Assessment of priority tobacco additives per the requirements of the EU Tobacco Products Directive (2014/40/EU): Part 3, Smoking behavior and plasma nicotine pharmacokinetics.

This publication is part of a series of 3 publications and describes the clinical assessment performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU under which Member States require manufacturers and importers of cigarettes and Roll Your Own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies (European Union, 2016). In our clinical study, two distinct end points were investigated, namely measuring plasma nicotine pharmacokinetics as a measure of nicotine uptake, and analyses of changes in smoker puffing behavior as a measure of cigarette smoke inhalation. This clinical study indicated that the inclusion of none of the priority additives either as single additive or as part of a chemical mixture, facilitated nicotine uptake. Furthermore, the data did not suggest that differences in the inhalation pattern of cigarette smoke of any of the Priority Additives tested occurred when compared to the additive-free reference cigarette. Finally, it is concluded that neither the scientific literature nor our study gave circumstantial indications of increased addictiveness for cigarettes containing these priority additives.

Assessment of priority tobacco additives per the requirements in the EU Tobacco Products Directive (2014/40/EU): Part 2: Smoke chemistry and in vitro toxicology.

This publication is part of a series of three publications and describes the non-clinical assessment performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU under which Member States shall require manufacturers and importers of cigarettes and Roll Your Own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies (European Comission, 2016). This publication contains the results of a literature search, comprehensive smoke chemistry, additive transfer, and in vitro toxicity studies for the 13 priority additives (carob bean extract, cocoa powder, fenugreek extract, fig juice concentrate, geraniol, glycerol, guaiacol, guar gum, liquorice extract powder, maltol, l-menthol (synthetic), propylene glycol, and sorbitol) commissioned by the members of the Priority Additives Tobacco Consortium to independent Contract Research Organizations. Comparisons of the 39 World Health Organisation smoke emissions in smoke from cigarettes with and without priority additives identified some differences that, with few exceptions, were minor and well within the inherent variability of the analytical method observed for the 3R4F monitor cigarette. Most differences were not statistically significant and did not show consistent additive-related increases or decreases. However, test cigarettes with guar gum showed a statistically significant, additive-related increase in formaldehyde and cadmium; test cigarettes with sorbitol showed a statistically significant, additive-related increase in formaldehyde and acrolein; test cigarettes with glycerol showed a statistically significant, additive-related decrease in phenols, benzo[a]pyrene and N-nitrosoanabasine; and test cigarettes with propylene glycol showed a statistically significant, additive-related decrease in phenol and m + p-cresols. These changes were not observed when the additives were tested as a mixture. None of the increases or decreases in smoke chemistry translated into changes in the in vitro toxicity. Comparisons of the in vitro toxicity of smoke from cigarettes with and without priority additives gave some differences that were minor, well within the inherent variability of the assays, not statistically significant, and did not show consistent additive-related increases or decreases. Thus, it can be concluded that the addition of priority additives had no effect on the in vitro toxicity of the cigarette smoke. The results obtained in our studies are consistent with those in scientific literature.

Assessment of priority tobacco additives per the requirements of the EU Tobacco Products Directive (2014/40/EU): Part 1: Background, approach, and summary of findings.

This paper is part of a series of 3 publications and describes the non-clinical and clinical assessment performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU; under which Member States shall require manufacturers and importers of cigarettes and roll-your-own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies. The Directive requires manufacturers and importers of cigarettes and Roll Your Own tobacco to examine for each additive whether it; contributes to and increases the toxicity or addictiveness of tobacco products to a significant or measurable degree; if it leads to a characterizing flavor of the product; if it facilitates inhalation or nicotine uptake, and if it results in the formation of CMR (carcinogenic, mutagenic and reprotoxic) constituents and if these substances increase the CMR properties of the respective tobacco product to a significant or measurable degree. This publication gives an overview on comprehensive smoke chemistry, in vitro toxicity, and human clinical studies commissioned by the members of the Priority Additives Tobacco Consortium to independent Contract Research Organizations (CROs) where the emissions of test cigarettes containing priority additives were compared to emissions emerging from an additive-free reference cigarette. Whilst minor changes in smoke chemistry parameters were observed when comparing emissions from test cigarettes with emissions from additive-free reference cigarettes, only two of the additives (sorbitol and guar gum) tested led to significant increases in a limited number of smoke constituents. These changes were not observed when sorbitol or guar gum were tested in a mixture with other priority additives. None of the priority additives resulted in increases in in vitro toxicity (Ames, Micronucleus, Neutral Red Uptake) or led to changes in smoking behavior or absorption (rate or amount) of nicotine measured during the human clinical study as compared to the additive-free reference cigarette.

Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas-vapor phase of cigarette smoke.

Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.

Cigarette smoke-induced morphological transformation of Bhas 42 cells in vitro.

In vitro cell transformation assays detect transformed cells that have acquired the distinct characteristics of malignant cells and thus model one stage of in vivo carcinogenesis. These assays have been proposed as surrogate models for predicting the non-genotoxic carcinogenic potential of chemicals. The Bhas 42 cell transformation assay, a short-term assay that uses v-Ha-ras-transfected Balb/c 3T3 cells, can detect the tumour promoter-like activities of chemicals, but has not previously been used with cigarette smoke. The particulate phase of cigarette smoke (total particulate matter [TPM]) is known to induce tumours in vivo in the mouse skin painting assay. Therefore, we investigated the ability of this Bhas cell assay to form morphologically transformed foci in vitro when repeatedly challenged with TPM from a standard research cigarette. TPM induced a dose-dependent increase in Type III foci, and a significant increase (up to 20-fold) in focus formation at moderately toxic concentrations between 5 and 60µg TPM/ml, with a peak at 20µg/ml. Three batches of TPM were tested in three independent experiments. Precision (repeatability and reproducibility) was calculated by using 0, 5, 10, and 20µg TPM/ml. Repeatability and reproducibility, expressed as the relative standard deviation obtained from the normalised slopes of the dose-response curves, were 17.2% and 19.6%, respectively; the slopes were 0.7402 ± 0.1247, 0.9347 ± 0.1316, and 0.8772 ± 0.1767 (increase factor∗ml/mg TPM; mean ± SD) ; and the goodness of fit (r2) of the mean slopes, each derived from n = 6 repeats, was 0.9449, 0.8198, and 0.8344, respectively. This in vitro assay with Bhas 42 cells, which are regarded as already initiated in the two-stage paradigm of carcinogenesis (initiation and promotion), is able to detect cell transformation induced by cigarette smoke in a dose-dependent manner with a high sensitivity and good precision. Because this assay is fast and yields reliable results, it may be useful in product assessment, as well as for further investigation of the non-genotoxic carcinogenic activity of tobacco smoke-related test substances.

Scientific assessment of the use of sugars as cigarette tobacco ingredients: a review of published and other publicly available studies.

Sugars, such as sucrose or invert sugar, have been used as tobacco ingredients in American-blend cigarettes to replenish the sugars lost during curing of the Burley component of the blended tobacco in order to maintain a balanced flavor. Chemical-analytical studies of the mainstream smoke of research cigarettes with various sugar application levels revealed that most of the smoke constituents determined did not show any sugar-related changes in yields (per mg nicotine), while ten constituents were found to either increase (formaldehyde, acrolein, 2-butanone, isoprene, benzene, toluene, benzo[k]fluoranthene) or decrease (4-aminobiphenyl, N-nitrosodimethylamine, N-nitrosonornicotine) in a statistically significant manner with increasing sugar application levels. Such constituent yields were modeled into constituent uptake distributions using simulations of nicotine uptake distributions generated on the basis of published nicotine biomonitoring data, which were multiplied by the constituent/nicotine ratios determined in the current analysis. These simulations revealed extensive overlaps for the constituent uptake distributions with and without sugar application. Moreover, the differences in smoke composition did not lead to relevant changes in the activity in in vitro or in vivo assays. The potential impact of using sugars as tobacco ingredients was further assessed in an indirect manner by comparing published data from markets with predominantly American-blend or Virginia-type (no added sugars) cigarettes. No relevant difference was found between these markets for smoking prevalence, intensity, some markers of dependence, nicotine uptake, or mortality from smoking-related lung cancer and chronic obstructive pulmonary disease. In conclusion, thorough examination of the data available suggests that the use of sugars as ingredients in cigarette tobacco does not increase the inherent risk and harm of cigarette smoking.

Toxicological assessment of cigarette ingredients.

Ingredients have been used in industrial manufacture of tobacco products since the early part of the 20th century. However, unlike other consumer goods, until now no regulatory authority has determined how tobacco ingredients should be assessed. Although there is currently no consensus on how added cigarette ingredients should be evaluated, this paper reviews some of the institutional guidance alongside published literature with a view to determining if there is a generally accepted approach in the absence of any strict regulation. Our aim was to review the recommendations, to compare them to the working practices as demonstrated from published studies, and to draw conclusions on currently used methodologies for testing ingredients added to cigarettes. The extent of testing is discussed in the light of practical and theoretical constraints and an example of an industry testing program is presented.

Smoke chemistry, in vitro and in vivo toxicology evaluations of the electrically heated cigarette smoking system series K.

The Electrically Heated Cigarette Smoking System Series K (EHCSS) produces smoke through the controlled electrical heating of tobacco. Evaluation of the EHCSS was accomplished by comparison with commercial and reference cigarettes, using International Organization for Standardization (ISO) and alternative puffing regimens based on nicotine exposures measured in a short-term clinical study. Using the alternative puffing regimen and compared with conventional cigarettes on a per cigarette basis, the EHCSS had 50-60% reductions in tar and nicotine; at least 90% reductions in carbon monoxide, nitrogen oxides, 1,3-butadiene, isoprene, acrylonitrile, polyaromatic hydrocarbons, hydrogen cyanide, aromatic amines, tobacco specific nitrosamines, and phenol; and least a 40% reduction in 2-nitropropane. Other important smoke constituents in EHCSS smoke were reduced as well. The in vitro studies showed similar large reductions in biological activity. Ames mutagenicity of total particulate matter (TPM) from the EHCSS was reduced by 70-90%; cytotoxicity of the TPM was reduced by approximately 82% and 65% for the gas-vapor phase. In vivo testing under ISO smoking conditions in the mouse skin painting assay demonstrated later dermal tumor onset, lower dermal tumor incidence, reduced dermal tumor multiplicity, and a lower proportion of malignant dermal tumors in EHCSS smoke condensate-exposed mice. Thirty-five day and 90-day nose-only inhalation studies in rats showed reductions in pulmonary inflammation and other biological activity, including histopathological endpoints. We conclude that under the conditions of these in vitro and in vivo studies, the EHCSS demonstrated significantly lower biological activity compared to conventional cigarettes, and may suggest the potential for reductions in human smokers.

Studies on the contributions of smoke constituents, individually and in mixtures, in a range of in vitro bioactivity assays.

Tobacco smoke is a complex mixture with over 8700 identified constituents. Smoking causes many diseases including lung cancer, cardiovascular disease, and chronic obstructive pulmonary disease. However, the mechanisms of how cigarette smoke impacts disease initiation or progression are not well understood and individual smoke constituents causing these effects are not generally agreed upon. The studies reported here were part of a series of investigations into the contributions of selected smoke constituents to the biological activity of cigarette smoke. In vitro cytotoxicity measured by the neutral red uptake (NRU) assay and in vitro mutagenicity determined in the Ames bacterial mutagenicity assay (BMA) were selected because these assays are known to produce reproducible, quantitative results for cigarette smoke under standardized exposure conditions. In order to determine the contribution of individual cigarette smoke constituents, a fingerprinting method was developed to semi-quantify the mainstream smoke yields. For cytotoxicity, 90% of gas vapor phase (GVP) cytotoxicity of the Kentucky Reference cigarette 1R4F was explained by 3 aldehydes and 40% of the 1R4F particulate phase cytotoxicity by 10 smoke constituents, e.g., hydroquinone. In the microsuspension version of the BMA, 4 aldehydes accounted for approximately 70% of the GVP mutagenicity. Finally, the benefits of performing such studies along with the difficulties in interpretation in the context of smoking are discussed.

Heterocyclic aromatic amines and their contribution to the bacterial mutagenicity of the particulate phase of cigarette smoke.

Heterocyclic aromatic amines (HAAs) rank among the strongest known mutagens. Approximately 30 HAAs have been found in cooked foods (broiled, fried, and grilled) and several HAAs have been characterized as animal carcinogens. Nine HAAs have also been reported to be constituents of cigarette smoke (CS) raising concerns that HAAs might contribute significantly to the known carcinogenicity of CS. As HAAs are found predominantly in the total particulate matter (TPM) of CS, an improved method for the quantification of HAAs in TPM is reported allowing detection and quantification of 8 HAAs in a single run. The mutagenic potency of these HAAs and that of TPM from the reference cigarette 2R4F was determined in the Salmonella Reverse Mutation Assay (Ames assay) with tester strain TA98 and a metabolic activation system. The 8 HAAs, when applied together in the Ames assay, showed a clear sub-additive response. Likewise, the combination of HAAs and TPM, if at all, gave rise to a slight sub-additive response. In both cases, however, the sub-additive response in the Ames assay was observed at HAA doses that are far above the amounts found in CS. The contribution of the individual HAAs to the total mutagenic activity of TPM was calculated and experimentally confirmed to be approximately 1% of the total mutagenic activity. Thus, HAAs do not contribute significantly to the bacterial in vitro mutagenicity of CS TPM.

Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke.

Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.

Towards the validation of a lung tumorigenesis model with mainstream cigarette smoke inhalation using the A/J mouse.

A generally accepted and validated laboratory model for smoking-associated pulmonary tumorigenesis would be useful for both basic and applied research applications, such as the development of early diagnostic endpoints or the evaluation of modified risk tobacco products, respectively. The A/J mouse is susceptible for developing both spontaneous and induced lung adenomas and adenocarcinomas, and increased lung tumor multiplicities were also observed in previous cigarette smoke inhalation studies. The present study was designed to collect data useful towards the validation of an 18-month mainstream smoke (MS) inhalation model. Male and female A/J mice were exposed whole-body at three MS concentration levels for 6h/day, and the results were compared to a previous study in the same laboratory and with a similar design. A linear MS concentration-dependent increase in lung tumorigenesis was observed with similar slopes for both sexes and both studies and a maximal 5-fold increase in multiplicity beyond sham control. The minimal detectable difference in lung tumor multiplicity for the current study was 37%. In the larynx, papillomas were detectable in all MS-exposed groups in a non-concentration dependent manner. No other extra-pulmonary MS-dependent neoplastic lesions were found. Gene expression signatures of lung tumor tissues allowed a clear differentiation of sham- and high dose MS-exposed mice. In combination with data from previous smoke inhalation studies with A/J mice, the current data suggest that this model for MS inhalation-induced pulmonary tumorigenesis is reliable and relevant, two crucial requirements towards validation of such a model.