TYLOSIN A review of pharmacokinetics,

residues in food animals and analytical methods1

Jacek Lewicki

Division of Pharmacology and Toxicology, Department of Preclinical Sciences Faculty of Veterinary Medicine, Warsaw Agricultural University Ciszewskiego 8, 02-787 Warsaw, Poland. E-mail: 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 15th 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 66th 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 66th JECFA meeting. INTRODUCTION Tylosin was previously evaluated at the twelfth meeting of JECFA in 1968 (JECFA, 1969) and the thirty-eighth 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 38th 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 re-evaluation: 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 body-weight gain in treated rats. 4. Studies to determine MIC values of inhibitory activity against micro-organisms 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 COMPOUND International nonproprietary name: Tylosin (INN-English) European Pharmacopoeia name: (4R,5S,6S,7R,9R,11E,13E,15R,16R)-15-[[(6-deoxy-2,3-di-O-

methyl-β-D-allopyranosyl)oxy]methyl]-6-[[3,6-dideoxy-4-O-(2,6-dideoxy-3-C-methyl-α-L-ribo-hexopyranosyl)-3-(dimethylamino)-β-D-glucopyranosyl]oxy]-16-ethyl-4-hydroxy-5,9,13-trimethyl-7-(2-oxoethyl)oxacyclohexadeca-11,13-diene-2,10-dione

IUPAC name: 2-[12-[5-(4,5-dihydroxy-4,6-dimethyl-oxan-2-yl)oxy-4-dimethylamino-3-hydroxy-6-methyl-oxan-2-yl]oxy-2-ethyl-14-hydroxy-3-[(5-hydroxy-3,4-dimethoxy-6-methyl-oxan-2-

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.

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yl)oxymethyl]-5,9,13-trimethyl-8,16-dioxo-1-oxacyclohexadeca-4,6-dien-11-yl]acetaldehyde

Other chemical names: 6S,1R,3R,9R,10R,14R)-9-[((5S,3R,4R,6R)-5-hydroxy-3,4-dimethoxy-6-methylperhydropyran-2-yloxy)methyl]-10-ethyl-14-hydroxy-3,7,15-trimethyl-11-oxa-4,12-dioxocyclohexadeca-5,7-dienyl}ethanal

Oxacyclohexadeca-11,13-diene-7-acetaldehyde, 15-[[(6-deoxy-2,3-di--methyl-b-D-allopyranosyl)oxy]methyl]-6-[[3,6-dideoxy-4-O-(2,6-dideoxy-3-C-methy-a-L-ribo-hexopyranosyl)-3-(dimethylamino)-b-D-glucopyranosyl]oxy]-16-ethyl-4-hydroxy-5,9,13-trimethyl-2,10-dioxo-[4R-(4R*,5S*,6S*,7R*,9R*,11E,13E,15R*,16R*)]-

Synonyms: AI3-29799, EINECS 215-754-8, Fradizine, HSDB 7022,

Tilosina (INN-Spanish), Tylan, Tylocine, Tylosin, Tylosine, Tylosine (INN-French), Tylosinum (INN-Latin), Vubityl 200

Registry numbers: CAS 1401-69-0 Table 1. Summary of physico-chemical properties of tylosin (Paesen et al., 1995abc;

McFarland et al., 1997; The Merck Index, 2001). Molecular formula: C46H77NO17

Molecular weight: 916.1

Appearance: An almost white or slightly yellow crystalline powder

Melting point: 128-132oC

Solubility: 5 mg/ml (water 25oC), soluble in lower alcohols, esters, ketones, chlorinated hydrocarbons, benzene, ether, chloroform

Stability: Solutions are stable at pH 4-9 (most stable at pH 7);

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

pKa: 7.73

log P (octanol-water): 1.63

UV Absorption: UVmax. at 282 nm, extinction coefficient (E1cm1%) is 245 at 282 nm

CONDITIONS OF USE Tylosin is a macrolide antibiotic that is active against certain Gram-positive and Gram-negative 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, 2001). Tylosin is a mixture of four macrolide antibiotics produced by a strain of Streptomyces fradiae (Figure 1). The main component of the mixture (> 80%) is tylosin A (Mr = 916; McGuire et al., 1961).

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Tylosin B (desmycosin, Mr = 772; Hamill et al., 1961), tylosin C (macrocin, Mr = 902; Hamill and Stark, 1964) and tylosin D (relomycin, Mr = 918; Whaley et al., 1963) may also be present. All four components contribute to the potency of tylosin, which is not less than 900 IU/mg, calculated with reference to the dried substance (European Pharmacopoeia, 2004). Relative antimicrobial activities of tylosin derivatives are: tylosin A – 1.0, tylosin B – 0.83, tylosin C – 0.75 and tylosin D – 0.35 (Teeter and Meyerhoff, 2003). Figure 1. Chemical structure of tylosin.

Tylosin A contains a polyketide lactone (tylactone) substituted with three 6-deoxyhexose sugars (Figure 2). The addition of D-mycaminose to the aglycone is followed by concurrent ring oxidation at C-20 and C-23 (to generate the tylonolide moiety) and substitution with L-mycarose and 6-deoxy-D-allose. Bis-O-methylation 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.

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Other pharmacologically active compounds, i.e., lactenocin, demecinosyl-tylosin (DMT) and O-mycaminosyl-tylonolide (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 22oC but approximately 10% of added tylosin was degraded within the first 200 hours in MilliQ water at pH 9.2 (Kolz et al., 2005). 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% (Halling-Sørensen et al., 2003). 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 50S ribosome subunit and inhibit mRNA-directed protein synthesis. Moreover, they stimulate the dissociation of peptidyl-tRNA from ribosomes during translocation. The precise mechanism of action has not been fully explained and many theories exist (Zhanel et al., 2001; Gaynor and Mankin, 2005). It has been suggested that 16-membered-ring macrolides inhibit protein synthesis by blocking elongation of the peptide chain, but the 14- and 15-membered-ring macrolides are only potent inhibitors of mRNA-directed peptide synthesis (Retsema and Fu, 2001). It was also demonstrated that the 16-membered-ring 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., 2000). Results of comparative antibacterial evaluation of tylosin and desmycosin showed that both compounds have almost identical antibacterial activity (Table 2). In the same study, tetrahydro-desmycosin and dihydro-desmycosin showed decreased antimicrobial activity, while the symmetric dimer of desmycosin was inactive (Iveković et al., 2003). Moreover, 4'-deoxy-10,11,12,13-tetrahydro-desmycosin, a derivative of tetrahydro-desmycosin, retained the antibacterial spectrum of tylosin with some improvement against tylosin-sensitive Staphylococci and Haemophilus influenzae (Narandja et al., 1995). Table 2. Antibacterial in vitro activity of tylosin, desmycosin, tetrahydro-desmycosin,

dihydro-desmycosin and dimeric desmycosin (Iveković et al., 2003).

MIC (µg/ml) Bacteria Tyl Des THDes DHDes DIMDes

Staphylococcus aureus B 0329 Staphylococcus aureus B 0538 (iMLS)a

Staphylococcus aureus B 0330 (cMLS)b

Streptococcus pneumoniae B 0541 Streptococcus pneumoniae B 0326 (M)c

Streptococcus pneumoniae B 0328 (cMLS) Streptococcus pyogenes B 0542 Streptococcus pyogenes B 0543 (iMLS)Streptococcus pyogenes B 0544 (cMLS) Escherichia coli B 0001 Haemophilus influenzae B 0529

0.5 1

>64 <0.125 <0.125

>64

<0.125 0.25

>64 >64 16

0.5 1

>64 ≤0.125 ≤0.125

>64

≤0.125 ≤0.125

>64 >64

4

0.5 2

>64 1 2

>64 0.5 1

>64 >64 32

2 4

>64 1 2

>64 ≤0.125

0.5

>64 >64 16

>64 >64 >64 >64 >64

>64 >64 >64

>64 >64 >64

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MIC – minimum inhibitory concentration; Tyl – tylosin; Des – desmycosin; THDes – 10,11,12,13-tetrahydro-desmycosin; DHDes – 10,11-dihydro-desmycosin; 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 thirty-second 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 thirty-sixth meeting of JECFA (JECFA, 1990), 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, 2004). Because it is slightly to moderately bound to plasma proteins (30-47% - Table 3) with a high degree of lipid solubility, tylosin is widely distributed in body fluids and tissues (Burrows, 1980). 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 50 mg/kg b.w. of tylosin base or tylosin tartrate in rats, tylosin peak serum concentrations of ≤ 1.0 µg/ml were seen after 1-2 hours. Within 7 hours serum concentrations decreased to less than the limit of detection (0.10 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 20, 50 and 100 mg/kg b.w. of tylosin base, peak serum concentrations (about 0.5-1.1 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 1-10 days of continuous medication revealed no detectable tylosin concentrations (< 0.1 µg/ml), while at the same time lung tissue contained 3.93-18.14 µg of tylosin/g (Carter et al., 1987).

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In general, higher blood concentrations of tylosin correspond to higher administered doses of tylosin. In dogs receiving tylosin orally by capsule (1, 10 or 100 mg/kg b.w./day for 8 days), tylosin blood concentrations determined 2 hours after the last dose ranged from < 0.15 µg/ml to 9.5 µg/ml. In dogs receiving 25 or 100 mg/kg b.w. of tylosin base orally by capsule daily for 29 days, peak serum concentrations (1.4-2.7 µg/ml) were seen 2 hours after dosing at 25 mg/kg b.w./day and the highest concentrations (2.7-4.6 µg/ml) were seen 2-5 hours after dosing at 100 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 30 mg/kg b.w., tylosin activity was present in blood plasma 10 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 110 mg/kg b.w. (as the granulated phosphate), tylosin serum activity peaked 1 hour after dosing (average 17.81 µg/ml); 24 hours after dosing tylosin was not detectable (< 0.1 µg/ml). Similar results were obtained after oral administration of tylosin phosphate (50 mg/kg b.w.) in water. Tylosin concentrations were detected in serum from 10 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 720 g), a single dose of 50 mg tylosin/bird (as tylosin tartrate) by stomach intubation, resulted in detectable tylosin activity in serum 0.5 hour after dosing. Peak concentrations of 0.6-4.0 µg/ml were found after 2 hours, declining to negligible after 24 hours. Repeated oral doses of 50 mg of tylosin to chickens (weight 2 kg, dosed at 1, 2, and 3 hours) resulted in peak serum concentrations at 4 hours (about 0.28 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 10 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 30-34% of the administered dose (Kowalski et al., 2002). When tylosin tartrate was added to the drinking water at 500 and 700 mg/l for 48 hours, average serum concentrations of tylosin were 0.12 and 0.17 µg/ml, respectively. However, peak concentrations (about 0.2-0.3 µg/ml) appeared after 24 hours (Ziv, 1980). In the oral bioequivalence study of two commercial products containing tylosin tartrate, the average tylosin concentration in serum of 5- and 7-week old broilers and 9-month old layers (treated for 5 consecutive days with medicated drinking water containing 750 mg tylosin tartrate/litre) was 0.20 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 1500 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 10 mg/kg b.w. as a 5% aqueous solution, tylosin peak serum concentrations (0.57-0.88 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 PEG-200 (10 mg/kg b.w.) solutions. Peak serum concentrations observed at 1 hour were 4.7-7.2 µg/ml and 0.96-1.25 µg/ml, respectively. Within 24 hours tylosin serum concentrations were below the limits of detection - 0.05 µg/ml for tylosin hydrochloride and 0.10 µg/ml for tylosin tartrate (van Leeuwen, 1991). Peak blood concentrations of tylosin were reached in 2-4 hours following intramuscular injection of tylosin base in 50% 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 17.6

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mg/kg b.w., tylosin peak concentrations (2.07-2.3 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 5.2-5.8 µg/ml were reached 1 hour after intratracheal administration, while peak concentrations of 2.7-4.7 and 1.25-1.8 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 5-6 hours after injection, with systemic bioavailability of 70-80% 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 10 mg/kg b.w., much lower than previously reported in cattle (Table 3), peak plasma concentrations (0.65 µg/ml in cattle at 1 hour and 0.47 µg/ml in buffaloes at 0.85 hour) were observed in both species (Saurit et al., 2002). Following intramuscular injection of tylosin base in pigs at a dose of 10 mg/kg b.w., tylosin peak plasma concentrations (0.4-1.9 µg/ml) were reached after 0.3-3 hours and the bioavailability was 95% of the dose (Prats et al., 2002a). In small ruminants after intramuscular injection of tylosin tartrate at a dose of 15 mg/kg b.w., tylosin peak plasma concentrations of 2.08 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 non-commercial avian species following intramuscular injection of tylosin base in 50% propylene glycol at a dose of 15-25 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 0.5 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, 1980). Calculated theoretical tissue:plasma ratios (k12/k21) of tylosin in cows and goats were 2.05 and 2.5 (Table 3), respectively (Baggot and Gingerich, 1976; Atef et al., 1991). In dogs, the tissue:plasma ratio for tylosin was only 0.68 (Weisel et al., 1977). In dogs the reported volume of distribution Vd = 1.7 l/kg (Weisel et al., 1977) was, however, similar to the Vd values calculated in other animal species: 2.2 l/kg in rats (Duthu, 1985), 1.1-2.27 l/kg in cows (Ziv and Sulman, 1973; Baggot and Gingerich, 1976; Gingerich et al., 1977; Cester et al., 1993), 1.59-2.74 l/kg in sheep (Ziv and Sulman, 1973; Taha et al., 1999) and 1.7-3.12 l/kg in goats (Atef et al., 1991; Taha et al., 1999). A lower volume of distribution was reported only in chickens - Vd = 0.69 l/kg (Kowalski et al., 2002). Higher Vd values for tylosin were calculated in young calves - 2.48-5.68 l/kg (Burrows et al., 1983; Burrows et al., 1986), pigs - 14.6 l/kg (Prats et al., 2002a) and camels - 11.93 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: 30% in chickens (Ziv, 1980), 33.5-44% in cows (Ziv and Sulman, 1972; Ziv and Sulman, 1973), 37.6% in goats (Atef et al., 1991), 38-45.4% in sheep (Ziv and Sulman, 1972; Ziv and Sulman, 1973) and 38.6-47.7% in camels (Ziv et al., 1995) – see Table 3. In studies in cows that received tylosin base intramuscularly at a dose of 6.8-7.3 mg/kg b.w., tylosin tissue:serum ratios measured 7-31 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 0.4 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 AUC48h:plasma

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AUC48h ratio of 16.64 (van Duyn and Folkerts, 1979). In six-week old calves with pneumonia that received three intramuscular injections of tylosin base every 12 hours at a dose of 10 mg/kg b.w., the tylosin tissue:serum ratios measured two hours after the last dose were: 2.0 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 (< 0.5 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 1-10 days of continuous medication revealed no detectable tylosin concentrations (< 0.1 µg/ml), while lung tissue contained 3.93-18.14 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 half-lives (t1/2el.) of tylosin after single intravenous administration in healthy animals were: 0.4 hour in rats, 0.52 hour in chickens, 0.9 hour in dogs, 0.92 hour in camels, 0.95-2.38 hours in young calves, 1.62-2.84 hours in cows, 2.05-4.75 hours in sheep, 3.04-4.24 hours in goats and 4.52 hours in pigs (Table 3). Slightly longer t1/2el. values were reported for tylosin after intramuscular injections: 2.4 hours in dogs, 2.24-3.2 hours in cattle and 3.71 hours in camels. In pigs however, the tylosin elimination half-life exceeded 24 hours after intramuscular administration of tylosin base at a dose of 10 mg/kg b.w. (Prats et al., 2002a). In quail, pigeons and cranes, the elimination half-life of tylosin after intramuscular injections of tylosin base was 1.2 hours; in emus, the elimination half-life 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 (ClB) after intravenous injection of tylosin tartrate was 239.28 ml/min/kg, while in water-deprived camels ClB decreased to only 39.73 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., 1990). Although only the AUC0-3h value (23.5 µg·h/ml) was given for mice, as calculated after intravenous administration of tylosin at a dose of 100 mg/kg b.w., using this value and the equation: ClB = Dose/AUCi.v., a clearance value of 70.9 ml/min/kg is calculated. In other animal species, ClB values were: 23.7-42.2 ml/min/kg in young calves, 26.8 ml/min/kg in pigs, 21.9 ml/min/kg in dogs, 7.43-8.7 ml/min/kg in cows, 6.8-8.66 ml/min/kg in goats and 6.89 ml/min/kg in sheep (Table 3). Allometric relationships between tylosin total body clearance (ClB) and animal body weight are presented in Figure 3. There is a statistically significant relationship (P-value = 0.0001) between tylosin clearance and body weight at 99% confidence level. The R-square 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 0.919, indicating a relatively strong relationship between the variables, especially when the data from chickens and camels were not included into the model ClB = 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, 2001).

Metabolism Primary metabolism of tylosin occurs within the liver. In contrast to some authors’ (Anadón and Reeve-Johnson, 1999) conclusions that tylosin is unable to produce cytochrome P450 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 P450-3A (9.67 nmol complex/nmol P450). This ability is lower than that for tiamulin, erythromycin and

8

roxithromycin, however. Moreover, the observed rate of the in vitro N-demethylation of tylosin was higher than for spiramycin and tilmicosin (Carletti et al., 2003). In microsomal fractions of goats and cattle, tylosin is a weak inhibitor of cytochrome P450-3A, as 12β-OH-testosterone formation was inhibited only with high (125 µM) tylosin concentrations (Zweers-Zeilmaker et al., 1998). In pig liver, at least four major metabolites of tylosin were isolated after oral treatment with medicated feed providing 110 mg of 14C-tylosin per kg twice daily for three days. Tylosin C and dihydrodesmycosin (DDM) represented 15% of the total liver residues, analysed using thin-layer chromatography (JECFA, 1991a). Significant amounts of tylosin D (50-250 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; 2000). 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 14C-tylosin to three pigs in feed at a dose of 220 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 (10.3% 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-desmethyl-tylosin A, tylosin D, N-desmethyl-dihydro-tylosin A and O-desmethyl-tylosin A (EMEA, 2000). Excretion Hepatic. - High concentrations of tylosin are present in the canine bile. In one dog given tylosin base at a dose of 10 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 1230 to 3780 (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 4.6-7.3 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, -/100.3 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 (10% of total 14C-residues), tylosin A (6% of total 14C-residues), and tylosin C and dihydrodesmycosin – DDM (4% of total 14C-residues). In one pig, given 14C-labelled tylosin, the greatest component of the excreted residues was found to be tylosin D (33% of total 14C-residues), DDM (8% of total 14C-residues) and tylosin A - 6% of total 14C-residues. 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 GLP-compliant study using 14C-tylosin 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

After intramuscular injection of 14C-tylosin to calves at a dose of 17.6 mg/kg b.w., less than 50% of the administered dose was recovered from excreta. Tylosin A (29.8%), tylosin D (11.4%), tylosin C (25.2%) and demethyl-tylosin D (10.8%) were found in faecal extracts, while cysteinyl-tylosin A was the main component (70%) 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 GLP-compliant study using 14C-tylosin 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 4.6-7.3 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: 147.5/230.7 µg/ml (at 1 hour), 84.5/158.4 µg/ml (at 2 hours), 27.0/32.1 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., 1991. In dogs that received tylosin base intravenously at a dose of 10 mg/kg b.w., urinary recovery was 18.8% of the dose during 6 hours after dosing (van Leeuwen, 1991). When chicken (5-7 weeks old) received 100 or 250 mg tylosin/kg b.w. (as tartrate) orally, peak tylosin concentrations in urine (< 100 µg/ml at the 25 mg/kg dose and > 1400 µg/ml at the 250 mg/kg dose) occurred 2-4 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 20 mg/kg b.w., peak concentrations of tylosin in milk (approx. 10 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 20 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 = 6.5-6.8) concentrations to peak serum concentrations of tylosin was approximately 2.5, while the ratio of peak mastitic milk (pH = 7.1-7.4) 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 60 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 20:1 (Gingerich et al., 1977). Similar milk:serum ratios (up to approx. 17.5:1) were observed in cows after a single intramammary infusion of 200 mg of tylosin/quarter. When mastitic cows received repeated intramuscular injections of tylosin base at a dose of 10 mg/kg b.w. every 12 hours for 5 days, peak milk concentrations increased gradually up to 18 µg/ml at the 5th day after the onset of therapy (El-Sayed 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), 10.4/5.1 µg/ml (at 4 hours), 8.1/6.8 µg/ml (at 6 hours), 1.8/5.6 µg/ml (at 12 hours) and 0.6/1.7 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 2.0 (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 10 mg/kg b.w., maximum concentrations (about 5-6 µg/ml) were observed 3 hours after injection, while after 50 hours tylosin concentrations in genital tract secretions were < 0.4 µg/ml (Cester et al., 1993).

10

Table 3. Pharmacokinetic parameters of tylosin in animals. Animals Route and

dose (mg/kg)

Protein binding

(%)

Maximum concentration - Cmax (µg/ml)

Time to reach maximum

concentration - Tmax (h)

Bioavailability F (%)

Elimination half-life - t1/2el. (h)

Total clearance - ClB

(ml/min/kg)

Volume of distribution -

Vd (l/kg)

Reference

Rats i.v., 25 0.4 86 2.2 Duthu, 1985 Rats p.o., 50a ≤ 1 1-2 van Leeuwen, et

al., 1991 Rats i.g., 20-100a ~ 0.5-1.1 2 Kietzmann, 1985 Rabbits i.m., 10a

i.m., 25b0.57-0.88

4.7-7.2 1.5 1

van Leeuwen, et al., 1991

Dogs i.v., 10a

i.m., 10a~ 1.5 0.5 0.9

2.4 21.9

1.7

tissue:plasma ratio - 0.68

Weisel, et al., 1977

Dogs p.o., 25a

p.o., 100a1.4-2.72.7-4.6

2 2-5

van Leeuwen, et al., 1991

Cows i.v., 12.5a

1.62

7.8

1.1

tissue:plasma ratio - 2.05

Baggot and Gingerich, 1976

Cows i.v., 20b

i.m., 20b33.5-44

~ 2.5

5 2.14

1.56 Ziv and Sulman,

1972; 1973 Cows i.v., 12.5a

i.m., 12.5a < 1

1.5-4 1.62 1.1 Gingerich, et al.,

1977 Cows i.m., 12.5a 0.85 6 70-80 Baggot, 1978Cows i.m., 10a 5.83d 0.75d 3.02d 5.46d El-Sayed, et al.,

1986 Cows i.v., 10a 2.77e

2.84f7.43e

8.78f1.84e

2.27fCester, et al., 1993

Calves i.v., 10a

2.32g

0.95h

1.53i

24.5g

42.2h

37.0i

4.4g

3.52h

5.68i

Burrows, et al., 1983

Calves i.v., 10a 1.22i

0.84i,j23.7i

26.4i,j2.48i

1.91i,jBurrows, et al., 1986

11

Table 3. continued Animals Route and

dose (mg/kg)

Protein binding

(%)

Maximum concentration - Cmax (µg/ml)

Time to reach maximum

concentration - Tmax (h)

Bioavailability F (%)

Elimination half-life

- t1/2el. (h)

Total clearance - ClB

(ml/min/kg)

Volume of distribution - Vd (l/kg)

Reference

Calves i.m., 25a

s.c., 25a

i.t., 25a

2.7-4.71.25-1.8 5.2-5.8

2 8 1

Hjerpe, 1979

Calf buffaloes

i.m., 10b

i.m., 10b 0.65

0.47 1.05 0.85

2.242.40

Sauri, et al., 2002

Sheep i.v., 20b

i.m., 20b38-45.4

~ 2.5

4 2.05 1.59 Ziv and Sulman,

1972; 1973 Sheep i.v., 15b

i.m., 15b 2.58

3.29

73 4.75

6.89

2.74

Taha, et al., 1999

Goats i.v., 15b

i.m., 15b 2.08

3.84

84 4.24 8.66 3.12 Taha, et al., 1999

Goats i.v., 15b

i.m., 15b37.6 2.4 4 72.6 3.04 6.8 1.7

tissue:plasma ratio – 2.5

Atef, et al. 1991

Camels i.v., 10b

i.m., 20b

38.6-47.7 26.8-45.8k

1.16 0.62k

~0.5 ~1.5k

88 41k

0.92 1.52 k

3.71 2.73 k

239.28 39.73 k

11.93 3.88 k

Ziv, et al., 1995

Pigs i.v., 10c

i.m., 10a1.0

1.5

95 4.52

24.49 26.8 14.6 Prats, et al.,

2002a Chickens

i.v., 10b

p.o., 10b30

1.2

1.5

30.7-34 0.52 2.07

5.3 0.69 Kowalski, et al., 2002

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 – water-deprived camels

12

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) 1.42a 21.5 12.98 197.1 124.02 129.2 589.6 960.4 1165.57 97,156 4563

Body weight (grams) 20 250 2450b 9000c 18,000d 19,000e 22,000f 39,200 49,180 406,000 585,000

References Cacciapuoti

et al., 1990

Duthu,

1985

Kowalski

et al., 2002

Weisel

et al., 1977

Taha

et al., 1999

Atef

et al., 1991

Prats

et al., 2002a

Burrows

et al., 1983

Burrows

et al., 1986

Ziv

et al., 1995

Baggot and

Gingerich, 1976

* - 2 days old, ** - 6 weeks old a – although the article specified only the AUC0-3h value (23.5 µg·h/ml) for mice, calculated after intravenous administration of tylosin at a dose of 100 mg/kg b.w., using this value and the equation: ClB = Dose/AUCi.v., a total body clearance value of 70.9 ml/min/kg is calculated (1.42 ml/min), b - estimated value; in original article: 2.3-2.6 kg, c - estimated value; in original article: 8.2-10 kg, d - estimated value; in original article: 16-20 kg, e - estimated value; in original article: 16-22 kg, f - estimated value; in original article: 20-23.7 kg Multiplicative model: ClB = 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 = 0.0463747; b = 0.925013; Analysis of variance (model): P-value = 0.0001; Correlation coefficient = 0.918526; R-squared = 84.3689% Model without camels - Estimated parameters: a = 0.120338; b = 0.789662; Analysis of variance (model): P-value = 0.0000; Correlation coefficient = 0.947446; R-squared = 89.7654% Model without chickens and camels - Estimated parameters: a = 0.179502; b = 0.763817; Analysis of variance (model): P-value = 0.0000; Correlation coefficient = 0.968067; R-squared = 93.7153%

13

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 20 up to 8000 g/ton). Moreover, the majority of the available reports were published between 1970 and 1985, when in most cases unspecific and low sensitive (LOD > 100 µg/kg) microbiological methods were used for residue analysis. The majority of these reports, however, were not included in the monograph prepared at the thirty-eighth 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 = 30-34% of the dose) was estimated only in pigs and chickens, respectively (van Leeuwen, 1991; Kowalski et al., 2002). Ruminants Edible tissues. - In newborn calves that received tylosin tartrate orally with a milk replacer at a dose of 22.2-27.8 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: 7500, 5500, 1600, 100, 200, 100 and < 100 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 10 mg/kg b.w., the highest tylosin concentrations measured by an agar-gel diffusion method 26 hours after the last dose were found in kidney (about 3300 µg/kg), while the lowest concentrations (< 500 µg/kg) were found in muscle and cerebrospinal fluid (Burrows et al., 1986). In older calves (weighing approximately 240 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 100 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 28-35 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 (290 µg/kg), although in other tissues the residues were below the limit of detection (JECFA, 1991a).

14

Table 4. Summary of tylosin residues (µg/kg) in calves (weighing approximately 240 kg) after receiving multiple intramuscular injections of tylosin (JECFA, 1991a).

Withdrawal time

(days) Residue concentration (µg/kg)

Study 1 – Microbiological assay*- dose: 8.8 µg/kg twice daily for 5 days Injection site – muscle Kidney Liver

0 1,800,000 44,100 7700 3 - - - 7 11,000 6000 310

10 6400 2900 200 14 4700 1700 160 21 1700 1800 < 100 28 600 180 < 100 35 190 100 - 42 ND ND - 49 ND - -

Study 2 – Microbiological assay*- dose: 17.6 µg/kg once daily for 5 days 0 7,633,000 17,400 10,000 3 - - - 7 - - -

10 - - - 14 4300 330 160 21 990 110 < 100 28 570 < 100 < 100 35 110 ND < 100 42 110 ND < 100 49 < 100 - -

Study 3 – HPLC assay**- dose: 10 µg/kg once daily for 5 days Inj. site muscle Muscle Kidney Liver Fat

0 1,337,000 470 7790 1960 230 3 323,000 280 460 170 ND 7 2750 ND 70 < 50 ND

10 - - - - - 14 1430 ND < 50 ND - 21 290 - ND - -

Note: * - detection limit of the microbiological assay was 100 µg/kg; ** - detection limit of the HPLC assay was 20 µg/kg; ND - not detected According to the information available in the CVMP Summary Report 3 (EMEA, 1997), following three daily intramuscular injections of 14C-tylosin 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 25210, 47810, 2870 and 1520 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, 705 and 940 µg/kg in liver, kidney, muscle and fat, respectively, corresponding to 10.5%, 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 70% 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 6.8-7.3 mg/kg b.w. Tylosin concentrations measured in renal cortex were: 8000 µg/kg (at 7 hours), 11500 µg/kg (at 24 hours) and 11600 µg/kg (at 31 hours). In

15

liver, corresponding concentrations of tylosin were lower than in kidney and amounted to: 2900, 4800 and 2600 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 0, 48, 72, 84 and 96 hours after the last injection were: 750, 350, 140, 80 and 50 µg/kg. At 108-144 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 10 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 200-630 µg/kg at the morning milking and 90-330 µg/kg at the afternoon milking. Three days after the end of the treatment, residues were found in one sample at the morning milking (60 µg/kg) and were below the limit of quantification (50 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 300-930 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 38-150 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 30 µg/kg five days after the last treatment with tylosin base at 10 mg/kg b.w. (Dudriková and Lehotský, 1998). When tylosin was intramuscularly injected at the same dose in ewes (10 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., 2001) – see Table 5. Table 5. Tylosin residues in milk (Dudriková and Lehotský, 1998; Nagy et al., 2001).

Residue concentration (µg/l) measured by an HPLC assay* Time of experiment (hours) Cows

10 mg/kg b.w. once daily for 5 days Ewes

10 mg/kg b.w. once daily for 5 days 0◄ - - 12 2220 632

24◄ 1080 128 36 870 1822

48◄ 690 471 60 1560 1647

72◄ 1790 263 84 3760 1046

96◄ 1650 161 108 1190 900

120(1) 1210 142 132 1010 31

144(2) 290 - 156 280 -

168(3 160 - 180 50 -

192(4 30 - 204 100 -

216(5) 30 - 228 30 -

240(6) - - Note: * - detection limit 10 µg/l; ◄- time of tylosin injection; (1-6)- days after the last injection

16

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 100, 200, 500 and 1000 grams of tylosin/ton for 14-105 days. It was demonstrated that a residue level of 200 µg/kg would not be exceeded in edible tissues when feeding tylosin at 100 g/ton feed, the recommended dose for pigs. Tylosin residues were detected in livers of two pigs (550 and 560 µg/kg) receiving tylosin at a dose of 1000 g/ton of feed (about 30 mg/kg b.w.) at zero hour withdrawal. However, no residues above the tolerance limit adopted in the USA (TL = 200 µg/kg; Code of Federal Regulation, 2005) were detected in tissues when tylosin was fed at a dose of 100, 200 and 500 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 0.025% and 0.05% for five days, doses equivalent to 121.6 mg/kg b.w. and 243.7 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: 600 µg/kg in bone marrow at day zero after the withdrawal and 420 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 40 and 100 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 200 and 400 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

90 kg b.w. and withdrawal times below three hours (Lauridsen et al., 1988).

Residue concentration (µg/kg) measured by a microbiological assay*

Pig No. Dose: g/ton of feed for 17 days

Kidney Liver Muscle 1-4 5-8 9

10 11 12 13 14 15 16

0 100 200 200 200 200 400 400 400 400

ND ND 50 30

< 30 < 30 30 40 50 30

ND ND 30 30 30 40 40 60 60 50

ND ND ND ND ND ND ND ND ND ND

* - detection limit of the microbiological assay was 30 µg/kg; ND - not detected

Similar data concerning tylosin residues in feeding experiments were reported by the CVMP and JECFA. After administration of 14C-tylosin in three male pigs at a dietary dose of 220 mg/kg for five days, mean residues of tylosin measured four hours after the slaughter were: 450, 460, 50 and 70 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 (50 µg/kg) in all samples. Low residues of tylosin A in liver and kidney (around 30 µg/kg) were occasionally found in pigs slaughtered six hours after tylosin administration in the feed (200 mg/kg) for 28 days or the drinking water (250 mg/l) for 10 days (EMEA, 1997). When feed containing 200 g/ton of tylosin was fed to pigs for 17 days, tylosin concentrations in both liver and kidney were 30 µg/kg when measured by a microbiological assay. The highest residue concentrations observed in pig tissues was 50 µg/kg in liver after administration of 400 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 80-110 kg treated with

17

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 < 100 µg/kg were below 200 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

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).

Tissue residue concentration (µg/kg) measured by an HPLC* or bioassay** Injection site

(Muscle) Sample 1a

Injection site (Muscle) Sample 2a

Contralateral Muscle Sample 1a

Contralateral MuscleSample 2a

Kidneyb LiverbAnimal

No. Withdrawal

time (hours)

HPLC Bioassay HPLC Bioassay HPLC Bioassay HPLC Bioassay HPLC Bioassay HPLC Bioassay204 205 206

207 208 209

210 211 212

4 4 4

24 24 24

48 48 48

92,000 27,000 58,000

270 ND

7300

ND ND ND

24,000 17,000 32,000

ND ND

7000

ND ND ND

58,000 46,000 52,000

50 90

8100 - - -

26,000 9000

32,000

ND ND

5000 - - -

1500 1300 1300

ND ND ND

- - -

ND 920 ND

ND ND ND

- - -

1300 650 560

ND ND ND

- - -

ND 680 ND

ND ND ND

- - -

530 530 590

ND ND ND

- - -

ND ND ND

ND ND ND

- - -

350 470 280

ND ND ND

- - -

ND ND ND

ND ND ND

- - -

* - detection limit of the HPLC assay was < 100 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 500 µg/kg; a- samples stored at minus 20oC for unknown time; b - samples stored at minus 20oC up to 2 months; ND - not detected

19

In storage stability studies, appreciable concentrations of tylosin (920-3130 µg/kg) determined by the bioassay procedure were observed in kidney samples (collected at 4 hours withdrawal) after 45 days of storage at minus 20oC. Residues were undetectable after 75 days of storage at minus 20oC. Moreover, detectable residues (1600 µg/kg) were found in liver in only one pig (collected at 4 hours withdrawal) after seven days of storage and were undetectable after 60 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 10 mg/kg b.w., tylosin concentrations at the injection site declined slowly from 6380 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 (20 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 (2002b). Following intramuscular injections of tylosin base at a dose of 10 mg/kg b.w., once daily for five days, the highest concentration of tylosin residues were found at the injection site three (110-2540 µg/kg) and seven days (100-4100 µg/kg) after the withdrawal, respectively. Residues at the injection site depleted below the limit of quantitation (50 µg/kg) of the HPLC assay at ten and fourteen days after the last dose (Prats et al., 2002b). 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 10 mg/kg b.w. for five days (Prats et al., 2002b).

Residue concentration (µg/kg) measured by an HPLC assay* Animals Withdrawal (days) Inj. site

(Muscle) Muscle Skin + fat Kidney Liver

group 1

group 2

group 3

group 4

3 3 3 3

7 7 7 7

10 10 10 10

14 14 14 14

440 110

1260 2540

120 310 100

4100

< 50 < 50 < 50 < 50

< 50 < 50 < 50 < 50

50 60

< 50 < 50

< 50 100 < 50 < 50

< 50 < 50 < 50 < 50

< 50 < 50 < 50 < 50

84 101 66 78

460 < 50 56

< 50

< 50 < 50 < 50 < 50

< 50 < 50 < 50 < 50

120 < 50 < 50 < 50

< 50 110 70

< 50

< 50 < 50 < 50 < 50

< 50 < 50 < 50 < 50

70 80

< 50 80

< 50 < 50 < 50 < 50

< 50 < 50 < 50 < 50

< 50 < 50 < 50 < 50

* quantification limit of the HPLC method was 50 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 2740000, 920, 870 and 120 µg/kg at 0, 7, 14 and 21 days after withdrawal,

20

respectively. Tylosin residues at the injection site depleted to below the quantitation limit of 100 µg/kg by 28 days following the last injection (JECFA, 1991a). Poultry Edible tissues. - Based on limited data on tylosin residues in poultry reviewed by JECFA in 1991, it was concluded that no tylosin residues were found in chicken tissues 24 hours after treatment withdrawal. When tylosin was administered in drinking water (500 mg/l) for eight days or in feed (1000 g/ton) for seven days, the highest residues of tylosin observed at zero withdrawal time were 1030 µg/kg in the liver and 432 µg/kg in the kidney. In turkeys that received tylosin in drinking water (5 g/gallon ≈ 1.319 g/l) for seven days, tylosin residues at zero withdrawal time were similar to those in chickens, 385 µg/kg in the liver and 240 µg/kg in fat (JECFA, 1991a). Low residues of tylosin in chicken tissues were found in other dietary experiments. When growing chickens received tylosin phosphate in feed at a dose of 2000-8000 g/ton for eight weeks, residues of tylosin were found only in liver (180-270 µg/kg) at 24 hours withdrawal (Yoshida et al., 1973a). In laying hens, however, after administration of tylosin phosphate in feed at a dose of 8000 g/ton for seven days, tylosin residues in liver were higher: 900-35600, 90-1000 and 460 µg/kg at zero, two and four days after withdrawal, respectively (Yoshida et al., 1973b). In laying hens, after administration of tylosin tartrate in drinking water at a dose of 1000 mg/l (twice the recommended dose) for five days, the mean residues of tylosin in breast muscles were: 27, 8 and 9 µg/kg at 1, 2 and 3 days after withdrawal, respectively. In thigh muscles, however, much higher tylosin residues were found: 114, 18 and 8 µg/kg, respectively (Nagy et al., 2000). After administration of tylosin tartrate in drinking water at a dose of 500 mg/l for three days in turkeys, residues of tylosin measured by an HPLC assay were below the detection limit of 50 µg/kg in all tissues at zero withdrawal (Montesissa et al., 1999). Eggs. - Residues of tylosin in eggs and their distribution between yolk and white have been studied by Kan and Petz (2000). Residues in egg white are reflected by the plasma concentrations. The time needed to achieve a constant concentration in egg white was 2-3 days. Drug residues in yolk reflect the plasma concentrations during the 10 days of its rapid growth. Thus, depending on the length and timing of the exposure relative to yolk growth, residue concentrations in yolk can increase, remain constant or decrease. In general, drug residues in yolk require exposure for about 8-10 days to reach a constant concentration and depletion from yolk generally takes about 10 days (Kan and Petz, 2000). In laying hens that received tylosin at a dose of 500 g/ton of feed for 14 days, tylosin concentrations in whole eggs, measured every 2 days, reached equilibrium (40-60 µg/kg) four days after the start of treatment up to 14 days. Tylosin transfer rate from the diet to eggs (percentage of the intake of the drug to the content of drug in the eggs) was only 0.005% (Furusawa, 2001). Similar transfer rates in whole eggs were reported in laying hens received tylosin tartrate in drinking water at a dose of 500 mg/l for five days (0.007%) or tylosin phosphate in feed at a dose of 400 g/ton for seven days (0.009%) (Roudaut and Moretain, 1990). In a dietary study of Yoshida and coworkers (1973b), after administration of tylosin phosphate in laying hens at a dose of 8000 g/ton in feed for seven days, tylosin residues in eggs were measured by a microbiological assay. One day after the last dose, the highest residues of tylosin amounted to 1350-6750 µg/kg in egg white and 1250-8500 µg/kg in egg yolk. Moreover, differences in residue distribution between egg white and yolk were observed up to six days after the last dose of tylosin (Table 9). It was also observed that the tylosin residues in yolk samples taken 2-6 days after the withdrawal were higher than the residues found in the liver (Yoshida et al., 1973b). The residue concentrations in eggs are summarized in Table 9.

21

Table 9. Tylosin residues in eggs laid by hens fed a diet containing 8000 g/ton of tylosin phosphate for 7 days (Yoshida et al., 1973b).

Residue concentration (µg/kg) measured by a microbiological assay*

Days on tylosin feeding Days after withdrawal Hen No. and Egg

part 3 7 1 2 3 4 5 6 7 Egg white:

2037 2084 2085 2116 2122

Egg yolk:

2037 2084 2085 2116 2122

4490 1500 3200 1000 3900

900 0

1200 5250 1750

7000 1150 3200 9750 4700

6000 950

3800 10,000 5000

1350 3400 6750 2950 5200

1250 4200 8500 5750 4500

- 0

790 1600

0 -

780 3100 7750 4000

0 0 0 0 0

4700 800

2800 5500 2580

0 0 0 0 -

3200 0

1900 5250

-

0 0 - 0 0

2000 1230

- 1800 1200

0 0 - 0 0

680 0 -

780 0

0 0 0 0 0

0 0 0 0 0

* threshold sensitivity of the assay method was 400 µg/kg Similar differences in residue distribution between egg white and yolk were observed in more recent in experiments comparing tylosin and other macrolide antibiotics (Roudaut and Moretain, 1990). After exposure of laying hens to tylosin tartrate in drinking water at a concentration of 500 mg/l for five days, only one hen excreted detectable residues into albumen and yolk as measured by a microbiological assay. Peak concentrations were 660 and 1710 µg/kg, respectively. In hens receiving tylosin tartrate in drinking water at a dose of 1000 mg/l for five days, residues of tylosin above the detection limit of the bioassay (150 µg/kg) were seen in whole egg up to five days after withdrawal. However, tylosin residues in egg white were detected only during the first day after the withdrawal (Table 10). Similar results were obtained in other studies. When laying hens were supplied with drinking water containing 0.05% tylosin tartrate for 1-5 days, no measurable tylosin residues were detected by a microbiological assay in most of eggs after withdrawal, except on the first day: 240 µg/ml and 1930 µg/ml and fourth day: 1120 µg/ml (Iritani et al., 1976). In a more recent study, where laying hens received tylosin tartrate in drinking water at a dose of 0.053%, no tylosin residues were observed in yolk samples during the seven days on treatment or the subsequent three days following cessation of treatment (McReynolds et al., 2000). According to the CVMP Summary Report 3, similar residues in eggs to those reported by Roudaut and Moretain (1990) were observed in radiometric study in laying hens supplied with drinking water containing 529 mg/l of 14C-tylosin for three consecutive days (EMEA, 2000). Peak residues in albumen were found in samples taken on the last day of treatment (mean = 423 µg equivalents of tylosin/kg) and in samples taken on the following day (mean = 415 µg equivalents of tylosin/kg). The mean total residues in albumen one and two days after the end of treatment were 157 and 43 µg equivalents of tylosin/kg, respectively, and were below the limit of detection (20 µg equivalents of tylosin/kg) in most of the eggs collected at later time points. Peak residues in yolk were found in samples taken two and three days after the end of treatment (mean values 334 and 344 µg equivalents of tylosin/kg, respectively) and declined to 192 and 74 µg equivalents of tylosin/kg at four and five days after the withdrawal, respectively. Calculated peak residues in whole eggs were found in samples taken on the last day of treatment (mean 333 µg equivalents of tylosin/kg) and in samples taken on the following day (mean 362 µg equivalents of tylosin/kg). Thereafter, the calculated mean residues in whole eggs depleted to 190, 134, 126 and 74 µg equivalents of tylosin/kg at 1, 2, 3 and 4 days after the withdrawal of treatment. When the same samples were analyzed by an HPLC method combined with electrospray ionization quadrupole mass spectrometry, residues of tylosin A and several active

22

metabolites were identified. Tylosin A was the most abundant residue present and accounted for 17% of the total radioactive residues in whole eggs. Metabolites present in lower concentrations included N-desmethyl-tylosin A, tylosin D, N-desmethyl-dihydro-tylosin A and O-desmethyl-tylosin A (EMEA, 2000). When laying hens received drinking water containing a tylosin formulation at a dose of 500 mg activity per litre of water, residues of tylosin A in eggs were below the quantification limit of the HPLC assay (50 µg/kg) one and two days after the withdrawal of treatment. Tylosin residues of 50 µg/kg were found in 2 eggs taken three days after the end of treatment, but no quantifiable residues were detected in eggs taken at later time points (EMEA, 2000). Table 10. Tylosin residues in egg white, yolk and whole eggs (µg/kg) measured by a

microbiological assay in hens administered tylosin tartrate at a dose of 1000 mg/l in the drinking water for 5 days (Roudaut and Moretain, 1990).

Days on feed Albumen Yolk Whole egg

1 < 150 (ND-750) ND - (520) 2 190 (ND-3060) < 200 (ND-510) 160 (0-890) 3 210 (ND-980) < 200 (ND-1320) 200 ((0-1090) 4 250 (ND-1340) 250 (ND-1800) 250 (0-1480) 5 220 (ND-870) 400 (ND-2480) 260 (0-1350)

Days after withdrawal 1 230 (ND-830) 650 (ND-2990) 370 (0-1460) 2 ND 470 (ND-2390) 140 (0-710) 3 - 390 (ND-2000) 130 (0-630) 4 - < 200 (ND-1280) 80 (0-423) 5 - < 200 (ND-660) - (190) 6 - < 200 (ND-230) - 7 - ND -

Detection limit of the microbiological assay was 150 µg/kg in albumen and 200 µg/kg in yolks; ND - not detected; ( ) - analytical range

ANALYTICAL METHODS A validated analytical method for the quantitation of the marker residue in target animal tissues is necessary for enforcement of MRLs and is required as part of the information for the evaluation of veterinary drug residues (JECFA, 2000). Many different analytical methods (screening or confirmatory) have been described for tylosin and other macrolide antibiotics. They include tylosin and/or its degradants/metabolites, detection in water solutions and fermentation media (Fish and Carr, 1986; Roets et al., 1993; Paesen et al., 1995b; van der Heyden et al., 1999), animal feeds (Houglum and Tasler, 1996; Markakis, 1996; van Poucke et al., 2003; van Poucke et al., 2004), environmental samples (Kumar et al., 2004) and excreta (Loke et al., 2000; Ingerslev and Halling-Sørensen, 2001; Teeter and Meyerhoff, 2003; González de la Huebra et al., 2005; Kolz et al., 2005). A number of methods for the detection of tylosin or other macrolides in biological fluids and animal tissues have been published (Moats, 1985; Kanfer et al., 1998; Botsoglou and Fletouris, 2001; González de la Huebra and Vincent, 2005). Microbiological assays are often used to screen samples for tylosin residues (Okerman et al., 1998; Tsai and Kondo, 2001; Caldwell et al., 2000; Althaus et al., 2003), although they lack specificity and are unable to identify the exact nature of the antibiotic residue. More specific methods, such as liquid chromatography coupled with ultraviolet (LC-UV) detection, have been proposed for tylosin determination in animal tissues (Table 11). Gas chromatography coupled to mass spectrometry (GC-MS) has also been described as a confirmatory method for tylosin residue analysis (Takatsuki et al., 1987). Several other methods based on a

23

combination of liquid chromatography with mass spectrometry (HPLC-MS) and tandem mass spectrometry (HPLC-MS-MS) have been reported for quantitation and confirmation of tylosin residues in animal tissues (Table 12). Some of the described HPLC-MS or HPLC-MS-MS methods are multiresidue methods, which allow for the simultaneous detection of several macrolide antibiotics or even other chemotherapeutic drugs (e.g., screening of macrolides and ionophores). These methods are preferred for regulatory confirmation methods. Moreover, these methods combine separation of analytes with structural information. Table 11. Overview of HPLC methods for residues of tylosin in foods of animal origin.

Method of detection

Matrix Detected compounds

LOD a,b

(µg/kg) Validation c Reference

HPLC-UV Bovine: muscle kidney liver

Tylosin 15 Yes Keng and Boison, 1992

HPLC-UV Bovine: muscle kidney Pig: muscle kidney

Tylosin

Tilmicosin

20 Not specified

Chan et al., 1994

HPLC-UV, -PDA

Bovine: milk

Tylosin 12.5-15 d Not specified

Usleber et al., 1997

HPLC-UV Pig: muscle kidney liver fat

Tylosin 5-16 Yes De Liguoro et al., 1998

HPLC-UV Bovine: muscle liver Pig: muscle kidney liver Chicken: muscle liver

Tylosin

Spiramycin Neo-

spiramycin Josamycin

Kitasamycin

50 Not specified

Horie et al., 1998

HPLC-UV Bovine: Pig: Poultry: muscle

Tylosin

Spiramycin Neo-

spiramycin Tilmicosin

15 Yes Juhel-Gaugarin et al., 1999

HPLC-UV Bovine: milk

Tylosin

< 50 d Not specified

Dudriková et al., 1999

24

Table 11. continued HPLC-UV

Bovine: Pig: Poultry: muscle kidney liver fat

Tylosin 50 e Yes Prats et al., 2001

HPLC-UV, -PDA

Chicken: muscle

Tylosin

Spiramycin Tilmicosin Josamycin

Kitasamycin

30 d Not specified

Leal et al., 2001

a - limit of detection (LOD) b - for multiresidue methods only a value for tylosin was specified c - declared by authors for the time of publication d - µg/l e - limit of quantification (LOQ)

Table 12. Overview of HPLC-MS and HPLC-MS-MS methods for residues of tylosin in foods of

animal origin.

Method of detection Matrix Detected compounds

LOD a,b

(µg/kg) Validation c Reference

PB/HPLC-MS Bovine: muscle

Tylosin 10 Not specified

Delépine et al., 1994

PB/HPLC-MS Bovine: muscle

Tylosin Spiramycin Tilmicosin Josamycin

Erythromycin

50 Not specified

Delépine, et al., 1996

MRM HPLC-ESI-MS-MS

Bovine: Pig: Hen: muscle kidney liver Bovine: milk Hen: eggs

Tylosin

Spiramycin Tilmicosin Josamycin

Erythromycin

Not specified

for tylosin0.01-37 for all

antibiotics

Yes Dubois et al., 2001

SRM micro-LC-API-MS-MS

Bovine: muscle kidney liver

Tylosin

Tilmicosin Erythromycin

30-40 e Yes Draisci, et al., 2001a

HPLC-ESI-MS-MS Pig: muscle kidney liver skin + fat

Tylosin A 0.2-0.8 5 e

Yes Cherlet et al., 2002

25

Table 12. continued HPLC-ESI-MS Chicken:

muscle Tylosin

Spiramycin Tilmicosin Josamycin

Kitasamycin Erythromycin Oleandomycin

1 d Not specified

Codony, et al., 2002

HPLC-ESI-MS-MS Screening method

Hen: eggs

Tylosin Erythromycin Novobiocin + 4 Ionophores

~1 d Yes (only for

screening)

Heller and Nochetto, 2004

MRM HPLC-ESI-MS-MS

Hen: eggs

Tylosin Spiramycin Tilmicosin

Erythromycin Oleandomycin

0.1 Yes Wang et al., 2005

PB - particle beam interface; MRM - multiple reaction monitoring ESI - electrospray ionisation SRM - selected reaction monitoring API - atmospheric pressure ionisation

a - limit of detection (LOD) b - for multiresidue methods only a value for tylosin was specified c - declared by authors for the time of publication d - µg/l e - limit of quantification (LOQ)

Other analytical methods (screening or confirmatory) e.g., electrochemical enzyme-linked immunosorbent assay (ELISA), high-voltage electrophoresis and thin-layer chromatography have also been described for tylosin and other macrolide residues (Petz et al., 1987; Krčmář and Růžičková, 1996; Draisci et al., 2001b; Ammida et al., 2004). Although many of these methods are able to detect tylosin at concentrations below 15 µg/kg (some HPLC methods) or even below 1 µg/kg (some HPLC-MS methods), almost none of them is specific for tylosin A, which is the marker residue within the European Union (Council Regulation, 1990). Currently, the only published method in the open literature that could be taken into consideration as part of recommending MRLs for tylosin is the reversed phase liquid chromatography-electrospray ionisation-mass spectrometry/mass spectrometry (HPLC-ESI-MS/MS) method described by Cherlet and coworkers (2002). Some details of this method are provided below. Tylosin A is extracted from the tissues using liquid extraction with methanol, followed by a solid-phase clean-up procedure on an SCX (strong cation-exchange) column. Spiramycin was used as the internal standard. After drying of the eluate, the residue was redissolved in water and further analyzed by reversed phase liquid chromatography-electrospray ionisation-mass spectrometry/mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation of the internal standard spiramycin I and tylosin A was achieved on a Nucleosil (5 µm) column using a mixture of 0.01 M ammonium acetate pH 3.5 in water and acetonitrile as the mobile phase. Tylosin A is identified from MS/MS detection and subsequently quantified. The method was validated according to the requirements of the European Community at 50, 100 and 200 µg tylosin A/kg for muscle, skin + fat, kidney and liver. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit < 10%). Limits of quantification of 5.0 µg/kg were obtained for the determination of tylosin A in all tissues. Limits of detection ranged between 0.2 and 0.8 µg/kg for the various tissues. The within-day and between-day precision and the accuracy were within the ranges specified. The method has been successfully used for the quantitation of tylosin A in tissue samples of pigs medicated with tylosin via intramuscular injection.

26

SUMMARY AND CONCLUSIONS Tylosin was evaluated by JECFA at the twelfth meeting in 1968 (JECFA, 1969) and thirty-eighth meeting in 1991 (JECFA, 1991a, 1991b). The Committee was not able to recommend MRLs for tylosin because no ADI was established. This review was prepared from a comprehensive literature search in absence of new data requested for evaluation of tylosin at the 66th meeting of JECFA. Important data on chemical properties and composition of tylosin have been published since 1991. For example, from a study of McFarland and coworkers (1997) it appears that the dissociation constant of tylosin (pKa = 7.73) is much higher than that previously reported for almost 40 years - 7.1. As a consequence, many theoretical calculations presented in pharmacokinetic studies would have to be verified. Tylosin is a mixture of four macrolide antibiotics. The main component of the mixture is tylosin A (> 80%). Tylosin B (desmycosin), tylosin C (macrocin) and tylosin D (relomycin) may also be present. All four components contribute to the potency of tylosin, which should not be less than 900 IU/mg, calculated with reference to the dried substance (European Pharmacopoeia, 2004). Relative antimicrobial activities of tylosin derivatives are as follows: tylosin A – 1.0, tylosin B – 0.83, tylosin C – 0.75 and tylosin D – 0.35 (Teeter and Meyerhoff, 2003). Other pharmacologically active compounds, i.e., lactenocin, demecinosyl-tylosin (DMT) and O-mycaminosyl-tylonolide (OMT) have also been isolated from fermentation media or aqueous commercial samples containing tylosin (Paesen et al., 1995abc). Some studies provided new data on the stability of tylosin in aqueous solutions (Halling-Sørensen et al., 2003; Kolz et al., 2005). In acidic media, tylosin B is the major degradation product of tylosin A. In neutral and alkaline media, tylosin A is decomposed into different products. Apart from tylosin B, tylosin aldol (TAD1 and TAD2) and polar degradation products were detected. Moreover, when tylosin solution was exposed to daylight, a photodegradation product - isotylosin A (isoTA) was formed (Paesen et al., 1995abc). Many pharmacokinetic studies were carried out with tylosin in different animal species. However, the HPLC assay of tylosin published after 1990 was used only by Prats et al. (2002a). Tylosin is quickly absorbed from the alimentary tract in monogastric animals as well as in poultry. After oral administration, peak plasma concentrations of tylosin appear after 1-4 hours. However, oral bioavailability of tylosin is moderate (F = 22.5% in pigs and F = 30-34% in chickens). Moreover, in contrast to results obtained in pigs, results of residue studies in chickens suggest that absorption of tylosin phosphate from the alimentary tract appears to be lower than tylosin tartrate. After intramuscular injections, tylosin peak plasma concentrations are observed after 2-6 hours, with systemic bioavailability of 70-95% of the administered dose. Tylosin was well distributed into organs and tissues of the animal species tested. Results of pharmacokinetic studies show that calculated tissue:plasma ratios are above 2 and volumes of distribution (Vd) are greater than 1 l/kg (even up to 14.6 l/kg in pigs). The extensive tissue distribution of tylosin was confirmed with tylosin concentrations observed in lungs, liver, kidneys and bile being several times higher than the corresponding concentrations in plasma. Tylosin was shown to be rapidly eliminated from the blood plasma in various animal species and elimination half-lives (t1/2el.) after single intravenous administration of tylosin are short, 0.4-4.5 hours. The highest rate of tylosin elimination from the blood plasma is observed in rodents and camels (clearance > 70 ml/min/kg), while the lowest rate was observed in adult cattle, sheep and goats (clearance < 10 ml/min/kg). There is a statistically significant relationship between tylosin clearance and body weight of animals. The R-squared value indicates that the multiplicative model as shown in Figure 3 explains 84.4% of the variability in tylosin clearance after transforming to a logarithmic scale to linearize the model. Moreover, high correlation coefficients (0.92-0.97) indicate a relatively strong relationship between the variables. Thus, presented models can be used for estimation of tylosin

27

clearances in other animal species, although caution is needed when allometric scaling is applied between mammals and birds or physiologically different ruminants (e.g., cows and camels). The primary metabolism of tylosin occurs in liver. In pig liver at least four major metabolites of tylosin were isolated. Tylosin C and dihydrodesmycosin (DDM) represented 15% of the total liver residues recovered by thin-layer chromatography (JECFA, 1991a). Significant amounts of tylosin D (50-250 µg/kg) were found in the liver of turkeys (Montesissa et al., 1999). No other studies on tylosin metabolism in animals were identified. A CVMP Summary Report states that, in pigs, residues of tylosin D and DDM were also found in the kidney. Smaller amounts of tylosin D, DDM and cysteinyl tylosin A (which readily converts to tylosin A) were 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). Several tylosin metabolites were found in eggs. Metabolites present in eggs at lower concentrations than the parent compound include N-desmethyl-tylosin A, tylosin D, N-desmethyl-dihydro-tylosin A and O-desmethyl-tylosin A (EMEA, 2000). However, the studies referred to in the CVMP Summary Report were not available to review. In rats and pigs, the majority (99%) of the metabolic residue of tylosin was excreted in the faeces and only 1% in the urine. In rats the greatest part of the excreted residues was found to be tylosin D (10% of total 14C-residues), tylosin A (6% of total 14C-residues), and tylosin C and dihydrodesmycosin – DDM (4% of total 14C-residues). In one pig, the greatest part of the excreted residues was found to be tylosin D (33% of total 14C-residues), DDM (8% of total 14C-residues) and tylosin A - 6% of total 14C-residues (JECFA, 1991a). In a recent GLP-compliant study using 14C-tylosin in three pigs, about 94% of the excreted radioactivity was found in faeces and 6% in urine. About 43% of the total radioactivity found in pig faeces was identified as tylosin D and 44% as DDM (EMEA, 1997). After intramuscular injection of 14C-tylosin to calves, less than 50% of the administered dose was recovered from excreta. Tylosin A (29.8%), tylosin D (11.4%), tylosin C (25.2%) and demethyl-tylosin D (10.8%) were found in faecal extracts, while cysteinyl-tylosin A was the major component (70%) in urine (EMEA, 1997). Significant amounts of tylosin were also found in milk (Gingerich et al., 1977; El-Sayed et al., 1986; Atef et al., 1991). Tylosin has been evaluated by the Committee for Veterinary Medicinal Products (CVMP) of the European Agency for the Evaluation of Medicinal Products (EMEA, 1997; 2000; 2002), and the following MRLs have been adopted by the European Union: marker residue - tylosin A; MRL = 100 µg/kg of tylosin A for muscle, fat, fat + skin (swine and poultry), kidney, and liver for all food-producing animals; MRL = 50 µg/kg of tylosin A for milk for all species; MRL = 200 µg/kg of tylosin A for eggs for all poultry species (Commission Regulation (EC) No 1181/2002). In the United States tylosin tolerance limits established by the Food and Drug Administration (FDA) and published in Title 21 of the Code of Federal Regulations (CFR), Part 556 (21 CFR 556.740) are as follows: marker residue – tylosin; TL = 200 µg/kg of tylosin for muscle, fat, kidney and liver for cattle, swine, chickens and turkeys; TL = 50 µg/kg of tylosin for milk; TL = 200 µg/kg for tylosin for eggs (Code of Federal Regulation, 2005). Results of residue studies presented in the current review confirm all previous conclusions of JECFA (JECFA, 1991b). However, some new information allow for certain additional conclusions. Most of them are based on CVMP Summary Reports. Because no new information was provided to the 66th

JECFA, no general conclusions concerning recommendations on MRLs for tylosin can be presented. Following three daily intramuscular injections of 14C-tylosin 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 25210, 47810, 2870 and 1520 µg of tylosin equivalents/kg in liver, kidney, muscle and fat, respectively. When the same

28

samples were analyzed by an HPLC method, the mean residues of tylosin A were: 2635, 6945, 705 and 940 µg/kg in liver, kidney, muscle and fat, respectively, corresponding to 10.5%, 14.5%, 24.6% and 61.8% of the total residues in these tissues. Based on results of microbiological assay of these samples, tylosin A represented 36.7%, 31% and 70% of the microbiologically active residues in kidney, liver and muscles, respectively (EMEA, 1997). Although tylosin is extensively metabolized, no single metabolite appears to be present in a concentration greater than the parent compound. Moreover, quantitative differences in the amounts of metabolites produced were observed between animal species. The parent compound, i.e., tylosin A, was considered the most suitable marker residue (EMEA, 1997). Based on the above, most of the tylosin-related residues detected in bovine tissues represent pharmacologically active metabolites (most probably tylosin D and DDM). Tylosin A, represents 10-15% of the total radiolabeled residues in liver and kidney and about 25% in muscle, and approximately 30% of the microbiologically active residues in liver and kidney and 70% in muscle. In a residue study in cows that received intramuscular injections of tylosin at a dose of 10 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 ranged from 200-630 µg/kg at the morning milking and 90-330 µg/kg at the afternoon milking. Three days after the end of the treatment, residues were found in one sample at the morning milking (60 µg/kg) and were below the limit of quantification of the HPLC assay (50 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 ranged from 300-930 µg/kg at the morning milking on first day. At the morning milking of the second day after the last dose, tylosin milk residues declined to 38-150 µg/kg (EMEA, 1997). In another residue study in cows, tylosin residues in milk measured by an HPLC assay were in the same concentration range and declined slowly to 30 µg/kg five days after the last treatment (Dudriková and Lehotský, 1998). Concentrations of tylosin A above 50 µg/kg were detected in bovine milk for the first 3-5 days after withdrawal. Because the analysis was conducted using an HPLC assay, no information on microbiologically active residues in milk is available. After administration of 14C-tylosin in three male pigs at a dietary dose of 220 mg/kg for five days, measured four hours after the slaughter, mean total residues of tylosin were 450, 460, 50 and 70 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 (50 µg/kg) in all samples (EMEA, 1997). In a CVMP Summary Report it is stated that following administration of 14C-tylosin to three pigs in the feed at a dose of 220 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 (10.3% 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 (EMEA, 1997). These results indicate that in pig tissues most of the tylosin-related residues represent other than tylosin A microbiologically active compounds (tylosin D and DDM). Tylosin A represents approximately 10% of the total radiolabeled residues in liver and kidney. This finding is similar to that seen in cattle. After administration of tylosin tartrate in drinking water to turkeys at a dose of 500 mg/l for three days, residues of tylosin measured by an HPLC assay were below the detection limit of 50 µg/kg in all tissues at zero withdrawal. Moreover, significant amounts of tylosin D (50-250 µg/kg) measured by an HPLC-MS-MS were found in the liver of turkeys at zero withdrawal (Montesissa et al., 1999). Tylosin A (17% of radiolabeled residues) and several tylosin metabolites were found in eggs. Metabolites present in eggs at lower concentrations than the parent compound included N-desmethyl-tylosin A, tylosin D, N-desmethyl-dihydro-tylosin A and O-desmethyl-tylosin A (EMEA, 2000). In laying hens receiving tylosin tartrate in drinking water at a dose of 1000 mg/l for five days, residues of tylosin above the detection limit of the bioassay (150 µg/kg) were seen in whole egg up to five days after withdrawal. Tylosin residues in egg white were detected only during the first day after withdrawal.

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The CVMP Summary Report indicates similar residue profiles in eggs as those reported by Roudaut and Moretain (1990) in a radiometric study in laying hens supplied with drinking water containing 529 mg/l of 14C-tylosin for three consecutive days. The mean total residues in albumen one and two days after the end of treatment were 157 and 43 µg equivalents of tylosin/kg, respectively, and were below the limit of detection (20 µg equivalents of tylosin/kg) in most of eggs collected at later time points. Peak residues in yolk were found in samples taken two and three days after the end of treatment (mean values 334 and 344 µg equivalents of tylosin/kg, respectively) and declined to 192 and 74 µg equivalents of tylosin/kg at four and five days after the withdrawal, respectively (EMEA, 2000). When laying hens received drinking water containing a tylosin formulation at a dose of 500 mg activity per litre of water, residues of tylosin A in eggs were below the quantification limit of the HPLC assay (50 µg/kg) one and two days after the withdrawal of treatment. Tylosin residues of 50 µg/kg were found in 2 eggs taken three days after the end of treatment, but no quantifiable residues were detected in eggs taken at later time points (EMEA, 2000). Although tylosin D is also detected in the liver of turkeys and in eggs, results of the above studies suggest that different pathways for tylosin metabolism exist in birds and mammals. Such a suggestion can also be justified (in part) based on clearance allometric scaling for chickens - see Figure 3. These results indicate that microbiologically active tylosin residues above 50 µg/kg persist in eggs for up to 5-6 days after treatment. Tylosin A, however, can be detected for only 2-3 days. Many different analytical methods have been described for tylosin detection since 1991. They include tylosin (or tylosin and its degradants/metabolites) detection in water solutions and fermentation media, animal feeds, environmental samples and excreta. A number of HPLC and HPLC-MS methods for the detection of tylosin in biological fluids and animal tissues have also been described. Although many of these methods are able to detect tylosin at concentrations below 15 µg/kg (some HPLC methods) or even below 1 µg/kg (some HPLC-MS methods), few of them are specific for tylosin A. Moreover, only some of these methods were validated. Currently, the only method published in the open literature, which could be taken into consideration as part of recommending MRLs for tylosin, is the HPLC-ESI-MS-MS method for the detection of tylosin A residues in pig tissues described by Cherlet and coworkers (2002). It is interesting that only the methods described by Prats et al. (2001) and De Liguoro et al. (1998) were used in published studies concerning residues of tylosin in animal tissues (Montesissa et al., 1999; Furusawa, 2001; Prats et al., 2002b). Based on results presented in this review, it can be concluded that there are certain data gaps precluding an evaluation of the tylosin residue profile. For a complete evaluation to be performed by JECFA it is recommended that the following information should be submitted together with previously requested information. 1. Additional information on tylosin stability in aqueous solutions and biological samples after

storage.

2. Additional information on microbiological activity of metabolites and degradation products of tylosin.

3. Studies on analytical methods for tylosin residue detection in edible tissues, milk and eggs.

4. Studies on the contribution of the major metabolites of tylosin to the total residues in edible tissues of poultry and in eggs and milk.

Acknowledgement The author acknowledges the technical review and advice from Dr. Lynn G. Friedlander, Division of Human Food Safety, Office of New Animal Drug Evaluation, Center for Veterinary Medicine, Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855, USA (E-mail: lynn.friedlander@fda.hhs.gov ) in the preparation of this manuscript.

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