INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
SAFETY EVALUATION OF CERTAIN FOOD
ADDITIVES AND CONTAMINANTS
WHO FOOD ADDITIVES SERIES: 44
Prepared by the Fifty-third meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
World Health Organization, Geneva, 2000
IPCS - International Programme on Chemical Safety
ZEARALENONE
First draft prepared by G.S. Eriksen1, J. Pennington2 & J.
Schlatter3 with contributions from J. Alexander4 & A. Thuvander5
1 Swedish University of Agricultural Sciences, Uppsala, Sweden; 2
National Institute of Health, Bethesda, United States; 3 Swiss
Federal Office of Public Health, Zürich, Switzerland; 4 National
Institute of Public Health, Oslo, Norway; and 5 National Food
Administration, Uppsala, Sweden
Explanation
Biological data
Biochemical aspects
Absorption, distribution, and excretion
Biotransformation
Effects on enzymes and other biochemical parameters
Toxicological studies
Acute toxicity
Short-term studies of toxicity
Long-term studies of toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Developmental toxicity
Special studies
Hormonal effects
Immune responses
Macromolecular binding
Genotoxicity of metabolites
Observations in humans
Occurrence and intake
Incidence and concentration of contamination
Variables that affect contamination
Weather and climate
Agricultural production methods
Varieties and cultivars
Storage conditions
Gamma irradiation
Grain preservatives and disinfectants
Food processing, preparation, and cooking
Residues in animal tissues
Regulation, control, and monitoring
Dietary intake
Estimates for Canada, 1987
Estimates for Canada, 1999
Estimates for Denmark, Finland, Norway, and Sweden
Estimates for the United States, 'eaters only'
Estimates for the United States, all persons
Limitations of estimates
Models of dietary intake
Comments
Evaluation
References
1. EXPLANATION
Zearalenone is a non-steroidal estrogenic mycotoxin produced by
several Fusarium spp. It has been implicated in numerous
mycotoxicoses in farm animals, especially in pigs. Zearalenone is
heat-stable and is found worldwide in a number of cereal crops, such
as maize, barley, oats, wheat, rice, and sorghum (Kuiper-Goodman et
al., 1987; Tanaka et al., 1988a) and also in bread (Aziz et al.,
1997). Zearalenone was shown to be produced on corn by Fusarium
isolates from Australia, Europe, and North America (Vesonder et al.,
1991) and in New Zealand (diMenna et al., 1997), the Philippines,
Thailand, and Indonesia (Yamashita et al., 1995). The occurrence of
zearalenone in food and feed was also demonstrated in South America
(Dalcero et al., 1997; Molto et al., 1997), Africa (Doko et al.,
1996), China and the former USSR (Ueno et al., 1986). Fusarium
isolates from bananas can also produce zearalenone (Jiménez et al.,
1997).
Zearalenone has not been evaluated previously by the Committee,
although a mammalian metabolite, alpha-zeralanol (zeranol), was
considered by the Committee at its twenty-sixth, twenty-seventh, and
thirty-second meetings (Annex 1, references 59, 62, and 80) for
use as a veterinary drug. The Committee allocated an ADI of 0-5 µg/kg
bw at the last meeting.
The chemical structures of zearalenone and some of its
metabolites are shown in Figure 1.
The concentrations in food and feed vary over a wide range,
depending on climatic conditions. Zearalenone was found in 11-80% of
samples of wheat and 7-68% samples of barley for feed use collected
randomly in south-west Germany in 1987 and 1989-93, with mean yearly
contents of 3-180 µg/kg in wheat (highest value, 8000 µg/kg) and 3-36
µg/kg in barley (highest value, 310 µg/kg) (Müller et al., 1997a,b).
Wheat for human consumption was collected from all regions of Bulgaria
(140 samples) after harvest in 1995, a year characterized by heavy
rainfall in spring and summer. The frequency of contamination with
zearalenone was 69%, with an average concentration in positive samples
of 17 µg/kg and a maximum of 120 µg/kg (Vrabcheva et al., 1996).
Zearalenone was found in 30% of 2271 maize samples collected in Buenos
Aires and Santa Fe provinces of Argentina in 1983-94, at an average
concentration of 165 µg/kg (yearly variation, 46-300 µg/kg) and a
maximum of 2000 µg/kg (Resnik et al., 1996). The concentrations in rye
and wheat produced by alternative or ecological methods were higher
than those in crops grown conventionally. Zearalenone was found in 40
out of 201 grain samples, with average concentrations of 24 µg/kg in
wheat and 51 µg/kg in rye in alternatively produced crops and 6 µg/kg
in wheat and 4 µg/kg in rye in conventionally produced samples. The
highest concentration of zearalenone was 199 µg/kg, found in
alternatively grown rye (Marx et al., 1995).
Zearalenone can be excreted into milk after lactating cows are
fed it in high doses. The maximum concentrations in the milk of one
cow given an oral dose of 6000 mg zearalenone (equivalent to 12 mg/kg
bw), 6.1 µg/L zearalenone, 4 µg/L alpha-zearalenol, and 6.6 µg/L
beta-zearalenol were found. Neither zearalenone nor its metabolites
were found in the milk (< 0.5 µg/L) of three lactating cows fed 50 or
165 mg zearalenone (equivalent to 0.1 and 0.33 mg/kg bw) for 21 days
(Prelusky et al., 1990). Zearalenone may be transmitted from
contaminated grains into beer at various stages of the brewing
process. Although high incidences (up to 58%) and high concentrations
of zearalenone have been found in beers brewed locally in Africa
(Nigeria, < 2 mg/L; Swaziland, < 53 mg/L; Zambia, < 4.6
mg/L), zearalenone and alpha-or beta-zearalenol have not been found in
Canadian, European, or Korean beers with the exception of one French
beer, which contained 100 µg/L (Okoye, 1987; Scott, 1996; Shim et al.,
1997).
A detailed review of 339 publications on zearalenone, including
physico-chemical data, isolation and purification, analytical methods,
mycology, laboratory and natural production, occurrence and stability
in foods and feeds, and toxicity is available (Kuiper-Goodman et al.,
(1987). The present monograph therefore covers only literature
published since 1986, with the inclusion of older publications when
they were considered highly relevant for the evaluation or when no
newer data were found.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
Most investigations of the distribution of zearalenone have
focused on tissue residues and metabolism rather than on
pharmacokinetics, and few data are available on kinetic parameters
such as absorption and biological half-life.
Both intestinal mucosa and gut microflora from pigs metabolize
zearalenone to alpha-zearalenol and to the glucuronides of both
compounds (Olsen et al., 1987; Kollarczik et al., 1994). Healthy human
intestinal microflora cultured in a continous flow system were unable
to degrade zearalenone (Akiyama et al., 1997).
Zearalenone is rapidly absorbed after oral administration.
Although the degree of absorption is difficult to measure owing to
extensive biliary excretion, it appears to be extensively absorbed in
rats, rabbits, and humans (reviewed by Kuiper-Goodman et al., 1987).
The uptake in a pig after a single oral dose of 10 mg/kg bw was
estimated to be 80-85% (Biehl et al., 1993).
When zearalenone dissolved in an isotonic solution was perfused
into the small intestine of rats, the concentration decreased rapidly,
only 4.5% remaining in the small intestine 20 min after injection. The
disappearance of zearalenone from the small intestine followed
first-order kinetics, with an average absorption rate constant of 9.3
per h (Ramos et al., 1996). Zearalenone and its metabolites were found
in the plasma of a pig < 30 min after the beginning of feeding
(Kuiper-Goodman et al., 1987; Olsen et al., 1991; Biehl et al., 1993).
Studies with radiolabelled zearalenone in mice showed that it is
distributed to estrogen target tissues such as the uterus,
interstitial cells of the testes, and ovarian follicles. Some
radiolabel was also found in adipose tissues, indicating that storage
in fat may take place (Kuiper-Goodman et al., 1987).
Zearalenone and its metabolites are excreted mainly in the bile
in most animal species except rabbits, in which urine is the main
route. Most of an administered dose is excreted within 72 h
(Kuiper-Goodman et al., 1987). The biological half-life of total
radiolabel in immature pigs given a single intravenous dose of 5 mg/kg
bw or a single oral dose of 10 mg/kg bw of radiolabelled zearalenone
was estimated to be 87 h. When the bile was removed through a cannula,
the half-life was reduced to 3.3 h. The authors attributed this
difference to enterohepatic cycling of zearalenone in intact pigs. In
pigs from which bile had been removed, about 46% of the total
radiolabel was recovered in the bile, which was a significantly higher
percentage than that recovered in the faeces of intact pigs treated
intravenously (6.6%) or orally (22%; p < 0.05). The concentration
of radiolabel in plasma declined in a multiphasic manner in intact
animals. In pigs exposed intravenously or orally, an initial
absorption and distribution phase was followed by a reduced plasma
concentration, a second maximal concentration, and an extended
elimination phase. No secondary peak or extended elimination phase was
observed in animals from which bile had been removed. In the pigs
dosed orally, 45% of the administered dose was recovered in the urine
during the first 48 h, 22% was recovered in the faeces, and the total
accumulated recovery in urine and faeces after 48 h was 67% (Biehl et
al., 1993).
No effect of dose was found on the routes of excretion of
zearalenone in Sprague-Dawley rats after a single oral dose of 1 or
100 mg/kg bw (Fitzpatrick et al., 1988).
The concentrations of zearalenone, alpha-zearalenol, and
beta-zearalenol in the urine of a male volunteer 6, 12, and 24 h after
a single oral dose of 100 mg zearalenone were 3.7 and 3 µg/ml and not
detected after 6 h; 6.9, 6, and 2.7 µg/ml after 12 h; and 2.7, 4 and 2
µg/ml after 24 h. As the total recovery of zearalenone in faeces was
not reported, the study gives no information on the relative
importance of different routes of excretion in humans (Mirocha et al.,
1981).
FIGURE 8;V44je194.BMP
In a study with the closely related compound alpha-zearalanol,
the peak fraction of the dose appearing in human plasma was several
times higher than in female rats, rabbits, dogs, and monkeys.
alpha-Zearalanol disappeared much more slowly from the blood of humans
and rabbits, the two species that excreted the compound mainly in
urine, than from that of the other species studied (Midgalof et al.,
1983).
2.1.2 Biotransformation
The main metabolites of zearalenone are alpha-and beta-zearalenol
and the glucuronide conjugates of both the parent compound and its
metabolites. In rat liver homogenate, rat microsomes, and rat
hepatocytes in vitro and in rats in vivo after oral exposure, most
of an administered dose of zearalenone is found as free zearalenone or
its glucoronide conjugate, and only small amounts of the zearalenols
and their conjugates are formed. Rat whole blood and erythrocytes can
metabolize zearalenone to alpha-zearalenol (Kuiper-Goodman et al.,
1987).
In gilts given zearalenone in feed, the concentrations of
alpha-zearalenol in plasma exceeded those of zearalenone in some
studies, while the concentra-tions of the parent compound exceeded
those of alpha-zearalenol in others (Bauer et al., 1987;
Kuiper-Goodman et al., 1987). In some studies, all of the zearalenone
detected in pigs was in the form of conjugated metabolites, while free
zearalenone was also found in others. A significant fraction of the
zearealenone in the urine of rabbits and pigs is in the form of
alpha-zearalenol or its glucuronide conjugate (Kuiper-Goodman et al.,
1987; Biehl et al., 1993).
In a comparative study of the metabolism of zearalenone,
significant differences between species were found in the metabolic
profile in urine and faeces. A higher proportion of the administered
zearalenone was metabolized to alpha-zearalenol in pigs than in rats
or cows. In both humans and pigs, zearalenone was found mainly as
glucoronide conjugates of zearalenone and alpha-zearalenol in urine.
All of the metabolites found in humans during the 24 h of sampling
were glucuronides (Mirocha et al., 1981).
The metabolic profile of zearalenone in Sprague-Dawley rats was
similar after a single oral dose of 1 or 100 mg/kg bw (Fitzpatrick et
al., 1988).
Further reduction of the C11-C12 double-bond leading to alpha-and
ß-zearalanol was demonstrated in sheep in vivo in a study in which gas
chromatography with mass spectrometry was used to determine
zearalenone and its metabolites. The authors suggested that the
failure to detect zearalanols in other species may be due to the use
of high-performance liquid chromatography with fluorescence detection
in those studies, as that method is much less sensitivity for
zearalanols than for the fluorescent zearalenols, reduction of the
C11-C12 double-bond leading to loss of fluorescence (Miles et al.,
1996).
Formation of alpha-zearalanol in bile in vivo has been
demonstrated by gas chromatography with mass spectrometry in cattle
given 10 mg of either zearalenone or alpha-zearalenol by gavage
(Kennedy et al., 1998).
2.1.3 Effects on enzymes and other biochemical parameters
Groups of 10 female Wistar rats and 20 controls received a single
intraperitoneal injection of zearalenone dissolved in sterile olive
oil at 0, 1.5, 3, or 5 mg/kg bw. The haematological parameters studied
48 h later that differed in treated and control groups were a lower
number of platelets and higher haematocrit and mean corpuscular volume
in treated animals; the leukocyte and haemoglobin counts were higher
in the groups given the two higher doses than in controls. The other
parameters studied--erythrocyte count, mean erythrocyte haemoglobin
concentration and mean haemoglobin--were not affected. The
concentration of creatinine in serum decreased, whereas the total and
conjugated bilirubin concentrations and alanine aminotransferase,
aspartate aminotransferase, and alkaline phosphatase activities were
all increased over control values. The authors concluded that the
observed changes indicated hepatic toxicity and and probably
impairment of blood coagulation processes (Maaroufi et al., 1996).
2.2 Toxicological studies
2.2.1 Acute toxicity
The results of studies of acute toxicity with zearalenone are
summarized in Table 1. When young female pigs were given single doses
of zearalenone orally in gelatine capsules at 0, 3.5, 7.5, or 11.5
mg/kg bw, vulva vaginitis and enlarged reproductive tracts were
observed in all animals one week after dosing (Farnworth & Trenholm,
1981).
2.2.2 Short-term studies of toxicity
Mice
Groups of 10 B6C3F1 mice of each sex were fed diets containing
zearalenone at 0, 30, 100, 300, 1000, or 3000 mg/kg of diet,
equivalent to 0, 4.5, 15, 45, 150, or 450 mg/kg bw per day, for 13
weeks. Two of the female mice fed 3000 mg/kg of diet died. The weight
gain of male mice receiving doses of > 300 mg/kg of diet was
depressed by 14% or more. Atrophy of the seminal vesicles and testes
and cytoplasmic vacuolization of the adrenals were found in males fed
1000 or 3000 mg/kg of diet, and squamous metaplasia of the prostate
was observed in males fed 3000 mg/kg. Endometrial hyperplasia of the
uterus was seen in all groups of treated females, but the incidence
was not dose-related. Osteoporosis was observed in animals of each sex
fed doses of > 100 mg/kg of diet, and myelofibrosis of the bone
marrow was seen in mice fed > 300 mg/kg of diet (National
Toxicology Program, 1982).
Diets containing zearalenone at 0 or 10 mg/kg (equivalent to 0 or
1.5 mg/kg bw per day) were fed to weanling female B6C3F1 mice (26
control and 8 exposed animals) for eight weeks, resulting in a total
intake of 2.2 mg/animal in the treated group. No differences between
treated animals and controls were seen in body-weight gain or feed
intake. Gross and histopathological evaluation of the thymus, spleen,
liver, kidney, uterus, small intestine, colon, heart, brain, lungs,
and bone marrow showed no alterations due to zearalenone, and the
organ weights of treated and control animals were similar.
Haematological examination revealed a statistically significant
increase (p < 0.01) in the number of erythrocytes in treated animals,
while other parameters were unchanged (Forsell et al., 1986).
In a study of the detoxification of zearalenone with
cholestyramine, groups of 12 female ICR mice, 15 days old and caged
four by four, were given diets containing zearalenone at 0 or 6 mg/kg,
equivalent to 0 or 0.9 mg/kg bw per day. After five days, the relative
weight of the uterus was higher in treated mice (p < 0.01) than in
controls (Underhill et al., 1995).
Rats
Groups of 9 or 10 Fischer 344/N rats of each sex were fed diets
containing 0, 30, 100, 300, 1000, or 3000 mg/kg zearalenone,
equivalent to 0, 3, 10, 30, 100, or 300 mg/kg bw per day, for 13
weeks. No treatment-related deaths occurred. Weight gain was depressed
by more than 17% in rats of each sex receiving doses > 100 mg/kg in
the feed. Atrophy of the seminal vesicles and fibromuscular
hyperplasia of the prostate were observed in rats fed 1000 or 3000
mg/kg zearalenone, and ductular hyperplasia of the mammary gland was
observed in animals of each sex at the highest dose. Endometrial
hyperplasia of the uterus was seen in rats fed > 100 mg/kg of diet.
Hyperplasia of the pituitary was seen in both males and females at the
two higher doses and in 1/10 females fed 100 mg/kg of diet.
Osteoporosis was observed in males at the two highest doses and in all
treated females (National Toxicology Program, 1982).
Rabbits
Groups of six four-month-old rabbits were given zearalenone in
the diet at concentrations of 0, 0.5, or 1 mg/kg of feed, equivalent
to 0, 0.15, or 0.03 mg/kg bw per day, and groups of six
eight-month-old animals were given 0, 1, or 4 mg/kg of feed,
equivalent to 0, 0.03, or 0.12 mg/kg bw per day, for 18 days. Some of
the treated animals died during the study. Histopathological
alterations due to zearalenone were observed in the liver, kidney,
lungs, heart, adrenal glands, spleen, and uterus of the
eight-month-old but not the four-month-old animals. The
histopathological alterations were not described quantitatively, and
the number of rabbits surviving to the end of the study was not
reported. The four-month-old rabbits showed a treatment-related
increase in body-weight gain, food and water consumption, haemoglobin
percentage, packed cell volume, and serum concentrations of calcium,
phosphorus, and vitamin C, but the eight-month-old animals showed
treatment-related decreases in these parameters. No explanation was
given for the differences in observed effects (Abdelhamid et al.,
1992).
Table 1. Results of studies of the acute toxicity of zearalenone
Species Sex Route LD50 Reference
(mg/kg bw)
Mouse M/F Oral > 2 000 National Toxicology
Program (1982)
Mouse F Oral > 20 000 Hidy et al. (1977)
Mouse F Intraperitoneal > 500 Hidy et al. (1977)
Rat M/F Oral > 4 000 National Toxicology
Program (1982)
Rat M/F Oral > 10 000 Hidy et al. (1977)
Rat M Intraperitoneal 5 500 Hidy et al. (1977)
Guinea-pig F Oral > 5 000 Hidy et al. (1977)
Guinea-pig F Intraperitoneal 2 500 Hidy et al. (1977)
M, male; F, female
Pigs
Two female pigs were fed diets containing zearalenone at 0.25
mg/kg of diet, equivalent to 10 µg/kg bw per day, for 11 days and then
feed without zearalenone for 5 days, and two other female pigs were
fed diets containing zearalenone at 0.05 mg/kg of diet, equivalent to
2 µg/kg bw per day, for 21 days; one pig was used as a control.
Treatment with 10 µg/kg bw per day resulted in redness and swelling of
the vulva, swelling of the mammary glands, and numerous vesicular
follicles and some cystic follicles on the ovaries. With the low dose,
no external changes were seen at the end of the experimental period,
but autopsy showed a greater number of vesicular follicles on the
ovaries in treated than in control animals (Bauer et al., 1987). The
Committee noted that the small number of animals used rendered this
study unsuitable for evaluating the toxicity of zearalenone.
Groups of 10 Yorkshire gilts of an average age of 70 days were
given diets containing zearalenone at a concentration of 2 mg/kg of
diet (equivalent to 0.08 mg/kg bw per day) for the first two weeks and
then 1.5 mg/kg of diet (equivalent to 0.06 mg/kg bw per day) for the
remainder of two identical studies for 0 (control), 45, or 90 days.
The feed was naturally infected, and no information was provided on
the presence of other mycotoxins. Vulvar swelling and reddening were
seen within seven days of exposure, but no difference was seen between
treated and control animals in body weight or depth of back fat
(Rainey et al., 1990).
Zearalenone mycotoxicosis in suckling piglets of each sex,
characterized by oedematous swelling and reddening of the vulva and
sometimes associated with reddening and/or necrosis of the tail, was
described in a case report. No signs of hyperestrogenism were seen in
sows given feed contaminated with zearalenone at 3-24 mg/kg,
equivalent to 0.1-1 mg/kg bw per day. Clinical signs usually appeared
in the prenatally exposed piglets two to three days after birth but
were apparent at birth in a few animals. The authors noted the poor
hygienic conditions of the breeding unit (Dacasto et al., 1995).
Six-week-old female pigs given Fusarium culmorum extracts
containing 80 mg/kg of zearalenone (equivalent to 3.2 mg/kg bw per
day) and 5 mg/kg of deoxynivalenol (experimental details not reported)
showed pathological alterations in the reproductive tract (Palyusik et
al., 1990), but the lack of details and multiple exposures made this
study unsuitable for evaluating the toxicity of zearalenone.
Ruminants
Seventy-one dairy cows and 25 replacement heifers were
accidentally given feed contaminated with 1.5 mg/kg of zearalenone and
1 mg/kg of deoxy-nivalenol for approximately 90 days. The daily feed
rations were 7-10 kg for the cows and 1-2 kg for the heifers. Episodes
of estrus were seen in most of the animals, starting about one week
after the onset of exposure. Mammary development occurred in the
prepubertal heifers, which were subsequently culled from the herd
because of sterility (Coppock et al., 1990). The Committee considered
this study to be of little value as it was not controlled, the dose
received per body weight is not clear, and there was exposure to
multiple mycotoxins.
2.2.3 Long-term studies of toxicity and carcinogenicity
Mice
Groups of 50 male and 50 female B6C3F1 mice, seven weeks old,
were fed diets containing zearalenone (purity, > 99%) at a
concentration of 0, 50, or 100 mg/kg of diet (maximum tolerated dose)
for 103 weeks. The average daily feed consumption as a percentage of
that of controls was 99% for males at the low dose, 97% for males at
the high dose, and 97% for females at both doses. The daily intake of
zearalenone was approximately 0, 8, and 17 mg/kg bw for males and 0,
9, and 18 mg/kg bw for females. No significant difference in survival
was seen between groups, and 64-88% of the mice survived to
termination of the study. No dose-related changes in body-weight gain
were seen. No treatment-related non-neoplastic lesions were found in
male mice, but females had estrogen-related effects in several
tissues, including fibrosis in the uterus and cystic ducts in mammary
glands, and myelofibrosis in the bone marrow. Hepatocellular adenomas
were found in 4/50 male controls, 3/50 at the low dose, and 7/49 at
the high dose and in 0/50 female controls, 2/49 at the low dose, and
7/49 at the high dose, the last of which was statistically
significantly different ( p < 0.006) from the incidence in the
control group. The incidence of hepatocellular adenomas in untreated,
historical control female B6C3F1 mice was 14/498. Statistically
significant trends in the incidence of pituitary adenomas were
observed for both males (control, 0/40; low dose, 4/45; high dose
6/44; p < 0.022) and females (control, 3/46; low dose, 2/43; high
dose, 13/42; p < 0.001), and the increased incidence was
statistically significant in males ( p < 0.032) and females
( p < 0.003) at the high dose. Pituitary carcinomas were found in
one male at the low dose and in two females at the high dose, but the
incidence of pituitary carcinomas was not statistically significantly
different in treated and control animals. The incidence of pituitary
adenomas and carcinomas in untreated historical controls at the
institute that conducted the study was 21/428 in females and 0/399 in
males (National Toxicology Program, 1982).
Rats
Groups of 90 FDRL Wistar rats of each sex and 140 rats of each
sex in the control group were fed diets containing zearalenone at
doses of 0, 0.1, 1, or 3 mg/kg bw per day from approximately 28 days
of age for 104 weeks. In order to maintain the appropriate daily
doses, the concentration of zearalenone in the diet was adjusted
weekly according to the body weights and food consumption measured in
the previous week. The rats were derived from F0 parents fed
equivalent concentrations for five weeks before mating and throughout
mating and gestation, but not during lactation. Zearalenone had no
effect on reproductive parameters in the parent generation, but
treated males of the F1 generation had a transient but significant
decrease in body-weight gain when compared with controls, although
this effect was not seen at the end of the study. No statistically
significant differences were seen among groups with respect to
haematological, clinical chemical, or urinary parameters measured in
10 animals per group sampled at weeks 13, 26, 65, and 104 or in the
remaining animals killed at weeks 108 (males) and 111 (females) after
the initiation of treatment. At the end of the study, significantly
increased liver weights were found in males and females exposed to 3
mg/kg bw, and the uterine weights were increased in females at the two
higher doses. Rats receiving the highest dose showed increased
trabeculation of the femur, but no histopathological changes were seen
and no treatment-related tumours were found (Becci et al., 1982a). The
Committee noted that survival rates and tumour incidences were not
reported.
Groups of 50 male and 50 female Fischer 344 rats, five weeks old,
were fed diets containing zearalenone (purity, > 99%) at 0, 25, or 50
mg/kg of diet (maximum tolerated dose) for 103 weeks. The average
daily feed consumption as a percentage of that of the controls was
102% for males at the low dose, 91% for males at the high dose, 96%
for females at the low dose, and 98% for females at the high dose. The
intake was estimated to be 1 mg/kg bw per day at the low dose and
about 2 mg/kg bw per day at the high dose. The mean body-weight gain
of treated rats was lower than that of controls, and the decreases of
19% in males and 11% in females at the high dose after 44 weeks of
exposure were dose-related. No significant difference in survival was
observed between groups, and 74-82% of the rats survived to
termination of the study. The non-neoplastic lesions observed were
inflammation of the prostate gland, testicular atrophy, cysts or
cystic ducts in mammary glands of males, an increased incidence of
hepatocellular cytoplasmic vacuolization in males, and an increased
incidence of chronic progressive nephropathy in animals of each sex.
Increased incidences of retinopathy and cataracts were observed in
males at both doses and in females at the low dose. No
treatment-related increase in tumour incidence was found. Male rats at
the low dose showed a significant ( p < 0.05) increase in the
incidence of pituitary adenomas but with no significant dose-related
trend. The combined incidence of pituitary adenomas and carcinomas
showed no indication of treatment-related change (National Toxicology
Program, 1982).
A working group convened by the International Agency for Research
on Cancer (IARC) in 1993 concluded on the basis of the three studies
described above that there was limited evidence for the
carcinogenicity of zearalenone in experimental animals (IARC, 1993).
2.2.4 Genotoxicity
The results of studies of the genotoxicity of zearalenone are
summarized in Table 2.
2.2.5 Reproductive toxicity
Mice
Newborn female C57BL/Crgl mice were injected subcutaneously with
1 µg of zearalenone daily for five days. Eight months after treatment,
25 of 34 treated mice and 3 of 33 control mice had no corpora lutea.
Treated mice also had dense collagen deposition in the uterine stroma,
56% of animals had no uterine glands, and 59% had squamous metaplasia
(Williams, B.A. et al., 1989).
Intraperitonal injection of 10-30 µg zearalenone to groups of
four to nine female ICR mice on days 1-3 or 1-5 after birth resulted
in delayed vaginal opening, persistent estrus in 60-80% of animals,
and sterility accompanied by thickening of the vaginal epithelium at
eight weeks of age. Vaginal opening was accelerated in animals given a
single dose of 30 µg on day 10 but was not affected in mice given the
same dose on day 1, 3, 5, or 8. The incidence of persistent estrus was
significantly increased in eight-week-old mice treated with
zearalenone on day 1, 3, or 5 but not in those treated on day 8 or 10
(Ito & Ohtsubo, 1994).
Rats
FDRL Wistar rats were given zearalenone at daily doses of 0, 0.1,
1, or 10 mg/kg bw in the diet. After four weeks of exposure, the F0
generation was bred to produce the F1a generation, and at sexual
maturity the F1a generation was bred to give the F2a generation.
The F0 and F1a generations were given zearalenone throughout
mating and gestation but not during lactation. Zearalenone reduced the
number of liveborn F1 pups per litter only at the highest dose while
the doses of 1 and 10 mg/kg bw per day reduced the number of liveborn
F2 pups per litter. Fertility was significantly decreased at the
highest dose in both the F1 and F2 generations. Feeding
zearalenone had no effect on the rate of survival of liveborn pups to
4 or 21 days of age (Becci et al., 1982b).
Guinea-pigs
In three experiments, groups of three or four pregnant
Murphy-Hartley guinea-pigs received diets containing zearalenone at 0,
7, 14, or 21 mg/kg bw per day on days 1-8 after mating (experiment 1),
0, 20, or 30 mg/kg bw per day on days 1-3, 4-5, or 6-8 after mating
(experiment 2), and 0, 60, or 90 mg/kg bw per day on days 4-5 after
mating (experiment 3). Blood samples were analysed for progesterone on
days 8, 15, and 21 in experiment 1 and on days 15 and 21 in the other
two experiments. All animals were killed with carbon monoxide on day
22 after mating and the numbers of corpora lutea and fetuses and fetal
length were determined. Histopathological examinations were made of
the ovary, both uterine horns, placenta, and fetuses from all pregnant
females. Only one of four animals receiving 21 mg/kg bw on days 1-8
after mating became pregnant 21 days after mating, while other animals
treated on those days became pregnant. No effect was seen on any
maternal parameters or on fetal development after exposure to 7 or 14
mg/kg bw per day on day 1-8 after mating. Three of five guinea-pigs
treated with zearalenone at 20 mg/kg bw per day and one of four given
30 mg/kg bw per day on days 1-3 after mating were found to be pregnant
on day 22. Female guinea pigs given 20 or 30 mg/kg bw per day on days
4-5 or 6-8 after mating and females treated with 60 or 90 mg/kg bw on
days 4-5 had normal pregnancies, and all of the observed differences
in progesterone concentrations between groups could be related to the
pregnancy of the animals (Long & Diekman, 1989).
Hamsters
Groups of six litters of neonatal golden Syrian hamsters received
zearale-none by subcutaneous injection at 0 (vehicle only) or 1 mg/pup
at two days of age, and one group was untreated. Vaginal opening was
accelerated in treated females, but administration of zearalenone did
not affect the age at first estrus or cycling in the females or the
mounting behaviour of males at 60-64 days of age. At 150 days of age,
the females were ovariectomized and a 25-mg pellet of testosterone was
implanted under the skin. At day 195, 67% of the exposed female
hamsters and only one of 30 control females mounted a sexually
receptive female. The mounting behaviour of the males was not affected
by zearalenone on days 225 and 280. The authors concluded that the
behaviour of females treated with zearalenone was masculinized but not
defeminized (Gray et al., 1985).
Pigs
Groups of 10 Yorkshire gilts of an average age of 70 days were
given diets containing zearalenone at a concentration of 2 mg/kg of
diet (equivalent to 0.08 mg/kg bw per day) for the first two weeks and
then 1.5 mg/kg (equivalent to 0.06 mg/kg bw per day) for the remainder
of two identical studies for 0 (control), 45, or 90 days. The feed was
naturally infected, and no information was provided on the presence of
other mycotoxins. Gilts treated with zearalenone reached puberty at a
younger age than controls, but the conception rates, ovulation rates,
and embryonic survival were not affected (Rainey et al., 1990).
Groups of six to eight prepubertal gilts of an average age of 178
days and weighing 94 kg were fed diets containing zearalenone at 0 or
10 mg/kg of diet, equal to 0.26 mg/kg bw per day, for two weeks in
three replicate experiments. Two weeks after withdrawal of the
zearalenone-containing diet, the gilts were exposed to boars for 15
min/day for three weeks. Blood samples were collected every 20 min for
4 h one week after the start of exposure and one week after withdrawal
of zearalenone. Blood samples were also taken twice a week and
analysed for progesterone to establish the age at puberty: no
difference was found between control and exposed animals. The mean
serum concentration of luteinizing hormone was reduced during exposure
to zearalenone, but no significant difference in serum concentrations
remained one week after the end of the exposure period. Zearalenone
did not change the frequency or amplitude of spikes of luteinizing
Table 2. Results of assays for genotoxicity with zearalenone
Test system Test object Concentration Results Reference
Reverse mutation S. typhimurium TA1535, 100 µg/platea Negative Kuczuk et al. (1978)
TA1537, TA1538
Reverse mutation S. typhimurium TA1535, TA1537, 400 µg/platea Negative Wehner et al. (1978)
TA98, TA100
Reverse mutation S. typhimurium TA1538, TA98, 50 µg/platea Negativeb Bartholomew & Ryan
TA100 (1980)
Reverse mutation S. typhimurium TA98, TA100 Not reporteda Negative Stark (1980)
Reverse mutation S. typhimurium TA1535, TA1537, 50 µg/platea Negativeb Ingerowski et al.
TA1538, TA98, TA100 (1981)
Reverse mutation S. typhimurium (strains not 1000 µg/platea Negative Tennant et al.
reported) (1987)
Reverse mutation S. typhimurium TA1535, TA1537, 1000 µg/platea Negative Mortelmans et al.
TA98, TA100 (preincubation) (1986)
Gene mutation S. typhimurium TA1535/pSK100 29.5 µg/L Negative Kasamaki & Urasawa
umu mutation (1993)
SOS repair E. coli C600 478 mg/L Positivec Ghedira-Chekir et al.
(1998)
SOS chromotest E. coli PQ37 30 mg/La Negative Krivobok et al. (1987)
Unscheduled DNA Rat hepatocytes 32 mg/L Negative Williams, G.M. et al.
repair (1989)
Point mutations/ S. cerevisiae D3 1000 µg/platea Negative Kuczuk et al. (1978)
mitotic recombination
Table 2. (continued)
Test system Test object Concentration Results Reference
Forward mutation Mouse lymphoma L5178Y 60 mg/La Negative McGregor et al. (1988)
Tk+/- cells
Forward mutation Mouse lymphoma L5178Y Tk+/- cells 65 mg/L Negative Tennant et al. (1987)
Chromosomal aberration Chinese hamster ovary cells 15 mg/L Positived,e Galloway et al. (1987)
50 mg/L Negativef
Sister chromatid Chinese hamster ovary cells 12.5 mg/L Positived,e Galloway et al. (1987)
exchange 40 mg/L Positivef
Chromosomal aberration Human fibroblasts (HAIN55 9.5 µg/L Weakly positive Kasamaki & Urasawa
and CPAE) (1993)
Chromosomal aberration Chinese hamster ovary cells 15 mg/L Positived, negativef Tennant et al. (1987)
Sister chromatid Chinese hamster ovary cells 12.5 mg/L Positived, negativef Tennant et al. (1987)
exchange
Chromosomal aberration Chinese hamster V79 cells 32 mg/L Negativea Thurst et al. (1983)
Sister chromatid Chinese hamster V79 cells 32 mg/L Negativea Thurst et al. (1983)
exchange
Cell cycle delay Chinese hamster V79 cells 32 mg/L Negativea Thurst et al. (1983)
Table 2. (continued)
Test system Test object Concentration Results Reference
Sister chromatid Human peripheral lymphocytes 3 mg/Lg Weakly positivea Kuiper-Goodman et al.
exchange (1987)
Gene mutation B. subtilis H17, M45 rec+/- 100 µg/disc Positiveh Ueno & Kubota (1976)
20 µg/disc Negative
a With and without metabolic activation
b Cytotoxic at next highest concentration
c 1-h preincubation with 6 mmol/L vitamin E prevented the effect.
d Without metabolic activation
e Tetraploidy and delayed cell cycle
f With metabolic activation
g Complete inhibition of DNA synthesis at 30 mg/L
h M45rec-, 2-3 mm and H17rec+, 0-1 mm growth inhibition at pH 6, 7, or 8
hormone secretion during exposure, and did not affect the numbers of
corpora lutea or live fetuses or the serum concentration of
follicle-stimulating hormone. Fetal weights were statistically
significant greater in gilts receiving zearalenone than in controls
(180 ± 8.4 g in treated and 150 ± 10 g in control animals) (Green et
al., 1990). The Committee noted a small difference in the age of
fetuses at examination (65 ± 2.6 days of gestation for exposed animals
and 63 ± 2.8 for controls) and that use of the usual conversion factor
for estimating dose from feed concentrations results in substantially
higher doses because of the assumption of a body weight of 60 kg. In
most studies in which the body weights of pigs are given, they are
approximately 100 kg. The dose used in this study would be 0.4 mg/kg
bw per day if the Committee's conversion factor were used.
Prepubertal gilts were fed a diet containing zearalenone at 0 or
10 mg/kg of diet ad libitum (equivalent to 0.4 mg/kg bw per day,
delivered dose not recorded) for 30 days from day 145 to day 193 of
age; they then received control diet and were exposed to a boar for 60
days. Vulvar swelling and reddening were observed throughout exposure
from day 3-5, but the symptoms disappeared slowly when zearalenone was
withdrawn. Exposed gilts had their first estrus significantly later
than controls, but there was no significant difference in the
proportion of animals reaching estrus within 60 days after withdrawal
of the zearalenone-containing feed. The length of the first estrus
cycle was not affected. In a second trial, sows were fed zearalenone
in the diet at 0 or 10 mg/kg of diet beginning 14 days before they
weaned their offspring and were then fed control diet and checked
daily for their first post-weaning estrus. The interval between
weaning and estrus was extended in sows given zearalenone, but the
incidence of pregnancy or farrowing and the numbers of liveborn and
dead pups per litter were not affected. No sign of hyperestrogenism
was observed in gilts or sows (Edwards et al., 1987b).
In sexually mature gilts given 2 kg of feed containing
zearalenone at 0, 1, 5, or 10 mg/kg of diet (equivalent to 0, 0.04,
0.2, and 0.4 mg/kg bw per day) on days 5-20 of estrus, the
inter-estrus interval increased significantly from 21 ± 0.3 days in
the control group to 29 ± 2.9 and 33 ± 3.3 days in gilts fed 5 and 10
mg/kg zearalenone in the diet. The inter-estrus interval was not
affected in gilts given 1 mg/kg in the diet. Increased plasma
concentrations of progesterone and prolonged maintenance of corpora
lutea were observed in the gilts with prolonged cycles. The corpora
lutea regressed when zearalenone was withdrawn from the diet (Edwards
et al., 1987a).
The frequency of pregnancy after mating was reduced in a pig
given zearalenone at a dose of 108 mg/animal, equal to 1.1 mg/kg bw,
in the diet daily on days 7-10 after mating, but not in a pig given
the same dose on days 2-6 or 11-15 after mating (Long & Diekman,
1986).
Groups of 16 pubertal gilts given zearalenone at 0, 3, 6, or 9
mg/kg of diet (equivalent to 0.12, 0.24, and 0.36 mg/kg bw per day)
from immediately after the first estrus throughout gestation showed an
increased incidence of pseudo-pregnancy, a decrease in breeding, and a
decrease in the number of liveborn per litter at the two higher doses.
Feeding of diets containing zearalenone to a limited (unspecified)
number of boars from 32 days to one year of age at 0, 3, 6, or 9 mg/kg
of diet (equivalent to 0.06, 0.12, and 0.18 mg/kg bw per day) had no
effect on growth rate, puberty, or libido, but there was an indication
of reduced sperm concentration and a small reduction in testicular and
epididymal weights (Young & King, 1984). The Committee noted the lack
of details.
Groups of 14-16 lactating Yorkshire × Landrace cross-bred gilts
were fed diets containing purified zearalenone at 0, 5, or 10 mg/kg of
diet (equivalent to 0, 0.2, or 0.4 mg/kg bw per day) in two trials
from day 7 of lactation until 40 days after the last breeding or until
40 days after weaning if no estrus was observed. The only difference
between the two trials was that the sows in trial 1 were first parity
and those in trial 2 were second parity. The sows were inseminated 8
and 30 h after observed estrus. Treatment with zearalenone did not
alter the proportion of sows returning to estrus, but the time from
weaning to estrus was significantly increased in trial 2, with a
similar trend at the highest dose in trial 1. The average number of
fetuses per pregnant sow decreased with increasing concentration of
zearalenone in trial 2 but not in trial 1. Embryonic mortality,
measured as the ratio of fetusus to corpora lutea in pregnant sows,
increased in trial 2 but not in trial 1. A trend to a lower incidence
of pregnancy was found at the end of trial 1, and there was great
variation in feed consumption among all groups in this trial, which
was attributed by the authors to the presence of 1.3-1.7 mg/kg
deoxynivalenol in the diet (Young et al., 1990). The Committee noted
that no information about Fusarium toxins other than zearalenone was
given for trial 2.
Groups of six mature cross-bred sows were fed diets containing
zearalenone at 0 or 2.1 mg/kg of diet, equivalent to 0.085 mg/kg bw
per day, from day 30 after mating throughout lactation. The piglets
were weaned and weighed at 21 days of age, and three male and three
female piglets from each group were kept for subsequent breeding and
maintained on control diet. Zearalenone had no effect on breeding
performance in the F0 generation, and no statistically significant
differences were observed between treated and control animals with
regard to parental body weight, litter size, number of livebirths per
litter, piglet sex ratio, birth weight, or weaning weight.
Furthermore, no significant differences were found in the ovarian or
uterine weights of sows in the F0 generation, although a trend to
ovarian atrophy and uterine enlargement was found. Sows in the control
group had numerous large follicles on the ovaries, while the ovaries
of the sows fed zearalenone contained only small, degenerated
follicles. No treatment-related histopathological alterations were
observed in the uterus or cervix of the sows. Examination of three
female piglets on day 21 resulted in similar findings: although they
had a slight, non-significant increase in age at first estrus, no
difference was found in the ovarian or uterine weights of surviving
female piglets. Male piglets had a small, non-significant reduction in
testicular weight at 21 days of age, but with no consistent
histopathological changes in the reproductive organs. The male
offspring of sows given zearalenone were significantly older at first
mount, but no differences were found in testicular weight at first
mount, terminal body weight, or the number of successful inseminations
of untreated sows (Yang et al., 1995).
2.2.6 Developmental toxicity
No teratogenic effect was found in CBA/Ca mice given single doses
of 5-20 mg/kg bw zearalenone by gavage on day 9 of gestation (Arora et
al., 1981, 1983). Zearalenone given at daily doses of 1-10 mg/kg bw by
gavage to female Wistar rats on days 6-15 of gestation caused minor
skeletal deformations, considered by the authors to be due to delayed
ossification (Ruddick et al., 1976).
In a two-generation study, FDRL Wistar rats were given
zearalenone in the diet at doses of 0, 0.1, 1, or 10 mg/kg bw per day
over both generations. After four weeks of exposure, the F0
generation was bred to produce the F1a generations, and rats of both
generations were given zearalenone throughout mating and gestation but
not during lactation. One week after weaning of the F1a generation,
the F0 generation was rebred. On day 20 of gestation, the F0
animals were killed, and the numbers of implants, resorptions, corpora
lutea, and viable fetuses were determined; fetuses were also examined
for gross abnormalities, weight, and sex. In females fed 10 mg/kg bw
per day, statistically significant reductions in the mean numbers of
viable offspring per litter, corpora lutea per dam, and implantations
per dam were found, with an increase in the number of resorptions per
dam on day 20 of gestation. At the highest dose, soft-tissue
abnormalities related to delayed fetal development were reported. At 1
mg/kg bw, minor skeletal abnormalities were observed which were
related to decreased growth. No unequivocal teratogenic effect was
found (Becci et al., 1982b).
Disruption of the development of growing blastocysts was observed
in vitro when zearalenone was added to a medium containing growing
mouse blastocysts or ovine oocytes (Long & Turek, 1989; Wallace &
Rajamahendran, 1993).
Three of four pigs given zearalenone in the diet at a dose of 110
mg/animal per day (equivalent to 1.1 mg/kg bw per day) on days 7-10
after mating did not become pregnant and had regressing corpora lutea,
while all animals dosed on days 2-6 or 11-15 became pregnant (Long &
Diekman, 1986). In mated pigs fed zearalenone at 1 mg/kg bw per day on
days 7-10 after mating, initial degeneration of blastocysts was
observed on day 11 and further degeneration and death on day 13
(Diekman & Long, 1989; Long et al., 1992). Changes in the intrauterine
environment, such as changes in the concentrations of Ca2+ or amino
acids, were observed in pigs exposed to zearalenone at 1 mg/kg bw per
day on days 7-10 after mating (Long et al., 1988).
In nine New Zealand white rabbits weighing 3-4 kg which were
given zearalenone orally at a dose of 12 mg/kg bw per day for 10 days,
the compound was detected in uterine tubal fluid from day 1.
Zearalenone caused an increase in the amount of intrauterine fluid,
reduced its pH, and changed the concentrations of various amino acids
and trace minerals. After the rabbits had been mated with untreated
males on the last day of exposure, no gross abnormalities were found
in fetuses examined 28-30 days after mating (Osborn et al., 1988).
In three experiments, groups of three or four pregnant
Murphy-Hartley guinea-pigs were given zearalenone orally at a dose of
0, 7, or 21 mg/kg bw per day on days 1-8 after mating (experiment 1),
0, 20, or 30 mg/kg bw per day on days 1-3, 4-5, or 6-8 after mating
(experiment 2), and 0, 60, or 90 mg/kg bw per day on days 4-5 after
mating (experiment 3). The incidence of pregnancy was reduced at the
highest dose in experiment 1 and in guinea-pigs treated on days 1-3
after mating in experiment 2, but no teratogenic effect was found
(Long & Diekman, 1989).
Studies of the reproductive and developmental effects of
zearalenone after oral treatment are summarized in Table 3.
2.2.7 Special studies
2.2.7.1 Hormonal effects
Several estrogenic effects of zearalenone have been observed in
short-term and long-term studies of toxicity and in studies of
reproductive toxicity in a number of mammalian species (see above).
Estrogen receptor subtypes and estrogen response elements:
Recent studies have shown that two subtypes of estrogen receptor (ER)
exist in rats, mice, and humans, ER-alpha and ER-beta, which differ in
the C-terminal ligand binding domain and the N-terminal
trans-activation domain (Kuiper et al., 1998). It has also been
shown that there are other subtypes of ER, namely the main form
ER-beta1 and a major variant called ER-beta2 (Lu et al., 1998;
Petersen et al., 1998). Analysis of competition for ligand binding
revealed that ER-beta2 has an eightfold lower affinity for
17beta-estradiol than ER-beta1 (Hanstein et al., 1999). ER-alpha and
ER-beta are differently distributed in the body and also in cells
within tissues such as the prostate and central nervous system
(Shughrue et al., 1996; Brandenberger et al., 1997; Kuiper et al.,
1997; Shughrue et al., 1997; Hrabovszky et al., 1998; Prins et al.,
1998). In some cells, the expression of ER-beta mRNA is regulated by
17beta-estradiol (Vladusic et al., 1998). In vitro the two receptors
can form heterodimeric complexes. Thus, the estrogenic signal could
bind to an ER-alpha homodimer, an ER-beta homodimer, or a heterodimer
complex, depending on whether the cell expresses only one or both
receptors (Pettersson et al., 1997). ER-beta2 may also form
heterodimers (Hanstein et al., 1999). Furthermore, some variation in
the estrogen response element has been found in different
estrogen-responsive genes, and the receptor subtypes vary in
activating ability (Pennie et al., 1998).
Studies on the estrogenicity of zearalenone and its derivatives
up to 1987 were reviewed by Kuiper-Goodman et al. (1987), but only a
few recent studies discriminate between the receptor subtypes.
Binding: The binding of zearalenone to ER in target tissues and
cells was < 1-10% that of 17beta-estradiol, whereas alpha-zearalanol
showed somewhat stronger binding and beta-zearalanol much less binding
(Kuiper-Goodman et al., 1987). The relative binding affinities of
zearalenone and its derivatives to the rat uterine cytoplasmic
receptor were in the order alpha-zearalanol > alpha-zearalenol >
beta-zearalanol > zearalenone > beta-zearalenol (Tashiro et al.,
1980).
Binding and activation of ER in cells: In an immortalized
pituitary cell line, zearalenone bound to the ER with an affinity of
0.01 relative to 17beta-estradiol and induced prolactin excretion
(Stahl et al., 1998).
In a comparison of the potency of zearalenone and
17beta-estradiol in two cell lines, MCF-7 which responds to
physiological concentrations of 17beta-estradiol with cell division
and protein synthesis and Le42 which are transfected with an
estrogen-responsive element coupled to a reporter gene, the relative
response was 2.5-5% (Mayr et al., 1992).
The binding and activation of ER-alpha and ER-beta by zearalenone
have been examined in cells transfected with human recombinant ERa and
ERb complementary DNA in the presence of an estrogen-dependent
reporter plasmid. In this model, 17beta-estradiol bound with high
affinity, with a Kd of 0.05-0.1 nmol/L. Zearalenone stimulated the
transcriptional activity of both receptors at concentrations of 1-10
nmol/L. In these experiments, zearalenone was found to be a full
antagonist for ER-alpha and a mixed agonist-antagonist for ER-beta
(Kuiper et al., 1998).
Activation in animals: Several studies have shown that
zearalenone and its derivatives initiate translocation of the receptor
complex to the nucleus, beta-zearalanol being more effective than
zearalenone; the latter was associated with a longer duration of
nuclear retention of the receptor complex than the former or of
17beta-estradiol. The studies also clearly demonstrated transcription
and synthesis of estrogen-induced protein in the uterus of rats after
zearalenone treatment, with a potency relative to that of
17 beta-estradiol of 0.07 for alpha-zearala-nol, 0.02 for
beta-zearalanol, and 0.001 for zearalenone (Katzenellebogen et al.,
1979). The relative potency of zearalenone with respect to 17
beta-estradiol and diethylstilbestrol in the uterotropic assay after
subcutaneous or oral administration was about 0.001, whereas the
potency relative to that of 17 beta-estradiol in the vaginal
cornification assay was 0.001 after subcutaneous injection and 0.01
after topical administration. alpha-Zearalanol had about the same
potency in this assay but is usually several times more active in the
uterotropic assay (Kuiper-Goodman et al., 1987).
Male 70-day-old rats treated orally with zearalenone at 20 mg/kg
bw per day for five weeks had increased serum prolactin values, but
other parameters such as body and testis weights, serum luteinizing
hormone and follicle stimulating hormone concentrations and volume
fractions of Sertoli cells, spermatogonia, early and late primary
spermatocytes, and long and round spermatids were not affected (Milano
et al., 1995).
Neonatal Charles River CD rats received 100 and 1000 µg of
zearalenone by subcutaneous injection on days 1-10 of life, were
castrated on day 21, and received gonadotropin-releasing hormone on
day 42, when luteinizing hormone was determined. Males and females
exposed to either dose of zearalenone had decreased pituitary
responsiveness to gonadotropin-releasing hormone. The highest dose of
zearalenone increased the volume of the sexually dimorphic nucleus of
the preoptic area in females, whereas no changes were seen in males
(Faber & Hughes, 1991).
In ovariectomized Charles River CD rats, subcutaneous injection
of zearalenone at 8 mg/kg bw or zearalenol at 0.8 or 8 mg/kg bw did
not inhibit tonic luteinizing hormone secretion and did not provide
estrogenic priming for progesterone-induced luteinizing hormone
secretion, but it did block gonadotro-pin-releasing hormone-induced
luteinizing hormone secretion (Hughes et al., 1991).
Daily injection of pregnant mice with 20 ng of 17 beta-estradiol
or 2 µg of zearalenone (equivalent to 10 µg/kg bw) on days 15-20 of
gestation increased the density of terminal end buds in the mammary
glands. Zearalenone also increased epithelial differentiation and
density (Hilakivi-Clarke et al., 1998).
Kuiper-Goodman et al. (1987) based a risk assessment on a study
on a 'no hormonal effect level' (NHEL) for alpha-zearalanol in
ovariectomized monkeys in which vaginal cornification was used as the
end-point. In rhesus monkeys treated orally for 10 days, the NHEL was
225 µg/kg bw per day (Parekh & Coulston, 1983), whereas a NHEL of
< 50 µg/ kg bw per day was found in a 90-day study with cynomolgus
monkeys (Griffin et al., 1984). Kuiper-Goodman et al. (1987) suggested
that zearalenone is less estrogenic than alpha-zearalanol and that the
NHEL for zearalenone is probably higher. In support of that
suggestion, effects were seen in pregnant mice at a dose of 80 µg/kg
bw per day (Hilakivi-Clarke et al., 1998).
Table 3. Reproductive and developmental effects observed after oral exposure of various species to zearalenone
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Mouse Adult 9, Gavage 20 1 day (day < 20 No malformation, Arora et al.
(CBA/Ca) (pregnant 10-20 8 or 9 of increased late (1981, 1983)
female) gestation) fetal deaths
Rat Adult (6-8 50 Diet 0.1, 1, Two-generation 0.1 Number of F2a2 Becci et al.
(Wistar) weeks) 10 study liveborn pups/litter (1982b)
decreased, increased
resorptions; increased
absolute and relative
adrenal, thyroid, and
pituitary weights in
Fo; skeletal
abnormalities related
to decreased growth
1 Maternal toxicity,
decreased fertility,
number of F1a1 live-born
pups/litter decreased,
increased resorptions;
soft-tissue
abnormalities (lack of
eyelids) related to
delay in fetal
development
10 Medullary trabeculation
increased
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Rat Adult 10 Gavage 1, 5, Days 6-15 < 1 Delayed Ruddick et al.
(Wistar) (pregnant 10 of gestation (0.3 ossification (1976)
female) according
to
un-
published
data)
Rat Adult approx. Diet (approx. 17 56 days < 0.85 Decreased Ruzsas et al.
(male 10 (un- 0.85) fertility of (1978, 1979)
and purified males and
female) in maize) females,
disturbed and
spermatogenesis,
disturbed
cycling,
decreased
fertility of
offspring
Rat Adult 5-7 Oral 20 5 weeks < 20 Increased serum Milano et al.
(Wistar) (70-day- prolactin concentration (1995)
old, male) > 20 No effect on
body or testis
weight, serum LH
or FSH, volume
fraction of Sertoli
cells, early and
late spermatocytes,
or long and round
spermatids
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Guinea- Adult, 3-4 Diet 7, 14, 21 1-8 (days 1-8 < 7 Reduced incidence of Long &
pig female after mating) pregnancy (21 mg/kg bw), Diekman
altered levels of (1989)
progesterone, no effect
on litter size,
fetal size
Chicken Female 4 Diet (approx. 10-800 56 days > 59 No effect on egg Allen (1980);
(210 days) 0.7-59) production or egg size Allen et
al. (1981)
Turkey Female 10 Diet (approx. 100 56 days < 4 Decreased egg Allen et
(225 days) 4) production (20%) al. (1983)
Mink Female 4 Diet 10, 20 21 days Increased weight of Cameron et
uterus, vulva size al. (1989)
Mink Female 8 Diet 10, 20 4 weeks Increased gestation Cameron et
before breeding period, increased al. (1989)
to 3 weeks mortality, reduced
after whelping litter size
Mink Female 12 Diet 20 2 months before Reduced whelping, no Yamini et
mating to 3 weeks effect on mating, al. (1997)
after whelping histopathological
changes in uterus and
ovarian follicles
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Mink Female 12 Diet 15.7 74-124 days Reduced whelping, Yang et
(mated day 48, increased gestation al. (1995)
49, 56) length, reduced no.
of live kits, ovarian
follicular atrophy,
endometrial hyperplasia,
endometrial glandular
and myometrial atrophy,
endometritis
Pigb Pregnant 7 Diet (approx. 2.2 Day 2 to < 0.09 Reduced relative Shreeve et
sow (mouldy 0.09) farrowing pituitary, thyroid, al. (1978)
wheat) and kidney weights,
increased relative
spleen and spinal
cord weights in
piglets. No maternal
toxicity, no increased
resorption, no bone
abnormalities, no
lesions
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Pigb Sow 4 Diet 0.28, 1.5, 7, 38, 64 Days 3-34 of < 0.28 Increased serum Long et
(culture 2.6 gestation progesterone al. (1982)
of F. 0.28 Decreased serum
deoxy- roseum with
nivalenol) progesterone and
serum estradiol,
decreased no. of sows
with fetuses, decreased
average fetal weight
1.5 Signs of
hyperestrogenism,
> 2.6 endometrial morphology
No effect on number
of pigs with corpora
lutea
Pig Sow 3-4 Diet (approx. 25, 50, Various < 1 Infertility, Chang et al.
1, 2, 4) 100 pseudogestation, (1979)
nymphomania, constant
estrus, decreased
offspring weight,
juvenile
hyperestrogenism
Pigc Pubertal 16 Diet (approx. 3, 6, 9 Throughout 0.12 Decreased breeding Young & King
gilt 0.12, gestation and live litters, (1984)
0.24, increased
0.36) pseudo-gestation,
no swollen vulvas
or abortions
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Boar, ? Diet (approx. 3, 6, 9 330 days > 0.36 No effect on growth Young & King
30 days 0.12, rate, libido, puberty, (1984)
0.24, or indications of
0.36) reduction in sperm
concentration,
testicular weight,
or epididymal weight
Pig Boar 4 Diet (approx. 56 days > 2 No effect on copulatory Ruhr et al.
0.02, behaviour or male (1983)
0.2, 2) reproduction
Pig Young 3 Gelatin 5, 10, 1 day > 5 Swollen and inflamed Farnworth &
male and capsules 15 vulvas, decreased Trenholm
female adrenal weights (1983)
Pig Young 9 Diet (1.2) 30 Various < 1.2 Precocious Vanyi & Szeky
male spermatogenesis, (1980)
damage to germinal
epithelium,
interstitial-cell
hyperplasia
Pig Pre- 24 Diet (approx. 10 14 days < 0.26 Reversible reduction Green et al.
pubertal 0.26) (exposure ended in serum (1990)
female 14 days before concentration of LH
breeding)
14 days before No effect on age
breeding at puberty, number of
corpora lutea or live
fetuses, fetus weight,
or fetus length
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Pigc Gilt 10 Diet (0.01, 0.36, 122-144 0.02 Swollen and inflamed Friend et al.
0.02, 0.47, days vulvas (1990)
0.05) 1.28
Pig Gilt 4 Diet 0.09 2.12 Day 30 of > 0.09 No statistical Yang et al.
gestation difference in weight (1995)
through at birth or weaning
weaning or ovarian, uterine,
or testicular weight
in offspring. Increased
age of F boars at first
mount but no effect on
precopulatory or
copulatory behaviour
Pig Gilt 4 Diet (approx. 108 mg/ 4-5 days (2-6, < approx. Reduced incidence of Long &
1.1) animal 7-10, 11-15 1.1 gestaion and regressed Diekman (1986)
days t after corpora lutea after
mating) exposure on days 7-10,
decreased LH on day 15
and in prolactin on days
10 and 15 after mating
Pig Sow 4 Diet 1 4 days (days < 1 Reduced frequency of Diekman &
7-10 after spikes in LH secretion, Long (1989)
mating) reduced mean serum LH
and FSH, death of
blastocysts on days
10-14
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Pig Sow 15 Diet 1 4 days < 1 Signs of degeneration Long et al.
(days 7-10 of blastocysts at day 11, (1992)
after advanced degeneration of
mating) blastocysts at day 13. No
changes in endometrium
Pig Gilt 10-15 Diet (approx. 1, 5, 15 days 0.04 Reversible increase in Edwards et al.
0.04, 10 (days 5-20 length of estrous cycle (1987a)
0.2, of estrous and prolonged luteal
0.4) cycle) maintenance
Pig Gilt 13-15 Diet (approx. 10 30 days < 0.4 Increased age at Edwards et al.
(pre- 0.4 ?) first estrus (1987b)
pubertal)
Pig Sow 15-17 Diet (approx. 10 14 days < 0.4 Extended weaning to Edwards et al.
0.4 ?) (before estrus interval, no (1987b)
weaning) effect on fertility, no
sign of hyperestrogenism
Pigb Gilt (pre- 10 Diet 0.06 2 for 2 45 or 90 0.06 Vulvar swelling, younger Rainey et al.
pubertal) (added to weeks, days age at puberty, reduced (1990)
naturally 1.5 response to estradiol.
infected there- No effect on body weight,
feed) after conception rate,
ovulation rate, number
of fetuses, or embryo
survival
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Pigc Gilt 6 Diet (approx. 2.2, 22 9 (day 1 < 0.09 Small decrease in number Kordic et al.
0.09, after mating of corpora lutea, number (1992)
0.88) to farrowing, of live embryos, small
3 gilts in increase in stillborn
each group piglets, increased weight
killed at day of uterus; only the latter
26-27) effect at low dose (no
statistical analysis)
Sheep Ewe 33 Oral (0.03, 1.5, 3, 10 days (from 0.03 Reduced relative Smith et al.
0.06, 6, 12, day 7 in estrus ovulation rate (1990)
0.11, or 24 before mating) (pretreatment vs
0.23, mg/animal post-treatment),
0.45) considered irrelevant
by Committee
0.06 Increased duration of
estrus, increased
uterine weight
0.11 Increased liver weight,
increased ovarian weight
0.23 Reduced incidence
of ovulation, reduced
fertilization
0.45 No effect on live weight,
number of ovulating ewes
yielding ova, number of
ewes with ova yielding
fertilized ova
Table 3. (continued)
Species Age No. Route Dose Duration NOEL Effects Reference
(strain) (mg/kg
mg/kg mg/kg bw per
bw daya feed day)
Sheep Ewe 50 Oral (0.03, 1.5, 3, 10 days (from 0.45 No effect on number of Smith et al.
0.06, 6, 12, day 5 after ovulations, ovulation (1990)
0.11, or 24 mating) rate, conception rate,
0.23, mg/animal incidence of gestation,
0.45) number of lambs born,
embryo or ova wastage
Sheep Ram 6 Diet 12 No effect on volume of Milano et al.
ejaculate or semen (1991)
concentration, motility,
or abnormalities
Cattle Bull 2 Diet (not 20 72 days Degeneration of germinal Vanyi et al.
purified) epithelium, 75% incidence (1980)
of sperm degeneration
Updated from Kuiper-Goodman et al. (1987); LH, luteinizing hormone; FSH, follicle-stimulating hormone
a In parentheses, estimated by the Committee on the basis of 1 mg/kg feed equivalent to 0.04 mg/kg bw per day
b The feed was naturally infected and no information was given on possible occurrence of other mycotoxins.
c The Committee noted the lack of details on the protocol, poor reporting, or poor experimental design.
In blood, zearalenone and zearalanol bind to human sex
hormone-binding globulin to some extent (Martin et al., 1978).
2.2.7.2 Immune responses
In vivo: Nine female B6C3F1 mice weighing 15-18 g were fed a
diet supplemented with zearalenone at 10 mg/kg of diet (equivalent to
1.5 mg/kg bw per day) for two weeks. After intravenous infection with
Listeria monocytogenes, the splenic bacterial count showed an
increasing trend on days 1 and 4 when compared with that in 11 control
animals. No adverse effects were seen after eight weeks of feeding.
The exposure did not affect the splenic plaque-forming response to
sheep red blood cells or the delayed hypersensitivity response to
keyhole haemocyanin after two or eight weeks (Pestka et al., 1987).
Female B6C3F1 mice received zearalenone subcutaneously at a
dose of 45 mg/kg bw, and 27 mice were then infected with 5 × 104
L. monocytogenes cells. No difference in survival rate was seen in
comparison with a group of 82 controls. When bacteria in the spleen
were counted after intravenous infection with 104 cells (number of
animals tested not given), no treatment-related differences were
reported (Pung et al., 1984).
When eight weanling female B6C3F1 mice were fed a diet
supplemented with zearalenone at 10 mg/kg of diet (equivalent to 1.5
mg/kg bw per day) for six weeks, no differences from 26 control
animals were seen in the serum concentrations of immunoglobulins G, M,
or A. Dietary administration of zearalenone had no effect on the
leukocyte count or on differential lymphocyte, polymorphonuclear
neutrophil, monocyte, or eosinophil counts (Forsell et al., 1986).
In vitro: Zearalenone at a concentration of 13 µg/ml inhibited
phytohaema-gglutinin-induced lymphocyte blastogenesis in human and PVG
rat peripheral blood lymphocytes, as measured by the incorporation of
[3H]thymidine into human lymphocytes, by 50%. To produce a similar
reduction in rat cell cultures, a concentration of 2.5 µg/ml was
required (Atkinson & Miller, 1984).
Incorporation of [3H]thymidine into the DNA of human peripheral
blood lymphocytes stimulated by phytohaemagglutinin was completely
inhibited by exposure of the cells to zearalenone at 30 µg/ml.
Exposure to 14 µg/ml inhibited DNA synthesis in mitogen-stimulated
lymphocytes by 50%. No alteration in toxicity was observed when rat
liver cells were present in the lymphocyte cultures (Cooray, 1984).
The concentrations of zearalenone and four metabolites required
to reduce [3H]thymidine uptake in mitogen-stimulated human
lymphocytes by 50% were 3.5 µg/ml for zearalenone, 6.3 µg/ml for
alpha-zearalenol, 36 µg/ml for beta-zearalenol, 3.8 µg/ml for
alpha-zearalanol, and 33 µg/ml for beta-zearalanol. The results
indicate that a keto group or an alpha-hydroxyl at position C-6
contributes to the lymphotoxicity. The concentration of each analogue
that caused a 50% reduction in [3H]thymidine uptake was similar for
all mitogens tested (leukoagglutinin, concanavalin A, and pokeweed
mitogen), suggesting that zearalenone and its metabolites can inhibit
mitogen-induced proliferation by both B and T lymphocytes (Forsell &
Pestka, 1985).
In order to study the effect of zearalenone on interleukin (IL)
production, T cells of the EL-4 murine thymoma cell line were
stimulated with phorbol-2-myristate-13-acetate and exposed for five
days to zearalenone or alpha-zearalenol at concentrations of 50, 500,
1000, 5000, or 10 000 ng/ml. Control cells were exposed to the vehicle
(ethanol) only. The production of IL-2 and IL-5 was significantly
increased in the presence of zearalenone or alpha-zearalenol at 5000
and 10 000 ng/ml. The two toxins did not affect cell proliferation or
viability, as shown in the 3-(4,5-dimethylthiazol-2-yl)
2,5-diphenyltetrazolium bromide cytotoxicity assay (Marin et al.,
1996).
2.2.5.3 Macromolecular binding
Several DNA adducts were detected by 32P-postlabelling in
female BALB/c mice treated intraperitoneally with a single dose of
zearalenone at 2 mg/kg bw in olive oil. A total of 1340 adducts/109
nucleotides were found in liver and 111 adducts/109 nucleotides in
kidney. Co-administration of 4 mg/kg bw alpha-toco-pherol significantly
decreased DNA adduct formation in liver and in kidney to 713 and 45
adducts/109 nucleotides, respectively (Grosse et al., 1997).
In weanling female Sprague-Dawley rats fed a diet containing
zearalenone at 0.05 mg/kg (equivalent to 5 µg/kg bw) for three weeks,
no DNA adducts were found by 32P-postlabelling in liver, kidney, or
uterus DNA (Li et al., 1992). The Committee noted the very low dose of
zearalenol used.
When six-week-old female BALB/c mice were treated
intraperitoneally or orally with zearalenone, 12-15 DNA adducts were
found by 32P-postlabelling in the kidney and liver, at levels of 114
± 37 adducts/109 nucleotides in kidney and 1393 ± 324 adducts/109
nucleotides in liver after intraperitoneal treatment and 548 ± 50
adducts/109 nucleotides in liver, after oral treatment with a single
dose of 2 mg/kg bw. Six DNA adducts were found in the ovary but only
after repeated doses of 1 mg/kg bw on days 1, 5, 7, 9, and 10, with a
total number of DNA adducts after 10 days of 17 ± 5 adducts/109
nucleotides. Some adducts were common to all organs, while others were
specific to one organ. In contrast, no DNA adducts were detected in
the organs of male and female Sprague-Dawley rats after
intraperitoneal treatment (no details given). The authors concluded
that these results confirm the genotoxicity of zearalenone and its
ability to induce hepatocellular adenomas rather than tumours of the
genital organs in mice (Pfohl-Leszkowicz et al., 1995). The Committee
disagreed, since the 32P-postlabelling method does not measure
direct covalent DNA binding, and other (indirect) mechanisms of action
may be involved.
2.2.7.4 Genotoxicity of metabolites
The results of studies of the genotoxicity of metabolites of
zearalenone are summarized in Table 4.
2.3 Observations in humans
Zearalenone was measured in endometrial tissue from 49 women and
found at a concentration of 48 ± 6.5 ng/ml in tissue from 27 women
with endometrial adenocarcinoma, at 170 ± 18 ng/ml in tissue from 11
women with endometrial hyperplasia, and at a concentration below the
limit of detection in tissue from 11 women with normal proliferative
endometrium. None was detected in eight samples of hyperplastic and
five samples of neoplastic endometrial tissue (Tomaszewski et al.,
1998).
Zearalenone or zearalanol was suspected to be the causative agent
in an epidemic of premature thelarche in girls aged six months to
eight years which occurred in Puerto Rico between 1978 and 1981 (Sàenz
de Rodriguez, 1984; Sàenz de Rodriguez et al., 1985), as these
compounds were detected in blood plasma. The authors reported that
homogenates of locally produced meat gave strong responses in a
cytosol receptor assay with rat uterus, indicating the presence of
substances that bind to estrogen receptors, although the United States
Food and Drug Administration later failed to detect any of the
estrogen growth promoters used in food (Anon., 1986). The involvement
of natural sources of estrogenic compounds, such as some plant
metabolites and mycotoxins, has not been ruled out. A statistically
significant correlation was found between the pubertal changes and
consumption of meat products and soya-based formula, but the
associations explained only 50% of the investigated cases, and the
authors suggested that better diagnosis and reporting or some
unsuspected factor accounted for the reported increase in precocious
pubertal changes (Freni-Titulaer et al., 1986).
An increased incidence of early thelarche was also reported from
southeastern Hungary, and zearalenone was found at concentrations of
19-100 µg/ml in serum and in samples of foods that had been consumed
by the patients (Szuetz et al., 1997); however, the report lacked
detailed information.
3. OCCURRENCE AND INTAKE
Few estimates of human exposure to zearalenone have been
published. This section provides information on the concentrations in
plant-based foods and animal tissues, estimates of human intake of
zearalenone, and guidelines for modelling dietary exposure to this
substance. Previous comprehensive reviews of publications on
zearalenone include those of Sundlof & Strickland (1986),
Kuiper-Goodman et al. (1987), and Krska (1999). Zearalenone is
determined in foods by high-performance liquid chromatography,
thin-layer chromatography, or gas chromatography with mass
spectrometry.
Sundlof & Strickland (1986) reviewed the literature on the
presence of zearalenone and alpha-zearalanol in animal tissues to
determine whether consumption of these products poses a threat to
human health. They summarized studies on the concentrations of
zearalenone in milk after exposure of cows to this compound, the
concentrations of zearalenone in tissue from cattle implanted with
alpha-zearalanol, and the concentrations of zearalenone in muscle and
liver from chickens exposed to [14C]zearalenone. They concluded that
milk is not a likely source of residues of zearalenone and that
because chicken muscle contained few binding sites for this substance,
the likelihood of human exposure to zearalenone residues due to
consumption of chicken was minimal. Because eggs accumulate a
zearalenone metabolite in yolks, the authors suggested that they might
be a source of exposure.
Kuiper-Goodman et al. (1987) reviewed the toxicology, chemistry,
mycology, natural occurrence, and stability of zearalenone in plant
and animal products and reported its occurrence in foods from 23
countries, including Australia, China, Mexico, South Africa, and the
United States and countries in northern Europe. The greatest
contamination was found in corn and corn products. The dietary intake
of young Canadian males was estimated on the basis of consumption of
corn breakfast cereals and popcorn, and that of children aged 1-4
years of age was estimated on the basis of consumption of corn
breakfast cereals, popcorn, and milk.
The most recent overview (Krska, 1999) covers the occurrence of
zearalenone in foods, the dietary intake estimates of Kuiper-Goodman
et al. (1987) in Canada, residues in animal products, the effects of
contamination on trade, and efforts to control contamination. The
highest prevalences of zearalenone are reported in Canada, central and
northern Europe, and the United States, although its occurrence was
also reported in foods in Egypt, Italy, New Zealand, South Africa, and
South America. Zearalenone occurs in many agricultural products,
including cereals, mixed feeds, rice, and corn silage. The reported
prevalences and concentrations in cereals and mixed feed vary
considerably. Zearalenone can occur concomitantly with the
trichothecenes nivalenol and deoxynivalenol, since the three compounds
are produced by the same Fusarium spp.
A considerable increase in the concentration of zearalenone was
found after treatment of three cereal samples and one barley sample
with ß-glucosidase, indicating the presence of glucosides (Gareis et
al., 1990), and zearalenone sulfate was isolated from cultures of
Fusarium spp. grown on rice (Plasencia & Mirocha, 1991). The
significance of such conjugates of zearalenone in plants, which are
not detected by routine analysis, is not known.
3.1 Incidence and levels of contamination
Table 5 gives published values for concentrations of zearalenone
in grains, grain products, legumes, nuts and seeds, fruits and
vegetables, spices and herbs, muscle and organ meats, milk, and
miscellaneous products. Because the purpose of this section is to
evaluate human dietary intake of zearalenone, Table 5 does not include
concentrations in animal feed. The data in Table 5 have a number of
limitations for estimating intake: Information on incidence is of
little value when it is based on few samples and when the samples were
not collected in a random fashion. If the samples were chosen
specifically because the food was of inferior quality (i.e. mouldy,
damaged, or off-colour), the incidence rates and concentrations of
zearalenone are likely to be high, and if those concentrations are
used to estimate dietary intake, it will be exaggerated. Another
limitation is that the information on zearalenone is derived from
studies in which different analytical methods were used
(high-performance liquid chromatography, thin-layer chromatography, or
gas chromatography with mass spectrometry), which have different
limits of detection. The incidence of positive samples is related to
the sensitivity of the method, since the more sensitive the method the
lower the detection limit and the greater the probability of a
positive reponse.
The reported incidence rates and concentrations of zearalenone in
grains and grain products vary according to type of grain and to
climatic, harvest, and storage conditions. Corn and wheat appear to be
the commodities of greatest concern with respect to zearalenone
contamination; other cereal grains appear to be less contaminated and
are less widely consumed. Of the 13 grains represented in Table 5, six
(acha, amaranth, buckwheat, millet, semolina, and tritical) were
mentioned in only one reference, whereas the data for oats, rice, rye,
and sorghum come from 4-11 countries in 4-12 references, and barley
(18 countries, 30 references), corn (26 countries, 43 references), and
wheat (25 countries, 43 references) were the best covered. In general,
the mean values for barley were quite low, those for wheat were
generally low, and those for corn were variable.
In most of the studies, it was not possible to determine if the
grain crops were intended for human consumption or for animal feed.
Thus, although the aim was to summarize information on human foods,
some of the crops listed in Table 5 may not have been grown for that
purpose. Since the authors of the papers did not distinguish between
corn and maize, the two products are listed together under 'corn',
although the term used in the references is given when it is of
interest. The term 'sweet corn', yellow or white corn used as a
vegetable in the USA, was used in one reference (Stoloff & Francis,
1980). Another paper (Abbas et al., 1988) referred to 'dent' corn. The
corn analysed in different countries may represent different
cultivars, subspecies, or commercial classifications.
Table 4. Results of assays for genotoxicity with metabolites of zearalenone
Metabolite Test system Test object Concentration Results Reference
Zearalanola Reverse mutation S. typhimurium TA1538, 250 µg/plateb Negativec Bartholomew & Ryan
TA98, TA100 (1980)
Zearalanone Reverse mutation S. typhimurium TA1535, 50 µg/plateb Negativec Ingerowski et al.
TA1537, TA1538, TA98, (1981)
TA100
Zearalanol Reverse mutation S. typhimurium TA1535, 250 µg/plateb Negativec Ingerowski et al.,
TA1537, TA1538, TA98, (1981)
TA100
Zearalanol SOS chromotest E. coli PQ37 106 mg/Lb Negative Scheutwinkel et al.
(1986)
Zearalanol Gene mutation B. subtilis H17, M45 Not reported Positived Scheutwinkel et al.
rec+/- (1986)
Zearalanol Sister chromatid Chinese hamster 32 mg/Lb Negative Scheutwinkel et al.
exchange V79 cells (1986)
alpha- and SOS chromotest E. coli PQ37 60 mg/Lb Negative Krivobok et al.
beta-Zearalenol (1987)
(1:1)
Zearalenol-alphae Gene mutation B. subtilis H17, M45 100 µg/disc Negative Ueno & Kubota
Zearalenol-betae rec+/- 100 µg/disc Positive (1976)
a Specified as 'low and high melting point zearalanol'
b With and without metabolic activation
c Cytotoxic at 500 µg/plate
d M45rec- 3 mm, H17rec+ 0 mm growth inhibition
e According to Kuiper-Goodman et al. (1987), zearalenol-beta is alpha-zearalenol and zearalenol-alpha
is beta-zearalenol
Table 5. Concentrations of zearalenone in foods
Food Geographical Incidencea Concentration Reference
location (µg/kg or µg/L)b
D/T % Mean Range
Grains
Acha (by season) Nigeria Gbodi et al. (1986a)
Dry, cold 4/9 44 248 200-309
Dry, hot 3/7 43 348 241-600
Humid, hot 1/8 13 18
Amaranth Argentina Bresler et al. (1998)
(by water
activity)
0.902 0
0.925 1500
0.950 11 100
Barley Sweden 23/329 7 18 Eriksen & Alexander (1998)
and Norway
Barley, six Southwest 7-68 3-36 max, 311 Müller et al. (1997b)
years Germany
Barley Canada 3/210 1 13 4-21 Scott (1997)
Barley and Uruguay 116/137 85 <100 Pineiro et al. (1996a)
malt
Barley and Uruguay 12/137 9 100-200 Pineiro et al. (1996a)
malt
Barley and Uruguay 8/137 6 > 200 Pineiro et al. (1996a)
malt
Barley Republic 0/30 0 0 Ryu et al. (1996)
of Korea
Barley Manitoba, 5/7 71 166 24-45 Usleber et al. (1996)
Canada
Barley Japan 7/17 41 4158 105-15 300 Yoshizawa & Jin (1995)
Barley Russian 0/NA 0 0 Zakharova et al. (1995)
Federation
Barley Papua 0/3 0 0 Yuwai et al. (1994)
New Guinea
Table 5. (continued)
Food Geographical Incidencea Concentration Reference
location (µg/kg or µg/L)b
D/T % Mean Range
Barley Republic 20/39 51 287 40-1416 Kim et al. (1993)
of Korea
Barley Canada 41/180 26 Most Stratton et al. (1993)
< 0.3
Barley Japan 13/18 72 24 2-97 Tanaka et al. (1993)
Barley
Undergrade Republic 10/37 27 Park et al. (1992)
of Korea
Husked Republic 183-1416 Park et al. (1992)
of Korea
Naked Republic 40-1081 Park et al. (1992)
of Korea
Barley Finland 2/30 7 26 21-30 Hietaniemi & Kumpulainen
Importedc 0/3 0 0 (1991)
Barley New Zealand 15/85 18 max, 170 Lauren et al. (1991)
Barley Germany 0/14 0 0 Ranfft et al. (1990)
Barley Netherlands 6/6 100 7 4-9 Tanaka et al. (1990)
Barley, Netherlands 0/1 0 0 Tanaka et al. (1990)
pearled
Barley Bavaria, 24/46 52 24 max, 320 Gleissenthal et al. (1989)
Germany
Barley USA 1/1 100 < 19 Bagneris et al. (1986)
Barley Japan Lee et al. (1986)
Husked 3/6 50 1-2
Unhusked 29/31 94 24 1-388
Barley malt Japan 5/5 100 23 3-48 Lee at al. (1986)
Barley Taiwan, 2/4 50 19 16-22 Ueno et al. (1986)
China
Barley Republic Lee et al. (1985)
of Korea
Polished 0/6 0 0
Unpolished 21/28 75 110 0-1281
Table 5. (continued)
Food Geographical Incidencea Concentration Reference
location (µg/kg or µg/L)b
D/T % Mean Range
Barley malt Republic 4/4 100 19 2-36 Lee et al. (1985)
of Korea
Barley Japan Tanaka et al. (1985)
Flour 6/6 100 2 1-4
Pearled 1/1 100 4
Polished 1/3 33 6
Barley Czechoslovakia 58 61-261 Bartos & Matyas (1981)
Barley Western Canada 0/NA 0 0 Prior (1976)
Barley, Scotland Gross & Robb (1975)
stored
<10 weeks 0/NA 0 0
12-51 weeks NA 2100-26,500
Buckwheat Beijing, C