Advancements and Challenges of Cigar Science, Testing and Regulation: A Review

All testing, whether content or yield related, are impacted by the tobacco and ultimately the growing conditions of that tobacco. There have been recent efforts to increase understanding in this area for cigar tobaccos. Some have argued that testing and reporting multiple constituents in cigar leaf and smoke without having in-depth knowledge of what drives the variability/variations will engender the submission of somewhat valueless and inconsequential data to the regulatory institutions (4). Generally, controls to minimize year-to-year variability from seed planting and harvesting to the finished tobacco leaf remain a challenge. Variability in cigar tobacco is a well-known issue and FDA has acknowledged that blend changes due to “natural variability” do not require a product to undergo premarket review

Deeming Tobacco Products to be Subject to the Federal Food, Drug, and Cosmetic Act, as Amended by the Family Smoking Prevention and Tobacco Control Act; Restrictions on the Sale and Distribution of Tobacco Products and Required Warning Statements for Tobacco Products, 81 Fed. Reg. 28,974, 28,996 (published May 10, 2016) (the “Final Deeming Rule”).

Cigar tobaccos, like cigarette tobaccos, have defined categories (such as dark air-cured and sun-cured tobaccos). Within each of the categories are numerous sub-types such as Sumatra, and Jatim), and varieties (such as Vuelta Abajo). Unlike cigarette tobaccos, cigar tobaccos have little to no standardization and are typically local varieties produced from suppliers and even farm-based selections. Unlike cigarette tobaccos, there are limited varieties produced through any type of seed certification process. So, though the total number of cigar tobacco varieties is much lower than that of cigarette tobaccos, standardization is significantly lower for cigar seeds and the range in seed sub-types and varieties is much greater (3, 4). In addition, the soil and climate conditions of the growing area are significant factors impacting variability of cigar tobacco physical and chemical properties (5). Knowledge of the relationship between the different types of soil, climate and the varieties of crops allows tobacco breeders to produce and distribute seeds specifically adapted to specific growing locations. For example, LUNDH affirmed that the strength, elasticity, thickness and shining quality of cigar wrappers strongly depends on the type of soil and climate in which the tobacco seed is planted. He claimed that even when Nicaraguan seed is planted in Ecuador, the tobacco wrapper produced is very different from a native Nicaraguan wrapper. He explained that the humidity from the constant cloud cover in Ecuador yields firm and elastic wrappers while the volcanic soil type in Nicaragua yields wrappers that are less elastic (6).

Lindegaard reported results for a controlled study whereby the same dark air-cured tobacco seed was planted in the same crop year by two different farmers in the same country and local area. A very significant natural variability was quantified including a 379% difference between the arsenic content, 115% difference in N-nitrosonornicotine (NNN) levels, 53% difference in ammonia content, 31% difference in 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) levels, 12% difference in cadmium content and 11% variation in the nicotine content (4).

Lindegaard's findings accentuated the existence of an innate variability within the cigar product due to variations in the tobacco itself. Additionally, Mukoyi et al. advised researchers, investors, leaf merchants, policy makers, and other stakeholders to trade and invest in the viable high-quality cigar wrappers, binders, and filler grown in Zimbabwe. They reported that conditions in the Burma valley area in Zimbabwe, such as ideal loamy to loam-clay well drained soils, high geographical altitude, high ambient relative humidity > 65%, high temperatures (28–35 °C), and high rainfall > 1000 mm/week, promote the growth of pliable fine textured, unique wrapper leaf, which favors the design of premium cigars. They demonstrated that the soil and environment in that precinct are also conducive to performing agronomic germ plasm holistic research to produce a various preeminent shade cloth-cured tobacco leaves capable of resisting foliar diseases like frog eyes, deformations from the scorching sun, droughts and hailstorms (7).

The effect of soil nitrogen content has been shown to impact certain quality attributes of Kentucky dark fire-cured tobacco. Sifola et al. showed that increasing the soil nitrogen impacted the brightness and increased “body” (thickness, density, and weight) of the leaf (8).

Another research group led by Borges performed experiments to establish the optimum nitrogen concentration for percent yield and quality of cigar tobacco based upon leaf chlorophyll content (9). Other researchers have found that nitrogen fertilizer impacted alkali metals like potassium but not alkali earth metals like calcium and magnesium (10). Over a threshold level of nitrogen, however, leaf quality declined rapidly (11) and potassium fertilizers may positively impact yield (12).

Monzón Herrera in collaboration with the University of Hohenheim, Germany, utilized greenhouse (hydroponic and pot farming) techniques to examine the effects of some micronutrients and macronutrients on the development and growth on four Cuban dark tobacco varieties used for “Habanos” cigars. Their extensive study showed that soil types deficient in micronutrients like boron, phosphorus, zinc, magnesium, potassium nitrogen and manganese resulted in various detrimental foliar diseases and defective stem growth patterns in the tobacco plant. They ascertained that the soil nitrogen content strongly impacted the composition of metallic cations in the green tobacco leaf. In conclusion the group advised that (i) early application of nitrogen is needed for intensive vegetative growth of the tobacco plant on landscapes that have compact soils which rarely undergo lixiviation of nitrates, and (ii) for farmers to obtain the best yield and quality harvest the nitrogen content must be increased to about twice the current recommended amount required for soil fertilization (13).

There has been significant research conducted in the area of Crop Protection Agents (CPAs) specifications and application in tobacco farms as well as Good Agricultural Practices (GAP). Vann and Fisher conducted a study on the effects of three CPAs (azoxystrobin, butralin, and flumetralin) on flue-cured tobacco grown in six different areas in North Carolina, USA. The goal of the researchers was to evaluate residue levels of the CPAs in order to administer their proper application rates as well as to determine the minimum preharvest interval application of specific CPAs. They gathered their data based on individual year, individual location, individual CPA, and individual stalk position (lower, middle, and upper) of the tobacco plant. They concluded that although residue concentration of azoxystrobin was quite high compared to butralin and flumetralin, azoxystrobin played a critical role in the control of target spot (Rhizoctonia solani), a foliar disease which was responsible for a 7% yield loss in North Carolina in the 2013 growing season (14).

In 2017 the CORESTA Agro-Chemical Advisory Committee (ACAC) developed and documented various trials performed by different companies across the globe to standardize and mandate specific Cigar Guidance Residue Levels (C-GRLs) for dark air-cured tobacco. Some of the mandates for farmers included strictly controlling fertilization of the soil, proper leaf variety selections, systematic curing strategies, proper topping and suckering as well as optimizing the fermentation process of the cigar leaves. To further bolster the standardization initiative, CORESTA also launched the Agrochemical Residue Field Trials Task Force (RFT-TF) which focused on the development of new agrochemical candidates for setting GRLs in terms of leaf quality and integrity to draw clear distinction between cigar leaf and cigarette tobacco leaves. The task force compared the yields and CPA residues data at different stalk positions between two crop protection programs (the local one, normally based on CPA application relative to when the pathogen is present and the worst-case scenario based on weekly CPA applications), to confirm which was the most effective for eradication of the three main fungal and insect-related tobacco diseases (15).

Differences in conventional cigarettes typically result from variations in tobacco blends and relatively small variations in cigarette construction and physical dimensions such as length, diameter and pressure drop (16). In the case of cigars, the physical parameters vary greatly within and across product categories. In fact, there are several categories of cigars; each consisting of tobaccos that are unique and different from each other. The two major categories which are premium and machine-made cigars (MMCs) are discussed below.

3.3.1

Premium cigar design characteristics

“Premium”, handmade, hand-rolled, (or long-filler) cigars consist of whole tobacco leaves that, when rolled, run the length of the cigar. Long-filler cigars are of a higher quality than short-filler or medium-filler cigars and tend to burn for a longer time. Most “premium” cigars are made entirely of long-filler tobacco, wrapped in a quality natural tobacco binder and wrapper. Tobacco is sorted, bunched, rolled, molded, and pressed by hand. Finally, the outer wrappers are added. During quality control evaluations, cigars are color matched for packaging (3).

Figure 2 shows a premium cigar design with sections labelled using typical vocabulary while Figure 3 shows the layers/parts of tobacco leaf used for making cigars (17, 18). Typically, the premium cigar body is composed of the wrapper (outer tobacco), binder, and the filler (inner tobacco). Generally, the wrappers are harvested from plants cultivated under shade (shade grown tobacco) whereas the fillers and binders are cultivated under full sunshine (sun grown tobacco). The binders are leaves selected from the lower part of the tobacco stem and should be wide, large, and undamaged as possible. During manufacturing, the binder is rolled around the filler leaves and are together referred to as the bunch. The filler leaves are composed of three proportions or varieties (from bottom to top positions of the stem, respectively) namely, volado (filler itself, which mainly contributes to combustibility), seco (dry, mainly contributes to aroma), and ligero (light, mainly contributes to strength). During the cigar hand rolling formulation process, the ligero leaves are sandwiched between the volado and seco leaves (7, 19). The wrapper leaf which is finally wrapped around the bunch must have excellent pliability and elasticity.

Figure 2

Figure 3

Figure 4 (a) shows the art of hand-made premium cigars and (b) an ideal cigar wrapper (15). It is reported and putative that the filler contributes about 85% of the total cigar weight, the binder 10%, and the wrapper the remaining 5% (20).

Figure 4

Much of the recent analytical research for cigars has been designed as a comparison to conventional cigarette results. Some research has been focused on smoking perception but much of the work has been in the area of comparing analytical content and yield or product variability. Two notable differences with regard to cigar smoke are (i) cigar smoke tends to be more alkaline than cigarette smoke and (ii) tobacco commonly used for cigars contains lower levels of reducing sugars than the rapidly dried varieties of tobaccos commonly used in cigarettes. Normal mouth (buccal) saliva is known to be neutral or slightly basic. The impact of the higher alkalinity of cigar smoke (pH 8.5) on nicotine absorption has been studied by Armitage and Turner. The group asserted that because of the higher concentration of unprotonated nicotine in alkaline medium, nicotine in cigar smoke is much more readily absorbed through the buccal mucous membranes than the protonated nicotine in acidic cigarette smoke (pH 5.3) (22). Extensive research by Leffingwell corroborated the complexity and variability in the chemical composition of tobacco leaf types (yellowed, flue-cured, Burley, Oriental, enzyme treated, Virginia, etc.) grown in different parts of the globe (23). They reported that Oriental leaf exclusively contained significant amounts of labdanoid (Z-abienol) and sugar tetraesters, which impact sensory attributes of the Oriental leaf. Quantitative examination of the cuticular tobacco components of cigar leaf and other leaf by Severson and associates outlined striking differences in the levels of analytes such as hydrocarbons, sucrose esters, docosanol and diterpenes (24). Table 1 (adopted from the Tobacco Monograph by Hoffmann and Hoffmann) points out unique chemical composition differences between cigar tobacco and four selected cigarette tobaccos. For example, the table depicts that cigar tobacco contains less than 0.1% polyphenols relative to tobacco blends used for conventional cigarettes (1.6–5.1%) (25). Conversely the cigar nitrate content appears higher than for cigarette tobaccos. Additionally, the burn characteristics of the cigar products differ from conventional cigarettes because cigars typically burn “inside – out” (tunneling) as opposed to the “outside – in” burn characteristics of conventional cigarettes (26). Clearly one would expect that the pyrolysis product profile for cigars will thus differ significantly from that of cigarettes.

Table 2 identifies differences between selected volatile components in the smoke of cigars, little cigars, and cigarettes. The concentrations of nitrogen oxides (NO_x) are significantly higher in cigar smoke compared to cigarette. This is attributed to the elevated nitrate content of the cigar tobacco, the incomplete combustion, and the naturally low porosity of cigar binders and wrappers (25). In contrast, the ammonia content of cigar smoke is more than three times less than the amount in cigarette smoke. The sources and physical attributes (e.g., full length, filler length, weight, etc.) of the cigars and cigarettes used in the above study are defined in a previous investigation by Hoffmann and Wynder (27).

While testing of cigar smoke is more akin to in-use testing, understanding of tobacco content and variability is an important area of research with significant recent focus. Several studies have focused on fundamental understanding of the leaf. For example, Lauterbach and Grimm extensively investigated the chemistry of cigar wrappers used on MMCs from various brands (28). They identified biomolecules like deoxyfructosazines and fructosazines (sugar-ammonia biomarkers) in the wrappers. They proposed to further study the interactions of the biomarkers and tobacco fillers. In a separate study, Lin et al. observed that the chemical composition of cigar wrappers varied significantly from topping to maturation of the tobacco leaf wrappers. For example, during the first 21 days (from topping to maturation) the ratio of total nitrogen to nicotine decreased constantly whereas the sugar, calcium and magnesium contents increased (29). These results are consistent with composition from topping to maturation of tobaccos used for cigarettes. The effect of curing temperature on the fatty acids profile and ability of amylase and invertase to regulate the carbohydrate content of cigar wrapper leaf has also been reported (30, 31).

On the other hand, many of the recent tobacco studies have focused on understanding content and variability of analytes of regulatory concern. Typically, this is with an underlying objective of determining relevance and feasibility of routine Harmful and Potentially Harmful Constituent (HPHC) testing for this product category.

For example, Lindegaard studied dark air-cured cigar tobacco of the same leaf grade (i.e., same seed, country, local area, texture, color) grown by the same farmer from 2013 to 2015. He noticed significant variability in the composition of ammonia, nicotine, NNN, NNK, arsenic, and cadmium.

It is interesting to note that the difference in nicotine concentration between 2013 and 2014 was 89% whereas the difference in the same analyte between 2014 and 2015 was a nominal 2%. As discussed earlier, this researcher found marked differences in tobacco analyte content in a study wherein the same seed was planted in the same crop year by different near-by farms (4). Wagner et al. also carried out a point-in-time variability study on the smoke and tobacco. With regard to tobacco analysis, the focus of the study was cigarette and smokeless tobacco HPHCs: ammonia, arsenic, cadmium, NNK, NNN, and nicotine. They inferred that for ammonia and nicotine, the %RSD was the same for cigarettes and cigars at approximately 2%. However, for the other analytes, the %RSD of the MMC fillers was twice that of the cigarettes (32). Tayyarah et al. tested different cigar products at multiple laboratories for tobacco HPHCs. The choice of analytes was based on FDA requirements for cigarettes and smokeless tobacco products since there were no specified HPHCs for cigars at the time of the study (33). The design of that study included evaluation of the results to compare range of content and variability between cigars of different design, by laboratories testing cigars from the same lots but using their own methods. The group ensured that participating laboratories were ISO 17025 accredited and used validated methods. With regard to tobacco analytes, there was a clear difference in content for different cigars.

For example, nicotine ranged from approximately 8.3 mg/g to approximately 30 mg/g. However, more interesting findings from the study were that the reported values from the different laboratories for the same samples were in some cases different enough that, in a blind study, one may conclude the results were from different samples. For example, for Sample F, the tobacco NNN values reported by Laboratory 1 and Laboratory 3 were in a similar range at 1748 ng/g and 2050 ng/g, respectively. Laboratory 2 reported a value of 4497 ng/g for the same sample batch, which was more than twice that of the other labs. The differences in standard deviation of the nicotine values between the laboratories were particularly conspicuous with %RSD values of 0.8%, 14%, and 4% for Laboratory 1, 2, and 3, respectively. This supports the essence of current initiatives to increase standardization of testing, including a validated, internationally recognized methodology. Other reported studies on tobacco constituents are consistent with these findings. They include investigations by Koszowski et al. wherein they described extensive variability of the nicotine content and physical dimensions of cigars and cigarillos in the cigar market (34). This substantive difference in cigars has been studied and confirmed by other research groups (35, 36, 37, 38).

Some researchers have focused attention on physical parameters as a direct and practical measure of product variability. Testing for weight and length is relatively inexpensive with high throughput and low measurement variability.

Testing for diameter and pressure drop may be less reliable given the range of product designs and lack of standardization for measurement technology; it is advisable to limit comparisons of results for these measures between products of different design and/or between laboratories using different analytical methodology.

Wagner et al. found striking differences in magnitude of weight and pressure drop (referred to as resistance to draw in their work) when comparing a set of 10 hand-made cigars, 77 machine-made cigars, and 10 typical cigarettes. The researchers found that the relative weight of 100 replicates varied as much as 48% for hand-made products and 70% for machine-made products in the study but was typically less than a 14% spread for cigarettes (39). Teillet, Vernon and Colard presented findings from a study of diameter, length, weight, and pressure drop for hand-made cigars. Teillet reported low variability for direct control measures (length and diameter). However, measures not directly controlled during the hand-making process had point-in-time %RSDs of 40% (weight) and 120% (pressure drop) (40). The findings highlighted in this review are supported by additional studies conducted by other researchers (41, 42, 43).

There is a rich body of literature, inter-laboratory studies, and significant hands-on experience for testing constituents of conventional cigarettes. For example, standard validated ISO methods for analyses of polyaromatic hydrocarbons (PAHs) tobacco-specific nitrosamines (TSNAs), polyaromatic amines (PAAs), ammonia, chlorides, volatile organic compounds (VOCs), “tar”, nicotine, and carbon monoxide (TNCO) and metals in conventional cigarette smoke are well-documented, established across the tobacco industry, and in use in ISO-accredited third party labs. In addition, several cigarette smoking regimes (ISO, HCI, Massachusetts, CORESTA) and cigarette references have been established, beginning as early as the 1960s (44). In contrast, expertise and standardization with cigar HPHCs testing is substantially limited. For instance, there is a standardized puffing regime and handling requirements (described in CORESTA Recommended Methods (CRM) 64 and 65) (21, 45), but application of that regime to cigars for constituents beyond “tar”, nicotine, and carbon monoxide (TNCO) methods needs optimization for both method development and testing consistency across labs. Within the past decade, study designs, presentations, and publications have revealed the challenges encountered and strides achieved in cigar testing method development. The challenges include optimization of smoke holder accessories needed to accommodate different cigar sizes, lack of in-house method development for cigar analysis and inter-lab proficiency studies for both MMC and premium cigar products.

Recent reports related to analytical testing of cigar smoke have focused on understanding yield differences across the product category, often in comparison to conventional cigarettes, inherent variability of smoke analytes, and challenges with regard to smoking parameters and technology.

Wagner et al. presented results for a TPM (total particulate matter) comparison between cigarettes and machine-made cigars smoked under standard regimes. The cigarettes were smoked using the standard regimes (ISO, HC (Intense)) and the cigars were smoked using the cigar smoking CRMs referenced herein. The holder used for cigar smoking, an ALCS smoke trap, was a custom design (39). Figure 5 presents a striking example with the simplest smoking measurement (the weight of trapped particulates), to support observations of inherent product variability and the challenges of machine-smoking cigar products (39). It can be inferred from Figure 5 that the variability in TPM of the cigars was substantially higher than the two cigarette regimes. While the cigarette ISO and HCI smoking regimes yielded approximately 5–25% and 30–70% TPM variability respectively, the cigar variability was 40–120% which was over 70% higher than that of the cigarettes. In another study, Tayyarah et al. compared mainstream cigarette smoke analytes tested in different laboratories using their own in-house methods. The analytes under study were HPHCs typically performed on cigarettes such as carbon monoxide, smoke nicotine, selected carbonyls, VOCs, tobacco nicotine, tobacco ammonia, TSNAs, PAAs, and PAHs (33). Despite the fact that all the labs were ISO 17025 accredited and used properly validated methods, their conclusions were similar to most findings for tobacco analytes i.e., the analyte levels varied greatly between samples and between reported results from different laboratories testing the same product lots.

Figure 5

Wagner et al. (32) also carried out a short-term variability study on the smoke and filler of 24 MMCs and 146 cigarettes products. They compared the variability between MMCs and cigarettes of 19 selected HPHCs under ISO, Intense and CORESTA smoking regimes. Their results affirmed that, under a specific smoke regime, variability between each quantified analyte was about 5–20% more pronounced in cigars than in cigarettes. For example, under ISO, the average %RSD of NNK for cigars was approximately 22% relative to about 5% for cigarettes. Similarly, under ISO Intense, the average %RSD of formaldehyde was 20% for cigars and about 8% for cigarettes.

For cigars, the CORESTA regime produced the highest HPHC variability while the ISO regime recorded the highest HPHCs variability amongst cigarettes.

Young et al. also investigated the extent of chemical composition and weight variations within some selected small (SM), large (LG), and sheet-wrapped (SW) cigar products under the CRM 64 guidelines. Their results in Table 3 summarize the changes in the mean %RSD that occurred in the carbonyl yields from 2016–2017. The values indicated represent the analytes that showed statistically significant differences between 2016 and 2017. For example, in 2016 the mean %RSD of formalde-hyde for Phillies Blunt (LG) was 9.6 and increased to 19.8 in 2017. This computes to a notable mean formaldehyde yield %RSD difference of about 106% within a year. Even products of similar size that were tested in the same year showed a wide range of %RSD values in parenthesis. They inferred that since no certified/qualified cigar reference was available to be used as a control, they could not confidently attribute the relative %RSD values to either the inter-sample or method variability (46).

The variability of tobacco-specific nitrosamines (TSNAs) which occurs during flue-curing and air-curing of cigar dark tobacco has been a contentious public health debate and well-studied. Cultivation of dark air-cured requires high quantity of fertilizers in nitrate NO₃⁻ form, which produces high concentration of this polyatomic anion in cured leaves. According to BUSH and coworkers, during curing the aerobic conditions cause the reduction of the nitrates to nitrites (NO₂⁻) which then react with the secondary alkaloids within the tobacco leaf to form the TSNAs (47). Richmond et al. studied the correlation between curing environment and TSNA accumulation in two barns about 200 miles apart. They found that although barn curing conditions like temperature and relative humidity impacted concentration of TSNAs, other crucial factors like the barn construction, inconsistency in microenvironments within the same barn and improper positioning of data loggers (which records/monitors the temperature and humidity) could also introduce more TSNA variability (48). Thus the impact of curing on analyte variability is an important consideration.

Another source of variability within this product category is sampling. The significance of monitoring analytes in cigar tobacco via product sampling and sample size considerations cannot be overemphasized. Borges Miranda et al. performed extensive characterizations of both raw materials and cigar products to underscore the inconsistencies of tobacco blends used for premium cigars as well as the variability that arises from different testing methodologies and analytes. In 2019, the group utilized the near-infrared-reflectance-spectroscopic technique to characterize 322 powdered samples (raw materials and products) of dark air-cured Cuban cigar tobacco that were processed the same way (19). Their study revealed significant statistical differences between the total alkaloids-nicotine, total nitrogen, and total ash concentration in the cigar tobacco. They processed the spectra and evaluated the variability of these analytes with several statistical regression models such as the PLS, PCR, and MLR models and ascertained that the PLS model exhibited better reproducibility, precision, and prediction statistics. To enhance standardization and mitigate some of the variability in premium cigar tobacco, the group recently analyzed about 3780 different cigars and proposed a chemosensory technique and methodology for selecting raw materials from specific lots and optimizing the aging time required for processed tobacco leaves. Their study also identified specific chemical constituents and independent variables in the raw materials that could be analyzed and used as indexes of the cigar strength (49). Borges Miranda et al. have once again reported that it is consequential to sample raw materials at the end of the stripping workshop, which is the phase where the low-quality leaves are separated from the production line for premium cigars. The group utilized a randomized sampling design and three estimation errors (difference between sample mean and actual population mean) to measure the nicotine content of different batches of tobacco produced in different geographical precincts. They concluded that although about 2016 samples a year could be analyzed (with an estimation error of 0.2 % w/w), the sample count should be increased to include and account for leaves pre-processed daily during the pre-processing season (50).

Odelin and Borges Miranda also inferred that the weight of cigars had a substantial influence on the concentration of the smoke analytes; and determined the minimal sample size required to estimate the weight of a single premium cigar (51). It is worth noting that CORESTA has embarked on crucial studies as part of international standardization efforts to address the TSNA variability and sampling predicament (52, 53). Other researchers have focused on agronomic and germplasm studies as means to standardize, optimize and homogenize the cigar leaf composition at the end of preprocessing to help minimize variability. In this regard, Morán Gómez et al. investigated the correlation between bacteria genera population density, the pH and nicotine concentration in cured tobacco leaves harvested from different locations of the stalk/stem. They identified and isolated bacterial microbiota such as Staphylococcus, Arthrobacter genus and the Bacillus genus. They found that although the Staphylococcus and Arthrobacter species are important indicators, the Bacillus genera were the most predominant in the leaves processed from all stalk positions. They also inferred that the bacteria population density was more dependent on the leaf nicotine levels than the changes in pH values. The group further emphasized that genomic technology could reduce the processing time of tobacco leaves, improve the quality of lower grade leaves, and ultimately promote a more homogeneous composition of the leaves at the end of pre-processing (54). Ye et al. conducted a similar study using genetic sequencing to identify beneficial microbial strains which could improve the quality of the cigar products (specifically the aging process of the cigar product itself) and hence reduce the end-product variability. Their study revealed quite a significant diversity of fungi and bacteria strains in ten different cigar products. The predominant bacterial genera were Staphylococcus, Acinetobacter, and Pseudomonas while that of the fungal genera was Aspergillus (55).

Another concern is that although there is availability of multiple reference cigarettes, internationally approved cigar testing/smoking references or monitors have not been established.

Fortunately, a project led by an industry team and the University of Kentucky in collaboration with CORESTA and accredited tobacco testing facilities to develop cigar monitors/references was completed in 2019 to fill this gap. A set of reference products, described in Table 5, were formulated with different tobacco composite blends and with varying design features that can represent most of the cigar shapes and sizes (71). The university has developed and marketed several tobacco references, including RT6, a flavored cigar ground filler, and RT8, an unflavored cigar ground filler (72). The University of Kentucky was recently awarded a U.S. federal grant to develop a set of certified reference products (73). Nonetheless, until this project is completed, gaps will exist in the literature for the definitive comparison of physiochemical composition of cigar tobacco leaf and smoke constituents. Additional studies related to analysis of cigar smoke in the recent past include work by Dethloff et al. and Mueller and Colard, among others (74, 75, 76, 77, 78).