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Journal of Animal Science logoLink to Journal of Animal Science
. 2019 Jul 6;97(9):3714–3726. doi: 10.1093/jas/skz231

Tissue residue depletion and estimation of extralabel meat withdrawal intervals for tulathromycin in calves after pneumatic dart administration

Zhoumeng Lin 1, Chunla He 1, Drew R Magstadt 2,3, Vickie L Cooper 2,2, Michael D Kleinhenz 1,2, Joseph S Smith 2, Patrick J Gorden 2, Larry W Wulf 2,3, Johann F Coetzee 1,2,3,✉
PMCID: PMC6736027  PMID: 31342061

Abstract

The objectives of this study were to evaluate the injection site pathology and determine tissue residue depletion of tulathromycin in calves following pneumatic dart administration and to calculate the associated extralabel withdrawal interval (WDI). Castrated male Holstein calves were injected with ~2.6 mg/kg tulathromycin via pneumatic dart administration. At 1 (n = 2), 6, 12, 18, and 24 d after drug injection (n = 3/time point), calves were euthanized, and muscle, liver, kidney, fat, and injection site samples were harvested and analyzed for tulathromycin concentrations using a LC-MS/MS method. Gross pathology and histopathology evaluations on the injection site samples were also performed. Pneumatic dart administration of tulathromycin caused severe localized lesions of hemorrhage and edema on days 1 and 6, as well as severe pathological reactions in the subcutaneous muscle on days 1, 6, and 12. Slight to moderate reactions were still observed in the majority of the skin or subcutaneous/muscle samples on day 24. Measured tulathromycin concentrations were converted to calculate the concentrations of the marker residue CP-60,300 by dividing a conversion factor of 1.4. The data were used to calculate extralabel WDIs based on the guidelines from U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The results showed that tulathromycin concentrations were the highest in the liver (4,877.84 ± 65.33 µg/kg), kidney (5,819.52 ± 1,087.00 µg/kg), muscle (1,717.04 ± 140.35 µg/kg), injection site (51,884.05 ± 7,529.34 µg/kg), and fat (161.69 ± 36.48 µg/kg) at 6, 1, 1, 1, and 1 d, respectively, after treatment. Tulathromycin concentrations remained above the limit of quantification of 5 µg/kg in all tissues at 24 d. The calculated WDIs based on kidney data were 26 d using EMA method, 36 d using FDA method based on CP-60,300 data, and 45 d using FDA method based on tulathromycin data. These results suggest that pneumatic dart administration of tulathromycin causes injection site reactions in calves and an extended WDI is needed. One limitation of this study was the small sample size of 3 that did not meet FDA guideline requirement. Therefore, the calculated WDIs should be considered as preliminary and additional studies that use a larger number of animals and directly measure the concentrations of the marker residue CP-60,300 are needed to make a more conclusive recommendation on the extralabel WDI.

Keywords: calves, extralabel withdrawal interval, pneumatic dart administration, remote drug delivery, tissue residue depletion, tulathromycin

INTRODUCTION

Remote drug delivery (RDD) technologies are commonly used in wildlife medicine to deliver tranquilizers for chemical restraint (Bush, 1992). In the past 5 yr, the use of RDD to treat livestock in extensive production systems with antimicrobials has become more common in the United States. The U.S. National Cattleman’s Beef Association (NCBA) has issued a Beef Quality Assurance (BQA) Advisory Statement highlighting concerns about the potential negative animal welfare and food safety implications of RDD in cattle (BQA, 2019). Specific issues that were identified included the risk of 1) inaccurate dosage or unapproved route of administration that may prolong tissue residues or promote antimicrobial resistance; 2) misidentification of treated animals resulting in violative tissue residues; 3) bruising, injection site lesions and damage to sensitive tissues or joints causing pain and distress; and 4) darts that remain attached to the animal for a period of time that may become a hazard to other livestock or personnel when they subsequently become dislodged in the environment. Recently, our group reported that RDD is associated with reduced total body exposure to the antimicrobial, tulathromycin and increased acute stress, muscle damage, and pain at the injection site (Coetzee et al., 2018). Furthermore, the failure of darts to deliver antimicrobial therapy to 5 of 15 animals highlighted potential animal welfare concerns in sick animals treated with RDD technologies. However, data on potential tissue damage at the injection site and tissue residue depletion of tulathromycin following RDD are deficient in the literature. To assist in addressing these concerns, the objectives of this study were to evaluate the injection site pathology and to determine the tissue residue depletion of tulathromycin following pneumatic dart administration in calves for the purpose of calculating the extralabel withdrawal interval (WDI).

MATERIALS AND METHODS

Animals, Treatments, and Tissue Collection

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Iowa State University (IACUC Log #: 11-15-8131-B). Detailed information on animals, housing conditions, acclimatization, baseline data collection, drug administration, post-treatment data collection, injection site and dart examination, plasma sampling, and analysis is provided in a companion paper published in the Journal of Animal Science (Coetzee et al., 2018). In brief, 23 Holstein male castrated calves (~10 mo of age with an average body weight of 378 ± 6.49 kg) that had no record of previous tulathromycin treatment for at least 2 mo prior to study commencement were used in the present study. Animals were randomly allocated to 2 treatment groups: subcutaneous injection group (n = 8) and pneumatic dart administration group (n = 15). The subcutaneous injection group was included mainly to characterize and compare the pharmacokinetics of tulathromycin between the 2 treatment groups and plasma pharmacokinetic results from these 2 groups of animals have been reported previously (Coetzee et al., 2018). In the pneumatic dart administration group, calves were administered with 10 mL (between 2.4 and 2.9 mg/kg depending on the body weight of individual calves; on average 2.6 mg/kg) of tulathromycin (Draxxin 100 mg/mL, Zoetis Animal Health, Kalamazoo, MI) using a Type U 10.0 mL 1.9-cm 14 GA Needle pneumatic dart (Pneu-Dart, Williamsport, PA) in the left neck. At 1, 6, 12, 18, and 24 d after treatment (n = 3 per time point), calves were euthanized by captive bolt followed by exsanguination, and then injection site, muscle, liver, kidney, and fat samples were harvested based on our recently reported tissue collection protocol (Coetzee et al., 2015).

Injection Site Gross Pathology Examination

Injection sites were photographed and tissue reactions were evaluated before tulathromycin administration and at 24 h after drug administration using a Westward Polycarbonate Caliper (Grainger International, Inc., Lake Forest, IL). The caliper had a measurement range of 0 to 150 mm with an accuracy of 0.20 mm and a resolution of 0.01 mm. Dorsal to ventral and cranial to caudal measurements were multiplied to calculate the area of the swelling at the injection site. Given that tulathromycin is only approved for subcutaneous injection in cattle, postmortem dissection, gross evaluation, and description of changes in all tissues at or near the injection site was performed by a single veterinary diagnostician with experience in gross examination of bovine tissues (DM). The goal of this examination was to attempt to establish if the site of drug administration after RDD was subcutaneous, which is the only approved route of administration for tulathromycin, or intramuscular, which would be an extralabel route of administration.

Injection Site Histopathology Examination

Formalin-fixed sections of the injection site skin and underlying musculature were trimmed and positioned in cassettes loaded into an automated tissue processor (Sakura VIP 5, Sakura Finetek, Torrance, CA) for overnight paraffin infiltration. Processed tissues in cassettes were then placed in a paraffin bath (Sakura Tissue-Tek TEC 5, Sakura Finetek, Torrance, CA) after which they were removed from the cassette and oriented in molds. The paraffin-embedded tissues were then fully exposed through sectioning on a microtome (HM 355S Automatic Microtome, Thermo Fisher, Waltham, MA). Tissue sections were cut at 4 microns from the cooled blocks. Paraffin ribbons with tissue were then laid out on a water bath and the floating tissue sections were collected onto microscope slides. The unstained tissue sections were then mounted on the slide and dried at 60 °C for 20 min. Finally, the tissue was deparaffinized and rehydrated for staining by transfer through xylene and a series of decreasing concentrations of alcohol to hematoxylin on an automated stainer (Sakura Tissue-Tek Prisma, Sakura Finetek, Torrance, CA). After a tap water rinse, the tissue on the slide was counterstained with eosin, dehydrated in an alcohol series, cleared in xylene and cover slipped (Sakura Tissue-Tek Glas g2) prior to blinded histological examination by a veterinary diagnostician with experience in the histological examination of bovine tissues (VC). The skin and subcutaneous/muscle reactions were evaluated on 1, 6, 12, 18, and 24 d postdrug administration (n = 3 per time point) and were categorized as one of the 4 scores: 0) normal, 1) slight reaction, 2) moderate reaction, and 4) severe reaction.

Tissue Sample Processing and Tulathromycin Analysis

Tissue concentrations of tulathromycin were determined using high-pressure liquid chromatography (Agilent 1100 Pump, Column Compartment and Autosampler, Agilent Technologies, Santa Clara, CA) with mass spectrometry detection (LTQ Ion Trap, Thermo Scientific, San Jose, CA) after acidic aqueous extraction and solid-phase clean-up. Tissue samples were thawed and homogenized in a Waring blender prior to extraction and analysis. Tissue samples, tissue spikes, and blanks, 1 g of tissue homogenate, were extracted with 10 mL of 0.04 M phosphoric acid in a 50 mL centrifuge tube. An internal standard, roxithromycin, was added to the tissue homogenate, prior to extraction with addition of 50 µL of a 100 ng/µL solution. The acidic extraction was performed on a multitube vortex mixer at 2,500 g for 10 min after the addition of the aqueous phosphoric acid. The samples were then centrifuged for 5 min at 3,000 g and decanted into a clean 50 mL centrifuge tube and 10 mL of a dibasic potassium phosphate buffer, 0.1 M, pH 6.8 was added. The tubes were again centrifuged for 5 min at 3,000 g and decanted into a clean 50 mL centrifuge tube. Solid-phase extraction (SPE) utilized 500 mg Bond Elut CBA cartridges (Agilent, Wilmington, DE), a weak cation exchange material. The SPE cartridges were washed with 2 mL of acetonitrile followed by 2 mL of 0.05 M K2HPO4 prior to passing the tissue extract through the SPE cartridge. The cartridges were then washed with 2 mL of 0.05 M K2HPO4 followed by 2 mL of water and then 2 mL of acetonitrile. The SPE cartridges were then eluted with 5 mL of 5% ammonium hydroxide in acetonitrile into 15 mL centrifuge tubes. Finally, 1 mL of each extract was pipetted into cell culture tubes and evaporated to dryness in a Turbovap at 48 °C. The tube contents were reconstituted with 100 µL of 25% acetonitrile and 50 µL of water with vortexing after each addition of liquid. The contents were transferred to autosampler vials equipped with 300 μL glass inserts. The samples were centrifuged at 2,500 g prior to LC-MS/MS analysis.

For LC-MS/MS analysis, a Hypersil Gold C18 column, 50 mm × 2.1 mm, 1.9 µm particles (Thermo Scientific) was utilized for LC separations. The injection volume was set to 10 μL and the column temperature was maintained at 45 °C. Mobile phases A and B consisted of 0.1% formic acid in water and acetonitrile, respectively. The solvent gradient was from 10% acetonitrile to 95% acetonitrile in 4 min at a flow rate of 0.275 mL/min with a 4.5-min column flush/re-equilibration. Tulathromycin eluted at 3.1 min and roxithromycin eluted at 4.1 min. The solvents used in the LC-MS/MS analysis were from Fisher Chemical (Fair Lawn, NJ). Solvent A was Optima LC-MS grade while solvent B was prepared from Optima grade acetonitrile plus LC-MS grade formic acid. Tulathromycin A was from Santa Cruz Biotechnology (Dallas, TX) and had a purity of 97.0%. Roxithromycin was sourced from Sigma Chemical Co. (St. Louis, MO) and its purity was 91.7%.

The LC-MS/MS analysis utilized positive electrospray ionization (ESI+) for precursor ion generation. The precursor ion for tulathromycin was at a mass-to-charge ratio (m/z) of 269.8 (triply charged) while the precursor ion for roxithromycin was at m/z of 837. Five fragment ions were used for detection and quantitation of tulathromycin. The fragment ions for tulathromycin were at 158, 231, 259, 289, and 325 m/z, while ions at 522, 540, 558, and 679 m/z were characteristic of roxithromycin fragmentation. Sequences consisting of tissue blanks, tissue calibration spikes, and porcine tissue samples were batch processed with a processing method developed in the Xcalibur software (Thermo Scientific). The processing method automatically identified and integrated each peak in each sample and calculated the calibration curve based on a weighted (1/X) quadratic fit. Seven calibration spikes were prepared in blank bovine tissue covering the concentration range of 25 to 5,000 µg/kg. Tissue concentrations of tulathromycin in unknown samples were calculated by the Xcalibur software based on the calibration curve. Results were then viewed in the Quan Browser portion of the Xcalibur software. Calibration curves exhibited a correlation coefficient (R2) exceeding 0.99 across the concentration range. The limit of quantitation (LOQ) of the analysis was 5 µg/kg (ng/g) with a limit of detection (LOD) of 0.5 µg/kg. The LOQ of the analysis was estimated from the response of the 25 µg/kg spike in each bovine tissue. The MS response to this spike level was a peak area of over 1 million. The intercepts of the calibration curves were equal to or less than the slopes of the curves so the curve at 5 µg/kg was still usable. The LOD was determined from the response in the blank control tissues which was about 2% of the response of the 25 µg/kg spike or an LOD of 0.5 µg/kg.

Statistical Analysis

Extralabel WDIs were calculated using 3 different methods based on the guidelines from the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA): 1) EMA method; 2) FDA method based on the marker residue CP-60,300 data; and 3) FDA method based on the parent drug tulathromycin data as detailed below (EMA, 1996; FDA, 2006; Damte et al., 2012; EMA, 2018; FDA, 2018). In this manuscript, we used the term “extralabel WDI” to represent the time when the predicted drug residue concentrations in a particular tissue was below the tolerance or maximum residue limit (MRL) for the 95% (EMA method) or 99% (FDA method) of the population with 95% confidence following extralabel drug administration; whereas the term “withdrawal period” was used to represent the official withdrawal period for an approved drug in an approved species following the labeled drug administration. In both the United States and Europe (EU), the marker residue for tulathromycin is CP-60,300 (EMA, 2003; Saito-Shida et al., 2019). The concentration of CP-60,300 was calculated via dividing tulathromycin concentrations by a conversion factor of 1.4 based on the respective molecular weight of the 2 compounds (i.e., 806.092 g/mol for tulathromycin and 576.772 g/mol for CP-60,300) (PubChem, 2019a, 2019b) as recommended by the manufacturer (Personal Communication, Zoetis, Inc., Kalamazoo, MI) and also based on a recent study (Saito-Shida et al., 2019). The concentration data on day 1 after drug administration were not included in the calculation of extralabel WDIs. This was because according to the observed kinetic profile of the concentrations in the target tissue liver (presented below), the concentrations on day 6 were higher than on day 1; thus, day 1 was still in absorption and/or distribution phases and was not during the elimination phase. If the calculated WDI was a fraction of a day, the estimated WDI was rounded up to the next day.

EMA method

We first calculated the extralabel WDIs by directly incorporating the calculated CP-60,300 concentrations in the liver, kidney, noninjection site muscle, fat, and injection site muscle into the withdrawal time calculation program WT 1.4, a program developed in Germany and adopted by the EMA’s Committee for Veterinary Medicinal Products (CVMP) of European Union (i.e., the EMA method) (Damte et al., 2012). The simulation results were compared to the MRL for each tissue to obtain the respective WDI.

FDA method based on the marker residue CP-60,300

Next, we calculated the extralabel WDIs using the tolerance limit method (coded in the “reschem” R package) developed by U.S. FDA (FDA, 2006; FDA, 2018). In the United States, the tolerance is used instead of MRL in the EU. The tolerance for the marker residue CP-60,300 in the target tissue liver is 5.5 ppm (5,500 µg/kg). Note that the unit of tolerances is ppm in the United States, but the unit of MRLs is µg/kg in the EU and the unit of the residue concentration data is µg/kg in the present study. To avoid confusion, the present study used the unit of µg/kg consistently based on the conversion factor of 1 ppm = 1,000 µg/kg. The tolerances for CP-60,300 in other edible tissues (i.e., kidney, muscle, and fat) are not available in the United States, so the MRLs for CP-60,300 from EMA were used in the calculation. The tolerance for CP-60,300 in the injection site was set to be 10-fold of the tolerance for the noninjection site muscle based on FDA guidelines (FDA, 2018).

FDA method based on the parent drug tulathromcyin

Even though in the United States the marker residue of tulathromycin is CP-60,300, in actual practice the Office of Public Health Science (OPHS) within the U.S. Department of Agriculture’s (USDA’s) Food Safety and Inspection Service (FSIS) does not quantify CP-60,300 directly. Instead, OPHS directly measures tulathromcyin concentrations. Therefore, we also calculated the extralabel WDIs based on the measured tulathromycin concentration data using the FDA tolerance limit method via the “reschem” R package (FDA, 2006; FDA, 2018). Since in the United States there is no information on the tolerances of tulathromycin in edible tissues, the tolerance of 5,500 µg/kg for CP-60,300 in the liver from U.S. FDA and the MRLs for CP-60,300 in the kidney, muscle, and fat from EMA were used in the calculation. The tolerance for tulathromycin in the injection site was set to be 10-fold of its tolerance in the noninjection site muscle based on FDA guidelines (FDA, 2018).

RESULTS AND DISCUSSION

Injection site and postmortem examination at 24 h after darting revealed that RDD was unsuccessful in calf 1462 that was euthanized at the first timepoint. This calf was therefore excluded from the tissue residue analysis. Injection site examinations at 24 h after RDD also revealed that 3 other calves (1460, 1468, and 1470) did not develop significant swelling at the injection site. Darts recovered from these calves were also found to weigh 24 g whereas darts recovered from calves that did develop swelling at the injection site were found to weigh 13.5 g. Therefore, it was concluded that RDD had also been unsuccessful in these 3 calves.

In order to minimize the impact of drug delivery failure on the integrity of the study, the remaining 9 calves that were successfully darted based on the presence of injection site reactions and changes in dart weights before and after delivery, were randomly assigned to the 12, 18, and 24-d euthanasia timepoints (n = 3 calves/timepoint). In the 3 calves in which RDD was unsuccessful, darting was repeated in the right neck at 13 d after the first dart attempt. In order to minimize the potential for these animals to influence the later tissue residue timepoints, these 3 animals were assigned to the 6-d euthanasia timepoint.

Injection Site Gross Pathology

The injection site locations and areas after pneumatic dart delivery of 10 mL of tulathromycin (Draxxin 100 mg/mL, Zoetis, Kalamazoo, MI) in calves were reported in our earlier manuscript (Coetzee et al., 2018). Eleven out of the 15 darts were successfully delivered inside the injection triangle for subcutaneous injection recommended by the Beef Quality Assurance (BQA) Manual (NCBA, 2014). The successful injection site area ranged from 59.86 to 187.2 (mean ± SD: 127.37 ± 46.70) mm2. On gross necropsy, lesions at the injection site were largest and most severe at 1 and 6 d postinjection and generally became smaller and less severe as time passed. On day 1, severe focal subcutaneous hemorrhage and edema were present with moderate to severe localized hemorrhage and edema present in the underlying skeletal muscle and facial planes; subcutaneous lesions ranged from 7.5 cm × 15.5 cm to 9 cm × 15 cm. Lesions were minimal in one calf in which the dart impacted the calf but the injection failed.

Similar lesions were present in the subcutaneous tissue on day 6, with lesions averaging 6.5 cm × 15 cm. Underlying skeletal muscle hemorrhage and edema were moderate. In one calf, the injection site was very near the superficial cervical lymph node; gross changes in this calf included edema surrounding the lymph node and moderate hemorrhage and edema within the lymph node parenchyma. Subcutaneous lesions present at the remaining necropsy time points were generally progressively smaller and less severe; skeletal muscle changes ranged from none observed to small areas of palor.

Injection Site Histopathology

The histopathology of the skin and subcutaneous/muscle samples from both the left and right neck was evaluated. With the exception of 3 calves where the dart failed to deliver the tulathromycin on the first attempt, the left neck was the injection site and the right (contralateral) neck was the noninjection site that served as the control. The scores of the skin and subcutaneous/muscle reactions of all samples are shown in Table 1. All the skin and subcutaneous/muscle samples from the control neck were normal with a score of 1. All the skin and subcutaneous/muscle samples from the darted neck displayed various degrees of abnormal pathological reactions with the score ranging from 2 to 4. In particular, on day 1, all 3 calves had severe subcutaneous/muscle reactions, including extensive muscle hemorrhage, deep dermal edema, necrosis, and sparse to extensive neutrophilic infiltrate, and vasocentric change. On days 6 and 12, all subcutaneous/muscle samples still exhibited severe pathological reactions. On day 18, 1 calf had normal reaction; 1 calf had severe reaction; and another calf had slight abnormal reaction. On day 24, none of the animals had severe reaction, but slightly to moderate reactions were still observed in the majority of the skin or subcutaneous/muscle samples.

Table 1.

Blinded microscopic examination of the skin and subcutaneous/muscle samples from the darted and contralateral neck of calves after exposure to approximately 2.6 mg/kg tulathromycin (Draxxin 100 mg/mL, Zoetis, Kalamazoo, MI) injected using a Type U 10.0 cc ¾ inch 14 GA Needle pneumatic dart (Pneu-Dart, Williamsport, PA) administered with a Model 178B breech loading projector (Pneu-Dart)1

Days postinjection Animal number Skin score Subcutaneous/muscle score
Contralateral Dart Contralateral Dart
1 1456 1 NA 1 4
1 1462* 1 4 1 3
1 1515 1 2 1 4
6 1460† 1 3 1 4
6 1468† 1 4 1 4
6 1470† 1 3 1 4
12 1469 1 2 1 4
12 1475 1 3 1 4
12 1561 1 2 1 4
18 1452 1 2 1 1
18 1464 1 4 1 4
18 1471 1 4 1 2
24 1455 1 2 1 1
24 1466 1 2 1 3
24 1480 1 2 1 3
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1NA, not available; Scoring criteria: 1, normal; 2, mild reaction; 3, moderate reaction; 4, severe reaction. *Pneumatic dart failed to deliver the tulathromycin resulting in removal from the study. †Pneumatic dart failed to deliver the tulathromycin at the first attempt resulting in repeat dart administration into the right neck at 13 d after the original treatment day.

Gross pathology and histopathology results suggest that pneumatic dart administration of tulathromycin can cause severe localized lesions of hemorrhage and edema on days 1 and 6, as well as severe pathological reactions in the subcutaneous muscle on days 1, 6, and 12. The observed severe localized lesions on days 1 and 6 suggest that pneumatic dart administration of tulathromycin may negatively impact animal welfare. The lesions of hemorrhage and edema imply vascular damage and alterations of regional subcutaneous blood flows, which may result in delayed/impaired/impacted uptake of tulathromycin at the injection site by systemic/lymphatic circulation compared to standard subcutaneous injections (KuKanich et al., 2005; Richter et al., 2012; Gradel et al., 2018). Delayed absorption at the injection site may, in turn, result in prolonged tissue residues and extended WDIs (KuKanich et al., 2005). These findings may explain the high variability in the plasma pharmacokinetics and drug residues in the injection site and potentially other edible tissues, resulting in extended WDI.

Tissue Residue Depletion Kinetics and Estimation of WDIs

The results of the tissue residue depletion for tulathromycin after a single pneumatic dart administration at ~2.6 mg/kg in calves are presented in Table 2. Mean ± SD tissue tulathromycin concentrations were the highest in the kidney (5,819.52 ± 1,087.00 µg/kg), muscle (1,717.04 ± 140.35 µg/kg), injection site (51,884.05 ± 7,529.34 µg/kg), and fat (161.69 ± 36.48 µg/kg) at 1 d after drug administration. Thereafter, concentrations decreased to 1,588.42 ± 1,014.88, 119.67 ± 62.95, 6,695.43 ± 1,840.03, 7.75 ± 3.01 µg/kg in the kidney, muscle, injection site, and fat, respectively, at 24 d after drug administration. In the liver, however, the highest concentration (4,877.84 ± 65.33) was observed at 6 d after drug administration. It should be noted that tulathromycin concentrations were still higher than the LOQ of 5 µg/kg in all tissues at 24 d after treatment. In particular, at 24 d after treatment, tulathromycin concentrations were 6,695.43 ± 1,840.03 µg/kg in the injection site, which were still higher than the injection site residue reference value (ISRRV) of CP-60,300 expressed as tulathromycin equivalents (EMA, 2015a), indicating that injection site is not suitable for human consumption or a substantially prolonged WDI is required if injection site is intended to be processed to become meat products.

Table 2.

Residue levels of tulathromycin in collected tissues from calves receiving around 2.6 mg/kg tulathromcyin via pneumatic dart administration1

Days postdose Animal number Liver Kidney Muscle Injection site Fat
1 1456 4,453.60 6,588.15 1,816.28 57,208.10 135.89
1515 3,921.50 5,050.90 1,617.80 46,560.00 187.48
Mean (SD) 4,187.55 (376.25) 5,819.52 (1,087.00) 1,717.04 (140.35) 51,884.05 (7,529.34) 161.69 (36.48)
6 1460 4,874.77 4,920.72 636.80 40,919.50 71.23
1468 4,814.10 5,150.47 750.26 8,906.10 24.20
1470 4,944.65 4,922.94 581.94 27,317.40 74.96
Mean (SD) 4,877.84 (65.33) 4,998.04 (132.01) 656.33 (85.84) 25,714.33 (16,066.79) 56.80 (28.29)
12 1469 3,010.96 2,333.08 300.60 13,164.00 62.40
1475 3,678.03 3,681.58 282.02 14,641.50 23.28
1561 3,176.78 2,783.79 322.22 13,750.90 22.19
Mean (SD) 3,288.59 (347.31) 2,932.82 (686.49) 301.61 (20.12) 13,852.13 (743.93) 35.96 (22.91)
18 1452 2,717.30 2,206.82 69.09 5,143.50 13.41
1464 2,176.05 2,340.46 99.08 668.50 7.85
1471 3,046.42 2,630.48 99.31 11,275.50 11.89
Mean (SD) 2,646.59 (439.48) 2,392.59 (216.59) 89.16 (17.38) 5,695.83 (5,325.03) 11.05 (2.87)
24 1455 2,466.45 2,614.86 191.95 7,889.10 11.21
1466 1,558.08 585.51 90.10 7,620.80 5.71
1480 2,959.67 1,564.87 76.95 4,576.40 6.33
Mean (SD) 2,328.07 (710.97) 1,588.42 (1,014.88) 119.67 (62.95) 6,695.43 (1,840.03) 7.75 (3.01)
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1The unit of residue levels is µg/kg or ng/g. Data in the table represent concentrations of tulathromycin. The concentrations of CP-60,300 were calculated by dividing the tulathromycin concentrations presented in this table by a conversion factor of 1.4 based on the respective molecular weight of the 2 compounds (PubChem, 2019a, 2019b), which was recommended by the manufacturer (Personal Communication, Zoetis, Inc., Kalamazoo, MI). Tissue residue data for Animal # 1462 were not available because the pneumatic dart failed to deliver the required dose of tulathromycin.

The results of the calculated extralabel WDIs using different methods are provided in Figs 1–3 and Table 3. According to the EMA method, the calculated WDIs were 12, 26, 21, and 5 d for the liver, kidney, muscle, and fat, respectively (Fig. 1). The longest calculated extralabel WDI of 26 d after a single pneumatic dart administration was selected as the WDI recommendation as this time allows drug residues in other edible tissues (except the injection site) to fall below the respective MRL. This calculated extralabel WDI of 26 d is longer than the labeled withdrawal period of 22 d after a single subcutaneous injection in calves in the EU (EMA, 2015a). However, according to the EMA guideline (EMA, 2015a), the final official withdrawal period should ensure that residues in the non-injection muscle, liver, kidney, and fat are below their respective MRLs and that residues at the injection site are below the ISRRV (6,000 µg/kg) (EMA, 2015b). In the present study, the results showed that CP-60,300 residues in the injection site were not below ISRRV until 136 d after drug administration (Fig. 1). Therefore, if pneumatic dart administration of tulathromycin in calves is used in the EU, an extended WDI of 26 d is needed and the injection site cannot enter into the food chain. If the injection site enters into the food chain, then an extended WDI of 136 d may be needed.

Figure 1.

Figure 1.

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Estimated withdrawal intervals (days) for tulathromycin in calves after administering 2.6 mg/kg tulathromycin via pneumatic dart administration based on the EMA (European Medicines Agency) method using WT 1.4 software based on the calculated CP-60,300 concentration data. MRL: maximum residue limits for CP-60,300 expressed as tulathromycin equivalents from the EMA (EMA, 2015a). If the calculated withdrawal interval was a fraction of a day, the estimated withdrawal interval was rounded up to the next day shown in the parenthesis. Note that the calculated withdrawal interval for the injection site showed a value of “-1” in the WT 1.4 interface, suggesting that the value was out of the typical calculation range. The value of >135 (136) was obtained by visual inspection of the produced figure.

Figure 2.

Figure 2.

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Estimated withdrawal intervals (days) for tulathromycin in calves after administering 2.6 mg/kg tulathromycin via pneumatic dart administration using the U.S. FDA method (coded in the “reschem” R package) based on the calculated CP-60,300 concentration data. The tolerance of 5.5 ppm (5,500 µg/kg) for CP-60,300 in the liver is from 21CFR556.745. FDA does not have tolerance levels for CP-60,300 in kidney, muscle, or fat. Therefore, the maximum residue limits (MRLs) for CP-60,300 in the kidney, muscle, and fat from the EMA (EMA, 2015a) were used in the calculation. If the calculated withdrawal interval was a fraction of a day, the estimated withdrawal interval was rounded up to the next day. The withdrawal interval for the injection site muscle was also calculated by setting the tolerance to be 10-fold of the tolerance for non-injection site muscle (FDA, 2018), but the value was out of the calculation range and the “reschem” program showed an error, so the result is not shown here.

Figure 3.

Figure 3.

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Estimated withdrawal intervals (days) for tulathromycin in calves after administering 2.6 mg/kg tulathromycin via pneumatic dart administration using the U.S. FDA method (coded in the “reschem” R package) based on the measured tulathromycin concentration data. In the United States, the marker residue of tulathromycin is CP-60,300 and the tolerance is 5.5 ppm (5,500 µg/kg) for CP-60,300 in the liver, but there is no information on the tolerance for tulathromycin in any other edible tissues in the United States. Therefore, the maximum residue limits (MRLs) for CP-60,300 in the kidney, muscle, and fat from the European Medicines Agency (EMA, 2015a) were used in the calculation. If the calculated withdrawal interval was a fraction of a day, the estimated withdrawal interval was rounded up to the next day. The withdrawal interval for the injection site muscle was also calculated by setting the tolerance to be 10-fold of the tolerance for non-injection site muscle (FDA, 2018), but the value was out of the calculation range and the “reschem” program showed an error, so the result is not shown here.

Table 3.

Calculated extralabel withdrawal intervals (days) for tulathromycin based on EMA and FDA methods1

Methods Tolerance or MRL, µg/kg Calculated extralabel withdrawal intervals, days
Liver Kidney Muscle Fat IS Liver Kidney Muscle Fat IS
EMA method based on calculated CP-60,300 data 4,500 3,000 300 200 6,000 12 26 21 5 1362
FDA method based on calculated CP-60,300 data 5,500 3,0003 3003 2003 3,0004 12 36 27 9 NA
FDA method based on tulathromycin data 5,500 3,0003 3003 2003 3,0004 20 45 31 11 NA
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1The tolerance of 5.5 ppm (5,500 µg/kg) for CP-60,300 in the liver is from 21CFR556.745. The maximum residue limits (MRLs) for CP-60,300 expressed as tulathromycin equivalents in the liver (4,500 µg/kg), kidney (300 µg/kg), muscle (300 µg/kg), fat (200 µg/kg), and injection site [6,000 µg/kg; injection site residue reference value (ISRRV)] are from the European Medicines Agency (EMA, 2015a). IS represents the injection site muscle. NA indicates that the calculated withdrawal interval was not available because the value was too large and the “reschem” program showed an error.

2The WT 1.4 program showed an value of “-1” indicating that the calculated withdrawal interval was out of the calculation range of the program, and the value of 136 d was obtained based on visual inspection of the produced figure.

3The tolerances for CP-60,300 or tulathromycin in kidney, muscle, and fat are not available in the United States, so the MRLs for CP-60,300 from EMA were used in the calculation.

4The tolerance for the injection site was set to be 10-fold of the tolerance for non-injection site muscle based on the FDA guideline (FDA, 2018).

Using the FDA method and assuming the conversion factor of 1.4 from tulathromycin to CP-60,300 is valid, it was estimated that the CP-60,300 level was below 5,500, 3,000, 300, and 200 µg/kg in the liver, kidney, muscle, and fat on day 12, 36, 27, and 9, respectively, after pneumatic dart administration for the 99% of the population with 95% confidence (Fig. 2). In addition, by directly calculating the extralabel WDIs using the FDA method and based on the measured tulathromycin concentrations, it was estimated that tulathromycin concentration was <5,500 µg/kg in the liver, <3,000 µg/kg in the kidney, <300 µg/kg in the muscle, and <200 µg/kg in the fat on day 20, 45, 31, and 11 after pneumatic dart administration, respectively, for the 99% of the population with 95% confidence (Fig. 3). The longest calculated extralabel WDIs of 36 to 45 d (depending on whether tulathromycin or CP-60,300 data were used) after a single pneumatic dart administration are much longer than the labeled withdrawal period of 18 d after a single subcutaneous injection in calves in the United States (VetGRAM, 2019). Furthermore, our results showed that the time needed for the concentrations of either CP-60,300 or tulathromycin to fall below the safe concentration in the injection site muscle (defined as 10-fold of the tolerance for the muscle (FDA, 2018)) was so large that it was outside the calculation range of the “reschem” package (data not shown). These results suggest that if pneumatic dart administration of tulathromycin is to be used in the United States, an extended WDI of at least 36 to 45 d (depending on whether the residue monitoring method is based on CP-60,300 or tulathromycin) should be recommended and the injection site muscle cannot enter into the food chain.

In the United States, the target tissue of tulathromycin is the liver for cattle. However, the calculated extralabel WDIs based on the kidney data were conservative (i.e., longer) than those calculated based on the liver data. This was, in part, due to the relatively high variability of the kidney concentration data, especially at the last time point of 24 d (Table 2). Additional studies are needed to establish tolerance level for the marker residue CP-60,300 in the kidney of cattle, which may help establish an updated withdrawal period for tulathromycin in cattle in the United States.

Tulathromycin is approved to treat and prevent bacterial and mycoplasmal infections administered by single subcutaneous injection in cattle and intramuscular injection in swine at 2.5 mg/kg in many countries, including United States, EU, Japan, and Australia, but the labeled withdrawal periods are different due to different regulatory guidelines (EMA, 2015a; Saito-Shida et al., 2019). The present study calculated the WDIs using 3 different methods based on different guidelines and regulatory standards from the United States and EU. In general, the calculated WDIs using the FDA method were more conservative than those based on the EMA method (e.g., 26 vs. 36 d for the kidney based on the CP-60,300 data). This is, in part, because FDA determines the withdrawal period using the 99th percentile tolerance limit with a 95% confidence, whereas EMA uses the 95th percentile tolerance limit with a 95% confidence (Damte et al., 2012). Regardless of which method used, the calculated extralabel WDI after pneumatic dart administration was much longer than the labeled withdrawal period for tulathromycin after subcutaneous administration (i.e., 26 vs. 22 d for the EU and 36 vs. 18 d for the United States for the Draxxin 100 mg/mL injectable solution) (EMA, 2015a; VetGRAM, 2019). Therefore, if pneumatic dart administration is to be used, an extended WDI is needed. The exact extralabel WDI will depend on different regulatory standards from different regulatory agencies.

This study has several limitations. According to the FDA guidelines (FDA, 2006; FDA, 2015; FDA, 2018), in order to calculate the withdrawal period, it is recommended to include at least 4 animals (evenly mixed as per sex) per each slaughter time at each of 4 appropriately distributed time intervals. However, the present study only had on average 3 animals per time point, thus it did not meet the FDA study design requirement. Also, the marker residue CP-60,300 data were calculated based on the measured tulathromycin data by using a conversion factor of 1.4. While this method was based on the molecular weights of the 2 compounds (PubChem, 2019a, 2019b) according to the manufacturer’s recommendation and has been used in a previous study (Saito-Shida et al., 2019), it would still be better if we establish a method to measure CP-60,300 directly in future studies. Therefore, while the present results provide a useful estimate of the likely extralabel WDI after pneumatic dart administration of tulathromycin, additional studies that use larger number of animals in compliance with the FDA guidelines and directly determine the marker residue CP-60,300 concentrations are needed to establish a more conclusive extralabel WDI for tulathromycin after pneumatic dart administration in calves. Besides the tolerance limit method used in this study, extralabel WDIs can also be calculated using other methods, including a nonparametric approach (Concordet and Toutain, 1997), withdrawal time calculator (a supplement to FDA’s tolerance limit method) (Udiani et al., 2018), half-life multiplier (Gehring et al., 2004), physiologically based pharmacokinetic (PBPK) modeling (Lin et al., 2016a; Henri et al., 2017; Li et al., 2019), and population pharmacokinetic modeling (Li et al., 2015; Lin et al., 2016b; Bon et al., 2018), which is a direction of our future study once more data become available.

In summary, the results of this study provide the first description of the tissue residue depletion kinetics of tulathromycin in beef calves following pneumatic dart administration. The newly collected data were used to calculate extralabel WDIs using 3 different methods based on the guidelines from both the United States and EU. These results will assist the U.S. Food Animal Residue Avoidance Databank (FARAD) program and the Global FARAD program in making an extralabel WDI recommendation for tulathromycin following pneumatic dart administration in calves (Riviere et al., 2017). Although RDD allows livestock producers to deliver medications in situations where handling facilities are not available or where it is perceived that RDD will reduce time to treatment and stress on the animal, our results suggest that pneumatic dart administration of tulathromycin can result in extended withhold times, injection site lesions up to 6 d and subcutaneous muscle histopathological reactions up to 24 d postadministration. Furthermore, the risk of dart failure described herein and in a previous report (Coetzee et al., 2018), creates significant challenges for an operator to confirm successful drug delivery to sick animals. Therefore, the failure of high-capacity pneumatic darts to consistently deliver antimicrobial therapy could have a negative impact on the welfare of sick animals treated with RDD technologies. These results will benefit bovine practitioners by allowing them to carefully consider and safely prescribe tulathromycin to treat bovine infectious diseases in an extralabel manner.

Conflict of interest statement. The authors declare no conflict of interest.

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