Effects of differing withdrawal times from ractopamine hydrochloride on residue concentrations of beef muscle, adipose tissue, rendered tallow, and large intestine.

Ractopamine hydrochloride (RAC) is a beta-agonist approved by the U.S. Food and Drug Administration (FDA) as a medicated feed ingredient for cattle during the final days of finishing to improve feed efficiency and growth. Maximum residue limits and U.S. FDA residue tolerances for target tissues have defined management practices around RAC usage in the U.S. However, many countries have adopted zero tolerance policies and testing of off-target tissues, presenting a major challenge for international export. Therefore, the objective this study was to determine the necessary withdrawal time among cattle group-fed RAC to achieve residue concentrations below tolerance levels in muscle and off-target tissues. Specifically, both total and parent RAC residues were quantified in muscle, adipose tissue, rendered tallow, and large intestines from animals group-fed RAC and subjected to withdrawal 2, 4, or 7 days before harvest. Ractopamine (parent and total) residues were below the assay limit of detection (< 0.12 ng/g) in all muscle and adipose tissue samples from animals in control groups (no RAC). However, RAC residues were detectable, but below the limit of quantitation, in 40% of tallow and 17% of large intestine samples from control animals. As expected, mean RAC residue concentrations in muscle, adipose tissue, and large intestine samples decreased (P < 0.05) as the RAC withdrawal duration (days) was extended. Irrespective of RAC withdrawal duration, mean parent RAC residue concentrations in muscle, adipose tissue, and large intestine ranged from 0.33 to 0.76 ng/g, 0.16 to 0.26 ng/g, 3.97 to 7.44 ng/g, respectively and all tallow samples were > 0.14 ng/g (detectable but below the limit of quantitation). Results of this study provide a baseline for the development of management protocol recommendations associated with withdrawal following group-feeding of RAC to beef cattle in countries that allow RAC use and intend to export to global markets which may be subject to zero tolerance policies and off-target tissue testing.

Introduction

Beef cattle producers have relied on growth and production enhancing technologies for over 50 years to increase production and improve feed efficiency [1]. While steroidal implants have been historically used for growth promotion through increased average daily weight gain and improvements in feed efficiency, beta-adrenergic receptor agonists (beta-agonists) were approved in the early to mid-2000s as a class of orally active growth enhancement technologies for use in beef cattle intended for harvest [1]. Beta-agonists are routinely used in some countries as medicated feed ingredients during the last three to six weeks of cattle finishing for improved feed efficiency and growth promotion [1, 2]. They bind to G protein-coupled beta-adrenergic receptors on cell surfaces, thus increasing muscle mass via hypertrophy and decreasing lipid synthesis and adipose tissue deposition [3-5]. Beta-agonists in livestock production stimulate skeletal muscle growth without increasing hormone levels, which eventually leads to heavier carcasses with fewer inputs [1] and, therefore result in an economic benefit to producers [6].

There are currently only two beta-agonists, ractopamine (RAC) and zilpaterol (ZIL), approved by the U.S. Food and Drug Administration (FDA) for use in food animal species for increased weight gain, improved feed efficiency, and increased carcass leanness [7]. Both RAC and ZIL were subjected to the New Animal Drug Approval (NADA) process, which is a robust registration system ensuring the safety and effectiveness of the compounds. RAC is approved for use in cattle, swine, and turkeys intended for slaughter while ZIL is only approved for use in cattle intended for slaughter [6-8]. Although these beta-agonists are approved for use in the United States and countries such as Brazil, Mexico, and Canada, they have been banned in other locations such as China and the European Union [7]. ZIL is not currently used in beef cattle in the United States for animal welfare concerns; however, at the current time RAC is still commonly used by U.S. beef producers.

Despite the extensive FDA approval process for beta-agonists and the adoption of FDA residue tolerances and maximum residue limits (MRL) by the Codex Alimentarius Commission (an intergovernmental food standard setting committee) the use of RAC remains contentious [9]. Some countries, including China and the EU, have adopted zero tolerance policies, which are more restrictive than the global standard for RAC use, creating challenges in certain export markets [9, 10] Furthermore, sample handling and testing methods are variable and not standardized, creating additional challenges for ensuring compliance with global standards or zero tolerance policies. Current FDA tolerances (defined as the “maximum concentration that can legally remain in a specific edible tissue of a treated animal”; [11]) for RAC residues in liver and muscle (target tissues) are 90 ng/g and 30 ng/g, respectively [9]. Current Codex MRLs (defined as the “maximum concentration legally tolerated in food obtained from an animal that has received a veterinary medicine”; [12]) in the edible tissues liver, muscle, kidney, and adipose tissue are 40 ng/g, 10 ng/g, 90 ng/g, and 10 ng/g, respectively. These safety standards are based on the acceptable daily intake which estimates the amount of veterinary drug that can safely be consumed by humans daily without adverse/deleterious health effects [11]. Importantly, these limits have been determined based upon detection of the parent RAC residues in specific target tissues. However, many countries have implemented residue testing based upon total RAC (ractopamine + ractopamine glucuronide metabolites) assays in both on- and off-target tissues. Thus, it is critical that livestock producers using RAC have accurate data available to enable production management decision making that will ensure compliance within this complex global landscape.

The objective of this study was to determine the concentration of RAC residues (both parent and total) in muscle, adipose tissue, tallow, and large intestines from steers fed RAC and subjected to 2, 4, or 7 days of withdrawal before harvest. The results presented here provide important information to guide the development of recommendations for withdrawal protocols to ensure adequate depletion of RAC in off-target variety meat tissues produced in the U.S. for export markets.

Materials and methods

Design

The present study was designed with a total of N = 75 experimental units consisting of British crossbred steers that were approximately 30 days from harvest at the end of finishing. Initial and final animal weights averaged 354 and 552 kg, respectively. The protocol was reviewed and determined to be exempt (exemption #2019-079-ANSCI) by the Colorado State University IACUC. Steers were initially housed in three pens to ensure equal opportunity for RAC exposure in their diets among negative controls (receiving no RAC and no feed-tallow), those receiving feed-tallow but not direct RAC supplementation, and those that received direct supplementation of RAC (approximately 250–300 mg/hd/d per label instructions, Actogain™ 45; Zoetis, Inc., Parsippany, NJ) plus feed tallow. The pen-based opportunity for exposure to RAC was meant to reflect commercial applications in large-scale feeding operations. During the dosing/no-dosing period, all cattle in the large RAC-receiving pen were exposed to dietary consumption as health and intake were monitored by feedlot personnel daily. Following the dosing exposure period, cattle from all three pens were then transferred to separate treatment pens to apply withdrawal time ‘treatment’ classifications. Animals were randomly assigned to five groups (15 animals per group): (i) a negative control (never fed RAC and never received feed-tallow during dosing; fed from verified clean feed trucks; “Control-No Tallow”); (ii) a control group that received feed-tallow (never receiving RAC, but received feed-tallow; “Control-With Tallow”); and cattle fed RAC plus feed-tallow, with withdrawal (iii) 2 days before harvest (“2 day”); (iv) 4 days before harvest (“4 day”); or (v) 7 days before harvest (“7 day”). The Control-No Tallow group was included in the study as previous work (unpublished) has shown that feed-tallow can recycle RAC in feeding systems and can therefore be a possible source of RAC that can be detected in bovine tissues.

At the time of harvest after dosing and subsequent withdrawal treatment, four tissue/matrix types (muscle, adipose tissue, rendered tallow, and large intestine) were collected from 15 carcasses per group. These tissues/products were selected to complement previous metabolic depletion studies in our labs that were designed to address applied beef export regulatory issues in importing countries; muscles and variety meats are frequently sampled and tested for presence of RAC in many importing countries. Adipose tissue was sampled and was also used to manufacture tallow so that the likelihood of recirculation in the feed supply could be measured. This led to a total of N = 15 × 5 groupings for 75 samples for each tissue/matrix type. All collected samples were tested for both parent and total RAC. All muscle tissue samples from animals subjected to a 2-day withdrawal had detectable RAC concentrations (both parent and total RAC, S3 Table), confirming effectiveness of the group-dosing exposure protocol.

Sample collection

Samples were collected at a commercial beef harvest facility under USDA-FSIS (U.S. Department of Agriculture, Food Safety and Inspection Service) inspection. Within a treatment withdrawal-time group, the sequence of cattle loaded for shipment and for slaughter was random. Steers were harvested in a balanced design (in which equal numbers of cattle were present in each group) in the following order: (i) Control-No Tallow; (ii) withdrawn from RAC 7 days before harvest; (iii) withdrawn from RAC 4 days before harvest; (iv) withdrawn from RAC 2 days before harvest; and (v) Control-With Tallow. As described, samples included muscle, adipose tissue, and large intestine from the 75 animal carcasses. Rendered tallow, which constituted the fourth tissue/matrix type to be tested for residue levels, was manufactured in-laboratory from adipose tissue (kidney, pelvic and heart region; KPH) collected during harvest (described below). Each tissue/matrix type was collected from each animal. As animals were harvested, tissues were identified via tag transfer and traced such that all tissues were collected from the same animal carcasses.

Samples were collected from carcasses as they were conveyed along the chain in the beef harvest facility. All samples were collected aseptically, using a new pair of gloves to prevent cross-contamination between samples. Never-tear tags were printed for each carcass, and tags were used for muscle, adipose tissue, tallow, and large intestine collection stations for identification as carcasses moved throughout the facility.

Muscle (hanging tender, also known as diaphragm) samples were trimmed by plant personnel on the carcass rail. Adipose tissue (subcutaneous) samples were also collected as carcasses were moving on the rail. Large intestine samples were identified and collected on the viscera table. At least 100 g was collected for each tissue sample type. Large intestine samples were collected from portions of the descending and sigmoid colon. Additional adipose tissue was collected from the KPH adipose tissue region for in-laboratory rendering. This protocol was the same for every animal. Collected samples were placed in individual Whirl-Pak bags (Nasco, Fort Atkinson, WI) and positioned in direct contact with ice in boxes or coolers. Samples were transported overnight to Colorado State University (Fort Collins, CO) where they were stored at -20°C until analysis.

Tissue homogenization

Collected muscle, adipose tissue, and large intestine samples were cryogenically homogenized prior to extraction. Large intestine samples were carefully cut and rinsed with water prior to homogenization to ensure no contamination from residual intestinal contents. Groups within each tissue type were identified based on tag transfer data and control samples were processed first to avoid cross-contamination. Approximately 100 g of each tissue was cut into small (approximately 3 × 3 cm) pieces, flash frozen in liquid nitrogen, and homogenized using a Nutribullet food processor (Capital Brands LLC, Los Angeles, CA). Two subsamples of tissue homogenate, each weighing 1 ± 0.05 g, were placed in separate 5 mL conical tubes and stored at -80°C until extraction. The Nutribullet, all cutting surfaces, and utensils were cleaned in between each sample using detergent followed by a hot water rinse (two times) and an additional final rinse with deionized water to remove all tissue and detergent residue.

In-laboratory tallow rendering

Tallow was generated by in-laboratory rendering [13] as the commercial beef harvest facility did not have its own rendering facility. Subcutaneous adipose tissue samples (> 100 g each) from carcasses were cut into approximately 3 × 3 cm pieces, placed in a sterile glass beaker, and microwaved for 6 min (Panasonic Countertop Microwave, The Genius Sensor 1250W, Panasonic Corp., Kadoma, Osaka Prefecture, Japan). After microwaving, remaining adipose tissue solids were removed using sterile forceps, and the temperature was obtained using an infrared thermometer. The average temperature after microwaving was 134.5°C ± 7.1 (standard deviation). The liquid portion was poured into two 50 mL centrifuge tubes. The first tube was filled to 30 mL and was centrifuged for 20 min (2,000 rpm, 20°C; Beckman Model TJ-6 Centrifuge, Beckman Coulter, Inc., Indianapolis, IN). This portion was used for ractopamine analysis while the second tube was stored at -20°C.

Materials for RAC analysis

Ractopamine HCl certified reference standard (1.0 mg/mL) was purchased from Sigma-Aldrich (St. Louis, MO). Ractopamine-d6 HCl internal standard (IS; 1 mg with exact weight packaging) was purchased from Toronto Research Chemicals (North York, ON, Canada). β-Glucuronidase (from Helix pomatia, type HP-d, aqueous solution, ≥100,000 units/mL) and sodium acetate (NaOAc) was purchased from Sigma-Aldrich. Ammonium formate was purchased from Sigma-Aldrich, water (LC-MS grade), methanol (LC-MS grade), formic acid (Pierce LC-MS grade), and acetonitrile (LC-MS grade) were purchased from Thermo Fisher Scientific (Waltham, MA).

Sample extraction

Samples were extracted in 4 mL methanol containing 25 ng/mL of the IS. Samples were quickly vortexed to suspend, mixed on a shaker plate for 10 min, sonicated for 30 min, and incubated at -80°C for 30 min. The samples were then centrifuged at 21,000 × g for 15 min to separate the solid from supernatant. One aliquot of 1 mL supernatant was transferred into a 1.7 mL Denville tube and stored at -80°C for analysis of parent RAC. A second aliquot of 1 mL supernatant was transferred into a separate 1.7 mL tube for total RAC analysis. The second aliquot was evaporated to dryness using a nitrogen dryer set at 50°C. The samples were resuspended in 200 μL of a master mix made of 10 mL of 25 mM NaOAc buffer (pH 5.2) and 200 μL β-glucuronidase from Helix pomatia. Samples were gently vortexed to mix and then incubated at 65°C for 2 h to activate the enzyme. Then, 800 μL of methanol was added, and the samples were mixed thoroughly by vortex and stored at -80°C until analysis. On the day of analysis, samples were taken out of the freezer, centrifuged at 21,000 × g for 30 min to remove any remaining particulates and transferred into microcentrifuge vials for analysis.

Instrumentation method

RAC residue concentration was measured and validated by UPLC-MS/MS as previously described [14]. Briefly, samples were analyzed on a PerkinElmer UHPLC system equipped with a PerkinElmer QSight LX50 Solvent Delivery Module (PerkinElmer, Inc., Waltham, MA). Two microliters of sample were directly injected onto a reverse phase 1.0 mm × 50 mm Waters Acquity UPLC HSS T3 column (1.8 μm particles; Waters Corporation, Milford, MA) for chromatographic separation. Mobile phase A consisted of LC-MS grade water with 2 mM ammonium formate, and mobile phase B consisted of LC-MS grade acetonitrile with 0.1% LC-MS grade formic acid. The elution gradient was initially set at 1.0%B for 0.2 min, which was increased to 30%B at 2.2 min and further increased to 99.0%B at 3 min until 4.5 min when B was decreased to 1%B until 6.5 min for a total run time of 6.5 min. The flow rate was set to 400 μL/min and the column temperature was maintained at 50°C. The samples were set held at 15°C in the autosampler. Detection was performed on a PerkinElmer QSight triple quadrupole tandem mass spectrometer (MS/MS) operated in selected reaction monitoring (SRM) mode using positive mode ionization. Prior to analysis, SRM transitions were optimized for RAC using an authentic standard. The quantitative transition for RAC was 302.5 m/z → 164.10 m/z at a collision energy of 20 V; the confirmatory ion was 302.5 m/z → 284.16 m/z at a collision energy of 16 V; and finally, the internal standard 308.0 m/z → 168.17 m/z at a collision energy of 22 V.

Data analysis

Peak picking and integration were performed using Simplicity 3Q software (Version 1.5, PerkinElmer, Inc.). Peak areas for each sample were normalized to the peak area of the internal sample in that sample. Quantification of the analytes and QCs were performed using a weighted linear regression against an external standard curve. The Limit of Detection (LOD) was calculated by multiplying the slope of the regression by three times the standard deviation of the blank signal and the Limit of Quantitation (LOQ) was calculated by multiplying the slope of the regression by ten times the standard deviation of the blank signal. These values were determined individually for each matrix (Table 1).

Table 1

Limits of Detection (LOD) and Quantitation (LOQ) of ractopamine residues in four tissue/matrix types.

Tissue/matrix	LOD (ng/g)	LOQ (ng/g)
Muscle	0.12	0.41
Adipose tissue	0.12	0.40
Tallow	0.04	0.14
Large intestine	0.32	1.08

Preparation of the calibration curve

Control tissue was obtained from carcasses of animals that were not fed RAC. Control tissue was homogenized and extracted as detailed above and then spiked with RAC and the IS. A serial dilution (using control tissue for each matrix) was performed to generate an 11-point standard curve ranging from 0.05–50 ng/mL. The standard curve range was optimized (ensuring at least a 6 point curve) for each tissue to capture the appropriate concentration of the samples.

Statistical analysis

Parent and total RAC residue concentrations were analyzed separately for each tissue/matrix (i.e., individually for muscle, adipose tissue, rendered tallow, and large intestine) using a general linear mixed model in SAS (version 9.4; Cary, NC). Analysis of variance included a fixed effect of withdrawal time (Control-No Tallow, Control-With Tallow, and 2-day, 4-day, or 7-day withdrawal). Data are reported as least squares means using a significance level of α = 0.05. Within treatment and tissue means were calculated using nominal values for all samples with detectable RAC (>LOD). The LOD value was used for samples with non-detectable RAC.

Results and discussion

Mean parent RAC residue concentrations ranged from 0.33 to 0.76 ng/g for muscle tissue samples collected from carcasses of cattle that received RAC and that were subjected to 2, 4, or 7 days of withdrawal (Fig 1A and S1 Table). Corresponding means of total RAC residue concentrations ranged from 0.41 to 1.22 ng/g (Fig 1A and S2 Table). Overall, mean RAC concentrations (parent and total) among the 2-, 4- and 7-day withdrawal groups were different (P < 0.05) and decreased with days of withdrawal (Fig 1A). The highest concentrations of parent and total RAC among the 45 individual muscle samples were 1.51 and 2.46 ng/g, respectively (S3 Table), with all samples testing below the current Codex MRL and FDA residue tolerance (10 and 30 ng/g, respectively). Importantly, all muscle tissue samples from animals subjected to a 2 day withdrawal had detectable RAC concentrations (both parent and total RAC, S3 Table), demonstrating the effectiveness of the group-dosing protocol.

Fig 1

Mean parent and total ractopamine (RAC) concentrations (ng/g) in muscle (A), adipose tissue (B), rendered tallow (C), and large intestine (D). Animals fed RAC were initially housed in the same, single pen to ensure equal group-dosing (approximately 250–300 mg/hd/day per label instructions) and then transferred to separate pens for withdrawal groups including 2 days, 4 days, and 7 days before harvest. Control-NT = cattle fed no RAC and no feed-tallow; fed from verified clean feed trucks. Control-WT = cattle fed no RAC but received feed-tallow. Significance (P < 0.05) for parent and total RAC concentrations is indicated within tissue type across groups. Dashed lines indicate matrix specific limits of detection (LOD) and limits of quantitation (LOQ). “A value of LOD/2 was used for visualization where mean RAC levels were below detection limits. Within treatment and tissue means were calculated using nominal values for all samples with detectable RAC (>LOD) and the LOD value for samples with non-detectable RAC”.

Mean RAC residue concentrations in adipose tissue samples from carcasses of steers fed RAC were numerically lower than in muscle samples (Fig 1A and 1B; S1 and S2 Tables) and 64% of the samples had total RAC residue concentrations above the assay LOD (S4 Table). Mean RAC residue concentrations in adipose tissue samples ranged from 0.16 to 0.26 ng/g (parent), and 0.18 to 0.50 ng/g (total) and decreased with days of withdrawal (Fig 1B; S1 and S2 Tables). Parent RAC residue concentrations in individual adipose tissue samples for the 2-, 4- and 7-day withdrawal groups ranged from < 0.12 (below LOD) to 0.64 ng/g, < 0.12 to 0.84 ng/g, and < 0.12 to < 0.40 (less than the LOQ), respectively (S4 Table). Corresponding total RAC residue concentration ranges in individual adipose tissue samples were < 0.12 to 1.34 ng/g (2-day), < 0.12 to 1.10 ng/g (4-day), and < 0.12 to 0.60 ng/g (7-day) (S4 Table).

Overall, RAC residue concentrations detected in rendered tallow manufactured in-laboratory from adipose tissue collected during harvest of cattle subjected to 2, 4, or 7 days of RAC withdrawal were numerically very low or not detectable (Fig 1C; S1, S2 and S5 Tables). However, of the 45 individual tallow samples tested, 67% had total RAC residue concentrations above the LOD (S5 Table). Three samples (from the 2-day and 4-day withdrawal groups) had total RAC residue concentrations above the LOQ with concentrations ranged from 0.15 to 0.19 ng/g (S5 Table).

Mean parent RAC residue concentrations in large intestine samples ranged from 3.97 (7-day withdrawal) to 7.44 (2-day withdrawal) ng/g, while mean total RAC residue concentrations ranged from 4.80 (7-day withdrawal) to 8.45 (2-day withdrawal) ng/g (Fig 1D; S1 and S2 Tables). Mean RAC residue concentrations (parent and total) for the 4-day and 7-day withdrawal groups were lower (P < 0.05) than those of the 2-day withdrawal group. RAC residue concentrations in individual large intestine samples ranged from below the LOD (< 0.32 ng/g) to 18.05 ng/g (parent), and from < 0.32 to 20.74 ng/g (total) (S6 Table).

RAC (parent and total) residues were not detected (< 0.12 ng/g; assay LOD) in any of the muscle and adipose tissue samples collected from carcasses of cattle that were not fed RAC, either with no feed-tallow (Control-No Tallow) or with feed-tallow (Control-With Tallow) (Fig 1A and 1B, S1–S4 Tables). For tallow from both control groups, RAC residues (total) were not detected (< 0.04 ng/g; assay LOD) in 60% of the samples, however, in the remaining 40% of samples, residue concentrations were detected but were below the LOQ (< 0.14 ng/g; S5 Table). For large intestines, RAC residues (total) were not detected (< 0.32 ng/g; assay LOD) in 67% of samples, and in 17% of the samples, residue concentrations were detected but were below the LOQ (< 1.08 ng/g). In the remaining 16% of large intestine samples, total RAC residue concentrations were detected and quantified with concentrations ranging from 2.33 and 4.00 ng/g (S6 Table).

Taken together, these results demonstrate that RAC (parent and total) residues were not detected (< 0.12 ng/g; LOD) in muscle and adipose tissue samples from control cattle (either without or with feed-tallow; Fig 1A and 1B, S3 and S4 Tables). This result is important as it demonstrates that it is possible to achieve a “negative for ractopamine residues” result for cattle not fed RAC. Moreover, tallow in feed was not a source of RAC in the current study. In the majority of large intestine and rendered tallow samples from control cattle, RAC residues were also not detected (< 0.32 ng/g for large intestines and < 0.04 ng/g for tallow); however, total RAC residues were detected above the LOD (but below the LOQ) in 5 large intestine and 12 tallow samples (S5 and S6 Tables) and an additional 5 large intestine samples had quantifiable values (> LOQ; S6 Table). While the detection (> LOD) of RAC residues in control samples was rare and limited to off-target tissues, these results indicate the potential for source contamination which could have ramifications for the industry. A larger number of samples had values that were detectable but not quantifiable suggesting that low concentrations could be quantified with more sensitive assays. Additionally, there are multiple methods that can be used to define assay limits [15] and thus these low concentrations could be considered “non-compliant” in a zero-tolerance scenario.

For cattle fed RAC, residue concentrations in muscle, adipose tissue, and large intestine tissue samples decreased as the withdrawal duration increased (Fig 1). Irrespective of withdrawal time, RAC residue concentration (parent and total) in muscle (a target tissue) were well below the current Codex MRL and FDA tolerance (10 and 30 ng/g, respectively). Nevertheless, even after a 7-day withdrawal, RAC residues were still detectable (and above the LOQ) in some of the tested tissues and particularly in the large intestine (Fig 1; S1, S2 and S6 Tables). As off-target tissues, adipose tissue and large intestines should not be officially held to the MRL set by Codex and the FDA tolerances. However, without available MRLs (or tolerances) for these tissues any detectable amounts are of potential concern, especially if testing is occurring in markets with zero tolerance policies. Using the results of this study, we can assess risk potential for a tissue to test above the regulatory limits. In this evaluation, an upper limit of 10 ng/g (the Codex MRL for muscle) was utilized. The probability (%) that total RAC residues would fall below the 10 ng/g was 100% for muscle, adipose tissue, and tallow, across all withdrawal groups. However, for large intestine samples, the probability of a total RAC residue result below the 10 ng/g cut-off was 67% (2 day), 73% (4 day) and 93% (7 day), indicating a higher risk associated with export of this product even with an extended withdrawal protocol.

It is expected that some countries, either for political or perceived food safety reasons, will continue to regulate the use of growth enhancing technologies and/or their residue levels in edible beef muscle and adipose tissue tissues and offal byproducts. In some cases, countries may forbid the use of the technology altogether and thus have an expectation for zero-tolerance relative to detection of residues. Still other countries may allow the approved use of such compounds but regulate compliance with the expectations by testing off-target tissues for residues. These scenarios apply to RAC as well as other growth enhancing technologies in both cattle and swine production [16] but use of RAC and consequential residues can also uniquely depend on the methods of testing used, i.e., assessment of total vs parent compound concentrations [17].

The results of the present study demonstrate that feedlot operations that are not feeding RAC can likely generate cattle with tissues that do not test positive for RAC if the operation manages cross-contact risk carefully. Additionally, the results support that withdrawal of RAC for 7 days substantially lowers the risk of detection in all tissues. However, detection in large intestine was evident even after extended withdrawal times (7 day) if the cattle were exposed to RAC. Therefore, large intestine (and possibly other offal items) from animals fed RAC should not be shipped to countries that have zero-tolerance or expectations for reduced concentrations of RAC in the tissue. Countries that test for residues using methods for total RAC rather than parent RAC pose a greater challenge to exports of all tissues evaluated in this study. Moreover, testing of off-target tissues for RAC residues, and its application to target tissue MRLs or tolerances, will not be appropriate given differing withdrawal time needs for each tissue type.

Least squares means of parent ractopamine (RAC) residue concentrations (ng/g) in muscle, adipose tissue, rendered tallow, and large intestine from steers in all five groups.

(DOCX)

Click here for additional data file.

Least squares means of total ractopamine (RAC) residue concentrations (ng/g) in muscle, adipose tissue, rendered tallow, and large intestine from steers in all five groups.

(DOCX)

Click here for additional data file.

Parent and total ractopamine (RAC) residue concentrations (ng/g) in individual muscle samples from steers in all five groups.

(DOCX)

Click here for additional data file.

Parent and total ractopamine (RAC) residue concentrations (ng/g) in individual adipose tissue samples from steers in all five groups.

(DOCX)

Click here for additional data file.

Parent and total ractopamine (RAC) residue concentrations (ng/g) in individual rendered tallow samples from steers in all five groups.

(DOCX)

Click here for additional data file.

Parent and total ractopamine (RAC) residue concentrations (ng/g) in individual large intestine samples from steers in all five groups.

(DOCX)

Click here for additional data file.

1 Oct 2020

PONE-D-20-27930

Effects of Differing Withdrawal Times from Group Treatment with Ractopamine Hydrochloride on Residue Concentrations in Beef Muscle, Fat, Rendered Tallow, and Large Intestine

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

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Reviewer #1: The experimental design and the data results shown in figures (1 A-D) and Tables (2-6) are very well presented.

Paper accepted in the current form.

Reviewer #2: The authors have conducted an extensive metabolic study to determine withdrawal time after treatment of ractopamine in cattle. The authors clearly understand the subject and explain it well, including presentation of excellent tables and figures, including Supplemental details. They designed and conducted a meaningful study, leading to sound conclusions that are important for scientific knowledge and global trade. I think the manuscript is acceptable for publication after the authors make the following minor revision.

Abstract and throughout manuscript: replace “ppb” with “ng/g” (ppb is not really a scientific unit, even if regulators may incorrectly use those units)

Reviewer #3: The reviewed manuscript deals with the withdrawal of ractopamine from beef cattle raising and its residues in tissues and fat which have been assessed by liquid chromatography-tandem mass spectrometry. It is well written and suitable for publication in Plos One, since its results are clearly presented and discussed. Few corrections are needed as pointed below:

- Change "ppb" for "ug/kg" in the whole manuscript;

- Provide details of animals used in the experiment: sex? age? breed?

- How RAC has been administered? Please indicate which commercial product has been tested and its presentation;

- Change "mM" for "mmol/L" (L.174 and L.186);

- Provide units for collision energies and SRM transitions;

- Please inform acceptance criteria for recovery, linearity, and precision;

- Chromatograms might be uploaded as supporting information.

Reviewer #4: Please see attached document for proper formatting of review (as written).

PONE-D-20-27930, “Effects of Differing Withdrawal Times from Group Treatment with Ractopamine Hydrochloride on Residue Concentrations in Beef Muscle, Fat, Rendered Tallow, and Large Intestine” is a generally well-written, but vague article describing residues of ractopamine in beef cattle. The manuscript is publishable but requires major revisions.

General Comments.

1. The authors have provided no explanation for the basis of tissue selection. Lung and kidney, in addition to intestinal tissue, are major offal tissues destined for export. Please provide a rationale for the rather limited selection of offal tissues.

2. The practical uses of limit of detection and limit of quantitation poorly defined.

a. The limit of detection for tallow is defined as 0.04 ng/g in Table 1 but is defined in Supplementary materials Tables 1 and 2 as 0.12 ppb.

b. The authors report values below the limit of quantitation, but above the limit of detection (i.e., detectable residues) as quantifiable residues. In addition, the authors quantify and report residues below the LOD. For example, Figures 1a and 1b show 4 means below lines representing the LOD. Thus, it appears that the authors are quantifying and reporting what is, by definition, not quantifiable. In other words, the authors appear to be quantifying and reporting background as measurable residue.

c. The authors have not provided how they calculate means from data sets having quantifiable, detectable, and non-detectable values. There are several methods for handling such data and I don’t really think it matters which method one uses, but the method should be disclosed to readers (in the materials and methods). For example, one could state something like “. . . within treatment and tissue, means were calculated using individual values of quantifiable residues, the nominal values returned for detectable residues, and the LOD value for non-detectable residues” (this is a fairly conservative method which will overestimate residues at the lower end of the scale). Again, there a lot of ways that values 3. Experimental Design. Whatever the authors state about experimental unit (lines 94 to 98), “animal” is not the experimental unit. As the manuscript reads, the ractopamine-fed animals were fed in a single pen and then segregated by withdrawal period. Therefore “pen” is the experimental unit (unless there are individual feed intake data; if there are, then the dose per animal on a mg/kg bw basis could be calculated for the beginning, mid-point, and end of the feeding period . . . then one might be able to justify the experimental unit argument). Having said that, the argument then becomes is the amount of “within pen” variation representative of the amount of variation if the study had been spread among, say 3 pens or locations. We’ll never know. Suggest reporting the study as designed, without the argument that “animal” was the experimental unit. Again, within treatment, single pens of animals were fed, not individual cattle.

4. Conceptual Design. The inclusion of tallow as a potential source of ractopamine residues is curious as adipose tissue is a poor reservoir for ractopamine residues (ractopamine is not lipophilic; at physiologic pH it is a rapidly excreted cation; the glucuronide metabolites are zwitterions). The authors should provide some context in the discussion as to why tallow is a concern as a source of ractopamine residues. I’m not saying it couldn’t be of concern, but the choice of tallow should be addressed, especially as the data of Gressler et al. (Journal of Chromatography B, 1015–1016 [2016] 192–200) and Aroeira (Carolina N. Aroeira, Vivian Feddern, Vanessa Gressler, Luciano Molognoni, Heitor Daguer, Osmar A. Dalla Costa, Gustavo J.M.M. de Lima & Carmen J. Contreras-Castillo [2019] Determination of ractopamine residue in tissues and urine from pig fed meat and bone meal, Food Additives & Contaminants: Part A, 36:3, 424-433, DOI: 10.1080/19440049.2019.1567942) demonstrate that meat and bone meal from ractopamine fed animals may contain ractopamine residues with potential for detectable ‘carry over’.

5. The authors should consider using “adipose tissue” in place of “fat” when they are describing the tissue that was sampled and assayed. Sometimes “fat” (the tissue) is conflated (or at least equated with “fat” tallow. Fat is a component of adipose tissue, but not its sole component.

6. The authors have failed to provide adequate descriptions of the test animals. What was the proportion of steers and heifers? What was the duration of ractopamine exposure (i.e., the length of the feeding period?)? What were the initial and final weights of the experimental animals?

7. There is quite a body of extant literature on ractopamine residues in the context of feed contamination, trade, illegal use, and safety. The authors have accessed almost none of this literature, but some of it is relevant to their study. At a minimum the Brazilian studies of Gressler and Aroeira (mentioned above) should be included in the introduction and/or discussion.

Specific Items. (line, comment)

12 Here and throughout the manuscript, be cautious of conflating fat and adipose tissue; they are not synonymous.

35-36 Tallow values are reported as 0.05-0.08 ng/g, but these values are below the limit of detection reported in the supplementary data tables 1 and 2.

52 “with fewer inputs” sounds like “animal science jargon”, but a broader audience will be accessing this journal. Please describe the economic, labor, energy, etc., “inputs” that are important for the reader to grasp.

57-58 Please provide a reference for the assertion that ractopamine is a pure beta-2 agonist. The authors may want to consider the papers by Mills et al (2003; Journal of Animal Science) which suggests that both beta-1 and beta-2 receptors are activated by ractopamine stereoisomers (i.e., commercial ractopamine is comprised of 4 stereoisomers in a roughly equimolar amounts).

77-70 The safety models used by the US FDA CVM assume that exposure to residues occurs daily over a lifetime (assumed to be 70 years). While this is sort of a technical detail, it has profound implications for setting tolerances (o,r in Europe, MRLs).

96-97 Suggest wording change; what exactly does “to reflect true application of the compound mean”? Perhaps “to reflect commercial application” is what the authors mean?

104-105 Fat is not a tissue type. Adipose tissue contains copious quantities of fat.

107-109 This sentence is out of place as it is essentially a result. It has nothing to do with how the study was conducted.

112-113 Suggest moving the IACUC approval to the beginning of the section where animal feeding is described. Chronologically, IACUC approval was obtained before the initiation of the experiment.

113-118 A random process is not described this section . . . far from it. Perhaps the sentence beginning at line 113 could be clarified by stating “Within treatment, the sequence of cattle loaded for shipment and for slaughter was random”.

130 “proceeded down the line” reads like animal science jargon. Suggest using different wording.

131-133 A better description of the large intestine collection/processing is needed. No mention is made of whether the analyzed sample contained tissue and intestinal contents or just tissue. Because ractopamine is provided in the diet at very high levels (10 ppm dietary ractopamine = 10 μg/g = 10,000,000 ng/g) it’s pretty important to know that intestinal contents were removed from the sampled tissue and how this process was accomplished. Is the measured ‘tissue’ ractopamine true residue incurred into intestinal tissue or unabsorbed gut lumen ractopamine of dietary origin?

133-134 Both subcutaneous (line 130) and KPH adipose tissue were collected. Why? KPH is not mentioned again in the manuscript. Which samples were used for adipose tissue analyses?

143 Is the 1 ± 0.5 g of tissue a typo? Read literally, the sample sizes analyzed ranged from 0.5 to 1.5 g . . . . this is a huge variance. If this is not a typo, then a better explanation of how differences in tissue mass were handled during the extraction method will be required. That is, was extraction solvent volume (and the amount of internal standard) adjusted for the sample weight?

167-168 It is implied that sample extract volume was dependent upon sample mass; if so, state explicitly. If not, then provide an explanation of how the 0.5 g samples were handled differently than 1.5 g samples. (see comment for line 143). “4 mL/g methanol” is ambiguous; read literally this means that solvent volume was added according to the grams of methanol added. Please reword.

180 Suggest rewording “analytical method” to read “instrumental method”. The “analytical method” encompasses sample processing trough instrumental analysis.

186 Mobile phase B is not a buffer. Please reword.

212-214 There are several problems with the description of the calibration curve.

a) Literally read, a “Serial dilution” of both ractopamine and the internal standard means that the standard curve would cover 11 orders of magnitude for an 11 point curve (which is impossible for a curve covering 0.05 to 50 ng/mL). Please reword describing to the reader the use of stock solutions and intermediate standards in the preparation of the calibration curve.

b) As described, the amount of matrix at each point in the standard curve would differ unless matrix were used as the diluent. If matrix was used as the diluent in the preparation of the standard curve, please state that fact.

c) As stated, the concentration of the internal standard would not be consistent across the points of the standard curve (i.e., a serial dilution of a matrix matched stock solution of ractopamine and internal standard). Please reword.

d) “The standard curve range was optimized for each tissue to capture the appropriate concentration of the samples”; what does this sentence mean? It implies that standard curve preparation was tissue specific. If that is the case, then the description of the standard curve and the dynamic range of the standard curve is inadequate. Please clarify.

e) There is no mention of quality control criteria used to establish assay validity. Such criterial commonly include recovery of analyte fortified into control tissue and precision (scatter) of fortified samples; linearity of calibration curves; etc.

261 Suggest rewording, “67% had total RAC residue concentrations above the LOD” to “had detectable, but not quantifiable, RAC residues”.

266-274 The steps used to remove intestinal contents from tissue were not described. Therefore, is not clear if the reported residues are residues from tissue or digesta. Please clarify.

281 The LOD provided is consistent with Table 1, but not with the LOD reported in the raw data (i.e. supplementary tables 1 and 2).

312-313 The FDA considers “fat” a traditionally edible tissue; it is not considered offal (Please see FDA CVM’s Guidance for Industry No. 3; General Principles for Evaluating the Human Food Safety of New Animal Drugs Used In Food-Producing Animals). So, when a drug sponsor conducts residue studies, residues in adipose tissue must be measured. Adipose tissue is not an appropriate target tissue for ractopamine use because the amount of residue in adipose tissue is very low relative to liver (see the FOI summary for ractopamine).

319-321 The reader needs to understand the preparation of the intestinal samples . . . how were contents removed?

Figures 1 & 2 How are means in figures 1 and 2 calculated with respect to data sets that included values above and below the LOD? The method used by the authors to calculate means and error used in graphics should be clearly explained.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Submitted filename: Pone 27930.docx

Click here for additional data file.

Submitted filename: PONE-D-20-27930 Review.pdf

Click here for additional data file.

28 Oct 2020

Response to reviewer comments

Reviewer #1: The experimental design and the data results shown in figures (1 A-D) and Tables (2-6) are very well presented.

Paper accepted in the current form.

Reviewer #2: The authors have conducted an extensive metabolic study to determine withdrawal time after treatment of ractopamine in cattle. The authors clearly understand the subject and explain it well, including presentation of excellent tables and figures, including Supplemental details. They designed and conducted a meaningful study, leading to sound conclusions that are important for scientific knowledge and global trade. I think the manuscript is acceptable for publication after the authors make the following minor revision.

Abstract and throughout manuscript: replace “ppb” with “ng/g” (ppb is not really a scientific unit, even if regulators may incorrectly use those units)

“ppb” has been changed to “ng/g” throughout the manuscript

Reviewer #3: The reviewed manuscript deals with the withdrawal of ractopamine from beef cattle raising and its residues in tissues and fat which have been assessed by liquid chromatography-tandem mass spectrometry. It is well written and suitable for publication in Plos One, since its results are clearly presented and discussed. Few corrections are needed as pointed below:

- Change "ppb" for "ug/kg" in the whole manuscript;

We have chosen to replace “ppb” with ng/g” as requested by Reviewer #2

- Provide details of animals used in the experiment: sex? age? breed?

The following statement has been added to clarify this information.

“The present study was designed with a total of N = 75 experimental units consisting of British crossbred steers that were approximately 30 days from harvest at the end of finishing. Initial and final animal weights averaged 354 and 552 kg, respectively.”

- How RAC has been administered? Please indicate which commercial product has been tested and its presentation;

This section of the methods has been extensively revised as follows:

“Steers were initially housed in three pens to ensure equal opportunity for RAC exposure in their diets among negative controls (receiving no RAC and no feed-tallow), those receiving feed-tallow but not direct RAC supplementation, and those that received direct supplementation of RAC (approximately 250-300 mg/hd/d per label instructions, Actogain™ 45; Zoetis, Inc., Parsippany, NJ) plus feed tallow. The pen-based opportunity for exposure to RAC was meant to reflect commercial applications in large-scale feeding operations.”

- Change "mM" for "mmol/L" (L.174 and L.186);

For brevity we have chosen to leave these units as mM. This is standard nomenclature to describe millimolar solutions.

- Provide units for collision energies and SRM transitions;

The units “V” for collision energy and “m/z” for SRM transitions have been added to the manuscript text.

- Please inform acceptance criteria for recovery, linearity, and precision;

Method validation was previously reported by our group in Davis et al, Journal of Animal Science, 2019, Vol. 97, No. 10. This is highlighted in the manuscript as: “RAC residue concentration was measured and validated by UPLC-MS/MS as previously described [14].”

- Chromatograms might be uploaded as supporting information.

We feel this is unnecessary given that the validated method has been previously published as described above.

Reviewer #4: Please see attached document for proper formatting of review (as written).

PONE-D-20-27930, “Effects of Differing Withdrawal Times from Group Treatment with Ractopamine Hydrochloride on Residue Concentrations in Beef Muscle, Fat, Rendered Tallow, and Large Intestine” is a generally well-written, but vague article describing residues of ractopamine in beef cattle. The manuscript is publishable but requires major revisions.

General Comments.

1. The authors have provided no explanation for the basis of tissue selection. Lung and kidney, in addition to intestinal tissue, are major offal tissues destined for export. Please provide a rationale for the rather limited selection of offal tissues.

The following statement has been added to provide justification of the tissues selected for this study:

“These tissues/products were selected to complement previous metabolic depletion studies in our labs that were designed to address applied beef export regulatory issues in importing countries; muscles and variety meats are frequently sampled and tested for presence of RAC in many importing countries. Adipose tissue was sampled and was also used to manufacture tallow so that the likelihood of recirculation in the feed supply could be measured.”

2. The practical uses of limit of detection and limit of quantitation poorly defined.

a. The limit of detection for tallow is defined as 0.04 ng/g in Table 1 but is defined in Supplementary materials Tables 1 and 2 as 0.12 ppb.

Thank you for catching this typo. In the supplemental tables the value of 0.12 ppb refers

to adipose tissue not tallow. This has been corrected.

b. The authors report values below the limit of quantitation, but above the limit of detection (i.e., detectable residues) as quantifiable residues. In addition, the authors quantify and report residues below the LOD. For example, Figures 1a and 1b show 4 means below lines representing the LOD. Thus, it appears that the authors are quantifying and reporting what is, by definition, not quantifiable. In other words, the authors appear to be quantifying and reporting background as measurable residue.

This is a very accurate observation. It is absolutely correct that below the LOQ is not quantifiable and below the LOD is not detectable. In the graphical representation we are using a value of LOD/2 as a placeholder value for visualization as we cannot claim the level is 0 just that it is below our LOD. Furthermore, you will notice that Figure 1C does not contain any markers of statistical significance because all samples were below the LOQ and thus could not be accurately quantified. This is meant to demonstrate that the tallow samples were detectable but not quantifiable – an important outcome of the study. To clarify, the following text has been added to the Figure caption:

“A value of LOD/2 was used for visualization where mean RAC levels were below detection limits. Within treatment and tissue means were calculated using nominal values for all samples with detectable RAC (>LOD). The LOD value was used for samples with non-detectable RAC.”

c. The authors have not provided how they calculate means from data sets having quantifiable, detectable, and non-detectable values. There are several methods for handling such data and I don’t really think it matters which method one uses, but the method should be disclosed to readers (in the materials and methods). For example, one could state something like “. . . within treatment and tissue, means were calculated using individual values of quantifiable residues, the nominal values returned for detectable residues, and the LOD value for non-detectable residues” (this is a fairly conservative method which will overestimate residues at the lower end of the scale). Again, there a lot of ways that values

The following statement has been added to the methods to clarify our approach: “Within treatment and tissue means were calculated using nominal values for all samples with detectable RAC (>LOD). The LOD value was used for samples with non-detectable RAC.”

3. Experimental Design. Whatever the authors state about experimental unit (lines 94 to 98), “animal” is not the experimental unit. As the manuscript reads, the ractopamine-fed animals were fed in a single pen and then segregated by withdrawal period. Therefore “pen” is the experimental unit (unless there are individual feed intake data; if there are, then the dose per animal on a mg/kg bw basis could be calculated for the beginning, mid-point, and end of the feeding period . . . then one might be able to justify the experimental unit argument). Having said that, the argument then becomes is the amount of “within pen” variation representative of the amount of variation if the study had been spread among, say 3 pens or locations. We’ll never know. Suggest reporting the study as designed, without the argument that “animal” was the experimental unit. Again, within treatment, single pens of animals were fed, not individual cattle.

We appreciate your comment and agree that our experimental design could be better described. Importantly, the initial pen-based opportunity for exposure to RAC was intentional to reflect commercial application in large-scale feeding operations. The “Design” section has been extensively revised to ensure clarity.

4. Conceptual Design. The inclusion of tallow as a potential source of ractopamine residues is curious as adipose tissue is a poor reservoir for ractopamine residues (ractopamine is not lipophilic; at physiologic pH it is a rapidly excreted cation; the glucuronide metabolites are zwitterions). The authors should provide some context in the discussion as to why tallow is a concern as a source of ractopamine residues. I’m not saying it couldn’t be of concern, but the choice of tallow should be addressed, especially as the data of Gressler et al. (Journal of Chromatography B, 1015–1016 [2016] 192–200) and Aroeira (Carolina N. Aroeira, Vivian Feddern, Vanessa Gressler, Luciano Molognoni, Heitor Daguer, Osmar A. Dalla Costa, Gustavo J.M.M. de Lima & Carmen J. Contreras-Castillo [2019] Determination of ractopamine residue in tissues and urine from pig fed meat and bone meal, Food Additives & Contaminants: Part A, 36:3, 424-433, DOI: 10.1080/19440049.2019.1567942) demonstrate that meat and bone meal from ractopamine fed animals may contain ractopamine residues with potential for detectable ‘carry over’.

Tallow was evaluated because it is the only bovine derived material that can be incorporated into cattle feed and the incorporation of tallow in cattle feed is a routine practice in commercial operations. Additionally, previous work from our group (unpublished) has indicated that feed-tallow can recycle RAC in the feeding system. The Ruminant Feed Ban prohibits cattle-derived meat and bone meal from ruminant feed (Federal Rule 21 CFR 589.2000). Thus, meat and bone meal was not considered as a potential for carry over in the context of this study.

The following statement has been added to the manuscript to improve clarity:

“The Control-No Tallow group was included in the study as previous work (unpublished) has shown that feed-tallow can recycle RAC in feeding systems and can therefore be a possible source of RAC that can be detected in bovine tissues.”

5. The authors should consider using “adipose tissue” in place of “fat” when they are describing the tissue that was sampled and assayed. Sometimes “fat” (the tissue) is conflated (or at least equated with “fat” tallow. Fat is a component of adipose tissue, but not its sole component.

The term “fat” has been replaced with “adipose tissue” throughout the manuscript text and supplemental data.

6. The authors have failed to provide adequate descriptions of the test animals. What was the proportion of steers and heifers? What was the duration of ractopamine exposure (i.e., the length of the feeding period?)? What were the initial and final weights of the experimental animals?

This has been addressed as described above in our response to Reviewer #3.

7. There is quite a body of extant literature on ractopamine residues in the context of feed contamination, trade, illegal use, and safety. The authors have accessed almost none of this literature, but some of it is relevant to their study. At a minimum the Brazilian studies of Gressler and Aroeira (mentioned above) should be included in the introduction and/or discussion.

We believe we have accurately captured relevant literature related to cattle, however, the studies mentioned by the reviewer are relevant from the perspective of international swine production. These references have been added in the discussion.

Specific Items. (line, comment)

12 Here and throughout the manuscript, be cautious of conflating fat and adipose tissue; they are not synonymous.

“fat” has been changed to “adipose tissue” throughout the manuscript text and supplemental data.

35-36 Tallow values are reported as 0.05-0.08 ng/g, but these values are below the limit of detection reported in the supplementary data tables 1 and 2.

Those values are actually above the LOD (0.04 ng/g) but below the LOQ (0.14 ng/g). This has been clarified in the abstract text as follows: “Irrespective of RAC withdrawal duration, mean parent RAC residue concentrations in muscle, fat, and large intestine ranged from 0.33 to 0.76 ng/g, 0.16 to 0.26 ng/g, 3.97 to 7.44 ng/g, respectively and all tallow samples were > 0.14 ng/g (detectable but below the limit of quantitation).”

52 “with fewer inputs” sounds like “animal science jargon”, but a broader audience will be accessing this journal. Please describe the economic, labor, energy, etc., “inputs” that are important for the reader to grasp.

We have added a reference [1] to point the reader to more information about the complexities regarding the benefits of beta-agonist use in livestock.

57-58 Please provide a reference for the assertion that ractopamine is a pure beta-2 agonist. The authors may want to consider the papers by Mills et al (2003; Journal of Animal Science) which suggests that both beta-1 and beta-2 receptors are activated by ractopamine stereoisomers (i.e., commercial ractopamine is comprised of 4 stereoisomers in a roughly equimolar amounts).

We have chosen to delete statements related to receptor binding as the mechanism of action is not the topic of this manuscript.

77-70 The safety models used by the US FDA CVM assume that exposure to residues occurs daily over a lifetime (assumed to be 70 years). While this is sort of a technical detail, it has profound implications for setting tolerances (o,r in Europe, MRLs).

We agree that there are more details related to how these important tolerances and thresholds are determined but as this is not the focus of the manuscript, we have chosen instead to provided appropriate citations for US FDA and Codex MRLs.

96-97 Suggest wording change; what exactly does “to reflect true application of the compound mean”? Perhaps “to reflect commercial application” is what the authors mean?

“true application” has been changed to “commercial application”

104-105 Fat is not a tissue type. Adipose tissue contains copious quantities of fat.

“fat” has been changed to “adipose tissue” throughout the manuscript

107-109 This sentence is out of place as it is essentially a result. It has nothing to do with how the study was conducted.

This sentence has been moved to the results section.

112-113 Suggest moving the IACUC approval to the beginning of the section where animal feeding is described. Chronologically, IACUC approval was obtained before the initiation of the experiment.

This statement has been moved to the beginning of the materials and methods.

113-118 A random process is not described this section . . . far from it. Perhaps the sentence beginning at line 113 could be clarified by stating “Within treatment, the sequence of cattle loaded for shipment and for slaughter was random”.

The manuscript was edited to the following statement: “Within a treatment withdrawal time group the sequence of cattle loaded for shipment and for slaughter was random.”

130 “proceeded down the line” reads like animal science jargon. Suggest using different wording.

The statement “as carcasses proceeded down the line” has been deleted.

131-133 A better description of the large intestine collection/processing is needed. No mention is made of whether the analyzed sample contained tissue and intestinal contents or just tissue. Because ractopamine is provided in the diet at very high levels (10 ppm dietary ractopamine = 10 μg/g = 10,000,000 ng/g) it’s pretty important to know that intestinal contents were removed from the sampled tissue and how this process was accomplished. Is the measured ‘tissue’ ractopamine true residue incurred into intestinal tissue or unabsorbed gut lumen ractopamine of dietary origin?

The intestine tissue was thoroughly cleaned of intestinal contents prior to analysis. The following statement has been added to clarify this important point:

“Large intestine samples were carefully cut and rinsed with water prior to homogenization to ensure no contamination from residual intestinal contents.”

133-134 Both subcutaneous (line 130) and KPH adipose tissue were collected. Why? KPH is not mentioned again in the manuscript. Which samples were used for adipose tissue analyses?

Subcutaneous tissue was used for adipose tissue analyses. KPH was used for tallow rendering. This has been clarified in the manuscript text.

143 Is the 1 ± 0.5 g of tissue a typo? Read literally, the sample sizes analyzed ranged from 0.5 to 1.5 g . . . . this is a huge variance. If this is not a typo, then a better explanation of how differences in tissue mass were handled during the extraction method will be required. That is, was extraction solvent volume (and the amount of internal standard) adjusted for the sample weight?

Thank you for catching this – yes that is a typo. Each tissue sample was weighed to 1 ± 0.05g. This has been corrected in the manuscript.

167-168 It is implied that sample extract volume was dependent upon sample mass; if so, state explicitly. If not, then provide an explanation of how the 0.5 g samples were handled differently than 1.5 g samples. (see comment for line 143). “4 mL/g methanol” is ambiguous; read literally this means that solvent volume was added according to the grams of methanol added. Please reword.

This has been changed to “4mL” to accurately reflect what was done in this experiment where all samples were 1 ± 0.05g of tissue.

180 Suggest rewording “analytical method” to read “instrumental method”. The “analytical method” encompasses sample processing trough instrumental analysis.

“Analytical” has been changed to “Instrumentation”

186 Mobile phase B is not a buffer. Please reword.

“buffer B” has been changed to “mobile phase B”

212-214 There are several problems with the description of the calibration curve.

a) Literally read, a “Serial dilution” of both ractopamine and the internal standard means that the standard curve would cover 11 orders of magnitude for an 11 point curve (which is impossible for a curve covering 0.05 to 50 ng/mL). Please reword describing to the reader the use of stock solutions and intermediate standards in the preparation of the calibration curve.

The standard curve was prepared as described using a serial dilution of 50 ng/mL to generate a range of concentrations to 0.05 ng/mL. No intermediate standards were required.

b) As described, the amount of matrix at each point in the standard curve would differ unless matrix were used as the diluent. If matrix was used as the diluent in the preparation of the standard curve, please state that fact.

The following text was added to further clarify that control tissue was used to generate matrix matched standard curves: “A serial dilution (using control tissue for each matrix) was performed….”

c) As stated, the concentration of the internal standard would not be consistent across the points of the standard curve (i.e., a serial dilution of a matrix matched stock solution of ractopamine and internal standard). Please reword.

The IS was added to the control tissue use for the matrix background and by definition of the serial dilution the amount of matrix added at each step is the same. This is adequately described in the text.

d) “The standard curve range was optimized for each tissue to capture the appropriate concentration of the samples”; what does this sentence mean? It implies that standard curve preparation was tissue specific. If that is the case, then the description of the standard curve and the dynamic range of the standard curve is inadequate. Please clarify.

This means that we adjusted the standard curve range that was utilized for each tissue based on values in the sample. For example, if the highest calculated value in the sample was only 2 ng/mL then we may truncate curve to remove the higher values. Although this is standard analytical practice, we have added the following statement to clarify:

“The standard curve range was optimized (ensuring at least a 6 point curve) for each tissue to capture the appropriate concentration of the samples.“

e) There is no mention of quality control criteria used to establish assay validity. Such criterial commonly include recovery of analyte fortified into control tissue and precision (scatter) of fortified samples; linearity of calibration curves; etc.

The assay has been previously validated and published in Davis 2019 which is cited in the methods.

261 Suggest rewording, “67% had total RAC residue concentrations above the LOD” to “had detectable, but not quantifiable, RAC residues”.

Some of the adipose tissue samples did have quantifiable RAC levels thus our statement is accurate.

266-274 The steps used to remove intestinal contents from tissue were not described. Therefore, is not clear if the reported residues are residues from tissue or digesta. Please clarify.

The intestine tissue was thoroughly cleaned of intestinal contents prior to analysis. The following statement has been added to clarify this important point:

“Large intestine samples were carefully cut and rinsed with water prior to homogenization to ensure no contamination from residual intestinal contents.”

281 The LOD provided is consistent with Table 1, but not with the LOD reported in the raw data (i.e. supplementary tables 1 and 2).

This was a typo in the supplementary tables and has been corrected.

312-313 The FDA considers “fat” a traditionally edible tissue; it is not considered offal (Please see FDA CVM’s Guidance for Industry No. 3; General Principles for Evaluating the Human Food Safety of New Animal Drugs Used In Food-Producing Animals). So, when a drug sponsor conducts residue studies, residues in adipose tissue must be measured. Adipose tissue is not an appropriate target tissue for ractopamine use because the amount of residue in adipose tissue is very low relative to liver (see the FOI summary for ractopamine).

We thank the reviewer for this thoughtful comment. As our efforts are focused on issues related to product consumption and export regulation it is important to note that under USDA inspection (FSIS), fat is only considered to be inedible (there are edible fats, but not in the classification sense). However, adipose tissue was included in this study - despite concerns related to the NADA for ractopamine - because there was evidence that recirculation of the compound in feed (through incorporation of adipose tissue as rendered tallow) might be a problem when fully negative tests for tissues were required at import.

319-321 The reader needs to understand the preparation of the intestinal samples . . . how were contents removed?

This has been addressed as presented above.

Figures 1 & 2 How are means in figures 1 and 2 calculated with respect to data sets that included values above and below the LOD? The method used by the authors to calculate means and error used in graphics should be clearly explained.

This has been addressed as described above.

Submitted filename: Response to reviewer comments.docx

Click here for additional data file.

9 Nov 2020

Effects of differing withdrawal times from ractopamine hydrochloride on residue concentrations of beef muscle, adipose tissue, rendered tallow, and large intestine

PONE-D-20-27930R1

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Reviewer #2: Again, the authors have appropriately answered all the reviewer comments and revised the manuscript accordingly for publication “as is” in my opinion.

Reviewer #3: The authors engaged in the correction of their manuscript and provided satisfactory answers to the reviewers' suggestions and corrections. However, I reiterate that "M" and "uM" are not concentration units recommended by IUPAC. There is no difficulty in making this correction.

Reviewer #4: The revised manuscript will make a nice addition to the growing body of literature on ractopamine residues in food animals. The authors did a nice job of addressing questions raised during the initial review.

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20 Nov 2020

PONE-D-20-27930R1

Effects of differing withdrawal times from ractopamine hydrochloride on residue concentrations of beef muscle, adipose tissue, rendered tallow, and large intestine

Dear Dr. Prenni:

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