PONE-D-19-21934

Phylogenic classification of flavors in cigarillos and little cigars and their variable cellular and acellular oxidative and cytotoxic responses

PLOS ONE

Dear Dr. Rahman,

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M. Firoze Khan, Ph.D.

Academic Editor

PLOS ONE

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2. Thank you for submitting this work to PLOS ONE. Please supply as a new Supplementary Information document a complete list of all the little cigars and cigarillos tested in this work. The document should include the brand name of the little cigar or cigarillo as well as the primary flavour and flavour subtype.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

This work was supported in part by a National Institutes of Health (NIH) Grants, NIH 1R01HL135613, NIH 1R01HL085613, and the Food and Drug Administration (FDA) Center for Tobacco Products (CTP). Also in part was supported by the National Cancer Institute of the National Institutes of Health (NIH) and the Food and Drug Administration (FDA) Center for Tobacco Products under Award Number U54CA228110.

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: 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 manuscript by Jackson et al presented a novel aspect of phylogenic classification of flavors in cigarillos and little cigars and their variable cellular and acellular oxidative and cytotoxic responses. Flavored and flavorings in tobacco products are emerging. This is the first ever classification of flavors based on phylogeny.

The authors have also studied the oxidative stress and cellular responses by different flavors based on their phylogenic classifications. Most importantly, they showed different flavored cigars/cigarillos had variability in tested parameters between flavors as well as brands of the same flavor with their particle size and cellular toxicity. They found that fruits/candy and drinks/beverages were more toxic which are interesting.

Overall, this is the first study which attempted to classify flavors based on phylogeny. The authors must be commended for their efforts. This classification may be useful for other tobacco products including e-cigarette flavors. Experimental design with data rigor and reproducibility are paramount to this study with solid data presentation, innovative design, and outstanding nature of studies.

Cellular and acellular data presented which are directly correlated with the classified flavors. Particle sizes from flavored cigars are presented using a standard approach. The manuscript is well-written. However, the manuscript needs some careful data organization and analyses.

Major Comments:

1. Table should contain more flavors based on the classified tree of flavors.

2. Particulate matter concentrations from different flavored cigars based on classified flavor tree should be presented in detail in table 1.

3. Figure 2: should have the level of significance by more repeats and careful consideration of flavors. Figures 2B and C should contain statistical analyses. Fruits and beverages drinks should be highlighted with their data in this figure and other figures based on their conclusion.

4. Fig 3B,C should contain statistical analyses.

5. Fig 4: should contain control cigar/cigarillos like in other data figures.

6. Fig 5: more flavors should be studied based on classified flavors.

Reviewer #2: The authors report phylogenetic classification of different flavors used in commercial cigars and cigarillos and their results on cellular and acellular oxidative and cytotoxic responses to these flavors in lung cell models. This is a novel study characterizing flavors using a novel strategy based on phylogenetic tree comparisons. Such analysis can be a prototype for testing other flavors like those for e-cigs. The study is based on logical measurements targeting ROS and cytotoxicity in relation to particulate size distribution. These data are striking in the sense that the flavors showed differential effects. This is the first report providing insights into and demonstrating health effects of flavor-infused cigars and cigarillos and may serve as a useful dataset for regulation of flavoring compounds.

Overall it’s a well written manuscript but attention is drawn to the following aspects:

1. Table 1. More flavors need to be added for their characterization as listed by the authors.

2. Figure 1. The phylogenetic tree needs more clarity and bigger font size.

3. Figures 2 and 3. Need detailed information on vendors and brands of cigars and their ROS production.

4. Figure 4. Information on production of ROS by different portions of cigars may be meaningful.

5. The authors should provide particulate matter data from other flavored cigars as listed in figures 1 and 6.

6. Figure 6: How do Cytotoxicity data correlate with ROS data and particulate data.

7. The authors may pick key flavors and correlate them with ROS generation potential.

Additionally, the authors should fix the following:

1. Replace the term ‘phylogenic’ with the correct term which is ‘phylogenetic’

2. Format the References list per the PLOSone format by cleaning up the extra information.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-21934R1

Phylogenic classification of flavors in cigarillos and little cigars and their variable cellular and acellular oxidative and cytotoxic responses

PLOS ONE

Dear Dr. Rahman,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The Academic Editor has reviewed your revised manuscript and notes that the revisions have appropriately addressed the reviewers' concerns. However, the PLOS ONE staff editors have identified one aspect of the study methodology and some minor concerns with the text that we think should be addressed before we proceed. Please note that we have discussed these concerns with the Academic Editor, who agrees that these minor issues should be fixed. They are as follows:

1) We note the use of the T-REX web server and iTOL tool to develop a phylogenetic tree of cigarillo and little cigar flavorings. We are a concerned about the development of a “phylogenetic tree” in this setting, as phylogenetic trees are generally used to show evolutionary relationships, which is not what is reported in this paper. We would propose that this is instead referred to as a “classification”. This would involve several edits to the text, as follows:

- Title: Change to “Classification of flavors in cigarillos and little cigars and their variable cellular and acellular oxidative and cytotoxic responses”

- Abstract: Change “A new phylogenic classification system…” to “A new classification system…”

- Introduction, p4: Delete the following sentences: “In biological classification, organisms are put into categories based on shared traits [20]. The closer organisms are in the tree, the more related they are to each other [20]. Although the amount of different cigar flavors is vast, the system used to classify these flavors must be able to convey a large amount of information clearly.”

- Results p9: Change “To understand how the flavorings in cigars can be grouped a phylogenic tree was created” to “To understand how the flavorings in cigars can be grouped a new classification system was created”

- Discussion p12: Change “Classification of flavoured cigars/cigarillos in a phylogenic tree should provide a convenient nomenclature/vocabulary in the scientific community as there was no scientific classification of cigar flavorings until the present” to “Classification of flavoured cigars/cigarillos should provide a convenient nomenclature/vocabulary in the scientific community as there was no scientific classification of cigar flavorings until the present”

- Discussion p12: Change “The tree created allows for the categorization of flavors…” to “The classification system created allows for the categorization of flavors…”

The tree presented in figure 1 should be changed to a different visual representation, for example a table showing to which flavour category and sub-category each flavour belongs. The legend to figure 1, as well as the Methods, should then be updated to remove the references to phylogenetic trees.

2) We would like to suggest some changes to the text for clarity:

- Abstract: We note that none of the flavouring compounds tested in this work was as cytotoxic as cigarette smoke (in the Results, the authors state “Almost all cigars tested had a reduced in cell viability compared with the control, but were not more cytotoxic than smoke cigarette”). We think this finding should be added to the Abstract, for example “A differential cytotoxic response was observed with cigarillo smoke extract treatments: “fruits/candy” and “drinks” were the most toxic, but were not more cytotoxic than smoke from cigarettes.”

- Discussion p12: The phrase “tobacco companies are creating more non-descriptive names to circumvent the regulation” may not be fully supported by the reference provided as the reference is from 2016 and that statement suggests that this practice is ongoing. We would suggest that this statement be removed; it should be sufficient to note that the introduction of additional flavourings will make comparison between categories more difficult.

3) As you may know, PLOS ONE does not copyedit text before publication. Thus, please ensure that the text is copyedited for grammar and usage before the manuscript is resubmitted.

We would appreciate receiving your revised manuscript by Dec 20 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Emily Chenette, Staff Editor, PLOS ONE

On behalf of

M. Firoze Khan, Ph.D.

Academic Editor

PLOS ONE

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Classification of flavors in cigarillos and little cigars and their variable cellular and acellular oxidative and cytotoxic responses

PONE-D-19-21934R2

Dear Dr. Rahman,

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

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With kind regards,

M. Firoze Khan, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Thank you for submitting your revised manuscript to PLOS ONE. As you prepare your manuscript for publication, we ask that you please incorporate the following edits to the text:

- Abstract - The phrase ‘have a shared vocabulary within the scientific community’ appears to suggest that the community has consulted on and accepted the classification system reported here. As this is the first publication reporting this classification, we suggest that this phrase be removed.

- Abstract – Because the study is performed in vitro, please add the word 'potential' to the following sentence: ‘Our study provides insight into understanding the potential health effects of flavor-infused cigars/cigarillos and the need for the regulation of those flavoring chemicals in these products’

Page 12 – We suggest that the phrase ‘Further, this could help not only researchers and public health officials to become more aware of which flavoring chemicals need to be regulated, but also users of cigars or any flavored tobacco product' be removed, as the chemicals per se were not tested in this work, and there is (as you note) high variability between batches and between flavours in the same category.

Page 15- We would suggest that the phrase ‘Our study highlights the importance of legislation and regulation of all flavored tobacco products. as well as increases the ease of communication in the scientific community' be removed as this is not supported by the analyses performed in this work.

Page 15 - Could you please rephrase the following sentences: ‘In conclusion, due to the high variability from one flavored cigar or cigarillo to the other (including batch to batch variations), fruit, candy and drink flavors show more deleterious effects. It is difficult to ascertain the consistent data due to the differences in chemical composition or flavors of the cigars and cigarillo.’ Currently, it is unclear how variability in batches would cause some flavors to show more deleterious effects, could this please be clarified?

Additional Editor Comments (optional):

Reviewers' comments: