TYLOSIN A review of pharmacokinetics, residues in food animals and analytical methods 1. Jacek Lewicki

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1 TYLOSIN A review of pharmacokinetics, residues in food animals and analytical methods 1 Jacek Lewicki Division of Pharmacology and Toxicology, Department of Preclinical Sciences Faculty of Veterinary Medicine, Warsaw Agricultural University Ciszewskiego 8, 2787 Warsaw, Poland. jacek_lewicki@sggw.pl A safety assessment of tylosin residues in poultry tissues and eggs to be performed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) was requested by the 15 th Session of the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF). As none of the requested information on tylosin was provided to the 66 th meeting of JECFA, the Committee did not include tylosin on its agenda for food safety assessment. A general review of the available information in the open literature concerning pharmacokinetics and tissue residues of tylosin in different animal species and analytical methods for detection of tylosin was carried out to provide FAO, WHO and/or Codex member governments with updated information. No evaluation regarding the toxicology of tylosin was prepared by the 66 th JECFA meeting. INTRODUCTION Tylosin was previously evaluated at the twelfth meeting of JECFA in 1968 (JECFA, 1969) and the thirtyeighth meeting in 1991 (JECFA, 1991ab). The review at the twelfth meeting was a general food safety assessment and prior to JECFA meetings held exclusively for residues of veterinary drugs in food. The 38 th meeting of JECFA was not able to recommend MRLs for tylosin as no ADI was established. At that time, the Committee requested specific information prior to reevaluation: 1. Detailed information on reproduction and teratogenicity studies. 2. Studies to explain the positive results in the mouse lymphoma genotoxicity assay in the absence of metabolic activation. 3. Studies designed to test the hypothesis that the increased incidence of pituitary adenomas in male rats was a consequence of the greater rate of bodyweight gain in treated rats. 4. Studies to determine MIC values of inhibitory activity against microorganisms representative of the human colonic microflora. 5. Detailed studies of residues in eggs using more sensitive analytical methods. 6. Detailed information on microbiologically active metabolites of tylosin. 7. Studies on the contribution of the major metabolites of tylosin to the total residues in edible tissues of cattle and pigs. DEFINITION OF THE COMPOU International nonproprietary name: Tylosin (INNEnglish) European Pharmacopoeia name: (4R,5S,6S,7R,9R,11E,13E,15R,16R)15[[(6deoxy2,3diO methylβdallopyranosyl)oxy]methyl]6[[3,6dideoxy4o(2,6 dideoxy3cmethylαlribohexopyranosyl)3(dimethylamino)β Dglucopyranosyl]oxy]16ethyl4hydroxy5,9,13trimethyl7(2 oxoethyl)oxacyclohexadeca11,13diene2,1dione IUPAC name: 2[12[5(4,5dihydroxy4,6dimethyloxan2yl)oxy4 dimethylamino3hydroxy6methyloxan2yl]oxy2ethyl14 hydroxy3[(5hydroxy3,4dimethoxy6methyloxan2 1 The views expressed in this publication are those of the author(s) and do not necessarily reflect the views of the Food and Agriculture Organization of the United Nations. 1

2 Other chemical names: yl)oxymethyl]5,9,13trimethyl8,16dioxo1oxacyclohexadeca4,6 dien11yl]acetaldehyde 6S,1R,3R,9R,1R,14R)9[((5S,3R,4R,6R)5hydroxy3,4dimethoxy 6methylperhydropyran2yloxy)methyl]1ethyl14hydroxy3,7,15 trimethyl11oxa4,12dioxocyclohexadeca5,7dienyl}ethanal Oxacyclohexadeca11,13diene7acetaldehyde, 15[[(6deoxy2,3di methylbdallopyranosyl)oxy]methyl]6[[3,6dideoxy4o(2,6 dideoxy3cmethyalribohexopyranosyl)3(dimethylamino)bd glucopyranosyl]oxy]16ethyl4hydroxy5,9,13trimethyl2,1dioxo [4R(4R*,5S*,6S*,7R*,9R*,11E,13E,15R*,16R*)] Synonyms: AI329799, EINECS , Fradizine, HSDB 722, Tilosina (INNSpanish), Tylan, Tylocine, Tylosin, Tylosine, Tylosine (INNFrench), Tylosinum (INNLatin), Vubityl 2 Registry numbers: CAS Table 1. Summary of physicochemical properties of tylosin (Paesen et al., 1995abc; McFarland et al., 1997; The Merck Index, 21). Molecular formula: C 46 H 77 NO 17 Molecular weight: Appearance: Melting point: Solubility: An almost white or slightly yellow crystalline powder o C 5 mg/ml (water 25 o C), soluble in lower alcohols, esters, ketones, chlorinated hydrocarbons, benzene, ether, chloroform Stability: Solutions are stable at ph 49 (most stable at ph 7); pka: 7.73 log P (octanolwater): 1.63 UV Absorption: COITIONS OF USE Below ph 4 tylosin B (desmycosin) is formed as a result of acid hydrolysis; In neutral and alkaline ph tylosin aldol (TAD) is formed together with polar degradation products of unknown identity; When tylosin solution is exposed to daylight, a photodegradation product isotylosin A (isota) is formed UV max. at 282 nm, extinction coefficient (E 1cm 1% ) is 245 at 282 nm Tylosin is a macrolide antibiotic that is active against certain Grampositive and Gramnegative bacteria, especially different members of Mycoplasma spp. Tylosin is registered exclusively for veterinary use in several countries, primarily for use in the chronic respiratory disease complex in chickens and infectious sinusitis in turkeys. Tylosin is also used to treat bovine respiratory and swine dysentery diseases. In some countries, tylosin is also registered for use as a growth promoter for poultry, pigs and cattle (Botsoglou and Fletouris, 21). Tylosin is a mixture of four macrolide antibiotics produced by a strain of Streptomyces fradiae (Figure 1). The main component of the mixture (> 8%) is tylosin A (M r = 916; McGuire et al., 1961). 2

3 Tylosin B (desmycosin, M r = 772; Hamill et al., 1961), tylosin C (macrocin, M r = 92; Hamill and Stark, 1964) and tylosin D (relomycin, M r = 918; Whaley et al., 1963) may also be present. All four components contribute to the potency of tylosin, which is not less than 9 IU/mg, calculated with reference to the dried substance (European Pharmacopoeia, 24). Relative antimicrobial activities of tylosin derivatives are: tylosin A 1., tylosin B.83, tylosin C.75 and tylosin D.35 (Teeter and Meyerhoff, 23). Figure 1. Chemical structure of tylosin. Tylosin A contains a polyketide lactone (tylactone) substituted with three 6deoxyhexose sugars (Figure 2). The addition of Dmycaminose to the aglycone is followed by concurrent ring oxidation at C2 and C23 (to generate the tylonolide moiety) and substitution with Lmycarose and 6deoxyDallose. BisOmethylation of the latter generates mycinose and completes the biosynthesis of tylosin (Baltz et al., 1983; Baltz and Seno, 1988). Figure 2. Chemical structure of tylosin A. 3

4 Other pharmacologically active compounds, i.e., lactenocin, demecinosyltylosin (DMT) and O mycaminosyltylonolide (OMT) have been isolated from fermentation media or aqueous commercial samples containing tylosin. In solutions for injections containing tylosin, an alkaline degradation product called tylosin aldol (TAD) has also been detected. Two epimers of this product called TAD1 and TAD2 and isotylosin A (isota) were recently isolated (Paesen et al., 1995abc). However, no information on antibacterial activity of TAD and isota has been presented. Recent studies have provided new data on stability of tylosin. Tylosin tartrate was stable for at least one month when stored in MilliQ water at ph 5.7 to 6.7 at 22 o C but approximately 1% of added tylosin was degraded within the first 2 hours in MilliQ water at ph 9.2 (Kolz et al., 25). When tylosin was dissolved in MilliQ water (ph = 5.5) and maintained under aerobic conditions for five days, only 56.1% (kept in dark) and 92.4% (exposed to light) of initial tylosin potency was retained. In the solution stored in the dark, tylosin concentration measured by a spectrophotometric assay remained stable in comparison to the initial tylosin concentration, while in solutions exposed to light, tylosin concentration was reduced by 13% (HallingSørensen et al., 23). In another study, when tylosin solution was exposed to daylight, a photodegradation product isotylosin A (isota) was formed (Paesen et al., 1995c). ANTIBACTERIAL ACTIVITY Macrolide antibiotics are bacteriostatic compounds that reversibly bind to the 23S rrna in the 5S ribosome subunit and inhibit mrnadirected protein synthesis. Moreover, they stimulate the dissociation of peptidyltrna from ribosomes during translocation. The precise mechanism of action has not been fully explained and many theories exist (Zhanel et al., 21; Gaynor and Mankin, 25). It has been suggested that 16memberedring macrolides inhibit protein synthesis by blocking elongation of the peptide chain, but the 14 and 15memberedring macrolides are only potent inhibitors of mrnadirected peptide synthesis (Retsema and Fu, 21). It was also demonstrated that the 16memberedring macrolides (carbomycin, spiramycin and tylosin) inhibit peptidyl transferase, and the presence of mycarose was correlated with peptidyl transferase inhibition. However, tylosin B did not inhibit peptidyl transferase (Poulsen et al., 2). Results of comparative antibacterial evaluation of tylosin and desmycosin showed that both compounds have almost identical antibacterial activity (Table 2). In the same study, tetrahydrodesmycosin and dihydrodesmycosin showed decreased antimicrobial activity, while the symmetric dimer of desmycosin was inactive (Iveković et al., 23). Moreover, 4'deoxy1,11,12,13tetrahydrodesmycosin, a derivative of tetrahydrodesmycosin, retained the antibacterial spectrum of tylosin with some improvement against tylosinsensitive Staphylococci and Haemophilus influenzae (Narandja et al., 1995). Table 2. Antibacterial in vitro activity of tylosin, desmycosin, tetrahydrodesmycosin, dihydrodesmycosin and dimeric desmycosin (Iveković et al., 23). Bacteria Staphylococcus aureus B 329 Staphylococcus aureus B 538 (imls) a Staphylococcus aureus B 33 (cmls) b Streptococcus pneumoniae B 541 Streptococcus pneumoniae B 326 (M) c Streptococcus pneumoniae B 328 (cmls) Streptococcus pyogenes B 542 Streptococcus pyogenes B 543 (imls) Streptococcus pyogenes B 544 (cmls) Escherichia coli B 1 Haemophilus influenzae B 529 MIC (µg/ml) Tyl Des THDes DHDes DIMDes > >64 >64 >64 >64 >64 >64 < >64 < >64 >64 < >64 >64 16 > >64 >64 4 > >64 >64 32 > >64 >64 16 >64 >64 >64 >64 >64 >64 4

5 MIC minimum inhibitory concentration; Tyl tylosin; Des desmycosin; THDes 1,11,12,13 tetrahydrodesmycosin; DHDes 1,11dihydrodesmycosin; DIMDes dimeric desmycosin; a imls, inducible resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics; b cmls, constitutive MLS resistance; c M, efflux mediated macrolide resistance EVALUATION OF VETERINARY DRUGS WITH A LONG HISTORY OF USE At the thirtysecond meeting of JECFA in 1987, the first to be exclusively devoted to veterinary drugs, the Committee began establishing criteria to be applied when evaluating toxicology and residue data for assessing the safety of veterinary products present in tissues of food animals (JECFA, 1988). From the Committee s deliberations on a variety of veterinary products, it became apparent that, for certain products with a long history of use (often referred to as older veterinary drugs e.g., tylosin), data that do not meet modern criteria may nevertheless be useful in the safety assessment of residues in human food. The Committee has developed an approach for evaluating veterinary drugs with a long history of use, including important requirements on toxicological and residue data (JECFA, 1993). In addition to information necessary to establish an ADI, the Committee has developed minimal data requirements for recommending maximum residue limits (MRLs) including: 1. A suitable analytical marker residue for the residues of toxicological concern; 2. At least two target tissues for recommending MRLs, one of which usually will be liver or kidney to accommodate current practices in national control programmes and the other in muscle or fat to facilitate testing in international trade; 3. A suitable analytical method for the marker residue that satisfies current scientific criteria. Moreover at the thirtysixth meeting of JECFA (JECFA, 199), the Committee agreed to consider good practice in the use of veterinary drugs and the applicability of analytical methods in recommending either MRLs or temporary MRLs (JECFA, 1993). PHARMACOKINETICS OF TYLOSIN Tylosin is a weak organic base (pka = 7.73) that readily forms salts and esters. Available forms of tylosin are: tylosin base, tylosin tartrate and tylosin phosphate (European Pharmacopoeia, 24). Because it is slightly to moderately bound to plasma proteins (347% Table 3) with a high degree of lipid solubility, tylosin is widely distributed in body fluids and tissues (Burrows, 198). Although comparative pharmacokinetics of tylosin in animals is poorly described in the scientific literature, certain reviews on tylosin pharmacokinetics are also available (Wilson, 1984; van Leeuwen, 1991). Absorption Oral administration Monogastric animals. Tylosin is quickly absorbed from the alimentary tract of monogastric animals; however oral bioavailability has not been estimated in most species (Table 3). After a single oral dose of 5 mg/kg b.w. of tylosin base or tylosin tartrate in rats, tylosin peak serum concentrations of 1. µg/ml were seen after 12 hours. Within 7 hours serum concentrations decreased to less than the limit of detection (.1 µg/ml) of the microbiological assay (van Leeuwen, 1991). Similar results were obtained in rats after intragastric administration of tylosin base solution. After a single dose of 2, 5 and 1 mg/kg b.w. of tylosin base, peak serum concentrations (about.51.1 µg/ml) appeared after 2 hours (Kietzmann, 1985). When rats were given water mixed with a commercially available preparation of tylosin base (final concentration about 71 mg/l), the bioassay of serum after 11 days of continuous medication revealed no detectable tylosin concentrations (<.1 µg/ml), while at the same time lung tissue contained µg of tylosin/g (Carter et al., 1987). 5

6 In general, higher blood concentrations of tylosin correspond to higher administered doses of tylosin. In dogs receiving tylosin orally by capsule (1, 1 or 1 mg/kg b.w./day for 8 days), tylosin blood concentrations determined 2 hours after the last dose ranged from <.15 µg/ml to 9.5 µg/ml. In dogs receiving 25 or 1 mg/kg b.w. of tylosin base orally by capsule daily for 29 days, peak serum concentrations ( µg/ml) were seen 2 hours after dosing at 25 mg/kg b.w./day and the highest concentrations ( µg/ml) were seen 25 hours after dosing at 1 mg/kg b.w./day (van Leeuwen, 1991). There was no evidence of tylosin accumulation in the serum after 2 years of continuous administration of tylosin in the diet (Anderson et al., 1966). In pigs receiving tylosin tartrate orally at 3 mg/kg b.w., tylosin activity was present in blood plasma 1 minutes after administration, with a peak concentration of 2.4 µg/ml at about 1.5 hours. By comparing the areas under the curve of the tylosin concentration in blood following the two routes of administration (i.v. and p.o.), a biological availability of 22.5% was determined. When tylosin was administered orally to pigs at a dose of 11 mg/kg b.w. (as the granulated phosphate), tylosin serum activity peaked 1 hour after dosing (average µg/ml); 24 hours after dosing tylosin was not detectable (<.1 µg/ml). Similar results were obtained after oral administration of tylosin phosphate (5 mg/kg b.w.) in water. Tylosin concentrations were detected in serum from 1 minutes to 8 hours after dosing and peaked (8.53 µg/ml) at 1 hour after dosing (van Leeuwen, 1991). Results of a comparative residue study in pigs suggest that absorption of tylosin phosphate from the alimentary tract is comparable to that of tylosin tartrate (Iritani et al., 1975). Poultry. In broiler chickens (weight 72 g), a single dose of 5 mg tylosin/bird (as tylosin tartrate) by stomach intubation, resulted in detectable tylosin activity in serum.5 hour after dosing. Peak concentrations of.64. µg/ml were found after 2 hours, declining to negligible after 24 hours. Repeated oral doses of 5 mg of tylosin to chickens (weight 2 kg, dosed at 1, 2, and 3 hours) resulted in peak serum concentrations at 4 hours (about.28 µg/ml) after dosing. Serum concentrations declined thereafter and were negligible at 24 hours after dosing (van Leeuwen, 1991). Similar results were obtained in chickens following a single oral dose of 1 mg/kg b.w. of tylosin tartrate. A peak plasma concentration of 1.2 µg/ml was observed 1.5 hours after tylosin administration with an oral bioavailability of 334% of the administered dose (Kowalski et al., 22). When tylosin tartrate was added to the drinking water at 5 and 7 mg/l for 48 hours, average serum concentrations of tylosin were.12 and.17 µg/ml, respectively. However, peak concentrations (about.2.3 µg/ml) appeared after 24 hours (Ziv, 198). In the oral bioequivalence study of two commercial products containing tylosin tartrate, the average tylosin concentration in serum of 5 and 7week old broilers and 9month old layers (treated for 5 consecutive days with medicated drinking water containing 75 mg tylosin tartrate/litre) was.2 µg/ml (Ziv and Risenberg, 1991). In contrast to results obtained from pigs (Iritani et al., 1975), tylosin phosphate was not as well absorbed as tylosin tartrate from the alimentary tract in chickens. Results of the residue study in chickens showed that no tylosin was detected in the blood and muscles of the chickens fed the diet containing tylosin phosphate up to 15 ppm for eight weeks (Yoshida et al., 1973a). Parenteral administration The usual route of parenteral administration of tylosin is intramuscular injection. In rabbits receiving tylosin base intramuscularly at a dose of 1 mg/kg b.w. as a 5% aqueous solution, tylosin peak serum concentrations ( µg/ml) were observed after 1.5 hours. A similar study was carried out using tylosin tartrate in aqueous (25 mg/kg b.w.) as well as in PEG2 (1 mg/kg b.w.) solutions. Peak serum concentrations observed at 1 hour were µg/ml and µg/ml, respectively. Within 24 hours tylosin serum concentrations were below the limits of detection.5 µg/ml for tylosin hydrochloride and.1 µg/ml for tylosin tartrate (van Leeuwen, 1991). Peak blood concentrations of tylosin were reached in 24 hours following intramuscular injection of tylosin base in 5% propylene glycol and the aqueous solution of the tartrate salt in cows, ponies and pigs (Sauter et al., 1962; Gingerich et al., 1977). In calves receiving tylosin base at a dose of

7 mg/kg b.w., tylosin peak concentrations ( µg/ml) were observed 2 hours after intramuscular injection (van Duyn and Folkerts, 1979). In calves receiving tylosin base at a dose of 25 mg/kg b.w., tylosin peak serum concentrations of µg/ml were reached 1 hour after intratracheal administration, while peak concentrations of and µg/ml were reached 2 and 8 hours after intramuscular or subcutaneous injection, respectively (Hjerpe, 1979). In another study peak serum concentrations in cattle following intramuscular injection of tylosin appeared about 56 hours after injection, with systemic bioavailability of 78% of the administered dose (Ziv and Sulman, 1973; Baggot, 1978). In cattle, slow absorption of tylosin base following intramuscular injections, with 16.9% absorption at 7 hours and 94% of total drug absorbed at 24 hours, has also been described (Nouws and Ziv, 1977b). Although results of the comparative pharmacokinetic study in cattle and buffaloes (Bubalus bubalis) did not show significant differences in the kinetics of tylosin administered intramuscularly (as tartrate) at a dose of 1 mg/kg b.w., much lower than previously reported in cattle (Table 3), peak plasma concentrations (.65 µg/ml in cattle at 1 hour and.47 µg/ml in buffaloes at.85 hour) were observed in both species (Saurit et al., 22). Following intramuscular injection of tylosin base in pigs at a dose of 1 mg/kg b.w., tylosin peak plasma concentrations (.41.9 µg/ml) were reached after.33 hours and the bioavailability was 95% of the dose (Prats et al., 22a). In small ruminants after intramuscular injection of tylosin tartrate at a dose of 15 mg/kg b.w., tylosin peak plasma concentrations of 2.8 µg/ml (Nubian goats) and 2.58 µg/ml (desert sheep) were observed after 3.8 and 3.3 hours, respectively. Bioavailabilities were 84% (N. goats) and 73% (d. sheep) of the dose (Taha et al., 1999). Another report showed lower intramuscular bioavailability of tylosin tartrate (72.6%) in goats (Atef et al., 1991). High intramuscular bioavailability of tylosin tartrate (88%) was also reported in camels (Ziv et al., 1995). In noncommercial avian species following intramuscular injection of tylosin base in 5% propylene glycol at a dose of 1525 mg/kg b.w., the average peak plasma concentrations of tylosin in quail, pigeons, cranes and emus were: 4.31, 5.63, 3.62 and 3.26 µg/ml, respectively. These peak concentrations were observed from.5 to 1.5 hours after tylosin administration (Locke et al., 1982). Distribution Tylosin is a weak organic base (pka = 7.73) with a high degree of lipid solubility, thus it is well distributed to the organs and tissues of animals (Burrows, 198). Calculated theoretical tissue:plasma ratios (k 12 /k 21 ) of tylosin in cows and goats were 2.5 and 2.5 (Table 3), respectively (Baggot and Gingerich, 1976; Atef et al., 1991). In dogs, the tissue:plasma ratio for tylosin was only.68 (Weisel et al., 1977). In dogs the reported volume of distribution V d = 1.7 l/kg (Weisel et al., 1977) was, however, similar to the V d values calculated in other animal species: 2.2 l/kg in rats (Duthu, 1985), l/kg in cows (Ziv and Sulman, 1973; Baggot and Gingerich, 1976; Gingerich et al., 1977; Cester et al., 1993), l/kg in sheep (Ziv and Sulman, 1973; Taha et al., 1999) and l/kg in goats (Atef et al., 1991; Taha et al., 1999). A lower volume of distribution was reported only in chickens V d =.69 l/kg (Kowalski et al., 22). Higher V d values for tylosin were calculated in young calves l/kg (Burrows et al., 1983; Burrows et al., 1986), pigs 14.6 l/kg (Prats et al., 22a) and camels l/kg (Ziv et al., 1995). The ability of tylosin to pass through the biological membranes is facilitated by low/moderate plasma protein binding. Results of animal studies showed that protein binding coefficients were: 3% in chickens (Ziv, 198), % in cows (Ziv and Sulman, 1972; Ziv and Sulman, 1973), 37.6% in goats (Atef et al., 1991), % in sheep (Ziv and Sulman, 1972; Ziv and Sulman, 1973) and % in camels (Ziv et al., 1995) see Table 3. In studies in cows that received tylosin base intramuscularly at a dose of mg/kg b.w., tylosin tissue:serum ratios measured 731 hours after the treatment were: 35.2 in kidney cortex, 13.9 in kidney medulla and 5.7 in liver. High tylosin concentrations at 24 hours were also found in bile (35.1 µg/ml) and urine (12.9 µg/ml), while muscle and plasma concentrations were below.4 µg/g or ml, respectively (Nouws and Ziv, 1977a; Nouws and Ziv, 1979). When calves less than 3 weeks of age received a single intramuscular injection of tylosin base at a dose of 17.6 mg/kg b.w., tylosin lung concentrations measured up to 24 hours ranged from 4.53 to 15.7 µg/g, with a lung AUC 48h :plasma 7

8 AUC 48h ratio of (van Duyn and Folkerts, 1979). In sixweek old calves with pneumonia that received three intramuscular injections of tylosin base every 12 hours at a dose of 1 mg/kg b.w., the tylosin tissue:serum ratios measured two hours after the last dose were: 2. for pneumonic lung, 1.6 for nonpneumonic lung, 2.1 for liver and 2.6 for kidney. The highest tylosin concentrations (about 3.3 µg/g) were found in kidney, while the lowest concentrations (<.5 µg/g) were found in muscle and cerebrospinal fluid (Burrows et al., 1986). When rats were given water containing a commercially available preparation of tylosin base (final concentration about 71 mg/l), bioassay of serum after 11 days of continuous medication revealed no detectable tylosin concentrations (<.1 µg/ml), while lung tissue contained µg/g of tylosin (Carter et al., 1987). Similar tissue:plasma ratios for tylosin were also found in various avian species. The highest tissue:plasma proportions were observed in liver (pigeons, cranes) and kidney (quails). Lower tylosin concentrations were found in lung tissues of all three species, but in all cases these concentrations were greater than the corresponding plasma concentrations (Locke et al., 1982). Elimination Tylosin is rapidly eliminated from the blood plasma in different animal species. Plasma elimination halflives (t 1/2el. ) of tylosin after single intravenous administration in healthy animals were:.4 hour in rats,.52 hour in chickens,.9 hour in dogs,.92 hour in camels, hours in young calves, hours in cows, hours in sheep, hours in goats and 4.52 hours in pigs (Table 3). Slightly longer t 1/2el. values were reported for tylosin after intramuscular injections: 2.4 hours in dogs, hours in cattle and 3.71 hours in camels. In pigs however, the tylosin elimination halflife exceeded 24 hours after intramuscular administration of tylosin base at a dose of 1 mg/kg b.w. (Prats et al., 22a). In quail, pigeons and cranes, the elimination halflife of tylosin after intramuscular injections of tylosin base was 1.2 hours; in emus, the elimination halflife of tylosin was 4.7 hours (Locke et al., 1982). The highest tylosin rate of elimination from the blood plasma was observed in camels. The total body clearance (Cl B ) after intravenous injection of tylosin tartrate was ml/min/kg, while in waterdeprived camels Cl B decreased to only ml/min/kg (Ziv et al., 1995). A very high rate of tylosin elimination was also observed in rodents. In rats, total body clearance was 86 ml/min/kg (Duthu, 1985). A similar elimination rate of tylosin was observed in mice (Cacciapuoti et al., 199). Although only the AUC 3h value (23.5 µg h/ml) was given for mice, as calculated after intravenous administration of tylosin at a dose of 1 mg/kg b.w., using this value and the equation: Cl B = Dose/AUC i.v., a clearance value of 7.9 ml/min/kg is calculated. In other animal species, Cl B values were: ml/min/kg in young calves, 26.8 ml/min/kg in pigs, 21.9 ml/min/kg in dogs, ml/min/kg in cows, ml/min/kg in goats and 6.89 ml/min/kg in sheep (Table 3). Allometric relationships between tylosin total body clearance (Cl B ) and animal body weight are presented in Figure 3. There is a statistically significant relationship (Pvalue =.1) between tylosin clearance and body weight at 99% confidence level. The Rsquare value indicates that the multiplicative model as fitted explains 84.4% of the variability in tylosin clearance after transforming to a logarithmic scale to linearize the model. Moreover, the correlation coefficient equals.919, indicating a relatively strong relationship between the variables, especially when the data from chickens and camels were not included into the model Cl B = a(b.w.) b, where a is a normalization constant known as the allometric coefficient, b.w. is the body weight, and b is the allometric exponent (West et al., 1997; Hu and Hayton, 21). Metabolism Primary metabolism of tylosin occurs within the liver. In contrast to some authors (Anadón and ReeveJohnson, 1999) conclusions that tylosin is unable to produce cytochrome P45 binding metabolites, results of the in vitro study with liver microsomes isolated from rabbits showed, that tylosin has a high ability to form metabolic intermediate complexes with cytochrome P453A (9.67 nmol complex/nmol P45). This ability is lower than that for tiamulin, erythromycin and 8

9 roxithromycin, however. Moreover, the observed rate of the in vitro Ndemethylation of tylosin was higher than for spiramycin and tilmicosin (Carletti et al., 23). In microsomal fractions of goats and cattle, tylosin is a weak inhibitor of cytochrome P453A, as 12βOHtestosterone formation was inhibited only with high (125 µm) tylosin concentrations (ZweersZeilmaker et al., 1998). In pig liver, at least four major metabolites of tylosin were isolated after oral treatment with medicated feed providing 11 mg of 14 Ctylosin per kg twice daily for three days. Tylosin C and dihydrodesmycosin (DDM) represented 15% of the total liver residues, analysed using thinlayer chromatography (JECFA, 1991a). Significant amounts of tylosin D (525 µg/kg) were also found in the liver of turkeys (Montesissa et al., 1999). No other results on tylosin metabolism in animals were found in the open literature. However, information on tylosin metabolism in different animal species is available in the EC Committee on Veterinary Medical Products (CVMP) Summary Reports (EMEA, 1997; 2). The CVMP Tylosin Summary Report 3 states that, in pigs, residues of tylosin D and DDM were also found in the kidney. Following administration of 14 Ctylosin to three pigs in feed at a dose of 22 mg/kg b.w. for five days, 12.3% and 7.6% of the residues present in liver and kidney consisted of tylosin A. Smaller amounts of tylosin D (1.3% in liver and 6.1% in kidney), DDM (5.4% in liver and 4.1% in kidney) and cysteinyl tylosin A (which readily converts to tylosin A) were also present. Moreover, tylosin was metabolized by similar metabolic pathways in rats, pigs and cattle, although quantitative differences in the amounts of produced metabolites were observed (EMEA, 1997). Tylosin A (17% of radioactive residues) and several tylosin metabolites were also found in eggs. Metabolites present in eggs were at lower concentrations than the parent compound and included N desmethyltylosin A, tylosin D, Ndesmethyldihydrotylosin A and Odesmethyltylosin A (EMEA, 2). Excretion Hepatic. High concentrations of tylosin are present in the canine bile. In one dog given tylosin base at a dose of 1 mg/kg b.w. intravenously, 13.7% of the dose was recovered from bile 5 hours after dosing. The bile:serum concentration ratio varied in this dog from 123 to 378 (van Leeuwen, 1991). High concentrations of unchanged tylosin were also found in the bile and eliminated in the faeces of cows. After a single intravenous/intramuscular administration of tylosin base at a dose of mg/kg b.w., measured (7, 24 and 31 hours after injection) tylosin concentrations in bile were: 59.1/56.3, /35.1 and /12.1 µg/ml, respectively. Respective bile:serum ratios for tylosin: 295.5/61.9, /1.3 and /48.4 (Nouws and Ziv, 1977a; Nouws and Ziv, 1979) were, however, much lower than in the dog (van Leeuwen, 1991). Faeces. In rats and pigs the great majority (99%) of the metabolic residues was excreted in the faeces. In rats, the greatest component of the excreted residues was tylosin D (1% of total 14 C residues), tylosin A (6% of total 14 Cresidues), and tylosin C and dihydrodesmycosin DDM (4% of total 14 Cresidues). In one pig, given 14 Clabelled tylosin, the greatest component of the excreted residues was found to be tylosin D (33% of total 14 Cresidues), DDM (8% of total 14 Cresidues) and tylosin A 6% of total 14 Cresidues. Moreover, at least ten minor metabolites of tylosin representing 5% or less of the total residues were isolated in the excreta of the pig. No tylosin B was identified in the metabolic profile for pig and rat (JECFA, 1991a). In a recent GLPcompliant study using 14 Ctylosin in three pigs, summarised in a CVMP Summary Report on tylosin, about 94% of the excreted radioactivity was found in faeces. About 43% of the total radioactivity found in pig faeces was identified as tylosin D and 44% as DDM. The seco acid of tylosin D (resulting from hydrolysis of the lactone ring) was also identified in pig excreta (EMEA, 1997). 9

10 After intramuscular injection of 14 Ctylosin to calves at a dose of 17.6 mg/kg b.w., less than 5% of the administered dose was recovered from excreta. Tylosin A (29.8%), tylosin D (11.4%), tylosin C (25.2%) and demethyltylosin D (1.8%) were found in faecal extracts, while cysteinyltylosin A was the main component (7%) in urine (EMEA, 1997). Renal. In rats and pigs only 1% of the metabolic residues of tylosin was excreted in the urine (JECFA, 1991a). In a recent GLPcompliant study using 14 Ctylosin in three pigs, about 6% of the radioactivity was found in urine (EMEA, 1997). Lower concentrations of tylosin are excreted in urine than in bile of cattle. After a single intravenous/intramuscular administration of tylosin base at a dose of mg/kg b.w., measured at 7, 24 and 31 hours after injection, tylosin concentrations in urine were 29.7/41.7, /12.9 and /17.7 µg/ml, respectively (Nouws and Ziv, 1977a; Nouws and Ziv, 1979). Similar results were also found in goats and camels. When tylosin tartrate at a dose of 15 mg/kg b.w. was administered intravenously/intramuscularly in goats, urine concentrations of tylosin (measured up to 24 hours after injection) were as follows: 147.5/23.7 µg/ml (at 1 hour), 84.5/158.4 µg/ml (at 2 hours), 27./32.1 µg/ml (at 6 hours), 11.8/13.8 µg/ml (at 12 hours) and 5.5/6.8 µg/ml at 24 hours (Atef et al., In dogs that received tylosin base intravenously at a dose of 1 mg/kg b.w., urinary recovery was 18.8% of the dose during 6 hours after dosing (van Leeuwen, 1991). When chicken (57 weeks old) received 1 or 25 mg tylosin/kg b.w. (as tartrate) orally, peak tylosin concentrations in urine (< 1 µg/ml at the 25 mg/kg dose and > 14 µg/ml at the 25 mg/kg dose) occurred 24 hours after dosing and declined rapidly thereafter (van Leeuwen, 1991). Milk. In general, lipophylic weak bases like tylosin easily pass from the blood plasma to the milk, which has a lower ph. This was confirmed in several experiments in different ruminant species. In cows receiving a single intravenous injection of tylosin tartrate at a dose of 2 mg/kg b.w., peak concentrations of tylosin in milk (approx. 1 µg/ml) were observed 4 hours after injections; corresponding plasma concentrations of tylosin were only around 3.5 µg/ml. Lower peak values (approx. 6 µg/ml) were observed in cow s milk 6 hours after a single intramuscular injection of tylosin tartrate at the same dose. When ewes received a single intramuscular injection of tylosin tartrate at a dose of 2 mg/kg b.w., peak concentrations of tylosin in milk (6.7 µg/ml) appeared 7 hours after injection. The ratio of peak normal milk (ph = ) concentrations to peak serum concentrations of tylosin was approximately 2.5, while the ratio of peak mastitic milk (ph = ) to peak serum concentrations was 1.6 (Ziv and Sulman, 1973). In cows receiving tylosin base intramuscularly at a dose of 12.5 mg/kg b.w. every 12 hours for 48 hours, the peak concentration of tylosin in milk (approx. 7 µg/ml) appeared after 6 hours and then rapidly decreased to 1.5 µg/ml at 72 hours. Milk:serum ratios corrected for differences in protein binding and calculated at various times ranged up to about 2:1 (Gingerich et al., 1977). Similar milk:serum ratios (up to approx. 17.5:1) were observed in cows after a single intramammary infusion of 2 mg of tylosin/quarter. When mastitic cows received repeated intramuscular injections of tylosin base at a dose of 1 mg/kg b.w. every 12 hours for 5 days, peak milk concentrations increased gradually up to 18 µg/ml at the 5 th day after the onset of therapy (ElSayed et al., 1986). Similar results as regards tylosin milk concentrations were obtained in goats. After a single intravenous/intramuscular administration of tylosin tartrate at a dose of 15 mg/kg b.w., milk concentrations of tylosin were: 2.4/1.8 µg/ml (at 1 hour), 3.8/3.3 µg/ml (at 2 hours), 1.4/5.1 µg/ml (at 4 hours), 8.1/6.8 µg/ml (at 6 hours), 1.8/5.6 µg/ml (at 12 hours) and.6/1.7 µg/ml (at 24 hours). Milk:serum ratios calculated at various times up to 12 hours after intravenous injections of tylosin ranged from 1.7 to 2. (Atef et al., 1991). Genital tract. High concentrations of tylosin are found in genital tract secretions in cows. After a single intravenous administration of tylosin base at a dose of 1 mg/kg b.w., maximum concentrations (about 56 µg/ml) were observed 3 hours after injection, while after 5 hours tylosin concentrations in genital tract secretions were <.4 µg/ml (Cester et al., 1993). 1

11 Table 3. Pharmacokinetic parameters of tylosin in animals. Animals Route and dose (mg/kg) Protein binding (%) Maximum concentration C max (µg/ml) Time to reach maximum concentration T max (h) Bioavailability F (%) Elimination halflife t 1/2el. (h) Total clearance Cl B (ml/min/kg) Volume of distribution V d (l/kg) Reference Rats i.v., Duthu, 1985 Rats p.o., 5 a 1 12 van Leeuwen, et al., 1991 Rats i.g., 21 a ~ Kietzmann, 1985 Rabbits Dogs Dogs Cows Cows Cows Cows Cows Cows Calves Calves i.m., 1 a i.m., 25 b i.v., 1 a ~ i.m., 1 a tissue:plasma ratio.68 p.o., 25 a p.o., 1 a i.v., 12.5 a tissue:plasma ratio 2.5 van Leeuwen, et al., 1991 Weisel, et al., 1977 van Leeuwen, et al., 1991 Baggot and Gingerich, e i.v., 2 b Ziv and Sulman, i.m., 2 b ~ ; 1973 i.v., 12.5 a Gingerich, et al., i.m., 12.5 a < i.m., 12.5 a Baggot, 1978 i.m., 1 a 5.83 d.75 d 3.2 d 5.46 d ElSayed, et al., 1986 i.v., 1 a 2.77 e 7.43 e Cester, et al., 2.84 f 8.78 f 2.27 f 1993 i.v., 1 a 2.32 g 24.5 g 4.4 g Burrows, et al.,.95 h 42.2 h 3.52 h i 37. i 5.68 i i.v., 1 a 1.22 i 23.7 i 2.48 i Burrows, et al.,.84 i,j 26.4 i,j 1.91 i,j

12 Table 3. continued Animals Route and dose (mg/kg) Calves Calf buffaloes Sheep Sheep Goats Goats Camels i.m., 25 a s.c., 25 a i.t., 25 a i.m., 1 b Protein binding (%) Maximum concentration C max (µg/ml) Time to reach maximum concentration T max (h) 2 8 Bioavailability F (%) Elimination halflife t 1/2el. (h) Total clearance Cl B (ml/min/kg) Volume of distribution V d (l/kg) 1 i.m., 1 b i.v., 2 b i.m., 2 b ~ i.v., 15 b i.m., 15 b i.v., 15 b i.m., 15 b i.v., 15 b i.m., 15 b tissue:plasma ratio 2.5 i.v., 1 b i.m., 2 b k k ~.5 ~1.5 k k Reference Hjerpe, Sauri, et al., Ziv and Sulman, 1972; Taha, et al., Taha, et al., k k Atef, et al Ziv, et al., k 3.88 k Pigs i.v., 1 c Prats, et al., i.m., 1 a a Chickens i.v., 1 b Kowalski, et al., p.o., 1 b Explanatory notes: i.v. intravenous; i.m. intramuscular; s.c. subcutaneous; p.o. oral; i.g. intragastric; i.t. intratracheal; a tylosin base; b tylosin tartrate; c tylosin phosphate; d cows with mastitis; e cows in oestrus; f cows in luteal phase; g newborn calves (2 days old); h 2 weeks old calves; i 6 weeks old calves; j calves with pneumonia, k waterdeprived camels 12

13 Figure 3. Allometric relationship between tylosin total body clearance and body weight in different animal species. Data Mice Rats Chickens Dogs Sheep Goats Pigs Calves* Calves** Camels Cows Clearance (ml/min) 1.42 a , Body weight (grams) b 9 c 18, d 19, e 22, f 39,2 49,18 46, 585, References Cacciapuoti Duthu, Kowalski Weisel Taha Atef Prats Burrows Burrows Ziv Baggot and et al., et al., 22 et al., 1977 et al., 1999 et al., 1991 et al., 22a et al., 1983 et al., 1986 et al., 1995 Gingerich, 1976 * 2 days old, ** 6 weeks old a although the article specified only the AUC 3h value (23.5 µg h/ml) for mice, calculated after intravenous administration of tylosin at a dose of 1 mg/kg b.w., using this value and the equation: Cl B = Dose/AUC i.v., a total body clearance value of 7.9 ml/min/kg is calculated (1.42 ml/min), b estimated value; in original article: kg, c estimated value; in original article: 8.21 kg, d estimated value; in original article: 162 kg, e estimated value; in original article: 1622 kg, f estimated value; in original article: kg Multiplicative model: Cl B = a(b.w.) b by West et al., 1997; Estimated parameters: a (intercept), b (slope) and analysis of variance from STATGRAPHICS PLUS 4.1. Model from the Figure 3. Estimated parameters: a = ; b =.92513; Analysis of variance (model): Pvalue =.1; Correlation coefficient = ; Rsquared = % Model without camels Estimated parameters: a =.12338; b = ; Analysis of variance (model): Pvalue =.; Correlation coefficient = ; Rsquared = % Model without chickens and camels Estimated parameters: a =.17952; b = ; Analysis of variance (model): Pvalue =.; Correlation coefficient =.96867; Rsquared = % 13

14 TYLOSIN TISSUE RESIDUES There are little published data on tylosin residue depletion in animals, especially those treated with therapeutic doses of tylosin. Many studies have measured tylosin residues following consumption of feed containing high doses of tylosin (from 2 up to 8 g/ton). Moreover, the majority of the available reports were published between 197 and 1985, when in most cases unspecific and low sensitive (LOD > 1 µg/kg) microbiological methods were used for residue analysis. The majority of these reports, however, were not included in the monograph prepared at the thirtyeighth meeting of JECFA in 1991 (JECFA, 1991a). From the summary evaluation published by the Committee (JECFA, 1991b), it is evident that residues of tylosin in different organs depend highly on the route of administration. When injectable formulations of tylosin were used, the highest and most persistent residues were found in kidney tissues (excluding injection site residues). After oral administration of tylosin preparations, the highest residue concentrations were found in liver. Moreover, the route of administration also determines the concentration of the residues of tylosin in animal tissues. In general, when tylosin was administered orally, lower residue concentrations were observed than after injections (JECFA, 1991b). This fact may be a result of low oral bioavailability of tylosin in animal species, although the oral bioavailability of tylosin (F = 22.5% and F = 334% of the dose) was estimated only in pigs and chickens, respectively (van Leeuwen, 1991; Kowalski et al., 22). Ruminants Edible tissues. In newborn calves that received tylosin tartrate orally with a milk replacer at a dose of mg/kg b.w., twice a day for 14 days, the highest residues of tylosin measured by a microbiological cylinder plate method were found in liver samples. Measured 1 hour, 1, 3, 5, 7, 9 and 12 days after the slaughter, tylosin concentrations in liver were: 75, 55, 16, 1, 2, 1 and < 1 µg/kg, respectively (JECFA, 1991a). In newborn calves with pneumonia that received three intramuscular injections of tylosin base (every 12 hours) at a dose of 1 mg/kg b.w., the highest tylosin concentrations measured by an agargel diffusion method 26 hours after the last dose were found in kidney (about 33 µg/kg), while the lowest concentrations ( µg/kg) were found in muscle and cerebrospinal fluid (Burrows et al., 1986). In older calves (weighing approximately 24 kg), tylosin residues were higher and persisted longer after intramuscular injections than in newborn calves. In studies 1 and 2 (Table 4), residues persisted at the injection site and only after 42 to 49 days all samples were below the assay limit of 1 µg/kg. Residues at the injection site persisted longer in calves that were treated with the higher dose once daily compared to the animals that were treated twice per day. Of the other tissues, tylosin residues were highest and depleted most slowly in kidney with residues below the detection limits at 2835 days withdrawal. In study 3, results obtained by the HPLC analysis showed lower concentrations of tylosin residues than those obtained using the microbiological method, although the daily dose of tylosin administered in this study was also lower than in study 1 and 2. However, the results in study 3 regarding depletion of tylosin residues in all tissues was similar with depletion occurring most slowly at the injection site (Table 4). Of the other tissues, the kidney was the main target tissue with residues present through 7 days after tylosin injections. After 21 days the injection site still contained high tylosin residues (29 µg/kg), although in other tissues the residues were below the limit of detection (JECFA, 1991a). 14

15 Table 4. Summary of tylosin residues (µg/kg) in calves (weighing approximately 24 kg) after receiving multiple intramuscular injections of tylosin (JECFA, 1991a). Withdrawal time Residue concentration (µg/kg) (days) Study 1 Microbiological assay * dose: 8.8 µg/kg twice daily for 5 days Injection site muscle Kidney Liver 1,8, 44, , < < Study 2 Microbiological assay * dose: 17.6 µg/kg once daily for 5 days 7,633, 17,4 1, < < 1 < < < 1 49 < 1 Study 3 HPLC assay ** dose: 1 µg/kg once daily for 5 days Inj. site muscle Muscle Kidney Liver Fat 1,337, , Note: * detection limit of the microbiological assay was 1 µg/kg; ** detection limit of the HPLC assay was 2 µg/kg; not detected According to the information available in the CVMP Summary Report 3 (EMEA, 1997), following three daily intramuscular injections of 14 Ctylosin in calves at a dose of 17.6 mg/kg b.w., the mean total residues of tylosin measured four hours after the slaughter were 2521, 4781, 287 and 152 µg of tylosin equivalents/kg in liver, kidney, muscle and fat, respectively. When the same samples were analyzed by an HPLC method, the mean residues of tylosin A were 2635, 6945, 75 and 94 µg/kg in liver, kidney, muscle and fat, respectively, corresponding to 1.5%, 14.5%, 24.6% and 61.8% of the total residues in these tissues. Based on results of microbiological assay of these samples, it was calculated that tylosin A represented 36.7%, 31% and 7% of the microbiologically active residues present in kidney, liver and muscle, respectively (EMEA, 1997). Similar tissue concentrations of tylosin were observed in cows that received a single intramuscular injection of tylosin base at a dose of mg/kg b.w. Tylosin concentrations measured in renal cortex were: 8 µg/kg (at 7 hours), 115 µg/kg (at 24 hours) and 116 µg/kg (at 31 hours). In 15

16 liver, corresponding concentrations of tylosin were lower than in kidney and amounted to: 29, 48 and 26 µg/kg, respectively (Nouws and Ziv, 1977a; Nouws and Ziv, 1979). Milk. In cows that received intramuscular injections of tylosin at a dose of 17.6 mg/kg b.w., once daily for five days, tylosin residues in milk measured by a microbiological plate assay (with a sensitivity of 25 µg/kg) at, 48, 72, 84 and 96 hours after the last injection were: 75, 35, 14, 8 and 5 µg/kg. At hours following the last tylosin injection, the residues were not detectable in milk samples (JECFA, 1991a). Similar results were obtained using an HPLC assay. In cows that received intramuscular injections of tylosin at a dose of 1 mg/kg b.w. once daily for three days immediately after the morning milking, the residues of tylosin A measured on the first day after the treatment were 263 µg/kg at the morning milking and 933 µg/kg at the afternoon milking. Three days after the end of the treatment, residues were found in one sample at the morning milking (6 µg/kg) and were below the limit of quantification (5 µg/kg) in all samples taken at the afternoon milking. In a similar study in cows that received an equal dose of tylosin for five days, milk residues of tylosin were 393 µg/kg at the morning milking on first day. At the morning milking of the second day after the last dose, tylosin milk residues had declined to 3815 µg/kg (EMEA, 1997). In a similar residue study in cows, tylosin residues in milk measured by an HPLC assay (Sokol et al., 1996) were in the same concentration range and declined slowly to 3 µg/kg five days after the last treatment with tylosin base at 1 mg/kg b.w. (Dudriková and Lehotský, 1998). When tylosin was intramuscularly injected at the same dose in ewes (1 mg/kg b.w. once daily for five days), milk residues of tylosin were not detected two days after the last dose (Nagy et al., 21) see Table 5. Table 5. Tylosin residues in milk (Dudriková and Lehotský, 1998; Nagy et al., 21). Time of experiment Residue concentration (µg/l) measured by an HPLC assay* (hours) Cows 1 mg/kg b.w. once daily for 5 days Ewes 1 mg/kg b.w. once daily for 5 days (1) (2) ( ( (5) (6) Note: * detection limit 1 µg/l; time of tylosin injection; (16) days after the last injection 16

17 Pigs A number of tylosin residue studies were carried out in pigs. In one study by Kline and Waitt (1971), tylosin was administered to pigs in feed containing 1, 2, 5 and 1 grams of tylosin/ton for 1415 days. It was demonstrated that a residue level of 2 µg/kg would not be exceeded in edible tissues when feeding tylosin at 1 g/ton feed, the recommended dose for pigs. Tylosin residues were detected in livers of two pigs (55 and 56 µg/kg) receiving tylosin at a dose of 1 g/ton of feed (about 3 mg/kg b.w.) at zero hour withdrawal. However, no residues above the tolerance limit adopted in the USA (TL = 2 µg/kg; Code of Federal Regulation, 25) were detected in tissues when tylosin was fed at a dose of 1, 2 and 5 g/ton (Kline and Waitt, 1971). In general, no tylosin residues were found in pig tissues when pigs received tylosin tartrate in drinking water at a dose of.25% and.5% for five days, doses equivalent to mg/kg b.w. and mg/kg b.w., respectively (Iritani et al., 1975). Small amounts of tylosin residues were found only in two pigs receiving the higher dose of tylosin: 6 µg/kg in bone marrow at day zero after the withdrawal and 42 µg/kg in duodenum one day after the withdrawal (Iritani et al., 1975). Similar results were obtained in another feeding experiment in pigs (Lauridsen et al., 1988). No residues of tylosin were found in pigs fed 4 and 1 g/ton of tylosin in the diet, even at zero withdrawal time. Residues of tylosin were detected only in liver and kidney from pigs fed 2 and 4 g/ton of tylosin in the diet for 17 days, and slaughtered within three hours after the last feeding (Table 6). Table 6. Residues of tylosin in tissues from pigs fed various amounts of tylosin until slaughter at 9 kg b.w. and withdrawal times below three hours (Lauridsen et al., 1988). Pig No Dose: g/ton of feed for 17 days * detection limit of the microbiological assay was 3 µg/kg; not detected Residue concentration (µg/kg) measured by a microbiological assay* Kidney Liver Muscle < 3 3 < Similar data concerning tylosin residues in feeding experiments were reported by the CVMP and JECFA. After administration of 14 Ctylosin in three male pigs at a dietary dose of 22 mg/kg for five days, mean residues of tylosin measured four hours after the slaughter were: 45, 46, 5 and 7 µg of tylosin equivalents/kg in liver, kidney, fat and muscle, respectively. However, when the same samples were analyzed by an HPLC method, the residues of tylosin A were below the limit of quantification (5 µg/kg) in all samples. Low residues of tylosin A in liver and kidney (around 3 µg/kg) were occasionally found in pigs slaughtered six hours after tylosin administration in the feed (2 mg/kg) for 28 days or the drinking water (25 mg/l) for 1 days (EMEA, 1997). When feed containing 2 g/ton of tylosin was fed to pigs for 17 days, tylosin concentrations in both liver and kidney were 3 µg/kg when measured by a microbiological assay. The highest residue concentrations observed in pig tissues was 5 µg/kg in liver after administration of 4 g/ton of tylosin in feed (JECFA, 1991a). There are little published data on the time required for depletion of tylosin from tissues of pigs treated intramuscularly with therapeutic doses. In crossbred pigs weighing 811 kg treated with 17

18 intramuscular injections of tylosin at a dose of 8.8 mg/kg b.w., tylosin residue concentrations measured by an HPLC assay with a detection limit < 1 µg/kg were below 2 µg/kg one day after withdrawal, except at the injection site in one pig. Tylosin residues were not detected in any tissue of any pig at two days after the treatment (Moats et al., 1985). It was also shown that results obtained by bioassay and HPLC methods differed considerably for tissue samples analyzed two months after the experiment (Table 7). 18

19 Table 7. Residues of tylosin in pig tissues after single intramuscular injection of tylosin base at a dose of 8.8 mg/kg b.w. (Moats et al., 1985). Animal No Withdrawal time (hours) Injection site (Muscle) Sample 1 a Tissue residue concentration (µg/kg) measured by an HPLC* or bioassay** Injection site Contralateral Muscle Contralateral Muscle Kidney b Liver b (Muscle) Sample 1 a Sample 2 a Sample 2 a HPLC Bioassay HPLC Bioassay HPLC 92, 24, 58, 26, 15 27, 17, 46, , 32, 52, 32, 13 Bioassay HPLC Bioassay HPLC Bioassay HPLC Bioassay * detection limit of the HPLC assay was < 1 µg/kg; tissue recoveries from spiked samples were: 82.8% (kidney), 88.8% (liver) and 96.8% (muscle); ** detection limit of the microbiological assay was about 5 µg/kg; a samples stored at minus 2 o C for unknown time; b samples stored at minus 2 o C up to 2 months; not detected 19

20 In storage stability studies, appreciable concentrations of tylosin (92313 µg/kg) determined by the bioassay procedure were observed in kidney samples (collected at 4 hours withdrawal) after 45 days of storage at minus 2 o C. Residues were undetectable after 75 days of storage at minus 2 o C. Moreover, detectable residues (16 µg/kg) were found in liver in only one pig (collected at 4 hours withdrawal) after seven days of storage and were undetectable after 6 days of storage. Based on these results, authors of the study concluded that tylosin residue analyses on stored liver and kidney samples appear somewhat unreliable (Moats et al., 1985). According to the CVMP Summary Report and the JECFA residue monograph on tylosin residues, after five intramuscular injections of tylosin in pigs at a dose of 1 mg/kg b.w., tylosin concentrations at the injection site declined slowly from 638 µg/kg (six hours after withdrawal) to 148 µg/kg three days after the last dose. After seven days withdrawal, tylosin residues at the injection site were below the limit of detection of the HPLC method (2 µg/kg). Tylosin residues in other swine tissues were detectable only at six hours after the last dose and amounted to: 92, 67, 61, 355 and 669 µg/kg in muscle, fat, skin, liver and kidney, respectively (EMEA, 1997; JECFA, 1991a). Similar results on tylosin residues in swine tissues (Table 8) were analysed by Prats and coworkers (22b). Following intramuscular injections of tylosin base at a dose of 1 mg/kg b.w., once daily for five days, the highest concentration of tylosin residues were found at the injection site three (11254 µg/kg) and seven days (141 µg/kg) after the withdrawal, respectively. Residues at the injection site depleted below the limit of quantitation (5 µg/kg) of the HPLC assay at ten and fourteen days after the last dose (Prats et al., 22b). In other tissues, tylosin residues declined faster than from the injection site. Table 8. Residues of tylosin in swine tissues after intramuscular injections of tylosin base in 16 pigs at a dose of 1 mg/kg b.w. for five days (Prats et al., 22b). Animals group 1 Withdrawal (days) Residue concentration (µg/kg) measured by an HPLC assay* Inj. site (Muscle) Muscle Skin + fat Kidney Liver group group group * quantification limit of the HPLC method was 5 µg/kg; detection limit was 25 µg/kg In another residue study, where tylosin was injected intramuscularly in pigs at a dose of 8.8 mg/kg b.w., twice a day for three days, tylosin residues, as measured by a microbiological assay method, at the injection site were 274, 92, 87 and 12 µg/kg at, 7, 14 and 21 days after withdrawal, 2