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

Flavored tobacco products are increasing in popularity but remain unregulated, with the exception of the ban on flavored conventional cigarettes. Lack of regulation of cigarillos and little cigars allows vendors to have their own version of popular flavors, each with different chemical components. A new classification system was created for flavored cigars in order to easily communicate our results with the scientific community. To understand the physicochemical characteristics of flavored little cigars and cigarillo smoke, size distribution and concentration of particulate matter in smoke were determined. Acellular reactive oxygen species generation was measured as an indirect measurement of the potential to cause oxidative stress in cells. In addition, cigarillo smoke extract treatment on bronchial epithelial (Beas-2b) cells were assessed to determine the flavor-induced cellular toxicity. Flavored cigars/cigarillos showed significant variability in the tested parameters between flavors as well as brands of the same flavor, but most of the cigars showed higher potential of generating oxidative stress, than research grade cigarettes. Flavored cigars produced maximum particle concentrations at 1.0μm and 4.0 μm compared with 3R4F reference research cigarettes. A differential cytotoxic response was observed with cigarillo smoke extract treatments, with "fruits/candy" and "drinks" being the most toxic, but were not more cytotoxic than smoke from cigarettes. These cigarillos with flavors must be well characterized for toxicity in order to prevent adverse effects caused by exposure to flavor chemicals. 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. Future research is directed to determine the flavoring toxicity of little cigars and cigarillos in vivo studies.

Introduction

In the current environment where the focus is on the dangers of cigarettes, cigars are often perceived to be safer than cigarettes due to the lack of public attention and scientific knowledge [1-3]. From the year 2000–2011, there was a 33% decrease in consumption in cigarettes and a 123% increase in the use of non-cigarette products, like cigars [4]. This change in behavior is also seen in youths where it was reported in 2012 that cigars were the second most common way for youths to use tobacco [5]. Smoking a cigar carries the same health risks as smoking a cigarette [6, 7].

The amount of legislation pertaining to cigars is much lower when compared to other tobacco products. In 2009, flavored cigarettes were banned by the Family Smoking Prevention and Tobacco Control Act; however, there is no such legislation for cigar products [5]. In association with this ban, there was a significant increase in cigar use seen in high school students, especially in flavored cigars/cigarillos [8]. Adding flavors reduces the harshness of tobacco, making flavored cigars an easy gateway to youth addiction and new tobacco user recruitment [1, 9]. These flavors are not just found within the filler of the cigar, but also extra sweetener is seen the wrappers and mouth tips that make direct contact with saliva [10]. Young adults, 18–24 years old, have the highest usage of flavored cigar products [5].

There is very little information available on what makes up these flavoring chemicals, how the flavor is added, and how these flavors differ by brand [11]. This can be attributed to flavors being a mixture of many compounds, where there is no universal combination method to create more complex tastes [11]. The same flavoring chemicals found in food and drink products are also seen in these flavored cigars [12]. Inhalation of a common flavoring chemical, diacetyl, is linked to the irreversible damage in the lung tissue causing “popcorn lung disease” or bronchiolitis obliterans[13].

A quantitative way of measuring oxidative stress-mediated toxicity is by determining the concentration of reactive oxygen species (ROS) produced by the flavored cigars [14]. ROS are cellular byproducts of the electron transport chain in the mitochondrial membrane and intracellular NADPH oxidase machinery [15]. In addition, ROS can be formed extracellularly through reactions among various radicals, including hydroxyl radicals. Oxidative stress is damaging to cellular components and biomolecules by causing DNA damage, inhibition of apoptosis, and activation of proto-oncogenes [16], leading to the pathogenesis of COPD and lung cancer [17].

Particulate matter (PM) released by cigars may contribute to tissue destruction of the lungs. When the cigar is lit, a combustion reaction occurs and produces vapor to be inhaled that contains PM, for instance:mainstream cigarette smoke contains 10,000–40,000 μg of PM [18]. PMs with respirable size, such as PM 2.5 and PM 1.0, are capable of depositing in the lungs, and can even be trapped in the alveolar region, thereby leading to damage of the lung tissue [18]. Further, extensive research has been conducted on mainstream cigarette smoke, but there is a lack of understating in flavored cigar/cigarillo smoke constituents.

Currently, there is no standard nomenclature for the classification of cigar flavors; this has become a challenge for communication among cigar users and in the scientific community [19]. This lack of organization does not allow for a definitive way to compare work from separate labs, and classify a vast and different cigar flavors available currently.

We hypothesize that the similarities in toxicity between similarly flavored cigars or even within a brand will be able to categorize which cigars are the most deleterious. This knowledge will help to alert not only flavored cigar smokers, but also to make a broader assumption to which flavoring chemicals, in general, are the most harmful. The objective of this study was to determine if the chemical composition in flavored cigars produces differential oxidative and cytotoxic responses. We tested the hypothesis by using various commercially available flavored little cigars and cigarillos in acellular and cellular systems.

Materials and methods

Ethics statements

All experiments performed in this study were approved and in accordance with the University of Rochester Institutional Biosafety Committee. And, all protocol, procedure and data analysis in this study were followed the NIH guildlines and standards of reproducibility and scientific rigor by an unbiased approach.

Animal or Human study protocol: None

Institutional biosafety approvals: Yes

Scientific rigor statement

The approach to creating the experiment was unbiased and analysis done on the results ensured that our data are reproducible.

Cigar procurment and categorization

Little cigars and cigarillos were purchased in Rochester, NY, at various locations and vendors, to be tested in this study (see ). The types of cigars used in the experiment are classified into six basic categories based on names and descriptions on the packaging. Flavor names are generated by the manufacturer and a visual representation of the flavors can be found in . Categories used are tobacco, menthol, fruit, candy, drinks, and spices. These categories were further sub-divided as needed which included, Black and Mild, berry, cherry, grape, mango, peach, pineapple, tropical, and alcohol. Some variation in which flavored cigar was used is present between each expirmental test due to the limitations in inventory.

Classificaton of cigar flavors.

The flavors listed are based on products available on the market but is not a fully inclusive list.The flavors are organized in a new classification system. The inner circle is shaded based on the primary flavor category of the cigar. The outer circle correspond to the sub-category of flavors within the primary category.

Cell-free ROS assay

A fluorogenic dye was created using 0.01N NaOH, 2’7’ dichlorofluorescein diacetate (H2DCF-DA,) (EMD Biosciences, CA) (Cat # 287810), PO4 buffer made of sodium phosphate monobasic (JT Baker, NJ) (Cat #3828–01) and sodium phosphate dibasic (Sigma- Aldrich, MO) (Cat #2–0751), and horseradish peroxide (HRP) (Thermo Fisher, Ma) (Cat # 31491). ROS were detected based on flouresent intensity at 495/529nm. Standards that ranged from 0 to 50 μM were created using a 1mM hydrogen peroxide stock that was reacted with the fluorogenic dye at 37°C for 15 minutes. Standards were measured on a spectrofluorometer (Turner Quantech fluorometer, Mo. FM109535) in fluorescence intensity units (FIU). Samples of cigar smoke extract were also given 15 minutes at 37°C to react with the fluorogenic dye, then measured immediately. Sample readings were based on the hydrogen peroxide standard curve and denoted as “hydrogen peroxide H2O2 equivalents”.

Three different methods were used for creating cigarillo smoke extract using an impinger mechanism. Multiple methods were tested to fully understand the cigar burning process. Method 1 used an SKC lab pump (model 224-PCXR8, Eighty-Four, PA, USA) with an average flow rate of 1 L/min. Little cigars or cigarillos were attached to a 50 mL conical tube containing 10 mL of freshly made fluorogenic dye. Cigarillos were lit and smoke was bubbled through the dye for 1 minute. A new cigarillo was used for each sample. Immediately following the bubbling of the dye, it was filtered through a sterile 0.45 μm polyethersulfone syringe filter to eliminate any debris. Method 2 used a standard lab vacuum to bubble the cigarillo smoke. A cigarillo was attached to a 50 mL conical tube containing 10 mL of freshly made fluorogenic dye and smoke was bubbled for 10 seconds. This process was repeated two to three times on the same cigarillo to create separate samples of extract. Immediately following the bubbling of the dye, it was filtered through a sterile 0.45 μm polyethersulfone syringe filter to eliminate any debris. Method 3 used a standard lab vacuum to bubble the cigarillo smoke. Each cigarillo was sectioned into three equal pieces by weight and attached to a 50 mL conical tube containing 10 mL of freshly made fluorogenic dye. Cigar smoke was bubbled through the dye at a constant rate as described above. Each portion of the split cigarillo was bubbled until completely burned and measured for ROS in H2O2 equivalents.

Cell culture and treatment with little cigar/cigarillo smoke extract (CSE)

Human bronchial epithelial cells (Beas-2b) were obtained from ATCC, USA. Cells were cultured and grown at 37°C in Corning® DMEM (Dulbecco’s Modified Eagle’s Medium) /Hams F-12 50/50 mix media (Cat # 16-405-CV) with added 5% fetal bovine serum (FBS), 1% penicillin streptavidin (P/S) (Corning, NY) (Cat # 30-002-Cl), and 15 mM HEPES (Alfa Aesar, MA) (Cat #J60712). At 85–90% confluency, cells were serum-deprived with DMEM media with added 1% FBS, 1% P/S, and 15 mM HEPES for 8 hours.

Beas-2b cells were seeded in six-well plates at a density of 400,000 cells per well. Cells were grown until 85–90% confluency and then serum-deprived for 8 hours. Cells were treated with 0.5% and 1.0% concentrations of CSE. The smoke extract was created by using method #1; however, in place of fluorogenic dye being bubbled, 10 mL of 1x PBS was used. The extract was then measured on a Beckman Coulter spectrophotometer (Model DU520). An absorbance value of 1.00 ± .05 was considered to be 10% concentration, and further dilutions were done to obtain that concentration. Cells were treated for twenty-four hours. Untreated cells remained in DMEM supplemented with 1% FBS.

The viability of the Beas-2b cells was measured using acridine orange (AO) and propidium iodide (PI) staining. 20 μL of AO/PI staining and 20 μL of live cells were combined, and then 20 μL of the mixture was inserted into the Nexcelom’s Cellometer (Model Auto 2000) and analyzed. The analysis included the number and concentration of live, dead, and total cells and the percent viability of the sample.

Particulate matter collection and its concentration distribution in cigar smoke

To characterize the particle size distribution in cigar smoke, TSITM‘s Dust Trak II (model 8530) was used with particle diameter cut-offs at 1 μm, 2.5 μm, 4 μm, and 10 μm at a 2 L/min sampling rate. Cigars were manually lit and puffed at 1.7 L/min for 1 minute using Scireq’s Inexpose system, with 2.5s puff duration, and 16.6s inter-puff interval. Cigar smoke particle sizes were measured in an Enzyscreen chamber (Cat. CR1601) with dimensions 22 cm x 14.5 cm x 16 cm and in a DSITM chamber with dimensions 44.8 cm x 30.1 cm x 29.6 cm [20].

Statistical analysis

Statistical analysis of significance was calculated using one-way ANOVA as well as Tukey’s post-hoc test for multiple comparisons by GraphPad Prism Software version 8.1.1. The results are shown as mean ± SEM of average n = 3 to 4. Data were considered to be statistically significant for P values <0.05.

Results

Categorization of cigars by flavor

To understand how the flavorings in cigars can be grouped, a new classification system was created (). This design was used to allow for categories and subtypes. Presently, there are no classifications that convey the flavors in a meaningful way. This figure attempts to do that in a manner which is easy to understand and follow for toxicological studies. There are six main flavor categories, drinks, fruit, tobacco, menthol, candy, and spices with sub-categories, alcohol, black and mild, berry, tropical, pineapple, peach, grape, watermelon, mango, and cherry. Fruit category contained the most flavors as well as the largest number of subtypes.

Acellular ROS production by little cigars and cigarillos

Little cigars and cigarillos produced differential H2O2 equivalents (Figs . For method #1, many of the flavored cigarillos showed a significant increase in acelluar ROS compared to air with the exeption of Black and Mild’s tobacco, Swisher Sweets’ blueberry, cherry from both Pt.Rillos and Jackpot, Swisher Sweets’ boozy mango, tropical fusion and caramel peach and finally Black and Mild’s wine. The highest aceulluar ROS produced was from Dutch Masters’ Mint Fusion, Game’s white grape and berry blast, White Owl grape, and Wine by Dutch Masters. Little cigars tested from Djarum, black and special, were both significantly higher in ROS than air and produced more ROS than a 3R4F. While each category has at least one low ROS producing cigar many of these cigars tested have higher ROS than a 3R4F ().

Generation of ROS by different flavors and brands.

Acellular ROS was measured in samples of various CSE (3R4F and others) using a hydrogen peroxide standard. The CSE was obtained using puffing method #1, where a SKC lab pump was used to continuously puff a cigar or cigarillo for 1 minute at 1 L/min. Names of cigars/cigarillos are listed below with their brand in parenthesis. Combined results of all the flavors tested by method #1. All flavors were compared to the control value of air. Data shown as mean ± SEM, and significance was determined by one-way ANOVA. * p< 0.05, ** p< 0.01, and *** p< 0.001 versus air controls.

Acellular ROS was measured in samples of various CSE (3R4F and 1R6F) using a hydrogen peroxide standard. The CSE was obtained using puffing method #2, where a general vacuum lab pump was used to continuously puff a cigar or cigarillo for 10 seconds. Names of cigars/cigarillos are listed below with their brand in parenthesis. Combined results of all the flavors tested by method #2. All flavors were compared to the control value of air. (. Data are represented as mean ± SEM, and significance was determined by one- way ANOVA. * p< 0.05, ** p< 0.01, and *** p< 0.001 versus air controls.

Generation of ROS by tobacco and menthol flavors and brands.

Acellular ROS was measured in samples of various cigarillos using a hydrogen peroxide standard. The puffing method #2 was used, where a general vacuum lab pump was used to continuously puff a cigar or cigarillo for 10 seconds. Names of cigars/cigarillos are listed below with their brand in parenthesis. Black and Mild, Tobacco, and Menthol flavors were compared to the control value of air. Data are represented as mean ± SEM.

Generation of ROS by fruit, candy, drink, and spice flavors and brands.

Acellular ROS was measured in samples of various cigarillos using a hydrogen peroxide standard. The puffing method #2 was used, where a general vacuum lab pump was used to continuously puff a cigar or cigarillo for 10 seconds. Names of cigars/cigarillos are listed below with their brand in parenthesis. Fruit, berry/cherry, candy, drinks, and spice flavors were compared to the control value of air. Data are represented as mean ± SEM.

Generation of ROS by different portions of a cigar.

Acellular ROS was measured in samples of CSE from 2 different cigarillos using a hydrogen peroxide standard. CSE was collected using method #3, where a cigar was partitioned into 3 segments and a general vacuum lab pump was used to continuously pull smoke until cigar was completely burned. Representative example includes Blueberry and Wild Rush cigarillos. Data are shown as mean ± SEM, no significant differences between the sections/segments.

Method # 2, similar to method #1 showed many of the cigars and cigarillos tested having signicantly higher acelluar ROS production than air (). Tested cigars were grouped based on their categories and depicted in . Highest significance was seen in the categories Black and Mild, Tobacco, and Spice with both Djarum cigars being the most significant (). Lowest ROS production was seen in categories Candy, Grape, Mango and Peach. Individual cigars and cigarillos tested for ROS productions which show a differential response (Figs ). Also, we are interested in how different segments of cigars would affect the ROS production. There was no significant difference among all the segment ( based on method #3 of ROS assay, which means cigar burned in a universal way, and oxidative stress generated continously and universally.

Most notably, between methods #1 and #2, it is easy to see that each method gives significantly different results even when testing the same cigarillo. Within method #1, many of the cigarillos burned down faster than others from the same brand and flavor. This resulted in a wide variations of ROS values for a single cigarillo. For example, PT Rillo’s wine flavor was tested four separate times using four separate cigarillos. The range of these tests was 49.6–10,788.80 H2O2 equivalents.

Fine particle emission and distribution by flavored little cigars and cigarillos

For all particle size measurements, research cigarettes (3R4F/1R6F) released lower concentration of particles, i.e., particulate matter (PM). The lowest PM from cigars/cigarillos tested was black by Djarum, and the highest was honey fusion by Dutch Masters, special by Djarum, and wine by Black and Mild (). Most of the cigar samples tested had the highes PM concentration in categories 1 μm and 2.5 μm (). shows the average PM concentration for each of the seven categories and two reference cigarettes only to be used as a visual representation of . depicts the PM concentrations of one cigar per category at 1.0 μm. Mint fusion by Dutch Masters and mango by Swisher Sweets have the highest concentrations, whereas a reference cigarette and tobacco by Swisher Sweets are the lowest.

Particulate matter from flavored cigars.

A) Maximum concentrations of particulate matter in 1.0 filter from representative cigars. Cigarillos and a 3R4F cigarette were each burned two separate times for one minute. Each time when the cigar was burned, the concentration of particles that were 1.0 μm or smaller was obtained and the maximum in that minute was plotted. Data are shown as mean ± SEM. B) The maximum concentration of particulate matter from key representative cigars. Measurements were taken for one minute for each filter size (1.0, 2.5, 4.0, and 10.0 μm). The average maximum concentration for each particle size within the minute of the burning of cigars was plotted.

Exposure to cigar flavorings induces variable cellular toxicity

To determine the cytotoxicity of flavored cigars, Beas-2b cells were treated 0.5% and 1% cigar/cigarillo extract. Overall, each cigar showed a decrease in cell viability from 0.5% treatment to 1% treatment except for Djarum’s black cigar (). Surprisingly, djarum cigars, tobacco flavor and menthol flavor do not show any significant decreasing in cell viability, while all other fruit, candy, and drink flavors showed reduction of viability. Most significant cytotoxicity can be seen in PT Rillos’ wine flavored cigarillo that had viability reduced to 43.9% with 0.5% extract treatment and with a 1% treatment, viability reduced further to 33.6%. At 1% extract treatments Swisher Sweet’s caramel peach had a viability reduction to 36.97%, PT Rillos’ Strawberry was reduced to 27.2%, and GAME’s pineapple reduced to 49.2%. Cell treatment with a 1% extract of a 3R4F cigarette showed a significant reduction in cell viability as well, with an average of 27.7%. Almost all cigars tested had a reduced in cell viability compared with the control, but were not more cytotoxic than a research cigarette ().

CSE treatment induces cell toxicity in human bronchial epithelium.

Beas 2B cells were treated with various flavored and unflavored CSE for 24 hours. Each cigarillo treatment was done in 2 concentrations, 0.5% and 1.0%, these are depicted respectively for each group. Flavor is listed with brand in parenthesis. A 3R4F reference cigarette was used for comparison and untreated cells under the same conditions were used as a control. Cell viability was measured using Nexcelom’s Cellometer and AOPI dye. Viability percentages are shown as mean ± SEM, and significance was determined by one-way ANOVA. * p< 0.05, ** p< 0.01, and *** p< 0.001 as compared with controls.

Discussion

This study intended to determine the toxicity of flavored cigarillos and little cigars. Selected little cigars/cigarillos from various flavor categories were combusted and acellular ROS, particle concentration/distribution, and the cell viability were assessed. Classification of flavored cigars/cigarillos should provide a convenient nomenclature/vocabulary in the scientific community as there was no scientific classification of cigar flavorings until the present. The classification system created, allows for the categorization of flavors in a way that groups like chemicals together, in an attempt to single out which flavors can be the most toxic. Otherwise, introduction of additional flavorings in cigars or any tobacco products will make comparison between categories more difficult.

The ROS produced by tobacco products are complex and not fully understood. However, it can be broken down into two phases, the gas and the tar phase [21]. Mainstream tobacco smoke constituents react with each other forming reactive oxygen and nitrogen species, including superoxide, hydrogen peroxide, hydroxyl radical, and peroxynitrites [22]. Oxidation of biomolecules, such as proteins and lipids and the formation of DNA lesions are mechanisms of toxicity of tobacco smoke exposure and disease progression [23]. It is widely known that cigarette smoke generates ROS and has significant carcinogenic properties as depicted in dose-dependent studies [24, 25]. In this study, we observe the ROS generated by most of the cigarillos tested in our study is significantly higher than conventional research cigarettes (University of Kentucky 3R4F or 1R6F). This suggests that cigarillos and cigarettes not only induce higher oxidative stresses, but different brands of cigarillos and little cigars showed variable toxicological effects dependent on different chemicals generated during combustion. Corroborating our data, another study showed a greater emission of total semivolative organic compounds (SVOC) and volatile organic compounds (VOC) in cigarillo smoke compared to regular cigar or cigarette smoke [26].

Most notably, between methods #1 and #2, it is easy to see that each method gives significantly different results even when testing the same cigarillo. Within method #1, many of the cigarillos burned down faster than others from the same brand and flavor, which might use multiple cigars during one run of measurement that might cause batch-batch variations. For example, wine flavors from methods one and two one can again see a wide variation, where PT Rillos is found to be significantly higher than Dutch Masters using method #2, but method #1 shows an opposite trend.Currently, regulations on cigarettes focus on very few ingredients when compared to the total 5,000 chemicals found in them, allowing for variation between brands and batches [27]. This variation is also seen in other tobacco products, such as e-cigarettes [28, 29]. In agreement with our findings, Hamad et al recently reported that the flavored cigars/cigarillos (Cheyenne menthol and Swisher sweets original and cherry) have more potentially harmful constituents (HPHCs) including volatile organic hydrocarbons, increased tobacco mass, and total particulate matter (TPM) compared with conventional research reference cigarettes (3R4F) [4]. Hence, HPHCs and particulate matter (PM) including ROS released by cigars would contribute to cellular/tissue injury in the lungs. Cigar smoke produces fine particles altering iron homeostasis, which would easily deposit in the lungs and leading to damage of nearby lung tissue [18]. Our data showed various ranges of particles from flavored cigars (from 1.0 μm to 4.0 μm with higher mass concentration) vs. research-grade research and filtered cigars. In flavored cigars, the particle distribution/concentration was significantly higher in compared to other filtered cigars or research cigarettes, suggesting that composition of flavored cigars generate broader particles with higher concentrations which might be even more harmful than cigarette smoke [30-32]. Further studies are required to determine the grading of various commercially available cigars for their HPHCs, volatile organic compounds (VOCs), and TPM, and their products chemical analyses to ascertain the toxicity in the realm of cellular toxicity especially with flavored cigars/cigarillos and flavoring chemicals used.

In a recent study, it has been shown that increased concentration of the cigar extract led to increased cell death [1, 33]. Although our study found that cigarettes had a higher percentage of cell death compared to most cigarillos or cigars, other studies have noted little cigars to be more toxic when compared to cigarettes [1, 33]. This could be due to the increased amount of chemicals that are found/released in little cigars as compared to cigarettes [33], or the differential interactions of various chemicals during combustion and cellular components. When compring at other tobacco products like e-cigarettes, cell damage still occurs even with the absence of nicotine [34, 35].

Other studies have shown, in particular, the flavorings cinnamaldehyde, O-vanillin, and pentanedione are significantly more cytotoxic on human cells than other flavoring chemicals [36]. Cinnamaldehyde and other aldehyde compounds are frequently used as flavorings, although the FDA and Flavors Extract Manufacturers Association (FEMA) both agree they have adverse effects on human health [37]. Grape and alcohol were common flavors for cigarillos and little cigars. The grape flavoring in food is often attributed to the compound methyl anthranilate, and alcohol flavors come from ethyl-decadienoate. These compounds are considered safe in food; however, there is little information on the combustion and inhalation toxicology effects of these chemicals [38]. Our data demonstrated that higher cytotoxic responses with fruits/candy and drinks/beverages/alcohols flavored cigars compared to tobacco flavor, which points the harmful possibility of the flavored chemicals. More detailed studies of those flavored chemicals, such as methyl anthranilate or ethyl-decadienoate, are needed to further understand the mechanisms.

Due to the high variability of each manufacturer’s/company’s flavorings (including batch to batch variations), future research should be directed with cellular methods and mouse models to take a further step in studying the respiratory toxicity [35]. In addition, it would also be beneficial to determine which chemical flavoring (in flavors) is the most deleterious. This has already been conducted in flavors present in e-liquids by studying barrier dysfunction and cellular toxicity in a cellular system [39]. Once more information on the chemical make-up of flavorings and cigars are obtained, the regulatory agencies should increase regulations and/or ban these flavored cigars. Our data showing differential responses due to the different flavors, manufacturers may limit the use of the chemical ingredients in the most toxic flavors (subject to products analysis), or remove flavors all together from cigar products.

In conclusion, despite the high brand-to-brand variability between flavored cigars or cigarillos, those with fruit, candy, and drink flavors tend to show more deleterious effects in our assays. Further, it is difficult to ascertain the consistent toxicity data due to the differences in chemical composition within a specific flavor of the cigar and cigarillo. Flavored cigars produced maximum particles/particulates compared with research cigarettes. The cellular assays resulted in variable cytotoxic responses with fruits/candy and drinks/beverages being more cytotoxic. Due to the irregular composition of flavors and flavoring chemicals, it is important to regulate flavored little cigars/cigarillos. Future research may be directed to determine the cellular methods as well as in vivo models to fully determine the flavoring toxicity in flavored little cigars/cigarillos.

A complete list of all little cigars and cigarillos tested based on flavor category and sub-category in this work.

(XLSX)

Click here for additional data file.

29 Aug 2019

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,

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.

We would appreciate receiving your revised manuscript by October 5th, 2019. 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,

M. Firoze Khan, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

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.

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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.

**********

6. 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

[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.

22 Oct 2019

Manuscript ID: PONE-D-19-21934

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

Authors: Gina R Lawyer*, Monica Jackson*, Melanie Prinz, Thomas Lamb, Qixin, Wang, Thivanka Muthumalage, and Irfan Rahman

Overall Response to Reviewers:

We are very grateful for the time the editors and reviewers took to review this manuscript and provided their valuable comments/suggestions. We made sure to review our manuscript thoroughly and answer each question or suggestion that is posed.

Comments and Responses:

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.

Response: We are extremely thankful for the kind comments and recognition. We have made sure our best effort to review the data organization and analysis to improve this manuscript.

Major Comments: Table should contain more flavors based on the classified tree of flavors

Response 1: We agree with the review’s comments, we also think more flavors based on our classification are needed and it’s really necessary for quantification and validation. Please check Figures 3 and 5, and Table 1, as suggested, we have ran several more cigars for ROS, and for particulate matter analyses in the revised manuscript.

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

Response 2. We are thankful for this kind suggestion regarding better understanding of physical properties of different flavored cigars. As showing in our revised Table 1, different flavored cigars are added, and physical particular matter concentrations are shown in 4 different diameters with units of mg/m3 (see Table 1 and Figure 5).

Major Comments 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.

Response 3: We agree with the reviewer’s comments and concerns. We have carefully analyzed more acellular ROS using different flavored cigars, and analyze the statistical significance accordingly. As we mentioned in our discussion, due to the batch-batch variation of the commercial available cigars, part of results are still scattered. However, most of the cigars showed significant harmful effects even compared to research cigarette smoke.

We are really thankful for the suggestion of highlight the fruits/drinks flavored cigars. We have revised it accordingly in our results and discussion in the main text of the revised manuscript.

Major Comments 4: Fig 3B, C should contain statistical analyses.

Response 4: We thank the reviewer for their suggestion. We have applied statistical analysis accordingly to Figures 3b and 3c.

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

Response 5: We thank the reviewer for their suggestion. We have added a control of air to figure 4.

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

Response 6: We thank the reviewer for their suggestion. More flavors have been tested for their particulate matter and acellular ROS. In Fig 3, more flavored cigars have been tested in ROS, several flavored cigars have been tested in this study. In table 1, several flavored cigars have been characterized with PM distribution and concentration; various flavored cigars have been measured.

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.

Response: We are extremely thankful for reviewer’s 2 encouraging comments and suggestions. We made sure to thoroughly address each suggestion or question.

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

Response 1: We agree with the reviewer’s comments. We have addressed this comment previously. Please refer to the earlier response (reviewer #1, comment #1).

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

Response 2: As suggested, we have increased the size of Figure 1 and the font within the figure so that it is clearer for readers.

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

Response 3: We agree with the reviewer’s comments. We have the details that described the information of each cigar used with their brand, flavor based on packaging, and which category they have been included in the revised manuscript.

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

Response 4: We agree with the review’s comments. We have cut the cigar in different portion and measured the ROS generation. As showing in Figure 4, there is no significant difference among different portions.

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

Response 5: As suggested, more cigars have been tested and included into Figure 5 and table 1. We have addressed the similar comments (see above); please check the previous responses (reviewer #1, comments #6)

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

Response 6: We agree and understand reviewer’s comments and concerns. Generation of particulate matter affects the cellular uptake of chemicals or particles from cigar combustion, and it does participate in the cellular toxicity responses, as well as acellular ROS produced by cigars. However, there are more factors affects the cigars, such as the chemical compounds and deposition of cigar particles, all have cytotoxic influence. Though, we cannot fully connect the ROS and particulate matter data to our cell viability results, more detailed studies, such as cell toxicity test with different flavored chemicals, are in progress to figure out what is the key points caused cytotoxicity.

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

Response 7: we thank for the reviewer’s comments/suggestions. We have picked up the key flavors that represent each flavoring category, as well as more cigars were used to test the ROS and particulate matter. Because of the cigars from different brands and even the different batch showed variations, there is no significantly specific trend across flavor and ROS. However, all the flavoring cigars showed higher ROS generation than research cigarette smoke.

Additional notes from Reviewer #2: 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.

Additional Notes Response: We thank the reviewer for his/her valuable suggestion, we have corrected and replaced accordingly.

Submitted filename: Lawyer et al responses.docx

Click here for additional data file.

5 Nov 2019

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

[Note: HTML markup is below. Please do not edit.]

[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.

10 Nov 2019

Manuscript ID: PONE-D-19-21934R2

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

Authors: Gina R Lawyer*, Monica Jackson*, Melanie Prinz, Thomas Lamb, Qixin, Wang, Thivanka Muthumalage, and Irfan Rahman

Academic Editor Comments:

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:

Specific Comments:

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”

Response: We have changed the title as suggested.

Comment:

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

Response: This has been changed.

Comment:

- 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.”

Response: This has been amended.

Comment:

- 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”

Response: This has been changed.

Comment:

- 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”

Response: This has been changed.

Comment:

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

Response: This has been changed.

Comment:

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.

Response: We have changed the Figure 1, and legend is amended as suggested.

Comment:

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.”

Response: This has been changed as suggested.

Comment:

- 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.

Response: This has been changed as suggested

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.

Response: we have now copyedited for grammar and usage in the revised manuscript.

Submitted filename: Lawyer et al responses R2.docx

Click here for additional data file.

20 Nov 2019

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.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

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:

2 Dec 2019

PONE-D-19-21934R2

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

Dear Dr. Rahman:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. M. Firoze Khan

Academic Editor

PLOS ONE

Table 1

Comparative particulate matter concentration of different particle size in flavored cigarillos comparing with research cigarettes.

Category	Name (Brand)	1.0 μm	2.5 μm	4.0 μm	10.0 μm
3R4F	Research grade cigarette	0.49± 0.71	1.91± 0.52	0.58± 0.42	0.80± 0.48
1R6F	Research grade cigarette	101.60± 3.40	117.90± 29.10	110.00± 5.00	98.30± 6.70
	Black (Djarum)	5.02± 3.05	4.02± 1.09	32.42± 29.78	47.86± 45.45
	Special (Djarum)	329.50± 22.50	400.00± 0.00	381.50± 18.50	397.00± 3.00
Black and Mild	Casino (Black and Mild)	150.50± 29.50	175.50± 5.50	117.50± 17.50	77.00± 3.00
	Crème (Black and Mild)	400.00± 0.00	400.00± 0.00	400.00± 0.00	396.00± 0.00
	Jazz (Black and Mild)	114.00± 20.00	160.00± 12.00	125.50± 3.50	154.00± 16.00
	Regular (Black and Mild)	180.00± 72.00	195.50± 48.50	158.00± 1.00	304.50± 75.50
Tobacco	Emerald (White Owl)	55.00± 16.00	114.00± 31.00	104.50± 20.50	151.50± 26.50
	Green Sweets (White Owl)	64.60± 23.00	143.50± 15.50	110.45± 22.55	80.90± 37.10
	Red Sweet (GAME)	72.00± 8.00	144.50± 51.50	89.00± 29.00	84.50± 14.50
	Sweet Aromatic (GAME)	14.90± 3.4	28.40± 0.40	23.45± 2.55	38.05± 0.35
	Tobacco (Swisher Sweets)	300.50± 61.50	248.50± 75.50	390.00± 10.00	363.00± 37.00
Menthol	Artic Ices (Swisher Sweets)	398.00± 0.00	275.00± 65.00	213.50± 65.50	371.00± 3.00
	Mint Fusion (Dutch Masters)	227.00± 40.00	216.00± 22.00	209.00± 39.00	154.05± 69.95
Fruit	Blueberry (Swisher Sweets)	114.45±21.55	86.65± 18.35	70.45± 9.95	108.50± 13.50
	Strawberry (White Owl)	146.85± 129.15	17.73± 17.06	116.90± 92.10	56.30± 15.60
	Cherry (Jackpot)	299.00± 14.00	286.00± 114.00	350.00± 18.00	281.00± 32.00
	Grape (GAME)	153.00± 16.00	110.50± 2.50	134.00± 4.00	144.50± 27.50
	Grape (Swisher Sweets)	260.50± 38.50	373.00± 27.00	379.00± 21.00	278.00± 41.00
	Grape (White Owl)	161.50± 26.50	108.00± 16.00	73.50± 17.50	154.00± 34.00
	Grape (Zig Zag)	55.70± 21.90	125.00± 21.00	216.50± 91.50	132.35± 62.65
	Mango (Swisher Sweets)	354.50± 27.50	368.00± 26.00	242.50± 156.50	310.50± 89.50
	Peach (Swisher Sweets	79.40± 15.60	106.50± 2.50	123.05± 23.95	128.05± 40.95
	Peach (White Owl)	92.20± 19.80	100.65± 6.35	108.85± 19.15	161.00± 6.00
	Pineapple (GAME)	73.55± 2.05	50.00± 1.70	52.20± 2.30	72.20± 14.00
	Pineapple (White Owl)	137.50± 4.50	117.50± 8.50	124.00± 15.00	75.15± 6.85
	Tropical Fusion (Swisher Sweets)	140.00± 4.00	129.00± 0.00	202.00± 0.00	197.00± 10.00
	Tropical Fusion (Swisher Sweets)	140.00± 4.00	129.00± 0.00	202.00± 0.00	197.00± 10.00
	Berry Fusion (Dutch Masters)	24.50± 5.50	89.00± 2.00	112.50± 35.50	75.50± 8.50
Candy	Brownie (GAME)	298.00± 59.00	292.00± 14.00	210.00± 53.00	290.00± 63.00
	Carmel Peach (Swisher Sweets)	286.00± 114.00	400.00± 0.00	316.50± 44.50	227.50± 14.50
	Chocolate (Swisher Sweets)	216.00± 126.00	297.00± 79.00	382.50± 17.50	219.00± 39.00
	Honey (GAME)	261.50± 24.50	266.50± 14.50	258.00± 20.00	320.00± 47.00
Drinks	Java Fusion (Dutch Masters)	303.50± 96.50	380.50± 19.50	268.50± 12.50	392.00± 6.00
	Honey Bourbon (White Owl)	371.50± 28.50	253.00± 11.00	397.50± 2.50	359.00± 41.00
	Sangria (White Owl)	273.00± 9.00	395.50± 4.50	398.00± 2.00	394.50± 5.50
	Spiked Lemonade (White Owl)	354.50± 45.40	365.00± 25.00	392.00± 8.00	400.00± 0.00
	Swerve (Swisher Sweets)	333.33± 66.66	221.50± 61.50	394.50± 5.50	400.00± 0.00
	Wine (Black and Mild)	316.50± 83.50	276.00± 123.50	386.00± 14.00	390.50± 9.50
Spices	Cream Vanilla (Swisher Sweets)	317.00± 6.00	307.00± 5.00	216.50± 61.50	234.00± 11.00
	Vanilla (White Owl)	151.00± 11.00	141.50± 15.50	246.00± 33.00	323.50± 70.50

The maximum concentration of particulate matter (sizes 1.0, 2.5, 4.0 and 10.0 μm) for each filter size over the course of a one minute test are reported in the table. Values are given in mg/m3. Each cigarillo or cigar tested in listed under their respective categories, their name and brand is reported as it appears on the wrapper. Data are reported as mean ± SEM.