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Residues of hormonal substances in foods of animal origin: a risk assessment
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PREVENTIVE VETERINARY MEDICINE
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Preventive Veterinary Medicine 20 (1994) 235-247
Residues of hormonal substances in foods of animal origin: a risk assessment D a v i d Waltner-Toews*, Scott A. M c E w e n Department of Population Medicine, Universityof Guelph, Guelph, Ont. N I G 2 W1, Canada Accepted 15 January 1994
Abstract
An assessment of the human health risks associated with residues of hormonal substances in foods of animal origin was undertaken. These compounds are used in the production of food animals to promote growth and increase the efficiency of feed utilization and in the case of bovine somatotropin, to increase milk production. The assessment is organized to combine information on the hazards posed by these residues with data on exposure to arrive at recommendations for risk management and avoidance.
1. Introduction Exogenous hormones first were used as growth promotants in cattle in the 1950s. Since that time, a variety of endogenous and synthetic (xenobiotic) hormonal substances has been developed and used in food animals (mainly ruminants) for the purposes of increasing the rate of growth, increasing the efficiency of feed utilization (feed conversion), regulating estrus, and (in the case of bovine somatotropin; BST) for increasing milk production. The anabolic agents as a group have been very effective in achieving these goals. The considerable controversy that has evolved over the use of these compounds in food animals is centred on two main points: ( I ) the effects of residues in foods on human health; (2) whether western society needs or wants to use these compounds at all. Widely disparate points of view on these matters are held. The controversy over the use of hormones in animal production began 20 years ago when diethylstilbestrol (DES) was banned from use as a growth promotant in cattle because clinical use of the compound in women was associated with *Corresponding author. 0167-5877/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIO167-5877(94)O3015-U
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vaginal cancer in some of their daughters (Health and Welfare Canada, 1973; Rodericks, 1986). The controversy has continued in recent years, with the European Economic Community ban on the use of all anabolic hormonal substances in food animals (Anonymous, 1985; Sundlof, 1989). In 1990, a moratorium was placed on the use of BST in lactating dairy cattle in the state of Wisconsin (USA), because of public concern over issues of milk production and the safety of milk products for human consumption, but the decision later was overturned (Reotutar, 1990). The purpose of this paper is to review the literature pertaining to the public health risk posed by residues of hormonal substances in foods from animals. This area has also been reviewed by Sundlof ( 1989 ). Placing residues found in Ontario, Canada (which we deem to be typical of many industrialized countries) into international contexts, we will discuss the risks that these residues might pose to consumers. Not considered in this review are the hormonal substances used for therapeutics and estrus synchronization in cattle. The safety of residues of these latter compounds in foods from animals has received little attention. The framework for this review, and for related reviews on residues of insecticides, industrial chemicals, and antibacterial drugs in foods of animal origin, is one generally used for risk assessment, as described in the first paper in this series (Waltner-Toews and McEwen, 1994a,b,c,d). The four elements of risk assessment are hazard identification and dose-response assessment (which we have grouped together), exposure assessment, and risk characterization. We have also added a section on residue avoidance, which is the primary risk management strategy for chemical residues in food.
2. Hazard identification and dose-response assessment Hormonal substances (anabolic agents) used in food animals fall into two groups: (1) those that are naturally occurring substances in animals (and are either extracted from animals or manufactured using recombinant DNA or some other technology); (2) those that are produced synthetically and do not occur naturally in animals (xenobiotic compounds). Among the first group are testosterone, estradiol, progesterone and somatotropin. Thyrotropin is a naturally occurring hormone not used intentionally as an anabolic agent, but residues of this substance can occur in foods and have been associated with human illness (Hedberg et al., 1987). Xenobiotics used in some countries (not legally in all cases) are trenbolone acetate, melangestrol acetate, zeranol and the stilbenes--most notably diethylstilbestrol (DES). Also included in this group are the fl-agonists (partitioning agents such as clenbuterol ). Estradiol (estrogen), testosterone and progesterone are sex hormones that occur naturally in mammals (including humans). Because of this, these hormones are normally present in food from animals and levels found in these foods vary widely depending upon the age, pregnancy status and physiological status of the animal (Hoffmann and Evers, 1986; Food and Agriculture Organization/World
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Health Organization (FAO/WHO), 1988 ). They are used mostly in cattle, either alone or in various combinations depending on the sex of the animal, by implantation of capsules of hormone into the subcutaneous tissues of the ear (an inedible portion of the carcass). The location of the implant influences the residue levels in plasma (Meyer et al., 1984); injection at the middle of the pinna results in lower levels than injection at the base. The possible hazards of residues of sex steroid hormones in edible tissues from animals have been previously reviewed (WHO, Regional Office for Europe, 1982; Meissonnier and Mitchell-Vigneron, 1983; Hoffmann and Evers, 1986; Sundlof, 1989). These sex steroid hormones are believed to playat least some role in human and animal cancer. For example, human castrates are at reduced risk of prostatic cancer, and epidemiological studies indicate that increased testosterone concentrations in blood are associated positively with increased risk of prostatic cancer (Meissonnier and Mitchell-Vigneron, 1983; WHO: International Agency for Research on Cancer (IARC), 1987). Most evidence indicates that steroid hormones can act as promoters of tumours in certain target organs that have hormone receptors, but the natural sex hormones are not genotoxic (Van Logten et al., 1981; Meissonnier and Mitchell-Vigneron, 1983; FAO/WHO, 1988). Examples of target organs for these compounds include the mammary gland, cervix, uterus and prostate gland. As promoters, these sex steroid hormones are capable of increasing the risk of cancer and therefore are classified as carcinogens. It is important to distinguish 'carcinogens' which promote tumours from those that have a genotoxic effect; the former exert their turnout enhancing effect by virtue of their hormonal activity. Compounds of this type are not carcinogenic at concentrations less than that which induces a measurable hormonal effect (WHO, Regional Office for Europe, 1982; Hoffmann and Evers, 1986; FAO/WHO, 1988 ). Acceptance of this concept is the basis for the establishment of so-called no-hormonal-effect-levels (NHEL) in animal models. These can be used to set safe acceptable daily intakes (ADI) (Foxcraft and Hess, 1986; Hoffmann and Evers, 1986). In contrast, many believe that the concept of safe levels does not apply to genotoxic carcinogens (Roe, 1984), although this is the subject of debate (see National Research Council (NRC), 1993; Waltner-Toews and McEwen, 1994a). Estradiol-17fl could be carcinogenic, but at levels greater than required for physiological purposes. In normal, untreated animals, highest levels are found in tissues of animals during late pregnancy. Treatment with hormone implants according to recommended procedures increased tissue levels by a factor of 2-5 (FAO/WHO, 1988). The amounts of estradiol-17fl produced daily by prepubertal boys (the lowest group) and pregnant women (highest) were estimated to be about 1000 to several million times, respectively, greater than the amount of hormone residue that would be present in one 500 g portion of meat from an animal implanted with this compound according to directions (Hoffmann and Evers, 1986; FAO/WHO, 1988). Levels of this hormone that would be present in edible tissues of treated animals could not exert any toxic effect in humans through hormonal action, and therefore the Joint FAO/WHO Expert Committee
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on Food Additives (JECFA; FAO/WHO, 1988) chose not to recommend an acceptable residue level. Therapeutic administration of the compound to cows produced higher-than-physiological levels (Vynckier et al., 1990). On the basis of this observation, a withdrawal period for therapeutic administration was suggested. Progesterone (in combination with estradiol-17//) is used in subcutaneous implants in steers and calves. It also is used for the regulation of the estrous cycle in ruminants and swine (Neff and Lauderdale, 1984). Like estradiol, progesterone exerts a carcinogenic effect by virtue of its hormonal activity (FAO/WHO, 1988 ). Consequently, it does not appear to be toxic at levels below those necessary for hormonal effect. As is the case with estradiol, residue levels of progesterone in tissues and milk of treated animals are much less than the daily production of the hormone even among prepubertal boys, and therefore are believed not to pose a risk to human health (WHO, Regional Office for Europe, 1982; FAO/WHO, 1988; Fitzpatrick, 1988; Sundlof, 1989). Similar comments apply to testosterone (in combination with estradiol-17//), used in implants in heifers (WHO, Regional Office for Europe, 1982; FAO/WHO, 1988). Trenbolone acetate (TBA), zeranol and melengestrol acetate (MGA) are xenobiotic anabolic agents, and any levels in tissues need to be considered contaminants (Roe, 1984). This requires that a somewhat different approach to the evaluation of their safety be taken from that of the natural sex hormones. TBA (a synthetic steroidal compound) is probably not a genotoxic substance (based on evidence from short-term genotoxicity assays and long-term feeding studies in laboratory animals (Meissonnier and Mitchell-Vigneron, 1983; Roe, 1984; Hoffmann and Evers, 1986; FAO/WHO, 1988; WHO, 1988, 1990). A proportion of the residues of TBA in tissue become bound to protein, but are of low toxicity and of no hormonal significance (Burgat-Sacaze et al., 1986 ). An acceptable daily intake (ADI) of TBA (0-0.02 #g kg-1 body weight day-~ ) was based on experiments in pigs that demonstrated a no-hormonal-effect-level (NHEL) of 2-3 #g kg -~ body weight day-l. Interim maximum residue levels (MRL) for trenbolone in bovine edible tissues have been set at 0.7 #g kg- ~of//-hydroxytrenbolone and 7.0 #g kg- ~of o~-hydroxytrenbolone (Sundlof, 1989 ). Zeranol is a non-steroidal anabolic agent used as an implant in cattle, and is derived from zearalenone, a mycotoxin produced by Fusarium spp. The compounds are similar in structure and are believed to share some toxic properties-particularly those of estrogenic effect (Baldwin et al., 1983; Sundlof and Strickland, 1986; FAO/WHO, 1988). The recommended ADI is 0-0.5 #g kg ~ body weight day -~ for humans (FAO/WHO, 1988; WHO, 1988). There is no evidence that zeranol is genotoxic (Meissonnier and Mitchell-Vigneron, 1983 ). Reports in the literature that describe precocious sexual development (thelarche) of children in Puerto Rico and Italy are sometimes cited as evidence of the human health risk posed by the use of anabolic hormonal substances in food animals (Fara et al., 1979; Saenez de Rodriguez and Toro-Sola, 1982; Bongiovanni, 1983; Sundlof and Strickland, 1986). A large number of cases were involved in the Puerto Rican outbreak while the Italian outbreak involved a smaller
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number of cases (Fara et al., 1979; Bongiovanni, 1983). In both outbreaks, the authors speculated that the cause of the problem could have been food contaminated with estrogenic substances. DES or zeranol were specifically named in association with the Puerto Rican outbreak. However, almost no evidence was presented that would support this hypothesis. The Italian outbreak was characterized by an increase in the occurrence of breast enlargement in girls and boys that attended a school in Milan (Fara et al., 1979). Samples of the school meals were tested for estrogen contamination, and none was found. In the Puerto Rican outbreak, some 'preliminary results' in the laboratory of one investigator were interpreted to suggest that there were quantities of estrogenic substances in meat and poultry, but gas-liquid chromatography of these foods for zeranol and DES was negative (Bongiovanni, 1983). Subsequent investigation of the outbreak by the Centers for Disease Control, USDA and FDA in the USA revealed that the outbreak could have been due to increased reporting and awareness of the problem of thelarche by physicians (Sundlof, 1989 ). USDA analyzed approximately 700 samples of animal products for estrogenic substances, and no violative residues were found. Melangestrol acetate (MGA; an orally active progestogen) is used in heifers in the form of a feed additive. A MRL of 25/tg kg -~ (25 ppb) in edible tissues has been set (Neff, 1983; Neff and Lauderdale, 1984), and although a 48 h withholding time was imposed, heifers fed MGA at prescribed doses have tissue levels less than MRL without feed withdrawal. Diethylstilbestrol (DES; a stilbene compound) was used in the 1950s and 1960s as implants for growth promotion in cattle. The compound was also used in human medicine--most importantly (in the context of this review) for the maintenance of pregnancy in women. Daughters of women who were treated with high levels of DES for this purpose had increased risk of a rare type of cancer of the vagina (Health and Welfare Canada, 1973; Jukes, 1974). Subsequent studies in laboratory animals established that high levels of DES fed to laboratory animals over a long period of time resulted in an increase in the incidence of tumours in these animals (Jukes, 1974; Van Logten et al., 1981 ); this presumably was a result of the hormonal effect of the compound, although there has been suggestion of mutagenicity or genotoxicity (Van Logten et al., 1981; WHO, Regional Office for Europe, 1982). Evidence that oral DES was absorbed readily and not destroyed readily by the liver after uptake also weighed in the decision to ban the substance from use in food animals in the early 1970s (Anonymous, 1982; Roe, 1984). Somatotropin (ST; also known as growth hormone; GH) has a variety of effects in animals, and scientists have known since the 1920s that it could increase the milk production of cows (Connaughton, 1989; Juskevich and Guyer, 1990). In cattle, the terms recombinant bovine growth hormone and recombinant bovine somatotropin are used interchangeably to describe the genetically engineered product. The production of this substance in commercial quantities is possible, and it is being used (or licensing is being sought for use) in dairy cows (Hart, 1987 ). ST also may find application as an anabolic agent in swine.
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As with other compounds, the human safety of ST use in animals has been addressed by evaluation of the biochemistry of the compound, its effects in laboratory animal species and humans, its effect on the nutritional composition of milk and the occurrence of residues in edible foods (especially milk) (Juskevich and Guyer, 1990). ST is a long polypeptide protein molecule that is cleaved into polypeptides and amino acids by the digestive process and therefore thought not to be absorbed intact through the gut. STs tend to have a very narrow species range of activity, and although bovine somatotropin (BST) is anabolic in rats by parenteral injection, it is not anabolic in rats when given orally, nor is it active in humans (Cherfas, 1990; Juskevich and Guyer, 1990). BST occurs normally in milk (at about 1 ppb; Juskevich and Guyer, 1990 ) and like other naturally occurring hormones, levels of BST in meat and milk from animals treated in the proper manner are no greater than levels in untreated animals (Connaughton, 1989; Juskevich and Guyer, 1990). Pasteurization reduces the amount of BST in milk by 90% (Juskevich and Guyer, 1990), but it does not reduce the quantity of insulin-like growth factor-1 (IGF-1; a mediator of GH action). IGF-1 is not species specific (Kronfeld, 1989), and levels in meat of BST-treated cows are approximately twice those of untreated cows. There is currently no evidence that these increased levels pose a health risk (Juskevich and Guyer, 1990; H a m m o n d et al., 1990). Although fat, protein and lactose concentrations of milk are not appreciably altered when BST is used in cows in a positive energy balance, BST can cause an increase in the relative amount of long-chain fatty acids and can increase the somatic cell counts of milk (Van Den Berg, 1991 ). Based on this type of evidence, most scientists believe that BST used in dairy cattle is safe for humans and that the composition and nutritional value of milk from treated animals are essentially the same as that from untreated cows (Anonymous, 1991 b). Other investigators however, believe that final judgement on the human safety of BST use is premature---especially until more is known of the importance of changes in IGF-1 and other hormones in meat and milk from BST treated cows (Reotutar, 1990; Van Den Berg, 1991 ). Unlike ST, thyrotropin (thyroid hormone, TH) from cattle is biologically active in humans, and is absorbed after ingestion. Although TH is not used as a therapeutic or growth-promoting agent in food animals, thyrotoxicosis as a result of ingestion of excessive quantities of bovine TH have been reported. In 1984 and 1985, 121 human cases of thyrotoxicosis were identified in Nebraska (Hedberg et al., 1987 ). These individuals were suffering from a variety of symptoms, including nervousness, headaches, heart palpitations and weight loss, and they had increased levels of thyroid hormones. Affected individuals were more likely than controls to have consumed ground meat partially composed of bovine laryngeal trimmings. The thyroid glands in cattle are in close proximity to the skeletal muscles of the larynx, making contamination with thyroid glandular tissue very easy. Clenbuterol is a fl-agonist (salbutamol is another) that has been used illegally as a so-called 'partitioning agent' for increasing the proportion of muscle in car-
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casses (i.e. leaner meat). The drug is not approved for use in food animals anywhere, but illegal use in cattle has been reported in the USA (Anonymous, 1991 a; Keller, 1991 ). In 1990, residues of clenbuterol in liver were linked epidemiologically to hospitalization due to increased heart rate, muscle tremors, headache, dizziness, fever and chills in 135 people in Spain (Keller, 1991 ). Beef liver specimens obtained from case families during the outbreak contained 160-291 ppb clenbuterol. Trace-back studies by health authorities indicated that the animals may have been given toxic doses of the drug inadvertently and when clinical signs arose, were slaughtered for salvage. Recently, the effect that anabolic agents may have on the disposition (and therefore residue levels) of other simultaneously administered drugs has been raised as a factor to be considered (Van Miert et al., 1988; Witkamp et al., 1990). Implantation of female goats with TBA and testosterone resulted in a decrease in the plasma elimination of antipyrine, sulfamethazine and trimethoprim. Additional research is needed to determine if these interactions have a substantial bearing on drug residue levels in foods.
3. Exposure assessment If properly administered as an implant in the inedible tissue of the ear, estradiol, progesterone and testosterone are released slowly from the injection site; direct human exposure to unabsorbed hormone is avoided by discard of the ear at slaughter. Residues of these hormones in foods from animals form an insignificant proportion of the total exposure of humans to these compounds. Estradiol17fl in fat is the last estrogen/tissue combination to return to endogenous levels after treatment of cattle with estrogen implants: this association could be used for monitoring (Henricks et al., 1983 ). Of the xenobiotic anabolic agents, melangestrol acetate (MGA) is used as a feed additive, is rapidly excreted and therefore requires only a brief withdrawal time (48 h) from feed of animals prior to slaughter. In contrast, zeranol is administered as an implant and has a rather long withdrawal time (65 days). Trenbolone acetate is an implant licensed for use in beef cattle in Europe and in the USA (Sundlof, 1989). Residues of zeranol can be found in liver up to 120 days after implantation, and are highest in edible tissues 14 days after administration (Horwitz et al., 1983; Dixon et al., 1986). An abattoir survey of growth promotant implants in cattle was conducted in 1984-1987 in Scotland and northern England (Maddox, 1988 ). Cattle were selected at random, and implants were detected by palpation: 6014 cattle were examined, and implants were found in 2% of dairy cows, 4% of beef cows, 8% of steers and 7% of heifers. Ninety-five percent of the implants in cows were trenbolone acetate, but 6% of implants in steers and heifers were zeranol (even though zeranol implants were less likely to be detected given their small size, and some of the implants were detected after an official ban on their use) (Maddox, 1988 ). DES has been banned in most countries for a number of years, but illegal resi-
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dues have been reported. Extensive screening has been credited with the nearly complete disappearance of illegal use of DES in The Netherlands (Stephany et al., 1985 ). However, injection sites in cattle containing other illegally used anabolic agents (such as nortestosterone, medroxyprogesterone, estradiol and testosterone) have been reported from The Netherlands (Jansen et al., 1985; Stephany et al., 1985 ). Of 573 injection sites examined during 1983-1985,462 were positive for one or more substance. Nortestosterone was found in 84% of samples, medroxyprogesterone in 14%, methyltestosterone in 2%, and trenbolone in 2%. Testosterone was detected in 29% of samples, estradiol in 66% and progesterone in 3%. Residues of DES, dienestrol, hexestrol and zeranol were detected in 2%, 3%, 0%, and 0% respectively, of samples. Up to four different substances were found in injection sites from some animals (Jansen et al., 1985 ). The illegal use of the natural sex hormones estradiol and testosterone resulted in a scandal in Germany where approximately 13 000 calves were seized in an effort to contain the problem (Sundlof, 1989 ). Violative residues of DES were found in veal calves from the state of New York in 1983. Thereafter until 1989, no DES residues were detected (Cordle, 1988; Food Safety and Inspection Service, US Department of Agriculture (USDA), 1990). USDA monitoring of ruminants for zeranol (622 animals in 1988, 378 in 1989) resulted in no cases of violative residues. Testing of 16/134 (1988) and 13/232 (1989) surveillance specimens (from suspect animals) were positive. All of the violative cases were calves (Food Safety and Inspection Service, USDA, 1990). In 1989, 373 specimens of fat from heifers were tested for MGA and none were violative.
4. Risk characterization
As discussed previously, the human safety of residues of the natural sex steroids (testosterone, progesterone and estrogen) in edible tissue is based on the premise that these compounds occur in animals naturally and tissue residues in treated animals are within established normal ranges for untreated animals (Craft, 1986 ). Accordingly, ADI and withdrawal times have not been established for the natural sex steroids. Improper administration of the capsules into an edible portion of the carcass could result in direct ingestion by a consumer and exposure to higher levels of hormone, but this has never been reported. There is one report that indicates that veal calves may be implanted repeatedly or with more than the approved number of implants (Agriculture Canada, 1987). This could raise the amount of hormone in tissue samples, but there is no evidence that these levels would be a risk to humans. The biological activity of the xenobiotic hormonal substances has been measured through assays of hormonal effect, toxicity and genotoxicity in laboratory animals. For the compounds legally available (TBA, MGA, zeranol), hormonal effects are the most significant. Quantities of hormone below the no-hormonaleffect-levels (NHEL) do not, by definition, result in a response in the target tis-
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sue (s) of that hormone. Properly administered, these substances should not leave residues in edible tissues and milk that could have an undesirable hormonal effect in humans. In theory, the risk of treated animals entering the food chain before the proper withdrawal times have been observed is probably greatest for zeranol. There are two reasons for this: the extent of its use (for example, zeranol is the most widely used hormonal substance for cattle in Canada) and the long (65 days for a 36 mg implant dose) withdrawal time. The risk of animals entering the food chain before the withdrawal time is completed may be greatest for animals culled for a medical reason (fracture, prolapse, etc. ) because these culls cannot be foreseen by farmers when cattle are implanted. The safe ADI of TBA is 0-0.02/tg kg -1 body weight day -1 (WHO, 1990). Appropriate maximum residue levels (MRL) of TBA are 2 #g kg- l in food animal skeletal muscle as fl-trenbolone and 10 #g kg- l in liver as a-trenbolone. The ADI of zeranol of 0-0.5/zg kg-~ body weight day-~ for humans (FAO/ WHO, 1988 ) corresponds to an MRL of 70 #g kg- ~of edible tissue. Since this is a higher level of residues than is necessary using good husbandry practices, MRL of 10/~g kg- l for bovine liver and 2 #g kg- 1 for bovine muscle have been recommended (FAO/WHO, 1988). In Canada, results of recent monitoring for tissue residues of the xenobiotics zeranol and MGA suggest that exposure of humans to violative levels of these compounds in meat is probably rare; only one specimen containing a violative level of zeranol has been detected. This, coupled with the relatively low toxicity of these compounds, suggests that the use of these compounds poses a very small risk to Canadians. Monitoring of food animal tissues for hormonal substances not licensed in Canada, including TBA, DES and clenbuterol, reveals that residues have either not been detected (TBA, DES) or, in the case of clenbuterol, are rare. In light of these data, and the fact that approved anabolic agents are legally available, risk of exposure and toxicity to TBA and DES is probably very low. The illegal use of clenbuterol in Canada (Agriculture Canada, 1989), Europe and (most recently) the USA (Keller, 1991 ) may be driven by a demand for partitioning agents not filled by a legal compound. The occurrence of illegal residues of this drug (along with the recent report of human toxicity attributed to residues of clenbuterol in liver) are of concern and argue for the continued monitoring of this drug. As was mentioned previously, DES is no longer used in food animals in North America because, in part, daughters of women treated with the compound were at increased risk of cancer and laboratory animals developed tumours after prolonged feeding of the substance (Health and Welfare Canada, 1973 ). Most scientists believe that the human health risk posed by DES use in cattle was very small (Jukes, 1974; Rodricks, 1986).
5. Risk avoidance
Although DES has been banned for use in food producing animals, the experience with this compound has left an important legacy. This was the first hor-
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monal substance that had widespread use in food animals, and was for years touted to be both effective and safe. The debate that followed the revelations that DES use in women was associated with cancer may have left the public with lasting mistrust and aversion to the use of any hormonal substance in food animals. It also may have resulted in a loss of public confidence in the ability of the scientific community to predict the safety of these compounds. In addition to this, high concentrations of illegally used DES were found in baby food in Europe in 1980 (Sundlof, 1989). This aggravated public concern, and played a role in the eventual banning by the European Economic Community (EEC) of the use of all hormones for growth promotion in food animals (Anonymous, 1985). The illegal use of hormonal substances in food animals has been reported in many countries and this is most often a problem when there are no substances legally available to suit the purpose (Anonymous, 1982 ). The natural sex steroids (progesterone, estradiol and testosterone) appear to be safe and the only conceivable human health risk associated with their use would be in the actual ingestion of implant sites. Limited evidence from monitoring of implant sites in Canadian cattle indicates that some implants are administered in improper locations, and some animals are treated repeatedly or given multiple implants. It would be prudent to ensure that users of these products in cattle are instructed in the proper techniques and the reasons for using the particular site. The widespread use of zeranol combined with its long withdrawal time could produce residues in treated animals prematurely culled to slaughter. Perhaps emergency cull animals should be monitored more closely than the general population of slaughter cattle. Also, since there is probably a seasonal trend to use of these compounds (most in the fall), this factor should be considered when devising a sampling plan for residue testing. Although not used intentionally in food animals, thyroid hormone is the one compound that has been shown to result in human disease after ingestion of meat contaminated with thyroid trimmings. The entire laryngeal area, including skeletal muscles of the larynx, the thyroid gland and other structures should be condemned at slaughter. Illegal use of clenbuterol and other fl-agonists is of some concern, especially in view of the report of human illness attributed to residues of clenbuterol in liver of treated animals (Keller, 1991 ). Monitoring of cattle for these compounds should be undertaken at a level sufficient to establish if at least 1% of the population of cattle is being treated illegally. Finally, for BST the issue is not public health, but socio-economics (Kneen, 1990; Roush, 1991; Kneen et al., 1991 ). Settling of the scientific and risk communication issues is just one part of determining safe and acceptable agricultural policies (NRC, 1983).
Acknowledgment Financial support for this project was provided by the Ontario Ministry of Agriculture and Food.
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References Agriculture Canada, 1987, 1989. Annual report on chemical and biological testing for agri-food commodities for the fiscal years 1987, 1988/89. Agri-food Safety Division, Food Inspection Directorate, Food Production and Inspection Branch, Agriculture Canada, Ottawa, 179 pp. Anonymous, 1982. Anabolics inl meat production. Lancet, I: 721-722. Anonymous, 1985. Residues in meat: How real are the dangers? Vet. Rec., 117: 353-354. Anonymous, 1991a. Agencies warn against clenbuterol use. American Association of Food Hygiene Veterinarians, News-o-Gram, 15, p. 11. Anonymous, 1991b. NIH sponsors conference on bovine somatotrophin. FDA Vet., 6: 3-6. Baldwin, R.S., Williams, R.D. and Terry, M.K., 1983. Zeranol: a review of the metabolism, toxicology, and analytical methods for detection of tissue residues. Regul. Toxicol. Pharmacol., 3: 9-25. Bongiovanni, A.M., 1983. An epidemic of premature thelarche in Puerto Rico. J. Pediatr., 103: 245246. Burgat-Sacaze, V., Rico, A. and Panisset, J.-C., 1986. Toxicological significance of bound residues. In: A.G. Rico (Editor), Drug Residues in Animals. Academic Press, Orlando, FL, pp. 1-31. Cherfas, J., 1990. Europe: Bovine growth hormone in a political maze. Science, 249: 852. Connaughton, D., 1989. Bovine somatotropins: benefits and risks. J. Am. Vet. Med. Assoc., 194: 1009-1012. Cordle, M.K., 1988. USDA regulation of residues in meat and poultry products. J. Anita. Sci., 66: 413-433. Craft, M., 1986. The EEC and growth promotants. Aust. Vet. J., 7:12. Dixon, S.N., Russel, K.L., Heitzman, R.J. and Mallinson, C.B., 1986. Radioimmunoassay of the anabolic agent zeranol. V, Residues of zeranol in the edible tissues, urine, faeces and bile of steers treated with Ralgro. J. Vet. Pharmacol. Ther., 9: 353-358. Fara, G.M., del Corvo, G., Bernuzzi, S., Bigatello, A., DiPietro, C., Scaghoni, S. and Chiumello, G., 1979. Epidemic of breast enlargement in an Italian school. Lancet, II: 295-297. Fitzpatrick, S., 1988. The use ofanabolic hormones for growth promotion. FDA Vet., VIII: 4-5. Food and Agriculture Organization/World Health Organization, 1988. Evaluation of certain veterinary drug residues in food. Vol. 763. WHO, Geneva, 40 pp. Food Safety and Inspection Service, US Department of Agriculture, 1990. Domestic Residue Data Book, National Residue Program: 1988-1989. USDA, Washington, DC. Foxcraft, G.R. and Hess, D.L., 1986. Hormonal no-effect studies: concepts versus realities. In: A.G. Rico (Editor), Drug Residues in Animals. Academic Press, Orlando, FL, pp. 147-174. Hammond, B.G., Collier, R.J., Miller, M.A., McGrath, M., Hartzell, D.L., Kotts, C. and Vandaele, W., 1990. Ann. Rech. Vet., 21 (Suppl. 1): 107s-120s. Hart, I.C., 1987. Biotechnology and production-related hormones. Proc. Nutr. Soc., 46: 393-405. Health and Welfare Canada, 1973. The facts behind the DES story. Educational Services, Health Protection Branch, Health and Welfare Canada, Ottawa, 2 pp. Hedberg, C.W., Fishbein, D.B., Janssen, R.S. et al., 1987. An outbreak of thyrotoxicosis caused by the consumption of bovine thyroid gland in ground beef. N. Engl. J. Med., 316: 993-998. Henricks, D.M., Gray, S.L. and Hoover, J.L., 1983. Residue levels of endogenous estrogens in beef tissues. J. Anim. Sci., 57: 247-255. Hoffmann, B. and Evers, P., 1986. Anabolic agents with sex hormone-like activities: problems of residues. In: A.G. Rico (Editor), Drug Residues in Animals. Academic Press, Orlando, FL, pp. 111-146.
Horwitz, C., Rozen, P. and Gilat, T., 1983. Should we worD' about animal promoters? Nutr. Cancer, 5: 51-54. Jansen, E.H.J.M., van Blitterswijk, H. and Stephany, R.W., 1985. Residues of anabolic agents in sites of administration in slaughtered cattle; period from October 1983 to January 1985. Tijdschr. Diergeneeskd., 110: 355-360. Jukes, T.H., 1974. Estrogens in beefsteaks. J. Am. Vet. Med. Assoc., 229: 1920-1921.
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Juskevich, J.C. and Guyer, C.G., 1990. Bovine growth hormone:human food safety evaluation. Science, 249: 875-884. Keller, W.C., 1991. Illegal use of clenbuterol in food animals. FDA Vet., 6:9-11. Kneen, B., 1990. Modern farming practices: a consideration of bovine growth hormone. The Ram's Horn, 74: 1-8. Kneen, B., Lapointe, L., Baldwin, B. and Wooden, M., 1991. Pure Milk Campaign (Pamphlet), Coxwell, Toronto, Ontario. Kronfeld, D.S., 1989. BST milk safety. J. Am. Vet. Med. Assoc., 195: 288-289. Maddox, J.G., 1988. An abattoir survey of growth promoter implants in cattle. Vet. Rec., 122:161. Meissonnier, E. and Mitchell-Vigneron, J, (Editors), 1983. Proceedings of a Symposium on Anabolics in Animal Production: Public Health Aspects, Analytical Methods and Regulation, Officc lnternationale des Epizooties, Paris, 15-17 February 1983, OIE, Paris, 570 pp. Meyer, G.W., Landwehr, M., Schopper, D. and Karg, H., 1984. Application of Synovex-H in veal calves: steroid release and residues. Food Addit. Contam., 1:261-275. National Research Council, 1983. Risk Assessment in the Federal Government: Managing the Process. National Academy Press, Washington, DC, t 91 pp. National Research Council, 1993. Issues in Risk Assessment. National Academy Press, Washington, DC, 356 pp. Neff, A.W., 1983. Analytical methods for MGA (melengestrolacctatc). In: E. Meissonnier and J. Mitchell-Vigneron (Editors), Proceedings of a Symposium on Anabolics in Animal Production: Public Health Aspects, Analytical Methods and Regulation, Office International des Epizooties, Paris, 15-17 February 1983, OIE, Paris, pp. 457-485. Neff, A.W. and Lauderdale, J.W., 1984. Progesterone and progestogen residues associated with commercial products used in animal agriculture. 10th Int. Congress on Animal Production and Artificial Insemination, University of Illinois, 4: Xll 9-XII 13. Reotutar, R., 1990. Bovine growth hormone raises national concern. J. Am, Vet. Med. Assoc., 196: 1018. Rodricks, J.V., 1986. FDA's ban on the use of DES in meat production: a case study. Agriculture and Human Values, Winter-Spring, 10-25. Roe, F.J.C., 1984. Anabolic agents: evaluation of hormono-mimetic agents for mutagenic and carcinogenic potential. In: DSA (Bureau European d'lnformation pour le Developpement de la Same Animale) (Editor), Safety and Quality in Food. Elsevier, Amsterdam, pp. 125-141. Roush, W., 1991. Who decides about biotech? The clash over bovine growth hormone. Technology Review, July, pp. 29-36. Saenez de Rodriguez, C.A. and Toro-Sola, M.A., 1982. Anabolic steroids in meat and premature telarche. Lancet, I: 1300. Stephany, R.W., Jansen, E.H. and Freudenthal, J., 1985. A quarter of a century of studies of the abuse of hormonal anabolics in slaughtering animals: no DES-illusion! Tijdschr. Diergeneeskd., 110: 654-661 (In German). Sundlof, S.F., 1989. Drug and chemical residues in livestock. Vet. Clin. North Am. Food Anim. Pract., 5:411-449. Sundlof, S.F. and Strickland, C., 1986. Zearalenone and zearanol; potential problems in livestock. Vet. Hum, Toxicol., 28: 242-250. Van den Berg, G., 1991. A review of quality and processing suitability of milk from cows treated with bovine somatotrophin. J. Dairy Sci., 74: 2-11. Van Logten, M.J., van Leeuwen, F.X.R. and Stephany, R.W., 1981. Toxicological aspects of the use of hormones diethylstilboestrol, trebolone, zeranol as anabolic agents residues in foods. Tijdschr. Diergeneeskd., 106:353-366 (In German ). Van Mien, A.S., Peters, R.H., Basudde, C.D., Nijmeijer, S.N., van Duin, C.T., van Gogh, H. and Korstanje, C., 1988. Effect of trenbolone and testosterone on the plasma elimination rates of sulfamethazine, trimetboprim, and antipyrine in female dwarf goats. Am. J. Vet. Res., 49: 20602064.
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Vynckier, L., Debackere, M., de Kruif, A. and Coryn, M., 1990. Plasma estradiol-17 beta concentrations in the cow during induced estrus and after injection of estradiol- 17 beta benzoate and estradiol- 17 beta cypionate--a preliminary study. J. Vet. Pharmacol. Ther., 13: 36-42. Waltner-Toews, D. and McEwen, S.A., 1994a. Chemical residues in foods of animal origin: overview and risk assessment. Prev. Vet. Med., 20:161-178. Waltner-Toews, D. and McEwen, S.A., 1994b. Insecticide residues in foods of animal origin: a risk assessment. Prev. Vet. Med,, 20:179-200. Waltner-Toews, D. and McEwen, S.A., 1994c. Residues of industrial chemicals and metallic compounds in foods of animal origin: a risk assessment. Prev. Vet. Med., 20: 201-218. Waltner-Toews, D. and McEwen, S.A., 1994d. Residues of antibacterial and antiparasitic drugs in foods of animal origin: a risk assessment. Prev. Vet. Med., 20: 219-234. Witkamp, R.F., van't Klooster, G.A.E. and van Miert, A.S.J.P.A.M., 1990. 'Anabolic' steroids and 'gender' as factors influencing the residue levels of simultaneously administered drugs. In: N. Haagsma, A. Ruiter and P.B. Czedik-Eysenberg (Editors), Residues of Veterinary Drugs in Food. CIP-gegevens Koninklijke Biblioteek, The Hague, Netherlands, pp. 415-419. World Health Organazation, 1988. Toxicological Evaluation of Certain Veterinary Drug Residues in Food. WHO Food Additives Series No. 23. Cambridge University Press, Cambridge, 174 pp. World Health Organization, 1990. Toxicological Evaluation of Certain Veterinary Drug Residues in Food. WHO Food Additives Series No. 25. WHO, Geneva, 168 pp. World Health Organization: International Agency for Research on Cancer, 1987. IARC monographs on the evaluation of carcinogenic risks to humans. Overall evaluations of carcinogenicity: an updating oflARC Monographs volumes 1 to 42. Vol. suppl. 7. WHO:IACR, Lyon, 440 pp. World Health Organization, Regional Office for Europe, 1982. Health Aspects of Residues of Anabolics in Meat. Rep. WHO Working Group, WHO, Copenhagen, 38 pp.
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Residues of hormonal substances in foods of animal origin: a risk assessment D a v i d Waltner-Toews*, Scott A. M c E w e n Department of Population Medicine, Universityof Guelph, Guelph, Ont. N I G 2 W1, Canada Accepted 15 January 1994
Abstract
An assessment of the human health risks associated with residues of hormonal substances in foods of animal origin was undertaken. These compounds are used in the production of food animals to promote growth and increase the efficiency of feed utilization and in the case of bovine somatotropin, to increase milk production. The assessment is organized to combine information on the hazards posed by these residues with data on exposure to arrive at recommendations for risk management and avoidance.
1. Introduction Exogenous hormones first were used as growth promotants in cattle in the 1950s. Since that time, a variety of endogenous and synthetic (xenobiotic) hormonal substances has been developed and used in food animals (mainly ruminants) for the purposes of increasing the rate of growth, increasing the efficiency of feed utilization (feed conversion), regulating estrus, and (in the case of bovine somatotropin; BST) for increasing milk production. The anabolic agents as a group have been very effective in achieving these goals. The considerable controversy that has evolved over the use of these compounds in food animals is centred on two main points: ( I ) the effects of residues in foods on human health; (2) whether western society needs or wants to use these compounds at all. Widely disparate points of view on these matters are held. The controversy over the use of hormones in animal production began 20 years ago when diethylstilbestrol (DES) was banned from use as a growth promotant in cattle because clinical use of the compound in women was associated with *Corresponding author. 0167-5877/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIO167-5877(94)O3015-U
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vaginal cancer in some of their daughters (Health and Welfare Canada, 1973; Rodericks, 1986). The controversy has continued in recent years, with the European Economic Community ban on the use of all anabolic hormonal substances in food animals (Anonymous, 1985; Sundlof, 1989). In 1990, a moratorium was placed on the use of BST in lactating dairy cattle in the state of Wisconsin (USA), because of public concern over issues of milk production and the safety of milk products for human consumption, but the decision later was overturned (Reotutar, 1990). The purpose of this paper is to review the literature pertaining to the public health risk posed by residues of hormonal substances in foods from animals. This area has also been reviewed by Sundlof ( 1989 ). Placing residues found in Ontario, Canada (which we deem to be typical of many industrialized countries) into international contexts, we will discuss the risks that these residues might pose to consumers. Not considered in this review are the hormonal substances used for therapeutics and estrus synchronization in cattle. The safety of residues of these latter compounds in foods from animals has received little attention. The framework for this review, and for related reviews on residues of insecticides, industrial chemicals, and antibacterial drugs in foods of animal origin, is one generally used for risk assessment, as described in the first paper in this series (Waltner-Toews and McEwen, 1994a,b,c,d). The four elements of risk assessment are hazard identification and dose-response assessment (which we have grouped together), exposure assessment, and risk characterization. We have also added a section on residue avoidance, which is the primary risk management strategy for chemical residues in food.
2. Hazard identification and dose-response assessment Hormonal substances (anabolic agents) used in food animals fall into two groups: (1) those that are naturally occurring substances in animals (and are either extracted from animals or manufactured using recombinant DNA or some other technology); (2) those that are produced synthetically and do not occur naturally in animals (xenobiotic compounds). Among the first group are testosterone, estradiol, progesterone and somatotropin. Thyrotropin is a naturally occurring hormone not used intentionally as an anabolic agent, but residues of this substance can occur in foods and have been associated with human illness (Hedberg et al., 1987). Xenobiotics used in some countries (not legally in all cases) are trenbolone acetate, melangestrol acetate, zeranol and the stilbenes--most notably diethylstilbestrol (DES). Also included in this group are the fl-agonists (partitioning agents such as clenbuterol ). Estradiol (estrogen), testosterone and progesterone are sex hormones that occur naturally in mammals (including humans). Because of this, these hormones are normally present in food from animals and levels found in these foods vary widely depending upon the age, pregnancy status and physiological status of the animal (Hoffmann and Evers, 1986; Food and Agriculture Organization/World
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Health Organization (FAO/WHO), 1988 ). They are used mostly in cattle, either alone or in various combinations depending on the sex of the animal, by implantation of capsules of hormone into the subcutaneous tissues of the ear (an inedible portion of the carcass). The location of the implant influences the residue levels in plasma (Meyer et al., 1984); injection at the middle of the pinna results in lower levels than injection at the base. The possible hazards of residues of sex steroid hormones in edible tissues from animals have been previously reviewed (WHO, Regional Office for Europe, 1982; Meissonnier and Mitchell-Vigneron, 1983; Hoffmann and Evers, 1986; Sundlof, 1989). These sex steroid hormones are believed to playat least some role in human and animal cancer. For example, human castrates are at reduced risk of prostatic cancer, and epidemiological studies indicate that increased testosterone concentrations in blood are associated positively with increased risk of prostatic cancer (Meissonnier and Mitchell-Vigneron, 1983; WHO: International Agency for Research on Cancer (IARC), 1987). Most evidence indicates that steroid hormones can act as promoters of tumours in certain target organs that have hormone receptors, but the natural sex hormones are not genotoxic (Van Logten et al., 1981; Meissonnier and Mitchell-Vigneron, 1983; FAO/WHO, 1988). Examples of target organs for these compounds include the mammary gland, cervix, uterus and prostate gland. As promoters, these sex steroid hormones are capable of increasing the risk of cancer and therefore are classified as carcinogens. It is important to distinguish 'carcinogens' which promote tumours from those that have a genotoxic effect; the former exert their turnout enhancing effect by virtue of their hormonal activity. Compounds of this type are not carcinogenic at concentrations less than that which induces a measurable hormonal effect (WHO, Regional Office for Europe, 1982; Hoffmann and Evers, 1986; FAO/WHO, 1988 ). Acceptance of this concept is the basis for the establishment of so-called no-hormonal-effect-levels (NHEL) in animal models. These can be used to set safe acceptable daily intakes (ADI) (Foxcraft and Hess, 1986; Hoffmann and Evers, 1986). In contrast, many believe that the concept of safe levels does not apply to genotoxic carcinogens (Roe, 1984), although this is the subject of debate (see National Research Council (NRC), 1993; Waltner-Toews and McEwen, 1994a). Estradiol-17fl could be carcinogenic, but at levels greater than required for physiological purposes. In normal, untreated animals, highest levels are found in tissues of animals during late pregnancy. Treatment with hormone implants according to recommended procedures increased tissue levels by a factor of 2-5 (FAO/WHO, 1988). The amounts of estradiol-17fl produced daily by prepubertal boys (the lowest group) and pregnant women (highest) were estimated to be about 1000 to several million times, respectively, greater than the amount of hormone residue that would be present in one 500 g portion of meat from an animal implanted with this compound according to directions (Hoffmann and Evers, 1986; FAO/WHO, 1988). Levels of this hormone that would be present in edible tissues of treated animals could not exert any toxic effect in humans through hormonal action, and therefore the Joint FAO/WHO Expert Committee
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on Food Additives (JECFA; FAO/WHO, 1988) chose not to recommend an acceptable residue level. Therapeutic administration of the compound to cows produced higher-than-physiological levels (Vynckier et al., 1990). On the basis of this observation, a withdrawal period for therapeutic administration was suggested. Progesterone (in combination with estradiol-17//) is used in subcutaneous implants in steers and calves. It also is used for the regulation of the estrous cycle in ruminants and swine (Neff and Lauderdale, 1984). Like estradiol, progesterone exerts a carcinogenic effect by virtue of its hormonal activity (FAO/WHO, 1988 ). Consequently, it does not appear to be toxic at levels below those necessary for hormonal effect. As is the case with estradiol, residue levels of progesterone in tissues and milk of treated animals are much less than the daily production of the hormone even among prepubertal boys, and therefore are believed not to pose a risk to human health (WHO, Regional Office for Europe, 1982; FAO/WHO, 1988; Fitzpatrick, 1988; Sundlof, 1989). Similar comments apply to testosterone (in combination with estradiol-17//), used in implants in heifers (WHO, Regional Office for Europe, 1982; FAO/WHO, 1988). Trenbolone acetate (TBA), zeranol and melengestrol acetate (MGA) are xenobiotic anabolic agents, and any levels in tissues need to be considered contaminants (Roe, 1984). This requires that a somewhat different approach to the evaluation of their safety be taken from that of the natural sex hormones. TBA (a synthetic steroidal compound) is probably not a genotoxic substance (based on evidence from short-term genotoxicity assays and long-term feeding studies in laboratory animals (Meissonnier and Mitchell-Vigneron, 1983; Roe, 1984; Hoffmann and Evers, 1986; FAO/WHO, 1988; WHO, 1988, 1990). A proportion of the residues of TBA in tissue become bound to protein, but are of low toxicity and of no hormonal significance (Burgat-Sacaze et al., 1986 ). An acceptable daily intake (ADI) of TBA (0-0.02 #g kg-1 body weight day-~ ) was based on experiments in pigs that demonstrated a no-hormonal-effect-level (NHEL) of 2-3 #g kg -~ body weight day-l. Interim maximum residue levels (MRL) for trenbolone in bovine edible tissues have been set at 0.7 #g kg- ~of//-hydroxytrenbolone and 7.0 #g kg- ~of o~-hydroxytrenbolone (Sundlof, 1989 ). Zeranol is a non-steroidal anabolic agent used as an implant in cattle, and is derived from zearalenone, a mycotoxin produced by Fusarium spp. The compounds are similar in structure and are believed to share some toxic properties-particularly those of estrogenic effect (Baldwin et al., 1983; Sundlof and Strickland, 1986; FAO/WHO, 1988). The recommended ADI is 0-0.5 #g kg ~ body weight day -~ for humans (FAO/WHO, 1988; WHO, 1988). There is no evidence that zeranol is genotoxic (Meissonnier and Mitchell-Vigneron, 1983 ). Reports in the literature that describe precocious sexual development (thelarche) of children in Puerto Rico and Italy are sometimes cited as evidence of the human health risk posed by the use of anabolic hormonal substances in food animals (Fara et al., 1979; Saenez de Rodriguez and Toro-Sola, 1982; Bongiovanni, 1983; Sundlof and Strickland, 1986). A large number of cases were involved in the Puerto Rican outbreak while the Italian outbreak involved a smaller
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number of cases (Fara et al., 1979; Bongiovanni, 1983). In both outbreaks, the authors speculated that the cause of the problem could have been food contaminated with estrogenic substances. DES or zeranol were specifically named in association with the Puerto Rican outbreak. However, almost no evidence was presented that would support this hypothesis. The Italian outbreak was characterized by an increase in the occurrence of breast enlargement in girls and boys that attended a school in Milan (Fara et al., 1979). Samples of the school meals were tested for estrogen contamination, and none was found. In the Puerto Rican outbreak, some 'preliminary results' in the laboratory of one investigator were interpreted to suggest that there were quantities of estrogenic substances in meat and poultry, but gas-liquid chromatography of these foods for zeranol and DES was negative (Bongiovanni, 1983). Subsequent investigation of the outbreak by the Centers for Disease Control, USDA and FDA in the USA revealed that the outbreak could have been due to increased reporting and awareness of the problem of thelarche by physicians (Sundlof, 1989 ). USDA analyzed approximately 700 samples of animal products for estrogenic substances, and no violative residues were found. Melangestrol acetate (MGA; an orally active progestogen) is used in heifers in the form of a feed additive. A MRL of 25/tg kg -~ (25 ppb) in edible tissues has been set (Neff, 1983; Neff and Lauderdale, 1984), and although a 48 h withholding time was imposed, heifers fed MGA at prescribed doses have tissue levels less than MRL without feed withdrawal. Diethylstilbestrol (DES; a stilbene compound) was used in the 1950s and 1960s as implants for growth promotion in cattle. The compound was also used in human medicine--most importantly (in the context of this review) for the maintenance of pregnancy in women. Daughters of women who were treated with high levels of DES for this purpose had increased risk of a rare type of cancer of the vagina (Health and Welfare Canada, 1973; Jukes, 1974). Subsequent studies in laboratory animals established that high levels of DES fed to laboratory animals over a long period of time resulted in an increase in the incidence of tumours in these animals (Jukes, 1974; Van Logten et al., 1981 ); this presumably was a result of the hormonal effect of the compound, although there has been suggestion of mutagenicity or genotoxicity (Van Logten et al., 1981; WHO, Regional Office for Europe, 1982). Evidence that oral DES was absorbed readily and not destroyed readily by the liver after uptake also weighed in the decision to ban the substance from use in food animals in the early 1970s (Anonymous, 1982; Roe, 1984). Somatotropin (ST; also known as growth hormone; GH) has a variety of effects in animals, and scientists have known since the 1920s that it could increase the milk production of cows (Connaughton, 1989; Juskevich and Guyer, 1990). In cattle, the terms recombinant bovine growth hormone and recombinant bovine somatotropin are used interchangeably to describe the genetically engineered product. The production of this substance in commercial quantities is possible, and it is being used (or licensing is being sought for use) in dairy cows (Hart, 1987 ). ST also may find application as an anabolic agent in swine.
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As with other compounds, the human safety of ST use in animals has been addressed by evaluation of the biochemistry of the compound, its effects in laboratory animal species and humans, its effect on the nutritional composition of milk and the occurrence of residues in edible foods (especially milk) (Juskevich and Guyer, 1990). ST is a long polypeptide protein molecule that is cleaved into polypeptides and amino acids by the digestive process and therefore thought not to be absorbed intact through the gut. STs tend to have a very narrow species range of activity, and although bovine somatotropin (BST) is anabolic in rats by parenteral injection, it is not anabolic in rats when given orally, nor is it active in humans (Cherfas, 1990; Juskevich and Guyer, 1990). BST occurs normally in milk (at about 1 ppb; Juskevich and Guyer, 1990 ) and like other naturally occurring hormones, levels of BST in meat and milk from animals treated in the proper manner are no greater than levels in untreated animals (Connaughton, 1989; Juskevich and Guyer, 1990). Pasteurization reduces the amount of BST in milk by 90% (Juskevich and Guyer, 1990), but it does not reduce the quantity of insulin-like growth factor-1 (IGF-1; a mediator of GH action). IGF-1 is not species specific (Kronfeld, 1989), and levels in meat of BST-treated cows are approximately twice those of untreated cows. There is currently no evidence that these increased levels pose a health risk (Juskevich and Guyer, 1990; H a m m o n d et al., 1990). Although fat, protein and lactose concentrations of milk are not appreciably altered when BST is used in cows in a positive energy balance, BST can cause an increase in the relative amount of long-chain fatty acids and can increase the somatic cell counts of milk (Van Den Berg, 1991 ). Based on this type of evidence, most scientists believe that BST used in dairy cattle is safe for humans and that the composition and nutritional value of milk from treated animals are essentially the same as that from untreated cows (Anonymous, 1991 b). Other investigators however, believe that final judgement on the human safety of BST use is premature---especially until more is known of the importance of changes in IGF-1 and other hormones in meat and milk from BST treated cows (Reotutar, 1990; Van Den Berg, 1991 ). Unlike ST, thyrotropin (thyroid hormone, TH) from cattle is biologically active in humans, and is absorbed after ingestion. Although TH is not used as a therapeutic or growth-promoting agent in food animals, thyrotoxicosis as a result of ingestion of excessive quantities of bovine TH have been reported. In 1984 and 1985, 121 human cases of thyrotoxicosis were identified in Nebraska (Hedberg et al., 1987 ). These individuals were suffering from a variety of symptoms, including nervousness, headaches, heart palpitations and weight loss, and they had increased levels of thyroid hormones. Affected individuals were more likely than controls to have consumed ground meat partially composed of bovine laryngeal trimmings. The thyroid glands in cattle are in close proximity to the skeletal muscles of the larynx, making contamination with thyroid glandular tissue very easy. Clenbuterol is a fl-agonist (salbutamol is another) that has been used illegally as a so-called 'partitioning agent' for increasing the proportion of muscle in car-
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casses (i.e. leaner meat). The drug is not approved for use in food animals anywhere, but illegal use in cattle has been reported in the USA (Anonymous, 1991 a; Keller, 1991 ). In 1990, residues of clenbuterol in liver were linked epidemiologically to hospitalization due to increased heart rate, muscle tremors, headache, dizziness, fever and chills in 135 people in Spain (Keller, 1991 ). Beef liver specimens obtained from case families during the outbreak contained 160-291 ppb clenbuterol. Trace-back studies by health authorities indicated that the animals may have been given toxic doses of the drug inadvertently and when clinical signs arose, were slaughtered for salvage. Recently, the effect that anabolic agents may have on the disposition (and therefore residue levels) of other simultaneously administered drugs has been raised as a factor to be considered (Van Miert et al., 1988; Witkamp et al., 1990). Implantation of female goats with TBA and testosterone resulted in a decrease in the plasma elimination of antipyrine, sulfamethazine and trimethoprim. Additional research is needed to determine if these interactions have a substantial bearing on drug residue levels in foods.
3. Exposure assessment If properly administered as an implant in the inedible tissue of the ear, estradiol, progesterone and testosterone are released slowly from the injection site; direct human exposure to unabsorbed hormone is avoided by discard of the ear at slaughter. Residues of these hormones in foods from animals form an insignificant proportion of the total exposure of humans to these compounds. Estradiol17fl in fat is the last estrogen/tissue combination to return to endogenous levels after treatment of cattle with estrogen implants: this association could be used for monitoring (Henricks et al., 1983 ). Of the xenobiotic anabolic agents, melangestrol acetate (MGA) is used as a feed additive, is rapidly excreted and therefore requires only a brief withdrawal time (48 h) from feed of animals prior to slaughter. In contrast, zeranol is administered as an implant and has a rather long withdrawal time (65 days). Trenbolone acetate is an implant licensed for use in beef cattle in Europe and in the USA (Sundlof, 1989). Residues of zeranol can be found in liver up to 120 days after implantation, and are highest in edible tissues 14 days after administration (Horwitz et al., 1983; Dixon et al., 1986). An abattoir survey of growth promotant implants in cattle was conducted in 1984-1987 in Scotland and northern England (Maddox, 1988 ). Cattle were selected at random, and implants were detected by palpation: 6014 cattle were examined, and implants were found in 2% of dairy cows, 4% of beef cows, 8% of steers and 7% of heifers. Ninety-five percent of the implants in cows were trenbolone acetate, but 6% of implants in steers and heifers were zeranol (even though zeranol implants were less likely to be detected given their small size, and some of the implants were detected after an official ban on their use) (Maddox, 1988 ). DES has been banned in most countries for a number of years, but illegal resi-
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dues have been reported. Extensive screening has been credited with the nearly complete disappearance of illegal use of DES in The Netherlands (Stephany et al., 1985 ). However, injection sites in cattle containing other illegally used anabolic agents (such as nortestosterone, medroxyprogesterone, estradiol and testosterone) have been reported from The Netherlands (Jansen et al., 1985; Stephany et al., 1985 ). Of 573 injection sites examined during 1983-1985,462 were positive for one or more substance. Nortestosterone was found in 84% of samples, medroxyprogesterone in 14%, methyltestosterone in 2%, and trenbolone in 2%. Testosterone was detected in 29% of samples, estradiol in 66% and progesterone in 3%. Residues of DES, dienestrol, hexestrol and zeranol were detected in 2%, 3%, 0%, and 0% respectively, of samples. Up to four different substances were found in injection sites from some animals (Jansen et al., 1985 ). The illegal use of the natural sex hormones estradiol and testosterone resulted in a scandal in Germany where approximately 13 000 calves were seized in an effort to contain the problem (Sundlof, 1989 ). Violative residues of DES were found in veal calves from the state of New York in 1983. Thereafter until 1989, no DES residues were detected (Cordle, 1988; Food Safety and Inspection Service, US Department of Agriculture (USDA), 1990). USDA monitoring of ruminants for zeranol (622 animals in 1988, 378 in 1989) resulted in no cases of violative residues. Testing of 16/134 (1988) and 13/232 (1989) surveillance specimens (from suspect animals) were positive. All of the violative cases were calves (Food Safety and Inspection Service, USDA, 1990). In 1989, 373 specimens of fat from heifers were tested for MGA and none were violative.
4. Risk characterization
As discussed previously, the human safety of residues of the natural sex steroids (testosterone, progesterone and estrogen) in edible tissue is based on the premise that these compounds occur in animals naturally and tissue residues in treated animals are within established normal ranges for untreated animals (Craft, 1986 ). Accordingly, ADI and withdrawal times have not been established for the natural sex steroids. Improper administration of the capsules into an edible portion of the carcass could result in direct ingestion by a consumer and exposure to higher levels of hormone, but this has never been reported. There is one report that indicates that veal calves may be implanted repeatedly or with more than the approved number of implants (Agriculture Canada, 1987). This could raise the amount of hormone in tissue samples, but there is no evidence that these levels would be a risk to humans. The biological activity of the xenobiotic hormonal substances has been measured through assays of hormonal effect, toxicity and genotoxicity in laboratory animals. For the compounds legally available (TBA, MGA, zeranol), hormonal effects are the most significant. Quantities of hormone below the no-hormonaleffect-levels (NHEL) do not, by definition, result in a response in the target tis-
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sue (s) of that hormone. Properly administered, these substances should not leave residues in edible tissues and milk that could have an undesirable hormonal effect in humans. In theory, the risk of treated animals entering the food chain before the proper withdrawal times have been observed is probably greatest for zeranol. There are two reasons for this: the extent of its use (for example, zeranol is the most widely used hormonal substance for cattle in Canada) and the long (65 days for a 36 mg implant dose) withdrawal time. The risk of animals entering the food chain before the withdrawal time is completed may be greatest for animals culled for a medical reason (fracture, prolapse, etc. ) because these culls cannot be foreseen by farmers when cattle are implanted. The safe ADI of TBA is 0-0.02/tg kg -1 body weight day -1 (WHO, 1990). Appropriate maximum residue levels (MRL) of TBA are 2 #g kg- l in food animal skeletal muscle as fl-trenbolone and 10 #g kg- l in liver as a-trenbolone. The ADI of zeranol of 0-0.5/zg kg-~ body weight day-~ for humans (FAO/ WHO, 1988 ) corresponds to an MRL of 70 #g kg- ~of edible tissue. Since this is a higher level of residues than is necessary using good husbandry practices, MRL of 10/~g kg- l for bovine liver and 2 #g kg- 1 for bovine muscle have been recommended (FAO/WHO, 1988). In Canada, results of recent monitoring for tissue residues of the xenobiotics zeranol and MGA suggest that exposure of humans to violative levels of these compounds in meat is probably rare; only one specimen containing a violative level of zeranol has been detected. This, coupled with the relatively low toxicity of these compounds, suggests that the use of these compounds poses a very small risk to Canadians. Monitoring of food animal tissues for hormonal substances not licensed in Canada, including TBA, DES and clenbuterol, reveals that residues have either not been detected (TBA, DES) or, in the case of clenbuterol, are rare. In light of these data, and the fact that approved anabolic agents are legally available, risk of exposure and toxicity to TBA and DES is probably very low. The illegal use of clenbuterol in Canada (Agriculture Canada, 1989), Europe and (most recently) the USA (Keller, 1991 ) may be driven by a demand for partitioning agents not filled by a legal compound. The occurrence of illegal residues of this drug (along with the recent report of human toxicity attributed to residues of clenbuterol in liver) are of concern and argue for the continued monitoring of this drug. As was mentioned previously, DES is no longer used in food animals in North America because, in part, daughters of women treated with the compound were at increased risk of cancer and laboratory animals developed tumours after prolonged feeding of the substance (Health and Welfare Canada, 1973 ). Most scientists believe that the human health risk posed by DES use in cattle was very small (Jukes, 1974; Rodricks, 1986).
5. Risk avoidance
Although DES has been banned for use in food producing animals, the experience with this compound has left an important legacy. This was the first hor-
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monal substance that had widespread use in food animals, and was for years touted to be both effective and safe. The debate that followed the revelations that DES use in women was associated with cancer may have left the public with lasting mistrust and aversion to the use of any hormonal substance in food animals. It also may have resulted in a loss of public confidence in the ability of the scientific community to predict the safety of these compounds. In addition to this, high concentrations of illegally used DES were found in baby food in Europe in 1980 (Sundlof, 1989). This aggravated public concern, and played a role in the eventual banning by the European Economic Community (EEC) of the use of all hormones for growth promotion in food animals (Anonymous, 1985). The illegal use of hormonal substances in food animals has been reported in many countries and this is most often a problem when there are no substances legally available to suit the purpose (Anonymous, 1982 ). The natural sex steroids (progesterone, estradiol and testosterone) appear to be safe and the only conceivable human health risk associated with their use would be in the actual ingestion of implant sites. Limited evidence from monitoring of implant sites in Canadian cattle indicates that some implants are administered in improper locations, and some animals are treated repeatedly or given multiple implants. It would be prudent to ensure that users of these products in cattle are instructed in the proper techniques and the reasons for using the particular site. The widespread use of zeranol combined with its long withdrawal time could produce residues in treated animals prematurely culled to slaughter. Perhaps emergency cull animals should be monitored more closely than the general population of slaughter cattle. Also, since there is probably a seasonal trend to use of these compounds (most in the fall), this factor should be considered when devising a sampling plan for residue testing. Although not used intentionally in food animals, thyroid hormone is the one compound that has been shown to result in human disease after ingestion of meat contaminated with thyroid trimmings. The entire laryngeal area, including skeletal muscles of the larynx, the thyroid gland and other structures should be condemned at slaughter. Illegal use of clenbuterol and other fl-agonists is of some concern, especially in view of the report of human illness attributed to residues of clenbuterol in liver of treated animals (Keller, 1991 ). Monitoring of cattle for these compounds should be undertaken at a level sufficient to establish if at least 1% of the population of cattle is being treated illegally. Finally, for BST the issue is not public health, but socio-economics (Kneen, 1990; Roush, 1991; Kneen et al., 1991 ). Settling of the scientific and risk communication issues is just one part of determining safe and acceptable agricultural policies (NRC, 1983).
Acknowledgment Financial support for this project was provided by the Ontario Ministry of Agriculture and Food.
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