); 
   PFP (pentafluoropropionyl ester); RIA (radioimmunoassay); ELISA (enzyme linked 
   immunosorbent assay).

Table 12.  Selected methods for the determination of deoxynivalenol and nivelenol in biological
Matrix       Extractiona  Clean-upb     Assayc         Detection  Toxins assayed     References
                                                       limit      -----------------
                                                       (µg/g)     T-2   DAS  Others
Wheat/corn   MeCN/H2O     char/alum     TLC            0.04-0.1   +     -    -       Trucksess
                                                                                     et al. 
Foods        MeCn/H2O     char/alum;    TLC            0.5        +     -    -       Trucksess 
                          Sep P                                                      et al.
Cereals      MeCN/H2O/    char/alum;    HPTLC          0.05       +     +    1       Trucksess
             MeOH         ppt                                                        et al.
Corn/rice    MeOH/H2O/    liq/liq extr  HPLC (UV)      O.005      +     +    2       Visconti &
             NaCl                                                                    Bottalico
Corn/rice    MeOH/H2O     none          HPLC (ED)      0.025      +     -    -       Sylvia et
                                                                                     al. (1986)
Corn         MeOH/H2O     prep. TLC     HPLC (UV)      0.01       +     -    -       Ehrlich et
                                                                                     al. (1983)
Cereals      MeCN/H2O     ion/char/     HPLC (UV)      0.05       +     +    -       Lauren &
                          alum                                                       Greenhalgh
Cereals      MeOH         flor.         LC/MS (micro)  0.01       +     +    -       Tiebach et
                                                                                     al. (1985)
Cereals      MeOH/H2O     sil.g         GC (ECD)-TMS   0.02       +     +    -       Scott et
                                                                                     al. (1986)
Cereals      MeCN/H2O     flor/Sep P    GC (ECD)-TMS   0.002      +     +    -       Tanaka et
                                                                                     al. (1986)
Wheat        chlor/EtOH   sil.g         GC (ECD)-HFB   0.1        +     -    -       Ware et
                                                                                     al. (1986)
Cereals      chlor/EtOH   sil.g         GC (ECD)-HFB   0.02       +     -    -       Mulders &
                                                                                     Peek (1986)
Milk         EtOAc        Sep P         GC (ECD)-TMS   0.001      +     -    -       Swanson et
                                                                                     al. (1986)

Table 12 (contd.)
Matrix       Extractiona  Clean-upb     Assayc         Detection  Toxins assayed     References
                                                       limit      -----------------
                                                       (µg/g)     T-2   DAS  Others
Corn/barley  MeOH/H2O     sil.g         GC/MS-TMS      0.01       +     -    -       Gilbert et
                                                                                     al. (1983a)
Foods        MeOH/H2O     XAD-2/flor/   GC/MS-TMS      0.02       +     +    -       Yoshizawa &
                          Sep P                                                      Hosokawa
Corn/wheat   MeCN/H2O     none          ELISA          0.01       +     -    -       Xu et al.
a  Extraction: MeCN (acetonitrile); H2O (water); MeOH (methanol); NaCl (sodium chloride soln);
   chlor/EtOH (chloroform/ethanol); EtOAc (ethyl acetate).
b  Clean-up: char/alum (charcoal/alumina column); Sep P (C18-Sep Pak); ppt (lead acetate
   precipitation); liq/liq extr. (liquid/liquid extraction); ion (ion exchange resin); 
   flor. (florisil); sil.g (silica gel); XAD-2 (Amberlite XAD-2 resin).
c  Assay: TLC (thin layer chromatography); HPTLC (high performance TLC); HPLC (high performance
   liquid chromatography); UV (ultraviolet detection); ED (electrochemical detection); 
   LC/MS (combined microbore-HPLC/mass spectrometry); GC (gas chromatography); ECD (electron 
   capture detector); TMS (trimethylsilyl derivative); HFB (heptafluorobutyryl ester 
   derivative); ELISA (enzyme linked immunosorbent assay).
    Early methods that may have involved the use of conventional 
silica gel columns or preparative TLC for clean-up, have to a large 
extent been superseded by methods using prepacked cartridges, such as 
silica gel and C18-Sep Paks, which are both more reliable and more 
convenient.  Florisil columns are widely used for clean-up (e.g., see 
Tanaka et al., 1985a), and the one step clean-up procedures using 
alumina/charcoal (Romer, 1986) or alumina/charcoal/Celite columns 
(Trucksess et al., 1984) have become widely adopted, particularly for 
DON and NIV assays. 

 (c)  Detection and quantification

 (i)  Thin-layer chromatography (TLC).  The lack of native 
fluorescence or UV absorbance of the trichothecenes means that TLC 
detection relies on the use of spray reagents for visualisation.  
Characteristic colours or fluorescence can be produced with sulfuric 
acid or  p-anisaldehyde followed by heating at 110-120 °C (Scott et 
al., 1970; Ueno et al., 1973c).  A general spray reagent for the 
12,13-epoxy function is 4-( p-nitrobenzyl)pyridine, which produces a 
blue coloration on heating and treatment with a base (Takitani et 
al., 1979).  A more sensitive, but somewhat elaborate, procedure, 
which again is specific for the epoxide function, involves reaction 
with nicotinamide and 2-acetylpyridine to produce fluorescent TLC 
spots (Sano et al., 1982).  Diphenylindenone sulfonyl esters of 
trichothecenes can be formed prior to TLC and, subsequently, when 
sprayed with sodium methoxide can yield fluorescent spots at high 
sensitivity (Yagen et al., 1986). 

    Most of the above spray reagents, though frequently demonstrated 
as useful for the determination of standards or of relatively high 
concentrations of toxins in culture extracts, have not been well 
developed in conjunction with clean-up procedures for the 
determination of trichothecenes in naturally contaminated cereal 
samples or other foods.  However, the exception has been the use of 
an aluminum chloride spray reagent for visualising the type B 
trichothecenes (Kamimura et al., 1981).  On spraying the plates, 
heating for 10 min at 110 °C and then treating with a base, blue 
fluorescent spots are produced for DON, NIV, and fusarenon X.  Using 
this approach, in conjunction with a clean-up utilising an alumina 
charcoal Celite column, a quantitative TLC procedure was developed 
using a fluorodensitometer for determining DON in wheat and corn 
(Trucksess et al. 1984); DON in processed grain products including 
breakfast cereals, corn syrup, and beer (Trucksess et al., 1986b); 
and for simultaneously monitoring DON, NIV, and fusarenon X in 
barley, corn, and wheat (Trucksess et al. 1987).  This TLC procedure 
was successfully collaboratively tested (Eppley et al., 1986) and was 
accepted as the AOAC first action method for DON in wheat. 

 (ii)  High performance liquid chromatography (HPLC).  HPLC has not 
proved particularly appropriate for the type A trichothecenes, which 
lack any significant UV absorption, making sensitive detection 
difficult.  Refractive index detection has been employed, but at 
relatively high toxin levels (Schmidt & Dose, 1984).  UV detection at 
short wavelength has been used for the determination of DON and NIV 
in cereals, and a number of methods have been reported that offer an 
advantage over alternative GC approaches in that they do not require 

derivatization of the toxins.  A post-column derivatization procedure 
for DON and NIV has been developed (Sano et al., 1987) involving 
alkaline decomposition of the trichothecenes to generate formaldehyde 
and then reaction with methyl acetoacetate and ammonium acetate to 
form a fluorescent derivative.  Although more sensitive and specific 
than UV detection, the approach does have the disadvantage of 
requiring rather elaborate instrumentation, in order to carry out the 
post column reaction.  Electrochemical detection (Sylvia et al., 
1986) looks particularly promising for the determination of DON by 
HPLC, offering both greater sensitivity and specificity than UV 
detection, and the possibility of analysis of sample extracts that 
have received minimal sample clean-up. 

 (iii)  Gas chromatography (GC).  A large number of GC methods have 
been published that differ in the approach to sample extraction, in 
sample clean-up, and in choice of derivative prior to GC analysis.  
Trichothecenes containing a hydroxyl group require derivatization, 
and the heptafluorobutyryl (HFB) ester and trimethylsilyl (TMS) ether 
derivatives have been most frequently used in conjunction with 
electron capture detection.  Formation of the HFB derivatives is 
relatively time-consuming, but complete derivatization is easily 
achieved and the derivative once formed is stable for at least 
several days.   However, poor reproducibility has been noted with HFB 
derivatives of DON (Mulders & Impelen-Peek, 1986) attributed to 
adsorption on to glass surfaces.  The relatively high mass of the HFB 
derivatives can cause difficulties if GC/MS confirmation is required.  
In contrast, TMS  derivatives  are easily prepared, and are of 
suitable mass for GC/MS, but, for some  type B  toxins,  they may 
require optimization  of  conditions for complete derivatization 
(Gilbert et al., 1985).  Scott & Kanhere (1986) have compiled 
retention data for 10 different trichothecenes as both TMS and HFB 
derivatives on capillary columns of different stationary phases.  
Trifluoroacetyl derivatives of trichothecenes are preferred by some 
workers (Kientz & Verweij, 1986), and pentafluoropropionyl esters 
have been used, particularly where detection has been by MS (Begley 
et al., 1986; Krishnamurthy & Sarver, 1986; Rood et al., 1988a). 

    An area where improvement in quantification of trichothecenes is 
still needed is in the selection of adequate internal standards.  
Compounds, such as methoxychlor (Romer et al., 1978), 
hexachlorobiphenyl (Blass et al., 1984), and alkanes (Ilus et al., 
1981), have been used, but these differ significantly in structure 
from the trichothecenes.  The deuterated TMS derivative of T-2 toxin 
has been used as an internal standard in GC/MS analysis (Rosen & 
Rosen, 1984), an isomer of T-2 toxin has been chemically synthesized  
(Stahr et al., 1981) as have 4-deoxyverrucarol and 16-hydroxyver-
rucarol for use as internal stan-dards (Krishnamurthy et al., 1986). 

    Despite the many difficulties, a GC method using the HFB 
derivative has been successfully collaboratively tested (Ware et al., 
1986) and subsequently adopted as an AOAC official first action 

    Other related trichothecenes have been determined in foods by GC 
methods, for example, the de-epoxidised metabolite of DON called 
DOM-1 has been determined in milk as both its TMS and HFB derivative 

(Swanson et al., 1986).  Also, an isomer of DON was detected by 
capillary GC, as its TMS derivative in bread and breakfast cereal 
products prepared from flour naturally contaminated with DON 
(Greenhalgh et al., 1984). 

 (iv)  Mass spectrometry (MS).  Mass spectrometry has been used for 
the structural characterization of novel trichothecenes; for 
identification and confirmation of trichothecenes in biological 
materials, and as a sensitive and specific means of detection with 
GC, LC, or supercritical fluid chromatography (SFC) sample 

    The usefulness of negative ion chemical ionization (NICI) mass 
spectrometry with hydroxide ion reagent gas has been demonstrated in 
relation to producing relative molecular mass information, as well as 
fragment ions indicative of structure for type A and B trichothecenes 
(Brumley et al., 1982).  The NICI technique has been applied to 
confirmation of the presence of DON in cereals and snack foods by 
rapid capillary GC introduction of the underivatized extract 
obtaining full scan spectra (Brumley et al., 1985), and by selected 
ion monitoring (Miles & Gurprasad, 1985). 

    NICI has also been used for selected ion monitoring GC/MS of the 
HFB derivatives of 13 different trichothecenes of both type A and 
type B in extracts from small samples of biological material  
(Krishnamurthy et al., 1986).  GC/MS is the preferred approach using 
SIM in electron ionization (D'Agostino et al., 1986) or NICI (Begley 
et al., 1986) modes for biological samples where only small sample 
sizes are available but high sensitivity is required.  For more 
routine surveys of cereal and food samples, particularly for DON and 
NIV, initial screening has normally been carried out using GC with an 
ECD and only selected positive samples have been confirmed by GC/MS 
(Tanaka et al., 1985a; Cohen & Lapointe, 1982). 

    Attempts have been made to determine the macrocyclic 
trichothecenes as TMS derivatives on short fused silica GC columns 
using GC/MS (Rosen et al., 1986).  The approach has also been adopted 
of alkaline hydrolysis of the ring system of the macrocyclic 
compounds to yield verrucarols, which can then be determined as HFB 
derivatives by NICI GC/MS (Krishnamurthy et al., 1987).  A similar 
approach using alkaline hydrolysis has been proposed (Rood et al., 
1988a,b) as a basis for a general method for the determination of 
trichothecenes and metabolites by conversion to their corresponding 
parent alcohols prior to derivatization and GC or GC/MS 

    Although LC/MS is really a research method, thermospray LC/MS is 
becoming more widely available and shows promise for the 
identification of low levels of trichothecenes in biological 
materials (Voyksner et al., 1985; Rajakyla et al., 1987).  Another 
promising research method for the determination of both the simple 
and the macrocyclic trichothecenes is supercritical fluid 
chromatography (SFC) combined with MS (Smith et al., 1985).  
Supercritical fluids can also be used for the on-line extraction from 
biological materials and direct introduction into the MS to monitor a 
number of different trichothecenes (Kalinoski et al., 1986). 

II.1.2.2  Immunological methods

    Chu et al. (1979) developed a radioimmunoassay (RIA) for T-2 
toxin.  The antibody preparation was obtained by immunizing rabbits 
with bovine serum albumin T-2 toxin hemisuccinate conjugate. The 
antibody had the greatest binding efficiency for T-2 toxin and less 
efficiency for HT-2 toxin.  Cross reaction with other trichothecenes 
was either very slight or absent, and the limit of detection of the 
assay ranged from 1 to 20 ng. 

    This RIA was applied to the determination of T-2 toxin in 
agricultural commodities, biological fluids, and animal organs (Lee & 
Chu, 1981a,b; Hewetson et al., 1987).  Xu et al. (1988) also reported 
an immunoassay using an antibody against triacetylated DON, for the 
determination of DON in wheat and corn with a limit of detection of 
about 0.02 µg/g. 

    A polyclonal enzyme-linked immunosorbent assay (ELISA) was 
developed for the rapid quantification of T-2 toxin in food and 
animal feed (Pestka et al., 1981).  This assay, which could be 
undertaken in 2 h, had a limit of detection of 2.5 pg/assay and was 
used for the detection of T-2 toxin in  Fusarium-infected corn 
(Gendloff et al., 1984).  More recently monoclonal ELISAs have been 
developed for T-2 toxin and DON (Feuerstein et al., 1985; Hunter et 
al., 1985; Chiba et al., 1988).  Although these monoclonal assays are 
of invariable specificity and the antibodies are available in 
unlimited supply, competitive indirect ELISAs using monoclonals are 
generally less sensitive than polyclonal-based ELISAs.  However, 
Chiba et al. (1988) developed a sensitive ELISA for the detection of 
T-2 toxin with monoclonal antibodies at a limit of 2.5 pg/assay.  
This assay has been applied to the detection of T-2 toxin in wheat 
flour (Chiba et al., 1988) and in fungal cultures (Nagayama et 
al., 1988). 

    Production of useful antibodies against DON has proved more 
difficult than for other trichothecenes.  Xu et al. (1988) adopted 
the approach of raising antibodies against triacetyl-DON, which 
requires acetylation of DON in an extract of the contaminated cereal 
prior to carrying out a direct ELISA.  This assay has the required 
sensitivity but also the disadvantage of determining the total 
concentration of DON plus any other acetylated derivatives that may 
be present in the extract.  A different approach to raising 
monoclonal antibodies to DON has been carried out by forming the 
hemisuccinyl derivative after protection of two of the available 
adjacent hydroxyls with a cyclic boronate ester (Casale et al., 
1988).  Some cross-reactivity to other trichothecenes and their 
de-epoxy metabolites was observed, but the assay does show 
considerable potential as a simple and rapid screening method for 
contaminated cereals and for detection in biological samples. 

II.1.2.3  Biological methods

    Biological methods are essential for working with field cases 
where the cause of an incident is unclear and evidence is sought to 
associate an observed biological effect with the presence of fungally 

contaminated material.  Biological methods are also important as 
monitoring procedures in the isolation and purification of new or 
previously unrecognized toxins, prior to characterization by 
spectroscopic and chemical methods. 

    The classical and commonly used bioassay for trichothecenes is 
the skin necrotization test.  Extracts prepared from samples (see 
section II.1.2.1) are applied in a single dose to the shaved back of 
a rabbit, rat, or guinea-pig.  Toxic preparations applied in this way 
produce erythema, oedema, intradermal haemorrhage, and necrosis.  The 
guinea-pig is the most sensitive of these animals to trichothecenes 
(Ueno et al., 1970).  As little as 0.2 µg of T-2 toxin and DAS were 
detected on the skin of the rabbit and guinea-pig (Chung et al., 
1974; Balzer et al., 1977).  Only 10 g of the corn sample are needed 
for testing with an extraction and clean-up procedure recommended by 
Eppley (1968).  Macrocyclic trichothecenes are highly irritant to the 
skin, but NIV and DON, which have a low skin-necrotizing potency, 
could be routinely missed in this assay (Ueno et al., 1970). 

    Brine shrimp ( Artemia salina L.) larvae also provide a useful 
biological system for monitoring trichothecenes, such as T-2 toxin,  
DAS, and the macrocyclic trichothecenes.  The limit of detection of 
this test for these toxins is approximately 0.04-0.4 mg/litre 
(Eppley, 1974). 

    The rabbit reticulocyte assay, based on the inhibition of uptake 
of 14C-leucine in eukaryote cells, is highly specific for 
trichothecenes; ID50 (50% inhibitory dose) values are 0.03 mg/litre 
for T-2 toxin and DAS, and 2.0-3.0 mg/litre for DON and NIV (Ueno et 
al., 1969a, 1973c).  By combining chemical methods with this assay, 
DON was successfully isolated from  Fusarium-infected corn (Ishii et 
al., 1975). 


II.2.1  Taxonomic considerations

    The vast majority of trichothecenes are produced by the  Fusarium 
species.  Production of these metabolites depends, of course, on many 
factors, including substrate, temperature, humidity, etc.  In 
general, the type A trichothecenes have been most frequently 
associated with the following  Fusarium species:  F. tricinctum, F. 
 sporotrichioides, F. poe, F. equiseti.  On the other hand, type B 
trichothecenes are usually associated with  F. graminearum (Gibberella 
 zeae), and  F. culmorum.  The type C trichothecenes, containing an 
additional epoxide function at the 7,8- or 9,10-positions, are 
produced by only a few species.  The Type D trichothecenes, i.e., 
those containing the macrocyclic ring between the 4,15-positions, are 
produced by several fungal genera, including  Myrothecium and 
 Stachybotrys.  Of these, the most important from an animal and human 
health standpoint, is  Stachybotrys atra.  The relationships between 
trichothecenes and fungal species are summarized in Table 13. 

Table 13.  The relationship between trichothecenes and fungal species
Trichothecenes       Fungal species         References
Type A
T-2, HT-2, DAS, NS    Fusarium tricinctum    Joffe & Yagen (1977)
                      F. sporotrichioides    Scott et al.  (1980)
                      F. poae                Marasas et al. (1984)
                      F. acuminatum          Ichinoe et al. (1985), 
                                            Rabie et al. (1986)
DAS                   F. equiseti            Brian et al. (1961)
                      F. semitectum          Suzuki et al. (1980), 
                                            Greenhalgh et al. (1984)

Type B
DON, 3-AcDON          F. graminearum         Yoshizawa & Morooka (1973)
NIV, F-X              Gibberella zeae        Marasas et al. (1984)
                     (anamorph)             Ichinoe et al. (1985)
DON, 3-AcDON          F. culmorum            Marasas et al. (1979b)
                                            Chelkowski et al. (1984)
Trichotecin           Trichothecium          Freeman & Morrison (1949)
                      roseum                 Ishii et al. (1986)

Type C
Baccharin             Baccharis              Kupchan et al. (1976)
                     (higher plant)

Type D
Roridin A, D, E       Mycothecium roridum    Bohner et al. (1965)
Verrucarin J          M. verrucaria          Harrach et al. (1981)
Satratoxin G, H       Stachybotrys atra      Jarvis et al. (1986)

    The taxonomy of the various fungal species is complex and has led 
to some confusion, particularly regarding the  Fusarium species 
responsible for the production of the various trichothecene 
metabolites.  Several excellent descriptions are now available of the 
Fusarium spp. and the metabolites produced by them (Nelson et al., 
1983;  Ueno, 1983; Marasas et al., 1984; Ichinoe et al., 1985; Joffe, 

II.2.2  Ecology of trichothecene-producing fungi

    Fusarium species are widely distributed throughout the 
environment.  There are over 50 recognized species commonly occurring 
in the soil (soil fungi), many of which are pathogenic to crop 
plants.  As a result, there are a number of frequently encountered 
plant diseases (wilts, blights, rots), such as  F. graminearum blight 
of wheat and barley (Akakabibyo in Japanese), pink ear rot of corn,  
pink scab (tombstone kernels) of wheat,  Fusarium snow blight, etc.  
 Fusarium graminearum, which produces DON and NIV, is a most important 

II.2.3  Natural occurrence

    It has become apparent in the past few years that whenever a 
trichothecene-producing  Fusarium species parasitizes a crop, food,  
or animal feed, it is highly probable that the metabolites of the 
trichothecene will be found as contaminants.  The chance of detecting 
the metabolite is then clearly a function of the efficiency of the 
sampling procedure and the capabilities of the analytical methods 
used (most importantly, the detection limit). 

    Because of its toxicity, analytical procedures for T-2 toxin were 
developed first, and consequently early surveys for trichothecenes 
tended to concentrate on T-2 toxin.  However, it soon became apparent 
that other trichothecenes, in particular, DON, NIV, and DAS, were 
more frequent contaminants of food and animal feed than T-2 toxin.  
As improved analytical procedures for these mycotoxins became 
available, surveys were conducted and additional data on the 
occurrence of these metabolites were published.  In reviewing this 
record, it should be recognized that the methodology used in 
developing the data was quite variable, so that only broad 
generalizations with respect to incidence/level may be drawn.  In the 
vast majority of cases, no evidence was included indicating that 
methods used were rigorously tested, that quality assurance 
programmes were in place, and that confirmation of identity was 
adequately obtained.  Much of the available survey data on the 
natural occurrence of trichothecenes in raw agricultural commodities, 
food, and animal feed is summarized in Tables 14-17.  Reports in 
which only a few samples were analysed have not been included. 

II.2.3.1  Agricultural products

 (a)  T-2 Toxin

    One of the first trichothecenes to be implicated in an episode of  
mouldy corn toxicosis was T-2 toxin; in 1972, it was reported that 
T-2 toxin at the level of 2 mg/kg was present in mouldy corn involved 
in lethal toxicosis in dairy cattle (Hsu et al., 1972).  This event, 
along with increasing information regarding the acute toxicity of T-2 
toxin, prompted considerable efforts to develop methods of analysis 
for T-2 toxin and the analysis of a wide range of agricultural 
commodities (Table 14).  Only occasional samples were found to 
contain T-2 toxin (incidence well below 10% in most cases), most 
frequently at levels <0.1 mg/kg.  Usually, other trichothecenes were 
also found (Vesonder, 1983).   On the other hand, there have been 
isolated reports of the finding of rather high levels of T-2 toxin, 
e.g., the finding of 25 mg T-2 toxin/kg in barley (Puls & Greenway, 
1976), and 38.9 mg T-2 toxin/kg in peanuts (Bhavanishankar & Shantha, 
1987).  These findings, as well as reports from India of the presence 
of T-2 toxin in safflower seed and sweet corn (Ghosal et al., 1977), 
and sorghum (Ruckmini & Bhat, 1978), and from Italy of its presence 
in barley, corn feed, oats, rice, and wheat (Cirilli, 1983) need to 
be further investigated. 

Table 14.  Natural occurrence of T-2 toxin in raw agricultural
Commodity  Levels      Incidence   Country       Reference     
           (mg/kg)     (+ve/total)
Corn       0.5-5.0     (5/150)     Hungary       Szathmary (1983)
           0.080-0.65  (9/118)     Taiwan        Tseng et al. (1983)
           0.01-0.2    (13/20)     New Zealand   Hussein et al. 
Feed       0.05-5.0    (28/464)    Hungary       Szathmary (1981)
Oats       0.01-0.05               Finland       Ylimacki et al. 
Peanuts    0.63-38.89  (6/87)      India         Bhavanishankar &
                                                 Shantha (1987)
Rice                   (3/64)      Egypt         Abdel-Hafez et al.
Sorghum    1.67-15.0   (4/84)      India         Bhavanishankar &
                                                 Shantha (1987)
Wheat      2.0-4.0     (3/12)      India         Bhat et al. (1989) 

    DON/NIV:  Deoxynivalenol (DON) and nivalenol (NIV) have been 
found to be the most frequent trichothecene contaminants of 
agricultural crops throughout the world (Table 15).  Extensive 
survey data indicate the common occurrence of these mycotoxins, 
particularly in corn and wheat, at levels usually below 1 mg/kg 
(Vesonder, 1983; Pohland & Wood, 1987; Jelinek et al., 1989).  
Perhaps the most extensive survey, and the one giving the most 
accurate picture of the global occurrence of DON and NIV, was 
reported recently by Tanaka et al. (1988). 

Table 15.  Natural occurrence of DON and NIV in raw agricultural 
Commodity      Toxin   Levels        Incidence   Country       Reference
                       (mg/kg)a      (+ve/total)
Barley         DON     t-40.4        (19/25)     Japan         Kamimura et
                                                               al. (1981)
               NIV     t-37.9        (19/23)
(unpolished)   DON     0.004-0.508   (26/28)     Korea         Lee et al.
               NIV     0.017-3.0     (28/28)     Republic of   (1985)
(polished)     DON     0.008-0.043   (5/6)
               NIV     0.085-0.328   (5/6)
               DON     0.006-2.14    (34/49)     Norway        Sundheim et
               NIV     0.013-1.56    (49/49)                   al. (1988)
               DON     0.02-0.36     (34/87)     United        Gilbert et
                                                 Kingdom       al. (1983b)

Corn           DON     1-20          (19/60)     Austria       Lew et al.
               DON     0.15-0.82     (9/9)       Canada        Scott et
                                                               al. (1981)

Table 15 (contd.)
Commodity      Toxin   Levels        Incidence   Country       Reference
                       (mg/kg)a      (+ve/total)
Corn (contd.)  DON     0.36-12.7     (100%)      China         Qiujie et
               NIV     0.054-2.67    (100%)                    al. (1988)
               DON     0.02-0.3      (11/20)     New Zealand   Hussein et 
                                                               al. (1989)
               DON     t-15.8        (20/72)     Transkei      Thiel et
               NIV     t-1.41        (43/72)                   al. (1982)
               DON     0.1-0.3       (37)        United        Gilbert et
                                                 Kingdom       al. (1984)
               DON     0.5-10.7      (24/52)     USA           Vesonder et
                                                               al. (1978)
               DON     0.1-2.47      (93/198)    USA           Wood & 

Oats           DON     20                        Germany,      Bauer et
                                                 Federal       al. (1980)
                                                 Republic of
               DON     0.02-0.1      (1/6)       United        Gilbert et
                                                 Kingdom       al. (1984)

Rye            DON     0.003         (1/5)       Korea         Lee et al.
               NIV     0.046-0.114   (5/5)       Republic of   (1985)

Wheat          DON     0.01-4.3      (51/52)     Canada        Scott et
                                                               al. (1981)
               DON     0.06-8.53     (24/52)     Canada        Trenholm et
                                                               al. (1981)
               DON     0.02-1.32     (55/199)    Canada        Osborne &
               DON     1.0           (1.36)      Denmark       Hald & Krogh
               DON     t-4.7         (15/18)     Germany,      Bauer et
               NIV     t-7.8                     Federal       al. (1980)
                                                 Republic of
               DON     0.008-3.19    (32/53)     Norway        Sundheim et
                                                               al. (1988)
               NIV     0.015-0.887   (53/53)
               DON     0.02 >0.5     (57/148)    United        Gilbert et
                                                 Kingdom       al. (1984)
               DON     0.12-5.5      (31/33)     USA           Hagler et
                                                               al. (1984)
               DON     0.2-9         (54/57)     USA           Eppley et
                                                               al. (1984)
               DON     0.1-2.65      (133/247)   USA           Wood &

Table 15 (contd.)
Commodity      Toxin   Levels        Incidence   Country       Reference
                       (mg/kg)a      (+ve/total)
Animal         DON     0.1-41.6      (274/342)   USA           Cote et
feed                                                           al. (1984)
               DON     <0.4-4.0                  USA           Vesonder
a  t = trace.

    This survey involved the analysis of 500 samples of cereal grains 
from 19 countries (Table 16) using a single analytical method. 

Table 16.  Natural occurrence of DON and NIV: worldwide survey
Commodity                 DON                         NIV        
               (Mean, ng/g)  (% + ve)      (Mean, ng/g)  (% + ve)
Barley         149           75            401           76
Corn           402           20            766           16
Oats           115           22            438           26
Rice           0                           22            22
Rye            183           33            47            33
Sorghum        0                           0
Wheat          488a          39            127           50
Othersb        135           44            3             6

Total:         292           45            267           48
a  A Beijing wheat sample containing 6 644 ng/g was excluded in 
   calculating this mean.
b  Wheat flour, 7; rye flour, 1; spice, 3; sesame, 7.

    It was found that 45-50% of random samples contained both DON and 
NIV, barley being most frequently contaminated.  Corn, although less 
frequently contaminated, contained the highest average amounts of 
NIV; of all the cereals examined, wheat was the most heavily 
contaminated with DON.  There were clear regional differences, not 
totally predictable, in the relative quantities of DON and NIV.  For 
example, in Canada and the USA, NIV was only rarely encountered 
whereas, in Japan, NIV was  more  frequently encountered than DON.   
Even  within  a country there were clear differences between regions; 
for example, in southern Japan, NIV was more frequently encountered 
than DON, whereas in northern Japan the reverse was true.  Similarly, 
levels of DON were generally lower in western than in eastern Canada 
(Jelinek et al., 1989). 

    The data indicate that crops parasitized by  Fusarium species will 
probably be contaminated with these mycotoxins.  In wheat, for 
example, there is an excellent correlation between the DON 
contamination level and the percentage of mouldy kernels, the 
percentage of total defects, and the degree of scab damage (Eppley et 
al., 1984; Shotwell et al., 1985).  It has been suggested that crop 
rotation might be a factor, i.e., planting wheat after corn tends to 

increase the DON levels in the resulting wheat crop (Teich & 
Hamilton, 1985).  Recently, it has been shown that the mean  
concentration of DON in wheat declines quite significantly in the 
2-week period immediately preceding harvest (Scott et al., 1984). 

 (b)  Miscellaneous trichothecenes

    There have been occasional reports of trichothecenes other than 
those discussed above (T-2 toxin, DON, NIV) in agricultural products, 
in particular, DAS, 15-acetyldeoxynivalenol (15-ADON), and 3-
acetyldeoxynivalenol (3-ADON) (Table 17).  Most of these reports 
involve corn.  There is also an unconfirmed report of the finding of 
T-2 toxin, DAS, and trichothecolone as well as the fatty acid esters 
of trichothecolone, scirpenetriol, and T-2 tetraol in banana fruit, 
which needs further investigation (Chakrabarti & Ghosal, 1986).  In 
1982, over 100 out of a flock of 1200 ewes on a Hungarian farm died 
after ingestion of bedding straw highly infected with  Stachybotrys 
 atra.  TLC, HPLC, and MS analysis of the methanol extract of the  
straw revealed the presence of the macrocyclic trichothecenes, 
satratoxins G and H (Harrach et al., 1983). 

Table 17.  Natural occurrence: miscellanceous trichothecenes in
agricultural products
Commodity   Toxin     Level     Incidence   Country       Reference
                      (mg/kg)   (+ve/total)
Corn        15-ADON   0.113                 China         Qiujie et al.
                      ave.                                (1988)
            3-ADON    0.495                 China         Qiujie et al.
                      ave.                                (1988)
            DAS       31.5                  Germany,      Siegfried (1977)
                                            Republic of

            DAS       0.01-     (6/20)      New Zealand   Hussein et al.
                      0.9                                 (1989)
            15-ADON   0-7.9                 USA           Abbas et al. 

Peanuts     DAS       0.41-     (7/87)      India         Bhavanishankar &
                      2.03                                Shantha (1987)

Wheat       ADON      0.6-2.4   (4/12)      India         Bhat et al.
flour                                                     (1989)

Bedding     satratoxin          not         Hungary       Harrach et al.
straw       G                   estimated                 (1983)
            satratoxin          not         Hungary       Harrach et al.
            H                   estimated                 (1983)

    The macrocyclic trichothecenes, such as satratoxin H, verrucarins 
B and J, and trichoverrins A and B, were also detected in air dust 
collected from a house in Chicago, where the occupants were subjected 
to a variety of recurring maladies including colds, dermatitis, and 
others (Croft et al., 1986).  These data indicated a possible 
airborne outbreak of macrocyclic trichothecene-induced toxicosis. 

    Undoubtedly, as good analytical methods become available, these 
trichothecenes will be found more frequently. 

II.2.3.2  Trichothecenes in human foodstuffs

    Given the widespread occurrence of trichothecenes in agricultural 
products, in particular DON and NIV, it is not surprising to find the 
compounds in human foodstuffs (Table 18).  The vast majority of the 
confirmed cases of contamination of foodstuffs by trichothecenes 
involve DON in wheat or wheat products.  Overall, the finding of DON 
in human foodstuffs at concentrations >1 mg/kg has been rare.  NIV 
has been detected in human foodstuffs, particularly in Japan, where 
an effort was made to develop and apply analytical methods capable of 
determining both DON and NIV. 

Table 18.  Natural occurrence of DON and NIV in commercial foods
Commodity   Toxin     Level         Incidence   Country       Reference
                      (mg/kg)a      (+ve/total)
Barley      DON       0.027-0.085   (6/6)       Japan         Yoshizawa &
flour       NIV       0.037-0.19    (6/6)                     Hosokawa 
(parched)                                                     (1983)
Barley      DON       0.008-0.039   (5/6)                     Tanaka et
flour       NIV       0.013-0.041   (6/6)                     al. (1985b)
Barley      DON       0.003-0.05    (10/14)                   Tanaka et
(pressed)   NIV       0.008-0.033   (13/14)                   al. (1985a)
Barley      DON       t-0.26        (27/147)                  Kamimura et
products    NIV       0.006-0.28                              al. (1981)
Corn meal   DON       0.025         (45/50)     USA           Trucksess et
                                                              al. (1986a)
Corn flour  DON       (0.18 ave.)   (27)        Canada        Scott et al.
meal        DON       (0.1 ave.)    (35)                      (1984)
products    DON       0.011-1.25    (7/7)
Popcorn     DON       0.012-0.25    (7/7)       Japan         Tanaka et al.
Job's tear  DON       0.048-0.5     (2/12)                    (1985b)
            NIV       0.003-0.92    (11/12)
Potatoes    DON                     (4/17)      Canada        El Banna et
                                                              al. (1984)
Rye flour   DON       (0.12 ave.)   (3)         Canada        Scott et al.
    bread   NIV       (0.058 ave.)  (4)                       (1984)
Wheat       DON       (0.4 ave.)    (43)
flour       DON       0.11-0.69     (5/5)       China         Ueno et al.
            DON       0.43-4.89     (9/12)      India         Bhat et al.
            NIV       0.03-0.1      (2/12)                    (1989)
            DON       0.002-0.239   (26/36)     Japan         Tanaka et
            NIV       0.004-0.084   (12/36)                   al. (1985b)
            DON       0-0.46        (44/50)     USA           Trucksess et
                                                              al. (1986a)

Table 18 (contd.)
Commodity   Toxin     Level         Incidence   Country       Reference
                      (mg/kg)a      (+ve/total)
Breakfast   DON       (0.086 ave.)  (36)        Canada        Scott et al.
cereals                                                       (1984)
Bread       DON       (0.08 ave.)   (21)
Baby cereal DON       (0.043 ave.)  (30)
Crackers    DON       (0.27 ave.)   (20)
Cookies     DON       (0.12 ave.)   (25)
Breakfast   DON       0-0.53        (35/60)     USA           Trucksess et 
cereals                                                       al. (1986a)
Bread       DON       0-0.24        (20/25)
Baby foods  DON       0-0.09        (14/39)
Snack foods DON       0-0.45        (25/44)
Bread       DON       0.013-0.24    (39/45)     USA           Wood & Carter
a  t = trace.

    The realization that trichothecene contamination of cereal grains 
was not uncommon prompted a series of studies on the fate of these 
compounds during the normal processing of such grains into consumer 
food products.  These studies can be summarized as follows: 

    In the case of corn, Collins & Rosen (1981) demonstrated that, 
during wet milling, roughly 66% of the T-2 toxin originally present 
was removed in the steeping water, about 4% remained in the starch, 
and the remainder was distributed evenly in the germ, gluten, and 
fibre fractions (a result clearly predictable in view of the 
considerable water solubility of T-2 toxin).  The same general 
observation was made about the fate of DON during the wet milling of 
corn (Scott, 1984b).  During the dry milling of corn, the major 
portion of contaminating DON was found in the germ meal, which is 
used for animal feed.  For any significant contamination of grits, 
the corn used in the manufacture would have to be highly contaminated 
(Gilbert et al., 1983a). 

    In the case of wheat, several studies have clearly demonstrated 
that cleaning and milling are not effective in completely removing 
DON (Hart & Baselton, 1983; Scott et al., 1983, 1984a;  Young et al., 
1984; Seitz et al., 1985).  The same was generally true for NIV (Lee 
et al., 1987).  Normal cleaning of wheat reduced average DON 
contamination levels by 6-19% (Abbas et al., 1985).  The remaining 
DON, after milling, is distributed in all fractions (bran, shorts, 
reduction flour, break flour), the greatest amounts being found in 
the bran and the smallest amounts in the flour, depending on the 
level of contamination of the wheat itself.  In a typical study, 
milling of whole wheat contaminated at the 2 µg/g level, resulted in 
approximately 65%  of the DON in the flour and 35% in the bran, red 
dogs, and shorts; i.e., there was little reduction in the DON 
concentration in the flour after milling (Hart & Baselton, 1983). 

    It has been found that the further processing of flour into baked 
or cooked products results in variable DON losses.  The overall range 
of reduction in DON levels from uncleaned wheat to bread was 24-71% 
(Abbas et al., 1985).  No reduction in DON levels was found in the 
preparation of Egyptian bread (El-Banna et al., 1983)  or  cookies  
from  hard  wheat  flour  (Scott et al., 1983), illustrating the 
importance played by the conditions of processing.  Kamimura et al. 
(1979) showed that bread and noodles prepared under conditions 
simulating commercial processes retained about 50% of the 
trichothecenes that had been previously added to the wheat flour.  
The trichothecene content of Japanese noodles was reduced by 30% in 
the boiling process. 

    Similarly, these authors found that 30% of NIV and DON was 
extracted from naturally contaminated wheat by water, while there was 
a 50% or more reduction in trichothecene levels in bread made from 
the same flour.  The preparation of Chinese noodles, where sodium 
carbonate is used as an ingredient, results in an even greater loss 
of DON than is observed in the preparation of Japanese noodles (Scott 
et al., 1984b; Nowicki et al., 1988).  Similarly, Young et al. (1984) 
observed a decrease of up to 35% in the production of cookies from 
biscuit and cake flours; in the process studied, the batter contained 
ammonium carbonate.  Finally, it has been found that, during the 
baking of bread, 3-13% of DON is converted into an isomer, iso-DON 
(Greenhalgh et al., 1984). 

    There have been some studies on possible detoxification 
procedures for corn and wheat contaminated with trichothecenes; 
agents studied include chlorine, ammonia (Young et al., 1984) and 
aqueous bisulfite (Young et al., 1986).  None of these processes are 
currently commercially feasible. 

    There has been some concern about the transmission of the 
trichothecenes, particularly DON, to milk.  Studies have shown that 
milk is only a minor excretion route in the lactating dairy cow; 
thus, the possibility that DON might contaminate milk and milk 
products is remote (Prelusky et al., 1984). 


II.3.1  Absorption and tissue distribution

II.3.1.1  Animal studies

    In general, trichothecenes absorbed from the alimentary tract are 
evenly distributed in many tissues and organs, without significant 
accumulation in specific organs.  However, at present, distribution 
studies have been limited mainly to T-2 toxin,  and the metabolism in 
animals of other naturally contaminating trichothecenes, such as DON, 
NIV, and DAS, remains to be elucidated. 

   In 6-week-old broiler chicks fed with a ration containing T-2 
toxin at 2 mg/kg for 5 weeks and then intubated with a single dose of  
3H-T-2 toxin at 0.5 mg/kg body weight, the radioactivity reached a 
maximum concentration in most tissues, 4 h after dosing; exceptions 

were the muscle, skin, and bile, in which the maximum level was  
reached after 12 h (Chi et al., 1978b).  After 48 h, chicks contained 
the equivalent of 39 µg T-2 toxin and/or its metabolites/kg in the 
muscle, and 40 µg/kg in the liver, as calculated on the basis of the 
specific activity of the radiolabelled T-2 toxin administered. 

    In chicken organs, 18 h after intraperitoneal injection of T-2 
toxin (3.5 mg/kg), considerable amounts of T-2 metabolites were found 
in the liver (1370 µg 3'-hydroxy-H-T-2 toxin/kg).  Smaller amounts of 
H-T-2 toxin, T-2 triol, and other metabolites were detected in the 
lungs (Visconti & Mirocha, 1985). 

    In weanling crossbred pigs (7.5-9.5 kg body weight) intubated 
with 0.1 or 0.4 mg 3H-T-2 toxin/kg body weight, the percentage of  
administered radioactivity (18 h after dosing) was 0.7% in the 
muscle, 0.29-0.43% in the liver, 0.08% in the kidney, and 0.06-0.14% 
in the bile (Robison et al., 1979b). In pigs intubated with 0.1 mg of 
the toxin/kg body weight, the calculated residue levels for T-2 toxin 
and/or its metabolites, based on the specific radioactivity of the 
tissues, were as follows: muscle, 3.1 µg/kg; fat, 0.49 µg/kg; liver, 
13.8 µg/kg; and kidney, 15.9 µg/kg.  The corresponding residue levels 
for T-2 toxin in the tissue of another pig intubated with 0.4 mg of 
the toxin/kg body weight were 11.5 µg/kg in the muscle, 37.7 µg/kg in 
the liver, and 61.4 µg/kg in the kidney. 

    Four hours after intravenous administration of T-2 toxin in 
swine, the greatest amount of radioactivity was located in the 
gastrointestinal tract (15-24% of the dose), and 4.7-5.2% of the dose 
was found in the remaining tissues, among which the muscle and the 
liver accounted for 2.9-3.2% and 0.7-1.7% of the dose, respectively 
(Corley et al., 1986). 

    The fate and distribution of 3H-T-2 toxin was studied in guinea-
pigs (Pace et al., 1985).  Except for the large intestine and bile, 
the radioactivity had peaked by 30 min and rapidly declined, with no 
measurable long-term accumulation.  In general, the distribution 
pattern in the guinea-pig during the first 12-24 h paralleled the 
distribution found in the chicken and swine. 

    The metabolic fate of T-2 toxin was investigated in a lactating 
Jersey cow weighing 375 kg, after daily oral administration, by 
capsule, of unlabelled toxin at 180 mg/day for 3 days, followed by 
administration of 3H-T-2 toxin at 156.9 mg (Yoshizawa et al., 1981).  
Although almost all of the administered dose was eliminated in 72 h, 
appreciable levels of tritium still remained in the bile, liver, and 
kidney (equivalent, repectively, to 27.2, 18.5, and 13.9 µg/kg 3H-T-2 
toxin) 3 days after dosing.  These levels are higher than in whole 
blood (13.3 µg/kg) and plasma (10.2 µg/kg) and in other tissues, 
including the spleen (9.4 µg/kg) heart (10.1 µg/kg), mammary gland 
(11.3 µg/kg), ovaries (10.7 µg/kg), muscle (8.8 µg/kg), and fat (4.7 

    Experimentally derived relationships between the residue of 
radioactivity in animal tissues or plasma and the toxin levels in the 
feed, calculated on the basis of the amount of 3H-T-2 toxin 
administered are summarized in Table 19 (Yoshizawa et al., 1981). 

Table 19.  The relationship between the level of 3H-T-2 toxin in the feed or the 
tritium residues in plasma and tritium levels in the edible tissues of the cow, chick, 
and piga
Tissue   Animal   Time after   Feed level   Tissue level   Tissue/feed   Tissue/plasma
                  dosing (h)b  (mg/kg)c     (µg/kg)d       ratio         ratioe
Muscle   Cow      72           31.38        8.8            0.0003        0.863
         Chick    24           1.26         17.3           0.0137        1.000
                               5.0          59.2           0.0118        0.938
                               18.95        228.6          0.0121        0.875
         Pig      18           1.25         3.1            0.002         0.775

Heart    Cow      72           31.38        10.1           0.0003        0.991
         Chick    24           1.26         13.7           0.011         0.792
                               5.0          49.4           0.010         0.783
                               18.95        207.7          0.011         0.795
         Pig      18           1.25         3.9            0.003         0.975

Liver    Cow      72           31.38        18.5           0.0006        1.863
         Chick    24           1.26         34.0           0.0270        1.965
                               5.0          107.3          0.0215        1.700
                               18.95        431.0          0.0227        1.649
         Pig      18           1.25         13.8           0.011         3.450

Milk     Cow      72           31.38        11.4           0.0004        1.118
a  From: Yoshizawa et al. (1981).
b  Animals were intubated with a single dose of 3H-T-2 toxin.
c  Estimates of feed levels were based on the assumption that each animal would consume
   the following amount of feed daily: cow, 5 kg; chick, 100 g; pig, 600 g.
d  Values were calculated from residual tritium levels in the edible tissues of animals
   given 3H-T-2 toxin and were expressed as equivalents of 3H-T-2 toxin (µg/kg).
e  The plasma levels were 10.2 µg/kg equivalents of T-2 toxin in the cow, and 17.3, 
   63.1, and 261.3 µg/kg equivalents of T-2 toxin in the chick at feed levels of 1.26,
   5, and 18.95 mg/kg, respectively.  The whole-blood level of residual tritium in the
   pig was 4 µg/kg equivalents of T-2 toxin.
    The tissue/feed ratio of tritium in the edible tissues of the 
cow, 72 h after dosing, ranges from 0.0003 to 0.0006.  These figures 
are 5-10% of those for swine, 18 h after dosing. The tissue/feed 
ratio for chickens, is higher than those in the cow and pig, ranging 
from 0.010 to 0.014 for the muscle and heart, and from 0.021 to 0.027 
for the liver, regardless of T-2 toxin dosage.  The tissue/plasma 
ratios of T-2 toxin metabolites, ranging from 0.8 to 1 in the muscle 
and heart, and from 1.6 to 3.5 in the liver, are independent of 
animal species, of T-2 toxin dosage, and of time after dosing.  The 
milk/plasma ratio of radioactivity in a cow treated with 3H-T-2 toxin 
increased linearly for 24 h and thereafter ranged from 1 to 1.3. 

II.3.2  Metabolic transformation

    The  in vivo metabolic transformation of trichothecenes in animals 
is reported in Table 20.  The trichothecene metabolites produced are 
less toxic than the corresponding parent toxins.  Both de-epoxidation 
and glucuronidation, in particular, are associated with remarkable 
detoxification of trichothecenes. 

Table 20.   In vivo (metabolic) transformation of trichothecenes in animals
Animal       Trichothecene     Transformation reaction            Reference
Chicken      T-2 toxin         hydrolysis, 3'-hydroxylation,      Yoshizawa et al. (1980b);
                               acetylation                        Visconti & Mirocha (1985)

Cow          T-2 toxin         hydrolysis, 3'hydroxylation,       Yoshizawa et al. (1981, 1982a,b)
                               7'-hydroxylation, de-epoxidation   Pawlosky & Mirocha (1984)
                                                                  Chatterjee et al. (1986)

             DON               de-epoxidation,                    Yoshizawa et al. (1986)
                               glucuronide conjugation            Côté et al. (1986)

Pig          T-2 toxin         hydrolysis, 3'-hydroxylation,      Corley et al. (1985, 1986)
                               de-epoxidation, glucuronide

             DAS               hydrolysis                         Bauer et al. (1985)

Sheep        DON               glucuronide conjugation            Prelusky et al. (1985)

Rat          DON               de-epoxidation                     Yoshizawa et al. (1983)
             T-2 metabolites   de-epoxidation                     Yoshizawa et al. (1985a,b)
             DAS               hydrolysis, de-epoxidation         Sakamoto et al. (1986)

Guinea-pig   T-2 toxin         hydrolysis, 3'-hydroxylation       Pace et al. (1985)

Dog          T-2 toxin         hydrolysis, glucuronide
                               conjugation                        Sintov et al. (1987)
    In  in vitro studies, HT-2 toxin was found to be the sole 
metabolite of T-2 toxin in the microsomes of the liver, kidney, and 
spleen of various animals (Ohta et al., 1977, Johnsen et al., 1986). 
The reaction of hepatic microsomes of different species in nmol/mg 
protein per 10 min were: rabbit, 3044; human, 331; mouse, 75; 
chicken, 55; rat, 36; and guinea-pig, 14.  Yoshizawa et al. (1984, 
1986) have proposed an  in vitro metabolic pathway for T-2 toxin that 
includes the hydrolysates at the C-4, C-8, and C-15 positions, and 
the hydroxylation at the C-3'position by liver homogenates from the 
rat, mouse, or monkey. This hydrolytic transformation was also 
observed in the hepatic homogenates of the rabbit, pig, and cow. 
However, HT-2 toxin was the sole metabolite in the homogenate of the 
chicken, suggesting species differences in the metabolic pathway of 
T-2 toxin (Yoshizawa & Sakamoto, 1982). In addition to the 
metabolites above, glucuronide conjugates were formed in a study 
using isolated perfused rat livers and T-2 toxin and DAS (Gareis et 
al., 1986). Various de-epoxidation metabolites were found by 
incubating intestinal and rumen microbes with T-2 toxin and its 
metabolites (Yoshizawa et al., 1985a; Swanson et al., 1987), DON 
(King et al., 1984; Côté et al., 1986; Swanson et al., 1987), and DAS 
(Swanson et al., 1987). 

II.3.3   Excretion

II.3.3.1  Animal studies

    The kinetics of T-2 toxin were determined in pigs and cattle 
(Beasley et al., 1986), and dogs (Sintov et al., 1986). Mean 
elimination phase half-lives were 13.8 and 17.4 min, and mean 
apparent specific volumes of distribution were 0.366 and 0.375 
litre/kg in intra-aortally dosed pigs and intravenously dosed calves, 
respectively.  In dogs, the following mean parameters were determined 
after intravenous administration of T-2 toxin and HT-2 toxin, 
respectively: half-life 5.3 and 19.6 min, clearance 0.107 and 
0.167 litre/min per kg, and volumes of distribution 0.86 and 4.47 

    The half-life of elimination of DON in sheep, ranged from 100 to 
125 min following oral administration, and it took 20-30 h to clear 
from the system. Glucuronidation after intravenous or oral 
administration of DON appeared to occur quite efficiently (iv, 21%; 
oral, 75%), with elimination half-lives of (150-200 min and 6.1-7.1 
h, respectively).  These were considerably longer than those of the 
parent toxin (Prelusky et al., 1985). 

II.3.3.2  Excretion in eggs and milk

    Radioactivity was transmitted into the eggs from laying hens that 
had been intubated gastrically with a single or several doses of 3H-
T-2 toxin (Chi et al., 1978a).  In birds dosed singly with  0.25 mg 
T-2 toxin/kg body weight, maximum residues in the eggs occurred 24 h 
after dosing; the yolk contained 0.04% of the total dose and the 
white contained 0.13%.  In birds dosed with 0.1 mg T-2 toxin/kg body 
weight per day for 8 consecutive days, the radioactivity in the egg 
accumulated until the 5th day of dosing, remained unchanged until the 

last day of dosing, and rapidly decreased thereafter.  Assuming that 
the birds weighing 1.6 kg consumed 100 g of the diet containing 1.6 
mg toxin/kg daily, the residues (T-2 toxin and/or its metabolites) in 
such contaminated eggs would be about 0.9 µg/egg. 

    Transmission of DON was studied in the eggs and meat of chickens 
(El Banna et al., 1983; Prelusky et al., 1987). Following a single 
oral dose of 14C DON, maximum radioactivity, which occurred in the 
first eggs laid after dosing (within 24 h), amounted to 0.087% of the 
dose: levels dropped rapidly in later eggs. 

    In a pregnant Holstein cow (third trimester) intubated daily with 
180 mg T-2 toxin for consecutive days (the toxin level corresponded 
to a concentration of 50 mg/kg in the feed), milk samples taken on 
the 2nd, 5th, 10th, and 12th days of intubation contained T-2 toxin 
concentrations ranging from 10 to 160 µg/kg (Robison et al., 1979a).  
Transmission of DON-1 (de-epoxydeoxynivalenol) to milk was confirmed 
in lactating dairy cows (Côté et al., 1986; Yoshizawa et al., 1986).  
Fresh and conjugated DON were also present in cow's milk following 
administration of a single oral dose of 920 mg DON, but only 
extremely low amounts (<4 µg/litre) were detected (Prelusky et al., 


II.4.1  Field observations

    In Hungary and other central European countries, pyosepticaemia 
has been reported sporadically, in the past, in horses after 
ingestion of mouldy hay and straw.  This disease was characterized by 
haemorrhages of the intestine and muscles, severe diarrhoea, and 
death.   Bacterium pyosepticum viscosum was detected in 1929 in the 
excreta, and the equine disease was diagnosed as a pyosepticaemia 
(Forgacs, 1965; Danko & Szerafin, 1976).  After the discovery of 
toxigenic  Stachybotrys atra and its metabolites, such as satratoxins 
G and H (Eppley & Bailey, 1973), the disease was presumed to be the 
same as stachybotryotoxicosis (Danko & Szerafin, 1976). 

    A field outbreak involving the death of 20% of a dairy herd was 
associated with prolonged ingestion of a diet containing 60% mouldy 
corn infested with  F. tricinctum.  The concentration of T-2 toxin in 
the feed was approximately 2 mg/kg dry weight (Hsu et al., 1972).  
The lesions in the cattle included extensive haemorrhages on the 
serosal surface of the internal viscera.  An outbreak of haemorrhagic 
syndrome in cows was associated with commercial feed containing T-2 
toxin or T-2-like toxin (concentration not determined).  The affected 
animals showed an extremely prolonged prothrombin time.  Necropsy 
findings in 2 adult cows were marked serosal, mucosal, and 
subcutaneous haemorrhages (Hibbs et al., 1975). 

    An outbreak of a disease, observed in poultry (ducks, geese), 
horses, and pigs, was associated with mouldy barley containing T-2 
toxin at approximately 25 mg/kg (Greenway & Puls, 1976).  Pigs fed 
the suspect barley exhibited signs of feed refusal, vomiting, and 

diarrhoea.  The horses became depressed and salivated excessively.  
The lesions in the geese included necrosis of the mucosa of the 
oesophagus, proventriculus, and gizzard.  No pathological lesions 
were described in other animals. 

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

    Equine leukoencephalomalacia reported from South Africa 
(Kellerman et al., 1972; Marasas et al., 1979a; Pienaar et al., 1981) 
and bean-hull toxicosis reported in horses in Hokkaido, Japan 
(Konishi & Ichijo, 1970) appear to be very similar diseases with 
nervous signs and hepatopathy as the major components.  The signs in 
these diseases were quite different from those caused by 
trichothecenes, though some fungal isolates from samples of bean-
hulls produced the trichothecenes, T-2 toxin, and neosolaniol (Ueno 
et al., 1972).   F. moniliforme was considered to be the causative 
fungus (Haliburton et al., 1979), but none of its metabolites, 
including moniliformin, have been established as the cause of the 
disease (Kriek et al., 1977). 

    Reports of field outbreaks of animal toxicoses associated with 
trichothecene-contaminated feed are summarized in Table 21. 

II.4.2  Effects on experimental animals

II.4.2.1  General toxic effects

    LD50 values for certain trichothecenes in several experimental 
animal species are summarized in Table 22 (Ueno et al., 1983).  The 
oral LD50 for T-2 toxin was 10.5 mg/kg body weight in mice, 3.06 
mg/kg in guinea-pigs, 5.2 mg/kg in rats, and 6.1 mg/kg in trout.  
The LD50 values for T-2 toxin in different species vary, but not 

    The LD50 values of fusarenon-X were compared using different 
routes of administration.  The LD50s (mg/kg) in mice were 3.4 (iv), 
3.4 (ip), 4.2 (sc), and 4.5 (oral).  These data indicate that the 
acute toxicity estimated for a single administration did not differ 
markedly when the toxin was administered by different routes (Table 
20) (Ueno et al., 1971). 

    Similar data were obtained with DON and acetyl-DON.  An 
interesting finding was that the ratio of the maximum lethal dose 
to the minimum lethal dose was approximately two, indicating a 
sharp dose-response curve for lethality. No marked differences in 
acute toxicity were observed between treated male and female 

    Newborn  animals  are  more  sensitive than  adults  to  the  
toxic effects of the trichothecenes.  For example, the LD50 values 
for toxin given sc (mg/kg body weight) in newborn mice were: T-2 
toxin, 0.15; DAS, 0.17; and fusarenon-X 0.23 (Ueno et al., 1973a). 

Table 21.  Field observations on animal toxicosis caused by trichothecenes
Mycotoxin                 Sample            Animal        Signs & lesions                Reference
(concentration in feed)
T-2 toxin (2 mg/kg)       mouldy corn       cattle        extensive haemorrhages         Hsu et al. 
                                                          (20% died)                     (1972)

T-2 toxin (25 mg/kg)      mouldy barley     poultry,      necrosis of mucosa in the      Greenway &
                                            horse,        proventriculus and             Puls (1976)
                                            pig           oesophagous of geese

T-2 toxin                 commercial feed   cow           haemorrhages                   Hibbs et 
                                                                                         al. (1975)

DAS                       maize             pig           vomiting                       Vesonder et
                                                                                         al. (1973)

T-2 toxin                 corn meal         horse         oral lesions, haemorrhages,    Szathmary 
                                                          (6 out of 58 died)             (1983)

T-2 toxin                 alfalfa           horse         inappetence, listlessness,     Szathmary 
                                                          (13 out of 31 died)            (1983)

T-2 toxin                                   poultry       oral lesions, inappetence,     Szathmary
                                                          death                          (1983)

DAS and T-2 toxin         corn              cattle        death                          Szathmary

DAS                       oat, sifting      pigeon        emesis, bloody stools          Szathmary

T-2 toxin (2.5 mg/kg)     chicken feed      broiler       inflammations, "atrophies"     Szathmary
                                            chicken                                      (1983)

DAS (150-300 mg/kg)                         cattle, pig   haemorrhagic syndrome          Cirilli 

T-2 toxin (50-150 mg/kg)                    pig, cattle   blood stools (swine, cattle)   Cirilli
                                                          ear necrosis (swine)           (1983)
                                                          intestinal lesions (poultry)
                                                          hepatic lesions (swine)

Table 22.  LD50 values (mg/kg) of trichothecenesa
Type   Trichothecenes                       Mouse                      Rat                   Guinea-pig    
                                  iv    ip     sc    oral   iv     ip     sc      oral   ip    sc    oral
A      T-2 toxin                        5.2          10.5                         5.2                3.06
       HT-2 toxin                       9.0
       Diacetoxyscirpenol (DAS)   12    23.0                1.3    0.75           7.3
       Neosolaniol                      14.5
       Monoacetoxyscirpenol                                               0.725

B      Nivalenol (NIV)            7.3   7.4    7.2   38.9
       Fusarenon-X                3.4   3.4    4.2   4.5                          4.4    0.5   0.1
       Diacetylnivalenol                9.6
       Deoxynivalenol (DON)             70.0         46.0
       3-Acetyldeoxynivalenol           49.0         34.0
       Trichothecin               300                                     250

D      Roridin A                  1.0
       Verrucarin A               1.5   0.5                 0.87
       Verrucarin B               7.0
       Verrucarin J                     0.5

Table 22 (contd.)
Type   Trichothecenes             Rabbit   Cat   Dog      Pig   Duckling   Day-old   Chick    Trout
                                  (iv)     (sc)  (iv)     (iv)  (sc)       chick     (oral)   (oral)
A      T-2 toxin                           0.5            1.21             1.75      4.0      6.1
       HT-2 toxin                                                          6.25
       DAS                        1.0            ca 1.1   0.37
       3'-OH HT-2 toxin                                                    8.5       5.0
       15-Acetyl-T-2 tetraol                                               10.0
       T-2 tetraol                                                         10.0

B      Fusarenon-X                         5.0                  ca 2.0
       DON                                                      27.0
       Acetyldeoxynivalenol                                     37.0

D      Verrucarin A               0.54
a  Adapted from: Ueno et al. (1983) and Ryu et al. (1988).
    The toxic potency of the trichothecenes varies depending on the 
modification of side chains in the molecule.  The acute lethal 
toxicity of certain trichothecenes was investigated using a single ip 
injection in mice and the LD50 values (mg/kg body weight) were:  
verrucarin A and B, 0.5; fusarenon-X, 3.4; NIV, 4.1; T-2 toxin,  5.2;  
HT-2 toxin, 9.0; diacetylnivalenol, 9.6; neosolaniol 
(8-hydroxydiacetoxyscirpenol), 14.5; DAS, 23.0; acetyldeoxynivalenol, 
49.0; DON, 70.0; and crotocin, 810.0. 

    Matsuoka et al. (1979) investigated the general effects of 
fusarenon-X on mice and rats.  Fusarenon-X induced hypothermia, but 
did not induce appreciable behavioural changes in mice.  In ether-
anaesthetized rats, it caused a rise in blood pressure and a decrease 
in respiratory rate, but did not induce any significant effects on 
cardiac rate, the muscle cell membrane, or nerve elements. 

    The administration of trichothecenes to some animals (rats, mice, 
and guinea-pigs) induces diarrhoea.  The mechanism of this sign was 
investigated using fusarenon-X and rats (Matsuoka & Kubota, 1981).  
The ip injection of fusarenon-X in rats caused watery diarrhoea 
within 36-60 h.  At necropsy, 24 h after injection of fusarenon-X, 
the small intestine was distended, but no blood was found in the 
lumen of the intestine.  The mycotoxin increased the absorption rate 
of D-xylose from the intestine  in vitro (Matsuoka & Kubota, 1981).  
The leakage of intravenously injected Evan's blue dye into the 
intestine also increased, but the sodium level in the serum 
decreased.  The intestinal villi were shortened and there was 
extravasation of erythrocytes in the intestinal lamina propria.  The 
diarrhoea induced by ip admin-istration of 1.0 mg fusarenon/kg in 
male Wistar rats was not mediated by the cyclic nucleotide system as 
the mycotoxin did not increase the cyclic GMP and AMP contents in the 
intestinal mucosa (Matsuoka & Kubota, 1987).  The permeability of 
abdominal blood vessels was increased in a dose-dependent manner in 
mice given an ip injection of fusarenon-X, and the peak was reached 
about 8 h after injection.  The increased permeability was not 
mediated by serotonin, histamine, norepinephrine, prostaglandins, 
leukotrienes, or thromboxanes (Matsuoka & Kubota, 1987). 

    The effects of fusarenon-X and T-2 toxin on intestinal absorption 
of monosaccharides were studied in rats.  The absorption of 3- O-
methyl-glucose was reduced 1-3 times after either toxin was injected 
into the jejunal lumen.  Absorption of 3- O-methyl-glucose was also 
reduced after the toxins were given by intravenous injection.  Both 
toxins impaired jejunal function by causing specific damage in the 
active transport and diffusional movement of monosaccharides (Kumagai 
& Shimizu, 1988). 

    Vomiting was one of the most significant signs of trichothecene-
induced toxicosis in the cat, dog, pig, and duckling (Ueno, 1980a).  
T-2 toxin and related trichothecene mycotoxins at doses of 0.1-10 
mg/kg induced vomiting (Vesonder et al., 1973; Sato et al., 1975; 
Yoshizawa & Morooka, 1977; Ueno, 1980b; Matsuoka & Kubota, 1981).  
The presence of the causal factor, DON, in mouldy corn was 

established using ducklings as the assay animal (Ueno et al., 1974).  
In pigs given 0.5 mg/kg body weight by infusion, vomiting commenced 
6-7 min after dosing.  Emesis or retching occurred at intervals of 2-
15 min and lasted from 0.5 to 2 min (Coppock et al., 1985).  It is 
strongly suggested that the mechanism of the vomiting of 
trichothecenes is their possible action on the chemoreceptor trigger 
zone (CTZ) in the medulla oblongata (Matsuoka et al., 1979).  The iv 
administration of 0.3 mg fusarenon-X/kg body weight to dogs induced 
emesis and vomiting, 5-15 min after injection.  Vomiting after 
injection of fusarenon-X was prevented by prior administration of 0.5 
mg metoclopramide hydrochloride/kg body weight or 1 mg 
chloropromazine hydrochloride/kg. 

    The cardiovascular effects of the trichothecenes have varied 
according to such factors as species, dose, and duration of exposure.  
In acute studies, T-2 toxin given intravenously at 1 mg/kg body 
weight produced a decline in blood pressure several hours after 
administration, the reduced blood pressure being accompanied by a 
decrease in heart rate (Smalley et al., 1970).  A single dose of T-2 
toxin administered to guinea-pigs and rabbits resulted in a decrease 
in systemic blood pressure and a decrease in heart rate (Parker et 
al., 1984; Wilson, 1984). 

    Using the  in vitro bovine ear perfusion system, it was determined 
that T-2 toxin can cause a dose-dependent vasoconstrictor response in 
peripheral vasculature, but that the toxin is a less potent 
vasoactive agent than either histamine or norepinephrine.  The 
presence of known histamine or noradrenergic anatogonist did not 
affect the response to the toxin (Wilson & Gentry, 1985).  T-2 toxin 
administered systemically produced a marked increase in peripheral 
vascular resistance in the conscious rat.  The cardiac output 
gradually decreased eventually resulting in cardiovascular collapse 
and death (Feuerstein et al., 1985). 

 (a) Swine 

     The acute and short-term toxicities of T-2 toxin, DAS, and DON 
were investigated in pigs (Weaver et al., 1978a,b; Coppock et al., 
1985).  A single dose LD50 of T-2 toxin dissolved in ethanol and 
administered iv was 1.21 ± 0.15 mg/kg body weight in normal, healthy, 
crossbred pigs weighing from 3 to 50 kg.  Soon after administration, 
emesis was followed by eager consumption of feed, moderate posterior 
paresis, staggering gait, extreme listlessness, and frequent 
defecation of normal stools.  Between 1 and 6 h, severe posterior 
paresis, knuckling-over of the rear feet, and extreme lethargy were 
observed.  These signs were followed by severe posterior paresis, 
frequent falling because of hind-quarter weakness, and the dragging 
of both rear legs while moving about.  Twenty-four hours after 
administration, the surviving pigs appeared normal.  Similar clinical 
signs were observed in pigs exposed to T-2 toxin through inhalation 
(Pang et al., 1988).  Pathologically, necrosis was present in the 
epithelial cells of the mucosa and in the crypt cells of the jejunum 
and ileum, the Peyer's patches of the ileum, the lymphoid elements of 
the caecum, the lymphoid follicles in the spleen, and the germinal 
centre of the mesenteric lymph node (Weaver et al., 1978a).  

    Young pigs were fed with T-2 toxin at 1, 2, 4, or 8 mg/kg 
standard pig ration for 8 weeks.  No statistically significant 
differences in body weight gain and feed consumption were observed 
between the treated animals and the controls.  Young pigs refused a 
ration containing 16 mg T-2 toxin/kg, but not a diet containing 10-12 
mg/kg.  The no-observed-effect level was estimated to be less than 1 
mg/kg, based on differences in body weight gain (Weaver et al., 
1978b).  In terms of clinical haematological changes, such as 
haemorrhaging, blood cell counts, serum-enzyme activities, and serum-
protein levels, the no-observed-effect level could not be accurately 
determined, but was higher than 12 mg/kg, based on the weight gain. 

    The intravenous administration of T-2 toxin to pigs at doses of 4 
or 8 mg/kg resulted in a shock syndrome characterized by reductions 
in cardiac output and blood pressure and increased plasma 
concentrations of epinephrine, norepinephrine, thromboxane B2, 6-
keto-PGF1a and lactate (Lorenzana et al., 1985).  The pigs in the 
high-dose group produced such signs as persistent vomiting, watery 
diarrhoea, abdominal straining, cold extremities, coma, and death. 

    Eighteen white cross-bred female pigs weighing 40-60 kg, 
immunized against erysepelas, were administered purified T-2 toxin 
dissolved in 70% ethanol, intravenously, in doses of 0 (5 pigs), 0.6 
(5 pigs), 1.2 (1 pig), 4.8 (5 pigs) and 5.4 (2 pigs) mg/kg.  The 
animals administered doses of 4.8 and 5.4 mg/kg died between 5 and 
10.5 h later and other groups were killed 12-24 h after treatment.  
Gross lesions were observed in pigs given 1.2 mg/kg or more and these 
consisted of oedema, congestion, and haemorrhages of the lymph nodes 
and pancreas and congestion and haemorrhages of the gastrointestinal 
mucosa, subendocardium, adrenal glands, and meninges.  Histological 
alterations confirmed the gross lesions.  Other lesions were 
widespread degeneration and necrosis of lymphoid tissue and the 
surface and crypt epithelium of the intestines.  Scattered foci of 
necrosis were present in the pancreas, myocardium, bone marrow, 
adrenal cortex, and the tubular epithelium of the renal medulla.  
Most lesions were dose dependent. The T-2 toxin-induced lesions in 
the lymphoid and gastrointestinal tract of pigs were similar to those 
described in other species.  The heart and pancreas were additional 
target organs in pigs (Pang et al., 1987b). 

    Male, castrated, crossbred, specific pathogen-free pigs (17 
controls and 17 treated),  9-11 weeks of age,  were used in a study 
to characterize the pulmonary and systemic responses to inhaled T-2 
toxin (nebulized dose of 9 mg/kg given by endotracheal tube) (Pang et 
al., 1987a).  The animals were exposed to the aerosol in pairs, one 
animal receiving the toxin, the other acting as a control.  From 20 
to 30% of the toxin was retained by the pigs (T-2 toxin was mixed 
with 100-200 µCi technetium for measurement).  Five pairs of animals 
each were killed 1, 3, and 7 days after dosing.  Two pairs were 
designated a 0.33 day group, when one treated  pig  died  and the 
other was killed in  a  moribund  state, 0-10 h after dosing.  
Clinically, the T-2 toxin-treated pigs vomited after exposure 
producing such signs as cyanosis, anorexia, and lethargy.  The pigs 
became laterally recumbent.  Alveolar macrophages showed reduced 
phagocytosis and the blastogenic responses to mitogen were reduced 

for pulmonary lymphocytes, but not for lymphocytes of the peripheral 
blood.  The lesions in pigs that died included multifocal 
interstitial pneumonia, necrosis of lymphoid tissue, 
necrohaemorrhagic gastroenteritis, oedema of gall bladder mucosa, and 
multifocal areas of necrosis in the heart and pancreas.  Inhalation 
exposure to T-2 toxin produced a clinical and morphological syndrome 
resembling that produced by intravenously administered T-2 toxin, at 
doses of 1.2 mg/kg (approximate LD50) or more, as well as death.  
Furthermore, the lesions produced by the inhaled toxin were more 

    The acute effects of DAS were studied using single, intravenous 
doses (range: 0.30-0.48 mg/kg body weight) in 13 crossbred pigs; 
there were 2 control pigs (Weaver et al., 1978b).  Seven of the 
toxin-exposed pigs developed emesis, frequent defecation, lethargy, 
staggering gait, and prostration by 10 h leading to death.  Severe 
haemorrhagic necrotizing lesions and mucosal congestion involved the 
jejunum and ileum and large intestines, portions of which were blood-
filled at necropsy.  Lymphoid follicular necrosis was present in the 
lymph nodes and spleen.  The LD50 value was found to be 0.376 ± 0.043 
mg/kg body weight. 

    Two  female crossbred pigs were administered DON by rapid 
intravenous infusion at a dose of 0.5 mg/kg body weight; there were 
two matching controls.  Vomiting was observed within a few minutes  
of dosing, the skin was flushed and the extremities became cold.  
Pigs had signs of diarrhoea, muscular weakness, tremors, and coma.  
Symptoms were progressive in severity reaching a maximum 6-7  h after 
the injection; recovery occurred after 12 h.  Necrosis of pancreatic 
acinar and islet cells was observed (Coppock et al., 1985).  Pigs can 
ingest up to 2 mg DON/kg feed without suffering any serious toxic  
effects (Trenholm et al., 1984).  T-2 toxin added to the ration at 18 
or 30 mg/kg caused the refusal of feed in pigs (Szathmary & Rafai, 
1978).  Feed refusal and emesis have been produced in other species  
and by other toxic metabolites of  Fusarium species (Kotsonis et al., 
1975; Vesonder et al., 1977). 

    The minimum emetic dose of DON in pigs weighing 9-10 kg was 0.05 
mg/kg body weight, when administered intraperitoneally, and 0.1-0.2 
mg/kg body weight, when given orally.  When this toxin was added to 
feed, the feed consumption of 20-45 kg pigs, was reduced by 20% at a 
dose of 3.6 mg/kg and by a 90% at a dose of 40 mg/kg (Forsyth et al., 
1977).  Pigs were about twice as sensitive to DON as rats (Vesonder 
et al., 1979).  DON in contaminated wheat reduced feed intake and 
weight gain, when it was fed to the pigs.  The intake of feed 
decreased linearly with increasing dietary concentration of DON 
(Friend et al., 1982).  The emetic activity of 15-acetyl DON in pigs 
was similar to that of DON, the minimum emetic doses being 75 and 50 
µg/kg, respectively (Pestka et al., 1987a). 

 (b) Poultry

    Chi et al. (1977b), reported that the single oral LD50 dose of 
T-2 toxin for one-day-old broiler chicks was 5 mg/kg body weight.  It 
was 5 and 6.3 mg/kg body weight for 8-week-old broiler chicks and 

laying hens, respectively.  Death of the birds occurred within 48 h 
of T-2 toxin administration.  Within 4 h of receiving the toxin, 
birds developed asthenia, inappetence, diarrhoea, and panting.  The 
abdominal cavities of birds given lethal doses contained  a white 
chalk-like material that covered much of the viscera. 

    In a study by Wyatt et al.(1972), chickens were fed a diet 
containing 1-16 mg T-2 toxin/kg feed for 3 weeks.  The birds with 
reduced growth at 4, 8, and 16 mg/kg developed yellow-white lesions 
in the mouthparts at all dietary concentrations.  The lesions 
consisted of a fibrinous surface layer and a heavy infiltration of 
the underlying tissues by granular leukocytes.   Escherichia coli and 
 Staphylococcus epidermis were isolated from the lesions. 

    Terao et al.(1978) observed the effects of T-2 toxin and related 
trichothecenes on the bursa of Fabricius of one-day-old chicks.  
After injection of 5 mg T-2 toxin/kg body weight into the residual 
yolk sac, cellular toxic effects were observed on the follicle-
associated epithelium, resulting in necrosis, which spread to the 
periphery.  The lesions induced by fusarenon-X and NIV were similar 
to those induced by T-2 toxin, but the toxins were less potent and 
their activity was estimated to be more than 40 times less than that 
of cyclophosphamide. 

    The acute toxicity of DAS and of T-2 toxin dissolved in 
dimethylsulfoxide in 7-day-old male broiler chicks was described by 
Hoerr et al. (1981).  The 72-h single oral LD50 doses of T-2 toxin 
and DAS were estimated to be 4 and 5 mg/kg body weight, respectively.  
Combination of the 2 toxins caused increased mortality in both the 
single-and multiple-dose tests.  Lesions produced by crop gavage with 
T-2 toxin and DAS were similar, but were more severe in chicks given 
T-2 toxin.  Necrosis of lymphoid tissue and bone marrow was observed 
in tissue taken 1 h after treatment followed by rapid depletion.  
Necrosis was observed in the liver, gall bladder, and gut.  

    Chi et al.(1977c), fed broiler chicks (36 per group), aged one 
day to 9 weeks, a diet containing T-2 toxin at concentrations of 0.2, 
0.4, 2, or 4 mg/kg.  Birds fed 4 mg T-2 toxin/kg showed reduced body 
weight gain and feed consumption and developed oral lesions  
characterized  by  circumscribed  proliferating  yellow  caseous  
plaques  at  the  margin  of  the  beak,  the  mucosa  of  the hard 
palate and the tongue, and the angle of the mouth.  No lesions were 
observed in the bone marrow or, to any significant extent, in the 
peripheral blood. The no-observed-effect doses of T-2 toxin were 0.2 
mg/kg for weight gain, and 0.2 mg/kg for oral lesions.  When one-day-
old broiler chicks were fed a diet containing 1, 2, 4, 8, or 16 mg T-
2 toxin/kg feed for 3 weeks the no-effect doses were estimated as 
follows:  growth rate, weight of pancreas, and weight of spleen: 2 
mg/kg; oral lesions: <1 mg/kg (Wyatt et al., 1973c).  

    T-2 toxin administered to laying hens at a concentration of 20 
mg/kg feed reduced egg production and resulted in the production of a 
thinner egg shell (Wyatt et al., 1975).  Speers et al.(1977), also 
observed cessation of egg production in hens fed diets  containing  
25-50 mg  monoacetoxyscirpenol/kg  or 16 mg T-2 toxin/kg.  It was 

reported by Chi et al. (1977a) that feed consumption, egg production, 
and shell thickness were significantly decreased in hens fed 8 mg of 
T-2 toxin/kg.  Furthermore, the hatchability of fertile eggs of hens 
fed 2 or 8 mg T-2 toxin/kg was lower than that of hens fed the 
control diet (Chi et al., 1977a). 

    Three groups of 1-day-old chicks (10 chicks per group) were each 
fed 0.5-15 mg T-2 toxin/kg for 3 weeks (Coffin & Combs, 1981).  
Plasma-vitamin E activity and hepatic-vitamin A content were 
measured.  Dose-dependent depression of plasma-vitamin E activity was 
observed, with a 65% decrease compared with controls in chicks fed a 
diet containing 15 mg T-2 toxin/kg.  This decrease was believed to be 
caused by a reduction in the plasma level of lipoproteins, which are 
required for the transport of vitamin E. 

 (c) Ruminants

    In a study by Pier et al. (1976), 4 calves received  0.08-0.6 mg 
T-2 toxin/kg body weight orally in capsules for 30 days.  The high-
dose calf developed a hunched stance and died on day 20.  At all 
levels, some evidence of mild enteritis with loose faeces was 
obtained.  Clinically apparent signs were confirmed at doses of 0.16 
mg/kg or more, and bloody faeces at doses of 0.32 mg/kg or more.  At 
necropsy, abomasal ulcers were present in the calf given 0.16 mg/kg 
and ruminal ulcers in calves given the 2 higher doses.  Prothrombin 
times and levels of serum GOT activity were increased in calves given 
the 2 higher doses. 

    Ten male Suffolk-Finn-Columbian lambs, in 2 groups of 5 animals 
each, were fed T-2 toxin at 0.3 or 0.6 mg/kg body weight for 21 days.  
There were 5 controls.  Experimental lambs developed focal hyperaemia 
and dermatitis at the mucocutaneous junction of the commissure of the 
lips, diarrhoea, leukopenia, lymphopenia and lymphoid depletion of 
the mesenteric lymph nodes and spleen (Friend et al., 1983b). 

    Sheep dosed with roridin A and verrucarin A (4 mg/kg) had severe 
and extensive haemorrhagic gastroenteritis.  Oedema was marked in the 
abomasum; the small intestine of the roridin-treated lamb had casts 
of clotted blood and necrotic debris.  Both small and large 
intestines contained grossly haemorrhagic areas and extensive mucosal 
erosions. In the lamb given verrucarin A at 4 mg/kg body weight, the 
lesions were sublobular haemorrhages in the liver, which had a nutmeg 
appearance, mucosal erosions, haemorrhages in the small intestine, 
and haemorrhages of the endocardium of the left ventricle of the 
heart (Mortimer et al., 1971). 

 (d) Cats

   Three studies have described the clinical and tissue alterations 
produced by administration of T-2 toxin to cats (Sato et al., 1975; 
Lutsky et al., 1978; Lutsky & Mor, 1981).  Lutsky et al. (1978) used 
20 cats in 4 groups of 4-6 animals each.  There were 4 controls.  The 
toxin was administered orally in gelatin capsules on alternative days 
at doses of 0.06, 0.08, or 0.10 mg/kg body weight, until death.  The 
survival time ranged from 6 to 40 days.  The signs included emesis, 

anorexia, bloody diarrhoea, and ataxia.  The cats lost weight and 
became emaciated.  Gross lesions included multiple petechiae to 
ecchymotic haemorrhages of the intestinal tract, lymph nodes, and 
heart.  The lumen of the gut contained copious amounts of dark red 
contents.  Microscopic lesions included haemorrhages in the gut, 
lymph nodes, heart, and mininges, necrosis of gastrointestinal 
epithelium and decreased cellularity of the bone marrow, lymph nodes, 
and spleen. 

 (e) Rodents

    The trichothecenes used in long-term studies were T-2 toxin and 
fusarenon-X.  Lesions were observed in the oesophageal region of the 
stomach of DDD mice fed T-2 toxin at 10 or 15 mg/kg diet for 12 
months.  The alterations included hyperplasia, hyperkeratosis, and 
acanthosis of the squamous epithelium.  Such changes were found 13 
weeks after the start of feeding the toxins and were consistently 
observed during the 12-month feeding period. However, most had 
subsided 3 months after cessation of feeding.  Similar gastric  
lesions were observed in Wistar rats fed T-2 toxin at concentrations 
of 5, 10, or 15 mg/kg feed for 4 weeks.  The lesions were diffuse and 
severe in the rats fed 15 mg/kg, focal but definite in those fed 10 
mg/kg, and negligible in the stomach of rats fed 5 mg/kg (Ohtsubo & 
Saito, 1977). 

    Six female Holtzman albino rats were fed T-2 toxin at 5 or 15 
mg/kg for 19 days and T-2 toxin at 10 mg/kg diet for 8 months.  No 
gastric lesions were observed in any of the animals in the 
experimental groups (Marasas et al., 1969).  

    Three groups of 12, six-week-old female Swiss ICR mice (15-20 g 
body weight) were administered T-2 toxin (by 10-minute aerosol 
exposure) Two control groups contained 8 mice each using nose-only 
exposure.  The aerosol mass concentration varied between 225 and 275 
µg T-2 toxin/litre of air.  Tissues from mice were microscopically 
examined 0.25, 1, 2, 4, 6, 8, 12, and 24 h after exposure.  Lymphoid 
necrosis was observed 1 h after exposure in the thymus, spleen, and 
lyphoid nodules of the intestinal tract.  Necrosis of intestinal 
crypt epithelial cells was present 2 h after exposure and necrosis of 
adrenal cortical cells 4 h after exposure (Thurman et al., 1988). 

    In male Sprague-Dawley rats, T-2 toxin, given intravenously, 
produced reduced blood flow and increased vascular resistance in 
hind-quarter, mesenteric, and renal vascular beds.  Mean arterial 
pressure and heart rate were not significantly altered.  A maximum 
drop in blood flow in mesenteric and renal vascular beds occurred 4 h 
after the T-2 toxin was injected (Siren and Feuerstein, 1986). 

II.4.2.2  Haematological and haemostatic changes

    A haemorrhagic syndrome was reported to be the characteristic 
feature of mouldy corn toxicosis in the cow (section II.4.1.). 
However, this hemorrhagic syndrome could not be produced in other 

    In a study by Patterson et al. (1979), 2 calves were administered 
0.2 mg T-2 toxin/kg body weight and one calf was given the same dose 
of DAS; both compounds were given by stomach tube,daily for 11 days.  
There were no controls.  The T-2-treated animals developed clinical 
signs of weakness, inappetence, and one died.  Prothrombin time was 
prolonged in both animals and one had marked neutrophilia.  No 
clinical signs or haematological changes were observed in the animal 
administered DAS.  No haemorrhagic syndrome was found in these 

    When pigs (9-10 weeks old, male, castrated, specific pathogen-
free) were exposed to a T-2 toxin aerosol (390 µg/litre, 15 µm mass 
median aerodynamic diameter) for a period that allowed an amount 
equivalent to 8 mg/kg to be nebulized, the haematological alterations 
included a decrease in lymphocyte and neutrophil counts, and 
decreased concentrations of serum-protein and haemoglobin (Pang et 
al., 1988). 

    Haematological changes were observed in mice, rats, cats, and 
guinea-pigs treated with T-2 toxin and related trichothecenes (Sato 
et al., 1975, 1978; Sato & Ueno, 1977; DeNicola et al., 1978).  In 
cats, leukocytosis occurred early after the administration of T-2 
toxin.  A similar change was observed in mice treated with T-2 toxin, 
neosolaniol, and fusarenon-X.  Among the leukocytes, lymphocytes 
showed the greatest increase followed by neutrophils; the 
leukocytosis was followed by marked leukopenia after short-term 
exposure to T-2 toxin.  This leucopenic state was also induced by DAS 
in mice (Conner et al., 1986), rats and dogs (Stahelin et al., 1968) 
and by verrucarin A in rats, dogs, guinea-pigs, and monkeys (Rusch & 
Stahelin, 1965).  Pancytopenia was also reported in cats administered 
T-2 toxin for 2 weeks (Lutsky et al., 1978; Lutsky & Mor, 1981).  In 
guinea-pigs treated with T-2 toxin (0.9 mg/kg body weight per day) 
for 27 days, erythropenia, leukopenia, and absolute lymphopenia were 
observed, with a marked decrease in the lymphocyte contents of the 
bone marrow (DeNicola et al., 1978).  

    Hayes et al. (1980) studied the effects of T-2 toxin on the 
haematopoiesis in mice.  Twenty-four male weanling outbred Swiss mice 
were fed a balanced semipurified diet, containing crystalline 
purified T-2 toxin at a level of 20 mg/kg dry diet. One group of 20 
animals received the toxin in the diet for 41 days and another group 
of 4 animals, for 21 days, followed by control diet for 7 days.  
Forty-eight animals in 3 groups served as controls and received the 
semipurified diet with restricted intake (20 animals for 41 days),  
and ad lib (8 animals for 28 days).  In addition, 12 animals were 
sacrificed at day 0.  Haematological studies were made at weekly 
intervals.  During the first 3 weeks of exposure to T-2 toxin, 
lymphoid tissues, bone marrow, and splenic red pulp became  
hypoplastic resulting in anaemia, lymphopenia, and eosinopenia.  
Subsequently, during continued exposure to T-2 toxin, there was 
regeneration leading to hyperplasia of the haematopoietic cells by 6 
weeks.  All animals also developed perioral dermatitis and ulceration 
of the gastric mucosa.  The above results indicate both the irritant 
and haematopoietic suppressive effects of the T-2 toxin.  However, 
the haematopoietic effects were transient at the dose administered 
and did not lead to haematopoietic failure. 

    The haemostatic derangements produced by T-2 toxin have been 
studied in the guinea-pig (Cosgriff et al., 1984), rabbit (Gentry, 
1982), chicken (Doerr et al., 1981), and monkey (Cosgriff et al., 
1986).  Guinea-pigs (Hartley strain, number not stated) admin-istered 
T-2 toxin dissolved in ethanol, by intramuscular injection, at a dose 
of 1 mg/kg body weight (LD50-24 h) developed decreased activities of 
all coagulation factors except fibrinogen.  Platelet aggregation in 
whole blood response to ADP and collagen was depressed.  The animals 
also showed an initial rise followed by a fall in the haematocrit 
level, leukocytosis, and a fall in platelet count.  These changes, 
which were found within a few hours of toxin administration, reached 
a maximal at 24 h and returned to normal over the next 2 days.  
Pretreatment with vitamin K1 did not prevent the effects of T-2 toxin 
on coagulation.  The addition of T-2 toxin to the plasma and blood of 
untreated guinea-pigs at a concentration of 1 mg/litre did not have 
any effect on clotting times or platelet aggregation, indicating that 
the T-2 toxin itself did not have any direct effect on the activ-ity 
of coagulation factors (Cosgriff et al., 1984). 

    Eight New Zealand White rabbits were administered T-2 toxin 
dissolved in dimethyl sulfoxide (DMSO) by intravenous injection at 
0.5 mg/kg body weight; 5 rabbits were given a single oral dose of 2.0 
mg/kg body weight.  In the rabbits treated intravenously, both the 
packed cell volume and the total leukocyte counts were reduced.  
However, no significant alterations occurred in the haematological 
parameters of rabbits given the T-2 toxin orally  (Gentry & Cooper, 

    In another study, 9 New Zealand White rabbits were given a single 
intravenous injection of T-2 toxin dissolved in DMSO at a dose of 0.5 
mg/kg body weight.  A second group of 5 animals received daily 
subcutaneous injections of vitamin K, at a dose of 0.5 mg/kg body 
weight, for 5 days prior to administration of a similar dose of T-2 
toxin and for a subsequent 4 days.  A total of 16 rabbits in 2 groups 
served as controls.  Blood samples were examined from each animal 
before toxin or DMSO treatment and 6-96 h later.  Several coagulation 
factors (VII, VIII, IX, X, XI) were decreased by about 40% within 6 h 
of toxin administration in the group administered toxin alone.  
Fibrinogen content was elevated  at  24 h.   However,  the reduction 
in  the  coagulation factors did not induce clinical haemorrhage and 
administration of vitamin K did not alter the effects of T-2 toxin 
administration, indicating that the mechanism of action of the toxin 
on coagulation was not as a vitamin K antagonist. 

    In a study by Cosgriff et al. (1986), 9 Cynomolgus monkeys 
received an intramuscular injection of an LD20 dose (0.65 mg/kg body 
weight) of T-2 toxin dissolved in ethanol.  Three monkeys served as 
controls.  Haematological studies were made before toxin  injection  
and at different intervals from 6 to 24 h and 2 to 7 days after 
treatment.  The animals were studied for signs of toxicity and 
particularly for evidence of haemorrhage.  Necropsy was performed on 
animals that died during study.  Leukocytosis levels in treated 
animals were 4-5 times pretreatment levels.  Prolongation of 
prothrombin, activated thromboplastin times, and a decrease in 
multiple coagulation factors were also observed.  These changes were 

detected within hours of toxin administration, reached a maximum at 
24 h, and returned to normal over the next 3 days.  Fibrin-fibrinogen 
degradation products were not detected at any time.  Platelet counts 
which were unchanged in treated animals, were significantly raised in 
control animals following repeated phlebotomies.  None of the animals 
developed the haemorrhagic syndrome.  Five animals that died during 
the study showed mild peticheal haemorrhages involving the colon and 
heart, as well as necrosis of lymphoid tissues. 

    Rukmini et al. (1980) conducted a study on adult rhesus monkeys 
in which 3 males and 2 females were administered  pure T-2 toxin in 
20 ml milk by stomach tube, daily, initially at 1 mg/kg body weight 
for 4 days, and then at 0.5 mg/kg body weight from day 5 to day 15.  
Three males and 3 females served as controls.  All 3 males in the 
treated group died of respiratory failure between days 0 and 15.  
Subsequently, after 30 days recovery, the 2 treated female and 2 
additional male monkeys received 0.1 mg T-2 toxin/kg body weight for 
15 days.  All monkeys given 1 mg T-2 toxin/kg per day showed signs of 
toxicity similar to those of alimentary toxic aleukia in man, i.e., 
vomiting, apathy, and weakness of lower limbs.  The signs were more 
severe in males, and they also developed peticheal haemorrhage on the 
face.  All male animals developed severe leukocytopenia, follicular 
atrophy of the spleen and lymph nodes, and pneumonia, suggesting 
involvement of the immune system.  At a dose of 0.1 mg/kg per day, 
both male and female animals developed leukocytopenia and mild 
anaemia after 15 days of treatment. 

    The effects of T-2 toxicosis on blood coagulation were studied in 
groups of 40, day-old chickens fed diets containing the toxin at 
concentrations of 1, 2, 4, 8, or 16 mg/kg.  Forty animals served as 
controls.  Factor X, and prothrombin and fibrinogen activities were 
reduced only at the highest dietary dose, whereas Factor VII was 
reduced at dietary doses of 4, 8, and 16 mg/kg and was the most 
sensitive of the clotting components to T-2 toxin toxicosis.  Thus, 
T-2 toxin toxicosis induced by high doses results in multiple-factor 
coagulopathy and mild toxicosis results in a deficiency of Factor VII 
(Doerr et al., 1981). 

II.4.2.3  Disturbances of the central nervous system

    Four-week-old male broiler chickens were intubated with a single 
dose of T-2 toxin at 2.5 mg/kg body weight, and the brain 
concentrations of dopamine, norepinephrine, and serotonin, and 
selected blood components were determined 4-48 h after 
administration.  There was a significant elevation in the brain-
dopamine concentration and a reduction in the brain-norepinephrine 
concentration.  The brain-serotonin contents did not change (Chi et 
al., 1981).  Batches of 40 broiler chickens fed graded concentrations 
of 1-16 mg T-2 toxin/kg feed for 3 weeks developed an abnormal 
positioning  of  the  wings, hysteroid seizures, and impaired 
righting reflex.  Neural toxicity, which occurred at levels above 4 
mg T-2 toxin/kg diet, might have been related to alterations in brain 
biogenic amines (Wyatt et al., 1973a,b; Chi et al., 1977a). 

    Signs of nervous system dysfunction (restlessness, dyspnoea, 
ataxia) were observed in rats after subcutaneous or intracerebral 
injection of T-2 toxin (10-20 µg toxin) or after intracerebral 
implantation of toxin adsorbed on talc (Bergmann et al., 1985).  
Weanling, male Wistar rats were administered T-2 toxin orally at 2.0 
mg/kg body weight and the concentrations of neurotransmitters 
determined.  The toxin increased concentrations of tryptophan, 
serotonin, and dopamine in the brain, but decreased concentrations of 
3,4-dihydroxyphenylacetic acid (MacDonald et al., 1988).  Male 
Sprague-Dawley rats (180 g) were dosed orally with DON or T-2 toxin 
at 21.5 mg/kg body weight.  Both the toxins significantly increased  
serotonin and 5-hydroxy-3-indoleacetic acid concentrations in all 
regions of the brain examined, whereas norepinephrine and dopamine 
concentrations were not altered (Fitzpatrick et al., 1988).  Male 
Sprague-Dawley rats received 1 mg T-2 toxin/kg body weight by 
intravenous injection.  Concentrations of vasopressin, oxytocin, and 
leucine enkephalin decreased in the posterior pituitary and 
concentrations of methionine enkephalin increased (Zamir et al., 

    Male Sprague-Dawley rats (180 g) and 4-week-old White Leghorn 
cockerels were dosed orally with DON at 2.5 mg/kg body weight.  Whole 
brain concentrations of monoamine neurotransmitters were not altered 
in either species. The treatment produced elevated concentrations of 
serotonin  and 5-hydroxy-3-indoleacetic acid in the rat, but not in 
the chicken (Fitzpatrick et al., 1988). 

II.4.2.4  Dermal toxicity

    After the discovery of the skin-necrotizing property of toxic 
metabolites of  F. sporotrichioides and related fungi (Joffe, 1962), 
the skin irritation test was introduced for the screening of toxins 
and metabolites of  Fusarium species (see section II.1.2.3).  T-2 
toxin, HT-2 toxin, and DAS were isolated from cultures of  F. 
 tricinctum using the skin test for selection of active fractions 
(Gilgan et al., 1966; Bamburg et al., 1968b).  Toxins, such as T-2 
toxin, HT-2 toxin, and DAS are extremely potent irritants while NIV 
and fusarenon-X are much less so (Bamburg et al., 1968b; Ueno et al., 
1970; Wei et al., 1972; Chung et al., 1974; Hayes & Schiefer, 1979; 
Bhavanishankar et al., 1988). 

    The mechanism of the skin toxicity of trichothecenes has not been 
established.  Results of studies with fusarenon-X have indicated that 
the vascular permeability of the skin of the back of the rabbit, 
estimated by exudation of a vital dye (pontamine sky blue), was 
biphasic reaching maxima 5 and 24 h after topical application.  These 
data indicate that increased vascular permeability is one of the 
early responses of the skin to these toxins and that some chemical 
mediators participate in the biphasic increase in vascular 
permeability (Ueno, 1980a). 

II.4.2.5  Impairment of immune response

    Experimental animal studies show that some trichothecenes affect 
the immune system and thereby modify the immune response.  The 
impairment comprises the following functions:  antibody formation; 

allograft rejection; delayed hypersensitivity; and blastogenic 
response to lectins.  As a consequence of the impairment, decreased 
resistance to microbial infection has been experimentally 
established.  It is likely that the impairment of the immune system 
is linked to the inhibitory effect of trichothecenes on macromolecule 

 (a) Antibody formation

    Rosenstein et al. (1979) showed that T-2 toxin and DAS inhibited 
responsiveness to sheep red blood cells in male Swiss IC and C5781/6 
mice.  In their first study, T-2 toxin or DAS was injected ip daily 
for 7 days at 0.75 mg/kg body weight, in 6 groups of 4 mice each, 
with matching controls.  Mice were immunized with sheep erythrocytes 
(SRBC) on day 3 after treatment and killed 5 days after immunization.  
Both toxins produced a fall in anti-SRBC titres measured by 
haemagglutination and reduced thymic weight.  In a second study, 7 
groups of 5 mice each were administered daily (ip) doses of T-2 toxin 
ranging from 0 (solvent alone) to 2.5 mg/kg body weight over a 7-day 
period.  The same number of mice received DAS under similar 
conditions.  Mice were immunized on day 3 and killed 5 days later.  
Antibody-producing cells from the spleen were counted by numbering 
the plaque-forming cells (PFC) on sheep erythrocytes.   A  dose-
dependent inhibition of PFC was observed in T-2 toxin-treated mice, 
with a total suppression of the immune response at 2.5 mg/kg.  The 
effects of DAS were less.  A subsequent follow-up of the evolution of 
the immune response in 36 mice administered T-2 toxin (ip) at 0.75 
mg/kg body weight daily for 7 days indicated that the immunosuppressive 
effect disappeared within 6 days followup cessation treatment. 

    The T-cell-independent-responses-production of anti-
polyvinylpyrrolidone and anti-dinitrophenol-ficoll-antibodies were 
enhanced by T-2 toxin and DAS.  T-2 toxin-treated mice produced 50% 
fewer plaque-forming cells against SRBC.  There was also a decreased 
response to phytohaemagglutinin in splenic cells from treated mice.  
However, Masuko et al. (1977), and Otokawa et al. (1979), reported 
that a single dose of 3 mg T-2 toxin/kg body weight in mice caused 
modification of delayed hypersensitivity responses without affecting 
antibody response.  This apparent contradiction was explained by a 
difference in the timing of the administration of T-2 toxin, mice 
receiving the toxin once, several days before or after antigen-
stimulation.  Results of studies with fusarenon-X indicated that both 
IgE and IgG anti-body responses to DNA-OVA were suppressed when male 
BALB/c mice were repeatedly dosed with mycotoxin at doses exceeding 
25 mg/day; the inhibition of antibody-formation was greater when 
given 7 days before antigen-stimulation.  In mice stimulated with 
pokeweed mitogen and lipopolysaccharides, the  in vitro antibody 
production by splenic cells from fusarenon-X-treated mice was 
suppressed (Masuda et al., 1982). 

    Sato et al. (1981), examined the effects of fusarenon-X on 
serological responses in chicks inoculated with Newcastle disease 
vaccines.  When chicks were fed 8 mg fusarenon-X/kg for 6 weeks, no 
significant reductions in body or organ weights were observed.  

However, haemagglutinin inhibitory antibody titres were reduced when 
chicks were immunized with live, but not with inactivated, vaccine. 

    Mann et al. (1983), reported alterations in the levels  
of several serum proteins in calves orally administered T-2 toxin 
(0.6 mg/kg per day over 43 days).  Total protein, albumin, and 
immunoglobulin fractions were decreased in toxin-treated calves, 
including the alpha-beta1-and beta2-globulin fractions.  IgA and IgM 
values and complement proteins were lower in treated calves. 

    Sublethal doses of DON (0.25, 0.50, or 1.0 mg/kg feed) were fed 
for 54 weeks, beginning at 21 days of age, to a total of 96 weanling 
male Swiss Webster mice divided into 4 groups.  There were 32 
controls.  The dose of 1.0 mg/kg reduced serum alpha1- and alpha2-
globulins, increased serum-albumin levels, and reduced feed 
consumption and body weight gain.  The dose of 0.5 mg/kg reduced 
alpha2- and beta-globulins (Tryphonas et al., 1986). 

    T-2 toxin was fed to 6 weaned pigs at 5 mg/kg feed for 25 days 
and the immune response evaluated by  in vitro testing for blast 
transformation, immune-rosette formation, and IF-detectable IgG-
positive cell counts.  T-2 toxin produced a 40-50% reduction in 
immune responsiveness and a decrease in total leukocyte count, but an 
increase in adrenocortical activity.  Neutralizing antibody titres to 
vaccination with enteritic B vaccine were lower in the treated pigs.  
It was concluded that T-2 toxin had a distinct immunosuppressive 
effect during the early phase of immune induction by altering the 
function of both T- and B-lymphocytes (Rafai & Tuboly, 1982). 

    Dietary DON (2, 5, or 25 mg/kg feed for 2 or 8 weeks) depressed 
the plaque-forming response to sheep erythrocytes in splenic cultures 
from B6C3F1 mice.  Some effect on the plaque-forming response was 
detectable with both the 2- and the 8-week period of feeding (Pestka 
et al., 1987b).  DON, given by gavage at 0.75, 2.5, or 7.5 mg/kg body 
weight also reduced serum-IgM response to sheep erythrocytes and 
plaque-forming cell numbers were lower in the treated groups 
(Tryphonas et al., 1984). 

 (b) Allograft rejection

    Observations on the inhibition of cellular immunity by 
trichothecenes have included responses to grafting.  According to 
Rosenstein et al. (1979), the mean survival time of the skin grafted 
from C57Bl/6 mice on to Swiss mice was 8.69 days in the control 
recipients.  However, when the recipients were treated with 0.75 mg 
T-2 toxin/kg per day for 7 days before skin graft and then 3 times a 
week for 20 days, the mean survival time of the graft was increased 
to 12 days, indicating that T-2 toxin suppressed certain steps of 
immunity resulting in allograft rejection.  The areas of the graft in 
T-2 toxin-treated mice lacked the typical cellular infiltrates of a 
cell-mediated immune response of macrophages and lymphocytes. 

 (c) Delayed hypersensitivity

Delayed hypersensitivity (DH) is an immune response mediated by 
sensitized T lymphocytes.  The possible impairment of T lymphocytes 
by T-2 toxin was studied in female BDF1 mice sensitized by the sc 
injection of sheep erythrocytes (SRBC) followed by estimation of foot 
pad swelling.  When mice received 3 mg T-2 toxin/kg body weight, 
before, or on the day of, sensitization, no appreciable effect on DH 
was observed.  However, when the toxin was administered 2 or 3 days 
after sensitization, marked enhancement of the delayed 
hypersensitivity response was seen (Otokawa et al., 1979).  This 
indicates that the timing of toxin exposure was critical for 
enhancement of the delayed hypersensitivity response.  Since the 
life-time of the effective T-2 toxin dose  in vivo was very short, 
and the optimal timing of toxin injection corresponded with the time 
of appearance of suppresser cells, it was presumed that trichothecenes 
might interfere with the proliferation of suppresser T cells that 
appear in DH-tolerant mice. 

    DON fed to B6C3F1 mice at 2, 5, or 25 mg/kg for 2 or 8 weeks 
depressed the delayed hypersensitivity response to keyhole limpet 
haemocyanin.  The effects on hypersensitivity were detectable in mice 
fed the mycotoxin for 2 weeks, but disappeared when the feeding 
period was extended to 8 weeks (Pestka et al., 1987b). 

 (d) Blastogenic response to lectins

    Certain mitogens, such as phytohaemagglutinin (PHA) and 
concanavalin A, stimulate  the proliferation of T cells  in vitro; 
lipopolysaccharide (LPS) causes the same phenomena in B cells.  
Lafarge-Frayssinet et al. (1979) investigated the responses of 
lymphocytes to PHA and LPS in mice treated with crude T-2 toxin and 
DAS.  Mice were treated with the mycotoxins at doses of one-quarter 
of the LD50 or one-twelfth of the LD50 for 15 days and the response 
of splenic or thymic cells to the mitogens was examined.  The data 
indicated that stimulation of both T and B cells was inhibited 
reversibly, and that the ability to synthesize anti-SRBC antibodies 
was suppressed.   In vitro effects on lymphocytes and fibrosarcoma 
cell cultures included a direct cytostatic action at high 
concentrations and a stimulating action at low concentrations.  
Histopathological observations included severe lymphoid damage in the 
thymus and spleen.  The results of these studies indicate that the 
immune system appears sensitive to the trichothecenes and is impaired 
at doses not inhibitory for other organs. 

    Swiss mice were fed a diet containing T-2 toxin at 5, 10, or 20 
mg/kg for 1, 2, 3, 4, or 6 weeks.  The ingestion of T-2 toxin (only 
at 20 mg/kg at 3 weeks) depressed total splenic cell counts.  T-2 
toxin at 20 mg/kg for 1-4 weeks decreased splenic proliferative 
responses to T-cell mitogen concanavalin A; however, the response to 
a lipopolysaccharide (LPS), B-cell mitogen, was decreased in mice fed 
T-2 toxin at 10 or 20 mg/kg for 1-4 weeks. (Friend et al., 1983a). 

    Lymphocytes from calves exposed to T-2 toxin at 0.6 mg/kg for as 
long as 43 days had a decreased response to the mitogen, PHA, on days 
1, 8, and 29 after toxin administration.  Lymphocyte responses to 
concanavalin A and pokeweed mitogen were also observed on day 29 
after dosing (Buening et al., 1982). 

    In a study to determine the effects of T-2 toxin on the bovine 
immune system, calves (5) were orally dosed with 0.3 mg/kg per day, 
for 56 days.  Neutrophil function was reduced by treatment with T-2 
toxin as was the cutaneous reaction to injected phytohaemagglutinin.   
In a second study, calves (6) were given T-2 toxin at a dose of 0.5 
mg/kg per day, for 28 days.  B-lymphocyte number and the response of 
the B-cell-enriched fraction to phytohaemagglutinin both increased 
after treatment.  The  in vitro exposure of mononuclear cells, B-cell-
enriched or T-cell enriched fraction, reduced the lymphoblastic 
response to mitogens.  A 50% reduction was induced by 1.4 ng T-2 
toxin/ml (Mann et al., 1984). 

    The effects of T-2 toxin on  in vitro mitogen response and 
antibody production by human peripheral blood lymphocytes were 
reported by Tomar et al. (1988).  The toxin inhibited the mitogen 
response to concanavalin A at a lower concentration (1.6 mg/ml) 
compared with phytohaemagglutinin (2-4 mg/ml) and pokeweed mitogen.  
In the presence of the toxin, inhibition reached a maximum during 
first 8 h.  The results indicate that various subpopulations of 
lymphocytes have different susceptibilities to T-2 toxin.  

    Mitogen-induced blastogenesis in cultured human lymphocytes was 
inhibited by T-2 toxin and its metabolites.  The concentrations of T-
2 toxin, HT-2, 3'-OH T-2,3'-OH HT-2, T-2 triol, and T-2 tetraol 
toxins that produced 50% inhibition of 3H-thymidine uptake in 
mitogen-stimulated human peripheral lymphocytes were 1.5, 3.5, 4.0, 
50.0, 150.0, and 150.0 ng/ml, respectively.  The initial hydrolysis 
of T-2 toxin to HT-2 toxin and the hydroxylation to 3'-OH T-2 did not 
significantly decrease the immunotoxicity (Forsell et al., 1985).  
Other trichothecenes were less toxic than T-2 toxin in this system.  
The doses that produced 50% inhibition of 3H-thymidine uptake in 
mitogen-stimulated human lymphocytes for fusarenon-X, NIV, DON, and 
15-AcDON were 18, 72, 140, and 240 ng/ml, respectively.  These 
results indicate that the lymphotoxicity of trichothecenes is related 
to the C-4 substituent (Forsell & Pestka, 1985). 

    T-2 toxin was examined for its effects on lymphocyte activation 
and interleukin-2 production by splenic cultures from mice.  Splenic 
cells were taken from female BALB/c mice given 2 mg T-2 toxin/kg body 
weight by stomach tube for 4 days or 4 mg T-2 toxin/kg by stomach 
tube in single dose.  Cells were incubated with 1 µg concanavalin A 
and the synthesis of cellular protein and DNA determined.  The single 
dose of 4 mg/kg did not alter lymphocyte activation, but the dose of 
2 mg/kg for 4 days produced a 50% reduction in activation.  The 
supernatant from these cells had 4 times greater interleukin-2 
activity (Holt et al., 1988a). 

    DON and 3-AcDON were evaluated  in vitro for their effects on 
mitogen-induced lymphocytic blastogenesis using rat or human 
peripheral blood lymphocytes.  Both mycotoxins produced a dose-
dependent reduction in lymphocytic proliferation and DON produced a 
greater inhibitory effect than the acetylated compound.  Thus, the 
concentrations of DON producing 50% inhibition of blastogenesis were 
90 and 220 ng/ml for rat and human lymphocytes, respectively.  The 
values for 3-AcDON were 450 and 1060 ng/ml, respectively (Atkinson & 
Miller 1984). 

 (e) Resistance to infection

    The immunosuppressive effect of trichothecenes has resulted in an 
increased incidence and severity of infection in animals in several 
studies.  According to Boonchuvit et al. (1975), an increased 
mortality rate was recorded when 40 chickens were fed a diet 
containing 16 mg T-2 toxin/kg for 1 week and were then inoculated 
orally with 1 x 108 cells of  Salmonella.  

    The depression of resistance to experimental tubeculosis by T-2 
toxin was studied in mice by Kanai & Kondo, (1984).  Groups of male 
mice, strain ddY, 18-20 g body weight, with 10-14 animals per group, 
were administered mycobacteria by intravenous injection in the tail 
vein.  In the first study, the doses injected consisted of 0.01 mg 
culture of tubercle bacteria per animal (species not indicated).  One 
group was administered 0.1 mg T-2 toxin per animal a total of 12 
times orally, starting on the day before the injection, 7 times with 
one-day intervals, and then 5 times daily.  For comparison, a second 
group of mice was administered 5 mg cortisone acetate per animal ip 
under a similar time schedule.  A third group was injected with 
tubercle control bacteria only.  At the end of the 20-day observation 
period, the mice in the first group had a lower spleen weight and a 
higher tubercle bacteria count in the spleen than those in the other 
2 groups, indicating a more pronounced depression of resistance by T-
2 toxin than by cortisone.  In a second study, two groups of animals 
were injected with 0.25 mg of a culture of  Mycobacterium bovis; one 
group was then administered 0.1 mg T-2 toxin per animal daily for 6 
days, starting 8 days after injection.  There were two groups of 
controls.  The average survival time in the T-2 toxin-treated group 
was reduced to 19 days, compared with 35 days in the untreated group, 
indicating decreased resistance. 

    Rats were injected ip with T-2 toxin in a single dose of 1 
mg/kg body weight or in doses of 0.5 mg/kg daily for 5 days.  
The rats were then inoculated with 0.1 ml of medium 
containing 109  Staphylococcus aureus/ml.  Rats given 
multiple intramuscular injections of T-2 toxin showed more 
oedema and myofibre necrosis at the injection site of the 
bacteria; the cellular infiltrate was sparse and bacteria 
were abundant.  Bone marrow myeloid cells were markedly 
decreased by multiple injections of T-2 toxin.  In  in vitro 
studies, small, non-lethal doses of T-2 toxin inhibited 
the chemotaxis of leukocytes and decreased phagocytosis of 
the bacteria by leukocytes (Yarom et al., 1984b). 

    Seven male rhesus monkeys  (Macaca mulatta) were dosed daily by 
stomach tube for 4-5 weeks with 100 µg T-2 toxin/kg body weight.  
This dose resulted in the death of 3 animals, 40% reduction in 
leukocyte counts, reduction in the bactericidal activity of 
neutrophils (phagocytosis of  E. coli), reduction in the 
transformation of lymphocytes by mitogens, and a reduction in numbers 
of C-cell and T-cell lymphocytes (Jagadeesan et al., 1982). 

    The immunotoxic effects of T-2 toxin on cell-mediated resistance 
were studied in female ICR mice infected with  Listeria monocytogenes.  
Mice in groups of 17 animals (10 animals in the control group) were 
inoculated ip with 4 x 105 (LD50) or 4 x 104 (non-lethal) doses of  L. 
 monocytogenes per animal, treated with a single oral dose of 4 mg T-2 
toxin/kg body weight, and observed for 15 days.  Bacterial 
multiplication was rapid in the spleen after T-2 toxin treatment and 
mortality was increased in both treated groups.  Necrosis and 
depletion of lymphoid tissue were observed in the thymus, the 
periarterioler lymphoid sheaths and the lymphoid follicles of the 
spleen.  Cellular response to  L. monocytogenes in the spleen and 
liver was decreased by treatment with T-2 toxin and the lesions were 
sparsely populated with mononuclear cells.  The foci of necrosis were 
larger with numerous colonies of bacteria.  The influx and number  
of lymphocytes and macrophages were greater in  Listeria-elicited 
peritoneal exudates.  The immunotoxic effects of T-2 toxin were 
comparable with those produced by cyclophosphamide and were 
attributed to depletion of T lymphocytes and subsequent failure of T-
cell-dependent macrophages to clear the host of bacteria (Corrier & 
Ziprin, 1986a).  In a continuation of the previous study, female ICR 
mice (17 animals per group) were inoculated ip on day 1 with 4 x 105 
(LD50) or 4 x 104 (non-lethal) bacteria per animal, treated orally on 
day 0, 1, 2, and 3 with 0, 1, or 2 mg T-2 toxin/kg, and observed for 
15 days.  The suppression of resistance by the mycotoxin was 
indicated by rapid multiplication of  Listeria in the spleen and 
increased mortality in mice in both exposed groups treated with 2 
mg/kg.  The thymuses and spleens of toxin-treated mice showed 
necrosis and depletion of lymphoid cells.  Foci of necrosis induced 
by  Listeria infection in the spleen and liver were larger in treated 
mice and the inflammatory reaction was sparse (Corrier & Ziprin, 

    Increased resistance to  L. monocytogenes infection was 
surprisingly observed by the same group (Corrier & Ziprin, 1986b) in 
mice administered T-2 toxin several days prior to the inoculation 
with bacteria.  Female ICR mice, 16 animals per group, were 
administered T-2 toxin by stomach tube at dosage levels of 2, 1, 0.5, 
or 0 mg toxin/kg body weight, on days -5, -4, -3, -2, -1, +1 and +3.  
On day 0 the two treated groups were inoculated ip with 106 (LD100)  
and 105 (LD50)  L. monocytogenes, respectively.  In addition, 20 mice 
were given 2 mg T-2 toxin/kg on the same days as above, and used to 
determine the effect of the toxin.  Although the cytotoxic effect of 
T-2 toxin on lymphoid tissue was marked, enhanced resistance to 
 Listeria infection was revealed by a decrease in mortality due to 
listeriosis (in both bacteria-exposed groups) in a T-2 toxin dose-
dependent way.  No specific cause for the increased resistance to 
listeriosis by T-2 toxin treatment prior to bacterial infection was 
identified by the authors. 

    ICR female mice were treated with the trichothecene mycotoxin DAS 
and subsequently inoculated ip with  Listeria monocytogenes.  The 
effect of the mycotoxin on the course of the infection was monitored 
by observing the resultant mortality and the bacterial content of the 
spleens from inoculated mice.  Mice given 3 mg DAS/kg body weight 
orally, on 2 and 1 days before inoculation, showed increased 
mortality and splenic  Listeria counts.  In these mice, thymus weights 
were reduced, and lymphocytes were depleted from the thymus cortex  
and from splenic lymphoid follicles and periarteriolar lymphoid  
sheaths.  A single dose of 4 mg DAS/kg given on day 6 before  
challenge exposure did not affect mortality compared with controls.  
Mice treated with DAS and subsequently inoculated with Listeria had  
significantly ( P = 0.006) higher levels of neutrophil populations  
than  Listeria-infected control mice (Ziprin  & Corrier, 1987). 

    Dietary DON decreased resistance of B6C3F1 mice to infection with  
 L. monocytogenes.  Resistance to the infection was similarly 
decreased in control mice fed restricted diets, comparable to dietary 
restriction caused by DON-induced feed refusal.  Resistance to  L. 
 monocytogenes was reduced to a greater extent by feed containing both 
DON and zearalenone (Pestka et al., 1987b).  DON in the diet at 0.50 
or 1.0 mg/kg was fed for 5 weeks.  Both doses resulted in a dose-
related decrease in time-to-death interval following challenge with 
 L. monocytogenes (Tryphonas et al., 1986).  

    T-2 toxin was fed to young male white Swiss mice at doses of 10 
or 20 mg/kg diet for 2-3 weeks.  The mice were then inoculated ip 
with herpes simplex virus (HSV-1).  Mice fed the high dose of T-2 
toxin were highly susceptible to HSV-1 infection and about 75% died 
with extensive hepatic and adrenal gland necrosis and with little or 
no inflammatory cellular reaction in affected tissues, such as the 
liver and adrenal glands, and the central nervous system.  No 
necrotizing encephalitis was found in treated mice.  Mice fed 10 mg 
T-2 toxin/kg had lesions of intermediate severity between those of 
the high-dose group and the virus-infected controls (Friend et al., 
1983c).  Feeding of T-2 toxin at 5, 10, or 20 mg/kg for 3-6 weeks did 
not reactivate the virus in mice latently infected with HSV-1 (Friend 
et al., 1983a). 

    Mice (male, Swiss strain weighing 25 g) received inoculations 
(route not stated) of  Cryptococcus neoformans (1 x 106 cells) and ip  
doses (1/8 or 1/4 LD50) of DAS on days 5, 6, and 7 after inoculation 
with the fungal cells.  No deaths occurred in either the  C. 
 neoformans-treated groups or the T-2-toxin group.  A marked additive 
effect on mortality was observed when mice received both  C. 
 neoformans and T-2 toxin (Fromentin et al., 1981). 

II.4.2.6  Carcinogenicity

    The IARC (1983) studied the experimental data on the 
carcinogenicity of T-2 toxin and concluded that no evaluation of the 
carcinogenic role of T-2 trichothecene in experimental animals could 
be made, because of the inadequacy of experimental data. 

    In a 16-month feeding study, groups of 50 male and 50 female 
weanling CD-1 mice were fed a semi-synthetic diet containing 1.5 or 
3.0 mg T-2 toxin/kg.  Survival was lowest in the control group.  No 
statistically recognizable differences were found in feed consumption 
or body weight gains among the groups.  Statistically significant 
differences were found in the incidence of pulmonary adenomas and 
hepatic adenomas in the males of the 3.0 mg/kg group and the 
controls.  Other treatment-related findings were an increased 
prevalence of epithelial cell hyperplasia and hyperkeratosis in the 
stomach of animals fed the T-2 toxin diets (Schiefer et al., 1987). 

    In an attempt to determine the carcinogenicity of NIV, a study 
was conducted in which mice were fed mouldy rice for 2 years.  Groups 
of 42, 7-week-old female C57BL/6CrSlc SPF mice were fed diets 
containing 6, 12, or 30 mg NIV/kg for 2 years, and were assessed for 
the effects on body weight gain, feed efficiency, terminal organ 
weights, haematological values, and lesions.  The mortality was 
lowest in the highest dose group, followed by the 12 mg/kg group; 
body weight gains and feed efficiency were dose-dependently reduced.  
No particular neoplasms attributable to treatment were found.  The 
incidence of naturally occurring neoplasms, mostly lymphomas, was 
similar in all groups.  On gross and microscopic examination of the 
liver, thymus, spleen, kidneys, stomach, small intestines with or 
without Peyer's patches, no alteration related to treatment was 
observed except for amyloidosis, which was lower in the two higher 
dose groups (Ohtsubo et al., in press). 

    Two groups of 16 or 18 DDD male mice received either 10 or 20 
weekly sc injections of 2.5 mg fusarenon-X/kg body weight.  No 
increase in tumour incidence was noted in treated animals, compared 
with the controls (Saito & Ohtsubo, 1974). 

    In a feeding study, fusarenon-X, at a dose of 3.5 or 7 mg/kg diet 
was fed to 151 male Donryu rats for 1-2 years.  Treatment with the 
mycotoxin reduced the growth rate, but did not induce any 
carcinogenic effects (Saito et al., 1980). 

    In studies to investigate skin tumour induction using T-2 toxin 
and DAS, the skin of the back of mice was painted with 0.1-1 mg of 
trichothecenes, twice a week, for one year.  A notable finding was 
necrosis of the skin, but no tumours were detected.  The skin tumour 
induction test was also carried out using the initiator-promotor 
procedure.  T-2 toxin and fusarenon-X were not promoting agents of 
dimethylbenz (a)anthracene-induced skin neoplasia.  According to 
Lindenfelser et al. (1974), neither T-2 toxin nor DAS served as 
initiating agents. 

II.4.2.7  Mutagenicity

    In a Rec-assay using  Bacillus subtilis, DNA damage was not 
induced by 20 or 100 µg of either T-2 toxin or fusarenon-X (Ueno & 
Kubota, 1976).  Such trichothecenes as T-2 toxin, DAS, and DON were 
not mutagenic to  Salmonella typhimurium strains TA98, TA100, TA1535,  
TA1537, and TA1538, with or without S-9 fraction from rat liver 
(Kuczuk et al., 1978;  Ueno et al., 1978; Wehner et al., 1978a). 

    T-2 toxin and DAS were not mutagenic in the D-3 mitotic 
recombination test with  Saccharomyces cerevisiae (Kuczuk et al., 

    Neither DNA-strand breakage nor induction of 8-azaguanine-
resistant mutation were detected with 1-32 mg fusarenon-X/litre in 
Hela cells and with the same toxin at 0.1-1.0 mg/litre in FM3A cells 
derived from C3H mouse mammary carcinoma cell line (Umeda et al., 
1972, 1977).  On the other hand, Lafarge-Frayssinet et al. (1981) 
reported that T-2 toxin induced single-strand breaks in the DNA of 
lymphoid cells  in vivo (3 mg/kg body weight) and  in vitro (0.7-5 
ng/ml).  Such DNA breakage was not observed in hepatic cells. 

    T-2 toxin, NIV, and fusarenon-X produced weak clastogenic effects 
in Chinese hamster V79-E cells (Thust et al., 1983).  Both T-2  toxin  
and  HT-2  toxin  inhibited  the  incorporation  of tritiated 
thymidine into the DNA of human fibroblasts in culture in a dose-
related fashion.  Non-toxic and toxic doses of DON (0.1-1000 
mg/litre), did not significantly increase unscheduled DNA synthesis 
in primary cultures of rat hepatocytes (Bradlaw et al., 1985). 

    Norppa et al. (1980) also reported weak induction of chromosomal 
aberrations by T-2 toxin (1.7, 2.7, or 3.0 mg/kg body weight) in  
Chinese hamster bone marrow cells.  The bone marrow micronucleus test 
was negative at a dose of 3 mg T-2 toxin/kg body weight resulting in 
a significant decrease in polychromatic erythrocytes. 

    Hamsters fed T-2 toxin for 6 weeks (2.5 mg/kg body weight) did 
not have more chromosomal aberrations than controls.  The clastogenic 
potential of T-2 toxin was very weak.  In hamster V79 cells, DON at 
levels of 2-3 µg/ml or more was cytotoxic, but was non-mutagenic at 
the hypoxanthine-guanine phosphoribosyl transferase locus, with or 
without hepatocytic mediated activation (Rogers & Heroux-Metcalf, 

    According to Reiss (1975), DAS induced various cytological 
abnormalities including shortened chromosomes, enlarged nucleoli, and 
a few chromosome breaks in the root tips of  Allium cepa (common 
onion) at concentration of 1000 and 100 µg/ml.  T-2 toxin and 
satratoxin H were C-mitotic poisons at concentrations exceeding 10 
µg/g.  Typical C-mitotic action on chromosomes morphology was 
produced by both toxins and was comparable to that of colchicine.  In 
addition, T-2 toxin induced polyploidy (Linnainmaa et al., 1979). 

    T-2 toxin and satratoxin H were not mutagenic in a sex-linked 
recessive lethal test in  Drosophila (Sorsa et al., 1980).  Lack of 
potency to produce recessive lethal mutations in  Drosophila was 
consistent with negative results obtained in certain bacterial assays 
in which trichothecenes were inactive as both base pair and frame 
shift mutagens (Kuczuk et al., 1978; Ueno et al., 1978a). 

II.4.2.8  Teratogenicity and reproductive effects

    T-2 toxin was embryotoxic and teratogenic in mice.  T-2 toxin 
dissolved in propylene glycol was injected intraperitoneally into 
pregnant mice on one of days 7-11 of gestation at doses of 0.5, 1, or 
1.5 mg/kg body weight.  T-2 toxin (doses of 1 or 1.5 mg/kg) caused 
significant maternal mortality, fetal death, and fetal body weight 
loss.  Approximately 37% of the fetuses from dams given 1 (8 litters) 
or 1.5 mg (4 litters) T-2 toxin/kg on day 10 were grossly malformed.  
The most frequent anomalies were bent, shortened, or missing tails, 
and limb malformations, including oligodactyly and syndactyly.  
Exencephaly, open eyes, retarded jaw, and skeletal malformations of 
the rib or vertebrae were also found in the fetuses (Stanford et al., 

    Pregnant CD-1 mice (18 litters/treatment group) were administered 
T-2 toxin dissolved in propylene glycol ip at 0.5 mg/kg body weight 
on gestation days 8 or 10.  The T-2 toxin produced grossly malformed 
fetuses, principally with tail and limb anomalies.  A higher 
incidence of malformations was observed when a T-2 toxin dose of 0.5 
mg/kg body weight was combined with an ochratoxin A dose of 4 mg/kg 
body weight.  An increase in fetocidal effects was found in offspring 
of dams in groups treated with the high-dose combination, on either 
day.  Few skeletal and visceral malformations were noted (Hood et 
al., 1978).  T-2 toxin (0.5 mg/kg body weight) dissolved in 1:1 
mixture of propylene glycol and 0.1N sodium bicarbonate was 
administered ip alone or in combination with rubratoxin B (0.4 mg/kg 
body weight) to pregnant CD-1 mice on day 1 of gestation.  Only T-2 
toxin resulted in gross malformations.  The combination of toxins 
increased the adverse effects on fetal body weight and mortality, but 
not the incidence or severity of the gross malformations (Hood, 

    The teratogenicity of orally administered T-2 toxin dissolved in
propylene glycol was evaluated in a study using 350 female CD-1
mice and doses of 0.5, 1.0, 2.0, 3.0, 3.5, or 4.0 mg/kg body weight 
given on day 9 of gestation with a single dose of 3.0 mg/kg on day 6,
7, 8, 10, 11, or 12 of gestation.  In the first study, the doses of 
3.5 and 4.0 mg/kg produced maternal deaths and toxicity; no fetuses 
were produced by the dams in the 4.0 mg/kg dose group and 
significantly fewer fetuses were produced by dams in the 3.5 mg/kg 
dose group.  More major and minor defects were seen in offspring in 
the 3.0 mg/kg dose group.  In the second study, the treated females 
had greater fetal loss than controls and the greatest number of dead
fetuses occurred among litters treated on day 9 of gestation.  Major
skeletal defects were more numerous in mice treated on day 7 of 
gestation.  The results indicated that a single oral dose of T-2 
toxin in propylene glycol was primarily maternally toxic and 
embryolethal; defective development was possibly secondary to  
maternal toxicity (Rousseaux & Schiefer, 1987). 

    Fusarenon-X was embryotoxic, but not teratogenic (Ito et al., 
1980).  Fusarenon-X dissolved in saline was given to pregnant DDD 
mice by subcutaneous injection at doses of 0.63, 1.0, 1.6, 2.6, or 
4.1 mg/kg body weight, or by feeding at concentrations of 5, 10, or 
20 mg/kg diet during pregnancy.  Two dams given a single sc dose of 

4.1 mg/kg died within 24 h of injection.  Abortion was induced in all 
females by a single injection of 2.6 mg/kg on day 10 of gestation.  
Smaller doses (0.63-1.6 mg/kg) produced a 16-20% abortion rate, when 
given on day 10.  Multiple doses (8-12 or 8-14 days of gestation) of 
1.0 or 1.6 mg/kg produced 100% abortion.  When the mice were fed a 
diet containing 5, 10, or 20 mg fusarenon-X/kg throughout the 
gestation period or in the early stages of gestation, the mycotoxin 
inhibited embryonal implantation.  Feeding fusarenon-X at 20 mg/kg 
for 7 days during the middle stages of gestation induced abortion in 
100% of dams.  Fetal body weight was significantly reduced by the 
administration of the mycotoxin, but no significant teratogenic 
effects were observed in the fetuses of dams in either the 
subcutaneous injection or feeding study (Ito et al., 1980). 

    The effects of NIV on fertilization, course of pregnancy, and 
fetuses were examined in ICR mice.  In a study by Ito et al. (1986), 
pure NIV was injected ip in pregnant mice (groups of 10 animals 
each), at dose levels of 0, 0.1, 0.5, or 1.5 mg/kg body weight per 
day, on days 7-5 of gestation.  The highest dose caused stillbirths 
after vaginal haemorrhage in 6 out of 10 animals.  High embryo 
lethality was recorded in the 2 highest dose groups (88 and 48%).  No 
fetal malformations were observed in the treated groups.  A single 
administration of 3 mg/kg on day 7 affected the embryo within 10 h,  
damaged the placenta within 24 h, and caused stillbirths at 48 h. 

    While NIV is embryotoxic, it is not teratogenic (Ito et al., 
1988).  Thirty ICR mice in 3 batches of 10 animals each were fed 
diets mixed with mouldy rice powder containing NIV at final levels of 
6, 12, or 30 mg/kg feed per day throughout gestation.  There were 11 
controls.  Purified NIV was also administered by gavage to 35 animals 
in 4 groups at doses of 1-20 mg/kg body weight on days 7-15  of  
gestation.  There were 10 controls.  Embryotoxicity associated 
with maternal weight loss was observed in the groups receiving 30 
mg/kg diet and 10 mg/ kg body weight per day, by gavage, whereas 
lower levels, such as 5 mg/kg body weight per day, by gavage, did not 
have any embryotoxic effects.  Intrauterine growth retardation was 
found at term in the fetuses of mice exposed to 12 mg/kg feed and 5 
mg/kg by gavage.  NIV did not have any significant adverse effects on 
the incidence of gross skeletal and visceral malformations.   As 5 
mg/kg body weight per day given by gavage corresponds to a feed level 
of approximately 35 mg/kg feed, the above data indicate that 
exposure to 30 mg NIV/kg feed throughout the gestation period results 
in embryotoxicity.  However, exposure to approximately 35 mg/kg feed 
during days 7-15 only of the gestation period does not induce 
embryotoxic effects, which shows the significance of constant 
exposure versus intermittent exposure to NIV. 

    DON was embryotoxic and teratogenic when dissolved in distilled  
water and given for 4 consecutive days (days 8-11 of gestation), by 
oesophageal intubation, to 15-19 pregnant Swiss-Webster mice (Khera 
et al., 1982).  The incidence of resorptions was 100% at doses of 10 
or 15 mg/kg body weight, and 80% at 5 mg/kg body weight.  The dose of 
5 mg/kg reduced the number of live fetuses and reduced the average 
fetal weight compared with the controls.  Low incidences of skeletal 

and visceral anomalies were found in the fetuses of the 1, 2.5, and 5 
mg/kg groups.  The skeletal malformations occurred in a dose-related 
manner and included lumbar vertebrae with fused arches or partly 
absent centra, and absent or fused ribs. 

    On the other hand, DON failed to produce embryotoxic and 
teratogenic effects when fed  ad lib at 0.5, 2.0, or 5.0 mg/kg to  
Fisher 344 rats during the entire course of pregnancy (Morrissey, 
1984).  No overt signs of toxicity were observed in the dams and no 
changes in maternal feed consumption were observed at any dose. 

    DON was fed to 71 adult female New Zealand White rabbits in 7 
batches of 6-14 animals during the entire period of gestation at 
doses of 0.3, 0.6, 1.0, 1.6, 1.8, or 2.0 mg/kg body weight.  There 
were 25 controls.  The fetal effects consisted of 100% incidence of 
fetal resorption in the females fed 1.8 and 2.0 mg/kg and reduced 
average body weight of fetuses from dams fed 1.0 and 1.6 mg/kg.  
These doses were not teratogenic (Khera et al., 1986). 

    Weanling F0 male and female mice were fed diets containing DON at 
a dose of 2.0 mg/kg body weight (15 male and 15 female animals) and 
0.375, 0.75, or 1.5 mg/kg body weight (7 males and 59 female 
animals).  There were 30 controls in the first study and 26 in the 
second.  After 30 days of dietary feeding, the mice were allowed to 
mate and the pregnant females were allowed to litter normally.  The 
F1a progeny were examined up to 21 days of age and discarded.  The F0 
mice were rebred.  The females bred to produce the F1b litters were 
killed on day 19 of gestation and the fetuses were examined for 
gross, visceral and skeletal malformations.  Reductions were 
observed in feed intake and in the body weight of F0 male and female 
mice, the number of live pups and postnatal survivors, postnatal body 
weight of F1a progeny, number of live fetuses, and fetal body weight 
of F1b.  No adverse effects on the fertility of F0 mice and no 
major malformations in F1b fetuses were found.  Results of cross-
fostering offspring between control dams and 1.5 mg/kg dams indicated 
that both postnatal survival and body weight were adversely affected 
by prenatal exposure as well as by combined pre- and post-natal 
exposure (Khera et al., 1984). 

    Male and female Sprague-Dawley rats (3 groups of 15 male and 15 
female animals each) were fed diets containing DON at levels of 0.25, 
0.5, or 1.0 mg/kg body weight.  Controls consisted of two groups of 
15 males and 15 females.  After 6 weeks of feeding, the rats were 
bred.  The mated females, maintained on their respective diets, were 
killed on the last day of pregnancy and fetuses were evaluated for 
effects on pre-natal development.  No adverse effects were observed 
except for dilatation of the renal pelvis and urinary bladder (Khera 
et al., 1984). 

    Male (20/group) and female Sprague-Dawley rats (25/group) were  
fed a diet containing 20 mg purified DON/kg for 60 and 15 days, 
respectively, before mating.  Rats consuming the DON-supplemental 
diet throughout gestation and lactation did not show any clinical 
signs of toxicity, but had reduced body weights.  Only 50% of the 
matings between toxin-fed rats resulted in pregnancy compared with 

80% in the controls.  No differences were detected among the groups 
in sex ratio, survival rate, or average litter number and weight.  
Pup weight gains in all groups were comparable up to post-natal day 
14.  From day 14 to 21, however, male and female pups of the control 
group showed significantly improved weight gains compared with pups 
from treated dams.  No treatment-related histological abnormalities 
were found in the testes or ovaries of treated pups (Morrissey & 
Vesonder, 1985). 

    A 2-generation reproduction and teratology study was carried out 
using 90 female CD-1 mice fed a semisynthetic diet containing the T-2 
toxin at 1.5 or 3.0 mg/kg, concentrations considered possible under 
field conditions.  Results indicated that continuous feeding of T-2 
toxin at low concentrations had minimal, if any, toxic effects on 
female reproduction and fetal development and that T-2 toxin fed 
continuously at 1.5 or 3.0 mg/kg was not teratogenic or fetocidal and 
produced minimal effects on the growth rates of CD-1 mice (Rousseaux 
et al., 1986). 

II.4.3  Biochemical effects and mode of action

II.4.3.1  Cytotoxicity

    In the early stages of research on trichothecenes, DAS was 
isolated from  F. scirpi as a phytotoxic principle (Brian et al., 
1961).  Subsequent surveys confirmed the phytotoxic nature of the 
trichothecenes using germination of seeds of  Brassica oleracea L. 
(Ueno et al., 1971c), germination of pea seedlings (Marasas et al., 
1971), growth of tobacco callus tissues (Helgeson et al., 1973), the 
germination of tobacco plant  (Nicotiana sylvestris) pollen 
(Siriwardana & Lafont, 1978), and the auxin-promoted elongation of 
soybean hypocotyl (Stahl et al., 1973). 

    Most of the trichothecenes tested have had fungistatic activity 
in a wide range of species (Bamburg et al., 1968a; Bamburg & Strong, 
1971; Reiss, 1973); DAS and verrucarin A were particularly potent in 
inhibiting growth and sporulation at concentrations of 0.5-1 

    Many of the trichothecenes have been cytotoxic to mammalian 
cells, both in  in vivo and  in vitro.  In animals administered 
trichothecenes, the mucosa of the stomach and small and large 
intestines had mucosal erosions, mucosal necrosis with ulceration, 
and severe haemorrhages; radiomimetic effects included necrosis of 
actively-dividing cells in the thymus, spleen, ovary, testis, and 
lymph nodes (Saito & Ohtsubo, 1974).  In a cell culture system, Grove 
& Mortimer (1969) demonstrated the cytotoxicity of DAS and its 
chemically modified compounds on hepatocytes of human origin and 
hamster kidney cells.  Ohtsubo et al. (1968), Ohtsubo & Saito (1970), 
and Bodon & Zoldag (1974) described the cytotoxicity of NIV and 
related trichothecenes for HeLa cells, and of T-2 toxin for 
epithelial cells of pig kidney, respectively.  The macrocyclic 
trichothecenes, verrucarins and roridins, were highly cytotoxic for 
0815 mouse tumour cells in the ng/ml range (Harri et al., 1962). 

    Tanaka et al. (1977), investigated the cytotoxicity of 20 types of 
trichothecenes (11 type A, 6 type B, 1 type C, and 2 type D) for 3 
cell lines, HeLa, HEK, and HL cells.  The type D trichothecenes,  
such as verrucarin A and roridin A had LC50s in the range of 0.003-
0.005 mg/litre; neosolaniol and NIV in the range of 0.1-1 mg/litre,  
and deoxynivalenol in the range of 1-5 mg/litre.  Trichodermol, 
calonectrin, monoacetyldeoxynivalenol, and tetraacetylnivalenol were 
weakly cytotoxic within the range of 5-10 mg/litre. 

    Chinese hamster ovary (CHO) and African green monkey kidney 
(VERO) cell cultures were exposed to 0.01 or 1.0 ng T-2 toxin/ml for 
1 or 12 h.  The cells exhibited  morphological changes considered to 
be related to inhibition of protein synthesis.  The alterations 
included disassociation of polysomes and matrix density, ballooning 
of intracristal space, and malalignment of cristae in mitochondria.  
The CHO cells had bleb formations of plasma membrane, a change 
produced by exposure to establish inhibition of protein synthesis 
(Trusal, 1985). 

II.4.3.2  Inhibition of protein synthesis

    The initial observation that the trichothecene mycotoxins 
inhibited protein synthesis in mammalian cells was made by Ueno et 
al. (1968).  They reported that NIV produced a dose-dependent 
inhibition of incorporation of several amino acids into protein  in  
rabbit reticulocytes (Ueno et al., 1968)  and  Ehrlich ascite tumour 
cells (Ueno & Fukushima, 1968).  This inhibitory effect of the 
trichothecene mycotoxins was observed in the whole animal (Ueno, 
1970), a protozoan (Ueno & Yamakawa, 1970), HeLa cells (Liao et al., 
1976), cultured mammalian cells (Ohtsubo et al., 1968; Ohtsubo & 
Saito, 1970), rat hepatocytes (Ueno et al., 1973b), hamster ovary 
cells (Gupta & Siminovitch, 1978), human tonsil (Carrasco et al., 
1973), yeast spheroplasts (Stafford & McLaughlin, 1973; Cundliffe et 
al., 1974; McLaughlin et al., 1977), rabbit reticulocytes (Ueno et 
al., 1968, 1969a, 1973b,c; Ueno & Shimada, 1974; Wei & McLaughlin, 
1974; Mizuno, 1975; Carter et al., 1976), and lymphocytes (Hartman et 
al., 1978).  No inhibitory effect on bacterial cells was observed 
(Ueno et al., 1973b). 

    The effects of T-2 toxin on protein and DNA synthesis were 
studied in Swiss mice and hepatoma cell cultures.  T-2 toxin was 
given as a single ip dose of 0.75 mg/kg and as a 3-day and a 7-day 
daily treatment.  The toxin inhibited protein synthesis after all 3 
schedules of treatment and inhibition was present in cells obtained 
from bone marrow, spleen, and thymus.  Protein synthesis was 
inhibited  in vitro in hepatoma cell cultures and PHA-stimulated 
lymphocytes (Rosenstein & Lafarge-Frayssinet, 1983). 

    The effects of T-2 toxin on rat hepatocytes were studied in 
culture by the addition of several doses at either 1 or 12 h of 
exposure.  A dose of 0.01 ng T-2 toxin/ml produced a 75% inhibition 
of protein synthesis within 1 h.  At a higher dose of 1.0 ng/ml,  
hepatocytes recovered from a 1-h but not a 12-h exposure.  Cell 
damage (release of lactate dehydrogenase) lagged behind inhibition of 
protein synthesis, which was 90% at the 1 ng/ml dose.  Ultrastructural 

alterations were present in the endoplasmic reticulum and 
mitochondria.  Degranulation involved the rough endoplasmic 
reticulum.  Mitochondria had translucent foci and electron dense 
cores (Trusal & O'Brien, 1986). 

    NIV inhibited poly-U, poly-A, and poly-C directed incorporation 
of phenylalanyl-tRNA into phenylalanine without affecting the 
activation of amino acids.  The inhibition was presumably caused by 
impairment of ribosomal function (Ueno et al., 1968).  Fusarenon-X 
caused breakdown of polysomes in rabbit reticulocytes (Ueno et al., 
1973b) and in mouse fibroblasts (L-cells), shortly after exposure 
(Ohtsubo et al., 1972).  The breakdown of polysomes was consistent 
with the action of an inhibitor of initiation of protein synthesis.  
T-2 toxin, DAS, and verrucarin A, but not trichodermin, also induced 
the disaggregation of polysomes in HeLa cells (Liao et al., 1976).  
These results were consistent with the effects of an inhibitor of 
prolongation or termination of protein synthesis (Stafford & 
McLaughlin, 1973).  An important point was that the trichothecenes, 
regardless of whether they were I-type or ET-type, interacted with 
the peptidyl transferase centre on the 60S ribosomal subunit and 
inhibited the transpeptidation of the peptide bond formation process. 

    Current research has focused on clarifying the molecular species 
of ribosomal protein that regulates the binding of trichothecene 
mycotoxins.  A mutant of yeast (Schindler et al., 1974; Jimenez & 
Vazquez, 1975; Jimenez et al., 1975; Grant et al., 1976; Carter et  
al., 1980) and Chinese hamster ovary cells, resistant to the effects 
of trichothecenes on protein synthesis (Gupta & Siminovitch, 1978), 
have been isolated.  Friend & Warner (1981) clearly demonstrated that 
the gene for trichodermin resistance in yeast specifies ribosomal 
protein L3, the largest of the yeast ribosomal proteins. 

    The ribosomal subunits of  Myrothecium verrucaria, a producer of 
macrocyclic trichothecenes, were resistant to T-2 toxin.  It can be 
assumed that the 60S subunits of eukaryotes are responsible for the 
sensitivity to the trichothecenes. (Hobden & Cundliffe, 1980). 

II.4.3.3  Inhibition of nucleic acid synthesis

    The dose-dependent inhibition by NIV of DNA and RNA synthesis in 
Ehrlich ascites tumour cells was first observed by Ueno & Fukushima 
(1968).  Inhibition (70%) of protein synthesis was induced by 1-10 mg 
NIV/litre, and thymidine incorporation into DNA was inhibited by as 
much as 60%; suppression (30%) of uracil incorporation into RNA was 
slight.  Similar results were obtained with several trichothecenes, 
including trichodermin, diacetoxyscirpenol, and fusarenon-X, in other 
cultured cells, such as KB cells (Ohtsubo et al., 1968), HeLa cells 
(Liao et al., 1976), mouse L-cell fibroblasts (Ohtsubo et al., 1972), 
hamster ovary cells (Gupta & Siminovitch, 1978), and lymphocytes 
(Hartman et al., 1978). 

    The inhibition of DNA and RNA synthesis by trichothecenes 
required higher concentrations of toxins than the inhibition of 
protein synthesis and the extent of the inhibition was much less.  

Thus, the observed inhibition of DNA and RNA synthesis in toxin-
treated animal cells was presumed a secondary effect of the 
trichothecenes.  This hypothesis was supported by the findings of 
Tashiro et al. (1979) who reported that, in  in vitro studies, a 
concentration of 0.0236-1.889 mmol fusarenon-X/litre did not inhibit 
DNA-dependent RNA hybridases of rat liver and  Tetrahymena pyriformis. 

    Munsch & Mueller (1980) reported that the incorporation of 3H-
thymidine into DNA in cell lines from thymus was strongly inhibited 
by over 10 ng T-2 toxin/ml and slightly inhibited  by 0.1-10 ng T-2 
toxin/ml.  A low concentration of 0.1-1 ng/ml toxin caused a 
transient increase in DNA polymerases, alpha- and beta-terminal 
deoxynucleotidyl transferases.  At a high dose of more than 1 ng T-2 
toxin/ml, these enzymatic activities were strongly inhibited.  
Rosenstein & Lafarge-Frayssinet (1983) described the depression of 
DNA synthesis  in vivo and  in vitro.  Three treatment schedules:  a 
single dose, 3 daily doses, or 7 daily doses of 0.75 mg T-2 toxin/kg 
inhibited DNA synthesis in cell cultures from the spleen, thymus, and 
bone marrow of treated mice.  The mycotoxin also inhibited DNA 
synthesis  in vitro in cultures of hepatoma cells and in PHA-
stimulated lymphocytes (Rosenstein & Lafarge-Frayssinet, 1983). 

    Agrelo & Schoental (1980) found that hydroxyurea did not alter 
unscheduled DNA synthesis in cells treated with T-2 toxin and HT-2 
toxin (6 ng/ml).  However, the combination of rat liver microsomes 
and hydroxyurea resulted in an increase in unscheduled DNA synthesis 
in cells exposed to 100 µg HT-2 toxin/ litre.  The data of Agrelo & 
Schoental (1980) suggest that the microsomal drug-metabolizing enzyme 
system may participate in the induction of DNA damage by 

    The effects of T-2 toxin and DAS on DNA synthesis in phyto-
haemagglutinin, stimulated in the peripheral blood lymphocytes of 
human beings, was assayed by incorporation of 3H-thymidine.  Total 
inhibition was obtained by 8 ng T-2 toxin and DAS and 80% was 
obtained with 1.5 ng T-2 toxin and 2.7 ng of DAS (Cooray, 1984).  

    Fusarenon-X and related toxins inhibited protein and nucleic acid 
synthesis in  Tetrahymena pyriformis in a manner similar to that 
observed in cultured mammalian cells (Ueno & Yamakawa, 1970).  In 
synchronously dividing  Tetrahymena cells (Iwahashi et al., 1982), the 
incorporation of radioactive amino acids, thymidine, and uracil into 
protein, DNA, and RNA, respectively, was achieved by nearly the same 
concentration of T-2 toxin.  Inhibition of protein synthesis was 
explained by the high affinity of T-2 toxin for 60S ribosomal 
subunits of  Tetrahymena, as is the case with cultured cells.  
However, neither DNA and RNA synthesis nor RNA hybridase activity 
were altered by T-2 toxin in an  in vitro system using isolated nuclei 
from normal cells or from cells pretreated with T-2 toxin.  The 
mechanism of inhibition of nucleic acid synthesis in the  in vivo 
system is not yet understood. 

II.4.3.4  Alterations of cellular membranes 

    The trichothecenes as a group inhibit protein synthesis, but the 
potency of the effect varies according to the trichothecene and the 

system ( in vitro or  in vivo) used to measure the inhibition.  T-2 
toxin was a potent inhibitor of protein synthesis both  in vitro and 
 in vivo (Ueno et al., 1973b; Rosenstein & Lafarge-Frayssinet, 1983). 

     In vitro experiments were conducted to determine the effects of 
T-2 toxin on the entry of sucrose into bovine erythrocytes, 
entrapment of sucrose and inulin in carrier erythrocytes, entrapment 
of T-2 and binding of T-2 toxin, and the measurement of cell 
permeability to the entrapped T-2 toxin.  At the highest 
concentration of T-2 toxin (20 µg), no entry of 14C-sucrose or 3H-
inulin was observed.  Very little 3H-T-2 toxin was bound to bovine 
erythrocytes and binding was independent of T-2 toxin concentration.  
The mycotoxin had no effect on the entrapment of sucrose or inulin.    
Carrier erythrocytes retained 85% of 14C-sucrose and only 18% of 
3H-T-2 toxin.  Thus, T-2 toxin diffused from carrier cells much more 
rapidly than sucrose.  It was concluded that the interaction of T-2 
toxin with bovine erythrocytes was minimal and intercalation with the 
inner bilayer was not likely, because the increase in cell volume 
that would have resulted did not occur (DeLoach et al., 1987). 

    The effects of T-2 toxin on membrane function were studied using 
L-6 myoblasts.  The minimal effective concentration (MEC) of T-2 
toxin for reduction in uptake of calcium and glucose and for 
reduction in uptake of leucine and tyrosine and their incorporation 
into protein was 4 pg/ml.  The uptake of rubidium was increased at 
0.4 pg/ml and reduced at 4 pg/ml or more.  Thymidine uptake and 
incorporation into DNA had a biphasic response with an increase at 
0.4 pg/ml and a reduction at 4 pg/ml for uptake and 40 pg/ml for 
incorporation.  Calcium efflux was reduced after 1, 5, and 15 min 
exposure to T-2 toxin at a concentration of 40 pg/ml.  These data 
indicate that T-2 toxin has multiple effects on all membrane function 
at very low concentrations and that these effects are independent of 
inhibition of protein synthesis (Bunner & Morris, 1988). 

II.4.3.5  Other biochemical effects

    Fusarenon-X inhibited the uptake of phosphate by  Tetrahymena 
cells (Chiba et al., 1972).  It caused a 50% inhibition of 
incorporation of acetate into phospholipids and a ten-fold 
stimulation of incorporation into triglyceride, effects that may be 
secondary to inhibition of phosphate uptake. However, in rabbit 
reticulocytes, fusarenon-X, T-2 toxin, and neosolaniol at 
concentrations of 100 mg/litre did not inhibit Na+-dependent glycine 
transport (Ueno et al., 1978b). 

    When SH-enzymes were pre-incubated with selected trichothecenes 
in the absence of substrates, the activities of such enzymes as 
creatine phosphokinase and lactate and alcohol dehydrogenases were 
reduced (Ueno & Matsumoto, 1975).  However, neither urease activity 
(Reiss, 1977) nor rat liver lysosomal cathepsin activity (Farb et 
al., 1976) was affected by diacetoxyscirpenol or T-2 toxin, 
respectively,  in vitro.  Foster et al. (1975), reported the 
reactivity of T-2 toxin with glutathione in the presence of epoxide-

    When mice were exposed to trichothecene mycotoxins, no detectable 
alterations were observed in hepatic and renal functions.  In chicks 
fed a diet containing 10 mg T-2 toxin/kg for 3 weeks, a hepatic 
microsomal aminopyrine, demethylase, was reduced by 29% compared with 
the control, while another mixed function oxidase,  aniline  
hydroxylase,  was  not  significantly  affected (Coffin & Combs, 
1981).  In mice receiving a sublethal dose of fusarenon-X ip, a rapid 
hypoglycemia was followed by depletion of hepatic glycogen (Shimizu 
et al., 1979).  The authors suggested that the toxin had induced a 
malfunctioning of glucose absorption in the intestines and had 
accelerated glycolysis.  However, the trichothecene mycotoxins are 
highly cytotoxic to the epithelia of the intestine, and impairment of 
carbohydrate metabolism and absorption may be a secondary effect of 
this cytotoxicity. 

II.4.4  Structure-activity relationships

    After the isolation and identification of numerous derivatives of 
the trichothecenes, the structure-activity relationship was 
investigated, on the basis of the information on lethal toxicity, 
dermal toxicity, cytotoxicity, inhibition of protein synthesis, and 
association with ribosomes. 

    The 12,13-epoxide of the trichothecenes is essential for their 
biological activity.  The de-epoxidation of DON and T-2 toxin by 
rumen-microorganisms (King et al., 1984) and in mammalian systems 
(Yoshizawa et al., 1983) results in loss of toxicity. 

    The macrocyclic ring contributes to the highly lipophylic 
property of the trichothecenes, and the macrocyclic trichothecenes, 
such as verrucarins, roridins, satratoxins, and baccharins, exhibit a 
potent cytotoxicity towards cultured mammalian cells (Kupchan et al., 
1976, 1977; Jarvis et al., 1978, 1980). 

    In contrast, the polyhydroxylated alcohols, such as verrucarol, 
NIV, and DON, are highly hydrophilic, resulting in a decrease in 
cytotoxicity and dermal toxicity compared with their parent 
trichothecenes (Ueno et al., 1970; Wei et al., 1974). 

II.4.5  Prevention and therapy of trichothecene toxicosis

    In studies to investigate effects of various dietary supplements 
on T-2 toxin toxicity, weanling male Wistar rats in groups of 10, 
with adequate controls, were fed diets containing 5% each of 
bentonite, anion exchange resin, cation exchange resin, or 
vermiculite-hydrobiotite with and without 3 µg T-2 toxin/g feed, for 
2 weeks.  Bentonite and anion exchange resin were most effective in 
reducing the growth depression and feed refusal caused by T-2 toxin.  
In a second study in which bentonite and anion exchange resin were 
administered at levels of 2.5, 5.0, 7.5, or 10% of diet, 10% 
bentonite in the diet was the most effective dietary supplement.   
Dietary bentonite reduced the absorption of 3H-T-2 toxin and 
increased faecal elimination (Carson & Smith, 1983).  

    Smectite, a clay composed of insoluble silicates of aluminum and 
magnesium, incubated with T-2 toxin for 24 h before the toxin was 
administered orally to mice (1 mg/kg per day), prevented the 
acceleration of gastric emptying and transit time for a milk meal, 
usually induced by administration of T-2 toxin.  Smectite given 
together with the toxin did  not prevent the gastrointestinal effects 
of T-2 toxin (Fioramonti et al., 1987).  Male Sprague-Dawley rats 
were given 15 or 5 mg/kg body weight of a platelet activating factor 
antagonist, with and without T-2 toxin, by intravenous injection.  
The drug prolonged the survival of conscious rats exposed to 0.65 mg 
T-2 toxin/kg (Feuerstein et al., 1987). 

    The effects of certain drugs and metabolic inhibitors on the 
toxicity of T-2 toxin have been studied in male ddY mice.  The toxin 
at a dose of 1.8 mg/kg was given subcutaneously and the drugs and 
other chemicals were given ip.  The lethal effects were reduced by 
the steroid drugs, prednisolone and dexamethasone; survival times 
were increased by the antihistaminic drug diphenhydramine, and an 
opioid antagonist, naloxone. Prednisolone also reduced the 
leukocytosis that developed after T-2 toxin treatment and decreased 
the increase in ear weight caused by the mycotoxin.  Other 
antihistaminic and/or antiserotonic drugs used in the treatment were 
found not to be effective in preventing the lethal effects of T-2 
toxin (Ryu et al., 1987). 

    Male ddY mice and male Wistar rats in several groups of 10 
animals each, pretreated with a radioprotective compound or with an 
anti-inflammatory agent, were given T-2 toxin or fusarenon-X (1.5 
mg/kg body weight).  Out of the 20 compounds evaluated, only 
prednisolone and hydrocortisone (100 mg/kg ip) were effective.  They 
reduced mortality from 90% in controls to less than 30% and they also 
reduced the trichothecene-induced increase in intestinal fluid volume 
(Mutoh et al., 1988). 

    Cutaneous irritation produced by T-2 toxin in 10 Porton female 
rats was largely reduced by application, within 10 min, of an aqueous 
soap solution when the dose was low (1.0 µg/cm2), but the solution was 
largely ineffective over a time span of 60 min when the dose was 
higher (100 µg T-2 toxin).  Washing the site of application with 
polyethylene glycol 300 was very effective in removing even large 
doses (100 µg) of T-2 toxin from the skin (Fairhurst et al., 1987). 

    The median effective dose of oral superactive charcoal in 
preventing deaths in batches of 5 female Sprague-Dawley rats was 
0.175 g/kg.  A dose of 1 g superactive charcoal/kg body weight 
increased survival times and rates in rats given the lethal dose of 8 
mg T-2 toxin/kg as long as 3 h after the toxin was administered by 
gavage (Galey et al., 1987). 


II.5.1  Contemporary episodes of human disease

    Two outbreaks of trichothecene-related disease have been 
reported, one in China in 1984/85 (Luo, 1988) and one in India in 
1987 (Bhat et al., 1989).  Each involved several hundred cases. 

    During the first incident, outbreaks of mouldy corn and scabby 
wheat poisoning were reported.  Out of approximately 600 persons who 
consumed mouldy cereals, there were 463 cases of poisoning (77% of 
the total).  The latency period for the onset of symptoms was 5-30 
min.  These included nausea, vomiting, abdominal pain, diarrhoea, 
dizziness, and headache.  No deaths occurred.  Pigs and chicks fed 
the same mouldy cereals were also affected (Luo, 1988).  GC-MS and 
RIA were used in the analysis of 5 samples of the mouldy corn.  DON 
was detected within a range of 0.34-92.8 mg/kg and zearalenone within 
a range of 0.004-0.587 mg/kg.  T-2 toxin and NIV were not found.  TLC 
was used in the analysis of 19 samples of scabby wheat collected from 
the affected and non-affected families.  The DON content was 1.0-40.0 
mg/kg, which was significantly higher than that in the non scabby 
wheat samples.  In addition to DON, zearalenone was detected in 2 
samples, the contents of which were 0.25 and 0.5 mg/kg, respectively.  
No T-2 toxin was found in the samples (Luo, 1988). 

    An analogous outbreak was reported in Kashmir, India, in 1987 
(Bhat et al., 1987, 1989).  It was ascribed to the consumption of 
bread made from flour that had become mouldy in storage following 
unseasonal rains in the wheat-harvesting season, from which  Fusarium 
sp. was grown, and which were found to contain mycotoxins.  Of the 
224 persons investigated on a random sample basis, 97 were affected 
with symptoms including abdominal pain (100%), throat irritation 
(63%), diarrhoea (39%), blood in stools (5%), and vomiting (7%).  
Symptoms developed 15 min to one hour after consumption of locally 
baked bread.  In 12 out of 24 samples of refined wheat flour used in 
the preparation of bread, the following mycotoxins were found:  DON 
(0.35-8.38 mg/kg), Ac-DON (0.64-2.49 mg/kg) (no details of estimation 
of this derivative were available), NIV (0.03-0.1 mg/kg) and T-2 
toxin (0.55-0.8 mg/kg).  Quantitative estimation of DON, Ac-DON, and 
NIV was obtained using HPLC, and that of T-2 toxin by TLC (Bhat et 
al., 1987) but no rigorous confirmation of identity was undertaken.  
Since no fatalities occurred in either of the above outbreaks, no 
information is available on the pathological changes, if any, at 

II.5.2  Historical  Fusarium-related diseases

    In the period 1931-47, a human disease known as alimentary toxic 
aleukia (ATA) occurred in the USSR that was suggested to be related 
to the presence of toxic  Fusarium species in mouldy over-wintered 
grain.  Data have been reviewed by Sarkisov et al. (1944) and more 
recently by Bilai (1977), Leonov (1977), and Joffe (1986).  An 
association was established with the ingestion of grain invaded by 
some moulds, in particular  Fusarium poae and  F. sporotrichioides.  
The dominant pathological changes were necrotic lesions of the oral 
cavity, the oesophagus, and stomach and, in particular, a pronounced 
leukopenia.  The primary lesion was bone marrow hypoplasia and 
aplasia.  The disease was lethal in a high proportion of cases. 

    The clinical symptoms reported in ATA, as well as the identified 
occurrence of  Fusarium in foodstuffs, suggest that it might have been 
associated with mycotoxins, identified years later in fungal cultures 
of  Fusarium species under laboratory conditions, such as T-2 toxin 
(Mirocha & Pathre 1973) or wortmannin (Mirocha & Abbas 1989). 

    Scabby grain toxicosis is a disease of human beings as well as 
farm animals, and was reported from Japan and Korea in the period 
1946-63 (Hirayama & Yamamoto 1948, 1950; Nakamura et al., 1951; 
Tsunoda et al., 1957; Cho 1964; Ogasawara 1965; Chung 1975).  The 
common clinical symptoms were nausea, vomiting, diarrhoea, and 
abdominal pain.  All cases were acute with recovery within a few 
days and no lethal cases were encountered.   Fusarium fungi,  F. 
 graminearum in particular, were isolated from suspected cereals 
(Tochinai, 1933; Tsunoda et al., 1957). 

    When compared with the symptoms observed in experimental 
animals, features of both the above human diseases were similar to 
trichothecene toxicosis, notably symptoms caused by DON and NIV, 
DAS, and T-2 toxin.  However, no epidemiological studies have been 
reported that link ATA and scabby grain toxicosis to these 

II.5.3  Skin irritation

    The Task Group was aware of several reports describing various 
effects on the skin of crude extracts of fungal cultures or 
solutions containing T-2 toxin, as well as other possible 
substances (Bamburg & Strong, 1971; Saito & Otshubo, 1974).  These 
cases, all of them accidental, involved a very limited number of 
persons who developed severe irritation, loss of sensitivity, and 
desquamation.  Despite the presence of T-2 toxin in the contact 
material, there is no evidence that the involvement of other 
compounds can be ruled out. 

II.5.4  Studies of haemostasis

    Platelet  function  and  electron  microscopic  morphological 
changes following T-2 toxin administration were studied on 
platelets isolated from 12 healthy human volunteers.  When 
platelets were incubated with T-2 toxin at doses of 5-500 µg/109 
platelets for 20 min, there was a dose-related inhibition of  
platelet aggregation with different activators, including 
epinephrine, arachidonic acid, and collagen, and a release of dense 
bodies consisting mainly of serotonin-containing granules.  There 
was also a change in membrane permeability, but no changes in 
shape.  No correlated inhibition of thromboxane synthesis, or 
significant alterations in platelet calcium content were observed.  
The microtubular system was unaffected.  It was suggested that the 
above observations , notably suppressed aggregation, played a 
contradictory role in the haemorrhagic phenomena associated with 
these toxins in man and animals (Yarom et al., 1984a). 

II.5.5  Airborne trichothecene-related diseases

    High concentrations of spores of  Stachybotrys atra were 
discovered in the air of living rooms of a suburban house in 
Chicago, USA (Croft et al., 1986).  Over a period of several years, 
the 5 occupants of the house had suffered a variety of non-specific 
symptoms including signs of colds, sore throats, diarrhoea, 
headaches, dermatitis, intermittent focal alopaecia, and 

generalized malaise.  Chemical analysis of building materials 
supporting the growth of  Stachybotrys atra confirmed the presence 
of the macrocyclic trichothecenes, verrucarin B and J, satratoxin 
H, and trichoverrins A and B.  Five weanling Sprague-Dawley rats 
and 5 mice, administered extracts of contaminated materials orally, 
died within 24 h of exposure, whereas control animals remained 
unaffected.  Histological lesions of the animals tissues were 
degeneration, necrosis and haemorrhage of the brain, thymus, 
spleen, intestine, lungs, heart, lymph nodes, liver, and kidneys.  
Although the clinical symptoms could have been related to allergic 
responses, the isolation of potent mycotoxins suggests that they 
were the causal factor of the illness observed. 

II.5.6  Toxicological information on man, obtained from 
therapeutic uses

    DAS (Anguidine) has been undergoing clinical trials as a 
chemotherapeutic agent in cancer patients.  During a weekly 
schedule of DAS, using healthy volunteers as controls, a dose of 5 
mg/m2 body surface infused over 3 h, produced nausea, vomiting, 
hypotension, neurological symptoms (confusion, hallucinations and 
psychomotor seizures), chills, fever, and diarrhoea (DeSimone et 
al., 1979).  A similar study carried out on 20 other cancer 
patients revealed gastrointestinal and neurological toxic effects 
(Belt et al., 1979).  A bolus administration or rapid infusion of 
the drug also caused gastrointestinal and neurological symptoms 
(Thigpen et al., 1981).  The human haematopoietic system appears to 
be extremely sensitive to DAS.  Myelosuppression was the dose 
limiting adverse effect of prolonged infusions over 8 h (Thigpen et 
al., 1981).  A mean myelosuppressive dose level in the above 
investigation was, 5 mg/m2 body surface by 8-h infusion or, roughly 
calculated in terms of body weight, 0.2 mg/kg (0.025 mg/kg per h). 


   On the basis of the data made available to the Task Group, there 
is a possible association between trichothecene exposure and 
episodes of human disease.  According to the limited data 
available, the most frequently reported trichothecenes involved in 
episodes of human exposure are DON and NIV.  In the episodes of 
alimentary toxic aleukia and scabby grain toxicosis reported in the 
past, a possible etiological role of trichothecenes cannot be 
excluded.  Exposure occurs through the ingestion of contaminated 
food,  mainly  cereals.  Processing, milling,  and  baking  are  
not effective in destroying DON, NIV, and T-2 toxin.  There is very 
limited evidence of exposure through inhalation, but such a 
possibility cannot be ruled out. 

    Among the naturally occurring trichothecenes in foods, T-2 
toxin is the most potent, followed by DAS and NIV; DON was the 
least toxic in acute toxicity studies.  In experimental animals, 
T-2 toxin and DAS produce acute systemic effects, with necrosis of 
epithelial tissues and suppression of haematopoiesis.  In 
contemporary outbreaks of disease, only gastrointestinal symptoms 
have been reported. 

    DON was shown to be teratogenic in mice but not in  rats.  
According to published chronic toxicity studies, NIV and T-2 toxin 
are not tumorogenic in animals.  No long-term carcinogenicity 
studies on DON have been published.  Certain trichothecenes, such 
as T-2 toxin and DON, have an immunosuppressive action in animals 
and have produced alterations in both cell-mediated and humoral 
immunity.  There is no evidence of immunosuppressive action in man. 

    Reported cases of human disease associated with trichothecene 
exposure are limited in number and information. Symptoms of 
digestive disorders and throat irritation develop rapidly after 
ingestion of food contaminated with trichothecenes.  At present, 
there is no evidence of human cancer caused by trichothecenes.  No  
reports were available to the Task Group on secondary infection, by 
bacteria, fungi, or viruses, in human beings following trichothecene 
exposure, as has been observed in experimental animal studies.  It 
appears that adequate studies elucidating such a sequence have not 
been made. 



III.1.1  Chemical properties

    Ergot is the French word for a rooster's spur and is used as 
the common term for sclerotia of fungal species within the genus 
 Claviceps, in particular  C. purpurea.  The fungi infect the florets 
of grasses and cereal, replacing the florets with compact fungal 
structures, sclerotia, 2-20 mm long, somewhat curved, tapering off  
at the ends, and strongly coloured, often purple-black, thus 
resembling rooster's spurs.  The sclerotia contain a large number 
of biologically active alkaloids, as well as amino acids, 
carbohydrates, lipids, and pigments, and, when the sclerotia are 
consumed by man and animals, toxicosis develops, called ergotism.  
The topic has been reviewed by Bove (1970), Van Rensburg & 
Altenkirk (1974), Lorenz (1979), and Berde & Schild (1978).  This 
section deals with ergot as a toxic food contaminant;  ergot 
compounds used as pharmaceutical drugs are excluded. 

    Ergot alkaloids (ergolines), of which more than 40 have been 
isolated from Claviceps sclerotia, are derivatives of lysergic acid 
(Fig. 4), and can be divided into 3 groups: 

Group I:    Derivatives of lysergic acid, e.g., ergotamine.

Group II:   Derivatives of isolysergic acid, e.g., ergotaminine. 

Group III:  Derivatives of dimethylergoline (clavines), e.g., 


    Physical and chemical properties of some ergolines are listed 
in Table 23. 

Table 23.  Physical and chemical properties of selected 
ergot alkaloids (ergolines)a
Ergoline               Molecular    Melting   [alpha]20
                       formula      point            D
Group I.  Derivatives of lysergic acid
ergotamine             C33H35O5N5   180       -160
alpha-ergocryptinine   C32H41O5N5   212-214   -190
ergocristine           C35H39O5N5   160-175   -183
ergosine               C30H37O5N5   220-230   -183
ergocornine            C31H39O5N5   182-184   -188
ergometrine            C19H23O2N3   162       +41

Group II.  Derivatives of isolysergic acid
ergocristinine         C35H39O5N5   226       +366
ergometrinine          C19H23O2N3   196       +414
ergosinine             C30H37O5N5   228       +420
ergocorninine          C31H39O5N5   228       +409
alpha-ergocryptinine   C32H41O5N5   240-243   +480
ergotaminine           C33H35O5N5   241-243   +369

Group III.  Derivatives of dimethyloergolines (clavines)
agroclavine            C16H18N2     206       -151
elymoclavine           C16H18ON2    249       -109
chanoclavine           C16H20ON2    222       -240
penniclavine           C16H18O2N2   222       +153
setoclavine            C16H18ON2    229-234   +174
a  From: Van Rensberg & Altenkirk (1974) and Lorenz (1979).

III.1.2  Analytical methods for ergot and ergot alkaloids

III.1.2.1  Ergot

    Sclerotia of  Claviceps species are all rich in triglycerides, 
but only a few species have a sufficiently distinctive fatty acid 
composition in the sclerotia to provide a basis for identification.  
Sclerotia of  C. purpurea are unique among microorganisms in 
containing a large (approximately 30%) triglyceride fraction in 
which ricinoleic acid is the principal (30-40%) component.  Thus, 
the presence of ricinoleate in a foodstuff known to be free from 
other sources of ricinoleic acid (e.g., castor oil) is diagnostic 
for the presence of  C. purpurea sclerotia.  The sample is 
saponified, the free fatty acids thereby released are methylated 
with diazomethane, and the resulting methyl esters are analysed 
using gas-liquid chromatography (Mantle, 1977a).  As little as 0.3% 
ergot in 1-2 g foodstuff has been detected by this procedure. 

III.1.2.2  Ergot alkaloids

    Sclerotia may contain up to 1% of total ergot alkaloids.  
Lysergic acid derivatives can easily be epimerized at C-8 to give 
isolysergic acid derivatives (Group III), thereby removing most of  
their biological activity.  As this may happen to a variable extent 

during extraction, it is difficult to know whether the small 
proportion of isolysergic acid derivatives, commonly found during 
analysis of extracts from sclerotia, has been generated in whole or 
in part during extraction (Mantle, 1977b). 

    A method for the determination of  C. purpurea ergot alkaloids 
in flour has been developed, based on liquid chromatography 
following extraction of the flour by a mixture of methylene 
chloride, ethyl acetate, methanol, and 28% ammonium hydroxide 
solution (Scott & Lawrence, 1980).  After filtration and 
evaporation to dryness, the residue is dissolved in methanol-ether 
and extracted with 0.5 N hydrochloric acid.  The acid layer is 
washed with hexane, made alkaline, re-extracted with methylene 
chloride, evaporated,  and  the residue dissolved in  methanol,  
ready  for liquid chromatographic analysis, with identification 
based on fluorescence at an excitation wavelength of 235 nm.  The 
method has a recovery of 66-93%, based on analysis of flour spiked 
with Group I alkaloids (ergotamine, ergocryptine, ergocristine, 
ergosine, ergocornine, ergometrine). 

    A method using high-performance liquid chromatography for the 
analysis of human blood has been reported (Zorz et al., 1985).  
Samples (5 ml) of human plasma are extracted with benzene-toluene-
ethyl-acetate-diethylamine, the solvent layer separated and 
evaporated to dryness.  High-performance liquid chromatography is 
performed with excitation wavelength at 285 nm for naturally 
occurring ergot alkaloids.  According to recovery studies, 
concentrations as low as 0.2 mg/litre plasma can be detected. 

    A radioimmunoassay has been developed for determination of 
ergotamine and ergocristine (Arens & Zenk, 1980).  The alkaloids 
were conjugated with bovine serum-albumin, and antibodies raised 
in rabbits.  Using 3H-labelled tracers, levels as low as 3.5 pmol 
of ergotamine and 0.8 pmol of ergocristine could be measured.  The 
antibodies were highly specific, and simple lysergic acid 
derivatives and clavines did not cross-react.  The procedure has 
been used in the detection of sclerotia of  C. purpurea with a high 
alkaloid concentration for industrial bioproduction. 

    A liquid chromatographic method for the detection of 
ergotamine, ergotaminine, and ergocristine in human plasma has been 
developed using extraction at pH 9, clean-up, and an ODS-hypersil 
reverse-phase column (Edlund, 1981).  Levels of ergolines as low as 
0.1 mg/litre in 3 ml plasma samples can be detected, with a 
recovery of 79-99% for the 3 ergolines. 

    Ergot contamination of pearl millet, due to infection by  C. 
 fusiformis, is characterized by the presence of clavine alkaloids 
(Group III).  A procedure for its determination has been developed 
using thin-layer chromatographic separation and spectrophotometric 
detection following colour reaction using Van Urk's reagent (Bhat 
et al., 1976; Krishnamachari & Bhat, 1976).  The procedure includes 
defatting of the grain sample, mixing of the defatted material with 
ammonium hydroxide, extraction with diethyl-ether followed by 
extraction of the diethyl-ether phase with 0.1 N sulfuric acid.  

The extract is made alkaline and extracted with chloroform, 
followed by thin-layer chromatographic separation. 


III.2.1  Fungal producers

    Sclerotia are compact hyphal structures that develop in the 
colonies of many fungal genera.  The sclerotia of species within 
the genus  Claviceps are unique in terms of size (length up to 
several cm), pronounced colour, and because the sclerotia of 
several species contain highly biologically active compounds, the 
alkaloids.  The sclerotia develop during the infection of plants; 
host plants for the  Claviceps species mainly belong to the grass 
family (Gramineae), which comprises the true cereals.  However, a 
few plant species within the family Juncaceae and Cyperaceae can 
also act as hosts.  Most  Claviceps species have a monogeneric host 
range, but  C. purpurea is unique in that it has a very wide host 
range (Van Rensburg & Altenkirk, 1974; Lorenz, 1979).  The eight 
leading cereals produced in the world are wheat, rice, corn, 
sorghum, rye, barley, oats, and millet, and they can all be hosts 
for  Claviceps species.  Man and animals are exposed to toxic 
sclerotia from two species,  C. purpurea and  C. fusiformis;  farm 
animals are also exposed to toxic sclerotia from  C. paspali, 
growing on grass.  Sclerotia of  C. purpurea have the dimension of 
2-20 x 1-6 mm and are purple-black in colour, whereas  C. fusiformis 
has small (2 x 4 mm) purple-red coloured sclerotia (Loveless, 1967;  
Siddiqui & Khan, 1973;  Mantle, 1977b).   C. purpurea primarily 
attacks cross-pollinated species in which the florets tend to 
remain open for a relatively long time and in which sterility 

    The cereals most commonly contaminated with ergot from  C. 
 purpurea are rye, wheat, triticale (the cross-breed between wheat  
and rye), barley, oats, and sorghum.  The ergot of  C. purpurea 
contain Group I and II ergolines as the principal ergot alkaloids.  
 C. fusiformis is a parasite of pearl millet  (Pennisetum typhoideum) 
in Africa and East Asia; in India, the cereal is called bajra.  
Ergot of  C. fusiformis contain predominantly Group III ergolines. 

    Ergot alkaloids have been isolated from fungi outside the genus 
 Claviceps (Aspergillus fumigatus, A. clavatus, A. nidulans, 
 Rhizopus nigricans, Penicillium chermesinum, P. concavo-rugulosum, 
 P. sizovae) and from higher plants  (Rivea corymbosa, Ipomoea 
 violacea, I. argyrophylla, I. hildebrandtii, I. tricolor) (Van 
Rensburg & Altenkirk, 1974; Kozlovsky & Reshetilova, 1984).  
Whether these sources represent human exposure is not known at 

    Successful attempts have been made to produce ergot alkaloids 
using  C. purpurea cultures in liquid media under laboratory 
conditions, and new alkaloids have been identified (Bianchi et al., 

III.2.2  Biosynthesis

    The ergoline ring system in ergot fungi is built up from L-
tryptophan and mevalonic acid  (Fig. 5).   The  N-methyl group of 
the ergot alkaloids is derived from methionine via a 
transmethylation reaction.  The precursors of tryptophan are 
indole, anthranilic acid, and indolpyruvic acid (Van Rensburg & 
Altenkirk, 1974; Mantle, 1977b).  It appears that Group III 
ergolines are intermediates in the production of Group I and II  
ergolines by  C. purpurea. 


III.2.3  Occurrence in foodstuffs

    Traditionally, contamination of grain with ergot has been 
expressed as a percentage on a weight basis, without measurement of 
total and individual amounts of ergolines.  Thus, it is generally 
recommended that feed containing more than 0.1% ergot should not be 
given to animals (Young, 1981a,b).  Quantitative analysis for total 
and individual ergolines in 14 samples of rye and  wheat  flour  
(Scott & Lawrence, 1980)  indicated  contamination with 6 Group I 
ergolines (ergometrine, ergosine, ergotamine, ergocornine, 
ergocryptine, ergocristine) at concentrations ranging from 0.3 to 
62 µg/kg for individual ergolines.  Ergocristine was the major 
ergoline present in the flours, 62 µg/kg being the maximal 
concentration found.  In 17 samples of rye grain collected  from 
health shops, 6 contained ergot;  ergoline concentration and 
composition were not indicated (Akerstrand, 1980).  In a survey of 
ergolines in ergot-contaminated cereals, in North America, the 

average total ergolines content was 0.24% (Young, 1981a,b; Young & 
Chen, 1982).  On average, the pooled ergoline composition in 
sclerotia in rye, wheat, and triticale was as follows:  
ergocristine (31%), ergocristinine (13%), ergotamine (17%), 
ergotaminine (8%), ergocryptine (5%), ergocryptinine (3%),  
ergometrine (5%),  ergometrinine (2%),  ergosine (4%), ergosinine 
(2%), ergocornine (4%), and ergocorninine (2%).  The individual 
ergoline composition was uniform throughout a single sclerotium or 
in different sclerotia from the same head, somewhat less uniform 
between different fields throughout a region, and highly variable 
from head-to-head in a given field.  In contrast, the total 
ergoline content was highly variable within-sclerotium, within-
head, head-to-head, and on a field-to-field basis.  In a 
comparative study of ergoline in ergots from rye and wheat (caused 
by infection with  C. purpurea) and in ergots from pearl millet 
(caused by infection with  C. fusiformis) in South-East Asia, it was 
found that the total ergoline content in the sclerotia of pearl 
millet was much lower (320 mg/kg) than that in the sclerotia of rye 
(700 mg/kg) and wheat (920 mg/kg) (Bhat et al., 1976).  The 
ergolines in the sclerotia of pearl millet were reported to 
comprise agroclavine, elymoclavine, chanoclavine, penniclavine, and 

    In a survey of cereals and cereal products on the Swiss market, 
using a high-performance liquid chromatography procedure, the 
following average concentrations of total ergolines were found: 
wheat flour, 4.2 µg/kg; wheat flour (coarse), 30.7 µg/kg; wheat 
flour (more coarse), 103.4 µg/kg; rye flour, 139.7 µg/kg; and 
"bioproducts", 10.2-22.7 µg/kg (Baumann et al., 1985).  The daily 
intake of total ergolines by human beings in Switzerland was 
estimated to be 5.1 µg/person. 

III.2.4  Fate of ergolines during food processing

    Treatment of sclerotia from wheat with chlorine (1%) and heat 
(150-200 °C) resulted in a 90% reduction in ergoline content within 
4 h (Young et al., 1983).  The reduction affected all the ergolines 
(ergotamine, ergocornine, ergocryptine, ergosine, ergometrine) in 
identical ways.  Autoclaving sclerotia at 121 °C for 30 min 
resulted in a 24.6% reduction in total ergoline content.  In the 
baking of bread and pancakes using grain that contained naturally 
occurring ergot, a 59-100% reduction in the individual ergolines 
(ergosine, ergocornine,  ergometrine, ergotamine, alpha-
ergocryptine, ergocristine) was observed in whole wheat bread, a 
50-86% reduction in all-rye flour bread, and a 25-74% reduction in 
triticale pancakes (Scott & Lawrence, 1982). 

    When bread made of rye flour spiked with finely ground 
sclerotia of  C. purpurea, (continuing a total ergoline 
concentration of 312.8 µg/kg), was baked, there was an overall 
reduction of 50% in the ergoline concentration, as measured by high 
performance liquid chromatography (Baumann et al., 1985). 


    No published information is available on the metabolism in 
animals or human beings of ergots containing ergolines and 
combinations of individual ergolines. 


III.4.1  Field studies

    An outbreak of bovine abortion associated with the ingestion of 
ergot was reported by Appleyard (1986). 

    Eleven out of 36 suckler cows, all in late pregnancy, aborted 
in 7-11 days following introduction to a rye grass pasture heavily 
infested with ergot.  At least 25% of the rye seed heads contained 
sclerotia of  C. purpurea, with up to 8 sclerotia present on any one 
seed head.  The sclerotia contained 1.57 mg total ergolines/g, 
consisting of 67% ergotamine and 17% ergotaminine, and smaller 
amounts of ergometrine.  Ergocryptine and ergosine were also 
present together with their corresponding -inine isomers. 

    Ten out of the 11 calves were delivered dead.  None of the 
aborting cows showed any premonitory signs of calving and, after 
parturition, there was almost complete agalactia.  The placenta was 
retained in each case, but there was no other evidence of ill 
health in the cows.  Any other cause of abortion of bacterial, 
viral, fungal, or toxic origin was ruled out, on the basis of 
laboratory and field investigations. 

III.4.2  Experimental animal studies

    This topic has been reviewed by Ainsworth & Austwick (1959) and 
Mantle (1977c).  It appears that most reports of field cases and 
experimental animal studies on ergotism do not contain any 
information on the contents of individual ergolines in the ergot 
associated with disease manifestations in animals. 

III.4.2.1  Cattle

    Lameness, sometimes leading to gangrene, is a common symptom 
observed in cattle when the feed contains more than 10 g ergot/kg.  
The symptoms are more pronounced when the animals are kept outside 
under cold weather conditions (Mantle, 1977c).  Four out of 6 
animals administered ergotamine tartrate, orally, at 1 mg/kg body 
weight per day, died within 10 days (Woods et al., 1966).  The 
animals became acutely ill within 1-2 days, the principal signs 
being anorexia, hyperventilation, cold extremities, salivation, 
and, occasionally, tongue necrosis.  Post-mortem examination of the 
most seriously affected animals revealed extensive intestinal 

III.4.2.2  Sheep

    Four lambs were administered aqueous suspensions of milled 
ergot (from  C. purpurea) through a stomach tube, over a 2-month 

period (Loken, 1984).  The doses ranged from 0.12 to 0.75 g 
sclerotia/kg body weight.  The sclerotia contained approximately 4 
g ergolines/kg, composed of ergotamine (15%), ergosine (35%), and 
ergocristine (5% each).  One lamb, dosed with 0.12 g sclerotia/kg 
body weight, was kept indoors at 15-17 °C and did not develop any 
symptoms.  The other 3 animals, given higher doses and kept 
outdoors, became ill after 2-6 days, with signs that included 
dullness, inappetence, high pulse rate, diarrhoea, edema of the 
hind legs and tail, and lameness.  Post-mortem findings included 
inflammation and necrosis of the forestomach and intestinal mucosa. 

III.4.2.3  Poultry

    Leghorn chickens were fed diets containing ergotamine tartrate 
at levels of up to 800 mg/kg in 7 to 10-day trials as well as in a 
51-day  trial  (Young & Marquardt, 1982).   In the short-term 
trials, only the highest level (800 mg/kg) had an effect on 
performance, in terms of a slight decrease in growth rate and a 
slight increase in feed consumption.  At the 250 mg/kg level, toe 
necrosis was observed, as well as cardiomegaly.  There were no 
pathological effects on the brain, liver, or muscle tissues, even 
at the highest level.  In the long-term study (51 days), effects 
were similar to those observed in the 7 to 10-day trials.  No 
residues of ergotamine were detected in tissues. 

III.4.2.4  Swine

    Ergot (from  C. purpurea) containing 0.3% ergolines 
(composition: ergotamine and ergosine) was used in a feeding study 
on pigs (Mantle, 1977c).  A diet containing ergot at 40 g/kg was 
well tolerated, and the only effect at 100 g ergot/kg diet was 
depression of the growth rate.  Agalactia in the sow was observed 
after feeding with rations containing ergot from  C. purpurea as 
well as from  C. fusiformis. 

III.4.2.5  Primates

    Male rhesus monkeys in groups of 2-4 animals were dosed with 
ergot from  C. fusiformis, either as part of the diet, or as an 
ergoline extract administered orally or intraperitoneally (Bhat & 
Roy, 1976).  The ergoline extract contained agroclavine (major 
components:  elymoclavine, chanoclavine, penniclavine, and 
setoclavine).  The period of treatment lasted from 2 days to one 
month.  There were no effects on the animals, with the exception of 
those injected ip with 5.44-11.1 mg total ergoline/kg body weight, 
who developed signs within 10 min including drowsiness, 
hyperexcitation, redness of face, and loss of response to thermal 
and tactile stimuli in the hind limbs and tail.  The animals 
recovered spontaneously in about 60 min.  Animals administered a 
total of 10 mg ergolines/kg body weight, orally, showed symptoms of 
hyperexcitation, but these were far less severe than those after 
intraperitoneal injection.  It was concluded that the signs in 
monkeys, in particular hyperexcitation, are different from those 
observed in human beings after ingestion of ergot in pearl millet. 


    The history of ergotism in man, following ingestion of ergot 
from  C. purpurea, was reviewed by Barger (1931).  Numerous 
epidemics in Europe occurred between the 9th and the 18th century.  
Two types of disease were noted:  gangrenous and convulsive 
ergotism.  In the first type, the affected part (arm or leg) 
shrank, became mummified and dry, and the gangrene gradually spread 
upwards.  In convulsive ergotism, the whole body was attacked by 
general convulsion, which returned at intervals of a few days.  The 
latest outbreaks of ergotism in Europe occurred in 1926-28 in the 
United Kingdom and the USSR.  A suspected episode in France in 1951 
turned out to be due to a different toxic substance (Gadiou, 1965). 

III.5.1  Ergometrine-related outbreaks

    In 1978, an epidemic was reported in Ethiopia (Demeke et al., 
1979;  King, 1979;  Pokrovskij & Tutelyan, 1982).  The episode 
occurred in the Wollo region, following two years of drought.  
During this time, the locally grown barley, the staple food, had 
become dominated by wild oats heavily contaminated with  C. purpurea 
sclerotia.  The grain consisted of 70% wild oats, 12% barley, and 
0.75% ergot; ergometrine was detected in the sclerotia by thin 
layer chromatography.  A total of 93 cases of ergotism was reported 
during the spring of 1978.  The male:female ratio was 2.5:1; more 
than 80% of affected persons were between five and 34 years of age.  
In addition to the 93 cases, 47 deaths were reported as having been 
due to ergotism.  Examination of 44 patients out of the 93 
registered revealed ongoing dry gangrene of the whole or part of 
one or more limbs (7.5%), feeble or absent peripheral pulses 
(36.4%), swelling of limbs (11.2 %), desquamation of the skin 
(12.8%), and loss of one or more limbs (21.5%).  It was noted that 
88% of patients had involvement of the lower extremities.  The most 
common general symptoms were weakness (78.5%), formication (15%), 
burning sensation (14.3%), nausea (7.2%), vomiting (5.6%), and 
diarrhoea (6.8%).  In addition, 50-60 infants and young children 
died from starvation due to failure of the mothers to lactate.  
This may have been related to the effect of ergot on lactation 
(Demeke et al., 1979).  No autopsies were performed, and thus there 
is no information on pathological changes in the viscera. 

III.5.2  Clavine-related outbreaks

    Intoxication following ingestion of ergot from  C. fusiformis in 
bajra or pearl millet has been reported from India.  Symptoms 
included nausea, vomiting, and giddiness.  Several outbreaks have 
been observed since 1958, when the first report was published;  the  
latest occurred in the autumn of 1975 in the state of Rajasthan 
(Krishnamachari & Bhat, 1976).  In 21 villages surveyed, 78 persons 
belonging to 14 households developed symptoms,  characterized  by  
nausea, repeated vomiting, and giddiness, followed by drowsiness 
and prolonged sleepiness, extending sometimes to over 24-48 h.  
There were no signs or symptoms suggesting vaso-occlusion.  The 
disease generally developed 1-2 h following a single meal.  
Domestic camels, offered the contaminated grain as feed, also 

developed sleepiness and signs suggesting abdominal discomfort.  
The pearl millet from affected villages  contained 15-174 g  
ergot/kg, resulting in a contamination of the grain with 15-199 mg 
total ergolines/kg.  The individual ergolines were identified as 
agroclavine, elymoclavine, chanoclavine, penniclavine, and 
setoclavine.  Pearl millet from villages with no cases of  
intoxication  contained 1-38 g ergot/kg with a total ergoline 
content of 15-26 mg/kg.  Since there were no deaths, no information  
on pathological changes is available.  The number of households 
studied was too small for no-effect levels to be calculated, but 
the authors suggested that the intake of 28 µg total ergolines/kg 
body weight would be non-toxic. 


    Human exposure to low levels of ergolines appears to be 
widespread.  Available data from the recent outbreaks in Ethiopia  
and India indicate that the  C. purpurea alkaloids (ergotamine 
group) produced more severe effects, including gangrene of the legs 
and death, than the alkaloids of  C. fusiformis (clavine group), 
which caused gastrointestinal symptoms without a fatal outcome.   
It is not known whether such differences can be accounted for by 
differences in the alkaloid content of the fungal species, in the 
toxicological or toxicometric properties of the alkaloids, or in 
the levels of intake by different types of populations. 

    Only low levels of ergolines remain in prepared foods as 
cleaning and milling processes remove the sclerotia; baking or 
other heat processing also destroys most alkaloids of the ergotamin 


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YAGEN, B. (1984b) T-2 toxin effect on bacterial infection and 
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1.  Ochratoxine A

1.1  Etat naturel

    Les ochratoxines sont produites par plusieurs espèces de 
champignons appartenant au genre  Aspergillus et  Penicillium. Il 
s'agit d'espèces ubiquistes, aussi le risque de contamination des 
denrées alimentaires destinées à l'homme et aux animaux est-il 
omniprésent.  Le principal composé, l'ochratoxine A se rencontre en 
Australie ainsi que dans certains pays d'Europe et d'Amérique du 
Nord.  La production d'ochratoxine par les espèces du genre 
 Aspergillus semble limitée aux environnements très chauds et 
humides alors que dans le cas de  Penicillium, au moins certaines 
espèces peuvent produire de l'ochratoxine à des températures 
n'excédant pas 5 °C. 

    Ce sont les céréales qui sont le plus fréquemment contaminées 
par l'ochratoxine A et à un moindre degré certaines fèves (café, 
soja, cacao).  L'ochratoxine B est très rare. 

1.2  Méthodes d'analyse

    On a mis au point des méthodes d'analyse permettant 
l'identification et le dosage de l'ochratoxine à des concentrations 
de l'ordre de 1 µg/kg. 

1.3  Métabolisme

    On trouve des résidus d'ochratoxine A intacte dans le sang, les 
reins, le foie et les muscles de porcs à l'abattoir ainsi que dans 
les muscles de poules et de poulets.  En revanche on ne trouve 
généralement pas de résidus chez les ruminants.   In vitro, 
l'ochratoxine A se fixe très solidement à l'albumine sérique 
bovine, porcine et humaine.  Des études expérimentales sur des 
porcins et des poules ont montré que c'est au niveau des reins que 
les concentrations d'ochratoxine A sont les plus élevées.  Chez le 
porc, le rat et l'homme, il pourrait y avoir détoxication par 
hydroxylation au niveau des microsomes.  L'expérience montre qu'on 
peut encore identifier des résidus dans les reins de porc un mois 
après la fin de l'exposition. 

1.4  Effets sur les animaux

    On a signalé des cas d'ochratoxicose chez des animaux d'élevage 
(porcs et volaille) dans plusieurs pays d'Europe, la manifestation 
essentielle étant une néphropathie chronique.  Les lésions se 
présentent sous la forme d'une atrophie tubulaire, d'une fibrose 
interstitielle et à un stade plus avancé, d'une hyalinisation des 
glomérules.  Au Canada, on a également trouvé de l'ochratoxine A 
dans du sang de porc recueilli à l'abattoir.  Des effets 
néphrotoxiques ont été relevés chez toutes les espèces d'animaux à 
estomac simple étudiées jusqu'ici, même aux doses les plus faibles 
qui aient été expérimentées (200 mg/kg de nourriture chez les rats 
et les porcs). 

    Des effets tératogènes ont été observés chez des souris 
exposées par voie orale à 3 mg d'ochratoxine A par kg de poids 
corporel.  On a observé une résorption des foetus chez des rats à 
partir de 0,75 mg/kg de poids corporel, le produit étant administré 
par voie orale.  Ces effets tératogènes, qui chez le rat sont 
accrus par un régime alimentaire pauvre en protéines, ont été 
également observés chez des hamsters. 

    Les épreuves à court terme n'ont pas permis de mettre en 
évidence d'activité mutagène (bactéries et levures).  Administré 
par voie orale à des rats, le produit a provoqué des ruptures 
monocaténaires dans l'ADN des tissus du rein et du foie.  
L'ochratoxine a provoqué l'apparition de cancers du rein chez des 
souris mâles et des rats des deux sexes qui recevaient le produit 
par voie orale.  La formation de carcinomes hépatocellulaires n'a 
été signalée que chez une souche de souris et pas du tout chez le 

    L'ochratoxine A est un inhibiteur de la synthèse protéique et 
de la tRNA-synthétase chez les microorganismes et dans les cellules 
hépatomateuses;  elle inhibe également le mARN chez le rat. 

    L'ochratoxine A peut inhiber la migration des macrophages.  
Chez la souris, une dose de 0,005 µg/kg de poids 
corporel a aboli la réponse immunitaire aux érythrocytes de mouton;  
toutefois d'autres résultats, contradictoires, ont été obtenus. 

    L'ochratoxine A s'est révélée cancérogène pour l'épithelium 
tubulaire rénal chez des souris mâles et des rats des deux sexes. 

1.5  Effets sur l'homme

    L'exposition humaine, qui ressort de la présence d'ochratoxine 
A dans les aliments, le sang et le lait humains, a été observée 
dans divers pays d'Europe.  Selon les données épidémiologiques 
disponibles, la néphropathie des Balkans peut être attribuée à la 
consommation de denrées alimentaires contaminées par cette toxine. 

    On a mis en évidence une relation tout à fait significative 
entre la néphropathie des Balkans et les tumeurs des voies 
urinaires, en particulier des tumeurs pyéliques et uretérales.  
Toutefois on n'a pas publié de données qui établissent une 
implication directe de l'ochratoxine A dans l'étiologie de ces 

2.  Trichothécènes

2.1  Etat naturel

    On connaît aujourd'hui 148 trichothécènes qui se caractérisent 
sur le plan chimique par la présence d'une structure de base 
commune, le système tétracyclique du scirpénol.  Ces composés sont 
principalement secrétés par des moisissures appartenant au genre 
 Fusarium, encore que d'autres genres notamment  Trichoderma, 
 Trichothecium, Myrothecium et  Stachybotrys produisent également des 

métabolites considérés désormais comme des trichothécènes.  
Quelques-uns seulement de ces trichothécènes contaminent les 
denrées alimentaires destinées à l'homme ou aux animaux, en 
particulier :  le désoxyvalénol (DON), le nivalénol (NIV), le 
disacétoxyscirpenol (DAS), ainsi que la toxine T-2 et, plus 
rarement, certains dérivés (3-Ac-DON, 15-Ac-DON, fusarenone-X et 
toxine HT-2).  Parmi ces substances, c'est le DON qui est de loin 
la plus fréquemment présente dans les denrées alimentaires 
destinées à l'homme et aux animaux;  à côté de quantités plus 
faibles de NIV.  Certains trichothécènes macrocycliques comme les 
satratoxines G et H et les verrucarines, se rencontrent de temps à 
autre dans les fourrages (paille, foin) mais il n'a pas été fait 
état de leur présence dans les produits alimentaires. 

    Les enquêtes sur la présence de trichothécènes ont révélé que 
le DON était présent dans le monde entier, essentiellement dans les 
céréales telles que le froment et le maïs à des concentrations 
pouvant parfois atteindre 92 mg/kg, encore que les teneurs moyennes 
soient beaucoup plus faibles et varient selon la denrée.  Des 
rapports isolés font état de la présence de DON dans de l'orge, des 
mélanges alimentaires pour aminaux, des pommes de terre, etc.  Le 
NIV, dont la présence n'est normalement pas signalée dans des 
céréales au Canada ou aux Etats-Unis se rencontre en revanche 
fréquemment au côté du DON dans les céréales originaires d'Asie ou 
d'Europe;  la concentration la plus forte enregistrée jusqu'ici est 
de 37,9 mg/kg.  On a signalé ça et là la présence de toxine T-2 et 
de DAS à des concentrations beaucoup plus faibles. 

    Des études portant sur les divers traitements subis par les 
produits alimentaires et notamment la mouture, montrent que, entre 
la céréale brute et le produit définitif, il n'y a guère de 
diminution des teneurs en DON.  De même la panification ne parvient 
pas à détruire le DON.  En général, les aliments destinés à l'homme 
que l'on trouve dans le commerce ne contiennent que rarement des 
quantités décelables de DON et de NIV. 

2.2  Methodes d'analyse

    On dispose, pour le dosage des quatre toxines les plus 
fréquemment rencontrées (DON, toxine T.2, DAS et NIV), des méthodes 
d'analyse basées sur la chromatographie en couche mince, la 
chromatographie en phase gazeuse ou la chromatographie liquide à 
haute performance ainsi que sur des réactions immunologiques;  les 
limites de détection se situent en-dessous de 1 µg/g.  Plusieurs de 
ces méthodes ont fait l'objet d'une expérimentation collective.  En 
outre, certaines méthodes utilisées en recherche comme la 
chromatographie gazeuse ou liquide associée à la spectrographie de 
masse peuvent être utilisées pour confirmer l'identité des 

2.3  Métabolisme

    On a procédé à des études métaboliques, essentiellement de la 
toxine T-2, mais dans quelques rares cas seulement du DON, chez 
l'animal.  Ces trichothécènes sont rapidement absorbés au niveau 
des voies digestives mais l'on ne dispose pas de données 

quantitatives.  Les toxines se répartissent de façon uniforme sans 
accumulation marquée au niveau d'un organe ou d'un tissu 
particulier.  Elles sont métabolisées en produits moins toxiques 
par hydrolyse, hydroxylation, désépoxydation et glucuronidation.  
La toxin T-2 et le DON sont rapidement éliminés par voie fécale et  

    Par exemple, une dose de toxine T-2 administrée par voie orale à 
des bovins a été éliminée presque à hauteur de 100 % dans les heures 
suivant l'administration;  des poulets ont éliminé 80 % de la 
substance dans les 48 heures suivant l'administration.  Chez le rat, 
25 % d'une dose de DON ont été éliminés dans les urines et 65 % dans 
les matières fécales 96 heures après l'administration.  Chez la poule 
pondeuse et la vache laitière on a constaté que moins de 1 % de la 
dose de toxine T-2 (et de ses métabolites) qui leur avait été 
administrée se retrouvaient dans les oeufs et le lait.  Après 
administration par voie orale de toxine T-2 à des poulets, on a 
constaté que les résidus présents dans la viande 24 heures plus tard 
représentaient moins de 2 % de la dose initiale. 

2.4  Effets sur les animaux

    C'est principalement par l'ingestion de fourrage contaminé que 
les animaux sont exposés aux trichothécènes.  La toxine T-2 et le DAS 
qui chez les animaux de laboratoire sont les plus actifs des 
trichothécènes couramment cités comme contaminants des denrées 
destinées aux animaux (toxine T-2, DAS, NIV et DON), provoquent des 
réactions toxiques analogues.  Le NIV est moins actif dans certains 
systèmes que les deux précédents composés et le DON est le moins 
toxique des quatre.  (Cette activité toxique peut s'évaluer au moyen 
de la DL50 pour la souris, par exemple dans le cas de la toxine T-2 
elle est égale à 10,5 mg/kg de poids corporel et dans le cas du DON à 
46,0 mg/kg). 

    Les trichothécènes les plus actifs, tels que la toxine T-2 et le 
DAS produisent des effets généraux aigus lorsqu'ils sont administrés 
expérimentalement à des rongeurs, des porcs et des bovins par voie 
orale, parentérale ou respiratoire (porc, souris).  Ces 
trichothécènes produisent une épithélionécrose par contact (une dose 
de 0,2 µg par touche dans le cas de la toxine T-2).  Avec les autres 
trichothécènes, il faut des doses plus élevées pour obtenir un effet 
irritant (dans le cas du NIV, 10 ug par touche).  Les trichothécènes 
cytotoxiques comme la toxine T-2 ont une action nécrosante sur 
l'épithélium des cryptes intestinales et sur les tissus lymphoïdes et 
hematopoïétiques après exposition par voie orale, parentérale ou 
respiratoire.  Après exposition à la toxine T-2 et au DAS on observe 
des anomalies hematologiques et des troubles de l'hémostase.  Dans 
les cas graves, la toxicose peut entraîner une pancytopénie.   Des 
études portant sur la toxine T-2, le DON et le DAS ont mis en 
évidence la suppression de l'immunité à médiation cellulaire et de 
l'immunité humorale et on a observé une réduction de la concentration 
des immunoglobulines ainsi qu'une dépression de l'activité 
phagocytaire des macrophages et des neutrophiles.  Des études sur 
animaux de laboratoire ont montré que l'effet immunodépresseur de 
trichothécènes tels que la toxine T-2, le DAS et le DON entraînait 

une moindre résistance aux infections secondaires par des bactéries 
(Mycobactérie,  Listeria monocytogenes) des levures ( Cryptococcus 
 neoformans) et des virus (virus de l'herpes simplex). 

    Il a été indiqué qu'après injection intrapéritonéale, la toxine 
T-2 était tératogène pour la souris (cette voie d'administration 
n'est pas courante dans les études de tératogénicité).  Le DON est 
tératogène pour la souris après intubation gastrique mais ne l'est 
pas chez le rat lorsque la toxine est administrée dans la nourriture 
de l'animal.  Le NIV ne s'est pas révélé tératogène pour la souris.  
La recherche du pouvoir mutagène de la toxine T-2, du DAS et du DON 
par une épreuve du type Ames n'a pas donné de résultat positif.  La 
toxine T-2 présente dans certaines épreuves une faible activité 
clastogène.  D'après les études de toxicité à long terme qui ont été 
publiées, rien n'indique que la toxine T-2, la fusarénone-X et le NIV 
ne soient tumorigènes chez l'animal.  Aucune étude de toxicité à long 
terme n'a été publiée sur le DON. 

    Les trichothécènes sont toxiques pour les cellules à forte 
activité mitotique telles que les cellules de l'épithélium des 
cryptes intestinales et les cellules hematopoïétiques.  Cette 
cytotoxicité proviendrait, soit d'une perturbation de la synthèse des 
protéines par une fixation des composés aux ribosomes des cellules 
eucaryotes, soit d'une dysfonction des membranes cellulaires.  
L'inhibition de la synthèse protéique serait due à l'induction de 
protéines régulatrices labiles telles que l'IL-2 dans les 
immunocytes.  A concentrations extrêmement faibles, les 
trichothécènes perturbent le transport des petites molécules à 
travers les membranes cellulaires. 

2.5  Effets sur l'homme

    L'ingestion de produits alimentaires contaminés d'origine 
végétale constitue la principale voie d'exposition aux tricho- 
thécènes mais d'autres voies ont été signalées à l'occasion, par 
exemple un contact cutané accidentel chez des chercheurs de 
laboratoire ou l'inhalation de trichothécènes présents dans des 
poussières aéroportées. 

    On n'a décrit que peu de cas de maladies attribuables à une 
exposition aux trichothécènes, sans d'ailleurs que la responsa- 
bilité de ces produits soit établie.  Toutefois, dans les deux 
flambées évoquées ci-dessous, il y a lieu de penser que leur 
responsabilité est en cause. 

    Une des flambées, qui s'est produite en Chine, a été attribuée à 
la consommation de blé moisi contenant 1,0 à 40,0 mg de DON par kg.  
La maladie se caractérisait par des symptômes gastro-intestinaux.  
Aucun décès n'a été à déplorer.  Les porcs et les poulets qui avaient 
mangé les restes de céréales ont également été affectés. 

    Une flambée du même genre a été signalée en Inde et attribuée à 
la consommation de pain fabriqué à l'aide de blé contaminé.  La 
maladie se caractérisait par des symptômes gastro-intestinaux et une 
irritation de la gorge qui apparaissaient 15 minutes à une heure 

après l'ingestion du pain.  On a décelé les mycotoxines suivantes 
dans des échantillons de farine raffinée utilisée pour la préparation 
du pain:  DON (0,35-8,3 mg/kg), acétyldésoxynivalénol (0,64-2,49 
mg/kg), NIV (0,03-0,1 mg/kg) et toxine T-2 (0,5-0,8 mg/kg).  
Toutefois, l'identité de ces trichothécènes n'a pas été confirmée.  
La présence de DON et de NIV à côté de la toxine T-2 est 

    Deux maladies d'intérêt historique, une aleucie alimentaire 
d'origine toxique en URSS et une toxicose due à du blé moisi au 
Japon et en Corée ont été attribuées à la consommation de céréales 
contaminées par des moisissures du genre  Fusarium.  Depuis lors, on 
a isolé en laboratoire, dans des cultures de  Fusarium isolés des 
céréales en cause, un certain nombre de trichothécènes.  Il n'avait 
pas été possible, au moment où ces maladies se sont déclarées, 
d'effectuer des recherches pour tenter de corréler l'aleucie 
toxique et la toxicose due au blé moisi à une exposition à des 
trichothécènes car on ne connaissait pas les toxines en question. 

3.  Ergot

3.1  Etat naturel

    Ergot est le nom que l'on donne aux sclérotes de certaines 
espèces de champignons appartenant au genre  Clavicepsa.  Ces 
sclérotes contiennent des alcaloïdes biologiquement actifs qui 
peuvent être à l'origine de toxicoses chez les personnes ou les 
animaux qui consomment des denrées contaminées. 

    Les alcaloïdes de l'ergot produisent deux types de maladies selon 
leur nature, qui dépend du champignon en cause ( C. purpurea ,  C. 
 fusiformis ).  L'ergotisme, dû à l'ergotamine et à l'ergocristine 
produites par  C. purpurea se caractérise principalement par une 
gangrène des extrémités et des symptômes gastro-intestinaux.  Quand à 
l'intoxication provoquée par du millet contaminé par  C. fusiformis, 
elle se caractérise principalement par des symptômes digestifs et 
elle est due aux clavines.  Aucun des signes ou symptômes observés ne 
sont révélateurs d'une oblitération vasculaire. 

3.2  Méthodes d'analyse

    Les alcaloïdes de l'ergot (ergolines) sont des dérivés de l'acide 
lysergique.  Ils ont une activité biologique variable selon leur 
nature.  Le dosage des alcaloïdes de  C. purpurea a été effectué par 
chromatographie liquide à haute performance avec détection par 
fluorescence.  On peut déterminer des concentrations de 0,2 µg 
d'ergolines par litre de plasma humain.  La détermination de 
l'ergotamine et de l'ergocristine peut s'effectuer de façon très 
spécifique par titrage radio-immunologique à des concentrations 
respectives de 3,5 et 0,8 picomoles. 

3.3  Effets sur les animaux

    Des flambées d'avortements chez des bovins ont pu être attribuées 
à l'ingestion d'ergoline, essentiellement de l'ergotamine et de 
l'ergotaminine.  Des moutons à qui l'on avait administré de 

l'ergotamine par voie orale sont tombés rapidement malades et 
présentaient une inflammation intestinale.  Chez des volailles, des 
porcs et des primates exposés par voie orale on a observé des effets 
légers.  Le groupe de travail ne disposait d'aucune donnée sur la 
mutagénicité, la tératogénicité et la cancérogénicité des ergolines. 

3.4  Effets sur l'homme

    L'homme peut être exposé aux ergolines par la consommation de 
céréales ergotées.  Dans la plupart des études toxicologiques qui ont 
été effectuées, on n'a pas procédé à l'identification précise des 
alcaloïdes en cause.  Les données qui ont été publiées au sujet d'une 
seule enquête effectuée en Suisse sur des céréales et des produits 
céréaliers indiquent que la consommation quotidienne totale 
d'ergolines se situe à environ 1,5 ug par personne, certaines denrées 
en contenant jusqu'à 140 µg/kg.  La panification réduit de 25 à 100 % 
la teneur en ergolines des farines contaminées. 

    En Ethiopie, une flambée d'ergotisme s'est produite en 1978 à la 
suite de l'exposition à des ergolines provenant de sclérotes de ce  C. 
 purpurea.  Les céréales comptaient jusqu'à 0,75 % d'ergot;  on a 
relevé la présence d'ergométrine.  Parmi les symptômes observés 
figuraient une gangrène sèche ayant entraîné la perte d'un ou 
plusieurs membres (29 % des cas), un pouls périphérique faible ou 
absent (36 %) et une desquamation.  Des symptômes digestifs n'ont été 
observés que dans quelques cas.  Chez 88 % des malades, les lésions 
intéressaient les membres inférieurs. 

    En Inde, plusieurs flambées se sont déclarées depuis 1958 à la 
suite de la consommation de millet contaminé par  C. fusiformis.  Ces 
symptômes consistaient en nausées, vomissements et vertiges.  Les 
symptômes toxiques étaient dus à la présence d'ergolines à des 
concentrations de 15 à 26 mg/kg.  Aucune autopsie n'ayant été 
effectuée, on ne dispose d'aucune information sur les effets 
pathologiques au niveau des viscères. 

4.  Evaluation des risques pour la santé humaine

4.1  Ochratoxine A

    L'exposition humaine, observée dans plusieurs pays d'Europe est 
objectivée par la présence d'ochratoxine A dans les denrées 
alimentaires et le sang.  Le groupe de travail n'a pas connaissance 
de tentatives qui ont effectuées dans d'autres régions du monde pour 
déceler la présence d'ochratoxine A dans le sang humain. 

    En s'appuyant sur l'étude de la maladie-naturelle ou provoquée  
par administration d'ochratoxine A-on a pu établir le rôle 
étiologique de l'ochratoxine A dans la néphropathie porcine.  A 
partir de ce modèle, on a pu émettre l'hypothèse que la néphropathie 
endémique des Balkans était due à une exposition à cette toxine. Les 
données immunologiques disponibles montrent que cette affection 
serait attribuable à la consommation de denrées alimentaires 
contaminées par de l'ochratoxine A.  Depuis la publication en 1980 du 
No 11 des Critères d'hygiène de l'environnement, des études 

épidémiologiques sur la concentration de l'ochratoxine A dans le sang 
humain dans les régions touchées et non touchées ont  conforté 
l'hypothèse d'une relation entre la néphropathie balkanique et 
l'exposition à l'ochratoxine A. 

    Ainsi, on a montré que les habitants des régions d'endémie 
présentaient plus fréquemment de l'ochratoxine A dans leur sang et à 
des concentrations plus élevées.  Cependant, ces études 
rétrospectives ne fournissent que des présomptions sur lesquelles on 
ne peut établir l'existence d'une relation causale directe.  On ne 
peut cependant pas l'exclure du fait de la longue période de latence 
entre l'exposition et l'apparition des symptômes. 

    On a montré que l'ochratoxine A exerçait, chez les souris mâles 
et les rats des deux sexes, des effets cancérogènes sur l'épithélium 
des tubules rénaux.  Il existe une relation tout à fait significative 
entre la néphropathie balkanique et la présence de tumeurs des voies 
urinaires, notamment de tumeurs pyéliques et uretérales.  Toutefois 
aucune donnée n'a été publiée qui établissent la responsabilité 
directe de l'ochratoxine A dans l'étiologie de ces tumeurs. 

4.2  Trichothécènes

    Sur la base des données dont disposait le groupe de travail, il 
est possible d'établir une relation entre l'exposition aux 
trichothécènes et certains épisodes toxiques chez l'homme.  Si l'on 
se réfère aux quelques données disponibles, ce sont le DON et le NIV 
qui sont les plus fréquemment cités dans les différents cas 
d'exposition humaine.  Dans le cas des flambées d'aleucie toxique 
alimentaire et de toxicose par ingestion de céréales moisies, on ne 
peut exclure la responsabilité des trichothécènes.  L'exposition se 
produit par ingestion de denrées contaminées, principalement des 
céréales.  Les différents traitements qu'elles subissent, notamment 
la mouture et la panification ne permettent pas d'éliminer le DON, le 
NIV, ni la toxine T-2.  L'existence d'une exposition par voie 
respiratoire est très peu documentée mais c'est une possibilité qu'on 
ne peut totalement écarter. 

    Parmi les tricothécènes qui se trouvent à l'état naturel dans les 
aliments, c'est la toxine T-2 qui est la plus active, suivie du DAS 
and du NIV;  les études de toxicité aiguë ont montré que le DON était 
la substance la moins toxique.  Chez l'animal d'expérience, la toxine 
T-2 et le DAS déterminent des symptômes généraux aigus, avec nécrose 
des tissus épithéliaux et suppression de l'hematopoïèse.  Lors des 
récentes flambées d'intoxications, il n'a été fait état que des 
symptômes digestifs. 

    Le DON est tératogène pour la souris mais pas pour le rat.  Selon 
les études de toxicité chroniques qui ont été publiées, le NIV et la 
toxine T-2 ne sont pas tumorigènes chez l'animal.  Aucune étude de 
cancérogénicité à long terme n'a été publiée sur le DON.  Certains 
trichothécènes, tels que la toxine T-2 et le DON ont une action 
immunosuppressive chez l'animal et produisent des modifications de 
l'immunité à médiation cellulaire et de l'immunité humorale.  En 
revanche, rien n'indique l'existence de tels effets chez l'homme. 

    On est mal informé sur les cas d'intoxication humaine due à une 
exposition aux trichothécènes, cas qui sont en nombre limité.  Les 
symptômes observés consistent en troubles digestifs et irritation de 
la gorge;  ils apparaissent peu après l'ingestion de denrées 
alimentaires contaminées par des trichothécènes.  A l'heure actuelle, 
on ne connaît pas de cas de cancer humain attribuable aux 
trichothécènes.  Le groupe de travail ne disposait d'aucune 
publication faisant état d'infections secondaires d'origine 
bactérienne, fongique ou virale chez l'homme, à la suite d'une 
exposition aux trichothécènes comme on en a observé chez l'animal 
d'expérience.  Il semble que l'on n'ait pas effectué d'études 
appropriées sur ce point. 

4.3  Ergot

    Il semble que l'exposition humaine à de faibles concentrations 
d'ergolines soit très répandue.  Les données relatives aux flambées 
qui se sont récemment déclarées en Ethiopie et en Inde montrent que 
les alcaloïdes de  C. purpurea (groupe de l'ergotamine) produisent 
les effets les plus graves, notamment une gangrène des membres 
inférieurs pouvant entraîner la mort, effets qui sont plus graves 
que ceux des alcaloïdes de  C. fusiformis (groupe de la clavine) qui 
entraînent des symptômes digestifs sans issue fatale.  On ignore si 
ces différences s'expliquent par la teneur des différentes espèces 
de champignons en alcaloïdes, par les propriétés toxicologiques de 
ces substances ou par des différences dans les quantités ingérées 
par les diverses populations. 

    Après nettoyage et mouture qui éliminent les sclérotes, il ne 
subsiste dans les produits alimentaires que de faibles quantités 
d'ergolines.  La panification ou d'autres traitements thermiques 
détruisent également la plupart des alcaloïdes du groupe de 

5.  Recommandations en vue de recherches futures

5.1  Recommandations generales

    Il conviendrait de créer un réseau de centres de référence pour 
aider les Etats Membres à confirmer l'identité des mycotoxines 
présentes dans les tissus humains et les denrées destinées à la 
consommation humaine.  Ces centres devraient également fournir sur 
demande des échantillons de référence de mycotoxines afin de 
faciliter la comparabilité des résultats d'analyse obtenus dans les 
différentes régions du monde. 

5.2  Ochratoxine A

    a)  Etudes épidémiologiques de grande ampleur à caractère 
        rétrospectif et études localisées à caractère prospectif sur 
        l'association entre l'ochratoxine A et la néphropathie 
        endémique des Balkans ainsi que les tumeurs des voies 
        urinaires :  ces études devraient être menées dans la 
        péninsule de Balkans et la Région méditerranéenne. 

    b)  Recherche et dosage de l'ochratoxine A dans le sang de 
        malades porteurs de tumeurs des voies urinaires, à 
        l'extérieur de la péninsule des Balkans. 

    c)  Recherche de sources d'exposition à l'ochratoxine A, par 
        analyse du sang, dans les pays situés en dehors de la 
        péninsule des Balkans. 

    d)  Elucidation du mécanisme à l'origine des différences entre 
        les sexes qui ont été relevées dans les anomalies rénales, 
        néoplasiques et non-néoplasiques produites par l'ochratoxine 
        A chez l'animal d'expérience. 

    e)  Etudes de grande envergure sur la teneur en ochratoxine A des 
        denrées alimentaires dans les différentes régions du monde.  
        Ce type d'enquêtes est particulièrement important dans les 
        régions où l'on observe une forte incidence de tumeurs des 
        voies urinaires, notamment rénales, ou de néphropathies. 

5.3  Trichothécènes

    a)  Il conviendrait d'effectuer des études longitudinales dans 
        les régions de l'Inde et de la République populaire de Chine 
        où ont été récemment observés des épisodes d'intoxication par 
        les trichothécènes.  Il conviendrait de mieux éclaircir la 
        présence inhabituelle de certains trichothécènes dans ces 

    b)  Il faudrait étudier les effets d'une exposition prolongée 
        d'animaux de laboratoire au DON, et notamment les effets 
        cancérogènes.  Comme la réaction au DON varie beaucoup selon 
        les espèces, l'espèce qui sera retenue devra être choisie 
        avec soin. 

    c)  Il faudrait étudier plus à fond les infections microbiennes 
        secondaires à une exposition aux trichothécènes chez l'animal 

    d)  Il convient d'étudier l'influence des conditions 
        environnementales, notamment la présence d'insecticides ou 
        autres produits chimiques industriels, sur la production de 
        trichothécènes par des champignons. 

    e)  Il faudrait élucider l'effet des différents modes de 
        préparation des denrées alimentaires sur les trichothécènes. 

    f)  Il conviendrait de mettre au point des espèces végétales qui 
        résistent aux champignons producteurs de trichothécènes, en 
        recourant aux biotechnologies. 

    g)  Il faudrait étudier sur l'animal les effets synergistiques 
        éventuels d'une exposition simultanée aux trichothécènes, aux 
        aflatoxines, à l'ochratoxine A et à d'autres mycotoxines. 

    h)  Il faudrait également déterminer l'apport de trichothécènes 
        d'origine alimentaire chez l'homme. 

    i)  Il faudrait mettre au point des méthodes de criblage des 
        trichothécènes qui soient à la fois rapides et sensibles, et 
        mener des enquêtes dans les régions tempérées du monde pour 
        rechercher la présence de trichothécènes dans les céréales et 
        les préparations alimentaires. 

5.4  Ergot

    a)  Il faudrait mettre au point des méthodes pour l'analyse des 

    b)  Il conviendrait de mettre à la disposition des pays en 
        développement des renseignements sur le repérage des semences 
        contaminées et sur les méthodes de mouture permettant de 
        réduire au minimum les problèmes posés par la présence de 

    c)  Il faudrait effectuer des études épidémiologiques sur les 
        effets éventuels que peut entraîner chez l'homme l'ingestion 
        de faible quantités d'ergolines. 

    d)  Il faudrait effectuer des études pharmacologiques et 
        toxicologiques sur les ergolines, seules ou en association, 
        chez l'animal d'expérience. 

    e)  Il conviendrait de déterminer si les ergolines peuvent se 
        transmettre de la mère à l'enfant par le lait maternel. 


1.  Ocratoxina A

1.1  Distribución natural

    Las ocratoxinas son producidas por varias especies de los 
géneros de hongos  Aspergillus y  Penicillium.  Esos hongos son 
ubicuos y hay amplias posibilidades de contaminación de alimentos 
para seres humanos y para animales.  La ocratoxina A, la más 
importante, se ha hallado en toda una serie de países de América 
del Norte, Australia y Europa.  La formación de ocratoxina por las 
especies del género  Aspergillus parece estar limitada a condiciones 
de humedad y temperatura elevadas, mientras que algunas especies, 
por lo menos, de  Penicillium pueden producir ocratoxina a 
temperaturas de sólo 5 °C. 

    Las mayores incidencias de contaminación con ocratoxina A se 
han hallado en cereales y, en menor medida, en algunos granos 
(café, soja, cacao).  La ocratoxina B es muy poco frecuente. 

1.2  Métodos de análisis

    Se han desarrollado técnicas de análisis para identificar la 
ocratoxina y determinar sus concentraciones en la gama de g/kg. 

1.3  Metabolismo

    Se han hallado residuos de ocratoxina A no modificada en la 
sangre, los riñones, el hígado y los músculos de cerdos en los 
mataderos y en los músculos de gallinas y pollos.  Sin embargo, por 
lo general no se han hallado residuos de ocratoxina A en los 
rumiantes.  El enlace  in vitro de la ocratoxina A con la 
seroalbúmina es especialmente importante en el ganado vacuno, el 
ganado ovino y el ser humano.  Estudios experimentales realizados 
con cerdos y gallinas han demostrado que las concentraciones más 
altas de ocratoxina A se hallan en los riñones.  La hidroxilación 
microsómica puede representar una reacción de detoxificación en los 
cerdos, las ratas y el ser humano.  En estudios experimentales, un 
mes después de terminada la exposición se localizaron aún residuos 
en riñones de cerdos. 

1.4  Efectos en animales

    Varios países europeos han notificado en animales de granja 
(cerdos, aves de corral) casos de ocratoxicosis, cuya principal 
manifestación era una nefropatía crónica.  Entre las lesiones había 
atrofia tubular, fibrosis intersticial y, en etapas ulteriores, 
hialinización de los glomérulos.  También se ha hallado ocratoxina 
A en sangre de cerdo recogida en mataderos canadienses.  La 
ocratoxina A ha producido efectos nefrotóxicos en todas las 
especies de animales provistas de un solo estómago que se han 
estudiado hasta el momento, incluso con las dosis más bajas con que 
se experimentó (200 µg/kg de alimentos en ratas y cerdos). 

    Se observaron efectos teratogénicos en ratones expuestos a 
dosis de 3 mg/kg de peso corporal administradas por vía oral.  En 
ratas que recibieron por vía oral dosis de 0,75 mg/kg de peso 
corporal se observó resorción fetal.  Los efectos teratogénicos, 
que en las ratas se acentuaron con una dieta baja en proteínas, se 
han observado también en hámsters. 

    No hay datos que demuestren la actividad de la ocratoxina A en 
pruebas a corto plazo de la mutagenicidad (bacterias y levaduras).  
Ratas expuestas a ocratoxina A administrada por vía oral mostraron 
roturas de una sola cadena del ADN en tejidos renales y hepáticos.  
Ocratoxina A administrada por vía oral provocó neoplasias de 
células renales en ratones machos y en ratas de uno y otro sexo.  
Se registraron neoplasias de células hepáticas en sólo una estirpe 
de ratones y no en la rata. 

    La ocratoxina A es inhibidora de la síntesis de proteínas y de 
la sintetasa del ARNt en microorganismos, células de hepatoma y el 
ARNm renal en la rata. 

    La ocratoxina A puede inhibir la migración de los macrófagos.  
En ratones, una dosis de 0,005 µg/kg de peso corporal suprimió la 
respuesta inmunitaria a eritrocitos de oveja; sin embargo, se han 
obtenido también resultados contradictorios. 

    Se ha demostrado que la ocratoxina A es carcinógena para el 
epitelio de los túbulos renales en ratones machos y de ratas de uno 
y otro sexo. 

1.5  Efectos en el ser humano

    En varios países de Europa se ha observado exposición humana a 
ocratoxina A, como lo demuestra la presencia de ésta en alimentos y 
en la sangre y la leche humanas.  Los datos epidemiológicos de que 
se dispone indican que la nefropatía de los Balcanes podría estar 
relacionada con el consumo de productos alimenticios contaminados 
por esta toxina. 

    Se ha observado una relación muy significativa entre la 
nefropatía de los Balcanes y tumores del tracto urinario, en 
particular los de la pelvis renal y los uréteres.  Sin embargo, no 
se han publicado datos que demuestren el papel causal de la 
ocratoxina A en la etiología de esos tumores. 

2.  Tricotecenos

2.1  Distribución natural

    Hasta ahora se conocen 148 tricotecenos, caracterizados 
químicamente por la presencia del mismo sistema básico de un anillo 
tetracíclico de scirpenol.  Son compuestos producidos 
principalmente por hongos pertenecientes al género  Fusarium, 
aunque otros géneros, entre ellos  Trichoderma, Trichothecium, 
 Myrothecium y  Stachybotrys, producen también metabolitos 
ahora reconocidos como tricotecenos.  Se ha visto que sólo un 

pequeño número de los tricotecenos conocidos contaminan los 
alimentos para seres humanos o para animales, entre ellos el 
deoxinivalenol (DON), el nivalenol (NIV), el diacetoxiscirpenol 
(DAS) y la toxina T-2 y, con menos frecuencia, ciertos derivados 
(3-Ac-DON,15-Ac-DON, fusarenon-X y toxina HT-2).  De todos ellos, 
el que se encuentra más a menudo en alimentos para seres humanos y 
para animales es el DON, por lo general con cantidades más pequeñas 
de NIV como co-contaminante.  Algunos tricotecenos macrocíclicos 
como las satratoxinas G y H y las verrucarinas se observan 
ocasionalmente en alimentos para animales (paja, heno) pero no se 
ha notificado su presencia en alimentos para seres humanos. 

    Estudios sobre la distribución de los tricotecenos han indicado 
que el DON se encuentra en todo el mundo, sobre todo en cereales 
como el trigo y el maíz, en concentraciones que pueden llegar hasta 
92 mg/kg, aunque las concentraciones medias son considerablemente 
inferiores y varían según el producto.  Hay notificaciones aisladas 
de la presencia de DON en la cebada, los piensos compuestos, las 
patatas, etc.  Habitualmente los cereales producidos en Canadá y 
los Estados Unidos de América no contienen NIV pero éste se ha 
encontrado en los cereales asiáticos y europeos, junto con DON; la 
mayor concentración de NIV registrada hasta la fecha fue de 37,9 
mg/kg. La presencia de toxina T-2 y DAS se ha notificado con escasa 
frecuencia y en concentraciones muy inferiores. 

    Estudios sobre la elaboración y la molienda indican que las 
concentraciones de DON apenas se reducen del cereal al producto 
acabado.  Tampoco se destruye cuando se cuece el cereal.  En 
general, los productos alimenticios para seres humanos que se 
encuentran en el comercio rara vez contienen concentraciones 
detectables de DON y NIV. 

2.2  Métodos de análisis

    Existen xistenmétodos de análisis basados en la cromatografía 
de capa fina, la cromatografía de fase gaseosa, la cromatografía de 
fase líquida de alto rendimiento y técnicas inmunológicas para la 
determinación de las cuatro toxinas más frecuentes (DON, toxina T-
2, DAS y NIV) con límites de detección inferiores a 1 µg/g.  
Algunos de esos métodos se han ensayado en colaboración.  Además, 
la identidad puede confirmarse mediante métodos de investigación 
como cromatografía de fase gaseosa/espectografía de masas y 
cromatografía de fase líquida/espectografía de masas. 

2.3  Metabolismo

    Se han realizado estudios metabólicos con animales, sobre todo 
administrando toxina T-2 y en unos pocos casos DON.  Estos 
tricotecenos  se  absorben  con  rapidez  por  el  tubo  digestivo, 
aunque no se dispone de datos cuantitativos.  Las toxinas se 
distribuyen bastante uniformemente, sin marcada acumulación en 
ningún órgano o tejido determinado.  Los tricotecenos se 
transforman en metabolitos menos tóxicos por reacciones como 
hidrólisis, hidroxilación, de-epoxidación y glucoronidación.  
Tricotecenos como la toxina T-2 y el DON se eliminan rápidamente en 

las heces y la orina.  Por ejemplo, casi el 100% de una dosis de 
toxina T-2 administrada a ganado por vía oral se eliminaba en unas 
horas; en pollos, alrededor del 80% se había eliminado a las 48 
horas.  En la rata, 96 horas después de la administración, el 25% 
del DON se había eliminado en la orina y el 65% en las heces.  Los 
resultados de la transmisión de toxina T-2 en gallinas ponedoras y 
vacas lactantes indicaron que pasaba a los huevos y a la leche 
menos del 1% de la dosis administrada de esa toxina y de sus 
metabolitos.  En la carne de pollo, 24 horas después de la 
administración de toxina T-2 por vía oral, los residuos de ésta y 
de sus metabolitos eran inferiores al 2% de la dosis. 

2.4  Efectos en animales

    La principal vía de exposición de los animales a tricotecenos 
es la ingestión de alimentos de origen vegetal.  La toxina T-2 y el 
DAS,   los más potentes en los experimentos de laboratorio con 
animales, de todos los tricotecenos que habitualmente se consideran 
contaminantes de alimentos para animales (toxina T-2, DAS, NIV y 
DON),   provocan una respuesta tóxica similar.  El NIV es menos 
potente en algunos sistemas que los dos compuestos anteriores y el 
DON es el menos tóxico de los cuatro (un ejemplo de la potencia es 
la DL50 por vía oral en el ratón:  toxina T-2, 10,5 mg/kg de peso 
corporal y DON, 46,0 mg/kg). 

    Los tricotecenos más potentes, como la toxina T-2 y el DAS, 
producen efectos generales agudos cuando se administran 
experimentalmente a roedores y ganado porcino y bovino por vía oral 
o parenteral o por inhalación (cerdo, ratón).  Una lesión producida 
por el contacto con tricotecenos potentes como la toxina T-2 y el 
DAS es la epitelionecrosis (dosis de 0,2 µg por zona en el caso de 
la toxina T-2).  En cuanto a otros tricotecenos, son necesarias 
dosis mayores para producir un efecto irritante (NIV, 10 µg por 
zona).  Los tricotecenos citotóxicos, como la toxina T-2, producen 
necrosis del epitelio de las criptas intestinales y de los tejidos 
linfoides y hematopoyéticos, tras exposición oral o parenteral o 
por inhalación.  La exposición a tricotecenos citotóxicos como la 
toxina T-2 y el DAS va seguida de alteraciones hematológicas y 
coagulopáticas.  Las toxicosis graves  pueden  originar  
pancitopenia.  En  estudios  con  toxina T-2, DON y DAS se ha 
demostrado la supresión de la inmunidad de base celular y humoral, 
y entre los efectos observados figuran la reducción de las 
concentraciones de inmunoglobulinas y la disminución de la 
actividad fagocítaria de macrófagos y neutrófilos.  Los  resultados 
de estudios experimentales con animales han indicado que el efecto 
inmunodepresor de tricotecenos como la toxina T-2, el DAS y el DON 
origina una disminución de la resistencia a la infección secundaria 
por bacterias (micobacterias,  Listeria monocytogenes), levaduras 
 (Cryptococcus neoformans) y virus (virus del herpes simplex). 

    Se ha comunicado que la toxina T-2 es teratogénica en el ratón, 
cuando se administra por inyección intraperitoneal (vía de 
administración poco corriente en los estudios de la 
teratogenicidad).  Se ha comunicado que el DON es teratogénico en 
los ratones tras intubación gástrica, pero no en las ratas cuando 

la toxina se administra con los alimentos.  El NIV no es 
teratogénico en el ratón.  La toxina T-2, el DAS y el DON no 
resultaron mutagénicos en una prueba de tipo Ames.  En algunas 
pruebas, se observó una débil actividad clastogénica de la toxina 
T-2.  En los estudios publicados sobre la toxicidad a largo plazo 
en animales, no se obtuvieron datos que indiquen que la toxina T-2, 
el fusarenón-X o el NIV sean oncogénicos en los animales.  No se 
han publicado estudios a largo plazo sobre la toxicidad del DON. 

    Los tricotecenos son tóxicos para las células que se dividen 
activamente como las del epitelio de las criptas intestinales y las 
hematopoyéticas.  La citotoxicidad se ha asociado con el trastorno 
de la síntesis de proteínas debido al enlace de los compuestos con 
los ribosomas de las células eucarióticas o bien con la disfunción 
de las membranas celulares.  La inhibición de la síntesis de las 
proteínas se ha relacionado con la inducción de proteínas lábiles y 
reguladoras como la IL-2 en los inmunocitos.  Concentraciones 
extremadamente bajas de tricotecenos alteran el transporte de 
pequeñas moléculas en las membranas celulares. 

2.5  Efectos en el ser humano

    La ingestión de alimentos contaminados de origen vegetal es la 
principal vía de exposición a tricotecenos, pero ocasionalmente se 
han notificado otras, por ejemplo el contacto accidental con la 
piel entre los investigadores de laboratorio y la inhalación de 
tricotecenos transportados por el polvo. 

    Los casos registrados de enfermedad asociados con la exposición 
a tricotecenos son escasos y en ninguno de ellos se ha demostrado 
la relación causal.  No obstante, los dos brotes que se describen a 
continuación parecen indicar su existencia. 

    En China se registró un brote de enfermedad asociado con el 
consumo de trigo mohoso que contenía de 1,0 a 40,0 mg de DON/kg.   
La enfermedad se caracterizó por síntomas gastro-intestinales.  No 
hubo defunciones.  Los cerdos y los pollos alimentados con restos 
de cereales también resultaron afectados. 

    Un brote análogo se registró en la India, asociado al consumo 
de pan hecho con trigo contaminado.  La enfermedad se caracterizó 
por síntomas gastrointestinales e irritación de la garganta, que 
aparecía de 15 minutos a una hora después de la ingestión del pan.  
En muestras de la harina de trigo refinada utilizada para su 
preparación se detectaron las siguientes micotoxinas: DON  
(O,35-8,3 mg/kg),  acetildeoxinivalenol (0,64-2,49 mg/kg), 
NIV (0,03-0,1 mg/kg) y toxina T-2 (0,5-0,8 mg/kg).  Sin 
embargo, no hubo confirmación de la identidad de los tricotecenos 
detectados.  La presencia simultánea de DON y NIV y de toxina T-2 
es insólita. 

    Dos enfermedades de interés histórico, la aleucia tóxica 
alimentaria (ATA) de la URSS y la toxicosis del trigo mohoso del 
Japón y Corea se han asociado con el consumo de cereales invadidos 
por hongos  Fusarium.  Desde entonces se han identificado en 

condiciones de laboratorio algunos tricotecenos en cultivos de 
mohos  Fusarium aislados en cereales encontrados en los incidentes.  
En la época en que apareció la enfermedad, no pudieron realizarse 
estudios que relacionaran la ATA o la toxicosis de los cereales 
mohosos con la exposición a tricotecenos porque las toxinas no eran 

3.  Cornezuelo

3.1  Distribución natural

    Cornezuelo es el nombre dado a los esclerocios de especies de 
hongos pertenecientes al género  Claviceps.  Alcaloides biológi-
camente activos contenidos en el esclerocio provocan toxicosis 
cuando éste es consumido por hombres o animales en alimentos 

    Los alcaloides del cornezuelo producen dos modalidades 
diferentes de enfermedad, según el hongo de que se trate ( C. 
 purpurea, C. fusiformis) y, por lo tanto, los alcaloides 
producidos.  El ergotismo, provocado por los alcaloides ergotamina- 
ergocristina producidos por  C. purpurea, se caracteriza sobre 
todo por gangrena de las extremidades, además de síntomas 
gastrointestinales.  La intoxicación resultante de la ingestión de 
mijo contaminado por  C. fusiformis se caracteriza principalmente 
por síntomas gastrointestinales y está relacionada con los 
alcaloides de tipo clavina.  No hay signos ni síntomas que indiquen 
la presencia de vaso-oclusión. 

3.2  Métodos de análasis

    Los alcaloides del cornezuelo (ergolinas) son derivados del 
ácido lisérgico.  Los diversos alcaloides difieren por la 
importancia de su actividad biológica.  La presencia de alcaloides 
del cornezuelo producidos por  C. purpurea se ha determinado por 
cromatografía de fase líquida de alto rendimiento con detección por 
fluorescencia.  Pueden medirse concentraciones de 0,2 µg de 
ergolina por litro de plasma humano.  La presencia de ergotamina y 
ergocristina puede determinarse con gran especificidad mediante 
pruebas de radioinmunovaloración en concentraciones de 3,5 
picomoles y 0,8 picomoles, respectivamente. 

3.3  Efectos en animales

    Las ergolinas, principalmente la ergotamina y la ergotaminina, 
se han asociado con brotes de abortos en el ganado bovino.  Ovejas 
a las que se administró ergotamina por vía oral enfermaron 
rápidamente y se observó en ellas inflamación intestinal.  Aves de 
corral, cerdos y primates expuestos por vía oral experimentaron 
efectos leves.  El Grupo Especial no dispuso de datos sobre la 
mutagenicidad, la teratogenicidad y la carcinogenicidad de las 

3.4  Efectos en el ser humano

    Los cereales infectados por  Claviceps son fuente de exposición 
humana a las ergolinas.  En la mayor parte de los estudios 
toxicológicos, no se han identificado los alcaloides específicos.  
La información resultante de un solo estudio sobre cereales y 
productos  de  cereales  indica  que  en  Suiza  la  ingesta  total  
de ergolinas por los seres humanos es de alrededor de 5,1 µg por 
persona, siendo el contenido de ciertos productos de hasta 140  
µg/kg.  La cocción reduce las ergolinas presentes en la harina 
contaminada un 25-100%. 

    En 1978 hubo en Etiopía un brote de ergotismo, resultante de 
exposición a ergolinas procedentes de esclerocios de  C. 
 purpurea.  El cereal contenía hasta un 0,75% de cornezuelo; se 
detectó específicamente ergometrina.  Los síntomas fueron gangrena 
seca, con pérdida de uno o varios miembros (29% de los casos), 
pulsos periféricos débiles o ausentes (36%) y desescamación de la 
piel.  Sólo hubo síntomas gastrointestinales en unos pocos casos.  
Trastornos de las extremidades inferiores se registraron en el 88% 
de los pacientes. 

    En la India ha habido varios brotes desde 1958 a consecuencia 
de la ingestión de mijo perlado que contenía cornezuelo de tipo 
clavina producido por  C. fusiformis.  Los síntomas comprendían 
náuseas, vómitos y mareos, y fueron causados por la ingestión de 
mijo perlado con un contenido de 15 a 26 mg de ergolina/kg. 

    Como en ninguno de los dos episodios se practicaron autopsias, 
no se dispone de información sobre los efectos anatomopatológicos 
en las vísceras humanas. 

4.  Evaluación de los riesgos para la salud humana

4.1  Ocratoxina A

    En diversos países de Europa se ha observado exposición humana 
a la ocratoxina A, como lo demuestra la presencia de ésta en 
alimentos y en la sangre.  El Grupo Especial no conoce ningún 
intento de detectar la ocratoxina A en la sangre humana en otras 
partes del mundo. 

    El papel causal de la ocratoxina A en la nefropatía porcina se 
ha demostrado sobre la base de estudios de casos sobre el terreno y 
de la reproducción de la enfermedad con ocratoxina A.  Utilizando 
el modelo porcino, se ha supuesto que la nefropatía endémica de los 
Balcanes podría obedecer a exposición a la ocratoxina A.  Los datos 
epidemiológicos de que se dispone indican que esa enfermedad podría 
estar asociada al consumo de alimentos contaminados por dicha 
toxina.  Desde la publicación de Criterios de salud ambiental 11 en 
1979, estudios epidemiológicos sobre concentración de ocratoxina A 
en la sangre humana en zonas afectadas y no afectadas han 
proporcionado un argumento más en favor de la relación entre la 
nefropatía de los Balcanes y la exposición a la ocratoxina A. 

    Se ha demostrado que tanto la presencia de ocratoxina A en la 
sangre como sus concentraciones son mayores en las personas que 
residen en las zonas de endemicidad.  No obstante, las pruebas 
indirectas que proporcionan los mencionados estudios retrospectivos 
no bastan para demostrar por sí solas la existencia de una relación 
causal directa.  Esta tampoco puede excluirse, dado el largo 
periodo de latencia entre la exposición y el comienzo de los 

    Se ha demostrado que la ocratoxina A es carcinogénica para el 
epitelio de los túbulos renales en los ratones machos y en las 
ratas de uno y otro sexo.  Se ha observado una relación muy 
significativa entre la nefropatía de los Balcanes y los tumores del 
tracto urinario, en particular de la pelvis renal y los ureteres.  
Sin embargo, no hay datos publicados que demuestren un papel causal 
directo de la ocratoxina A en la etiología de esos tumores. 

4.2  Tricotecenos

    Sobre la base de la información facilitada al Grupo Especial, 
es posible asociar la exposición a tricotecenos con episodios de 
enfermedad en los seres humanos.  Según los limitados datos 
disponibles, los tricotecenos que intervienen más frecuentemente en 
episodios de exposición humana son el DON y el NIV.  En los 
episodios de aleucia tóxica alimentaria y toxicosis por ingestión 
de cereales mohosos registrados en el pasado, no puede excluirse el 
posible papel etiológico de los tricotecenos.  La exposición se 
produce por la ingestión de alimentos contaminados, principalmente 
cereales.  La elaboración, la molienda y la cocción no logran la 
destrucción del DON, el NIV y la toxina T-2.  Los datos en favor de 
la exposición por inhalación son muy limitados, pero no puede 
excluirse esa posibilidad. 

    Entre los tricotecenos naturalmente presentes en alimentos, el 
más potente es la toxina T-2, seguida por el DAS y el NIV; el DON 
resultó el menos tóxico en los estudios de la toxicidad aguda.  En 
los animales experimentales, la toxina T-2 y el DAS tienen efectos 
generales agudos, con necrosis de tejidos epiteliales y supresión 
de la hematopoyesis.  En los brotes de enfermedad contemporáneos, 
sólo se han registrado síntomas gastrointestinales. 

    Se ha demostrado que el DON es teratogénico en los ratones pero 
no en las ratas.  Según los estudios de la toxicidad crónica 
publicados, el NIV y la toxina T-2 no son oncogénicos en los 
animales.  No se han publicado estudios sobre la carcino-genicidad 
a largo plazo del DON.  Ciertos tricotecenos, como la toxina T-2 y 
el DON, tienen una acción inmunosupresora en los animales y han 
producido alteraciones de la inmunidad, tanto de base celular como 
humoral.  No hay datos que demuestren la existencia de una acción 
inmunosupresora en el hombre. 

    Los casos de enfermedad humana asociados con la exposición a 
tricotecenos que se han notificado son limitados, tanto por su 
número como por la información proporcionada.  Tras la ingestión de 
alimentos contaminados con tricotecenos aparecen rápidamente 

síntomas de trastornos digestivos e irritación de la garganta.   En  
la actualidad  no  hay  nada  que  demuestre  que los tricotecenos 
causen cáncer en el ser humano.  El Grupo Especial no tuvo a su 
disposición informes sobre infección secundaria por bacterias, 
hongos o virus en los seres humanos tras la exposición a 
tricotecenos, como las observadas en estudios experimentales con 
animales.  Al parecer, no se han realizado estudios suficientes 
para aclarar esa sucesión. 

4.3  Cornezuelo

    La exposición humana a bajas concentraciones de ergolinas 
parece estar muy extendida.  Los datos de que se dispone sobre los  
recientes  brotes  en Etiopía y en la  India  indican  que  los 
alcaloides producidos por  C. purpurea (grupo de la ergotamina) 
tienen efectos más graves-entre ellos gangrena de las piernas y 
muerte-que los alcaloides producidos por  C. fusiformis (grupo de la 
clavina), que provocaron síntomas gastrointestinales sin desenlace 
mortal.  No se sabe si esas diferencias corresponden a diferencias 
en el contenido de alcaloides de las especies de hongos, en las 
propiedades toxicológicas o toxicométricas de los alcaloides o en 
las dosis ingeridas por diferentes tipos de poblaciones. 

    Cuando la limpieza y la molienda eliminan los esclerocios, sólo 
quedan en los alimentos preparados bajas concentraciones de 
ergolinas; la cocción y otras aplicaciones de calor destruyen 
también la mayor parte de los alcaloides del grupo de la 

5.  Recomendaciones para ulterior investigación

5.1  Recomendaciones generales

    Debe establecerse una red de centros de referencia, que ayuden 
a los Estados Miembros a confirmar la identidad de las mico-toxinas 
halladas en alimentos y tejidos humanos.  Esos centros deben 
proporcionar también muestras de referencia de micotoxinas, cuando 
se les solicite, a fin de aumentar las posibilidades de comparar 
los resultados de análisis realizados en distintas partes del 

5.2  Ocratoxina A

    a)  En los Balcanes y en la región del Mediterráneo deben 
        realizarse estudios epidemiológicos retrospectivos extensos 
        y prospectivos focales sobre la asociación de la ocratoxina 
        A con la  nefropatía  endémica  de tipo balcánico y  con  
        tumores  del tracto urinario. 

    b)  Fuera de los Balcanes, deben realizarse, en enfermos con 
        tumores del tracto urinario, análisis de sangre para 
        detectar la presencia de ocratoxina A. 

    c)  En países no situados en la zona de los Balcanes, debe 
        aclararse la fuente de la exposición a ocratoxina A, cuando 
        los análisis de sangre humana indiquen que la misma existe. 

    d)  Debe aclararse la manera en que actúa la diferencia de sexo 
        en las enfermedades renales, neoplásicas y no neoplásicas, 
        causadas por la ocratoxina A en animales de 

    e)  Son necesarios extensos estudios sobre el contenido de 
        ocra-toxina A de los alimentos en distintas partes del 
        mundo.  Es especialmente importante realizar estudios de 
        ese tipo en las regiones con una elevada incidencia de 
        tumores del tracto urinario, tumores renales o nefropatías. 

5.3  Tricotecenos

    a)  Deben realizarse estudios de seguimiento en las zonas de la 
        India y de la República Popular China en que ha habido 
        recientemente episodios de intoxicación de seres humanos 
        por tricotecenos.  Debe aclararse más la modalidad no usual 
        de presencia de tricotecenos en esos episodios. 

    b)  Deben estudiarse los efectos de la exposición a largo plazo 
        de animales de experimentación al DON, incluidos los 
        efectos carcinogénicos.  Como la respuesta de distintas 
        especies al DON varía mucho, deben elegirse cuidadosamente 
        las especies utilizadas para el estudio. 

    c)  Debe aclararse más la aparición de infecciones microbianas 
        secundarias en animales de experimentación tras la 
        exposición a tricotecenos. 

    d)  Debe estudiarse la influencia de las condiciones 
        ambientales, incluida la presencia de insecticidas y de 
        otros productos químicos artificiales, en la producción de 
        tricotecenos por los hongos. 

    e)  Deben aclararse los efectos de la elaboración de los 
        alimentos en los tricotecenos. 

    f)  Deben desarrollarse, mediante métodos biotecnológicos, 
        cultivos agrícolas resistentes a la infección por hongos 
        productores de tricotecenos. 

    g)  Deben estudiarse en animales de experimentación los 
        posibles efectos sinérgicos de la exposición combinada a 
        tricotecenos, aflatoxinas, ocratoxina A y otras 

    h)  Deben realizarse estudios sobre la ingesta de tricotecenos 
        por seres humanos. 

    i)  Deben elaborarse métodos rápidos y sensibles de detección 
        de los tricotecenos y deben realizarse estudios para 
        localizar los tricotecenos en cereales y alimentos tratados 
        en las zonas templadas. 

5.4  Cornezuelo

    a)  Deben hallarse métodos de análisis que permitan detectar la 
        presencia de agroclavinas. 

    b)  Debe proporcionarse a los países en desarrollo información 
        sobre el empleo de procedimientos de detección de semillas 
        patológicas y de molienda para reducir al mínimo los 
        problemas causados por el cornezuelo. 

    c)  Deben realizarse estudios epidemiológicos sobre los 
        posibles efectos de bajas concentraciones de ergolinas en 
        la población humana. 

    d)  Deben efectuarse estudios farmacológicos y toxicológicos en 
        los que se administren a animales de experimentación 
        distintas ergolinas, aisladas y en combinación. 

    e)  Debe aclararse la posible trasmisión de ergolinas a los 
        lactantes a través de la leche materna. 

    See Also:
       Toxicological Abbreviations