Total residue depletion study

Proper conduct of the total residue depletion study is important not only because the results define the depletion of total drug-related residue from the edible tissues of treated animals, but also because this data will be utilized to identify the target tissue (the edible tissue selected to monitor total residue - usually the last tissue in which residues deplete to the safe concentration) and marker residue (residue, i.e. drug and/or metabolite(s), selected to monitor the concentration of the total residue in the target tissue). These results will be utilized to establish the relationship between depletion of total residue and the marker residue in the target tissue. Identification of major metabolites in the urine and feces is important because these products must undergo environmental impact assessment. Tissue and excreta profiles will also be used... 

Initial Total Residue Depletion Study with Flu xin in ( ttle. 

In the initial total residue depletion study, C-flunixin NMG was administered once daily for two consecutive days by intravenous injection to three lactating cows and three steers at a dose of 2.2 mg/kg/day (based on flunixin free acid). One cow and one steer per time point were sacrificed at 24, 72 and 120 hours after the final dose, and selected tissues, including liver, kidney, muscle and fat, were collected and analyzed for radioactive content. Highest levels of total radioactivity were noted in the liver and kidneys. Average values of radiolabeled residues in the liver at 24, 72 and 120 hours were 530 226,... 

Second Total Residue Depletion Study vith Flunixin in Cattle. 

Profiling and identification of flunixin and its metabolites in the liver and kidneys of male and female feeder cattle was also carried out during the second total residue depletion study. Initially, liver tissue from cattle treated intravenously with... 

Following development of the assays for the marker residue (flunixin), liver tissue from feeder cattle dosed intravenously daily for three days with C-flunixin NHG (second total residue depletion study) was assayed by the surveillance method. Based on this assay, mean values of flunixin in the liver at 12 and 24 hours post final dose were 531 and 36 ng/g tissue, respectively. Liver samples collected at 72 and 120 hours contained flunixin concentrations below the limit of quantitation of the assay. The flunixin concentrations at.12 and 24 hours represented less than one-third of the total u residues, and although no fluni n was detected at 72 and 120 hours, there were still detectable residues at these sacrifice intervals. 

Residue depletion studies in pigs after intramuscular administration of ceftiofur showed total residue concentrations of 590, 1190, 250, 400, and 1320 ppb in liver, kidney, muscle, skin/fat, and injection site, respectively, at 12 h after dosing. In cattle, intramuscular administration of radiolabeled ceftiofur resulted in total residue concentrations of 1294, 250, 60, and 60 ppb equivalents in liver 3508, 853, 159, and 159 ppb equivalents in kidney 208, 20, 10, and 10 ppb... 

Residue depletion studies with radiolabeled furazolidone have shown that the almost complete degradation of the drug in the body resulted in formation of a variety of protein-bound metabolites that were not solvent-extractable. Thus, when pigs were given radiolabeled furazolidone orally at 16.5 mg/kg bw/day for 14 days (123), total residual radioactivity in liver, kidney, muscle, and fat accounted for 41.1 ppm, 34.4 ppm, 13.2 ppm, and 6.2 ppm furazolidone equivalents, respectively, at zero withdrawal (132). Total residues were substantially lower by 21 days withdrawal, but were still in the ppm range at 45 days withdrawal. Extraction of the incurred muscle tissue at 0 and 45 days withdrawal with organic solvents led to removal of 21.8 and 13.7% of the total radioactivity, respectively. In contrast, 44 and 8.3% of the total radioactivity was extracted from liver on days 0 and 45, respectively. 

When pigs and calves were subcutaneously given marbofloxacin, residues persisted in liver and kidney for up to 4 days posttreatment. Almost all of the residues detected in muscle and fat were due to the parent drug, whereas residues in liver and kidney were also due to drug-related metabolites as well. Residue depletion studies in dairy cows similarly treated showed that a proportion of 73-89% of the total residues in the milk was due to the parent marbofloxacin. 

Baquiloprim residue depletion studies in cattle treated by oral and parenteral route and in swine treated by parenteral route showed that 14-42 days after administration, the parent compound amounted to a very small proportion of the total residues in liver, kidney, and at the injection site. This was also the case with all identified metabolites. The concentrations of the residues in fat and normal muscle were too low to permit examination of their presence. However, pig skin contained a relatively high proportion of the parent compound. Pigs generally showed a faster degradation and elimination profile than cattle at comparable times after administration, resulting in lower total and parent drug residue levels. 

Residue depletion studies in cattle, sheep, swine, and horses showed that residue levels in tissues rapidly decreased with time (27). Total residue levels in muscle and fat, although higher than in liver and kidneys, were generally lower than 0.1 ppm at 4 days postdosing. Levels of extractable residues were also lower than 0.1 ppm at 7 days after treatment. 

Residue depletion studies in cattle following subcutaneous treatment with a single dose of 0.3 mg radiolabeled abamectin/kg bw showed total residues in liver and fat to be significantly higher than those in kidney and muscle at any... 

Radiolabeled residue depletion studies in swine also showed that the maximum concentrations of total olaquindox residues at 2 days after its oral administration occurred in the kidney (110 ppb) and liver (52 ppb) much lower concentrations could be seen in the plasma (10 ppb) and muscle (9 ppb), whereas fat did not contain detectable residues (7). These residue levels declined with time so that at 28 days postdosing they were negligible in kidney (1 ppb) and liver (2 ppb) and nondetectable in muscle and fat. 

Metabolism and residue depletion studies of avilamycin in swine and rats showed that oral doses are excreted rapidly and nearly quantitatively, with only 5% of the dose excreted in urine and the remainder in feces (23). Most of the parent compounds were metabolized or degraded, since only about 8% of the total residues in feces was parent avilamycin. 

Residue depletion studies (21) in calves orally given a single dose of 3 mg/kg bw radiolabeled clenbuterol showed that total urinary, fecal, and carcass radioactivity averaged 41.5%, 2.4%, and 52.3% of the dose, respectively. Radioactive residues detected in carcass at 2 days postdosing averaged 0.6 ppm in blood, 1.4 ppm in heart, 8.4 ppm in lungs, 2.6 ppm in spleen, 5.0 ppm in liver,... 

Radiolabeled residue depletion studies in target animals from zero withdrawal time to periods beyond the recommended withdrawal time (these studies should provide information on total residues, including free and bound residues, and major residue components in order to select a marker residue and target tissue). 

If little or no previous data on the metabolism of the compound in the target species is available, it is prudent to consider conducting a probe residue depletion study in three animals sacrificed at three widely spaced intervals following the last dose. For example, one animal might be sacrificed at 12 hours (zero withdrawal) and another each at 72 and 120 hours post final dose (the exact times selected will depend on the tissue clearance of the drug under development). The information obtained will allow one to select more accurately sacrifice intervals for the definitive study which encompass proposed safe concentrations of total drug-related residue. Additionally, the probe study residue data will allow for adjustments in the specific activity of the radiolabeled dose to ensure tissue concentrations of total radioactivity at later sacrifice intervals which are sufficient for metabolite profiling and isolation. 

The mean concentration of flunixin in the liver was 389 ng/g after 12 hours, 53 ng/g after 24 hours, and 13 ng/g after 48 hours of withdrawal from treatment. At 48 hours, only two of the five animals treated had residues above the limit of quantitation (8 ng/g). No flunixin was detected in the 72-hour withdrawal liver samples. Confirmation analyses by GC-MS indicated that detected residues were flunixin. As shown in Figure 2, the flunixin concentrations detected in the livers of treated cattle at 12 and 24 hours post final dose in both the total and final residue depletion studies are similar. However, in both studies, the concentrations of detected flunixin are low with respect to total radiolabeled residue. These results suggest that a significant portion of the total residue may be bound and/or the existing surveillance and confirmatory assays do not adequately extract flunixin residues from the tissue of treated animals. The need for further work on the bound residues and the existing assays will depend on the final safe concentration assigned to the drug. 

Residues are evaluated to determine the extent of uptake of the veterinary drug, its distribution throughout the body, and its elimination. Normally, contemporary residue depletion studies establish tissue concentrations in a radiolabeled drug study, in which total residues and parent compound are determined at several pre-determined times between zero time and a time beyond the proposed withdrawal time. As well as total residues, which include free and bound components, the study quantifies major metabolites. These are compounds contributing 10% or more of total radioactivity or that are present at a concentration of > 0.10 mg/kg. Metabolism studies enable identification of the marker residue and target tissue. The marker residue must give assurance that, when its concentration is at or below the MRL, total residues satisfy ADI requirements. 

To ensure compliance with the withdrawal period, an assay is needed to monitor total residues in the edible tissues. Because it is impractical to develop assays for each residue in each of the edible tissues, the concept of a marker residue and a target tissue is introduced. The marker residue is a selected analyte whose level in a particular tissue has a known relationship to the level of the total residue of toxicological concern in all edible tissues. Therefore, it can be taken as a measure of the total residue of interest in the target animal. The information obtained from studies of the depletion of the radiolabeled total residue can be used to calculate a level of the marker residue that must not be exceeded in a selected tissue (the target tissue) if the total residue of toxicological concern in the edible tissues of the target animal is not to exceed its safe concentration. 

At 168 h postadminisfration, the parent drug represented 48% of the total radioacUvity in muscle, 87% in fat, 61% in liver, and 78% in kidney. In a pertinent radiometric depletion study (64) carried out in horse, moxidectin residues in fat were found to be 221, 165, 130, and 131 ppb at 28, 35, 42, and 49 days, respectively. In all other edible tissues, residue concentrations were below 10 ppb even at the first sampling. 

Metabolism Studies. Having determined the likely choice for a target tissue, the sponsor examines the metabolite profrles during residue depletion to select a marker residue that may serve to monitor the total residue during residue depletion in the target tissue. Metabolic profiles should be examined in tissues other than the target tissue to determine that no additional metabolites are present. One of the residues (metabolite or parent compound) in the target tissue is chosen to be the marker residue and its... 

The safe concentration of drug-related residue must be known in order to determine the withdrawal period for a veterinary product. Often the toxicity data is incomplete and an estimate must be made to progress with requisite residue studies. One approach is to conduct a total residue study with sufficiently widely-spaced sacrifice intervals to assess the rate of depletion of total residue over the projected range of probable safe concentrations. A zero-withdrawal sacrifice interval should be included. The target tissue and marker residue are identified and surveillance/confirmatory assays developed. If a major portion of residue is non-extractable (bound) and the marker is undetectable at times when total residue is still significant, a residue bioavailability study may be necessary. To complete the data package, final residue and comparative metabolism studies are conducted. Studies on the metabolism of flunixin in cattle will illustrate this approach. 

Radiotracer Studies in Poultry. The radiotracer study characterized the depletion of total residues in edible tissues of 37-day old broiler chickens that received 7-day s ad libitum access to feed medicated with 25 ppm l4C-semduramicin sodium. Residues in edible tissues were determined at 6,12,24,48 and 120 hours after treatment. The tissue containing the highest total residues at all withdrawal times was liver. Liver residues were 1.8 or more times higher than residues in the next highest tissues, fat or skin. Total residues in liver were depleted from a mean value of 0.273 pg/g at 6 hours to 0.058 pg/g at 24 hours, and eventually to 0.018 pg/g at 120 hours (Table II). 

ADME studies. Twelve cows were administered two doses of " C-pirlimycin at a dose rate of 200 mg/quarter into all 4 quarters at a 24-hour interval. This dose rate was selected as the highest potential dose rate before the final efficacious dose of 50 mg/quarter had been firmly established. This treatment rate thus resulted in a 4-fold overdose. Blood, milk, urine and feces were collected at various times following the first dose. Combustion analysis of whole blood produced the time course of total residue, as illustrated in Figure 3 for three of the cows. There was a slow absorption of pirlimycin across the udder membrane/blood barrier with maximum concentrations occurring in the 6- to 12-hour posttreatment period. The terminal depletion of the... 

Tissue residues. The concentrations of total residues resulting from the 200 mg/quarter/ dose study in the various tissues at various withdrawal times are presented in Table II. Muscle and fat contained little or no detectable residue beyond day 6. Liver is clearly the target tissue for residue analysis and showed a first order depletion (r =. 995) with a t of 5.7 days. Kidney depleted at a faster rate, with a of 3.3 days. Pirlimycin was not sequestered in the udder as demonstrated by the relatively low concentration of total residue detected in udder. 

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