There has been a recent increase in the use of tobacco- and nicotine-containing products, such as electronic cigarettes (e-cigarettes) and heated tobacco products (HTPs), which have been the subject of extensive research [[1], [2], [3]]. Numerous studies have compared the chemical properties of e-cigarette and HTP aerosols with those of cigarette smoke (CS), consistently demonstrating significantly lower levels of toxicants in e-cigarette and HTP aerosols [[4], [5], [6], [7], [8], [9]]. In vitro toxicological evaluations have indicated almost no mutagenic and genotoxic activities in e-cigarette and HTP aerosols [4,[7], [8], [9], [10]].

Previous studies have employed exposure concentrations of 5 mg/plate for the Ames test and 2 mg/mL for the in vitro micronucleus test and mouse lymphoma assay, unless significant cytotoxicity was observed [4,7,8,10]. These concentrations align with the guidelines set forth by the Organization for Economic Co-operation and Development [[11], [12], [13]]. In some previous experiments according to the relevant guidelines, the concentration at which toxicity was found in vitro was reported for several HTP aerosols as well as CS, so that the toxic concentrations found can be directly compared with the calculation of how low the toxicity of HTP aerosols was compared to CS [4,8,10]. However, in the other previous experiments, even at the highest concentrations tested, no significant toxicological change was observed, thus the toxic potency of e-cigarette or HTP aerosols relative to CS has not been achieved [7,14]. For such e-cigarettes and HTPs, relative comparison of toxic potency could be improved if higher concentrations of the test solution were prepared, and toxic concentration was achieved. To accurately compare the toxicity levels between CS and e-cigarette or HTP aerosols, it is crucial to employ feasibly higher exposure concentrations that in turn will make more probable the detection of toxicity in in vitro tests for e-cigarette and HTP aerosols. This will allow for a meaningful comparison of toxicity levels between CS and e-cigarette or HTP aerosols. Furthermore, such improved preparation or extraction methods may allow toxicity comparison not only with CS but also between novel tobacco product categories, such as e-cigarettes and HTPs.

In this study, we introduce a novel solvent-free extraction method for preparing highly concentrated aerosol collected mass (ACM) extracts, to assess higher treatment concentrations in in vitro tests for e-cigarette and HTP aerosols. The conventional method for preparation of ACM extracts typically involves the use of dimethyl sulfoxide (DMSO) as an extraction solvent, as recommended by the International Organization for Standardization (ISO) 20768 [15] and Health Canada Official Methods T-501 [16] and T-503 [17]. However, DMSO can produce cytotoxicity in the bacterial strains used in the Ames test [18,19] and mammalian cell lines commonly used [20], thus limiting the amount of organic solvent that can be employed. Instead of using organic solvents, we have developed a novel method that involves squeezing and extracting the components on the Cambridge filter pad (CFP) collected as ACM using liquid components originally present in ACM, such as propylene glycol and glycerol. These liquid components, traditionally used in e-cigarettes, are expected to be effective as solvents for dissolving various types of components. Moreover, they have been classified as "generally recognized as safe" (GRAS) by the FDA. A study has also indicated a reduced cytotoxicity, along with the absence of mutagenicity and genotoxicity when tested base e-liquid [21]. This method aims to generate concentrated extracts suitable for evaluating the toxicological activity of e-cigarette and HTP aerosols at higher concentrations. We compared the efficiencies of different extraction methods, including solvent-free squeezing, solvent-free centrifuging and conventional solvent extraction, to obtain various components, such as propylene glycol, glycerol and nicotine as well as the volatile flavor compounds and fatty acid esters.