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, China 0/1 0 0 Ueno et al. (1986) flour Corn Indonesia 2/16 13 11, 12 Ali et al. (1998) Cornd United Kingdom Scudamore et al. (1998) Baby 4/4 100 55 40-80 Flaked 3/3 100 93 80-110 Germ 7/7 100 67 50-80 Germ/bran 6/6 100 330 160-540 Gluten 8/40 20 270 80-480 Meal 3/3 100 1080 640-1500 Screen 4/4 100 1450 1300-1800 Cornd Botswana 1/20 5 40 Siame et al. (1998) and meal Corn Italy 14/15 93 46 4-150 Visconti & Pascale (1998) Corn Egypt 15/50 30 22.3 Abd Alla (1997) Corn, Canada Scott (1997) by year 1978-81 21/77 27 105 30-475 1986-93 87/126 69 65 5-647 Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Cornd Hungary Fazekas et al. (1996) At harvest 17 30 6-79 Mouldy, stored 88 1260 10-11 800 Corn, Wisconsin, NA/98 904 Park et al. (1996) mouldy USA Cobs NA/98 21 000 Kernels NA/98 500 Corn Uruguay 71/76 93 < 100 Pineiro et al. (1996a) Corn Uruguay 2/76 2 100-200 Pineiro et al. (1996a) Corn Uruguay 4/76 5 > 200 Pineiro et al. (1996a) Cornd, Argentina Resnik et al. (1996) by year 1983 9/126 7 154 140-350 1984 54/138 39 46 25-150 1985 17/35 49 114 95-332 1988 40/108 37 158 100-1200 1989 16/162 10 301 200-2000 1990 195/491 40 120 100-350 1991 121/288 42 151 100-800 1992 127/349 36 168 97-1108 1993 8/294 3 152 97-820 1994 89/280 32 293 210-1500 Cornd Republic of 1/15 7 71 Ryu et al. (1996) Korea Cornd South Africa 3/161 2 NA Dutton & Kinsey (1995) Corn, Egypt 13/22 59 9800-38,400 El-Maghraby et al. (1995) yellow, white, popcorn Cornd, Brazil 0/36 0 0 Hennigen & Dick (1995) stored Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Corn Kansas, Hooshmand & Klopfenstein Three moisture USA 4875-4930 (1995) levels With 5 4480-4700 or 7.5 kGy With 10 3175-3921 or 20 kGy Corn Philippines 2/50 4 282 59-505 Yamashita et al. (1995) Corn Thailand 1/27 4 923 Yamashita et al. (1995) Corn Indonesia 0/12 0 0 Yamashita et al. (1995) Corn Taiwan 2/32 6 25 Rheeder et al. (1994) from South Africa Corn Papua New 0/3 0 0 Yuwai et al. (1994) Guinea Corn Republic of 8/46 17 151 4-388 Kim et al. (1993) Korea Corn Central 7/40 18 NA max, 3 L'vova et al. (1993) Russian Federation Cornd Bavaria 0 Abramson et al. (1992) Cornd New Zealand 69/91 76 max, 500 Lauren et al. (1991) Cornd, Bulgaria 0/264 0 0 Petkova-Bocharova et al. (1991) home-stored Corn Minnesota, USA 10/339 3 NA Russell et al. (1991) Cornd South India NA 1454 Sivaswamy et al. (1991) Corn Linxian, 16/27 59 44 14-169 Luo et al. (1990) China Corn Shangqiu, 1/20 5 39 Luo et al. (1990) China Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Cornd India 2/22 9 NA Pande et al. (1990) Cornd Germany 6/7 86 12 5-35 Ranfft et al. (1990) Corn, Netherlands 1/1 100 677 Tanaka et al. (1990) yellow Cornd New Zealand 15/20 75 100-16 000 Hussein et al. (1989) Corn Brazil 15/328 5 653-9830 Sabino et al. (1989) Corn, Minnesota, 17/19 89 2700 0-13 200 Abbas et al. (1988) mouldy USA Corn Linxian, 5/5 100 NA Hsia et al. (1988) China Corn Canada 0/1 0 0 Tanaka et al. (1988b) Corn Indonesia 7/26 27 6 1-14 Widiastuti et al. (1988a) Composite 11/52 21 7 1-14 Damaged 0/52 0 0 Good 0/52 0 0 Green-yellow NA/52 580 fluorescence Mouldy 0/52 0 0 Purple NA/52 1840 50-13 500 Corn, Minnesota 2/2 100 100, 5000 Abbas et al. (1986) refused and Indiana, USA Corn USA Bagneris et al. (1986) Shelled 13/31 42 117 21-480 Unshelled 6/7 86 982 19-3656 Cornd, Nigeria max, 17 500 Gbodi et al. (1986b) mouldy Corn Illinois, 5/5 100 1376 114-3008 Bennett et al. (1985) USA Corn and Canada Williams (1985) products Domestic 23/81 28 13-475 Imported 1/61 2 200 Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Cornd Czechoslovakia 7 105 Bartos & Matyas (1981) Corn Western Canada 2/19 11 NA Prior (1981) Corn Argentina 16/55 29 200-750 Lopez & Tapia (1980) Corn, USA 0/263 0 0 Stoloff & Francis (1980) sweet, canned/frozen Corn Ontario, Canada 266/2022 13 3850 <10-141 000 Funnell (1979) Corn Transkei 2/4 50 max, 1100 Marasas et al. (1979) Corn Zambia NA max 1800 Senti (1979) Corn Yugoslavia 3/100 3 5100 43-10 000 Balzer et al. (1977) Cornd Zambia NA/17 290 100-800 Lovelace & Nyathi (1977) for beer NA/13 680 brewing Corn Mexico 6/139 4 NA Shotwell et al. (1977) Corn Western Canada 0/6 0 0 Prior (1976) Corn NA/5 431-7622 Shotwell et al. (1976) Corn USA 19/315 6 117 38-204 Stoloff et al. (1976) Corn USA 6/26 23 200-500 Eppley et al. (1974) Corn, stored wet France NA 2350 Jemmali (1973) Cornd New Zealand 2200-4800 Lauren & Ringrose (1997) germ, fibre, gluten Corn flour United Kingdom NA/4 6.5-41 Patel et al. (1996) Corn flour Papua New 0/1 0 0 Yuwai et al. (1994) Guinea Cornd malt Zambia NA/13 680 max, 4000 Lovelace & Nyathi (1977) Cornmeal/flour Canada Scott (1997) 1978-81 0/28 0 0 1986-93 14/126 11 26 5-178 Cornmeal Michigan, USA 3/11 27 38 8-100 Abouzied et al. (1991) Cornmeal Mexico 12/50 24 NA Argumedo et al. (1985) Cornmeal USA 9/11 82 10-70 Ware & Thorpe (1978) Popcorn Michigan, USA 1/8 12 10 10-10 Abouzied et al. (1991) Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Popcorn, Canada 1/1 100 25 Scott (1997) popped Millet Papua New 1/1 100 440 Yuwai et al. (1994) meal Guinea Oats Sweden and 14/233 6 26 Eriksen & Alexander (1998) Norway Oats Finland 3/21 14 63 30-86 Hietaniemi & Kumpulainen (1991) Oats New Zealand 10/29 34 max, 90 Lauren et al. (1991) Oats Germany Müller et al. (1998) Oats Germany 2/7 29 10 8-11 Ranfft et al. (1990) Oats Netherlands 3/3 100 22 16-29 Tanaka et al. (1990) Oats, Netherlands 0/1 0 0 Tanaka et al. (1990) unhusked Oats Bavaria, 2/7 29 3 max, 8 Gleissenthal et al. (1989) Germany Oats USA 1/1 100 18 Bagneris et al. (1986) Oats Tbilisi, 0/1 0 0 Ueno et al. (1986) Georgia Oats Czechoslovakia 0 Bartos & Matyas (1981) Oats Finland 2 300 000 Kallela & Saastamoinen (1981a) Oats Western Canada 0/NA 0 0 Prior (1976) Rice Egypt 4/45 9 15.5 Abd Alla (1997) Rice United Kingdom Patel et al. (1996) Basmati NA/4 5-16 Chinese 0/4 0 0 Rice Uruguay 39/42 93 < 100 Pineiro et al. (1996a) 0/42 0 100-200 3/42 7 > 200 Rice Russian L'vova et al. (1993) Federation Central 1/24 4 NA South 3/12 25 NA Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Rice South India 0/NA 0 0 Sivaswamy et al. (1991) Rice India 0/30 0 0 Pande et al. (1990) Rice Russian 0/280 0 0 L'vova et al. (1984) Federation Rice, Papua New 1/1 100 3060 Yuwai et al. (1994) brown Guinea Rice Brazil Soares & Parboiled NA 0 Rodriquez-Amaya (1989) Polished NA 0 Rye Sweden and 0/31 0 0 Eriksen & Alexander (1998) Norway Rye Germany Marx et al. (1995) Conventional production NA/100 4 Alternative production NA/100 51 max, 199 Rye Russian 0/NA 0 Zakharova et al. (1995) Federation Rye Finland 0/31 0 0 Hietaniemi & Kumpulainen Importedc 0/10 0 0 (1991) Rye Germany 2/6 33 8 7-9 Ranfft et al. (1990) Rye Netherlands 1/4 25 11 Tanaka et al. (1990) Rye Bavaria, 15/31 48 17 max, 100 Gleissenthal et al. (1989) Germany Rye Canada 0/1 0 0 Tanaka et al. (1988b) Rye, Republic of 3/5 60 2 3-4 Lee et al. (1985) polished Korea Semolina South India 0/NA 0 0 Sivaswamy et al. (1991) Sorghum Botswana 0/19 0 0 Siame et al. (1998) and meal Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Sorghum South Africa 0/7 0 NA Dutton & Kinsey (1995) Sorghum USA 5/5 100 504 47-1280 Bagneris et al. (1986) Sorghum malt Zambia NA/8 < 100 Lovelace & Nyathi (1977) Triticale South Africa 1/2 50 NA Dutton & Kinsey (1995) Wheat Sweden and 7/101 7 5 Eriksen & Alexander (1998) Norway Wheat Egypt 5/40 13 8.8 Abd Alla (1997) Wheat Egypt 10/NA 28-42 Aziz et al. (1997) Wheat Southwest Müller et al. (1997a) Germany 1987 67/84 80 178 1-8036 1989 11/78 14 3 1-6 1990 9/80 11 5 1-15 1991 10/80 13 20 1-109 1992 15/78 19 4 1-20 1993 28/45 62 11 2-52 Wheat, Ontario, 9/95 10 14 5-33 Scott (1997) soft Canada Wheat, Western Canada 1/88 1 4 Scott (1997) hard Wheat Switzerland NA/92 > 60 Bucheli et al. (1996) Wheat Uruguay 101/106 95 < 100 Pineiro et al. (1996a) 2/106 2 100-200 3/106 3 >200 Wheat Bulgaria 69 17 max, 120 Vrabcheva et al. (1996) Wheat South Africa 0/5 10 NA Dutton & Kinsey (1995) Wheat Sao Paulo, 3/NA 40-210 Furlong et al. (1995a) Brazil Wheat, Brazil 0/12 0 0 Furlong et al. (1995b) stored Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Wheat, Brazil stored, from Argentina 0/4 0 0 Furlong et al. (1995b) Wheat, Brazil stored, from Uruguay 0/4 0 0 Furlong et al. (1995b) Wheat Japan 4/17 24 677 53-1690 Yoshizawa & Jin (1995) Wheat Russian 3/154 2 NA Zakharova et al. (1995) Federation ('low') Wheat, Papua New 1/1 100 1040 Yuwai et al. (1994) ground Guinea Wheat Papua New 0/1 0 0 Yuwai et al. (1994) Guinea Wheat Russian L'vova et al. (1993) Federation Central 1/31 3 NA South 4/6 67 NA max, 22 Wheat Southwest Müller & Schwadorf (1993) Germany Wheat Canada 30/201 15 Most, < 0.3 Stratton et al. (1993) Wheat Bavaria, 0 Abramson et al. (1992) Germany Wheat Finland 2/40 5 22 12-32 Hietaniemi & Kumpulainen Importedc 0/10 0 0 (1991) Wheat New Zealand 48/151 32 max, 460 Lauren et al. (1991) Wheat South India NA 4744 Sivaswamy et al. (1991) Wheat Linxian, 6/15 40 <10 Luo et al. (1990) China Wheat Shangqiu, 6/15 40 <10 Luo et al. (1990) China Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Wheat India 0/30 0 0 Pande et al. (1990) Wheat Germany 7/21 33 27 4-64 Ranfft et al. (1990) Wheat Netherlands 7/13 54 45 2-174 Tanaka et al. (1990) Wheat Bavaria, 61/106 58 80 max, 1560 Gleissenthal et al. (1989) Germany Wheat Canada 9/10 90 9 Tanaka et al. (1988b) Wheat Japan 5/9 56 141 3-1254 Lee et al. (1986) Wheat United Kingdom 4/31 13 1 Tanaka et al. (1986) and barley Wheat Scotland 10/10 100 9 Tanaka et al. (1986) and barley Wheat Beijing, 0/5 0 0 Ueno et al. (1986) China Wheat Shanghai, 1/1 100 2 Ueno et al. (1986) China Wheat Taiwan 9/22 41 16 4-32 Ueno et al. (1986) Wheat Tbilisi, 0/2 0 0 Ueno et al. (1986) Georgia Wheat, Republic 2/10 20 8, 40 Lee et al. (1985) polished of Korea Wheat, Kansas and 3/33 9 35, 90, 115 Hagler et al. (1984) scabby Nebraska, USA Wheat Bavaria, 0/NA 0 Abramson et al. (1982) Germany Wheat Czechoslovakia 46 61-182 Bartos & Matyas (1981) Wheat India 73/85 86 max, Neelakantan et al. (1979) and rice 600 000 Wheat Virginia, 19/42 45 360-11 050 Shotwell et al. (1977) USA Wheat Western Canada 0/NA 0 0 Prior (1976) Wheat bran Papua New 0/2 0 0 Yuwai et al. (1994) Guinea Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Wheat bran Shanghai, 1/1 100 3 Ueno et al. (1986) China Wheat germ Papua New 0/1 0 0 Yuwai et al. (1994 Guinea Wheat flour Papua New Yuwai et al. (1994 Guinea Fine-ground 0/1 0 0 Raw 1/1 100 250 Whole grain 4/4 100 1893 1400-2570 Wheat flour Michigan, USA 2/17 12 13 12-14 Abouzied et al. (1991) muffin mix Wheat flour Egypt 4/NA 34 Aziz et al. (1997) Wheat flour Beijing, 0/3 0 0 Ueno et al. (1986) China Wheat flour Shanghai, 0/1 0 0 Ueno et al. (1986) China Wheat flour Japan 3/27 11 3 1-6 Tanaka et al. (1985) Bread United Kingdom Patel et al. (1996) Chapatti 0/4 0 0 Nan 0/4 0 0 Pitta 0/4 0 0 Bread, wheat Egypt 4/NA 95 Aziz et al. (1997) Bread, Papua New 2/2 100 500 250-750 Yuwai et al. (1994) wheat crumbs Guinea Breakfast United Kingdom 8/56 14 < 51 Norton et al. (1982) cereals Corn Michigan, USA 7/8 88 12 5-20 Abouzied et al. (1991) cerealse Corn Canada 0/60 0 0 Scott (1997) cerealse Corn chips Michigan, USA 0/6 0 0 Abouzied et al. (1991) Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Grain products Corn flakes Canada 1/1 100 10 13-20 Scott et al. (1978) Corn products Brazil 0 Soares & Rodriquez-Amaya (1989) Corn products, USA 0/119 0 0 Stoloff & Dalrymple (1977) dry-milled Crackers Michigan, USA 3/18 17 12 10-16 Abouzied et al. (1991) and cookies, wheat and oat Mixed-grain Michigan, USA 2/3 67 31 12-50 Abouzied et al. (1991) cerealse Noodles United Kingdom Trace Patel et al. (1996) Oat cerealse Michigan, USA 3/5 60 16 9-22 Abouzied et al. (1991) Poppadoms United Kingdom 0/4 0 0 Patel et al. (1996) Rice Michigan, USA 1/4 25 12 12-12 Abouzied et al. (1991) cerealse Wheat Michigan, USA 2/12 16 28 27-30 Abouzied et al. (1991) cerealse Wheat noodles United Kingdom 0/4 0 0 Patel et al. (1996) Legumes Beans, red Papua New 0/1 0 0 Yuwai et al. (1994) Guinea Beans, Bulgaria 0/260 0 0 Petkova-Bocharova et al. (1991) home-stored Beans, dried Brazil 0 Soares & Rodriguez-Amaya (1989) Beans Yugoslavia 1/50 2 160 Pepeljnjak (1984) Legumes Western Canada 0/NA 0 0 Prior (1981) Legumes Western Canada 0/NA 0 0 Prior (1976) Soya beans Canada 6/97 6 24 5-39 Scott (1997) and productsf Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Soya beans Uruguay 15/17 88 < 100 Pineiro et al. (1996b) 0/17 0 100-200 2/17 12 > 200 Soya bean South Africa 0/14 0 NA Dutton & Kinsey (1995) meal Soya beans South Africa 13/417 3 Dutton & Kinsey (1995) Soya beans Papua New 0/3 0 0 Yuwai et al. (1994) Guinea Soya beans Egypt 0/100 0 0 El-Kady & Youssef (1993) Soya beans USA 0/180 0 0 Shotwell et al. (1977) Nuts and seeds Areca nuts South India 0/NA 0 0 Sivaswamy et al. (1991) Almonds Egypt 0/NA 0 0 Abdel-Gawad & Zohri (1993) Almonds Spain 0/34 0 0 Jimenez et al. (1991) Cashews Egypt 0/NA 0 0 Abdel-Gawad & Zohri (1993) Cashews South India 0/NA 0 0 Sivaswamy et al. (1991) Chestnuts Egypt 0/NA 0 0 Abdel-Gawad & Zohri (1993) Coconut South India 0/NA 0 0 Sivaswamy et al. (1991) Cottonseeds South Africa 0/3 0 NA Dutton & Kinsey (1995) Fennel United Kingdom NA/3 7 Patel et al. (1996) seeds Hazelnuts Egypt 0/NA 0 0 Abdel-Gawad & Zohri (1993) Hazelnuts Egypt 0/20 0 0 Abdel-Hafez & Saber (1993) Hazelnuts Spain 0/29 0 0 Jimenez et al. (1991) Oilseeds Uruguay 58/64 90 <100 Pineiro et al. (1996b) 3/64 5 100-200 3/64 5 > 200 Peanut Botswana 0/15 0 0 Siame et al. (1998) butter Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Peanuts South Africa 0/10 0 NA Dutton & Kinsey (1995) Peanuts Spain 0/38 0 0 Jimenez et al. (1991) Ground South India 0/NA 0 0 Sivaswamy et al. (1991) nuts Peanuts Egypt 1/40 3 NA El-Maghraby & El-Maraghy (1987) Pistachios Egypt 0/NA 0 0 Abdel-Gawad & Zohri (1993) Pistachios Spain 0/32 0 0 Jimenez et al. (1991) Sesame United Kingdom 0/3 0 0 Patel et al. (1996) seeds Sunflower South Africa 0/1 0 NA Dutton & Kinsey (1995) seeds Sunflower Spain 0/35 0 0 Jimenez et al. (1991) seeds Sunflower Russian NA/58 L'vova et al. (1993) seeds Federation Walnuts Egypt 0/NA 0 0 Abdel-Gawad & Zohri (1993) Walnuts Egypt 1/20 5 125 Abdel-Hafez & Saber (1993) Walnuts France 3/60 5 50-450 Jemmali & Mazerand (1980) Fruits and vegetables Banana, India NA 17 000 Chakrabarti & Ghosal (1986) infected Apricot, Egypt 0/3 0 0 Zohri & Abdel-Gawad (1993) dried Fig, Egypt 0/4 0 0 Zohri & Abdel-Gawad (1993) dried Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Job's Japan 8/12 67 39 6-116 Tanaka et al. (1993) tears Job's Japan 7/7 100 133 10-440 Tanaka et al. (1985) tears Mung Papua New 0/1 0 0 Yuwai et al. (1994) beans Guinea Fruit, Uruguay 148/154 96 < 100 Pineiro et al. (1996b) dried 3/154 2 100-200 3/154 2 > 200 Plum, Egypt 0/3 0 0 Zohri & Abdel-Gawad (1993) dried Raisins Egypt 0/3 0 0 Zohri & Abdel-Gawad (1993) Tamarind South India 0/NA 0 0 Sivaswamy et al. (1991) Tomato Egypt 4/15 27 80 000 El-Morshedy & Aziz (1995 Aldicarbg 0/15 0 0 Carbofurang 1/15 7 10 000 Fenamiphosg 2/15 13 25 000 Tomato South India 0/NA 0 0 Sivaswamy et al. (1991) Vegetables, United Kingdom NA/8 6 Patel et al. (1996) tinnedh Vegetables, Uruguay 98/99 99 < 100 Pineiro et al. (1996b) dried 0/99 0 100-200 1/99 1 > 200 Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Spices and herbs Asiseeds South India 0/NA 0 0 Sivaswamy et al. (1991) Chili United Kingdom Patel et al. (1996) Pickle 0/4 0 0 Powder NA/4 4.5-15 Powder, hot NA/3 1-11 Sauce NA/4 7 Coriander United Kingdom NA/3 4-7 Patel et al. (1996) seeds Coriander South India 0/NA 0 0 Sivaswamy et al. (1991) Curry United Kingdom Patel et al. (1996) Mix, dry NA/4 5.2 Paste NA/4 3-4 Powder 0/3 0 0 Curry South India 0/NA 0 0 Sivaswamy et al. (1991) leaves Five-spice United Kingdom NA/4 3-5 Patel et al. (1996) powder Garlic United Kingdom 0/4 0 0 Patel et al. (1996) Garlic United Kingdom NA/4 4 Patel et al. (1996) pickle Ginger United Kingdom 0/4 0 0 Patel et al. (1996) Ginger South India 0/NA 0 0 Sivaswamy et al. (1991) Mint South India 0/NA 0 0 Sivaswamy et al. (1991) leaves Mustard India NA 36 000 Chakrabarti & Ghosal (1987) seed Tandori United Kingdom 0/3 0 0 Patel et al. (1996) Spices, Egypt 0/120 0 0 El-Kady et al. (1995) 24 kinds Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Animal muscle and organs Meat Uruguay 58/58 100 < 100 Pineiro et al. (1996b) products Pig 78-310i James & Smith (1982) liver Chicken Minnesota, USA 59-103j Mirocha et al. (1982) muscle Chicken Minnesota, USA 681k Mirocha et al. (1982) liver Animal Western Canada 0/24 0 0 Prior (1981) tissue Liver Western Canada 0/10 0 0 Prior (1976) and kidney Milk Buttermilk South India 0/NA 0 0 Sivaswamy et al. (1991) Cows' milk South India NA 25 Sivaswamy et al. (1991) Cows' milk Minnesota, USA Mirocha et al. (1981) 7 days after 25 mg/kg diet 1/1 100 210 1 day after 250 mg/kg diet 1/1 100 45 2 days after 250 mg/kg diet 1/1 100 62 Sheep's Minnesota, USA 1-2 Hagler et al. (1980) milkl Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Miscellaneous products Beer, Korean Republic of 0/54 0 0 Shim et al. (1997) and imported Korea Beer Canada 1/NA Scott (1996) Beer, Canada 0/50 0 0 Scott et al. (1993) Canadian and imported Beer Lesotho 17/140 12 300-2000 Martin & Keen (1978) Beer Nigeria 4/4 100 153 49-264 Okoye (1987) µg/brew µg/brew Beer, Zambia NA/23 920 90-4600 Lovelace & Nyathi (1977) opaque maize Cassava Brazil 0 Soares & Rodriguez-Amaya (1989) flour Chili/almond United Kingdom NA/4 5 Patel et al. (1996) oil Cocoa beans Uruguay 69/69 100 < 100 Pineiro et al. (1996b) Corn oil New Zealand 1/1 100 4600 Lauren & Ringrose (1997) Cottonseeds Egypt 0 Mazen et al. (1990) Cottonseed Egypt 0 Mazen et al. (1990) meal Cottonseed Egypt 0 Mazen et al. (1990) cake Fermented Swaziland 6/55 11 8000-5300 Martin & Keen (1978) products Infant Canada 9/60 15 4.2 Roscoe (1998) foods Infant Japan 0/27 0 0 Isohata et al. (1986) foodsm Table 5. (continued) Food Geographical Incidencea Concentration Reference location (µg/kg or µg/L)b D/T % Mean Range Infant Canada 8/13 62 < 3-4.5 Roscoe (1998) mixed cereals Infant Canada 7/8 88 < 3-6.9 Roscoe (1998) soya cereal Phanen Botswana 0/20 0 0 Siame et al. (1998) Ragi South India NA 2056 Sivaswamy et al. (1991) Sesame United Kingdom 0/3 0 0 Patel et al. (1996) oil Sugar Bosch & Mirocha (1992) beets Mouldy field 6/25 24 12-391 Commercial stockpiles 0/10 0 0 Tea, Croatia 0/7 0 0 Halt (1998) herbal Toddy South India NA 765 Sivaswamy et al. (1991) NA, not available a Incidence presented as number of times zearalenone was detected (D) in the total number of samples analysed (T). The percent incidence (D/T × 100) was rounded to the nearest whole number, 0.5 being rounded upwards. b Concentrations above the limit of detection are given as mean values and ranges. The concentrations of zearalenone in foods were reported in several units, including ng/g, ppb, µg/g, mg/kg, ppm, µg/L, and µmol/kg. To allow comparisons of values from different references, most of the values are expressed in µg/kg. c Barley imported from Canada and Sweden; rye imported from Germany, Hungary, the Russian Federation, Sweden, and the USA; wheat imported from Canada, Germany, Hungary, Saudi Arabia, and the USA Table 5. (continued) d Referred to as maize in the cited reference. e Breakfast cereals f Soya products included soya flour, soya bean protein, tofu, and soya sauce. g Tomatoes grown on soil treated with these nematicides h Not further described i Zearalenone at 40 mg/kg feed for four weeks j Zearalenone at 100 mg/kg feed for eight days k Zearalenone at 10 mg/day (5 mg/kg bw) l Sheep received a dose of 1.8 g zearalenone. m Rice sticks, two samples; biscuits, eight samples; gruel, eight samples; rice cake, one sample; apple juice, two samples; vegetable soup, one sample; mixed fruit juice, two samples; tomato juice, one sample; peach juice, one sample; mandarin orange juice, one sample n Larval stage of the Emperor moth The concentrations of zearalenone in the grain products in Table 5, which include breads, breakfast cereals, crackers, cookies, and noodles from six countries, were low or below the limit of detection. The concentrations in legumes, available from 12 references representing nine countries, and in nuts, from 12 references representing 11 countries, were mostly below the limit of detection. The values for fruits and vegetables, available from nine references representing seven countries, were mostly below the limit of detection or low, but a high concentration was found in infected bananas in India. The authors (Chakrabarti & Ghosal, 1986) reported that infected bananas are preferred by the population because of their sweet taste. The concentrations of zearalenone in a number of spices and herbs (four references from three countries) were usually at or below the limit of detection. In five references from three countries, the concentrations of naturally occurring zearalenone in animal tissues were below the limit of detection or low, although the values can be temporarily elevated when animals have been treated with zearalenone (James & Smith, 1982; Mirocha et al., 1982; Annex 1, reference 82; see below for more discussion.) The concentrations in milk are described in only three references from two countries. Zearalenone and its metabolites may occur in the milk of animals fed or dosed with the substance, but the normal concentrations are usually below the limit of detection or low (see below for more discussion). Miscellaneous products were reported in 16 references representing 15 countries, with values below the limit of detection. Of particular interest are the concentrations in beers made from contaminated grains in Africa (Martin & Keen, 1978; Okoye, 1987; Lovelace & Nyathi, 1977). Zearalenone and alpha-and beta-zearalanol have not been found in beers from Canada, Europe (except at 100 µg/L in one French beer), or the Republic of Korea (Okoye, 1987; Scott, 1996; Shim et al., 1997). 3.2 Variables that affect contamination The concentrations of zearalenone may increase in moist grains during storage. The factors that favour its production in foods are generally the same as those that favour the development and growth of Fusarium mould in crops during growth, harvest, and storage. The variables that affect the incidence and concentration of zearalenone in foods are described below. 3.2.1 Weather and climate The results of reports on the effects of rainfall, temperature, and humidity on the concentration of zearalenone in foods are inconsistent, probably because the combination of these variables is difficult to control for. In general, zearalenone production by Fusarium spp. is greater in mouldy samples and is favoured by wet climates (high rainfall) and especially by wet, cool weather. Cliver (1990) reported that the production of zearalenone on corn and other cereals is favoured by temperatures near freezing for an extended time and by cycles of temperature from low to moderate. The effects of moisture content, relative humidity, temperature, and rainfall on mycotoxin production were determined in 130 samples of post-harvest and stored corn in Sao Paulo, Brazil, throughout one year (Pozzi et al., 1995). Fusarium spp. were the main contaminants, and a significant positive correlation was found for the presence of Fusarium with the moisture content of grains and a significant negative correlation with minimum and medium temperatures, rainfall, and relative humidity. The occurrence of mycotoxins (including zearalenone) in wheat, rye, rice, corn, and sunflower seeds collected from commercial batches in four grain-producing areas of the Russian Federation was greater in the humid, southern regions and in Kazakhstan and Uzbekistan (L'vova et al., 1993). The 1990 barley crop in the southern part of the Republic of Korea was reported to have been heavily contaminated with Fusarium mycotoxins because of the high rainfall and humidity during that year (Park et al., 1992). A combined effect of water activity and temperature on zearalenone synthesis in corn has been reported. A constant temperature of 25 °C was most favourable, but both F. graminearum growth and zearalenone production at this temperature were inhibited at a water activity of 0.90. With short incubation times, toxin accumulation was greater at a water activity of 0.97 than at 0.95, but this relationship was inverted with longer incubation (Montani et al., 1988). When Fusarium isolates from wheat were tested for their ability to produce trichothecenes and zearalenone, 12 of 13 isolates of F. culmorum produced zearalenone, with particularly high yields in cultures of seven pathogenic isolates. A higher temperature (20 °C) during the first week of incubation increased the yield (Chelkowski et al., 1984). 3.2.2 Agricultural production methods In 1993, 1.7% of the cereal deliveries in 24 cereal-collecting centres in Switzerland were affected by Fusarium, but only 0.2% were affected in 1994. The concentrations of mycotoxins were not affected by whether the cereals were grown conventionally or without fungicides, insecticides, and bioregulators (Bucheli et al., 1996). The organophosphate nematicide fenamiphos and the carbamate nematicides carbofuran and aldicarb reduced the occurrence of Fusarium spp. on the roots and fruits of tomato plants, and zearalenone production at harvest was inhibited or reduced in comparison with controls (El-Morshedy & Aziz, 1995). In 100 samples of German rye and 101 samples of wheat grown conventionally or ecologically, zearalenone was found in 40 samples, with average concentrations of 6 µg/kg in conventionally grown wheat, 24 µg/kg in alternatively grown wheat, 4 µg/kg in conventionally grown rye, and 51 µg/kg in alternatively grown rye. The highest concentration of zearalenone was 199 µg/kg in alternatively grown rye (Marx et al., 1995). 3.2.3 Varieties and cultivars When grains from 14 inbred and 4 single-cross hybrids of corn were inoculated with three isolates of Gibberella zeae, the hybrids appeared to have less resistance to toxin formation than the inbred varieteies. Analysis of variance indicated a highly significant variation between corn varieties and fungal isolates (Shannon et al., 1980). The zearalenone content of corn subspecies may vary, but no information was available in the literature about the zearalenone content of different types of corn or its products. Corn, also called maize and Indian corn, belongs to the family Gramineae (grass) and is of the genus and species Zea mays. The corn grain consists of an outer hull, the soft endosperm being used for corn flour, the hard endosperm to make corn meal and corn grits, and the soft oily germ for corn oil. A number of subspecies or commercial classifications of corn are based on kernel texture (Yamaguchi, 1983): Dent corn (indentata) has a depression in the crown of the kernel caused by unequal drying of the hard and soft starch making up the kernel. The grains have a corneous endosperm with soft white starch. Flint corn ( indurata) is the field corn characterized by a starchy endosperm. The kernels are large and broad with rounded tops. When they are harvested at the immature stage, they are called roasting ears and are used as a vegetable. Flint corn contains little soft starch. Flour or soft corn ( amylacea) kernels have a soft or floury rather than a vitreous endosperm. This corn is composed largely of soft starch and has soft, mealy, easily ground kernels. Sweet corn ( sacchorata) has grains with a sweetish endosperm at the immature stage. Starch accumulation occurs with maturity, but less sugar is converted to starch than in the other corn types. There are a number of sweet corn varieties, with yellow, white, or black kernels. Popcorn ( everta) is an extreme type of flint corn with small, hard kernels, and a large portion of the endosperm is horny. The ears and kernels are small. When the kernels are heated, the moisture turns to steam and causes an explosion, bursting the seed coat and exposing the white fluffy endosperm. It is devoid of soft starch. 3.2.4 Storage conditions Zearalenone can grow on corn not only in the field but also during storage, especially when the corn has too much moisture when harvested and is not dried properly before storage (Cliver, 1990). In Hungary, 88% of mouldy stored corn samples contained zearalenone; the incidence of contamination and the mycotoxin concentrations were markedly lower in samples that were not mouldy. During harvest, only 17% of samples contained zearalenone (Fazekas et al., 1996). Zearalenone production was inhibited almost completely in high-moisture corn grains kept under atmospheres enriched with 20-60% carbon dioxide with 20 or 5% oxygen. Less carbon dioxide was needed to inhibit fungal development and toxin formation in the presence of less oxygen (Paster et al., 1991). Zearalenone was not detected in amaranth grains at 25 °C with a water activity of 0.902, and maximum accumulation occurred at a water activity of 0.92 at 35 days and 0.95 at 29 days (Bresler et al., (1998). The disappearance of zearalenone from contaminated corn was dependent on the concentration of water, temperature, and the length of exposure. A degradation rate of 84% was seen with 10% water at 80 °C for 16 h and 75% degradation under the same conditions for 8 h; only 3% degradation was seen with 3% water at 50 °C for 2 h (Abd Alla, 1997). In a study of the production of zearalenone in corn by seven isolates of Fusarium under different conditions of water activity, temperature, and incubation time, two isolates of F. graminearum were the most active. The culture conditions that resulted in the highest yields were a water activity of 0.97, two weeks' incubation at 28 °C, and 40 days' incubation at 12 °C. These conditions were also optimal for two isolates of F. oxysporum, but for the remaining isolates the maximum concentrations of zearalenone were obtained at room temperature and 30 days' incubation. At 37 °C, zearalenone was not detected under any of the conditions assayed (Jimenez et al., 1996). Six of 25 mouldy sugar beet rot samples collected in the field contained zearalenone at concentrations of 12-390 µg/kg, whereas 10 samples from commercial stockpiles showed no activity (Bosch & Mirocha, 1992). Stored rice was comparatively resistant to contamination with mycotoxins, none being found in 208 samples; however, zearalenone was detected in some samples subjected to experimental self-heating (L'vova et al., 1984). The only mycotoxin found in wheat maintained at 15 and 22 °C for 10 weeks was ochratoxin A (Abramson et al., 1982). Zearalenone was found in both cobs and grain of freshly harvested corn with 26-35% humidity. Maximum amounts were found in cobs when the temperature reached 30-45 °C due to self-heating, and the highest concentra-tion of zearalenone was found on days 8-12 after the onset of self-heating (L'vova et al., 1981). 3.2.5 Gamma irradiation Gamma-irradiation greatly reduced the natural occurrence of Fusarium mycotoxins in wheat, flour, and bread, the zearalenone concentrations being reduced from 28-42 µg/kg to 20 µg/kg in wheat and from 95 to 45 µg/kg in flour after exposure to 4 kGy. A sharp drop in Fusarium toxin concentrations occurred at 5 kGy, and all were eliminated at 6 or 8 kGy (Aziz et al., 1997). Significant reductions in the zearalenone concentration of corn were found after gamma irradiation at 10 or 20 kGy, with no significant interaction between radiation dose and grain moisture level (Hooshmand & Klopfenstein, 1995). At 9 kGy, neither mycotoxin growth nor toxin production could be detected in corn or rice inoculated with F. graminearum or F. tricinctum (Halasz et al., 1989). 3.2.6 Grain preservatives and disinfectants Strains of F. semitectum that produced zearalenone were found in amaranth grains both before and after surface disinfection (Bresler et al., 1995). In a study of the effects of the grain preservatives Luprosil (propionic acid) and Gasol (organic acids with other compounds) on the growth of mycelium and the zearalenone content of stored oats infected by Fusarium, both preservatives completely prevented the growth of a visible mycelium. Luprosil had no influence on the toxin content of the oats, but Gasol decreased the percentage of the total toxin by 60% in three days, 85% in 14 days, and 90% in 28 days (Kallela & Saastamoinen, 1981a). Luprosil and Gasol completely prevented visible mycelium growth of F. graminearum on contaminated oats, wheat, and barley, and Gasol, but not Luprosil, reduced the amount of zearalenone in the crops (Kallela & Saastamoinen, 1981b). The degree to which zearalenone in milled grains was destroyed by Gasol depended on the dose applied. A dose twice that recommended destroyed all the toxin in the grains, whereas one-eighth of the recommended dose prevented the growth of the fungus but only slightly reduced the amount of toxin at onset. Later, the growth of the fungus was more vigorous and significantly more toxin was present in the treated grain than in the grain that had not been treated with Gasol (Kallela & Saastamoinen, 1982). No aflatoxin or zearalenone was reported in grains that had been treated with dichlorvos at 20 mg/kg, whereas untreated samples contained zearalenone at an average concentration of 150 000 µg/kg (Rao & Harein, 1973). 3.2.7 Food processing, preparation, and cooking Like most mycotoxins, zearalenone is heat-stable, and decomposition during cooking or processing is therefore unlikely (Fink-Gremmels, 1989). No change was observed when pure zearalenone was heated for 4 h at 120 °C, and when it was present in ground corn no decomposition was seen after 44 h at 150 °C (Gilbert, 1989). Wet milling of contaminated corn concentrated zearalenone in the gluten fraction by two-to sevenfold; some remained in the soluble fraction but almost none in the starch fraction (Bennett et al., 1978a,b). After corn grown in New Zealand had been passed through a commercial wet-milling plant, only 600 µg/kg was present in concentrated steep liquor whereas 2200-4800 µg/kg were present in the germ, fibre, and gluten fractions (Lauren & Ringrose, 1997). Dry-milling of corn resulted in recovery of 10-20% zearalenone in grits (Bennett et al., 1976). Processing of rice grain resulted in a substantial reduction in the concentration of zearalenone, by 88% in rice groats and 37% in cooked rice (L'vova et al., 1984). About 60% of zearalenone survives bread baking, 40-50% survives noodle manufacture, and 80% remains after biscuit manufac-ture (Gilbert, 1989). Zearalenone has been reported in beers from Lesotho, Swaziland, and Zambia (Lovelace & Nyathi, 1977; Martin & Keen, 1978; Okoye, 1987). The mean concentration of zearalenone that passed from mouldy guinea-corn into native Nigerian beer ( burukutu) was about 51% of that in the starting mixture, suggesting moderate stability during fermentation. About 12% of the zearalenone was discarded in the solid residue (Okoye, 1987). 3.2.8 Residues in animal tissues The amount of detectable zearalenone in animal tissues depends on the contamination of feed, treatment of animals with zearalenone or alpha-zearalanol, duration of exposure to the toxin, the persistence of zearalenone in the animal, and species variation in response to the mycotoxin. Few attempts have been made to detect zearalenone in animal products or to determine residue rates (Kuiper-Goodman et al., 1987; Gilbert, 1989). 3.2.8.1 Muscle and organ meats Plasma clearance of implanted alpha-zearalanol in cattle was rapid and the drug did not accumulate appreciably in any edible tissue. After 65 days, no residues could be detected (Sharp & Dyer, 1972). The concentration of zearalenone in liver from a pig given feed containing zearalenone at 40 mg/kg for four weeks was 78-128 µg/kg (James & Smith, 1982). Zearalenone was found in 23 samples of piglet liver and 16 pig milk samples from animals with mycotoxicosis or fed mouldy feed (Sandor, 1984). Chickens fed feed containing 100 mg/kg zearalenone for eight days had concentrations of 59-103 µg/kg in muscle and up to 681 µg/kg in liver (Mirocha et al., 1982). Substantial residues of zearalenone and its metabolites were found in the livers of chickens during the first 24 h after exposure to [14C]zearalenone, but the amount of radiolabel declined rapidly thereafter. The lowest concentrations were found in skeletal muscle (Dailey et al., 1980). The maximum residue limits recommended by the Committee at its thirty-second meeting for use of zeranol (alpha-zearalanol) as a veterinary drug were 10 µg/kg in bovine liver and 2 µg/kg in bovine muscle (Annex 1, reference 80). When recommending maximum residue limits for veterinary drugs, the Committee uses food factors of 100 g/day for liver and 300 g/day for muscle in calculating a theoretical maximum daily intake to ensure that, when used according to good practice in the use of veterinary drugs, the intake would not exceed the ADI. On the basis of the recommended maximum residue limits and these food factors, the theoretical maximum daily intake of alpha-zearalanol is 1.6 µg/day. 3.2.8.2 Eggs Eggs accumulated metabolites of zearalenone in the yolks, even after 94% of the dose had been eliminated in excreta (Dailey et al., (1980). In an experiment to determine the effects of corn and grain sorghum on the performance of laying hens, egg production decreased significantly and lesions were more severe in hens fed grain sorghum than in those fed corn. Analysis of the grains revealed the presence of low concentrations of zearalenone and other mycotoxins in the sorghum (Branton et al., 1989). 3.2.8.3 Milk Experimental studies have shown some transmission of zearalenone and alpha-and beta-zearalanol into the milk of sheep (Hagler et al., 1980), cows (Mirocha et al., 1981), and pigs (Pullar & Lerew, 1937; Miller et al., 1973; Kurtz & Mirocha, 1978; Palyusik et al., 1980; Vanyi et al., 1983) given high concentrations of zearalenone. Once administration was stopped, the concentrations in milk dropped sharply, although the compound was still detectable after five days in sheep milk (Hagler et al., 1980) and pig milk (Palyusik et al., 1980). Dairy cattle fed a diet containing 25 mg/kg of zearalenone for seven days excreted only 1.3 mg/kg of zearalenone and its metabolites in milk, indicating that milk is not a likely source of zearalenone residues (Mirocha et al., 1981, 1982). The milk of one cow given 6000 mg zearalenone (equivalent to 12 mg/kg bw) contained maximum concentrations of 6.1 µg/L zearalenone, 4 µg/L alpha-zearalenol, and 6.6 µg/L beta-zearalenol. Neither zearalenone nor its metabolites was found in the milk (< 0.5 µg/L) of three lactating cows fed 50 or 165 mg zearalenone (equivalent to 100 or 330 µg/kg bw per day) for 21 days (Prelusky et al., 1990). No residue of zearalenone was found in animal products after administration of lower dietary concentrations (Shreeve et al., 1979; Young et al., 1982). None was detected in several normal pig milk samples (Palyusik et al., (1980), and zearalenone has not been detected in normal retail milk samples, although only limited surveillance has been undertaken (Gilbert, 1989). Only minimal transmission of zearalenone to bovine milk has been demonstrated under realistic concentrations of exposure (Krska, 1999). 3.3 Regulation, control, and monitoring Owing to the huge amounts of corn that are found in world trade, contamination by zearalenone is of economic relevance, and reliable means of control are needed. As the risk of contamination is a criterion of quality in trade, especially for corn and corn products, many cereal companies include analysis for zearalenone in their internal quality control of corn production. Livestock producers and food and feed processors are concerned by the presence of zearalenone in corn because their competitiveness and profitability depend on control of mycotoxins in animal diets. Because mould-damaged corn is often used in animal feed, the risk for zearalenone intoxication is highest for farm animals. Six countries--Austria, Brazil, France, Romania, the Russian Federation, and Uruguay--have set maximum tolerated concentrations of zearalenone at 30-1000 µg/kg in some or all foods, and three countries--Cyprus, Hungary, and The Netherlands-- have set maximum tolerated concentrations for all mycotoxins at 0-0.5 µg/kg in some foods. Questions have been raised, however, about the rationale used by governments to regulate zearalenone and about the implementation of guidelines in different countries (Krska, 1999). The only country that has provided a rationale for setting limits for mycotoxins (other than aflatoxins) in human foods and animal feeds is Canada, where risk assessments have been performed for deoxynivalenol, zearalenone, and ochratoxin A (Van Egmond, 1993). A pilot study for monitoring mycotoxin contamination of foods and feeds was implemented in Uruguay with technical assistance from FAO to determine the potential hazard of food and feed contaminants (Pineiro et al., 1996b). The principal commodities were wheat, barley, rice, corn, soya, dairy products, feeds, dried fruits, dried legumes, oil seeds, cocoa beans, and organ meats, and zearalenone was included among the mycotoxins analysed. The results for 1993-95 showed that feed had the highest concentrations of mycotoxins, but the regulatory limits for toxins were exceeded by less than 3% in wheat, 9% in barley, and 7% in rice samples. 3.4 Dietary intake Only three reports are available of human dietary intake of zearalenone: two from Canada (Kuiper-Goodman et al., 1987; Canada, 1999) and one from the Nordic countries (Eriksen & Alexander, 1998). Estimates are provided here for the US population and for the five regional diets established by the WHO Global Environment Monitoring System-Food Contamination Monitoring and Assessment Programme (GEMS/Food). Guidelines are given for a model of exposure to zearalenone. 3.4.1 Estimates for Canada, 1987 Kuiper-Goodman et al. (1987) estimated the intake of zearalenone by Canadians on the basis of the assumption that the mean concentration of zearalenone in corn used for corn-based breakfast cereals was 39.3 µg/kg. The estimated daily intake of zearalenone from the consumption of corn cereals containing 33 µg/kg, popcorn containing 18.6 µg/kg, and beefsteak by the highest consumption group, 12-19-year-old males, is shown in Table 6. The estimated average daily exposure to zearalenone from corn cereals for the whole group and for eaters and 90th percentile consumers was 0.12, 1.2, and 2.5 µg/person, respectively, and the intake from popcorn was estimated to be 0.06, 0.52, and 1.3 µg/person, respectively. The total from all three sources for the whole group was 0.19 µg/day or 0.003 µg/kg bw per day. Kuiper-Goodman et al. (1987) also calculated the intake of zearalenone for 1-4-year-old children (Table 7). On the basis of body weight, the children's greatest exposure to zearalenone was from corn cereals. Exposure to zearalenone in other foods such as wheat, flour, or milk could increase the estimates, and exposure to estrogens from other sources would add to the estrogenic burden. The authors recommended that exposure to other sources of related estrogens (such as alpha-zearalanol in milk) be estimated. Table 6. Zearalenone intake of Canadian males aged 12-19 years, 1987 Food Zearalenone Food Zearalenone intake concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Corn cereals 33 All persons 3.6 0.12 0.002 Eaters only 37 1.2 0.020 90th percentile eaters 76 2.5 0.042 Popcorn 19 All persons 3 0.06 0.001 Eaters only 28 0.52 0.009 90th percentile eaters 72 1.3 0.022 Beefsteak 1.0 All persons 13 0.01 0.000 Eaters only 150 0.15 0.003 90th percentile eaters 340 0.34 0.006 Total 0.19 0.003 Adapted from Kuiper-Goodman et al. (1987); body weight, 60 kg The consumption figures for cereals and beefsteak were obtained from the Nutrition Canada Survey conducted by Health and Welfare Canada, and those for popcorn were obtained from a food consumption survey of the US Department of Agriculture. 3.4.2 Estimates for Canada, 1999 The estimated intake of zearalenone by 60-kg Canadian adults (Table 8) is based on the concentrations of zearalenone in 10 products and the estimated consumption of six of those foods. The estimated mean intake of zearalenone was < 0.98 µg/day or < 0.016 µg/kg bw per day. The contributions of the foods to the daily intake of zearalenone were 30% from hard wheat, 30% from amber durum wheat, 20% from corn, 15% from rice, 4% from barley, 4% from soft wheat, 3% from cornmeal products, and none from oats, soya beans, or tinned beans. The estimated intake of zearalenone by infants (Table 9) is based on the concentrations of zearalenone in infant cereal, infant formula, and creamed corn and the consumption of these products by infants aged 6-9 months. The daily estimated mean intake was < 0.52 µg or < 0.06 µg/kg bw. The intake comprises 60% from infant formula, 23% from infant cereals, and 17% from creamed corn. 3.4.3 Estimates for Denmark, Finland, Norway, and Sweden Eriksen & Alexander (1998) calculated the average daily intake of zearalenone on the basis of the intake of wheat, rye, barley, and oats derived from food balance sheets. The intakes were 0.48 µg/day (0.01 µg/kg bw per day) in Denmark; 1.2 µg/day (0.02 µg/kg bw per day) in Sweden; 1.3 µg/day (0.02 µg/kg bw per day) in Finland; and 1.5 µg/day (0.02 µg/kg bw per day) in Norway. Because the information on food intake on balance sheets reflects the national concentration per capita, results based on this information are probably over-estimates. When food consumption data derived from individual quantitative questionnaires were used to calculate the daily intake of zearalenone from the same commodities in Denmark and Norway, the average daily intakes were 1.2 µg/day (0.02 µg/kg bw per day) in Denmark and 1.1 µg/day (0.02 µg/kg bw per day) in Norway. It is curious that the average daily intake for Denmark was higher when individual intake data were used than when the data from balance sheets were used. Table 7. Zearalenone intake of Canadian children aged 1-4 years, 1987 Food Zearalenone Food Zearalenone intake concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Corn cereals 33 All children 2.2 0.07 0.005 Eaters only 21 0.69 0.050 90th percentile eaters 76 2.5 0.042 Popcorn 19 All children 1.1 0.02 0.001 Eaters only 11 0.21 0.015 90th percentile eaters 18 0.33 0.023 Beefsteak 1.0 All children 3.4 0.00 0.000 Eaters only 46 0.05 0.003 90th percentile eaters 110 0.11 0.008 Milk 1.0 All children 380 0.38 0.027 Eaters only 670 0.67 0.047 90th percentile eaters 950 0.95 0.066 Total intake of all 0.47 0.033 children Total intake of eaters 1.6 0.12 only Adapted from Kuiper-Goodman et al. (1987); body weight, 14 kg. Consumption figures for cereals and beefsteak were obtained from the Nutrition Canada Survey conducted by Health and Welfare Canada, and those for popcorn were obtained from a food consumption survey of the US Department of Agriculture. Table 8. Zearalenone intake of 60-kg Canadian adults, 1999 Food Zearalenone Food Zearalenone intake concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Barley < 8.3 4.7 < 0.039 < 0.001 Beans, tinned < 10 NR Cornmeal or flour productsa < 13 2.0 < 0.025 0.000 Corn, kernel or cob < 41 4.8 < 0.2 < 0.003 Oats < 5.0 NR Rice < 7.4 20 < 0.15 < 0.003 Soya beans < 10 NR Wheat, amber durum < 3.7 64 < 0.24 < 0.004 Wheat, hard < 5.6 53 < 0.3 < 0.005 Wheat, soft < 7.4 5.3 < 0.039 < 0.001 Table 8. Zearalenone intake of 60-kg Canadian adults, 1999 Food Zearalenone Food Zearalenone intake concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Total < 0.98 < 0.016 Adapted from Canada (1999); NR, not reported; assumed to be insignificant a Includes tortillas, natchos, and other corn snacks and products 3.4.4 Estimates for the USA (eaters only) Estimates for the intake of zearalenone in US diets are based on food intake data from the US Department of Agriculture Continuing Survey of Food Intakes by Individuals for 1989-91 (Krebs-Smith et al., 1997). The survey derived information on dietary intake information from a one-day recall and from a two-day food record for 11 488 individuals. Table 10 gives the daily intake of foods likely to contain zearalenone for individuals who reported eating the food on one or more days, as mean intakes for eaters who were 2 years of age and older and for men aged 20-39 years and the 90th percentile intakes of men aged 20-39. In the absence of more reliable data, the concentrations of zearalenone in foods in the 1999 Canadian estimates were also used for the USA, with the addition of a value of 10 µg/kg for popcorn since no value for this commodity was included in the Canadian estimates. The concentration of zearalenone in durum wheat was used for pasta, the value for soft wheat was used for cakes, doughnuts, or sweet rolls, and the value for hard wheat was used for the other grain products. Because wheat is the primary ingredient in biscuits, yeast bread, pasta, and rolls, the value for wheat was used directly. Since the other grain products contain ingredients other than wheat, such as sugar, shortening, eggs, milk, fruit, cheese, and meat, the consumption figures were reduced by one-half in order to avoid overestimating the zearalenone intake from these products. For several of the grain products (cooked cereal, ready-to-eat cereals, and tortillas), the type of grain was not specified. Cooked cereals in the USA include wheat and rice, oatmeal being counted separately; ready-to-eat cereals are made of wheat, corn, oats, and rice; and tortillas are made of either flour or corn. The concentrations of zearalenone in hard wheat were used for these products as it was suspected that most are wheat-based. 3.4.5 Estimates for the USA, all persons Table 11 shows the average daily intakes of foods in the USA by all persons aged 2 years or older and for men aged 20-39 years, on the basis of the food composition values used for eaters only. The zearalenone intakes of all persons are additive, whereas those for eaters only are not since they would not be expected to consume all the foods listed on a given day. Table 9. Zearalenone intake of 8.7-kg Canadian infants, 6-9 months of age, 1999 Food Zearalenone Food Zearalenone intake concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Infant cereal < 3.4 35 < 0.12 < 0.014 Infant formula < 3.0 100 < 0.31 < 0.036 Creamed corn (vegetable) < 3.0 30 < 0.089 < 0.010 Total < 0.52 < 0.060 Adapted from Canada (1999) 3.4.6 Limitations of estimates The estimates of dietary intake of zearalenone given by Kuiper-Goodman et al. (1987) were based on only three foods for men and four for young children and did not include values for cornmeal or cornflour products, corn kernels, rice, or wheat products. The estimates are therefore probably low, as indicated by comparing these results with those in the more recent Canadian study (Table 12), which include seven foods in adult diets and three in infant diets. Although the figures from the 1999 Canadian study were used for the US estimates, these are higher than the ones for Canada, perhaps because more food products were included and as a result of the overestimates of zearalenone in finished grain products, especially those of wheat. The zearalenone concentration in wheat was assigned to the full weight of some wheat products and half the weight of products with other ingredients, which may have been too generous an assumption, but the fraction of wheat in all finished wheat products is difficult to estimate. Accurate estimates of dietary intake of zearalenone are hampered by the following limitations: * Lack of reliable data on the zearalenone content of foods as consumed, as the values for zearalenone given in the literature (Table 5) are usually for raw commodities. It is difficult to predict the percent of zearalenone that will remain in a finished product and to estimate the contribution of a cereal grain to a finished grain product, as finished products have added ingredients like water and sugar that add to their weight and dilute the zearalenone. * Lack of plans for random sampling of commodities to be analysed for zearalenone and inadequate numbers of samples. If the foods to be analysed are selected because they are mouldy, damaged, or off-color or because of a particularly wet or cool harvest season, the incidence and concentration of zearalenone in the foods will be higher than under normal circumstances. * In many studies, it is not clear if the values given are for the raw or prepared food or for the entire grain or only the edible portion, and it is often impossible to determine the subspecies or cultivar of rice, wheat, rye, barley, or corn or the extent of processing or milling. * No guideline or standard protocol is available for using the percent incidence and the mean concentration of positive samples in estimating the potential intake of zearalenone from a food. If the incidence of contamination is high, the mean concentration of positive samples may be used in making estimates, but if the incidence is low or if only a few samples were analysed, use of the mean of positive samples may overestimate the intake of zearalenone. Furthermore, the percent incidence of contamination with zearalenone is related to the sensitivity of the analytical method used. * Lack of information on the effects of processing and cooking on the zearalenone content. Although the heat stability and water insolubility of zearalenone are indicated in several references, the effects on the concentration of zearalenone of fractionating grains and using only portions for food are not clear. Data from various references (Table 5) generally show lower concentrations of zearalenone in grain products and in milled and polished grains than in the raw commodities. Table 13 shows that corn, corn products, and wheat products are important sources of zearalenone in Canada and the USA. Although the concentrations are probably higher in corn and corn products, wheat products are consumed in larger amounts in both countries and thus contribute more zearalenone to the daily intake. In the Nordic countries, the main sources of zearalenone were wheat, rye, and oats. Kuiper-Goodman et al. (1987) did not consider the contributions of wheat products and rice to zearalenone intake in their estimates of dietary intake. Table 14 shows the dietary intake of zearalenone in the five WHO GEMS/Food regional diets, Middle Eastern, Far Eastern, African, Latin American, and European, the last covering the diets in Australia, Europe, and the USA. Only the grain and legume groups were considered in assessing the dietary intake of zearalenone because it does not occur to any large extent in the other food groups. As the zearalenone concentrations for the foods were the same as those used for the Canadian dietary assessment presented in Table 8 and the US dietary assessment presented in Tables 10 and 11, the variables in the regional diets were different intake levels of grains and legumes. Table 14 shows that the daily intake of zearalenone is < 3.5 µg in the Middle East, < 3.3 µg in the Far East, < 2.5 µg in Africa, < 2.2 µg in Latin America, and 1.5 µg in Europe. The European intake (< 1.4 µg/day) is comparable to that estimated for the USA (< 1.7 µg/day) in Table 12. The higher intakes in the other regions are probably due to higher intakes of grains and legumes, as shown in Table 14. The intakes, expressed per kilogram of body weight, are < 0.059 µg for the Middle East, < 0.056 µg for the Far East, < 0.041 µg for Africa, < 0.036 µg for Latin America, and < 0.025 µg for Europe. The European intake is comparable to that of Canada (< 0.016 µg/kg per day), the Nordic countries (0.02 µg/kg per day), and the USA (< 0.030 µg/kg per day). Table 15 shows the contributions of the grain and legume commodities to the total intake of zearalenone in the regional diets. The main sources were maize or corn (31%) and wheat (52%) in the Middle Eastern diet; rice (62%) in the Far Eastern diet; maize or corn Table 10. Estimates of zearalenone intake of 70-kg men in the USA, eaters only Food Zearalenone Food Zearalenone intake Age (years) concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Corn < 41 All > 2 86 < 3.5 Men 20-39 110 < 4.5 < 0.064 Men 20-39, 90th 230 < 9.3 < 0.13 percentile Corn chips < 13 All > 2 42 < 0.53 Men 20-39 54 < 0.69 < 0.010 Men 20-39, 90th 100 < 1.3 < 0.019 percentile Popcorn approx. 10 All > 2 37 0.37 Men 20-39 49 0.49 0.007 Men 20-39, 90th 86 0.86 0.012 percentile Oatmeal < 5 All > 2 250 < 1.2 Men 20-39 320 < 1.6 < 0.023 Men 20-39, 90th 490 < 2.4 < 0.035 percentile Table 10. (continued) Food Zearalenone Food Zearalenone intake Age (years) concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Rice < 7.4 All > 2 170 < 1.2 Men 20-39 220 < 1.6 < 0.023 Men 20-39, 90th 410 < 3.0 < 0.043 percentile Wheat productsa Biscuits < 5.6 All > 2 58 < 0.32 Men 20-39 76 < 0.43 < 0.006 Men 20-39, 90th 120 < 0.69 < 0.010 percentile Bread, yeast < 5.6 All > 2 72 < 0.40 Men 20-39 92 < 0.52 < 0.007 Men 20-39, 90th 180 < 0.98 < 0.014 percentile Wheat productsb Pasta < 3.7 All > 2 120 < 0.46 Men 20-39 180 < 0.65 < 0.009 Men 20-39, 90th 370 < 1.4 < 0.019 percentile Wheat productsc Cake < 7.4 All > 2 45d < 0.33 Men 20-39 52d < 0.38 < 0.005 Men 20-39, 90th 95d < 0.70 < 0.010 percentile Table 10. (continued) Food Zearalenone Food Zearalenone intake Age (years) concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Doughnuts and < 7.4 sweet rolls All > 2 41d < 0.30 Men 20-39 50d < 0.37 < 0.005 Men 20-39, 90th 85d < 0.63 < 0.009 percentile Wheat productse Cookies < 5.6 All > 2 21d < 0.12 Men 20-39 30d < 0.17 < 0.002 Men 20-39, 90th 66d < 0.37 < 0.005 percentile Crackers < 5.6 All > 2 12d < 0.07 Men 20-39 14d < 0.08 < 0.001 Men 20-39, 90th 24d < 0.13 < 0.002 percentile Pancakes and waffles < 5.6 All > 2 50d < 0.28 Men 20-39 75d < 0.42 < 0.006 Men 20-39, 90th 120d < 0.67 < 0.010 percentile Pie < 5.6 All > 2 80d < 0.45 Men 20-39 87d < 0.49 < 0.007 Men 20-39, 90th 140d < 0.80 < 0.011 percentile Table 10. (continued) Food Zearalenone Food Zearalenone intake Age (years) concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Pizza < 5.6 All > 2 100d < 0.57 Men 20-39 160d < 0.87 < 0.012 Men 20-39, 90th 280d < 1.6 < 0.022 percentile Quickbreads and muffins < 5.6 All > 2 43d < 0.24 Men 20-39 54d < 0.30 < 0.004 Men 20-39, 90th 110d < 0.62 < 0.009 percentile Mixed grainsa Cereal, cooked < 5.6 All > 2 250 < 1.4 Men 20-39 340 < 1.9 < 0.027 Men 20-39, 90th 500 < 2.8 < 0.040 percentile Cereals, < 5.6 ready-to-eat All > 2 50 < 0.28 Men 20-39 73 < 0.41 < 0.006 Men 20-39, 90th 110 < 0.63 < 0.009 percentile Tortillas < 5.6 (wheat and corn) All > 2 60 < 0.34 Men 20-39 87 < 0.49 < 0.007 Men 20-39, 90th 190 < 1.0 < 0.015 percentile a Zearalenone value for hard wheat used b Zearalenone value for durum wheat used c Zearalenone value for soft wheat used with one-half of the consumption value d One-half of the consumption figure reported by the US Department of Agriculture e Zearalenone value for hard wheat used with one-half of the consumption value Table 11. Estimates of zearalenone intake in the USA, all persons Food Zearalenone Food Zearalenone intake Age (years) concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Corn, vegetable < 41 All > 2 12 < 0.49 Men 20-39 13 < 0.53 < 0.008 Corn chips < 13 All > 2 2 < 0.025 Men 20-39 4 < 0.050 < 0.001 Popcorn approx. 10 All > 2 2 0.020 Men 20-39 2 0.020 0.000 Oatmeal < 5 All > 2 12 < 0.060 Men 20-39 8 < 0.040 < 0.001 Rice < 7.4 All > 2 25 < 0.18 Men 20-39 38 < 0.28 < 0.004 Wheat productsa Biscuits < 5.6 All > 2 3 < 0.017 Men 20-39 4 < 0.022 0.000 Bread, yeast < 5.6 All > 2 59 < 0.33 Men 20-39 76 < 0.43 < 0.006 Rolls < 5.6 All > 2 15 < 0.084 Men 20-39 23 < 0.13 < 0.002 Wheat productsb Pasta < 3.7 All > 2 31 < 0.12 Men 20-39 44 < 0.16 < 0.002 Wheat productsc Table 11. (continued) Food Zearalenone Food Zearalenone intake Age (years) concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Cake < 7.4 All > 2 4d < 0.03 Men 20-39 4d < 0.03 0.000 Doughnuts and sweet < 7.4 rolls All > 2 3d < 0.022 Men 20-39 3d < 0.022 0.000 Wheat productse Cookies < 5.6 All > 2 4d < 0.022 Men 20-39 4d < 0.022 0.000 Crackers < 5.6 All > 2 1d < 0.006 Men 20-39 1d < 0.006 0.000 Pancakes and waffles < 5.6 All > 2 3d < 0.017 Men 20-39 3d < 0.017 0.000 Pie < 5.6 All > 2 4d < 0.022 Men 20-39 3d < 0.017 0.000 Pizza < 5.6 All > 2 10d < 0.056 Men 20-39 18d < 0.10 < 0.001 Quickbreads and < 5.6 muffins All > 2 3d < 0.017 Men 20-39 2d < 0.011 0.000 Table 11. (continued) Food Zearalenone Food Zearalenone intake Age (years) concentration intake (µg/kg) (g/day) µg/day µg/kg bw per day Mixed grainsa Cereal, cooked < 5.6 All > 2 20 < 0.11 Men 20-39 14 < 0.078 < 0.001 Cereals, ready-to-eat < 5.6 All > 2 14 < 0.078 Men 20-39 14 < 0.078 < 0.001 Tortillas (wheat < 5.6 and corn) All > 2 5 < 0.028 Men 20-39 9 < 0.050 < 0.001 Total All > 2 < 1.7 Men 20-39 < 2.1 < 0.030 a Zearalenone value for hard wheat used b Zearalenone value for durum wheat used c Zearalenone value for soft wheat used with one-half of the consumption value d One-half of the consumption figure reported by the US Department of Agriculture e Zearalenone value for hard wheat used with one-half of the consumption value Table 12. Summary of estimates of dietary intake of zearalenone Country Body Zearalenone Reference weight (kg) µg/day µg/kg bw per day Canada Kuiper-Goodman 12-19-year-old males 60 0.19 0.003 et al. (1987) 1-4-year-old children 14 0.47 0.033 Canada Canada (1999) Adults 60 < 0.98 < 0.016 6-9-month-old infants 8.7 < 0.52 < 0.060 Nordic countriesa Eriksen & Alexander Denmark 48 0.48 0.01 (1998) Finland 66 1.3 0.02 Norway 73 1.5 0.02 Sweden 60 1.2 0.02 Nordic countriesb Eriksen & Alexander Denmark 60 1.2 0.02 (1998) Norway 60 1.1 0.02 USA This monograph All aged > 2 years < 1.7 20-39-year-old men 70 < 2.1 < 0.030 a Data from balance sheets b Data on individual intake (55%) and rice (31%) in the African diet; maize or corn (27%), rice (30%), and wheat (30%) in the Latin American diet; and wheat (67%) in the European diet. 3.4.7 Models of dietary intake In order to improve calculations of dietary intake of zearalenone, more reliable data are needed on the incidence and concentration of the toxin in foods, especially in grain products as consumed and in foods that have high concentrations and are commonly consumed by some populations, such as corn-based beer in Africa and infected bananas in India. Differences in the zearalenone content of foods in western and developing countries must also be determined. The following conclusions can be drawn with regard to dietary exposure to zearalenone. * The concentrations of zearalenone in animal products (meat, fish, poultry, milk, and eggs) are probably not significant. * There appears to be only minimal transmission of zearalenone into the milk of dairy cows exposed to zearalenone. * The concentrations of zearalenone in fruits, vegetables, and nuts are not significant, with the exception of infected bananas consumed in India. * Humans do not usually eat mouldy foods except in conditions of poverty or famine. * Unless legumes are consumed in large amounts or are known to be contaminated, they probably will not affect estimates significantly. * Grains and grain products are probably the main sources of dietary zearalenone intake. * Because the intake of grains and grain-based products may be higher in developing countries, intake of zearalenone may be higher than in developed countries. The grains that should be included in an intake assessment are those consumed in fair to large amounts by the population, but grains that are known or suspected to be contaminated should also be included even if they are consumed in small amounts. The data in Table 5 indicate that the zearalenone concentrations in barley, oats, rice, and rye are low, and they could be omitted from intake calculations unless they are consumed in large amounts or are contaminated. Table 14. Dietary intake of zearalenone in regional diets Food item Zearalenone Middle Far Eastern African Latin American European concentration Eastern (µg/kg) Food Zearalenone Food Zearalenone Food Zearalenone Food Zearalenone Food Zearalenone (g/day) (µg/day) (g/day) (µg/day) (g/day) (µg/day) (g/day) (µg/day) (g/day) (µg/day) Grains Barley < 8.3 1.0 < 0.008 3.5 < 0.029 1.8 < 0.015 6.5 < 0.054 20 < 0.16 Buckwheat approx. 0 0.0 approx. 1.0 approx. 0.0 approx. 0.0 approx. 0.0 approx. 0.000 0.000 0.000 0.000 0.000 Maize < 41 16 < 0.67 0.0 0.000 0.0 0.000 1.5 < 0.061 0.0 0.000 Maize < 13 32 < 0.40 31 < 0.40 110 < 1.3 40 < 0.51 8.8 < 0.11 flour Sweet approx. 0 0.0 approx. 0.0 approx. 7.7 approx. 0.0 approx. 14 approx. corn 0.000 0.000 0.000 0.000 0.000 Popcorn approx. 10 0.2 < 0.002 0.2 < 0.002 0.2 < 0.002 0.2 < 0.002 0.2 < 0.002 Millet approx. 0 2.5 approx. 9.3 approx. 52 approx. 0.0 approx. 0.0 approx. flour 0.000 0.000 0.000 0.000 0.000 Oats < 5.0 0.0 0.000 0.0 0.000 0.2 < 0.001 0.8 < 0.004 2.0 < 0.010 Rice < 7.4 49 < 0.36 280 < 2.1 100 < 0.76 86 < 0.64 12 < 0.087 Rye approx. 0 0.0 approx. 1.0 approx. 0.0 approx. 0.0 approx. 1.5 approx. 0.000 0.000 0.000 0.000 0.000 Sorghum approx. 0 2.0 approx. 9.7 approx. 27 approx. 0.0 approx. 0.0 approx. flour 0.000 0.000 0.000 0.000 0.000 Triticale approx. 0 0.0 approx. 1.0 approx. 0.0 approx. 0.0 approx. 0.0 approx. flour 0.000 0.000 0.000 0.000 0.000 Wheat, < 5.6 0.3 < 0.002 0.0 0.000 0.0 0.000 0.0 0.000 0.0 0.000 bulgur Wheat, < 3.7 1.0 < 0.004 0.3 < 0.001 0.0 0.000 2.8 < 0.010 1.3 < 0.005 pasta Wheat < 7.4 3.0 < 0.022 0.5 < 0.004 0.0 0.000 2.0 < 0.015 1.0 < 0.007 pastry Wheat approx. 0 0.1 approx. 0.1 approx. 0.0 approx. 0.0 approx. 0.0 approx. germ 0.000 0.000 0.000 0.000 0.000 White < 5.6 220 < 1.2 76 < 0.43 19 < 0.11 37 < 0.21 120 < 0.66 bread Table 14. (continued) Food item Zearalenone Middle Far Eastern African Latin American European concentration Eastern (µg/kg) Food Zearalenone Food Zearalenone Food Zearalenone Food Zearalenone Food Zearalenone (g/day) (µg/day) (g/day) (µg/day) (g/day) (µg/day) (g/day) (µg/day) (g/day) (µg/day) Wholemeal < 5.6 110 < 0.60 38 < 0.21 9.4 < 0.053 75 < 0.42 59 <0.33 bread Total, 430 < 3.3 450 < 3.1 330 < 2.3 250 < 1.9 240 < 1.4 grains Legumes Soya < 10 4.5 < 0.046 2.0 < 0.020 0.5 < 0.005 0.0 0.000 0.0 0.000 beans Other < 10 20 < 0.20 18 < 0.18 17 < 0.17 23 < 0.23 12 <0.12 legumesa Total, < 0.25 < 0.20 < 0.18 < 0.23 < 0.12 legumes Overall < 3.5 < 3.3 < 2.5 < 2.2 < 1.5 total Total < 0.059 < 0.056 < 0.041 < 0.036 < 0.025 in µg/kg bw per dayb Concentration assumed to be zero (approx. 0) or below detection limit in the other food groups a The zearalenone value for tinned beans was used for other legumes b Based on 60 kg * Focus should therefore be directed to zearalenone in corn kernels, cornmeal and cornflour products, popcorn, rice, and wheat flour products. If possible, wheat flour products should be separated from those of durum wheat (pasta), soft wheat (cakes and pastries), and hard wheat (bread, crackers, cookies) because different wheat flours have different concentrations of zearalenone. * It is important to determine whether other foods consumed by a population might contain zearalenone. Examples include infected bananas, beer brewed from contaminated grain, and tissues from alpha-zearalanol-injected animals. * The intake of zearalenone by infants might be greater if they consume grain-or legume-based formula instead of human milk or a milk-based formula. The following equation could be used to estimate the intake of zearalenone by a population: the average daily intake of zearalenone (ZEA) is equal to the sum of the average daily gram intake (I) of seven foods (corn, cornmeal/cornflour products, popcorn, rice, pasta, cake/pastry, and bread/crackers/other hard-wheat flour products) by a defined age/sex group of a population multiplied by the average zearalenone concentration (µg/kg; C) of each food: ZEA = (I)(C) corn + (I)(C) cornmeal/flour products + (I)(C) popcorn + (I)(C) rice + (I)(C) pasta + (I)(C) cake/pastry + (I)(C) bread/crackers/other hard-wheat flour products Table 15. Contributions of various commodities to total zearalenone intake in the five regional diets (% of intake) Commodity Middle Far African Latin European Eastern Eastern American Barley 0 1 1 3 11 Maize or corn 31 12 55 27 8 Oats 0 0 0 0 1 Rice 10 62 31 30 8 Wheat 52 19 6 30 67 Legumes 7 6 7 11 8 Total 100 100 100 101 103 4. COMMENTS The average dietary intakes of zearalenone from cereals and legumes in the GEMS/Food regional diets were estimated to be 1.5 µg/day in the European diet and 3.5 µg/day in the Middle Eastern diet. If a mean body mass of 60 kg is assumed, these intakes correspond to 0.03 and 0.06 µg/kg bw per day, respectively. The average dietary intakes of zearalenone estimated on the basis of individual dietary records are < 0.98 µg/day (0.02 µg/kg bw per day) for Canada, 1.2 µg/day (0.02 µg/kg bw per day) for Denmark, 1.1 µg/day (0.02 µg/kg bw per day) for Norway, and < 2.1 µg/day (0.03 µg/kg bw per day) for the United States. The theoretical maximum daily intake of alpha-zearalanol when used as a veterinary drug was calculated to be 1.6 µg/day (0.02 µg/kg bw per day) on the basis of the recommended maximum residue limits of 10 µg/kg in cattle liver and 2 µg/kg in cattle muscle (Annex 1, reference 80). Studies of the pharmacokinetics and metabolism of zearalenone indicate that it is extensively metabolized by intestinal tissue in pigs, and possibly in humans, during its absorption, with the formation of alpha-and beta-zearalenol and alpha-and beta-zearalanol, which are subsequently conjugated with glucuronic acid. The existence of this pathway limits the value of studies conducted by parenteral administration for assessing the risk associated with dietary intake. Biliary excretion with enterohepatic circulation occurs in rats and mice, while urinary excretion predominates in rabbits. Urinary excretion is also the main route of elimination in pigs, in spite of the demonstrated enterohepatic circulation of zearalenone, owing to a high degree of reabsorption in the gut. The very limited data in humans (one individual) suggest that urinary excretion is also significant. Differences between species in the metabolism of zearalenone were found: a higher proportion of an administered dose of zearalenone was metabolized to alpha-zearalenol in pigs than in rats or cattle. In humans as in pigs, zearalenone was found mainly in urine as glucuronide conjugates of the parent compound and alpha-zearalenol. Zearalenone has little toxicity after administration of single oral or intraperitoneal doses. In studies of oral administration for up to 90 days, the effects appeared to be dependent on the estrogenic activity of zearalenone and/or its metabolites. Pigs and sheep were more sensitive than rodents; in controlled studies with well-defined exposure to multiple doses, the NOEL in pigs was 40 µg/kg bw per day on the basis of estrogenic effects in responsive tissues and reproductive performance, compared with a NOEL of 3 mg/kg bw per day in rats. Zearalenone has been tested for genotoxicity in a variety of test systems covering several end-points, including point mutations, unscheduled DNA synthesis, and chromosomal aberrations. The results were negative, except for the induction of chromosomal aberrations after exposure of mammalian cells in vitro to very high concentrations. Evidence from a 32P-postlabelling assay that zearalenone modifies DNA was reported, but the Committee concluded that the results do not unequivocally demonstrate covalent binding of zearalenone and/or its metabolites to DNA and most likely reflect oxidative damage to DNA, since the DNA damage was greatly reduced by co-administration of the antioxidant alpha-tocopherol. Hepatocellular adenomas and pituitary tumours were observed in studies of long-term toxicity and carcinogenicity in mice, but only at doses greatly in excess of the concentrations that have hormonal effects, i.e. at 8-9 mg/kg bw per day or more. The Committee concluded that these tumours were a consequence of the estrogenic effects of zearalenone. A similar conclusion was drawn by the Committee at its thirty-second meeting with regard to a-zearalanol. In rats, there was no treatment-related increase in the incidence of tumours at doses of 1-3 mg/kg bw per day. 5. EVALUATION The Committee concluded that the safety of zearalenone could be evaluated on the basis of the dose that had no hormonal effect in pigs, the most sensitive species. Using a safety factor of about 100, the Committee established a provisional maximum tolerable daily intake (PMTDI) for zearalenone of 0.5 µg/kg bw. This decision was based on the NOEL of 40 µg/kg bw per day in the 15-day study in pigs. The Committee also took into account the lowest-observed-effect level of 200 µg/kg bw per day in this study and the previously established ADI of 0-0.5 µg/kg bw for the metabolite alpha-zearalanol, evaluated as a veterinary drug. 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See Also: Toxicological Abbreviations ZEARALENONE (JECFA Evaluation)