Illegal use of beta-adrenergic agonists: European Community.

H A Kuiper, M Y Noordam, M M van Dooren-Flipsen, R Schilt and A H Roos J ANIM SCI 1998, 76:195-207.

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Illegal Use of b-Adrenergic Agonists: European Community1,2

H. A. Kuiper, M. Y. Noordam, M.M.H. van Dooren-Flipsen, R. Schilt, and A. H. Roos
State Institute for Quality Control of Agricultural Products (RIKILT-DLO), P.O. Box 230, 6700 AE Wageningen, The Netherlands

ABSTRACT: The use of veterinary medicinal products within the European Community is governed by a series of directives and regulations that describe the requirements for safety, quality, and efficacy of these products. Veterinary therapeutic use of bagonists has only been approved in the case of clenbuterol for bronchodilatation in horses and calves and for tocolysis in cows. No b-agonists have been permitted in the European Community for growthpromoting purposes in farm animals. Surveillance for the presence of residues of veterinary agents in foodproducing animals and meat is regulated by the Directive 86/469/EEC containing specific guidelines for sampling procedures on farms and in slaughterhouses. The level and frequency of sampling is dependent on the category of compounds and animal species. When positive samples have been identified (above certain action levels), sampling intensity is increased. Results of monitoring programs in EU member states during 1992 and 1993 for the occurrence of residues of b-agonists in food-producing animals vary substantially with respect to the percentages of positive samples, ranging from 0 to 7%. The variability is partly explained by differences in

sampling strategies, detection methods, and action levels applied. Identification of the proper matrices for sampling and detection of b-agonists is important. In the case of clenbuterol, hair and choroid retinal tissue are appropiate tissues because clenbuterol accumulates in these matrices. A clear decrease in the use of clenbuterol in cattle has been observed in The Netherlands, Germany, Northern Ireland, and Spanish Basque Country over the last 3 yr. This is partly due to intensified surveillance activities at farms and slaughterhouses by governmental agencies and production sector organizations. There are data on human intoxication following consumption of liver or meat from cattle treated with b-agonists. At the concentrations of clenbuterol measured in contaminated liver and meat samples, pharmacological effects may be expected in humans after consuming 100 to 200 g of product. The use of highly active b-agonists as growth promoters is not appropriate because of the potential hazard for human and animal health, as was recently concluded at the scientific Conference on Growth Promotion in Meat Production (Nov. 1995, Brussels).

Key Words: Regulations, Surveillance, b-Adrenergic Agonists, Safety, Detection

1998 American Society of Animal Science. All rights reserved.

J. Anim. Sci. 1998. 76:195207

Introduction
The use of growth promoters in the production of animal products for human consumption has been an issue of scientific debate and public concern for many

1Presented at a Symposium titled Pharmacology, Toxicology, and the Illegal Use of b-Adrenergic Agonists July 1996, at the ASAS 88th Annu. Mtg., Rapid City, SD 2We would like to acknowledge the Quality Programme Agricultural Products (KAP) for providing residue data for several products and the Dutch Cattle Quality Inspection (KCR) for provision of data on surveillance activities. Irene Konig-Lamers is acknowledged for technical assistance with the preparation of the manuscript. Received October 21, 1996. Accepted July 8, 1997.

years. Since the European ban on anabolic steroids was established in 1986, alternative substances such as b-agonists, the physiological analog of adrenalin, have been introduced as growth-promoting agents. These substances, in addition to their regular role in veterinary medicine as bronchodilatory and tocolytic agents, increase lean meat to fat ratio (repartitioning) and improve feed conversion efficiency (Allen et al., 1987; Moloney and Beermann, 1996). None of the European Member States has authorized the use of bagonists for repartitioning purposes. Illegal use of these pharmacologically active substances has resulted in severe cases of food poisoning (MartinezNavarro, 1990; Pulce et al., 1991), which underlines the necessity of developing proper detection methods and control systems. 195

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This paper describes the legal framework in European Union ( E U ) Member States concerning the detection and control of illegally applied substances for growth-promoting purposes. Member States have designed national surveillance programs for the detection and control of residues in animals and meat, and production sector organizations have initiated specific control activities. Results of these activities, and of a survey organized by European consumers organizations, are presented.

Legal Control of Veterinary Drugs and Growth-Promoting Agents in European Union Member States Marketing Authorization of Veterinary Drugs
Marketing veterinary drugs in a Member State is preceded by a lengthy registration procedure governed by several EU Regulations and Directives. Two Directives, 81/851 and 81/852, concerning the harmonization of laws, and pharmaceutical quality, efficacy, safety, and residue data requirements have been issued, followed by various updates (90/676, 90/ 677, 87/20, and 92/18). Since January 1992, active substances of veterinary drugs are evaluated according to EU Regulation 2377/90, which describes a Community procedure for the establishment of maximum residue limits ( MRL) of veterinary medicinal products in foodstuffs of animal origin. Before market authorization, the active substances must be listed on one of the three annexes of the Regulation: Annex I (definite MRL), Annex II (no MRL necessary), or Annex III (provisional MRL). Annex IV contains a list of substances for which it was not possible to establish a MRL, and which therefore are not allowed to be administered within the EU. The use of stilbenes, stilbene derivatives, their salts and esters, and thyreostatic substances in all animal species has been prohibited in the EU since 1981, as well as the use of natural or synthetic hormones for fattening purposes (Council Directives 81/602/EEC and 88/146/EEC). Administration of substances with estrogenic, androgenic, or gestagenic action is only allowed for therapeutic reasons under specified conditions, such as fertility disorders, in those member states where these substances are authorized.

for tocolysis. Some European authorities adopted national MRL, such as the United Kingdom, with a MRL of .5 mg clenbuterol/kg edible tissue, and The Netherlands, with a MRL of 1 mg/kg liver. None of the Member States has authorized the use of clenbuterol or any other b-agonist for repartitioning purposes. Dose levels of clenbuterol used for these purposes are 5 to 10 times higher and the duration of dosing is more prolonged than in therapeutic treatment (Godfrey, 1996; Witkamp et al., 1996). Recent information indicates that lower doses, even therapeutic ones, have a positive influence on growth and carcass quality of calves and cattle.

National Monitoring Plans

Each European Member State is obliged to monitor animals and meat for residues of legally and illegally used veterinary drugs, including hormones and other substances such as pesticides and environmental contaminants (Directive 86/469/EEC), and to present a National Residue Monitoring Plan that takes into account the specific situation in each Member State (Gaudot, 1993). Aspects to be covered in a National Plan are a description of the organizations and laboratories involved in the implementation and execution of the National Plan, substances to be measured, analytical methods, action levels, animal species, and number of samples to be taken (in relation to the number of slaughtered animals in the previous year). Frequency of sampling is dependent on the group of compounds and animal species. With regard to bagonists, provisions in Directive 86/469 are rather vague. According to Annex I of this Directive bagonists belong to group B. For substances belonging to group B (ecto- and endoparasitica, tranquillizers and b-blockers and other veterinary drugs, contaminants in animal feed, environmental contaminants and other substances), a minimum of 700 samples are to be taken. Sampling frequency should reveal a 1% abuse with a statistical confidentiality of 95%. Differences exist between Member States in terms of the species sampled, number of samples taken, sampling methods (e.g., random sampling vs targeted sampling), matrices for analysis, methods of analysis, range of b-agonistic substances, and action/ detection levels used in determining positive results.

Registered Use of -Agonists

The only b-agonist registered for veterinary use in cattle, horses, and pets in almost all European countries is clenbuterol (Boenisch and Quirke, 1992). Registered therapeutic oral or parenteral dose levels for clenbuterol in The Netherlands are 1.5 mgkg body weight1d1 for a maximum of 10 d for treatment of bronchospasms in horses and nonlactating cattle, and a single dose of 300 mg/cow ( .5 mg/kg body weight)

Intensification of Control on Illegal Use of -Agonists

Recently (March 1996), the EU ban on hormone use for growth promotion has been extended to bagonists (Directive 96/22/EC), and controls and sanctions on illegal use have been tightened (Directive 96/ 23/EC). Only therapeutic treatment with b-agonists of equidae, domestic pets, and pregnant cows under strict veterinary supervision will be allowed.

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Any residue of b-agonists found in animals not specified in the exemption will be a violation. In other animals, a drug prescription record to be kept by the responsible veterinarian will illustrate whether withdrawal periods were respected. Moreover, more details concerning residue control procedures are prescribed. Most important is the introduction of targeted sampling for banned substances or banned usage (e.g., sampling where an illegal application of substances is most likely). Species to be examined for b-agonists residues are cattle, sheep, goats, swine, horses, poultry, rabbits, and game. Samples should be taken without prior notice. The minimum number of samples to be checked for substances of group A of Annex I of 96/23/EC (subgroups: stilbenes and derivatives, antithyroids, steroids, resorclic acid lactones, b-agonists, and substances of Annex IV of 2377/90) is expressed in percentages of the number of animals slaughtered the previous year or production volume in the Member State concerned (Table 1). A minimum of 5% of the total number of samples collected should be investigated for residues of each subgroup of group A. In the case of cattle, 25% of the farm samples for bagonist control may be taken from suspected matrices such as feed and milk replacer. The Directives 96/22 and 96/23 must be implemented by Member States July 1, 1997.

Table 1. Sampling frequencies for residues of Group A (Appendix IV 96/23/EC) as percentage of the number of animals slaughtered in the previous year or related to production volume in the Member State concerned
Animals Number of samples to be collected .25% .02% Farm Slaughterhouse

Cattle Swine Sheep/goat Horse Poultry

50% 50% Min. 1 per 100,000 animals .01% Not specified Not specified Not specified Not specified Not specified 1 per 400,000 20% 80% kg production

Confirmation Methods
Results of screening analysis should be confirmed by gas chromatography-mass spectrometric analysis ( GC-MS) , a common practice in many countries. Sample extraction is necessary and may be performed in different ways depending on the b-agonist(s) to be determined. The broad immunochemical screening methods only show the possible presence of a substance, but not the (chemical) identity. Thus, a similarly broad confirmatory analysis must be performed, otherwise a false negative result will be obtained. Confirmatory analysis must meet the EU identification criteria (93/256/EEC). In short, preferably four (diagnostic) ions must be measured by MS and the relative abundance ratios must be within 10% compared with the standard analyte. For the most often used monotrimethylsilyl (TMS) derivative of clenbuterol, however, the mass spectrum contains a number of diagnostic ions with low abundances (86, 262, 264, 277, 333, and 348), making it difficult to fulfill the criteria described above at low residue levels. Extensive sample clean-up is needed to achieve these requirements (Hooijerink et al., 1994). Several alternative derivatization procedures have been developed; however, they are applicable to a smaller number of b-agonists, which limits their use (Polettini et al., 1991; Montrade et al., 1993; Van Rhijn et al., 1993).

Methods
It has been observed that the levels of residues of bagonists detected in urine and liver have decreased over the years due to optimalization of administration schemes to avoid detection. Also, application of comedication may lower residue levels of b-agonists (Schilt et al., 1994a). Many laboratories now use a combination of screening and confirmatory methods to cope with relatively large numbers of samples and to increase the reliability of the final result (Schilt et al., 1990).

Screening Methods
A number of commercial enzyme immunoassays are now available. Most kits are designed for clenbuterol or salbutamol and may detect other cross-reacting bagonists (mabuterol, mapenterol, clenproperol, cimaterol, bromobuterol, and terbutaline). For example, in some clenbuterol kits, salbutamol with a crossreactivity of 10% can be determined at 10 mg/L in urine, compared to 1 mg/L for clenbuterol. Most important for the screening step is an acceptable low number of false negative results. Performance of the kits is not always clearly described by the manufacturers (Hahnau and Julicher, 1996), and validation of the actual performance of an immunoassay for the analysis of urine or liver samples should be performed by the analyzing or reference laboratory.

Developments
The rapid changes in the development of new bagonists have shown the need for analytical procedures capable of screening and confirmation of a larger range of b-agonists. Some of the known bagonists and their synthesized analogs exhibit only minor differences in their chemical structures, and the number of possible structures is very large (Keck et al., 1972). A common property of b-agonists is their binding to a receptor. Clenbuterol is a known stimulant of the b(2)-receptor (smooth muscle relaxation), although at a higher dosage also b( 1 ) stimula-

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tion (cardiac effects) are seen. Furthermore, pharmacochemical studies suggested that clenbuterol also interacts with the b( 3 ) or intermediate b-receptors (lipolysis) (Hollenga et al., 1990). Several research groups have developed radioreceptor assays for b( 2 ) agonists as a screening tool (Helbo et al., 1994). Trachea tissue containing b(2)-receptors may be used to detect residues of b-agonists by measuring relaxation (Schilt et al., 1994b). One advantage of this approach is the actual measurement of the effect instead of the measurement of the affinity, because b(2)-blocking drugs may also bind to the receptor. Future developments include development of dipstick technology to analyze samples directly at the farm or slaughterhouse (Ploum et al., 1990). The confirmatory methods must be able to identify the b-agonist detected during screening, and therefore ranges of detected analytes must match. Gas chromatographymass spectrometric confirmation is widely used but requires specific sample clean-up and derivatization for the different subgroups of b-agonists. Using broad solid-phase extraction and clean-up procedures with mixed phases or other specific ligand-containing materials, obtaining extracts for many b-agonists is possible. For identification, liquid chromatographymass spectrometry ( LC-MS) that does not need any derivatization is a promising alternative. Using the atmospheric pressure chemical ionization (APCI) interface, identification is possible at low micrograms per liter (ppb) levels (Doerge et al., 1995).

Matrices for Detection of b-Agonists

For residue surveillance of b-agonists, it is essential to identify the proper matrices for detection of residues, with respect to potential accumulation of the drug, ease of sampling and handling, and extractability of residues. Difficulties have been encountered in distinguishing between legal and illegal treatment. Control at the farm level has been focused on analysis of urine samples, but other matrices such as blood and hair are considered now as target sample material. Furthermore, samples of animal feed, drinking water, or milk replacer are being examined. In calves, plasma and urine concentrations during oral treatment with clenbuterol at growth-promoting dosages varied widely, but they were well above detection limits; concentrations decreased rapidly in both matrices after treatment was discontinued (Sauer et al., 1995). Therefore, these matrices are of limited value for the detection of illegal use of clenbuterol. Melanincontaining tissues, such as retina and hair, are known to accumulate clenbuterol (Meyer and Rinke, 1991; Elliott et al., 1993b; Sauer and Anderson, 1994; Sauer et al., 1995). In black hair of male veal calves orally treated with clenbuterol at a therapeutic dose level (.8 mg/kg body weight, twice daily for 10 d), residues could be detected 60 d after treatment. This was also

the case for fair-colored hair, although at much lower levels. Once incorporated into hair, no depletion of clenbuterol occurs, making hair a promising matrix to monitor for abuse in live animals (Gleixner et al., 1996; Godfrey et al., 1996). For inspection in slaughterhouses, liver is the appropriate target organ because residues of clenbuterol persist longest in this tissue after repartitioning treatment of calves (Table 2 ) (Meyer and Rinke, 1991; Sauer et al., 1995) and sheep (Elliott et al., 1993a). Sheep treated orally with clenbuterol at 1, 10, or 25 times the therapeutic dose level for 15 d displayed a tissue distribution similar to that seen in calves (Elliott et al., 1993a). In the retina/choroid tissue of treated animals, levels of clenbuterol far exceeded those in liver. Choroid retinal epithelium/liver ratios in calves increased from 11 to 52 over the withdrawal period of 6 h until 16 d after discontinuation of treatment (Sauer et al., 1995). In cattle orally treated with clenbuterol at a therapeutic dose level, the only tissue containing detectable residues beyond d 14 of drug withdrawal was the eye, with a mean level of 6.3 mg/kg eye tissue 42 d after treatment (Elliott et al., 1995). In two heifers fed 10 times the therapeutic dose level (16 mgkg body weight1d1) for 30 d retina/liver ratios 56 d after drug withdrawal were 183 in one and 308 in the other animal, respectively (Elliott et al., 1993c). It is not known whether other b-agonists also accumulate in eye tissue. Melanin binding studies indicate that the affinity of salbutamol for melanincontaining tissues may be less than that of clenbuterol (Howells et al., 1994; Sauer and Anderson, 1994). The limited quantity of retinal material available ( 2 to 3 g per pair of cattle eyes) may pose an analytical problem (Elliott et al., 1993b). Co-medication may influence the detection of b-agonist residues. A recent study of combinations of b-agonists and estradiol, methylthiouracil or dexamethasone administered to calves indicated higher concentrations of b-agonists in urine, feces, and liver when estradiol was co-administered, and lower concentrations when dexamethasone was present (Groot et al., RIKILT-DLO, personal communication).

Results of Surveillance of the Use of b-Agonists in European Union Member States Eurpoean Union Surveillance Programs
Since 1987, the European Commission has received the monitoring plans of EU Member States concerning anabolic steroids and thyreostats, and since 1988 concerning other substances. Results of the 1992 and 1993 monitoring program on b-agonists in randomly sampled cattle are presented in Table 3. At present approximately 30,000 animals in EU member countries are examined each year for the presence of residues of b-agonists. Results of the monitoring vary

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Table 2. Ratios of clenbuterol levels in calves orally treated with 10 mg/kg body weight twice daily for 10 days at different withdrawal intervals (mean of five animals per withdrawal period). Mean concentration in liver at day 16 of drug withdrawal was 9.8 mg/kg livera
Withdrawal period Tissue/tissue ratio Liver/kidney Liver/diaphragm Liver/bile Liver/urine
aSauer et al. (1995). bDenotes very high liver-diaphragm

6 h 1 15.9 2.2 1.8

1 d .6 26.3 2.5 5.0

2 d 1.5 25.2 3.2 3.0

4 d 1.3 39.4 6.2 10.6

8 d 2.3 b 31.2 24.0

16 d 2.5 41.1 34.5

ratio of clenbuterol; concentrations in diaphragm were not different from zero.

substantially between Member States; positive samples taken at the farm or in the slaughterhouse range from 0 to 7%. The percentage of random samples taken at the farm in which b-agonists were detected in Belgium in 1993 (17.5%) is questionable, because results of investigations in live suspect animals were much lower (10%). Percentages of positive samples containing b-agonists taken in the farm or slaughter phase during 1992 and 1993 are presented in Figure 1. It should be noted that the number of countries reporting positive samples and the percentage of positive samples taken in the slaughter phase in most countries are decrasing. In Denmark, no positive samples were found during this period. Comparison of results between Member States is difficult due to differences in residue sampling plans and sampling methods, laboratory analysis methods, action levels, and reporting formats. For example, in Italy and Luxembourg, samples were only taken from slaughtered animals, and Germany and France did not report separate results for samples taken at the farm or in slaughterhouses. There are also differences with respect to applied action limits; in Denmark the action level is .1 mg/kg tissue, and in Spain it is 1 to 2 mg/kg tissue. The 1992 and 1993 monitoring results are based on analyses of urine and liver, matrices that may not be the most proper targets for monitoring of the use of b-agonists, as discussed above.

Surveillance Activities on the Use of -Agonists in The Netherlands Annual National Program. Results of random sampling are obtained by the National Inspection Service for Livestock and Meat (RVV). The number of samples to be taken for examination of b-agonists should be 1% of the slaughtered cattle younger than 1 yr, 100 of slaughtered cattle older than 1 yr, and 100 pigs. Clenbuterol, salbutamol, mabuterol, mapenterol, cimaterol, and terbutalin have been assayed with enzyme immunoassay ( EIA) screening and GC-MS confirmation (Ploum et al., 1990; van Rhijn et al., 1995). Since 1993, samples have also been screened for the presence of bromobuterol and since 1995 for clenproperol. b-Agonists have been detected in sam-

ples from veal calves, young cattle, and cows, but not in samples from pigs, sheep, and horses at a level 1 mg/kg. Most of the positive samples with b-agonists contained clenbuterol, and other substances occurred marginally. The percentage of samples of urine and liver of cattle containing clenbuterol 1 mg/kg is presented in Figure 2. Since 1990, a clear decrease in positive urine samples was observed for cattle. A similar decrease in the number of positive liver samples has been observed since the time systematic monitoring started in 1992. Targeted Sampling Activities. In addition to random monitoring programs on the illegal use of b-agonists and hormones, targeted sampling on suspect farms is performed in The Netherlands by governmental services. Since 1989 farms in different regions of the country have been visited. Urine samples from veal calves, young cattle, and cows have been taken, as well as samples from animal feed, pharmaceutical preparations, and manure. The samples are screened using an EIA specific for a broad group of b-agonists such as clenbuterol, salbutamol, mabuterol, mapenterol, bromobuterol, and clenproperol (Haasnoot et al., 1990) and are confirmed in case of a positive response by GC-MS (van Rhijn et al., 1995). Between 1993 and 1995, more than 250 farms were inspected, and more than 4,000 urine samples and 400 feed preparations were examined. During this period, the percentage of positive urine samples from suspected animals decreased from 30 to < 5%, and the percentage of positive feed samples and preparations varied between 11 and 15%. In most positive samples, clenbuterol was detected, and in some cases mabuterol, bromobuterol, salbutamol, and clenproperol. In a few cases more than one b-agonist has been detected in urine (clenbuterol + mabuterol), and in feed samples and preparations various synthetic anabolic hormones were found.

Surveillance Activities by the Production Sectors.

Control of the illegal use of growth promoters in the veal calf and beef sector is also performed by production sector organizations in The Netherlands (e.g., Foundation for Quality Guarantee of the Veal Calf Sector [SKV] and the Cattle Quality Inspection KCR). The SKV carries out inspections at producers of

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Figure 1. Percentage of random positive samples of bovine animals containing b-agonists in European Union Member States in 1992 and 1993 (European Commission). NL = The Netherlands, B = Belgium, UK = United Kingdom, ES = Spain, F = France, IR = Ireland, PO = Portugal, DK = Denmark, I = Italy.

calf feed, calf farmers, and calf slaughterhouses. These investigations involve sampling of urine, feed, and visual controls. By December 1994, all nine calf slaughterhouses in The Netherlands and 626 feed producers and farmers had joined SKV. With respect to the veal calf sector, since 1991, urine samples have been taken at random at farms from approximately 35% of all veal calf groups, approximately 25,000 samples (Arts et al., 1996). In cases of illegal treatment, the probability of being found positive was calculated to be between 25 and 35%. Samples are analyzed for the presence of bagonists, estrogenic and androgenic steroids, and corticosteroids. For screening, immunoassays (sensitivity .2 mg/L) and receptor binding assays are used. Suspected samples are further analyzed by GC-MS, LC-MS, or LC-MS-MS. The SKV system has reduced the use of clenbuterol, salbutamol, mabuterol, mapenterol, and bromobuterol; the number of positively identified groups of calves dropped from 4 to .4% during the period 1991 to 1995. In 1991, mainly clenbuterol, but some mapenterol, were detected, and in 1995 only clenbuterol was found. In the beef sector, a similar control system was organized that became effective in 1995 (KCR, Annual Report, 1995). In 1995, urine samples were taken from approximately 3,000 farms, and at approxi-

mately 13,000 farms visual inspections were carried out. The percentage of farms with cattle positively identified for b-agonists dropped from .6% during the first quarter to .1% during the last quarter of 1995.

Surveillance of -Agonists in Germany

Random monitoring and targeted surveys of the presence of b-agonists in veal calves, young cattle, cows, pigs, poultry, and lambs carried out during 1994 and 1995 in Germany has been intensified. Besides clenbuterol, salbutamol, cimaterol, cimbuterol, mabuterol, mapenterol, carbuterol, pirbuterol, bromobuterol, terbutaline, orciprenaline, isoproterenol, fenoterol, ractopamine, and etilefrine have been screened for by ELISA, followed by GC-MS in the case of a positive finding. The sensitivity of the ELISA method was < .5 mg/kg. At farms, urine and blood samples were taken, and since 1995 also samples from feces, drinking water, and milk. At slaughterhouses urine, blood, liver, bile, kidney, muscle, and the retina/choroid and other parts of the eye were collected. Furthermore, samples have been taken at suspected farms and from suspected animals in slaughterhouses. The incidence of positive samples containing clenbuterol was highest in veal calves, followed by young

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and blood taken at farms is observed, as well as of samples taken at slaughterhouses with respect to urine and liver from 1994 through 1995. Clenbuterol was detected in eye tissue from which the retina was removed, but surprisingly not in the retina/choroid fraction. These data are difficult to compare with previously reported results in the framework of the EU monitoring programs in 1992 and 1993, because no separate data are available over that period with respect to animal species and farm or slaughter phase.

Surveillance of Clenbuterol in Northern Ireland

Several sampling programs were developed during the period of 1989 to 1994 in Northern Ireland to monitor clenbuterol abuse in the local meat industry (Elliot et al., 1996). The National Surveillance (NSS) aims at random survey of veterinary drug residues and of clenbuterol in samples taken from pigs, sheep, and cattle, with a number of samples according to the United Kingdom sampling plan, for the EU monitoring program. In the framework of the Meat Inspection Scheme ( MIS) , cattle suspected of being treated with growth promoters are further examined for the presence of clenbuterol. Between 1989 and 1994, urine and bile were collected, and later, liver, retina, and hair. Specific on-farm surveys (OFFUS) are carried out, and samples of suspected animals, medicines, and feedstuffs may be taken. Moreover, a voluntary quality assurance program has been set up by cattle farmers (Farm Quality Assurance Scheme, FQAS). An EIA method was used for screening of urine, bile, liver, feedstuffs, retina, and hair. In the case of a positive screening result LC-MS was used for confirmative purposes. Since 1992, high-resolution GC-MS has also been applied. Results of the National Monitoring Programme (NSS) indicated only a low incidence of clenbuterol in cattle liver in 1992, three positive samples out of 151 liver samples tested and in 1994 one positive out of 219. Results of targeted sampling from cattle (MIS) indicated high percentages of positively identified carcasses for clenbuterol during 1990 and 1991 (35.5 and 48.6%, respectively), and in the following years a marked decline was observed (6.2% in 1992 and 5.6% in 1993) despite a substantial increase in the number of samples taken (Table 5). Abuse of clenbuterol in Northern Ireland is clearly declining. However, followup monitoring of farms indicated that in 1994 all tested urine samples ( n = 51) were negative for clenbuterol, but 17 out of 61 hair samples were positive (27.9%), indicating that clenbuterol abuse has still not been abandoned.

Figure 2. Percentage of positive urine and liver samples containing b-agonists (at least 1 mg/kg) during 19891995 in The Netherlands. (National Inspection Service for Livestock and meat, Central Laboratory CLRVV).

cattle, and samples from cows and pigs were negative. b-Agonists other than clenbuterol have not been detected in these species. The number of samples containing clenbuterol taken from veal calves at farms and slaughterhouses is presented in Table 4. A decrease in the percentage of positive samples of urine

National Surveillance Programs in the United Kingdom

Random sampling in the United Kingdom in 1994 and 1995 of young cattle liver and urine, and cows,

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Table 3. Beta-agonists in bovine animals (veal calf, young bovine, cows) from random samplings in the farm phase and slaughter phase in 1992 and 1993a
Farm phase Level of action in matrices 1993 Number of samples 1992 798 107 108 0 (0) 0 (0) 300 301 806 61 (7.64) 142 (17.51) 99 168 1993 1992 1993 1992 1993 Number of samples 1 mg/kg 250/100 ppt Clenbuterol 5/1 mg/kg Salbutamol 3/1 mg/kg Other bagonists 1 mg/kg 1 mg/kg 12 mg/kg 5,294 3,988 59 (1.11) 36 (.90) 19 20 0 (0) 20 6,515 (1.1)* 20 7,040 Number positive samples ( % ) Slaughter phase Number positive samples ( % ) 1992 7 (7.07) 0 (0) 1993 9 (5.36) 9 (0)

Country

Matrix

Laboratory methodb

Belgium

Liver

EIA HPLC

Denmark

Liver Urine

ELISA

Germany

Urine

EIA GC-MS HPLC

0 (0) 268 (4.39) 343 (3.45)

Greece

Urine Liver

HPLC HPTLC

Spain

Urine Liver Feed 1 mg/kg 1 mg/kg .25 mg/kg 0 0 0 69 0 1,013 1 (0.10) (3.8)* (0)

HPLC

France

Urine

GC-MS

953 781 7,121

210 5,883

40 (4.20) 10 (1.28)

0 (0) 264 (3.71) 397 (6.75)

KUIPER ET AL.

Ireland

Urine

RIA

Italy

Urine

TLC RP HPLC GC-MS 2 mg/kg 848 920 6 0 0 (.71)

LuxembourgUrine

7 (.76)

24 2,069

20 2,091 20

(.97)

0 (0) 41 (1.96)

Netherlands Liver

GC-MS

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Urine 1 mg/kg Detection limit: .51 mg/ kg 156 132 97 175

1 mg/kg Clenbuterol, salbutamol, mabuterol, mapenterol 2 mg/kg Cimaterol, terbutaline, broombuterol

Portugal

Urine

RIA/HPTLC

(0) 0 (0)

0 0

(0) (0)

1,112 509

821 1,242

60 (5.40) 2 (.39)

21 (2.56) 0 (0)

United Kingdom

Liver

ELISA

Urine

Commission des communautes: Plans residues etats membres 1993. Resultats des analyses pour la recherche de residus chez les animaux vivants et dans les viandes fraiches 1992, 1993 etats membres. bEIA = enzymeimmunoassay; HPLC = high performance liquid chromatography; ELISA = enzyme-linked immunoassay; GC-MS = gas chromatography-mass spectrometry; RIA = radioimmunoassay; TLC = thin-layer chromatography; RP = reversed phase; HPTLC = high prformance thin-layer chromatography. * Percentage of positive total number of farm and slaughter phase samples including pigs, sheep, and goats.

aSource:

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sheep, and pig liver for analysis for clenbuterol and for analysis for salbutamol of young cattle liver did not reveal samples with concentrations above the action level of .5 mg/kg (MAVIS, 1996). Non-statutory surveillance in 1995 of b-agonists in cattle liver, in calf liver, and in retail products of cattle liver, calf liver, and liver pate revealed no samples with concentrations of b-agonists above the action level.

Monitoring of the Use of Clenbuterol in Spanish Basque Country

In Spain, outbreaks of food poisoning in humans presumably due to the ingestion of residues of bagonists has led to the modification of the sampling plan for monitoring clenbuterol residues in cattle in Spanish Basque Country (Hidalgo et al., 1996). In 1991 and 1992, control of clenbuterol residues was fully random, and a very limited number of samples were taken (29 and 21, respectively). Since 1992, the number of samples has increased, up to 1,484 in 1994, with specific attention for suspected origins. In 1994, 1,016 samples were taken at slaughterhouses, of which 149 were taken under suspicion, 125 samples at random at farms, and 331 samples at refrigerated storehouses/retailers. Some 114 samples were from suspected animals. The number of positive samples, presumably analyzed by immunoassay, increased from 1 out of 29 (3.4%) in 1991 to 70 out of 957 (7.3%) in 1993, and declined to 47 out of 1,484 (3.2%) in 1994.

Europe were analyzed for the presence of anabolic hormones and b-agonists. The number of samples per country was determined on the basis of the size of the country, but a minimum of 60 was considered necessary to provide a representative picture of the use of growth promoters in each country. Tests for the presence of b-agonists were carried out jointly by two laboratories in Belgium, using ELISA kits, the first one a general kit for b-agonists and the second one specific for clenbuterol. For beefsteaks, 19 positive samples were identified (1.6%) containing acetoxyprogesterone ( 1 ) , boldenone ( 2 ) , megestrolacetate ( 1 ) , methylboldenone ( 2 ) , methyltestosterone ( 8 ) , and nortestosterone ( 5 ) . No synthetic estrogens were found. For livers tested for b-agonists, 92 (10%) were positive. In each case, clenbuterol was identified as the residue. The relative incidences of positive samples containing b-agonists in the EU Member States is presented in Figure 3. In 11 of the 12 EU Member countries, positive samples have been identified, with the exception of Denmark.

Cases of Human Intoxication with Residues of Clenbuterol

Several cases of foodborne clenbuterol poisoning have been described. In Spain, outbreaks were reported after the ingestion of cows liver; one outbreak occurred in the central part of the country between October 1989 and July 1990, affecting 135 persons, and 232 cases were reported in the northern part of the country between January and April 1992 (Anonymous, 1992a,b). Adverse effects such as palpitation, tachycardia, agitation, tremors, and headaches

Surveillance on Growth Promoters by the European Consumer Organizations

An extensive survey on growth promoters was carried out in 1994 by the Belgian Consumers Organization in 12 EU Member States in cooperation with National Consumers Organizations (Remy and de Debeuckelaere, 1994). Beefsteaks ( n = 1,183) and livers ( n = 936) purchased in retail stores across

Table 5. Results of targeted testing programs for bagonist abuse in cattle in Northern Irelands National Surveillance Scheme (Meat Inspection Scheme [MIS] and On-Farm Follow-up Sampling [OFFUS])
Location and year Slaughterhouse (MIS) 1990 Matrices No. of carcasses Percentage positive

Table 4. Random monitoring of clenbuterol concentrations in veal calves in 1994 and 1995 in Germanya
Number of positive samples (total number of samples) 1994 Farm phase Urine Blood Slaughter phase Urine Liver Retina/choroid Eye (other parts) 70 (449) 39 (270) 59 (393) 15 (159) 0 (23) 7 (46) % 15.5 14.4 12.8 9.4 9 15.2 1995 37 (379) 9 (534) 17 (324) 2 (211) 0 (36) 13 ( 5 3 ) % 9.8 1.7 5.2 .9 0 24.5

1991 1992 1993 1994 On farm (OFFUS) 1991 1992 1993 1994

Urine, bile, liver Bile, liver Liver, retina Retina, liver Retina, liver

121 286 1,831 1,861 973 No. of samples

35.5% 48.6% 6.2% 5.6% 1.5%

aMethod of detection = ELISA/gas chromatography-mass spectrometry (sensitivity < .5 mg/kg).

Urine Urine Urine Urine Hair

276 853 237 51 61

19.2% 3.8% 8.4% 0% 27.9%

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have been recorded. There are indications that in some of the latter cases clenbuterol was added to animal feed together with antithyroid substances such as thiouracil and methimazole, used to promote water retention and increase weight (Martinez-Frias et al., 1992). The 135 cases reported during 1989 to 1990 are described in more detail by Martinez-Navarro (1990). Epidemiologists located the cases using the pharmacological profile of clenbuterol and the appearance of symptoms 30 min to 6 h after ingestion, lasting for approximately 40 h. Consumption of liver was identified as the common item in the 43 families affected. Symptoms were observed in 97% of the family members who consumed liver. Clenbuterol residues ranging from 160 to 291 mg/kg liver were found. Of the 232 cases reported in 1992, 113 occurred in Catalonia. Salleras et al. (1995) described the outcome of an epidemiologic study on the outbreak of clenbuterol poisoning in this region. Based on food consumption data obtained from patients, the origin of incriminated foodstuffs could be determined. Symptomology in more than half of the patients included tachycardia, muscle tremors, nervousness, myalgia, and headache. Intervals between exposure and onset of symptoms ranged from 15 min to 6 h and the duration varied from 90 min to 6 d. In urine samples from patients ( n = 47), clenbuterol concentrations ranged from 11 to 486 mg/L, and in serum samples ( n = 2 ) clenbuterol was not detectable (< 5 mg/L). A total of 61 of those afflicted had eaten at the same restaurant or company canteen. All patients had consumed veal liver, veal tongue, or cannelloni before onset of symptoms. Concentrations of clenbuterol in samples of veal liver originating from one slaughterhouse ranged from 19 to 5,395 mg/kg liver ( n = 9), and in 7 of the liver samples no residues were found (< 5 mg/kg liver). Fifteen cases of poisoning in this period in Barcelona were also reported separately by Tomas et al. (1995). The source of another incident in Spain (Aragon) in December 1991 affecting 59 persons was found to be cinnamon contaminated with clenbuterol that was used in locally made desserts (Anonymous, 1992a,b; Salleras et al., 1995). In France, an incident of food poisoning by residues of clenbuterol in veal liver was described by Pulce et al. (1991). In the fall of 1990, the Poison Centers of Lyon and Clermont-Ferrand were consulted by physicians treating persons with symptoms typical of bagonists use. A total of 22 persons from eight families at two sites (Roanne and Clermont-Ferrand) were affected. Symptoms appeared 1 to 3 h after consuming veal liver dishes, and all patients recovered within 1 to 3 d. A woman with a prior heart disease developed marked palpitations, whereas her son, who consumed the same dish, did not, indicating that heart conditions can make a person more susceptible. The veal liver originated from a slaughterhouse in Roanne. In

two samples, relatively high residue concentrations of clenbuterol were found, 375 and 500 mg/kg liver. Clenbuterol exerts a bronchodilatory action at an oral dose as low as 1 mg ( .02 mg/kg body weight) in patients with chronic pulmonary diseases. A statistically significant ( P < .05) dose-response in bronchodilation is observed in the dose range of 5 to 25 mg per person (Nolte and Laumen, 1972). In countries such as Austria, Germany, and Mexico, clenbuterol is prescribed in humans for treatment of bronchial asthma at an oral dosage of .02 to .03 mg (.3 to .5 mg/ kg body weight) twice daily. Contraindications include cardiovascular disorders, diabetes, and hypersensitivity to symphaticomimetics (Prather et al., 1995). Adverse effects such as palpitation, tremors, and headaches have been reported in humans after ingestion of clenbuterol at an oral dose rate of 10 mg per person, four times daily. The pharmacological and toxicological profiles of clenbuterol indicate that it interacts with b(2)-receptors in the bronchial airways, with b(1)-adrenoreceptors in the myocardium (resulting in palpitation), and b(2)-adrenoreceptors in the central nervous system (resulting in tremors and headaches) (Drennan, 1994). The food intoxication cases described here demonstrate clearly that safety evaluation of pharmacologically active compounds to which consumers may be exposed as residues in food must be based on toxicological criteria and on the pharmacological potency of a compound to arrive at an acceptable daily intake (ADI) for humans. Consumption of 100 g of liver contaminated with clenbuterol in the concentration range as measured in contaminated samples of 160 to 500 mg/kg liver would exceed the pharmacological effect level of 5 mg per person.

Figure 3. Incidence of liver samples containing clenbuterol (Survey Belgian Consumers Organization in 12 EU Member-States, 1994). NL = The Netherlands, B = Belgium, UK = United Kingdom, ES = Spain, F = France, IR = Ireland, PO = Portugal, DK = Denmark, I = Italy, D = Germany, GR = Greece, L = Luxemburg.

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Discussion
The role of b-agonists, in particular of clenbuterol as repartitioning agents, became important in the late 1980s in Europe. Detection and control of abuse of these compounds in EU Member States is performed at the national level and varies substantially between Member States with respect to testing schemes, animal species to be investigated, target matrices, analytical test methods, and action levels. The Directive 86/469 EC has not functioned fully because of the rather vague descriptions of sampling strategies and testing schemes, in particular with respect to sampling frequency, target matrices, and random or targeted sampling at the farm and in slaughterhouses. The quality of the data reported for 1992 and 1993 in the framework of EU monitoring programs is questionable because of a lack of information on sampling strategies and analytical detection methods applied, making a comparison of results from different member states hardly possible. Conditions for monitoring and surveillance of drug abuse have been specified in the new Directive 96/23 EC, which will certainly tighten detection and control of residues at the European level. Surveillance programs as carried out in The Netherlands, Germany, Northern Ireland, and Spanish Basque Country during the last 4 to 5 yr indicate that the abuse of clenbuterol is declining, presumably due to intensified sampling programs and targeted inspection at farms and to the start of the activities of production sector organizations in the Netherlands, (SKV and KCR) and in Northern Ireland (FQAS). The obvious question remains whether other unknown agents are being administered for growth-promoting purposes. Results from the study of the Belgian Consumers Organization carried out in 12 EU Member States in 1994 indicated a relatively high percentage of livers purchased in retail stores contained clenbuterol residues. It should be noted that these results were obtained using an immunoassay method without confirmation by GCMS. This may explain the difference in percentages observed in other monitoring programs, which are generally lower. Continuous improvement of detection methods is necessary to keep pace with the rapid development of new unknown substances used for growth promotion. It is of great importance to apply a validated combination of screening and confirmatory methods to deal with relatively large numbers of samples and to guarantee high reliability of results. The development of functional screening tests such as receptor assays and biosensors constructed of genetically modified cell lines offer new possibilities for a rapid and specific detection of residues at relatively low costs. The documented cases of clenbuterol poisoning of humans through ingestion of contaminated food demonstrate that safety evaluation of pharmacologi-

cally active compounds to which consumers may be exposed must be based not only on toxicological but also on pharmacological criteria to establish safe levels. Large-scale application of pharmacologically potent agents for growth-promoting purposes should not be recommended because misuse may readily lead to adverse health effects in humans. A similar conclusion was recently drawn at the EU Scientific Conference on Growth Promotion in Meat production (Brussels, 1995).

Implications
Intensive control programs and targeted surveillance at farms and in slaughterhouses by regulatory agencies and production sector organizations have proven effective in reducing the illegal use of bagonists as growth promoters during animal production. Rapid, robust, and economical detection methods based on the functional properties of the applied compounds that are currently under development should be applied at farms and slaughterhouses as first the screening tools to eradicate drug abuse. Continuing active involvement and participation of the production sector is a prerequisite for maintaining consumers confidence in the quality of meat and meat products.

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