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Toxicological Assessment

Philip Morris International’s toxicological assessment aims to confirm whether the reduced formation of harmful and potentially harmful constituents (HPHCs) leads to reduced toxicity in laboratory models. Toxicological studies were conducted both in vitro and in vivo. Philip Morris International (PMI) conducts both standard toxicology assessment as well as advanced systems toxicology studies.

Standard Toxicology Assessment

First, three commonly used in vitro assays (Neutral Red Uptake, Ames and Mouse Lymphoma) were used to assess the cytotoxicity and genotoxicity of the THS aerosol in comparison with the smoke from the 3R4F reference cigarette.

The Neutral Red Uptake assay results showed that the relative in vitro cytotoxicity of the THS aerosol fractions was reduced by approximately 95% when expressed on a per-mg total particulate matter (TPM) basis, compared with the 3R4F reference cigarette. The in vitro cytotoxicity of THS aerosol fractions, expressed on a per-mg nicotine basis, was reduced by 85–90% compared with the 3R4F reference cigarette. In the second test (Ames Assay), no bacterial mutagenicity was observed at the tested dose range for THS, whereas reproducible mutagenic responses were observed for cigarette smoke. In the third test (Mouse Lymphoma Assay), which investigates mammalian mutagenicity, the aerosol from THS was at least eight-fold less mutagenic than smoke from a reference cigarette.1

Reduced in vitro toxicity of THS aerosol. The relative in vitro toxicity of THS aerosol is shown by the yellow bars and is compared with the 100% of smoke from the 3R4F reference cigarette (Red). The level of reduction for each test is shown in blue numbers.

Second, the inhalation toxicity of THS aerosol was analyzed in vivo according to the testing guidelines from the Organization for Economic Co-operation and Development (OECD 2009). The study was conducted in rats and the effects of increasing doses of THS aerosol and 3R4F smoke were compared after 90 days of nose-only inhalation.

The study results show that the reduced exposure to HPHCs achieved with THS aerosol exposure leads to significantly reduced measures of lung inflammation and respiratory toxicity of the THS aerosol compared with cigarette smoke.2

Further, to assess the combined chronic toxicity and carcinogenicity upon exposure of mice to THS aerosol, compared with 3R4F smoke, PMI conducted a lifetime study with A/J mice, which have been shown to be susceptible to develop pulmonary emphysema and an increase in lung tumors upon exposure to cigarette smoke. While the analysis of the 18-month dissection time point is ongoing, key data from the interim dissections at months 1, 5, and 10 have shown no lung function impairment in the THS exposed animals, which supports the overall reduced respiratory tract toxicity of the THS aerosol.

Systems Toxicology Assessment

PMI’s systems toxicology approach helps determine whether reduced toxicity leads to reduced risk in laboratory models. Systems toxicology allows a detailed assessment of the disease-relevant biological mechanisms affected by exposure to toxicants.3 Systems toxicology heavily relies on state-of-the-art high-throughput experimental technologies and advanced computational sciences.

First, systems toxicology is applied to identify the biological mechanisms that are altered by cigarette smoke, capturing this knowledge in biological network models. These models are then used to analyze the datasets for product assessment, allowing comparisons between the network alterations caused by the aerosols of the THS and those caused by cigarette smoke. Furthermore, the approach allows quantitative comparison of the overall biological impact of these exposures in the context of toxicological and disease endpoints.4

PMI studied the impact of direct exposure to whole 3R4F smoke and THS aerosol of human organotypic tissue cultures of oral, nasal, and bronchial epithelia grown at the air-liquid interface as well as in two in vivo studies in rats, which combined a standard toxicology study design with high-throughput analyses such as transcriptomics, proteomics, and lipidomics.5

These two 90-day rat inhalation studies were conducted according to the OECD Test Guideline 413. One study was conducted on rats exposed to 3R4F smoke or THS aerosol6; the other study used a mentholated THS (mTHS) product and a mentholated reference cigarette (MRC)7.The effects on the classical inhalation toxicology endpoints were considerably less pronounced in THS aerosol exposed rats compared with cigarette smoke exposed rats. The systems toxicology analysis demonstrated that the THS8 and mTHS aerosols9 caused significantly reduced perturbations of all biological networks affected by 3R4F and MRC smoke. For instance, the relative biological impact factors (RBIF) of THS aerosol exposure in the lung were reduced by 94% and 96% in male and female rats, respectively, compared with 3R4F smoke exposure at the higher comparable concentration. Similar results were obtained for mTHS compared with cigarette smoke.

Further, PMI conducted a systems toxicology study in an animal model (Apoe-/- mouse) that develops atherosclerotic plaque and emphysema when exposed to cigarette smoke. In this study, mice were exposed to either 3R4F smoke or THS aerosol for 8 months. A group of mice was first exposed for two months to 3R4F smoke and then randomized to either THS aerosol (switching) or fresh air (cessation) to mimic the framework illustrated in the figure below. Switching to THS aerosol following two months of cigarette smoke exposure was shown to reduce the development of both atherosclerosis and emphysema in a manner similar to smoking cessation.10

Disease endpoints in a mouse switching study: Lung emphysema (A) and atherosclerotic plaque volume (B) in Apoe-/- mice that were exposed for 8 months to either 3R4F smoke (red bars) or THS aerosol (magenta bars). A group of mice was first exposed for two months to 3R4F smoke and then switched to either THS aerosol (orange bars) or fresh air (green bars). The fresh air control is depicted by blue bars. Lung emphysema scores were assessed by histopathology after 8 months of exposure, Atherosclerotic plaque volumes were measured by micro-CT after 7 months of exposure.

A detailed analysis of the molecular mechanisms affected by smoke exposure in the lung showed that switching to THS aerosol reduced the overall biological impact in a way that approached cessation and that long-term exposure to the THS aerosol had only little effect on these mechanisms compared with 3R4F smoke exposure (figure below).

The graph above shows the network-based relative biological impact factor (RBIF) analysis of the lung. RBIF for each treatment group compared to air. The percentages show the relative biological impact, which is derived from the cumulated network perturbations caused by the treatment relative to the air control.

In summary, PMI conducted multiple in vitro and in vivo toxicology studies using a range of relevant biological test systems, as well as a systems toxicology study in an animal model of disease. Taken together, the results indicate that THS aerosol, compared with cigarette smoke, may have a reduced impact on biological mechanisms linked to disease and switching from cigarette smoke to THS aerosol exposure may lead to favorable changes that approach those of cessation across all levels of biological organization (from molecular changes to organ-level changes). Importantly, PMI has not detected any new toxicological effect with THS, in either classical or systems toxicology studies.

1. Schaller J-P, Keller D, Poget L, Pratte P, Kaelin E, McHugh D, Cudazzo G, Smart D, Tricker AR, Gautier L, Yerly M, Pires RR, Le Bouhellec S, Ghosh D, Hofer I, Garcia,E, Vanscheeuwijck P and Maeder S (2016) Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol. Regulatory Toxicology and Pharmacology, e-pub ahead of print. (PMID: 27720919).

2. Wong E, Kogel U, Veljkovic E, Martin F, Xiang Y, Boue S, Vuillaume G, Leroy P, Guedj E, Rodrigo G, Ivanov NV, Hoeng J, Peitsch MC and Vanscheeuwijck P. Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarettes smoke. Regulatory Toxicology and Pharmacology. 2016; in press.

3. Sturla SJ, Boobis AR, FitzGerald RE, Hoeng J, Kavlock RJ, Schirmer K, Whelan M, Wilks MF and Peitsch MC (2014) Systems Toxicology: from basic research to risk assessment. Chemical Research in Toxicology, 27:314-329. (PMID: 24446777).

4. Hoeng J, Kenney RD, Pratt D, Martin F, Sewer A, Thomson TM, Drubin DA, Waters CA, de Graaf D and Peitsch MC (2012) A Network-Based Approach to Quantify the Impact of Biologically Active Substances. Drug Discovery Today, 17:413-418. doi:10.1016/j.drudis.2011.11.008 (PMID: 22155224).

5. Kogel U, Schlage WK, Martin F, Xiang Y, Ansari S, Leroy P, Vanscheeuwijck P, Gebel S, Buettner A, Wyss C, Esposito M, Hoeng J and Peitsch MC (2014) 28-day rat inhalation study with an integrated molecular toxicology endpoint demonstrates reduced exposure effects for a prototypic modified risk tobacco product compared with conventional cigarettes. Food and Chemical Toxicology, 68:204-217. (PMID: 24632068).

6. Wong E, Kogel U, Veljkovic E, Martin F, Xiang Y, Boue S, Vuillaume G, Leroy P, Guedj E, Rodrigo G, Ivanov NV, Hoeng J, Peitsch MC and Vanscheeuwijck P (2016) Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarettes smoke. Regulatory Toxicology and Pharmacology, e-pub ahead of print. (PMID: 27793746).

7. Oviedo A, Lebrun S, Kogel U, Ho J, Tan WT, Titz B, Leroy P, Vuillaume G, Bera M, Martin FT, Rodrigo G, Esposito M, Dempsey R, Ivanov NV, Hoeng J Peitsch MC and Vanscheeuwijck P (2016) Evaluation of the Tobacco Heating System 2.2. Part 6: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of a mentholated version compared with cigarette smoke. Regulatory Toxicology and Pharmacology, e-pub ahead of print. (PMID: 27818348).

8. Wong E, Kogel U, Veljkovic E, Martin F, Xiang Y, Boue S, Vuillaume G, Leroy P, Guedj E, Rodrigo G, Ivanov NV, Hoeng J, Peitsch MC and Vanscheeuwijck P (2016) Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarettes smoke. Regulatory Toxicology and Pharmacology, e-pub ahead of print. (PMID: 27793746).

9. Kogel, U., Titz, B., Schlage, W. K., Nury, C., Martin, F., Oviedo, A., Lebrun, S., Elamin, A., Guedj, E., Trivedi, K., Ivanov, N. V., Vanscheeuwijck P, Peitsch, MC and Hoeng J (2016) Evaluation of the Tobacco Heating System 2.2. Part 7: Systems toxicological assessment of a mentholated version revealed reduced cellular and molecular exposure effects compared with cigarette smoke. Regulatory Toxicology and Pharmacology, e-pub ahead of print. (PMID: 27818347).

10. Phillips B, Veljkovic E, Boué S, Schlage WK, Vuillaume G, Martin F, Titz B, Leroy P, Buettner A, Elamin A, Oviedo A, Cabanski M, Guedj E, Schneider T, Talikka M, Ivanov NV, Vanscheeuwijck P, Peitsch MC and Hoeng J (2016) An 8-Month Systems Toxicology Inhalation/Cessation Study in Apoe-/- Mice to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified Risk Tobacco Product, THS 2.2, Compared with Conventional Cigarettes. Toxicological Sciences, 149:411-432. (PMID: 26609137). Corrected Suppl. Table 1 in Toxicol. Sci., 151:462-4 (PMID: 27225756).

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