V. DISCUSSION. in food animals and poultry. Quinoline compounds are one among the several

Transcription

1 Discussion

2 V. DISCUSSION Use of antimicrobial substances is one of the age old practices to augment growth in food animals and poultry. Quinoline compounds are one among the several antimicrobial agents employed in poultry farming as feed additive. Quinoline derivatives were introduced way back in 1960s for the treatment of amoebic dysentery in man. Historically, an oxyquinoline derivative, iodochlorhydroxyquinoline was banned since its prolonged usage caused a neurological syndrome described as sub-acute myelo-optic neuropathy in Japanese people. Alternatively halquinol, a related drug having antibacterial, antifungal and antiprotozoal activity (Botsoglou and Fletouris, 2001) was introduced across the world to overcome common challenges of modern poultry and swine farming, like pathogenic microbial infections and growth promotion aspects. In Asian countries including India, halquinol is being extensively used as growth promoter in poultry and to control intestinal infections in ruminants. However, European union (EU) and several other western countries have restricted the use of halquinol in food producing animals and in poultry on account of insufficient or conflicting toxicological data, thus protecting public health. Further, from the year 1900 onwards the committee for veterinary medicinal products (CVMP) while reviewing the maximum residual limit (MRL) of some of the older substances found their way to annexure-iv of regulation (EEC) No 2377/90 (i.e: substances withdrawn from the EU MRL procedure or substances which the CVMP could not make a recommendation) and halquinol is one among them (Woodward, 2009).

3 81 As there is paucity of studies regarding tissue distribution and residues of halquinol following dietary exposure in broiler chickens when used as growth promoter, the pre-slaughter withdrawal time in poultry is not being specified, posing public health hazard. Therefore the present study was undertaken with the objective to determine tissue distribution of halquinol following dietary inclusion and to assess tissue depletion profile following its withdrawal in order to establish pre-slaughter withdrawal time. Halquinol is a mixture obtained by chlorinating quinolin-8-ol. It contains not less than 57 per cent and not more than 74 per cent of 5,7-dichloriquinilin-8-ol, not less than 23 per cent and not more than 40 per cent of 5-choloroquinolin-8-ol and not more than 4 per cent of 7-chloroquinolin-8-ol and the total content of the three components is not less than 95 per cent and not more than 105 per cent (Anon., 1980). The oxyquinoline derivative halquinol-halquinol BP 80, composed of % of 5,7- dichloroquinolin-8-ol, % of 5-chloroquinolin-8-ol and 0 % of 7-chloroquinolin-8- ol, the total content of the two components being 98 % w/w, manufactured and supplied by M/s. Provimi Animal Nutrition India Pvt. Ltd., Bangalore was used in the present experimental study. Halquinol was incorporated in the feed of broiler chickens at different levels viz: 60, 120 and 240 ppm for a period of 40 days and few birds (n=6 each) in each treatment group at regular intervals were sacrificed to determine tissue concentration of 5,7- dichloroquinolin-8-ol and 5-chloroquinolin-8-ol, the constituent of molecules of halquinol. The remaining birds were serially sacrificed (n = 6 at a time) at regular intervals from all groups following withdrawal of experimental exposure to halquinol and

4 82 vital edible tissues viz: liver, kidney and breast muscles were collected and subjected to high performance liquid chromatography (HPLC). The detection and quantification of 5,7-dichloroquinolin-8-ol and 5- chloroquinolin-8-ol in tissues was carried out by using HPLC fitted with photo diode array detector set at a wavelength of 247 nm. Reference standards of 5,7- dichloroquinolin-8-ol and 5-chloroquinolin-8-ol from Sigma were used for standardization, recovery and assay precision studies. The tissue clean-up and analytical procedures to quantify 5,7-dichloroquinolin-8-ol and 5-chloroquinolin-8-ol, the constituent molecules of halquinol with permissible limits of repeatability, precision and sensitivity were standardized in the present study. However the plasma or tissue concentration of 5-chloroquinolin-8-ol was less the detection limit of the assay system (20 ng/ml plasma [or] g tissue). Therefore, the interpretations of halquinol residues made in the present study were limited to 5,7-dichloroquinolin-8-ol only. 5.1 Tissue distribution of 5,7-dichloroquinolin-8-ol In the present study, 5,7-dichloroquinolin-8-ol was detected in the liver and kidney samples belonging to all the three treatment groups throughout the experimental period, while its residue concentration was not found in breast muscles except in those belonging to 240 ppm group. Similarly, the plasma levels of 5,7-dichloroquinolin-8-ol were below the limit of sensitivity of the developed assay system, except in four samples (out of six) belonging to 240 ppm group. Thus, the absorption and bioavailability of halquinol is poor in the present study. The bioavailability of halquinol was reported to be 5-10 per cent (Botsoglou and Fletouris, 2001). There exist a wide variation in lipid

5 83 solubility among 8-hydroxyquinoline derivatives (Musiol et al., 2010 ). The pka value for 5,7-dichloroquinolin-8-ol is 1.81 and 7.62 and the log p value of 5,7- dichloroquinolin-8-ol is Further, poor bioavailability of halquinol in the present study may be due to its glucuronide conjugation in the intestine itself as in the case of closely related compound clioquinol (Kotaki et al., 1983). The 8-hydroxyquinolines are known to form chelation with several metal ions, especially with iron to a significant amount (Beltran et al., 1985). The poor bioavailability observed in the present study may be due to combined effect of poor solubility and chelation and glucuronide metabolism with in the gastrointestinal tract. In the present study, the significant increase (p 0.05) in concentrations of 5,7- dichloroquinolin-8-ol in liver and kidney on each successive sampling days of 5, 10, 20, 30 and 40 in the three treatment groups were dose dependent (Table 1). On day 40 of halquinol exposure the tissue concentration of 5,7-dichloroquinolin-8-ol was relatively high in kidney ( ng.g -1 ) in experimental birds which received 240 ppm. However, maximum residue concentration of 5,7-dichloroquinolin-8-ol was present in breast muscles ( ng.g -1 ) of birds belonging to 240 ppm group on day 40. Bound residues may arise from incorporation of residues of the drug in to endogenous compounds, chemical retention of proven drug or its metabolites in to macromolecules or physical encapsulation in to tissue matrices (WHO, 1989). A metabolic study with 36 Cl labeled mixture of 5,7-dichloroquinoline-8-ol and 5- chloroquinoline-8-ol showed that distribution of 36 Cl in the urine and faeces to the extent of 39.3 and 60.4 per cent respectively, in rats following per-oral administration through

6 84 stomach intubation, while their excretion were 80.5 and 10.5 per cent respectively in two month old French Friesian calf fed in capsule form (Bories and Tulliez, 1972). Thus, urinary excretion is predominant in the calf while faecal excretion is predominant in the rat. Urinary and faecal withdrawal of several quinoline compounds were very rapid with more than 90 per cent being eliminated within 48 h (Bories and Tulliez, 1972; Kotaki et al., 1983). The present study was limited to distribution of halquinol (5,7- dichloroquinolin-8-ol) in edible tissues in poultry. However, pharmacokinetic studies following single large bolus dose (per os) of halquinol delivered through an appropriate vehicle might have been a better option to assess the plasma kinetics as zero or first order and to study biliary, faecal and urinary excretion pattern. In breast muscle samples, 5,7-dichloroquinolin-8-ol was not detected throughout the experimental period in birds exposed to 60 and 120 ppm in the present study. However, residues were detected in 240 ppm group and the concentration was found to be ng.g- 1 (Table 1). When compared to liver and kidney tissue (60 and 120 group), the mean residue concentration of 5,7-dichloroquinolin-8-ol in breast muscles was relatively less despite the experimental birds were fed 240 ppm. This may be attributed to less vascular nature of skeletal muscles when compared to highly perfused organs (liver and kidney) in the present study. In the residue depletion study, the highest concentration of 5,7-dichloro-8- quinolin-ol was observed in kidney ( ng.g -1 ) in the 240 ppm group on day three following withdrawal of halquinol (Table 2). Halquinol is generally incorporated in

7 85 the poultry 30 or 60 ppm. The metabolic profile of halquinol in poultry is not understood, however, radio labeled studies in rat indicated that halquinol is excreted as glucoronide conjugate in the bile (Borries and Tulliez, 1972) similar to that of one of the closely related compound clioquinol which essentially undergo glucuronidation in the intestine (Kotaki et al., 1983). In addition to poor absorption and metabolism in gastrointestinal tract, quinolines undergo aromatic hydroxylation and N-oxygenation based on regioselectivity among them (Dowers et al., 2004). 5.2 Tissue depletion of 5,7-dichloroquinolin-8-ol following withdrawal of halquinol Following a day after withdrawal of halquinol given either therapeutic dose (60 ppm) or extra-label dose (120 and 240 ppm), the concentrations of 5,7-dichloroquinolin- 8-ol fell significantly (p<0.05) in both liver and kidney tissues and their concentration was not detectable (< 20 ng.g -1 ) on day 4 in these tissues after withdrawal. Relatively, the depletion of 5,7-dichloroquinolin-8-ol from breast muscles of experimental birds fed 240 ppm was rapid when compared to other edible tissues subjected to analysis in the present study. There are no reports of either pharmacokinetic profile of halquinol or metabolic site (s) in poultry following extra - vascular administration. Depletion of tissue residues depends on terminal elimination phase of the drug in question. It has been reported that clioquinol, a quinoline compound mostly metabolised to clioquinol glucuronide and sulfate and these metabolites were excreted in urine and bile (Kotaki et al., 1983). Rapid depletion of 5,7-dichloroquinolin- 8-ol in all the treated birds might be due to combined effect of intestinal and/or hepatic metabolism and urinary or biliary excretion in the present study.

8 Pre-slaughter withdrawal period Fixation of meat withdrawal period (WP) requires quantification of tissue residues of drug or its metabolite/s quantified according to specified analytical procedures as specified from time to time by enforcement agencies. However, partly because of complexity in structural chemistry of halquinol, tissue or plasma kinetics of individual constituent molecules of halquinol has not been determined. In the present study, tissue residues of 5,7-dichloroquinolin-8-ol was determined, however the concentration of 5- chloroquinolin-8-ol was below the detection limit of assay system. Hence, the WPs for halquinol in broiler chickens were made based on the tissue depletion profile of 5,7- dichloroquinolin-8-ol obtained in the present study. By whatever procedures are applied, separate withdrawal periods (WPs) must be estimated from each edible tissue and the longest of these individual tissues WPs then becomes overall withdrawal period for the marketed product (Woodward, 2009). US- FDA requires that log-linear kinetics and least square regression to be used to calculate withdrawal time in edible tissue/s after deriving a statistical tolerance limit (Reeves, 2010). An important feature of the statistical tool is that it takes account of both acceptable daily intake (ADI) and kinetic behavior of the residue in muscle, fat, liver and kidney. Generally, MRL and tolerance values are employed to derive WPs for marketed veterinary medicines. Hence it can be derived by examining the time-dependent tissue depletion preferably in key tissues (viz: liver, muscle, kidney and fat) against the MRL or tolerance values. Upon extensive literature survey, it was found that there is lack of

9 87 information on either residue kinetic data or other parameters of regulatory toxicological importance (viz: tolerance, ADI, NOAL, NOAEL) with respect to halquinol or its constituent molecules. However, in the absence of MRL, particularly keeping in view of developing countries where veterinary drug assessment and MRL values are difficult to generate, FAO/WHO joint expert committee on food additives (JECFA) has permitted the use of veterinary medicine products and trade at least based on scientific approach or those that develop and use their own national standards. However, there is no standard for halquinol residues in foods of animal origin set by Agricultural and Processed Food Products Export Development Authority or by Government of India under Prevention of Food Adulteration Act (GOI, 2004), thus, complicating the fixation of WPs. Present regulatory standards require that drugs be regulated on the basis of total residues. Total residues resulting from drug administration to an animal consist of the parent drug and all metabolites, conjugates, and residues bound to endogenous macromolecules. To ensure compliance with the withdrawal period, an assay is needed to monitor total residues in the edible tissues. However, it would be impractical to subject all known metabolites of a drug to analysis, since some drugs can give rise to numerous metabolites (Woodward, 1992). Maximum residue limits (MRLs) for a commodity are set for residues measured by a valid method of analysis. This method may measure the chemical or a derivative of the chemical and may include metabolites originating from the parent compound or other chemicals. In some cases, the nominal concentration of the parent compound is calculated from the measured concentration of a metabolite, but in other cases a derivative or metabolite is used as a measure of the residue. Therefore, the residue to which the MRL applies to each chemical compound (Botsoglou and Fletouris,

10 ). Admittedly, all the indirect ways employed for establishing pre-slaughter WP cannot guarantee absolutely that the poultry meat is free from antimicrobial substance. Halquinol is derived from a mixture of compounds obtained by the chlorination of 8-quinolinol and it is composed of 5,7-dichloroquinolin-8-ol (57 74%), 5-chloro-8- quinolinol (23 40%), and 7-chloro-uinolinol-8-ol (not more than 3%), however the stability of the third component is weak. In the present study, commercial product of halquinol (Provimi Animal Nutrition India Pvt. Ltd., Bangalore) which was certified to contain respectively and per cent of 5,7-dichloroquinolin-8-ol and 5-chloroquinolin-8-ol was used. In the present study, the residue profile of 5,7-dichloroquinolin-8-ol (major component of halquinol) was established and the withdrawal period was established based on its concentration equal or near to the analytical sensitivity (20.0 ng.g -1 ). The residue concentration of 5,7-dichloroquinolin-8-ol found in liver, muscle and kidney were much below the levels of feed additives like apramycin, oxolinic acid and chlortetracycline used in poultry (Botsoglou and Fletouris, 2001). In the present study, neither the first order kinetics of halquinol nor its homogenous distribution at equilibrium in tissues was confirmed. Thus deriving withdrawal periods based on half life derived from the tissue residue data alone may not be scientifically justifiable as opined by Riviere and Sundlof (2001) and Woodward (2009). However, the analytical method employed for detection of 5,7-dichloroquinolin- 8-ol in the present study (HPLC) was satisfactory. In the case of substances for which MRL has not been established the issue of regulator may be proven based on presence or

11 89 absence of residue in a food rather than the quantity present (Ellis, 2004). In such cases critical issues for validation may be the selectivity and sensitivity of the screening test or the lowest concentration at which a confirmatory test can detect and confirm the presence of residues. Due to complexity and high standard procedure of using MRL some countries have not established MRL (Ellis, 2004). The procedure for establishing MRLs for veterinary drugs is not consistent amongst the jurisdictions, or between the jurisdictions and the JECFA. Keeping in view of the minimum required performance limit (MRPL) of the analytical device (Ellis, 2008) to detect residues of enrofloxacin and other agents (Botsoglou and Fletouris, 2001), the sensitivity, precision and repeatability of the present assay system to detect 5,7-dichloroquinolin-8-ol is highly acceptable. The toxic potential of halquinol residues is relatively negligible when compared to considering clioquinol, a 8-hydroxyquinoline derivative which was withdrawn from the market on account of its sub-acute myelo-optic neuropathy in human beings despite such effects are observed in animal experiments including poultry (Gad, 2004). Similarly any clinical signs of toxicity in experimental birds fed with 60 and 120 ppm were not observed. In the extra vascular administration of a drug, the pharmaceutical formulation can condition the rate of absorption and consequently the final elimination phase. According to this, a formulation may require longer WPs when the drug is slowly depleted from tissues. Otherwise, shorter WPs can be used when faster depletion is adequately proven (Kukanich et al., 2005). Further, the differences among pharmaceutical formulations of a drug must also be considered as well as the MRLs while fixing the WPs (Martin et al.,

12 ). In the present study, halquinol was employed in powder form and similar practice is followed in commercial poultry farms. Normally halquinol is 30 and 60 ppm in poultry. The FDA has established that the edible tissue from which residues deplete most slowly could be considered as the target tissue (Ellis, 2004). It has been reported that WPs required to reach MRL could be five days for oxytetracycline and sulphadimidine and six days for ampicillin in chickens (Alhendi et al., 2000). Considering the poor bioavailability (~5-10 %) of halquinol (Botsoglou and Fletouris, 2001) and the depletion profile residues of 5,7-dichloroquinolin-8-ol in the present study, it can be hypothesized that even upon the higher dose levels (120 and 240 ppm) of halquinol, did not result in extension of tissue depletion time when compared to recommended dose (60 ppm). In spite of its extensive use in India as a feed additive in broiler chickens there were no incidence of reported adverse effects. In the absence of systemic pharmacovigilance studies in India and continued use of halquinol in broiler chickens, the present study suggests that three days pre-slaughter withdrawal time for halquinol is the best option when used as feed additive at 60 ppm.