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

PLOS ONE

Dear Dr. Prenni,

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Academic Editor

PLOS ONE

Additional Editor Comments:

As appended below, the reviewers have raised major concern/critique (Reviewer # 4) and suggested further justification/work to consolidate the findings. Do go through the comments and amend the MS accordingly

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

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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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

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 <loq and="">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.</loq>

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Attachments

Attachment

Submitted filename: Pone 27930.docx

Attachment

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

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

Dear Dr. Prenni,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Nice piece of work. Congratulation for having your work being published in PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

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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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No