Comparative review of chemical composition and toxicological evaluation of heated tobacco products compared to regular combustible cigarettes

Comparative review of chemical composition and toxicological evaluation of heated tobacco products compared to regular combustible cigarettes Georgi Momekov1, Magdalena Kondeva-Burdina1, Simeon Slavchev2 1 Department of Pharmacology, pharmacotherapy and toxicology, Faculty of Pharmacy, Medical University-Sofia, 2 Dunav str., 1000, Sofia, Bulgaria 2 Department of Social and Preventive Medicine and MES, Faculty of Public Health, Medical University-Sofia, 8 Byalo more str., 1527, Sofia, Bulgaria Corresponding author: Magdalena Kondeva-Burdina (ma[email protected]m) Received 9 October 2025♦ Accepted 21 October 2025♦ Published 28 November 2025 Citation: Momekov G, Kondeva-Burdina M, Slavchev S (2025) Comparative review of chemical composition and toxicological evaluation of heated tobacco products compared to regular combustible cigarettes. Pharmacia 72: 1–16. https://doi.org/10.3897/pharmacia.72.e174427 Abstract Heated tobacco products (HTPs) are a new category of tobacco products that deliver nicotine by heating tobacco at temperatures of 300–350 °C, thereby avoiding the combustion process typical of conventional cigarettes (CCs). A comprehensive comparative review of the scientific literature was conducted, including independent studies and regulatory assessments by the U.S. Food and Drug Administration (FDA) and European agencies (BfR, RIVM), as well as data from international laboratories that are members of the WHO TobLabNet network. In addition, data from peer-reviewed studies conducted by the THS manufacturer—approved for publication and available in the ClinicalTrials.gov database—were examined. Emission data for harmful and potentially harmful constituents (HPHCs) were analyzed under standardized testing conditions. HTPs demonstrate a significantly reduced chemical and toxicological profile compared to CCs due to the absence of combustion. These findings support the potential of HTPs as an alternative with markedly reduced risk for adult CC smokers who are unable or unwilling to quit tobacco use completely. Keywords 3R4F, conventional cigarettes, harmful and potentially harmful constituents, heated tobacco products, IQOS, THS, tobacco harm reduction, toxicological assessment Introduction Tobacco use is a global health problem affecting more than one billion people worldwide, and smoking (predominantly CC use) has been proven to be the largest cause of morbidity and mortality associated with tobacco use (Abrams et al. 2018a). Epidemiological studies have found significantly increased risks of lung cancer, cardiovascular disease, and chronic obstructive pulmonary disease (COPD) among CC smokers (Ramstrom and Wikmans 2014). The high level of toxicity of CC smoke is mainly due to combustion at temperatures from 650 °C to 900 °C (Baker 2006), which generates more than 6,000 chemical compounds, including about 100 identified HPHCs causally associated with smoking-related diseases (Fowles and Dybing 2003; WHO 2008; Rodgman and PerCopyright: This is an open access article distributed under the terms of the CC0 Public Domain Dedication. Pharmacia 72: 1–16 DOI 10.3897/pharmacia.72.e174427 Review Article Momekov G et al.: Chemical composition and toxicological evaluation of heated tobacco products2 fetti 2016). Various constituents of tobacco leaves, such as carbohydrates, biopolymers, waxes, and proteins, degrade at the high temperatures of the self-sustaining smoldering process, releasing toxic compounds into cigarette smoke (Torikai et al. 2004; Torikaiu et al. 2005). In response to these health challenges, heated tobacco products (HTPs) have been developed as a new technological solution to tobacco use, reducing toxic exposure while maintaining nicotine delivery. THS heats tobacco at temperatures of 300–350 °C, thereby excluding the combustion process and thus reducing the formation of and exposure to toxic substances, while maintaining a satisfactory amount of nicotine inhaled by users in the form of aerosol (Forster et al. 2015), not smoke. Regulators have abandoned strategies aimed at reducing the toxicity of CCs, having recognized the failure of “light” cigarettes in this regard. Hence, attention has shifted to the potential of non-combustible alternatives such as HTPs and other smokeless nicotine substitutes for CCs, which can provide significantly lower exposure to toxic substances for smokers who are unable or unwilling to quit tobacco completely (EU 2014; Abrams et al. 2018b). The WHO Tobacco Regulation Study Group has identified 39 priority toxic substances to be monitored, recommending 9 of them as key compounds for mandatory reduction: acetaldehyde, acrolein, benzene, benzopyrene, 1,3-butadiene, carbon monoxide, formaldehyde, NNK, and NNN (Burns et al. 2008; WHO 2008, 2019; Piade et al. 2013). In parallel, the FDA has compiled a list of 93 HPHCs, requiring reporting for 18 of them in cigarette smoke (FDA 2012a). Although independent regulatory agencies and scientific institutes, including the German BfR (Mallok et al. 2019), the Dutch RIVM (Dutch RIVM 2018), and laboratories from the WHO TobLabNet network (Bekki et al. 2017; Li et al. 2019), have carried out their own assessments of HTPs, a comprehensive systematic analysis of the scientific evidence on comparative toxicity between HTPs and CCs has not yet been made available. This study addresses this gap through a comprehensive comparative review of the scientific literature, including independent peer-reviewed studies, regulatory documents from the FDA (US FDA 2016, 2020, 2021), European government agencies (Dutch RIVM 2018; Mallok et al. 2019; Superior Health Council Belgium 2020), data from accredited international laboratories (WHO 2021), and HTP manufacturer’s studies validated by independent research groups. The analysis focused on HPHC emissions and toxicological profiles under standardized testing conditions (ISO and Health Canada Intense regimes) (Health Canada 2000), comparing the most widely studied tobacco heating system, THS/IQOS, with the reference conventional cigarette 3R4F (Smith and Hansch 2000). The review results revealed that HTPs demonstrated an 85–95% reduction in most HPHCs (Jaccard et al. 2017; Farsalinos et al. 2018; Poget et al. 2021), including key carcinogens and the nine toxic substances in cigarette smoke recommended by the WHO for mandatory reduction (Hecht 1999), while maintaining comparable nicotine levels. This analysis provides a necessary scientific basis for understanding the comparative risk between HTPs and CCs, with potential applications in regulatory policy through tobacco harm reduction strategies, in addition to smoking abstinence strategies (Abrams et al. 2018a, 2018b). Materials and methods Literature search strategy A comprehensive comparative review of the scientific literature for the period 1997–2024 was conducted (Witschi et al. 1997; Smith and Hansch 2000; Valavanidis et al. 2009; Rodgman and Perfetti 2016) by searching the following databases: PubMed, Web of Science, Google Scholar, and regulatory archives. The English keywords used were “heated tobacco products,” “IQOS,” “THS,” “HPHC,” “harmful and potentially harmful constituents,” “3R4F,” “cigarette emissions,” and “toxicological assessment.” Additionally, publications from the list of PMI (Philip Morris International) studies, comprising 20 peer-reviewed scientific articles (2015–2024), as well as fundamental studies on the characterization of harmful constituents in cigarette smoke from the late 1990s, were analyzed (Witschi et al. 1997; Smith and Hansch 2000). Inclusion criteria The comprehensive comparative review includes: • Peer-reviewed scientific publications comparing emissions and/or toxicological profiles of HTPs and conventional cigarettes (CCs). • Fundamental research on toxic constituents in cigarette smoke (1997–2000) (Witschi et al. 1997; Kunze et al. 1998; Hecht 1999; Smith and Hansch 2000). • Official regulatory documents and assessments by the FDA (FDA 2012a, 2012b; US FDA 2016, 2020, 2021), European health agencies (BfR, RIVM) (Dutch RIVM 2018; Mallock et al. 2019), and other national health authorities (Committee on Toxicity 2017; Public Health England 2018) (15 documents from 2017–2022). • Studies by accredited laboratories that are members of the WHO TobLabNet network (Bekki et al. 2017; Li et al. 2019). • Independent academic research (41 third-party publications, 2017–2024) (Stephens 2017; Farsalinos et al. 2018; Karkela et al. 2022). • Papers analyzing emissions of harmful and potentially harmful constituents (HPHCs) under standardized testing conditions (ISO and Health Canada Intense regimes) (Health Canada 1999). • Toxicological studies (in vitro, in vivo) and cohort studies for biomarkers of exposure (Gonzalez-Suarez et al. 2015; Wong et al. 2016; Polosa et al. 2021). • WHO regulatory frameworks for the assessment of tobacco products, including the WHO Technical Report Series from 2008 to 2023 (WHO 2005, 2008). Pharmacia 72: 1–16 3 Exclusion criteria Excluded were: • Research focused exclusively on e-cigarettes without comparison to HTPs or CCs. • Studies on other smokeless tobacco products (snus, chewing tobacco) without relevance to products delivering nicotine by inhalation. • Unpublished or incomplete papers without independent peer review. • Studies lacking clear methodology or standardized testing conditions, or without comparative data between HTPs and CCs. • Abstracts or conference presentations containing partial or incomplete information. Regulatory documents analyzed Special attention was given to: • FDA Modified Risk Tobacco Product (MRTP) and Premarket Tobacco Product Application (PMTA) assessments of THS/IQOS with HTPs (2019–2022). • WHO TobReg guidance on toxic constituents prioritized for monitoring and reduction. • National health agencies: German Federal Institute for Risk Assessment (BfR), Dutch National Institute for Public Health and the Environment (RIVM), Japanese National Institute of Public Health, and the Committee on Toxicity (COT) of the UK. • European regulatory standards for tobacco products (TPD 2014/40/EU). • Report by Public Health England/Office for Health Improvement and Disparities (PHE/OHID). Data analytics methodology The analysis is structured into three main areas: 1. Chemical composition of emissions—comparison of HPHC concentrations between HTP aerosols and CC smoke under standardized testing conditions, including the characterization of major classes of toxic compounds (aromatic amines, polycyclic aromatic hydrocarbons, and tobacco-specific nitrosamines) based on classic cigarette smoke studies from the late 1990s to the present. 2. Toxicological assessment—analysis of independent regulatory assessments, in vitro toxicological tests (Ames, micronuclei, cytotoxicity), in vivo studies, and systemic toxicology (Schaller et al. 2016a, 2016b). 3. Cohort and clinical studies—evaluation of biomarkers of exposure, short-term clinical studies, and long-term follow-up. Focus of the study The main focus is on the tobacco heating system THS/ IQOS due to its extensive scientific documentation (a total of 76 publications analyzed) and regulatory assessment compared to the reference 3R4F cigarette, which serves as the standard for CCs in laboratory studies (Smith and Hansch 2000). Both scientifically validated data from industry and independent studies were analyzed to provide a comprehensive and balanced assessment of the available scientific evidence. Results 1. Chemical composition and emissions Analysis of the chemical composition of HTP emissions compared to CCs reveals significant quantitative differences in the content of the main aerosol components (Peitsch and Hoeng 2021). As shown in Table 1, standardized measurements according to ISO protocols demonstrate the characteristic features of aerosols from both types of tobacco products. Main aerosol components A comparative analysis of the main aerosol components between the reference 3R4F cigarette and HTP with THS shows the following key differences (Schaller et al. 2016a): • Glycerol—HTPs demonstrated an increased content (5.02 ± 0.101 mg/unit) compared to 3R4F (2.08 ± 0.113 mg/unit), reflecting the specific composition of the tobacco substrate in HTPs. Glycerol is present in HTPs as an aerosol-forming chemical and is not classified as a toxicant (HPHC). • Nicotine—HTPs provide comparable amounts of nicotine to CCs (1.29 ± 0.047 mg/unit for HTP versus 1.74 ± 0.039 mg/unit for 3R4F), ensuring satisfactory nicotine delivery to users. • Water—The water content of HTP aerosol (30.2 ± 2.17 mg/unit) is significantly higher than that of 3R4F smoke (14.7 ± 2.10 mg/unit). Table 1. Main suppliers of aerosols for HTP and CC–3R4F. Capturing of aerosols defined in accordance with ISO 4387; 8454; 10315 Unit CC (3R4F) HTPS (THS/IQOS) Average SD nAverage SD n Glycerin mg/product 2.08 0.113 25.02 0.101 3 Nicotine mg/product 1.74 0.039 21.29 0.047 3 Water mg/product 14.7 2.10 230.2 2.17 3 Momekov G et al.: Chemical composition and toxicological evaluation of heated tobacco products4 Reduction of harmful and potentially harmful constituents (HPHCs) The most significant conclusion of the chemical analyses is the consistent and substantial reduction of HPHCs in aerosols from HTPs compared to CCs. PMI studies demonstrated an average decrease of about 85–95% in 58 HPHC substances (including those from the WHO list of nine and the FDA list of 18 HPHCs) in HTP aerosol (version THS 2.2) compared to HPHC levels in CCs. This decrease in HPHCs has been confirmed by several independent studies (Jaccard et al. 2017; Davigo et al. 2023; Ghazi et al. 2024). Specific data on reduction by constituent The levels of carbonyl compounds were shown to be significantly reduced in HTPs compared to CCs (Farsalinos et al. 2018). Under the Health Canada Intense Testing Regime (Health Canada 1999), HTPs emit markedly lower levels of all major carbonyls: • Formaldehyde—reduced by approximately 91.6% • Acetaldehyde—reduced by approximately 84.9% • Acrolein—reduced by approximately 90.6% • Propionaldehyde—reduced by approximately 89.0% • Crotonaldehyde—reduced by approximately 95.3% Independent studies confirm these findings, also showing that HTPs emit about half the nicotine and significantly lower levels of harmful substances compared to cigarettes (Davigo et al. 2023). Toxic gases also show a drastic decrease (Schaller et al. 2016a; Bekki et al. 2017): • Carbon monoxide (CO)—decreased by 99% (one hundredth of the levels in CCs; CO is a marker of combustion, practically negligible). • Volatile organic compounds (VOCs)—total reduction of 81.2%. • Carcinogenic compounds show significant decreases (Fig. 1) (Stephens 2017; Bekki et al. 2017; Slob et al. 2020; Karkela et al. 2022; Davigo et al. 2023): • Benzene, 1,3-butadiene, benzo[a]pyrene—reduced by more than 80%. • Tobacco-specific nitrosamines (TSNAs)—the concentrations of NNK and NNN are reduced to onefifth (an 80% decrease). Numerous independent studies confirm these data (Mallok et al. 2018; Uchiyama et al. 2018; Hidayat and Alayyannur 2021; Lu et al. 2021; Perezhogina et al. 2021; Szparaga et al. 2021; Wang et al. 2021a, b). The WHO Study Group on Tobacco Regulation (TobReg) has identified priority substances whose concentrations in tobacco product emissions are an important criterion for regulation. Despite thousands of compounds in tobacco product emissions, including hundreds of toxic substances—some of which are carcinogenic—TobReg designates 39 substances for ongoing monitoring and recommends mandatory reductions in the levels of nine of them in cigarette smoke as the most toxic: acetaldehyde, acrolein, benzene, Figure 1. Comparative emissions of HPHCs from reference cigarettes 3R4F (CCs) and HTPs. The values are presented in μg/unit. Pharmacia 72: 1–16 5 Figure 2. Reduced emissions for the nine toxic substances (CCs and HTPs). benzo[a]pyrene, 1,3-butadiene, carbon monoxide (CO), formaldehyde, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosonornicotine (NNN) (WHO 2008). The most important criterion for selecting toxic compounds to be regulated is evidence of their toxicity, with particular attention to compounds associated with cardiovascular toxicity, pulmonary toxicity, and carcinogenicity. The list of nine toxic substances may apply to all tobacco products for smoking or inhalation, including non-combustible tobacco products (Fig. 2; Table 2) (WHO 2008). Stability and consistency of the reductions under different conditions Studies conducted under extreme climatic conditions confirm the consistent reduction of HPHCs. Under varying temperature and humidity conditions (warm and dry climates—30 °C and 35% relative humidity; warm and very humid climates—30 °C and 75% relative humidity), the HPHC levels in the HTP aerosol remain reduced by at least 90% on average compared to the concentrations in the mainstream smoke of a 3R4F cigarette (Poget et al. 2021). Solid particles and aerosol structure Due to the combustion of tobacco, cigarette smoke contains high levels of carbon-based solid particles (approximately 0.5 trillion/cigarette). A significant difference between HTPs and CCs is observed in the structure of the generated particles in cigarette smoke and HTP aerosol. Because combustion is absent, the HTP aerosol is composed of gases and liquid droplets but contains no solid particles (Pratte et al. 2017). Studies have shown that no solid particles are detected in HTP aerosol after passing through a thermal thermodenuder at 300 °C, which is consistent with the fact that there is no combustion process in HTPs. In contrast, about 80% of the total particulate matter in 3R4F smoke consists of solid particles and/or low-volatile liquid droplets. Non-targeted analytical approach A complex non-targeted analytical comparison was conducted to identify unique or increased compounds in HTP aerosol versus cigarette smoke. By applying a wide range of LC-MS and GC×GC-MS methods, it was found that only a few compounds were significantly more abundant in HTP aerosol than in cigarette smoke, and some were unique to THS. Overall, the results showed that cigarette smoke is far more complex in composition than HTP aerosol (Bentley et al. 2020; Lang et al. 2024). Advanced analytical techniques, including chromatographic–mass spectrometric methods, confirm these findings and provide a detailed characterization of aerosol components (Heide et al. 2021; Sussman et al. 2023). A significant portion of these compounds have been identified as reaction products of glycerol. 2. Toxicological assessment The toxicological assessment of HTPs includes an extensive research program covering in vitro tests, preclinical animal studies, and clinical studies involving humans. The results of these studies have been evaluated by both independent scientific groups and regulatory authorities in different countries. Momekov G et al.: Chemical composition and toxicological evaluation of heated tobacco products6 Independent studies In vitro cytotoxicity and genotoxicity Standard in vitro toxicology tests demonstrate consistent differences between HTPs and CCs. When using a set of standard in vitro toxicology tests (Ames, Micronucleus, and Neutral Red Uptake tests), HTPs showed no observable responses, while a significant toxic response from a 3R4F cigarette was observed. Specifically, the Ames test showed no observable response for HTP aerosol compared to 3R4F cigarette smoke, and a significant reduction of 90% in cytotoxicity and 95% in genotoxicity was observed in HTP aerosol compared with cigarette smoke (Buratto et al. 2018). Various studies have confirmed the reduced cytotoxicity of HTP aerosols (Zagoriti et al. 2020; Caruso et al. 2021; Niu et al. 2023; Merritt et al. 2024; Sood et al. 2024). When examining different methods for assessing cytotoxicity (NRU, MTT, Annexin V apoptosis, high-content screening, and real-time cell analysis), all tests showed reduced cellular viability after exposure to 1R6F reference cigarette smoke and little or no reduction for HTPs within 24 hours (Fig. 3) (Gonzalez-Suarez et al. 2015; Schaller et al. 2016a; Esposito et al. 2022). The study of 55 HPHCs in different HTPs shows that the levels generated by them were significantly reduced compared to those of 3R4F (Schaller et al. 2016b; Poynton et al. 2017). Epigenetic effects Studies on epigenetic effects reveal significant differences between HTPs and CCs. Only CC extract (3R4F at 0.5% concentration) significantly reduced cell proliferation of A549 and BEAS-2B cell lines, while 0.5% extracts from commercially available HTPs did not affect cell growth (Hattori et al. 2020). The TUNEL test showed that apoptosis occurred in the A549 and BEAS-2B cell lines 24 hours after exposure to 3R4F, while HTPs did not produce similar effects. Only the extract of 3R4F reduced histone H2A phosphorylation in both cell lines, indicating an epigenetic disorder not seen in HTPs (Hattori et al. 2020). Oxidative stress HTPs demonstrated significantly lower oxidative toxicity compared to CCs (Lyu et al. 2022). The effects of HTPs, when observed, occurred only at very high, non-physiological concentrations, supporting the reduced harm profile under normal use. Systemic toxicological assessment When using a systemic toxicology approach, HTP aerosol induced lower levels of cytotoxicity and smaller changes in secreted pro-inflammatory mediators than 3R4F smoke (Wong et al. 2016). A meta-analysis of systemic toxicology in three types of human organotypic cultures of the aerodigestive tract (buccal, bronchial, and nasal epithelium) showed lower toxicity in all cultures after exposure to HTP aerosol compared to 3R4F smoke at comparable nicotine concentrations (Iskandar et al. 2017). Long-term animal studies An 18-month carcinogenicity study in A/J mice conducted in accordance with OECD Guideline 453 showed that chronic exposure to HTP aerosol did not increase the incidence or multiplicity of bronchioloalveolar adenomas or carcinomas compared to the control group, while exposure to cigarette smoke increased these rates (Wong et al. 2020). In contrast to mice exposed to HTP aerosol, mice exposed to cigarette smoke showed increased heart weight, changes in red blood cell profiles, and serum parameters Table 2. Reductions of the toxicants included in the WHO list of nine toxic substances in the HTP aerosol compared to 3R4F cigarette smoke. HPHCs (WHO 9) Unit of measurement 3R4F reference cigarette THS 2.2* THS 3.0** Average SD Average SD Red. (%) Average SD Red. (%) Carbon monoxide (CO) [mg/stick] 30.2 2.76 0.447 0.0893 98.5% 0.324 0.0501 98.9% 1,3-Butadiene [μg/stick] 98.5 9.8 0.233 0.0185 99.8% 0.14 0.0162 99.9% Formaldehid [μg/stick] 85.2 16.7 8.89 1.17 89.6% 9.03 0.448 89.4% Acetaldehyde [μg/stick] 1641 258 215 22.5 86.9% 158 5.28 90.4% Acrolein [μg/stick] 156 25.4 11.8 2.61 92.4% 7.95 0.393 94.9% Benzene [μg/stick] 81.1 8.78 0.533 0.0198 99.3% 0.348 0.0273 99.6% N-Nitrozonornicotine (NNN) [ng/stick] 283 27.8 12.3 0.869 95.7% 10.6 0.847 96.3% 4-(methyl-nitrosamine)-1-(3-pyridyl)- 1-butanone (NNK) [ng/stick] 264 26.4 8.72 0.554 96.7% 8.95 0.708 96.6% Benzo[a]pyrene [ng/stick] 15 1.3 0.621 0.0317 95.9% 0.6 0.057 96.0% The values of the nine toxic substances in cigarettes, THS 2.2, and THS 3.0. SD – standard deviation; Nam (%) – reduction expressed in percentages. *THS 2.2 is the initial version of the PMI THS (blade THS), which uses a blade to heat the tobacco through internal resistive heating. It is commercialized as IQOS 3 or IQOS Originals and designed to be used with HTPs (heatsticks) sold under the brand name HEETS. **THS 3.0 is the new version of the PMI THS (induction THS), commercialized as IQOS ILUMA. It features internal induction heating technology and uses specially designed HTPs sold under the brand name TEREA. Pharmacia 72: 1–16 7 of liver function. Increased lung inflammation, altered lung function, and emphysematous changes were observed only in mice exposed to cigarette smoke (Phillips et al. 2015; Wong et al. 2020). 3. Regulatory assessments FDA (USA) – U.S. Food and Drug Administration On July 7, 2020, the FDA authorized the IQOS version THS 2.2 with three types of consumables (heatsticks) as a Modified Risk Tobacco Product (MRTP), concluding that scientific evidence demonstrated it was “appropriate for promoting public health and is expected to benefit the health of the general population.” The FDA confirmed the product’s potential to reduce harm and risk of disease compared to continued cigarette smoking (US FDA 2016). Specifically, the FDA found a significant reduction in the formation of harmful and potentially harmful substances compared to cigarette smoke and stated that “it is reasonable to expect that subsequent studies will establish a measurable and substantial reduction in morbidity and mortality in individual users.” Independent laboratory analyses confirmed more than a 90% reduction in most HPHCs and over an 80% reduction in other substances compared to 3R4F (Bekki et al. 2017; Dutch RIVM 2018; Karkela et al. 2021; Maeder and Jeannet 2025). In one study (Maeder and Jeannet 2025), 105 out of 108 evaluated HPHCs showed an average reduction of more than 91.6% in HTPs compared to 3R4F. Similar results were reported in comparisons of gas-phase and particulate matter between HTPs and 3R4F, with significantly lower emissions and only sporadic particulate matter in the aerosol (Karkela et al. 2021). BfR (Germany) – Federal Institute for Risk Assessment Germany’s Federal Institute for Risk Assessment (BfR) published an assessment of HTPs in 2018, concluding that they contain fewer harmful substances than CCs but still pose a health risk. The BfR emphasized the need for further long-term studies for a complete risk assessment (Mallock et al. 2019). RIVM (Netherlands) – National Institute for Public Health and the Environment The National Institute for Public Health and the Environment of the Netherlands (RIVM) confirmed that IQOS THS emits significantly fewer harmful substances than CCs (Dutch RIVM 2018). RIVM developed a method—Change in Cumulative Exposure (CCE)—to estimate the reduction in cancer risk based on toxicant emissions. The CCE was estimated to be 10to 25-fold lower when using HTPs instead of cigarettes. Such a reduction indicates a substantially smaller decrease in expected lifespan, based on available dose–response data in smokers (Slob et al. 2020). WHO TobLabNet laboratories Member laboratories of the WHO TobLabNet network in Japan and China confirmed significant reductions in HPHCs in HTPs compared to CCs (Bekki et al. 2017; Li et al. 2019). The Japanese laboratory found that the concentration of TSNAs was one-fifth and the carbon monoxide level only one-hundredth of those in CCs. The Chinese laboratory confirmed that, in addition to some carbonyls, ammonia, and N-nitrosoanabasine, the levels of several HPHCs in HTPs were at least 80% lower than those of 3R4F (Bekki et al. 2017). Figure 3. In vitro toxicological evaluation of HTPs and reference cigarettes 3R4F (CCs). The results show the relative levels of toxic response, where 3R4F is defined as 100 relative units (reference standard). Momekov G et al.: Chemical composition and toxicological evaluation of heated tobacco products8 UK Committee on Toxicity (COT) The British Committee on Toxicity (COT) published a statement on the toxicological assessment of new HTPs, acknowledging the significant reduction in toxicants but highlighting the need for more data on long-term effects (Committee on Toxicity 2017). Consensus in regulatory assessments An extensive review of 17 independent and industry-funded studies shows that the results of these studies and regulatory assessments are approaching a general consensus that HTP aerosols expose consumers to significantly lower levels of toxicity than tobacco smoke (Caruso et al. 2021). Regulatory agencies in different countries reach similar conclusions regarding the harm reduction potential of HTPs, despite their different methodological approaches and assessment criteria. All emphasize the need for ongoing monitoring and long-term research for full risk characterization (Caruso et al. 2021). 4. In vivo carcinogenic studies The 18-month study in A/J mice, conducted in accordance with OECD Guideline 453, represents the gold standard for assessing carcinogenic potential (Wong et al. 2020). The results demonstrate fundamental differences between HTPs and CCs in terms of tumorigenesis. Key findings from long-term studies: • Chronic exposure to HTP aerosol did not increase the incidence or multiplicity of bronchioloalveolar adenomas or carcinomas compared to the control group (Wong et al. 2020). • Cigarette smoke significantly increased tumor incidence and multiplicity (Wong et al. 2020). • The gene profile of tumors in mice exposed to HTP aerosol was similar to that of spontaneous tumors and differed distinctly from the profile of tumors induced by cigarette smoke (Xiang et al. 2021). • A 13-gene signature was developed that reliably distinguishes tumors associated with exposure to cigarette smoke from spontaneous tumors (Xiang et al. 2021). Carcinogenic risk assessment Margin of exposure (MOE) analysis shows significant advantages of HTPs in the carcinogenic risk profile (Auer et al. 2018): • The carcinogenic risk for CCs indicates elevated risk levels when consuming as few as 10 cigarettes per day (Inoue-Choi et al. 2016). • Incremental lifetime cancer risk (ILCR) analysis confirmed these findings, with CCs exceeding the safe value of 10-4 in some cases (Esposito et al. 2022). • Cancer potency modeling shows that HTPs have at least one order of magnitude lower carcinogenic potential than tobacco smoke (Stephens 2017). Effects on specific tissues Studies on specific tissues reveal consistent patterns of reduced toxicity: • Bone tissue—HTPs are significantly less toxic to bone cells than CCs when analyzing mitochondrial and esterase activity (P < 0.001). Harmful effects of HTPs on bone cell function are observed only at very high, non-physiological concentrations (Aspera-Werz et al. 2020). • Vocal cords—Short-term in vitro exposure of human fibroblasts from vocal cords to HTPs does not result in cytotoxicity and reduces the gene expression of measured inflammatory mediators (Grossmann et al. 2024). • Adipose tissue—Only CC extract significantly disrupts the differentiation of preadipocytes into beige adipocytes, while HTP extracts do not affect this process (Zagoriti et al. 2020). Consistent reduction of HPHCs in HTPs Studies conducted under extreme climatic conditions have demonstrated the robustness of THS technology with HTPs in reducing HPHCs (Poget et al. 2021): • Under hot and dry conditions (30 °C, 35% RH) and hot and very humid conditions (30 °C, 75% RH), reductions in HPHCs remain above 90%. • Fifty-four HPHCs show consistent reduction regardless of climate conditions. • The volumetric-corrected approach confirms a sustained reduction for all investigated HPHCs. General safety profile The body of evidence from long-term, clinical, and specialized studies has consistently demonstrated significantly reduced harm from HTPs compared to CCs, supporting their harm reduction potential through heating technology. 5. In vitro toxicological assessment Basic cytotoxic tests Standard genotoxic tests demonstrate fundamental differences between HTPs and CCs. When using the Ames test (bacterial reverse mutation test), in vitro micronucleus test, and mouse lymphoma assay, HTPs showed no observable responses, whereas CCs induced significant toxic reactions. Multiple cytotoxic screenings using different methods (NRU, MTT, Annexin V apoptosis, high-content screening assays, real-time cell analysis) in human adenocarcinoma lung epithelial cells (H292) show: • Decreased cellular viability after exposure to the reference cigarette 1R6F (similar to 3R4F). Pharmacia 72: 1–16 9 • Little or no reduction in viability for HTPs within 24 hours. • Time-resolved analyses reveal the kinetic dependence of the toxicity of chemicals in smoke/aerosol. Systemic toxicology with organotypic cultures The most advanced in vitro studies use three-dimensional human organotypic cultures of the aerodigestive tract that accurately reproduce the physiological characteristics of human tissues under exposure to smoke or aerosol (Iskandar et al. 2017). Buccal epithelial cultures Exposure of human organotypic oral epithelial tissue cultures (EpiOral, MatTek Corporation) to an aerosol of 3R4F or HTPs for 28 minutes at comparable nicotine concentrations (0.32–0.51 mg nicotine/L) revealed (Zanetti et al. 2016): • A greater impact of 3R4F smoke compared to HTP aerosol in terms of cytotoxicity, morphological tissue alterations, and secretion of inflammatory mediators. • Significantly impaired network patterns in smoke exposure (apoptosis, necroptosis, aging, xenobiotic metabolism, oxidative stress). • Markedly reduced stress responses after exposure to HTP aerosol, with exposed cultures recovering more fully. Bronchial epithelial cultures In a model of human small airways using a systems toxicology approach that combines functional tests with omics technologies (Iskandar et al. 2017): • Aerosol from HTPs induces lower levels of cytotoxicity and smaller changes in secreted pro-inflammatory mediators compared to 3R4F smoke. • The effects of HTPs, when observed, are mostly transient and decrease more rapidly after exposure. • A higher transcriptome-induced biological impact was observed with 3R4F smoke. Nasal epithelial cultures Three-dimensional nasal cultures demonstrated consistent patterns of reduced toxicity in HTPs, with causal network enrichment analysis supporting a similar mechanistic impact of cigarette smoke across all three cultures (Iskandar 2016). Air–liquid interface (ALI) studies ALI studies using different human cell lines (A549, BEAS-2B, NCI-H292, NHBE, and NHLF) have shown a consistent reduction in cytotoxicity and inflammatory responses when exposed to HTP aerosol compared to conventional cigarette smoke. Cellular functionality remained preserved at physiologically relevant concentrations for HTPs (Caruso et al. 2021). • A 90–95% reduction in cytotoxicity in HTPs compared to CCs. • Significantly lower levels of pro-inflammatory mediators after exposure to HTPs. • Preserved viability and metabolic activity of cells at physiologically relevant doses of HTPs. 6. Specialized tissue models Epigenetic and apoptotic effects A comparative study of the cell lines A549 (human lung carcinoma) and BEAS-2B (normal bronchial epithelium) revealed key differences (Choukrallah et al. 2018; Hattori et al. 2020): • Conventional cigarette smoke (3R4F extract, 0.5%) significantly reduces cell proliferation and induces apoptosis in both cell lines. • HTPs (0.5% extracts) do not affect cell growth. • Smoke from 3R4F alone reduces histone H2A phosphorylation, affecting transcriptional regulation. • When using 3R4F, increased activity was reported in 339 genes, whereas for HTPs, only 103–107 genes showed increased activity. Effects on the bone cell system In human mesenchymal stem cells and primary human preosteoblasts, HTPs showed significantly lower toxicity in mitochondrial and esterase activity analyses (P < 0.001), while harmful effects occurred only at nonphysiologically high concentrations. At the same time, CC extracts reduced alkaline phosphatase twofold and matrix mineralization fourfold at low doses (Aspera-Werz et al. 2020). Adipocyte differentiation In a study on 3T3-L1 preadipocytes differentiating into beige adipocytes, cigarette smoke extract alone induced a doseand time-dependent decrease in cell viability. Exposure to HTPs did not disrupt the differentiation process, and lipid accumulation and marker expression remained unchanged (Zagoriti et al. 2020). Oxidative stress and free radicals The levels of free nicotine in the aerosol from HTPs and in CC smoke are similar (Shein et al. 2019). The emission of reactive oxygen species (ROS) from HTPs is 85% lower than that from CCs (Salman et al. 2018), and the carbonyl compounds in HTP aerosol are 77% lower (Farsalinos et al. 2018). 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