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.

Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol.

The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under "desert" or "tropical" conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine-smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen.

Nucleation of ethanol, propanol, butanol, and pentanol: a systematic experimental study along the homologous series.

We present homogeneous vapor-liquid nucleation rates of the 1-alcohols (C(n)H(2n+1)OH, n = 2-4) measured in the well-established two-valve nucleation pulse chamber as well as in a novel one-piston nucleation pulse chamber at temperatures between 235 and 265 K. The nucleation rates and critical cluster sizes show a very systematic behavior with respect to the hydrocarbon chain length of the alcohol, just as their thermo-physical parameters such as surface tension, vapor pressure, and density would suggest. For all alcohols, except ethanol, predictions of classical nucleation theory lie several orders of magnitude below the experimental results and show a strong temperature-dependence typically found in nucleation experiments. The more recent Reguera-Reiss theory [J. Phys. Chem. B 108(51), 19831 (2004)] achieves reasonably good predictions for 1-propanol, 1-butanol, and 1-pentanol, and independent of the temperature. Ethanol, however, clearly shows the influence of strong association between molecules even in the vapor phase. We also scaled all experimental results with classic nucleation theory to compare our data with other data from the literature. We find the same overall temperature trend for all measurement series together but inverted and inconsistent temperature trends for individual 1-propanol and 1-butanol measurements in other devices. Overall, our data establishe a comprehensive and reliable data set that forms an ideal basis for comparison with nucleation theory.

Homogeneous nucleation of a homologous series of n-alkanes (C(i)H(2i+2), i=7-10) in a supersonic nozzle.

Homogeneous nucleation rates of the n-alkanes (C(i)H(2i+2); i=7-10) were determined by combining information from pressure trace measurements and small angle x-ray scattering (SAXS) experiments in a supersonic Laval nozzle. The condensible vapor pressure p(J max), the temperature T(J max), the characteristic time Deltat(J max), and supersaturation S(J max) corresponding to the peak nucleation rate J(max) were determined during the pressure trace measurements. These measurements also served as the basis for the subsequent SAXS experiments. Fitting the radially averaged SAXS spectrum yielded the mean droplet radius r, 5

Using small angle x-ray scattering to measure the homogeneous nucleation rates of n-propanol, n-butanol, and n-pentanol in supersonic nozzle expansions.

In our earlier publication [M. Gharibeh et al., J. Chem. Phys. 122, 094512 (2005)] we determined the temperatures and partial pressures corresponding to the maximum nucleation rate for a series n-alcohols (C(i)H(2i+l)OH; i=3-5) during condensation in a supersonic nozzle. Although we were able to determine the characteristic time Deltat(Jmax) corresponding to the peak nucleation rate, we were unable to measure the number density of the aerosol and, thus, unable to directly quantify the nucleation rate J. In this paper we report the results of our pioneering small angle x-ray scattering (SAXS) experiments of n-alcohol droplets formed in a supersonic nozzle together with a new series of complementary pressure trace measurements. By combining the SAXS and pressure trace measurement data we determine the nucleation rates as a function of temperature and supersaturation.

Definition and prediction of the full range of transcription factor binding sites--the hepatocyte nuclear factor 1 dimeric site.

In animals, transcription factor binding sites are hard to recognize because of their extensive variation. We therefore characterized the general relationship between a specific protein-binding site and its DNA sequence and used this relationship to generate a predictive algorithm for searching other DNA sequences. The experimental process was defined by studying hepatocyte nuclear factor 1 (HNF1), which binds DNA as a dimer on two inverted-repeat 7-bp half sites separated by one base. The binding model was based on the equivalence of the two half sites, which was confirmed in examples where specific modified sites were compared. Binding competition analysis was used to determine the effects of substitution of all four bases at each position in the half site. From these data, a weighted half-site matrix was generated and the full site was evaluated as the sum of two half-site scores. This process accurately predicted even weak binding sites that were significantly different from the consensus sequence. The predictions also showed a direct correlation with measured protein binding.

Transcription factor therapeutics: long-shot or lodestone.

Many drugs, both approved and in development, exert their effects through transcription factors. The complexity of the biology of transcriptional regulation, however, presents challenges to the effective design of therapies that directly target transcription factors. This review focuses on the different approaches that are currently pursued in therapeutics and drug discovery aimed at targeting transcription factors and signaling molecules, and their areas of effect in transcription factor biology. A relatively recent approach termed "transcription therapy" has been applied for a number of drugs in development, which may potentially provide a new avenue for targeting transcription factors for therapeutic purposes.

Homogeneous nucleation of n-propanol, n-butanol, and n-pentanol in a supersonic nozzle.

We have measured the nucleation conditions of n-propanol, n-butanol, and n-pentanol in a supersonic Laval nozzle, and estimated that the maximum nucleation rate J is 5 x 10(16) cm(-3) s(-1) with an uncertainty factor of 2. Plotting the vapor pressures p(J(max) ) and temperatures T(J(max) ) corresponding to the maximum nucleation rate as ln(p) versus 1T, produces a series of well separated straight lines. When these values are scaled by their respective critical parameters, p(c) and T(c), the data lie close to a single straight line. Comparing the experimental data to the predictions of classical nucleation theory reveals much higher experimental rates, and the deviation increases with increasing alcohol chain length and decreasing temperature. A scaling analysis in terms of Hale's scaled nucleation model [Phys. Rev. A 33, 4156 (1986); Metall. Trans. A 23, 1863 (1992)], clearly shows that our data are consistent with experimental nucleation rates measured using other devices that have characteristic rates many orders of magnitude lower.